WO1996015266A2 - Eukaryotic cell assay using ribosomal rna detection - Google Patents

Eukaryotic cell assay using ribosomal rna detection Download PDF

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WO1996015266A2
WO1996015266A2 PCT/US1995/014579 US9514579W WO9615266A2 WO 1996015266 A2 WO1996015266 A2 WO 1996015266A2 US 9514579 W US9514579 W US 9514579W WO 9615266 A2 WO9615266 A2 WO 9615266A2
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seq
sequence
nucleic acid
cells
ribosomal rna
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PCT/US1995/014579
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French (fr)
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WO1996015266A3 (en
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Janice T. Brown
Yu Ping Maguire
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Dade International Inc.
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Priority to AU42331/96A priority Critical patent/AU4233196A/en
Publication of WO1996015266A2 publication Critical patent/WO1996015266A2/en
Publication of WO1996015266A3 publication Critical patent/WO1996015266A3/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/136Screening for pharmacological compounds

Definitions

  • This invention relates to methods for quantitating eukaryotic cell viability or eukaryotic cell proliferation using detection of ribosomal
  • This invention also relates to detection probes, capture substances and articles of manufacture for quantitating cell viability or cell proliferation using detection of ribosomal RNA.
  • the purpose of many biological assays is to determine the effect of an exogenous agent on viability or proliferation of eukaryotic cells.
  • Such assays are used to test the effects of, for example, chemotherapeutic agents, suspected carcinogens or growth supplements and are essential in many fields such as medicine and pharmacology.
  • Examples of biological assays to measure eukaryotic cell viability or proliferation are mitogen stimulation or antigen stimulation assays, in which lymphocyte proliferation is measured in response to exposure to a known or suspected antigenic or a mitogenic agent.
  • mitogen stimulation or antigen stimulation assays in which lymphocyte proliferation is measured in response to exposure to a known or suspected antigenic or a mitogenic agent.
  • Basic and Clinical Immunology Stites, D., Stobo, J., Fudenberg, H. and Wells, J., eds., Lange Medical Publications, Los Altos, CA, 4th edition, 1982, pp. 369- 371.
  • Another cell viability or proliferation assay is a mixed leukocyte reaction (also termed mixed lymphocyte culture) assay, wherein lymphocytes of a first sample are exposed to "stimulating" lymphocytes of a second sample. The first sample lymphocytes will undergo blast transformation and/or be stimulated to proliferate if foreign HLA alleles are present on the "stimulating" lymphocytes.
  • Mixed leukocyte reaction also termed mixed lymphocyte culture
  • Chemotherapy can be used to treat more than 16 tumor types, including hematological neoplasms, sarcomas, testicular, gestational, trophoblastic, Wilm's tumors, small cell lung and ovarian cancers. Other tumors curable in the adjuvant setting are breast and colon cancers.
  • tumor chemotherapeutic treatment is based upon standard practices resulting from empirical drug selection or established protocols. Von Hoff, D.D., L. Weisenthal, Advances in Pharmacology and Chemotherapy, 17:133-156, 1980; Woltering, Eugene A., Laboratory Medicine, 21 :82, 1990.
  • the response of a particular tumor type to chemotherapy is not always predictable. Variations in response can render unattainable the primary goals of chemotherapy, which include maximizing the beneficial effects on the tumor and minimizing side effects for the patient.
  • the traditional in vitro method of growing human tumor cells in semi-solid agar developed by Salmon and Hamburger is referred to as the human tumor cloning assay (HTCA).
  • HTCA human tumor cloning assay
  • solid tumors or source The tumor specimens are mechanically and enzymatically dissociated to obtain a suspension of single cells.
  • the single cell suspension is incubated in media with or without the therapeutic drug. After the cells are exposed to the drug for one hour, control and treated cells are plated on agar.
  • the drug is added directly to the top agar layer containing cells in a 2-layer system. In both cases, the cells are incubated for 14-21 days and observed for colony formation.
  • the subrenal capsule assay utilizes human tumor specimens as first- generation transplant xenografts in athymic mice.
  • the subrenal capsule assay method differs from the traditional HTCA by utilizing tumor fragments, measuring the responsiveness of multiple cell populations rather than single-cell suspensions.
  • the predictability of drug resistance with the subrenal capsule assay (SCA) has not been found to be superior to the HTCA.
  • SCA subrenal capsule assay
  • the greatest advantage is that more tumor specimens can be successfully grown using the SCA than the HTCA, Woltering, Eugene A., supra.
  • the subrenal capsule assay has not become a routine test.
  • Rotman and coworkers have developed an in vitro chemosensitivity method (RIVCA) also referred to as the fluorescent cytoprint assay cultured.
  • RIVCA in vitro chemosensitivity method
  • the viability of the tumor cells is measured by their ability to hydrolyze fluorescein diacetate and retain the fluorescein.
  • the difference in the number of fluorescent fragments before and after drug treatment is used as a measure of drug response.
  • RIVCA is not amenable to fluid specimens or to solid specimens yielding small cell aggregates, since only aggregates larger than 50-100 cells can be photographically recorded.
  • the predictability of drug resistance with RIVCA is similar to that of HTCA, but a greater number of tumors can be grown in vitro and evaluated using RIVCA, Woltering, Eugene A., supra.
  • Another chemotherapeutic response assay uses the incorporation of tritiated thymidine into DNA as an indication of cell viability and proliferation.
  • Tumor preparations are exposed to a chemotherapeutic drug to be tested, either short-term or continuously, and cultured in liquid medium.
  • Tritiated thymidine is added and the culture incubated for 16-24 hours.
  • Cellular DNA is harvested and the amount of incorporated radioactivity is determined, e.g., with a scintillation counter.
  • a decrease in the uptake of tritiated thymidine by tumor cells exposed to cancer chemotherapeutics compared to tumor cells not so exposed indicates that the tumor is sensitive to the drug.
  • This assay has several advantages over HTCA, SCA, and RIVCA: a shorter culture period (4-6 days) is required, a smaller sample size can be assayed, and the strict requirement for single-cell suspensions -- a goal often unachievable for solid tumors - is eliminated. Another benefit of this assay is that the determination of tumor growth is more readily quantitated and automated, in contrast to many other known chemotherapeutic response assays, which involve counting of colonies by a tissue culture technician.
  • the clinical relevance of a radionucleotide detection system Kern, D., et al., Cancer Res. 45:5435-5441 (1985); Daidone, M. et al..
  • a method for determining the effect of an agent on eukaryotic cell viability or proliferation comprises the step of quantitating the amount of ribosomal RNA in a sample of eukaryotic cells exposed to the agent, by specific hybridization of a detection probe to the ribosomal RNA. The amount of hybridization is then correlated with the effect of the agent.
  • the agent may be, for example, a chemotherapeutic drug, an antigenic or mitogenic compound, or a population of lymphocytes.
  • a probe according to the method may hybridize to 18S or to 28S ribosomal RNA. If a probe hybridizes to 18S rRNA, the nucleic acid 4, SEQ. ID NO: 6, SEQ. ID NO: 7 or SEQ. ID NO: 8. Such a probe also may be a sequence that hybridizes to a sequence complementary to SEQ. ID NO: 4, SEQ. ID NO: 6, SEQ. ID NO: 7 or SEQ. ID NO: 8. If a probe hybridizes to 28S rRNA, the nucleic acid component of the probe may comprise a sequence such as SEQ. ID NO: 1 , SEQ. ID NO: 2, or a sequence that hybridizes to a sequence complementary to SEQ. ID NO: 1 or SEQ. ID NO: 2.
  • a probe may, if desired, have a label coupled to the nucleic acid, which often facilitates detection and quantitation of the amount of hybridization.
  • a label may be, for example, horseradish peroxidase, alkaline phosphate, ⁇ -galactosidase or ATPase.
  • the quantitating step may involve the inclusion of a capture substance, which hybridizes to rRNA and binds to an immobilizing compound on a solid phase.
  • the capture substance thereby immobilizes rRNA on the solid phase, which often facilitates analysis of the amount of probe hybridization.
  • the capture substance may comprise a nucleic acid that hybridizes to 18S rRNA, such as those comprising sequences SEQ. ID NO: 5 or SEQ. ID NO: 3.
  • the capture substance also may comprise a sequence that hybridizes to a sequence complementary to SEQ. ID NO: 3 or SEQ. ID NO: 5.
  • a kit is disclosed herein for determining the effect of an agent on eukaryotic cell viability or proliferation.
  • a kit comprises packaging material and a detection probe accompanying the packaging material; the probe is effective for quantitating ribosomal RNA in a sample of eukaryotic cells exposed to the agent.
  • the probe may comprise a nucleic acid effective for hybridization to 18S or to 28S rRNA and also may have a label coupled to the nucleic acid.
  • the kit may also comprise a capture substance effective for binding may comprise a nucleic acid effective for hybridization to 18S ribosomal RNA.
  • the kit may further comprise a plurality of vessels accompanying the packaging material, and the vessels may further comprise a solid phase, such as the inner surfaces of a microwell plate.
  • a selected number of the vessels may contain a pre-determined amount of a chemotherapeutic drug or an antigenic or mitogenic compound.
  • FIGURES Figure 1 shows a slot blot of RNA isolated from two different tumor cell lines that were probed with 28S ribosomal RNA detection probe BB193.
  • Figure 2 shows a slot blot of RNA isolated from two different tumor cell lines that were probed with 28S ribosomal RNA detection probe BB200.
  • Figure 3 shows a slot blot of RNA isolated from 6 different tumor cell lines and probed using 18S ribosomal RNA detection probe DET231.
  • Figure 4 shows a slot blot of RNA isolated from 6 different tumor cell lines and probed using 18S ribosomal RNA detection probe DET212.
  • Figure 5 shows a slot blot of RNA isolated from SW480 cells grown in the presence of bleomycin sulfate or no chemotherapeutic agent and probed using 28S rRNA detection probe BB200.
  • Figure 6 shows a slot blot of RNA isolated from SW480 cells grown in the presence of cis-diamminedichloro platinum or etoposide and probed using 28S rRNA detection probe BB200.
  • Figure 7 is a graph of a 3 H TdR assay of SW480 cells grown in the presence of different concentrations of chemotherapeutic agents.
  • Figure 8 is a graph of detector probe concentration versus A 450 in Figure 9 is a graph of A 450 versus cell number of line SW480 in an rRNA assay.
  • Figure 10 is a graph of A 4S0 versus cell number of line HTB158 in an rRNA assay.
  • Figure 11 is a graph showing doxorubicin hydrochloride chemoresponse assays for cell line A549.
  • Figure 12 is a graph showing bleomycin sulfate chemoresponse assays for cell line A549.
  • Figure 13 is a graph showing cis-diamminedichloroplatinum chemoresponse assays for cell line A549.
  • Figure 14 is a graph showing mitomycin-C chemoresponse assays for cell line A549.
  • Figure 15 is a graph showing doxorubicin hydrochloride chemoresponse assays for an ovarian cancer.
  • Figure 16 is a graph showing bleomycin sulfate chemoresponse assays for an ovarian cancer.
  • Figure 17 is a graph showing carboplatin chemoresponse assays for an ovarian cancer.
  • Figure 18 is a graph showing taxol chemoresponse assays for an ovarian cancer.
  • Figure 19 is a graph showing 5-Fluorouracil chemoresponse assays for an ovarian cancer.
  • RNA ribonucleic acid
  • RNA mRNA
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • Messenger RNA molecules are the templates for protein synthesis and account for less than about 1 % of the total cellular RNA in a typical activated form to the ribosome for peptide synthesis and account for about 15% of cellular RNA.
  • Ribosomal RNA molecules are a major component of ribosomes and account for about 85% of cellular RNA.
  • a method according to the invention comprises the step of quantitating ribosomal RNA in a sample of eukaryotic cells exposed to an agent to be tested for its effect on cell viability or proliferation. The amount of substantially intact rRNA in the exposed sample is determined by hybridization of a detection probe to the sample, and the amount of substantially intact ribosomal RNA in a cell sample is correlated with the effect of the agent.
  • the amount of rRNA within a viable eukaryotic cell depends upon the developmental stage, mitotic activity and cell type. Under conditions of balanced growth, the amount of rRNA present in a cell population is expected to be relatively constant on a per cell basis. However, the amount of rRNA in a population of cells exposed to a harmful agent will be less than the rRNA amount in a corresponding population not exposed to the agent. Exposure to a harmful agent results in a population composed primarily of dead, non-proliferating and/or physiologically stressed cells. rRNA amounts in dead and dying cells decrease within a short period of time, e. g., a few days and rRNA amounts in non-proliferating cells does not increase and may also decrease.
  • a population of cells exposed to an agent having a beneficial effect results in a population that has proliferated more rapidly of such a population will be greater than the rRNA amount in a corresponding sample not exposed to the agent and the rRNA amount can be correlated with the effect of the agent.
  • Eukaryotic cells that are suitable for the invention include cells grown in vitro or cells obtained from an in vivo source, e.g., a surgical specimen, biopsy or fluid sample, and may comprise a clonal population or a cell mixture.
  • an in vivo source e.g., a surgical specimen, biopsy or fluid sample
  • cells maybe from any eukaryotic species, such cells are preferably from a vertebrate, more preferably from a mammal.
  • Cells according to the invention have been exposed to an agent for which it is desired to determine the effect upon cell viability or cell proliferation.
  • the term cell viability comprises the capacity of such cells to live and/or be metabolically active even though in a resting state or a terminally differentiated state.
  • Cells according to the invention preferably have been exposed to an agent for time sufficient to exert an effect, if any, upon viability and/or proliferation.
  • the time to obtain an effect will depend upon, for example, the cell type, a known or suspected mode of action for the agent and a known or suspected type of effect (harmful or beneficial).
  • the exposure may have been in vivo or in vitro. In some instances, cells may be cultured in vitro or grown in vivo for a period of time after exposure to an agent.
  • the amount of substantially intact ribosomal RNA in exposed eukaryotic cells is measured by means of a detection probe that hybridizes to rRNA.
  • the rRNA-specific probe is hybridized to a sample of the cell population and the amount of hybridization is used to quantitate the amount of substantially intact rRNA.
  • a probe of the invention may comprise a single nucleic acid fragment or may comprise a plurality of nucleic acid fragments.
  • a probe may have some proportion of nucleotide mismatches compared to the rRNA sequence to which it must be sufficiently specific and of a suitable length so that only binding or hybridization to substantially intact rRNA is detectable under the hybridization conditions employed.
  • Ribosomal RNA sequences to which a probe specifically hybridizes typically are in a non-base paired region, e.g., an "open” or “loop” structure. Such regions generally have less base-pairing and less secondary or tertiary structure than other regions of the molecule. A region of "open” structure is believed to be more accessible for probe hybridization than base-paired and/or highly structured regions of the same molecule. The general location of "open" regions may be determined from rRNA secondary structure models. Gonzalez, I.L., et al., Proc. Natl. Acad. Sci. USA 82:7666-7670 (1985); McCallin, S.S., et al., Biochem. J.
  • Probes complementary to all of a region, to a portion of a region, or to sequences adjacent to such regions may be synthesized and one or more suitable probes may be selected, for example, by analysis of Northern blots of serial dilutions of rRNA preparations.
  • a probe according to the invention hybridizes to sequences in 28S, 18S, or 5S rRNA molecules of eukaryotes. It is preferred that a probe is capable of recognizing 18S rRNA molecules of vertebrate species, particularly of mammals. However, a probe according to the invention typically is not species-specific, and may hybridize to species as diverse as frogs and humans. Illustrative examples of nucleic acid sequences in detection probes are shown in Table 1 as SEQ. ID Nos: 1 , 2, 4, 6, 7 and 8.
  • Hybridization takes place under conditions that permit discrimination specific hybridization to other nucleic acids in the sample.
  • the stringency conditions under which hybridization may be carried out are generally understood by the skilled artisan and are set out in widely recognized protocols for nucleic acid hybridization. See, e.g., Sambrook et al, Molecular Clonino: A laboratory manual (2nd Edition), Cold Spring Harbor Laboratory Press (1989), pp. 1.101 - 1.104; 9.47 - 9.58 and 11.45 - 11.57.
  • An rRNA-specific probe may comprise a nucleic acid component with a ribose phosphate backbone, a deoxyribose phosphate backbone, or a modified ribose phosphate backbone, e.g., methyl phosphonates, phosphorothioates, or phosphorodithioates.
  • a probe may comprise a peptide nucleic acid. Egholm, M., et al., Nature 365:566- 568 (1993): Egholm, M., et al., J. Am. Chem. Soc, 1 14:9677-9678 (1992); Nielsen, P., et al., Science 254:1497-1500 (1991 ). If a peptide nucleic acid is selected for use in an rRNA probe, the number of mismatches and the hybridization conditions are adjusted to achieve the desired degree of specificity. Egholm, M., et al., Nature, supra.
  • a probe may further comprise a label-that is coupled, e.g., covalentiy linked, to a component of the probe.
  • a label typically facilitates detection and quantitation of the amount of substantially intact rRNA.
  • a label may comprise, for example, radioactive atoms incorporated into a nucleic acid component of a probe or may comprise a compound that can be directly or indirectly detected and that is coupled to a nucleic acid component.
  • a probe need not be labeled.
  • rRNA amounts may be measured by using two unlabeled oligonucleotide primers that hybridize specifically to rRNA in spaced apart relationship, followed by amplification of the intervening template and quantitation of the amplified product.
  • the unlabeled Generally it is more convenient to include a label that can be directly or indirectly detected, e.g., by chemiluminescence, bioluminescence, phosphorescence or fluorescence.
  • Suitable fluorophores include, without limitation, fluorescein-5-isothiocyanate, sulforhodamine 101 sulfonyl chloride (also known by the name Texas Red , Molecular Probes, Eugene Oregon), 7-diethylaminocoumarin-3-carboxylic acid succinimidyl ester (DECCA), 5 (and 6)-carboxytetramethylrhodamine succinimidyl ester (CTMR), 5 (and 6)-carboxydiaminorhodamine and N- (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacenepropionic acid succinimidyl ester (DMBP).
  • Chromophore labels for indirect visual detection include, without limitation, horseradish peroxidase, alkaline phosphate, ⁇ -galactosidase and ATPase. Chromophore labels typically are conjugated through an amino group to the 3' end of the oligonucleotide.
  • Hybridization of a probe to substantially intact ribosomal RNA in a sample may be carried out by solid phase hybridization, i.e., by immobilizing the rRNA on a solid phase such as a nitrocellulose membrane, a nylon membrane, controlled pore size glass beads, plastic beads, or a microwell. Alternatively, hybridization may be carried out in solution. Hybridized probe/rRNA complex may be immobilized after solution hybridization, if desired, in order to facilitate detection.
  • Hybridized probe/rRNA may be immobilized in solid phase hybridization by an rRNA-specific capture substance.
  • An rRNA-specific capture substance comprises a nucleic acid component that hybridizes to the same type of ribosomal RNA molecule of eukaryotic cells as the detection probe but at a sequence other than the sequence to which the detection probe hybridizes.
  • a capture substance also comprises a binding component, coupled to the nucleic acid, that binds to an allows hybridized probe/ribosomal RNA to be captured on the solid phase and facilitates measurement of the amount of probe that has hybridized.
  • a suitable binding component is biotin.
  • Other immobilizing compounds may be substituted for avidin or streptavidin.
  • an antibody may be used as an immobilizing compound, in which case an antigen specifically recognized by the antibody may be coupled to a nucleic acid to form a capture substance.
  • nucleic acid sequences suitable for use in a capture substance are shown in Table 1 as SEQ. ID Nos.: 3 and 5.
  • the appropriate concentration of capture substance may be readily determined in conjunction with the appropriate concentration of probe. Capture substance concentrations can be adjusted by the skilled artisan to suit the particular conditions under which rRNA amounts are being measured.
  • a capture substance comprising a biotinylated nucleic acid having the sequence of SEQ. ID No.: 5 maybe used at less than about 10 picomoles per 100 &l, preferably from about 3 pmoles to about 6 pmoles per 100 &l of hybridization mixture.
  • Hybridized probe is detected in a manner appropriate to the particular probe being used.
  • a radioactively labeled probe may be detected by autoradiography, scintillation counting and the like.
  • a fluorescently labeled probe may be detected by fluorimetry and a chromophore-labeled probe may be detected by incubation with a substrate for the label and spectrophotometrically measuring formation of a reaction product.
  • an rRNA detection probe depends effect of the agent and the label (if present) used in the detection probe.
  • the appropriate concentration of probe may be readily determined by a skilled artisan and may be adjusted to suit the particular conditions under which rRNA is being measured. For example, if the probe comprises a horseradish peroxidase label and rRNA amounts are measured in about 200 to about 50,000 cultured human cells, an appropriate concentration is from about 0.1 to about 15 pmoles of probe per 100 &l of hybridization mixture, preferably from about 1 to about 10 pmoles of probe per 100 &t of hybridization mixture.
  • the effect of the agent on the cell population may be correlated to the rRNA amount by inspection of tabular data, by plotting rRNA amounts versus agent concentration and the like.
  • curve-fitting algorithms may be used, if desired, to calculate an ID 50 .
  • Statistical analyses such as linear regression may be used.
  • a user may choose to evaluate a panel of agents for their effect on a eukaryotic cell population. In this case, the effect of any particular agent may be determined with reference to the effect of at least one other agent tested in the panel.
  • An agent to be tested in an assay may be in essentially pure form or may be a component in a mixture of ingredients.
  • the amount of agent in an assay may be pre-determined or otherwise be known by the user.
  • the method may comprise exposure of cells to an unknown amount of an agent, in which case, assays according to the invention may be used to determine the amount of agent in an unknown by comparison to a control that has known amounts of the same or a similar agent.
  • a ribosomal RNA-specific detection probe may be packaged in a kit or laboratories. Additional, optional components may be included within packaging material of the kit. For example, pre-weighed aliquots of agents to be tested, lysing buffer components, hybridization buffer components, wash solutions or vessels (e. g., microwell plates) may be included as desired, in order to make the kit more convenient to use by a technician or clinical practitioner.
  • a vessel suitable for use in a kit according to the invention comprises a solid phase.
  • the solid phase typically is the inner surface of the vessel and preferably is the inner surface of a well of a microwell plate.
  • Other suitable solid phases include, without limitation, controlled pore size glass beads, polystyrene beads, or nitrocellulose membrane.
  • An rRNA-specific capture substance effective for binding substantially intact rRNA to an immobilizing compound may also be included in a kit.
  • Methods and articles according to the invention provide numerous advantages compared to known assays of cell viability and/or proliferation. Many known assay methods are time consuming, cumbersome and expensive. Assay methods involving enumeration of cells in a cell counter or under a microscope are considered to be subjective, in that counting errors and difficulties in interpreting microscope data may lead to problems in quantitation of results. It has not been recognized heretofore that quantitating rRNA amounts in a cell sample exposed to an agent results in simple, reliable methods and articles that are amenable to automation and, in many cases, are less subjective and more sensitive.
  • a preferred embodiment of a method according to the invention comprises quantitating the amount of substantially intact rRNA in a population of tumor cells exposed to a pre-determined amount of a chemotherapeutic agent. If the tumor cells are sensitive to the will die, not proliferate, or be physiologically stressed. There will be a relatively small amount of substantially intact rRNA at the end of the exposure period compared to a population of the same tumor cells not so exposed. If the tumor cells are relatively unaffected by the agent, the amount of substantially intact rRNA in the tumor cell population will be significant and may even approach the amount in an unexposed control population. By measuring the amount of substantially intact rRNA present in populations exposed to different levels of a chemotherapeutic agent, it is possible to correlate the amount of substantially intact rRNA in the exposed sample to the sensitivity of the tumor cells.
  • Tumor samples are collected and transported to a laboratory according to standard practices and may be obtained from, for example, surgical specimens, malignant fluids, bone marrow or blood.
  • a sample may be preserved, e.g., frozen in liquid nitrogen, provided that such preservation permits subsequent recovery of viable tumor cells.
  • a sample is preserved to allow later analysis, e.g., at a laboratory distant from the site where the sample was taken or when a sufficient number of samples have been accumulated for efficient analysis of chemosensitivity.
  • a sample may be a fresh preparation, i.e, a specimen that is to have tumor cells exposed to a chemotherapeutic agent within a short period of time after the specimen is taken, without an intervening preservation step.
  • the sample may be processed using mechanical and enzymatic digestion procedures known in the art.
  • specimens typically are minced and enzyme treated.
  • cells typically are washed and resuspended in culture medium.
  • a differential cell count using trypan blue may be agents.
  • tumor cells can be concentrated with the use of Ficoll and Percoll density gradient separation methods.
  • the number of cells present in the sample may be enumerated to facilitate dispensing an appropriate number of cells into a vessel for incubation.
  • the cells may be allowed to recover, for example, in culture medium for 24 hours in a 37°C, 5% C0 2 , humidified incubator, before they are incubated with a chemotherapeutic agent.
  • Tumor cell samples are cultured by means known in the art. For example, populations may be cultured in vessels such as tissue culture bottles with or without a surface coating. However, it is preferred that samples be cultured in plastic microwell plates such as those containing 12, 24, 48 or 96 wells, e.g., Costar 3524 plates, or Falcon 3072 plates. Each well in a microwell plate serves as a vessel for incubating one sample. Plates are available in the form of strip plates or as conventional culture plates. The use of microwell plates facilitates automation of the method, since rRNA amounts in each well can be determined using known plate reader machines suitable for the type of rRNA detection probe being used. A substratum of extracellular matrix may be used to preferentially enhance the growth of the tumor cells from primary specimens. A preferred matrix is bovine cornea endothelial cell-generated extracellular matrix (BC-ECM), U.S. Patent 5,242,806, incorporated herein by reference.
  • BC-ECM bovine cornea endothelial cell-generated extracellular matrix
  • Tumor cells are aliquoted into a plurality of vessels, preferably with the same number of cells in each vessel.
  • the number of cells in each vessel will depend upon the size of the vessel, the growth rate expected for the tumor cells and the volume of medium used for culturing the cells.
  • the number of cells in each well may be from about 10 1 cells to about 10 6 cells, preferably from about cells.
  • the volume of medium per well maybe from about 20 ⁇ l to about 150 ⁇ l, preferably about 100 ⁇ l.
  • Tumor cells are cultured in a medium containing a pre-determined amount of a chemotherapeutic drug.
  • Culture medium may be any medium that is suitable for the growth of the cells being assayed, for example, Roswell Park Memorial Institute (RPMI) medium containing 1 % fetal bovine serum (FBS) and a growth supplement.
  • a growth supplement is a combination of growth factors and hormones that is suitable for the type of cell population being tested.
  • An example of a growth supplement is Cyto-Gro 289 (Scientific Products) which contains insulin, transferrin, selenium, ⁇ -estradiol, hydrocortisone, prostaglandin F2a, and epidermal growth factor.
  • CEM Computed Eagle's fetal calf serum
  • F12 fetal calf serum
  • McCoy's fetal calf serum
  • CMRL CMRL
  • a range of pre-determined drug concentrations is preferably tested, one of the concentrations generally being zero (to serve as a control).
  • the number of different concentrations and the concentration range tested depend upon previous assay experience with the chemotherapeutic drug, clinical prognosis for the tumor, assay costs and the like. For example, a drug for which there is considerable clinical experience may be tested over a narrower range and at fewer concentrations than an drug for which there is less clinical experience.
  • the drug is added before or after adding the tumor cell population, as desired.
  • a chemotherapeutic agent can be lyophilized or dried in appropriate-sized, disposable vessels such as the wells in a microwell strip, each well of the strip containing a pre-determined amount of the drug.
  • appropriate-sized, disposable vessels such as the wells in a microwell strip, each well of the strip containing a pre-determined amount of the drug.
  • Such strips fit into a housing that has a configuration similar to a microwell plate.
  • the strip format allows the test, and facilitates transfer of medium with a multiple channel pipettor.
  • the drug may be then be reconstituted with culture medium containing tumor cells.
  • Tumor cell populations are exposed to a drug for a period of time sufficient to allow substantially intact rRNA amounts in sensitive cells to decrease significantly.
  • the length of the incubation period will depend upon factors such as tumor type and culture conditions (media, temperature and the like). If desired, duplicate or triplicate samples may be used, each sample incubated for a different time to ensure that an appropriate incubation period is selected. For most tumors, the incubation period is from about 1 to about 10 days at 37°C, typically from about 3 to about 6 days at 37°C.
  • culture medium is removed and tumor cells are disrupted to release substantially intact ribosomal RNA.
  • the amount of substantially intact ribosomal RNA in each of the tumor cell samples is quantitated by hybridization to a detection probe specific for eukaryotic rRNA.
  • a horseradish peroxidase label is coupled to a nucleic acid component of the probe.
  • Hybridization is carried out with probe, disrupted cells and biotinylated capture substance present during the hybridization.
  • the hybridized mixture is transferred to a streptavidin-coated microwell and incubated for a period of time sufficient to bind the capture substance/substantially intact rRNA/detection probe complex to the microwell solid phase.
  • Bound complex is then determined by adding a horseradish peroxidase substrate such as 3,3',5,5'-tetramethyl benzidine and measuring the amount of colored product formed spectrophotometrically in a plate reader available from, for example, Baxter PRIMA (Baxter Diagnostics, Deerfield, Illinois).
  • the chemosensitivity of the tumor is determined by correlating the amount of rRNA to the effect of each drug. The amount of rRNA is plotted versus the amount of chemotherapeutic drug and the plots for each drug are compared in order to determine which drug, if any, is toxic to the tumor cells.
  • This information is provided to the clinician responsible for treatment of the patent and is used to choose a course of treatment for the patient, taking into account such factors as the concentrations of each drug achievable in plasma, the type of tumor, clinical experience with each drug, and patient medical history.
  • the assay is useful either alone, or in combination with other chemoresponse assays, in selecting a chemotherapy regimen for a patient.
  • Another embodiment of a method according to the invention comprises quantitating the amount of substantially intact rRNA in a sample of lymphocytes exposed to a pre-determined amount of at least one compound known or suspected to be an antigenic or mitogenic agent. If lymphocytes are stimulated to proliferate at the concentration of the compound to which they have been exposed, there will be a larger amount of substantially intact rRNA compared to a population of the same cells not so exposed. If lymphocytes do not respond to exposure by proliferating, the amount of substantially intact rRNA in the sample will be not be significantly larger and may even be about equal to the amount in a sample not exposed the compound. By measuring the amount of substantially intact rRNA present in samples exposed to different levels of an antigenic compound and for different times, it is possible to correlate the effect of the compound to the amount of substantially intact rRNA in the exposed samples.
  • a typical sample to be used for antigen or mitogen stimulation gradient centrifugation on Ficoll ⁇ -Hypaque ⁇ from peripheral blood of an individual About 10 4 to 10 ⁇ cells in a microwell plate are mixed with an antigenic or mitogenic agent to be tested, and incubated in a suitable medium at 37°C, 5% C0 2 for 2-8 days. Aliquots may be removed at various times during the exposure period or at the end of the exposure period. Substantially intact rRNA is determined on the aliquots as described above. The amount of substantially intact rRNA measured in the aliquots is correlated to stimulation of lymphocyte proliferation.
  • the amount of substantially intact rRNA is a sensitive measure of specific antigen-mediated cellular hypersensitivity.
  • Such measurements may be used to assess cellular immunity and/or to evaluate lymphocyte function in patients known or suspected of immune suppression or deficiency, autoimmunity, infectious disease and the like; the measurements may be used in conjunction with other tests (e.g., delayed hypersensitivity skin tests) for assessing cellular immunity.
  • Another embodiment of a method according to the invention comprises quantitating the amount of substantially intact rRNA in a first sample of "responding" lymphocytes exposed to a pre-determined amount of a second sample of lymphocytes ("stimulating" lymphocytes), which second sample serves as an agent to be tested for its effect on the responding lymphocytes.
  • the stimulatory lymphocyte preparation may be, if desired, subjected to mitomycin or irradiation before use, to prevent proliferation of stimulatory cells during the exposure period.
  • the stimulatory lymphocyte preparation may be from a potential organ donor and the responding lymphocyte preparation may be from a potential organ recipient.
  • Another embodiment of a method according to the invention comprises quantitating the amount of substantially intact rRNA in a sample of eukaryotic cells exposed to a pre-determined amount of one or more compounds to be incorporated into a growth supplement.
  • Any eukaryotic cell sample for which it is desired to quantitate the effect of a growth supplement may be used.
  • the compound to be tested is incorporated into a culture medium and the sample is incubated for a period of time sufficient to allow the presence or absence of the compound to exert an effect upon cell proliferation or viability.
  • rRNA amounts are then quantitated on the cell sample and are correlated to the effect of the compound.
  • kits suitable for chemoresponse assays comprises a detection probe, a capture substance and pre-weighed, dried, aliquots of chemotherapeutic agents in appropriate-sized, disposable vessels within a suitable packaging material.
  • Such vessels may be microwell strips that fit into a housing with a configuration similar to a microwell plate. This configuration facilitates transfer of channel pipettor.
  • Drug-containing microwell strips can be packaged in aluminum foil pouches or other air- and light-tight packaging material with desiccant, and sealed.
  • the strip format allows the user flexibility in the selection of different chemotherapeutic agents to test. Suitable embodiments of such microwell strips are disclosed in, for example, U.S. Patent No. 5,242,806, issued September 7, 1993, and incorporated herein by reference.
  • kits suitable for lymphocyte assays comprises a detection probe and a capture substance within a suitable packaging material.
  • a kit may further comprise pre-weighed, dried, aliquots of antigenic or mitogenic agents in appropriate-sized, disposable vessels such as microwell strips or a microwell plate.
  • EXAMPLE 1 Level of rRNA in Different Tumor Cell Lines An experiment was performed to determine the level of rRNA in various tumor cell lines. The experiment also evaluated the efficacy of 18S and 28S rRNA detection probes.
  • Oligonucleotides were synthesized on a Milligen 8700 DNA synthesizer using standard phosphoramidite chemistries. (Millipore, Marlborough, MA).
  • the CAP215 and CAP207 capture substances contained a biotin molecule at the 5' end of the DNA. Both capture substances were synthesized using biotin phosphoramidite (Glenn Research, Sterling, VA).
  • detector oligonucleotide sequences contained an amino group at the 3' end o'f (Glenn Research, Cat. No. 20-2957-42). Amine-containing detector oligonucleotides were conjugated through the amino group to horseradish peroxidase (HRP).
  • HRP horseradish peroxidase
  • K562 and SiHa cells were grown in RPMI medium containing 1 % fetal bovine serum (FBS) and 1X Cyto-Gro 289.
  • FBS fetal bovine serum
  • CaHa is a HPV- infected cervical carcinoma cell line. Lines were obtained from American Type Culture Collection (Rockville, Maryland).
  • RNA was isolated using RNAzol B (Teltest, Friendswood, Tx) according to the manufacturers instructions. Two different preparations of K562 RNA were used, one of which had been stored frozen until use. Serial dilutions of the target RNA from the preparations were transferred to a membrane in a slot blot apparatus. Northern blots were performed as described in U.S. Patent Application No. 08/264,556, June 23, 1994, which application is incorporated herein by reference. The probes used were 32 P-labeled 28S rRNA detection oligonucleotides (BB193 and BB200). The hybrids formed by these probes have melting temperatures (Tm) of about 54°C (BB193) and about 52°C (BB200).
  • Tm melting temperatures
  • the cell lines used were KG1 , SiHa, HeLa, CEM/s, HL-60 and K562 (ATCC, Rockville, MC).
  • KG1 is an acute myelopathic leukemia line from a 59 year old male.
  • HeLa is an HPV-18 infected cervical carcinoma cell line.
  • CEM/s is a T-cell lymphoma blastoid cell line.
  • HL-60 is a promyelocytotic cell line from a 36 year old female with acute promyelocytotic leukemia.
  • Serial dilutions of the RNA were applied to a slot blot apparatus and Northern blots were performed as described above.
  • EXAMPLE 2 Ribosomal RNA Detection Assay This example teaches that the effect of a chemotherapeutic agent on a tumor cell line can be determined by measuring the amount of rRNA present after the cell line is grown in the presence of the agent.
  • SW480 tumor cells were grown in RPMI medium, 1 % FBS and 1 % 1X Cyto-Gro 289.
  • SW480 is a coleorectal adenocarcinoma cell line.
  • About 10 4 cells in 200 ⁇ l of medium were dispensed into wells containing bleomycin sulfate (Sigma Chemical Co. St. Louis, MO, Cat. No. B507), cis-platinum diamminedichloride (Sigma, Cat. No. P4394, also known as Platinol ® or cisplatinum), and etoposide (Sigma, Cat. No. E1383, also known as VP-16).
  • the concentration of each drug ranged from 0.01 ⁇ g to 10 ⁇ g per well, as indicated in Figures 5 and 6. Cells were incubated at 37°C for 3 days and were analyzed in duplicate at each drug concentration.
  • RNA was isolated from each well using RNAzolr B.
  • the RNA was applied to a slot blot apparatus and the blot probed as described in Example 1 , using 32 P-labeled BB200 28S rRNA detection oligonucleotide.
  • An autoradiogram of the rRNA slot blot is shown in Figures 5 and 6.
  • a companion plate using the same cell line was grown under the same conditions and the same concentrations of chemotherapeutic drugs.
  • the cells on the companion plate then were pulsed with tritiated thymidine ( 3 H TdR, 1 ⁇ C ⁇ per well) for 16 hours as described in U.S. Patent 5,242,806, incorporated herein by reference.
  • Acid-precipitable radioactivity was determined using a scintillation counter; the results are plotted in Figure 7 as a percentage of the radioactivity in no-drug control wells.
  • Streptavidin-coated microwell plates were prepared by incubating in
  • Probes DET212, DET216, DET218 and DET231 were tested again as described above using a hybridization temperature of 50°C. The results are shown in Table 3. The two detector probes that had the highest absorbance were DET231 and DET212. DET218 also produced a high absorbance but the background absorbance was high with this Table 2.
  • a biopsy of an ovarian cancer (Sample No. 92018S) was obtained from a patient.
  • the specimen was minced and resuspended in RPMI medium containing 1 % FBS and 1X Cyto-Gro 289.
  • Foil packaged microwell drug strips were allowed to reach room temperature before opening the package in a laminar flow hood.
  • the microwell strips, containing pre-determined amounts of the indicated drugs, were removed from their foil pouches, placed in a plate frame and snapped firmly into place.
  • a strip label at the bottom of the frame was coded according to the particular drug present in that strip.
  • the drugs used in this experiment were doxorubicin hydrochloride (also known as Adriamycin ® ), mitomycin-C, 5-fluorouracil, L-Pharm, bleomycin sulfate, cis-platinum and VP16.
  • the amounts used were 0, 0.01 , 0.1 and 1.0 ⁇ g per well.
  • Tumor cells (about 10 4 cells) in 100 ⁇ l of medium were then transferred into each well in a laminar flow hood using sterile techniques. Transfer was facilitated by the use of a multi-channel pipettor and V-shaped reservoir (Costar, Cambridge, MA). The dispensing procedure was completed in less than one minute to ensure an even distribution of cells.
  • the vessels were covered and incubated at 37°C, 5% C0 2 for 3 days in a fully humidified incubator. At the end of the incubation period, the cells were centrifuged, the supernatant was discarded and 50 ⁇ l of lysing buffer (0.3% SDS and 5 mM vanadyl ribonucleoside complex, VRC) were added to each well of the incubation plate.
  • the cells were incubated in the lysing solution for about 5 minutes at about room temperature. About 50 ⁇ l of the hybridization solution of Example 3, containing 4 picomoles each of horseradish peroxidase-conjugated DET231 and DET212 and 4 picomoles of CAP215 were added to each well. The lysed cells and hybridization solution were gently mixed by tapping the corner of the microwell plate and incubated at 50°C for 30 minutes.
  • the lysed cell/hybridization mixtures was transferred to corresponding streptavidin-coated wells of an assay plate, the assay plate was rinsed three times with wash buffer (2X SSC, 0.05% Tween- 20 ® and 0.01 % thimerosal) and two drops (about 100 ⁇ l) of 0.04% 3,3 * ,5,5'-tetramethyl benzidine and 0.02% hydrogen peroxide were added to each well. The plate was incubated at room temperature for 15 minutes, avoiding light. After the 15 minute period, about 100 ⁇ l of 1 M H 3 PO 4 was added to each well. The A 450 of each well was measured and recorded. The results are shown in Table 7.
  • a 450 and detection probe concentration was examined at a constant capture substance concentration and constant cell number.
  • the cells were obtained from a primary ovarian cancer (Specimen 920069). The specimen was minced and trypsinized, cultured overnight in RPMI-1640, 1 % FBS and frozen in liquid nitrogen.
  • lysing buffer (0.3% SDS and 5 mM VRC) were added to each well of the incubation plate. The cells were incubated in the lysing solution for about 5 minutes at room temperature. Hybridization solution (50 ⁇ l), containing 5 picomoles of
  • streptavidin-coated microwell plates were blocked with 300 ⁇ l of blocking buffer (5X SSC, 5X Denhardt's solution, 1 % BSA, 0.1 % Tween-20 and 0.2 ng/ml sheared salmon sperm DNA). The blocking buffer was removed before transferring the hybridization mixture.
  • blocking buffer 5X SSC, 5X Denhardt's solution, 1 % BSA, 0.1 % Tween-20 and 0.2 ng/ml sheared salmon sperm DNA.
  • each mixture was transferred to a streptavidin- coated assay well plate and incubated for 45 minutes at 50° C.
  • the assay plates then were rinsed four times with wash buffer (2X SSC, 0.05% Tween-20).
  • Two drops (about 100 ⁇ l) of 0.04% 3,3', 5,5'- tetramethyl benzidine and 0.02% hydrogen peroxide were then added to each well and the mixture incubated at room temperature for 15 minutes, avoiding light. After the 15 minute period, about 50 ⁇ l of 1 M H 3 PO 4 was added to each well. The A 450 of each well was measured and recorded. The results are shown in Figure 8.
  • EXAMPLE 8 Correlation Between Cell Number and A- ⁇ The relationship between the number of tumor cells and A 450 was examined for 2 different tumor cell lines.
  • SW480 cells were aliquoted into microwells at 400, 2,000, 10,000 and 50,000 cells per well.
  • HTB158 cells were aliquoted into microwells at 300, 600, 1 ,200, 2,500, 5,000, 10,000, 20,000 and 40,000 cells per well.
  • rRNA amounts were measured as described in Example 7, using 2.5 pmoles of CAP215 and 1 pmole of DET212.
  • the results for the SW480 line are shown in Figure 9.
  • the results for the HTB158 line are shown in Figure 10.
  • Tumor cells were obtained from a human lung cancer cell line, A549 (ATCC, Rockville, MD). Cells were washed with RPMI, 1 % FBS and resuspended in RPMI, 1 % FBS, IX Cyto-Gro 289TM The cells were plated at about 1.5 X 103 cells per well in BC-ECM coated microwell plates and cultured in the presence of chemotherapeutic drugs for 4 days as described in Example 6. BC-ECM coated microwell plates were prepared as described in U.S. Patent 5,242,806, issued Sept. 7, 1993 and incorporated herein by reference.
  • the drugs used in the experiment were: Adriamycin ® , bleomycin sulfate, Platinol ® and mitomycin-C at concentrations from about 0.01 ⁇ g/ml to about 10 ⁇ g/ml.
  • an rRNA assay was carried out as described in Example 7, using 5 pmoles CAP215 and 1 pmole of DET212.
  • A549 cells were exposed to chemotherapeutic drugs as described above in two separate plates.
  • an MTT assay was carried out by adding 25 ⁇ l of MTT solution to each well.
  • MTT solution contained 5 mg/ml MTT in phosphate buffered saline (PBS), stored at -20°C. After incubating for 4 hours at 37°C, the blue formazan product was dissolved in DMSO and the absorbance at 570 nm was measured.
  • PBS phosphate buffered saline
  • DMSO DMSO
  • a 3 H TdR assay was carried out as described in U.S. Patent 5,242,806, issued September 7, 1993, incorporated herein by reference. The 3 H TdR (specific activity 6.7 //Ci/ml) was added at 1 Ci/well.
  • This example compares an rRNA assay to a 3 H TdR assay and an MTT assay in measuring the effect of a chemotherapeutic drug upon cell viability and/or cell proliferation of a clinical tumor specimen.
  • Tumor cells were obtained from a primary ovarian cancer. The specimen was minced, trypsinized, counted, and incubated overnight in 1 % DMEM. Cells were plated and cultured in the presence of chemotherapeutic drugs for 4 days as described in Example 9.
  • the drugs used in the experiment were: Adriamycin ® , bleomycin sulfate, cis- diammine[1 ,1-cyclobutane-dicarboxylato] platinum (also known as carboplatin), paclitaxel from Taxus brevifolia (also known as taxol) and 5-fluorouracil.
  • Adriamycin ® bleomycin sulfate
  • cis- diammine[1 ,1-cyclobutane-dicarboxylato] platinum also known as carboplatin
  • paclitaxel from Taxus brevifolia
  • 5-fluorouracil 5-fluorouracil.
  • Carboplatin and taxol were obtained from Sigma or Bristol-Myers.

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Abstract

An assay to determine the effect of an agent on eukaryotic cell viability or cell proliferation is disclosed. The assay involves quantitating the amount of ribosomal RNA in cells by hybridizing the ribosomal RNA in the sample with a detection probe. The amount of rRNA present in the sample is correlated with the effect of the agent on the viability or proliferation of the cells. The assay may be used to test the effect of chemotherapeutic agents or growth supplements on eukaryotic cells, or to measure histocompatibility in a mixed lymphocyte response assay.

Description

EUKARYOTIC CELL ASSAY USING RIBOSOMAL RNA DETECTION
F1EIP OF THE INVENTION
This invention relates to methods for quantitating eukaryotic cell viability or eukaryotic cell proliferation using detection of ribosomal
RNA. This invention also relates to detection probes, capture substances and articles of manufacture for quantitating cell viability or cell proliferation using detection of ribosomal RNA.
BACKGROUND OF THE INVENTION
The purpose of many biological assays is to determine the effect of an exogenous agent on viability or proliferation of eukaryotic cells. Such assays are used to test the effects of, for example, chemotherapeutic agents, suspected carcinogens or growth supplements and are essential in many fields such as medicine and pharmacology.
Examples of biological assays to measure eukaryotic cell viability or proliferation are mitogen stimulation or antigen stimulation assays, in which lymphocyte proliferation is measured in response to exposure to a known or suspected antigenic or a mitogenic agent. Basic and Clinical Immunology, Stites, D., Stobo, J., Fudenberg, H. and Wells, J., eds., Lange Medical Publications, Los Altos, CA, 4th edition, 1982, pp. 369- 371.
Another cell viability or proliferation assay is a mixed leukocyte reaction (also termed mixed lymphocyte culture) assay, wherein lymphocytes of a first sample are exposed to "stimulating" lymphocytes of a second sample. The first sample lymphocytes will undergo blast transformation and/or be stimulated to proliferate if foreign HLA alleles are present on the "stimulating" lymphocytes. Basic and Clinical Immunology, supra, pp. 56-58, pp. 189-192 and pp. 371-372.
Another example of a biological assay to measure cell viability or proliferation is a chemotherapy response assay. Chemotherapy can be used to treat more than 16 tumor types, including hematological neoplasms, sarcomas, testicular, gestational, trophoblastic, Wilm's tumors, small cell lung and ovarian cancers. Other tumors curable in the adjuvant setting are breast and colon cancers.
Currently, tumor chemotherapeutic treatment is based upon standard practices resulting from empirical drug selection or established protocols. Von Hoff, D.D., L. Weisenthal, Advances in Pharmacology and Chemotherapy, 17:133-156, 1980; Woltering, Eugene A., Laboratory Medicine, 21 :82, 1990. However, it is known in the art that the response of a particular tumor type to chemotherapy is not always predictable. Variations in response can render unattainable the primary goals of chemotherapy, which include maximizing the beneficial effects on the tumor and minimizing side effects for the patient.
The development of an in vitro assay which could predict the response of a tumor to chemotherapeutic agents has been a longstanding objective in cancer research. Current in vitro chemotherapy response assays include: the cloning of human tumors on double layer soft agar, i.e., human tumor cloning assay (HTCA), subrenal capsule assay method, fluorescent cytoprint assay (Rotman RIVCA) and tritiated thymidine uptake assay. Shoemaker, R., et al.. Cancer Res. 45:2145 (1985); Woltering, E., Laboratory Medicine 2:82 (1990); Kern, D., et al., Cancer Res. 45:5435 (1985).
The traditional in vitro method of growing human tumor cells in semi-solid agar developed by Salmon and Hamburger is referred to as the human tumor cloning assay (HTCA). In this method, solid tumors or source. The tumor specimens are mechanically and enzymatically dissociated to obtain a suspension of single cells. When short-term drug treatment is being evaluated, the single cell suspension is incubated in media with or without the therapeutic drug. After the cells are exposed to the drug for one hour, control and treated cells are plated on agar. For continuous drug treatment, the drug is added directly to the top agar layer containing cells in a 2-layer system. In both cases, the cells are incubated for 14-21 days and observed for colony formation. The different in the number of colonies counted in plates containing drug treated cells and in control plates is used to determine drug responsiveness. Shoemaker, R. H., et al.. Cancer Research, 45:2145- 2153, 1985, Woltering, Eugene A., Laboratory Medicine, 2:82-84, 1990. Due to difficulties in obtaining single-cell suspensions from solid tumors, long incubation times, and poor tumor growth in the HTCA, alternative methods would be desirable.
The subrenal capsule assay utilizes human tumor specimens as first- generation transplant xenografts in athymic mice. The subrenal capsule assay method differs from the traditional HTCA by utilizing tumor fragments, measuring the responsiveness of multiple cell populations rather than single-cell suspensions. The predictability of drug resistance with the subrenal capsule assay (SCA) has not been found to be superior to the HTCA. The greatest advantage is that more tumor specimens can be successfully grown using the SCA than the HTCA, Woltering, Eugene A., supra. However, due to the expense involved in maintaining mouse colonies and the technical expertise required, the subrenal capsule assay has not become a routine test.
Rotman and coworkers have developed an in vitro chemosensitivity method (RIVCA) also referred to as the fluorescent cytoprint assay cultured. The viability of the tumor cells is measured by their ability to hydrolyze fluorescein diacetate and retain the fluorescein. The difference in the number of fluorescent fragments before and after drug treatment is used as a measure of drug response. RIVCA is not amenable to fluid specimens or to solid specimens yielding small cell aggregates, since only aggregates larger than 50-100 cells can be photographically recorded. The predictability of drug resistance with RIVCA is similar to that of HTCA, but a greater number of tumors can be grown in vitro and evaluated using RIVCA, Woltering, Eugene A., supra.
Another chemotherapeutic response assay uses the incorporation of tritiated thymidine into DNA as an indication of cell viability and proliferation. Tumor preparations are exposed to a chemotherapeutic drug to be tested, either short-term or continuously, and cultured in liquid medium. Tritiated thymidine is added and the culture incubated for 16-24 hours. Cellular DNA is harvested and the amount of incorporated radioactivity is determined, e.g., with a scintillation counter. A decrease in the uptake of tritiated thymidine by tumor cells exposed to cancer chemotherapeutics compared to tumor cells not so exposed indicates that the tumor is sensitive to the drug. This assay has several advantages over HTCA, SCA, and RIVCA: a shorter culture period (4-6 days) is required, a smaller sample size can be assayed, and the strict requirement for single-cell suspensions -- a goal often unachievable for solid tumors - is eliminated. Another benefit of this assay is that the determination of tumor growth is more readily quantitated and automated, in contrast to many other known chemotherapeutic response assays, which involve counting of colonies by a tissue culture technician. The clinical relevance of a radionucleotide detection system Kern, D., et al., Cancer Res. 45:5435-5441 (1985); Daidone, M. et al.. Cancer 56:450-456 (1985); Tanigawa, N., et al., Cancer Res. 42:2 59 (1982); Wilson, A., et al., Brit. J. Cancer 49:57-63 (1984). An in vitro chemoresponse assay is not in general use because of a number of obstacles which have contributed to the lack of clinical feasibility of the assay. Currently, in vitro drug response assays are performed in university hospitals and a few specialized service centers. These institutions generally require the transportation of the specimen, resulting in a loss of often more than 24 hours before specimen processing can begin. Within this 24 hour period, specimen viability declines significantly. Additionally, an in vitro assay is not in general use because of difficulties in trying to model in vivo pharmacokinetics. Certain of these assays require single-cell suspensions, which are difficult or impossible to obtain from solid tumors. Further, certain assays are time-consuming, expensive, technically demanding or may require a large tumor sample.
SUMMARY OF THE INVENTION
A method is disclosed for determining the effect of an agent on eukaryotic cell viability or proliferation. The method comprises the step of quantitating the amount of ribosomal RNA in a sample of eukaryotic cells exposed to the agent, by specific hybridization of a detection probe to the ribosomal RNA. The amount of hybridization is then correlated with the effect of the agent. The agent may be, for example, a chemotherapeutic drug, an antigenic or mitogenic compound, or a population of lymphocytes.
A probe according to the method may hybridize to 18S or to 28S ribosomal RNA. If a probe hybridizes to 18S rRNA, the nucleic acid 4, SEQ. ID NO: 6, SEQ. ID NO: 7 or SEQ. ID NO: 8. Such a probe also may be a sequence that hybridizes to a sequence complementary to SEQ. ID NO: 4, SEQ. ID NO: 6, SEQ. ID NO: 7 or SEQ. ID NO: 8. If a probe hybridizes to 28S rRNA, the nucleic acid component of the probe may comprise a sequence such as SEQ. ID NO: 1 , SEQ. ID NO: 2, or a sequence that hybridizes to a sequence complementary to SEQ. ID NO: 1 or SEQ. ID NO: 2.
A probe may, if desired, have a label coupled to the nucleic acid, which often facilitates detection and quantitation of the amount of hybridization. Such a label may be, for example, horseradish peroxidase, alkaline phosphate, β-galactosidase or ATPase.
The quantitating step may involve the inclusion of a capture substance, which hybridizes to rRNA and binds to an immobilizing compound on a solid phase. The capture substance thereby immobilizes rRNA on the solid phase, which often facilitates analysis of the amount of probe hybridization. The capture substance may comprise a nucleic acid that hybridizes to 18S rRNA, such as those comprising sequences SEQ. ID NO: 5 or SEQ. ID NO: 3. The capture substance also may comprise a sequence that hybridizes to a sequence complementary to SEQ. ID NO: 3 or SEQ. ID NO: 5.
A kit is disclosed herein for determining the effect of an agent on eukaryotic cell viability or proliferation. A kit comprises packaging material and a detection probe accompanying the packaging material; the probe is effective for quantitating ribosomal RNA in a sample of eukaryotic cells exposed to the agent. The probe may comprise a nucleic acid effective for hybridization to 18S or to 28S rRNA and also may have a label coupled to the nucleic acid.
The kit may also comprise a capture substance effective for binding may comprise a nucleic acid effective for hybridization to 18S ribosomal RNA.
The kit may further comprise a plurality of vessels accompanying the packaging material, and the vessels may further comprise a solid phase, such as the inner surfaces of a microwell plate. A selected number of the vessels may contain a pre-determined amount of a chemotherapeutic drug or an antigenic or mitogenic compound.
BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows a slot blot of RNA isolated from two different tumor cell lines that were probed with 28S ribosomal RNA detection probe BB193.
Figure 2 shows a slot blot of RNA isolated from two different tumor cell lines that were probed with 28S ribosomal RNA detection probe BB200.
Figure 3 shows a slot blot of RNA isolated from 6 different tumor cell lines and probed using 18S ribosomal RNA detection probe DET231.
Figure 4 shows a slot blot of RNA isolated from 6 different tumor cell lines and probed using 18S ribosomal RNA detection probe DET212. Figure 5 shows a slot blot of RNA isolated from SW480 cells grown in the presence of bleomycin sulfate or no chemotherapeutic agent and probed using 28S rRNA detection probe BB200.
Figure 6 shows a slot blot of RNA isolated from SW480 cells grown in the presence of cis-diamminedichloro platinum or etoposide and probed using 28S rRNA detection probe BB200.
Figure 7 is a graph of a 3H TdR assay of SW480 cells grown in the presence of different concentrations of chemotherapeutic agents.
Figure 8 is a graph of detector probe concentration versus A450 in Figure 9 is a graph of A450 versus cell number of line SW480 in an rRNA assay.
Figure 10 is a graph of A4S0 versus cell number of line HTB158 in an rRNA assay. Figure 11 is a graph showing doxorubicin hydrochloride chemoresponse assays for cell line A549.
Figure 12 is a graph showing bleomycin sulfate chemoresponse assays for cell line A549.
Figure 13 is a graph showing cis-diamminedichloroplatinum chemoresponse assays for cell line A549.
Figure 14 is a graph showing mitomycin-C chemoresponse assays for cell line A549.
Figure 15 is a graph showing doxorubicin hydrochloride chemoresponse assays for an ovarian cancer. Figure 16 is a graph showing bleomycin sulfate chemoresponse assays for an ovarian cancer.
Figure 17 is a graph showing carboplatin chemoresponse assays for an ovarian cancer.
Figure 18 is a graph showing taxol chemoresponse assays for an ovarian cancer.
Figure 19 is a graph showing 5-Fluorouracil chemoresponse assays for an ovarian cancer.
DETAILED DESCRIPTION OF THE INVENTION Living cells contain three types of ribonucleic acid (RNA): messenger
RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA). Messenger RNA molecules are the templates for protein synthesis and account for less than about 1 % of the total cellular RNA in a typical activated form to the ribosome for peptide synthesis and account for about 15% of cellular RNA. Ribosomal RNA molecules are a major component of ribosomes and account for about 85% of cellular RNA. A method according to the invention comprises the step of quantitating ribosomal RNA in a sample of eukaryotic cells exposed to an agent to be tested for its effect on cell viability or proliferation. The amount of substantially intact rRNA in the exposed sample is determined by hybridization of a detection probe to the sample, and the amount of substantially intact ribosomal RNA in a cell sample is correlated with the effect of the agent.
The amount of rRNA within a viable eukaryotic cell depends upon the developmental stage, mitotic activity and cell type. Under conditions of balanced growth, the amount of rRNA present in a cell population is expected to be relatively constant on a per cell basis. However, the amount of rRNA in a population of cells exposed to a harmful agent will be less than the rRNA amount in a corresponding population not exposed to the agent. Exposure to a harmful agent results in a population composed primarily of dead, non-proliferating and/or physiologically stressed cells. rRNA amounts in dead and dying cells decrease within a short period of time, e. g., a few days and rRNA amounts in non-proliferating cells does not increase and may also decrease. If an agent is harmful, it is believed that within the exposed sample some rRNA molecules may be intact while some are partially or completely degraded due to physiological stress or cell death. The result is that the overall level of hybridization to a probe is lower compared to the level of hybridization in an unexposed sample.
Conversely, a population of cells exposed to an agent having a beneficial effect results in a population that has proliferated more rapidly of such a population will be greater than the rRNA amount in a corresponding sample not exposed to the agent and the rRNA amount can be correlated with the effect of the agent.
Eukaryotic cells that are suitable for the invention include cells grown in vitro or cells obtained from an in vivo source, e.g., a surgical specimen, biopsy or fluid sample, and may comprise a clonal population or a cell mixture. Although cells maybe from any eukaryotic species, such cells are preferably from a vertebrate, more preferably from a mammal. Cells according to the invention have been exposed to an agent for which it is desired to determine the effect upon cell viability or cell proliferation. In this sense, the term cell viability comprises the capacity of such cells to live and/or be metabolically active even though in a resting state or a terminally differentiated state. Cells according to the invention preferably have been exposed to an agent for time sufficient to exert an effect, if any, upon viability and/or proliferation. The time to obtain an effect will depend upon, for example, the cell type, a known or suspected mode of action for the agent and a known or suspected type of effect (harmful or beneficial). The exposure may have been in vivo or in vitro. In some instances, cells may be cultured in vitro or grown in vivo for a period of time after exposure to an agent.
The amount of substantially intact ribosomal RNA in exposed eukaryotic cells is measured by means of a detection probe that hybridizes to rRNA. The rRNA-specific probe is hybridized to a sample of the cell population and the amount of hybridization is used to quantitate the amount of substantially intact rRNA. A probe of the invention may comprise a single nucleic acid fragment or may comprise a plurality of nucleic acid fragments. A probe may have some proportion of nucleotide mismatches compared to the rRNA sequence to which it must be sufficiently specific and of a suitable length so that only binding or hybridization to substantially intact rRNA is detectable under the hybridization conditions employed.
Ribosomal RNA sequences to which a probe specifically hybridizes typically are in a non-base paired region, e.g., an "open" or "loop" structure. Such regions generally have less base-pairing and less secondary or tertiary structure than other regions of the molecule. A region of "open" structure is believed to be more accessible for probe hybridization than base-paired and/or highly structured regions of the same molecule. The general location of "open" regions may be determined from rRNA secondary structure models. Gonzalez, I.L., et al., Proc. Natl. Acad. Sci. USA 82:7666-7670 (1985); McCallin, S.S., et al., Biochem. J. 232:725-733 (1985); Raue, H.A., et al., Prog. Biophys. Mol. Biol. 51 :77-129 (1988); Noller, H.F., et al., Ann. Rev. Biochem. 53:119-162 (1984); Gonzalez, I.L, et al.. Am. J. Hum. Gen. 38:419- 427 (1986). Probes complementary to all of a region, to a portion of a region, or to sequences adjacent to such regions may be synthesized and one or more suitable probes may be selected, for example, by analysis of Northern blots of serial dilutions of rRNA preparations. A probe according to the invention hybridizes to sequences in 28S, 18S, or 5S rRNA molecules of eukaryotes. It is preferred that a probe is capable of recognizing 18S rRNA molecules of vertebrate species, particularly of mammals. However, a probe according to the invention typically is not species-specific, and may hybridize to species as diverse as frogs and humans. Illustrative examples of nucleic acid sequences in detection probes are shown in Table 1 as SEQ. ID Nos: 1 , 2, 4, 6, 7 and 8.
Hybridization takes place under conditions that permit discrimination specific hybridization to other nucleic acids in the sample. The stringency conditions under which hybridization may be carried out are generally understood by the skilled artisan and are set out in widely recognized protocols for nucleic acid hybridization. See, e.g., Sambrook et al, Molecular Clonino: A laboratory manual (2nd Edition), Cold Spring Harbor Laboratory Press (1989), pp. 1.101 - 1.104; 9.47 - 9.58 and 11.45 - 11.57.
An rRNA-specific probe may comprise a nucleic acid component with a ribose phosphate backbone, a deoxyribose phosphate backbone, or a modified ribose phosphate backbone, e.g., methyl phosphonates, phosphorothioates, or phosphorodithioates. Alternatively, a probe may comprise a peptide nucleic acid. Egholm, M., et al., Nature 365:566- 568 (1993): Egholm, M., et al., J. Am. Chem. Soc, 1 14:9677-9678 (1992); Nielsen, P., et al., Science 254:1497-1500 (1991 ). If a peptide nucleic acid is selected for use in an rRNA probe, the number of mismatches and the hybridization conditions are adjusted to achieve the desired degree of specificity. Egholm, M., et al., Nature, supra.
A probe may further comprise a label-that is coupled, e.g., covalentiy linked, to a component of the probe. A label typically facilitates detection and quantitation of the amount of substantially intact rRNA. A label may comprise, for example, radioactive atoms incorporated into a nucleic acid component of a probe or may comprise a compound that can be directly or indirectly detected and that is coupled to a nucleic acid component. On the other hand, a probe need not be labeled. For example, rRNA amounts may be measured by using two unlabeled oligonucleotide primers that hybridize specifically to rRNA in spaced apart relationship, followed by amplification of the intervening template and quantitation of the amplified product. The unlabeled Generally it is more convenient to include a label that can be directly or indirectly detected, e.g., by chemiluminescence, bioluminescence, phosphorescence or fluorescence. Suitable fluorophores include, without limitation, fluorescein-5-isothiocyanate, sulforhodamine 101 sulfonyl chloride (also known by the name Texas Red , Molecular Probes, Eugene Oregon), 7-diethylaminocoumarin-3-carboxylic acid succinimidyl ester (DECCA), 5 (and 6)-carboxytetramethylrhodamine succinimidyl ester (CTMR), 5 (and 6)-carboxydiaminorhodamine and N- (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacenepropionic acid succinimidyl ester (DMBP). Chromophore labels for indirect visual detection include, without limitation, horseradish peroxidase, alkaline phosphate, β-galactosidase and ATPase. Chromophore labels typically are conjugated through an amino group to the 3' end of the oligonucleotide. Hybridization of a probe to substantially intact ribosomal RNA in a sample may be carried out by solid phase hybridization, i.e., by immobilizing the rRNA on a solid phase such as a nitrocellulose membrane, a nylon membrane, controlled pore size glass beads, plastic beads, or a microwell. Alternatively, hybridization may be carried out in solution. Hybridized probe/rRNA complex may be immobilized after solution hybridization, if desired, in order to facilitate detection.
Hybridized probe/rRNA may be immobilized in solid phase hybridization by an rRNA-specific capture substance. An rRNA-specific capture substance comprises a nucleic acid component that hybridizes to the same type of ribosomal RNA molecule of eukaryotic cells as the detection probe but at a sequence other than the sequence to which the detection probe hybridizes. A capture substance also comprises a binding component, coupled to the nucleic acid, that binds to an allows hybridized probe/ribosomal RNA to be captured on the solid phase and facilitates measurement of the amount of probe that has hybridized.
If the immobilizing compound is streptavidin, a suitable binding component is biotin. Other immobilizing compounds may be substituted for avidin or streptavidin. For example, an antibody may be used as an immobilizing compound, in which case an antigen specifically recognized by the antibody may be coupled to a nucleic acid to form a capture substance. Illustrative examples of nucleic acid sequences suitable for use in a capture substance are shown in Table 1 as SEQ. ID Nos.: 3 and 5. The appropriate concentration of capture substance may be readily determined in conjunction with the appropriate concentration of probe. Capture substance concentrations can be adjusted by the skilled artisan to suit the particular conditions under which rRNA amounts are being measured. For example, if a probe is hybridized to a sample at about 1 picomole per 100 &l, a capture substance comprising a biotinylated nucleic acid having the sequence of SEQ. ID No.: 5 maybe used at less than about 10 picomoles per 100 &l, preferably from about 3 pmoles to about 6 pmoles per 100 &l of hybridization mixture.
Hybridized probe is detected in a manner appropriate to the particular probe being used. For example, a radioactively labeled probe may be detected by autoradiography, scintillation counting and the like. A fluorescently labeled probe may be detected by fluorimetry and a chromophore-labeled probe may be detected by incubation with a substrate for the label and spectrophotometrically measuring formation of a reaction product.
The appropriate concentration of an rRNA detection probe depends effect of the agent and the label (if present) used in the detection probe. The appropriate concentration of probe may be readily determined by a skilled artisan and may be adjusted to suit the particular conditions under which rRNA is being measured. For example, if the probe comprises a horseradish peroxidase label and rRNA amounts are measured in about 200 to about 50,000 cultured human cells, an appropriate concentration is from about 0.1 to about 15 pmoles of probe per 100 &l of hybridization mixture, preferably from about 1 to about 10 pmoles of probe per 100 &t of hybridization mixture. Once rRNA amounts have been measured, the effect of the agent on the cell population may be correlated to the rRNA amount by inspection of tabular data, by plotting rRNA amounts versus agent concentration and the like. When using graphs to correlate an effect to rRNA amounts, curve-fitting algorithms may be used, if desired, to calculate an ID50. Statistical analyses such as linear regression may be used. Furthermore, a user may choose to evaluate a panel of agents for their effect on a eukaryotic cell population. In this case, the effect of any particular agent may be determined with reference to the effect of at least one other agent tested in the panel. An agent to be tested in an assay may be in essentially pure form or may be a component in a mixture of ingredients. The amount of agent in an assay may be pre-determined or otherwise be known by the user. On the other hand, the method may comprise exposure of cells to an unknown amount of an agent, in which case, assays according to the invention may be used to determine the amount of agent in an unknown by comparison to a control that has known amounts of the same or a similar agent.
A ribosomal RNA-specific detection probe may be packaged in a kit or laboratories. Additional, optional components may be included within packaging material of the kit. For example, pre-weighed aliquots of agents to be tested, lysing buffer components, hybridization buffer components, wash solutions or vessels (e. g., microwell plates) may be included as desired, in order to make the kit more convenient to use by a technician or clinical practitioner.
A vessel suitable for use in a kit according to the invention comprises a solid phase. The solid phase typically is the inner surface of the vessel and preferably is the inner surface of a well of a microwell plate. Other suitable solid phases include, without limitation, controlled pore size glass beads, polystyrene beads, or nitrocellulose membrane.
An rRNA-specific capture substance effective for binding substantially intact rRNA to an immobilizing compound may also be included in a kit.
Methods and articles according to the invention provide numerous advantages compared to known assays of cell viability and/or proliferation. Many known assay methods are time consuming, cumbersome and expensive. Assay methods involving enumeration of cells in a cell counter or under a microscope are considered to be subjective, in that counting errors and difficulties in interpreting microscope data may lead to problems in quantitation of results. It has not been recognized heretofore that quantitating rRNA amounts in a cell sample exposed to an agent results in simple, reliable methods and articles that are amenable to automation and, in many cases, are less subjective and more sensitive. A preferred embodiment of a method according to the invention comprises quantitating the amount of substantially intact rRNA in a population of tumor cells exposed to a pre-determined amount of a chemotherapeutic agent. If the tumor cells are sensitive to the will die, not proliferate, or be physiologically stressed. There will be a relatively small amount of substantially intact rRNA at the end of the exposure period compared to a population of the same tumor cells not so exposed. If the tumor cells are relatively unaffected by the agent, the amount of substantially intact rRNA in the tumor cell population will be significant and may even approach the amount in an unexposed control population. By measuring the amount of substantially intact rRNA present in populations exposed to different levels of a chemotherapeutic agent, it is possible to correlate the amount of substantially intact rRNA in the exposed sample to the sensitivity of the tumor cells.
Tumor samples are collected and transported to a laboratory according to standard practices and may be obtained from, for example, surgical specimens, malignant fluids, bone marrow or blood. A sample may be preserved, e.g., frozen in liquid nitrogen, provided that such preservation permits subsequent recovery of viable tumor cells. A sample is preserved to allow later analysis, e.g., at a laboratory distant from the site where the sample was taken or when a sufficient number of samples have been accumulated for efficient analysis of chemosensitivity. Alternatively, a sample may be a fresh preparation, i.e, a specimen that is to have tumor cells exposed to a chemotherapeutic agent within a short period of time after the specimen is taken, without an intervening preservation step.
The sample may be processed using mechanical and enzymatic digestion procedures known in the art. For primary human tumors from surgery, specimens typically are minced and enzyme treated. For malignant fluid specimens, cells typically are washed and resuspended in culture medium. A differential cell count using trypan blue may be agents. If desired, tumor cells can be concentrated with the use of Ficoll and Percoll density gradient separation methods. The number of cells present in the sample may be enumerated to facilitate dispensing an appropriate number of cells into a vessel for incubation. The cells may be allowed to recover, for example, in culture medium for 24 hours in a 37°C, 5% C02, humidified incubator, before they are incubated with a chemotherapeutic agent.
Tumor cell samples are cultured by means known in the art. For example, populations may be cultured in vessels such as tissue culture bottles with or without a surface coating. However, it is preferred that samples be cultured in plastic microwell plates such as those containing 12, 24, 48 or 96 wells, e.g., Costar 3524 plates, or Falcon 3072 plates. Each well in a microwell plate serves as a vessel for incubating one sample. Plates are available in the form of strip plates or as conventional culture plates. The use of microwell plates facilitates automation of the method, since rRNA amounts in each well can be determined using known plate reader machines suitable for the type of rRNA detection probe being used. A substratum of extracellular matrix may be used to preferentially enhance the growth of the tumor cells from primary specimens. A preferred matrix is bovine cornea endothelial cell-generated extracellular matrix (BC-ECM), U.S. Patent 5,242,806, incorporated herein by reference.
Tumor cells are aliquoted into a plurality of vessels, preferably with the same number of cells in each vessel. The number of cells in each vessel will depend upon the size of the vessel, the growth rate expected for the tumor cells and the volume of medium used for culturing the cells. For a 96 well microwell plate, the number of cells in each well may be from about 101 cells to about 106 cells, preferably from about cells. The volume of medium per well maybe from about 20 μl to about 150 μl, preferably about 100 μl.
Tumor cells are cultured in a medium containing a pre-determined amount of a chemotherapeutic drug. Culture medium may be any medium that is suitable for the growth of the cells being assayed, for example, Roswell Park Memorial Institute (RPMI) medium containing 1 % fetal bovine serum (FBS) and a growth supplement. A growth supplement is a combination of growth factors and hormones that is suitable for the type of cell population being tested. An example of a growth supplement is Cyto-Gro 289 (Scientific Products) which contains insulin, transferrin, selenium, β-estradiol, hydrocortisone, prostaglandin F2a, and epidermal growth factor. Other media that may be used CEM (Microbiological Associates), DMEM, F12, McCoy's, or CMRL. A range of pre-determined drug concentrations is preferably tested, one of the concentrations generally being zero (to serve as a control). The number of different concentrations and the concentration range tested depend upon previous assay experience with the chemotherapeutic drug, clinical prognosis for the tumor, assay costs and the like. For example, a drug for which there is considerable clinical experience may be tested over a narrower range and at fewer concentrations than an drug for which there is less clinical experience. The drug is added before or after adding the tumor cell population, as desired. For example, a chemotherapeutic agent can be lyophilized or dried in appropriate-sized, disposable vessels such as the wells in a microwell strip, each well of the strip containing a pre-determined amount of the drug. Such strips fit into a housing that has a configuration similar to a microwell plate. The strip format allows the test, and facilitates transfer of medium with a multiple channel pipettor. The drug may be then be reconstituted with culture medium containing tumor cells.
Tumor cell populations are exposed to a drug for a period of time sufficient to allow substantially intact rRNA amounts in sensitive cells to decrease significantly. The length of the incubation period will depend upon factors such as tumor type and culture conditions (media, temperature and the like). If desired, duplicate or triplicate samples may be used, each sample incubated for a different time to ensure that an appropriate incubation period is selected. For most tumors, the incubation period is from about 1 to about 10 days at 37°C, typically from about 3 to about 6 days at 37°C. At the conclusion of the incubation period, culture medium is removed and tumor cells are disrupted to release substantially intact ribosomal RNA. The amount of substantially intact ribosomal RNA in each of the tumor cell samples is quantitated by hybridization to a detection probe specific for eukaryotic rRNA. A horseradish peroxidase label is coupled to a nucleic acid component of the probe. Hybridization is carried out with probe, disrupted cells and biotinylated capture substance present during the hybridization. The hybridized mixture is transferred to a streptavidin-coated microwell and incubated for a period of time sufficient to bind the capture substance/substantially intact rRNA/detection probe complex to the microwell solid phase. Bound complex is then determined by adding a horseradish peroxidase substrate such as 3,3',5,5'-tetramethyl benzidine and measuring the amount of colored product formed spectrophotometrically in a plate reader available from, for example, Baxter PRIMA (Baxter Diagnostics, Deerfield, Illinois). The chemosensitivity of the tumor is determined by correlating the amount of rRNA to the effect of each drug. The amount of rRNA is plotted versus the amount of chemotherapeutic drug and the plots for each drug are compared in order to determine which drug, if any, is toxic to the tumor cells. This information is provided to the clinician responsible for treatment of the patent and is used to choose a course of treatment for the patient, taking into account such factors as the concentrations of each drug achievable in plasma, the type of tumor, clinical experience with each drug, and patient medical history. The assay is useful either alone, or in combination with other chemoresponse assays, in selecting a chemotherapy regimen for a patient.
Another embodiment of a method according to the invention comprises quantitating the amount of substantially intact rRNA in a sample of lymphocytes exposed to a pre-determined amount of at least one compound known or suspected to be an antigenic or mitogenic agent. If lymphocytes are stimulated to proliferate at the concentration of the compound to which they have been exposed, there will be a larger amount of substantially intact rRNA compared to a population of the same cells not so exposed. If lymphocytes do not respond to exposure by proliferating, the amount of substantially intact rRNA in the sample will be not be significantly larger and may even be about equal to the amount in a sample not exposed the compound. By measuring the amount of substantially intact rRNA present in samples exposed to different levels of an antigenic compound and for different times, it is possible to correlate the effect of the compound to the amount of substantially intact rRNA in the exposed samples.
A typical sample to be used for antigen or mitogen stimulation gradient centrifugation on Ficollφ-Hypaqueφ from peripheral blood of an individual. About 104 to 10β cells in a microwell plate are mixed with an antigenic or mitogenic agent to be tested, and incubated in a suitable medium at 37°C, 5% C02 for 2-8 days. Aliquots may be removed at various times during the exposure period or at the end of the exposure period. Substantially intact rRNA is determined on the aliquots as described above. The amount of substantially intact rRNA measured in the aliquots is correlated to stimulation of lymphocyte proliferation. In the case in which the agent is an antigen, the amount of substantially intact rRNA is a sensitive measure of specific antigen-mediated cellular hypersensitivity. Such measurements may be used to assess cellular immunity and/or to evaluate lymphocyte function in patients known or suspected of immune suppression or deficiency, autoimmunity, infectious disease and the like; the measurements may be used in conjunction with other tests (e.g., delayed hypersensitivity skin tests) for assessing cellular immunity.
Another embodiment of a method according to the invention comprises quantitating the amount of substantially intact rRNA in a first sample of "responding" lymphocytes exposed to a pre-determined amount of a second sample of lymphocytes ("stimulating" lymphocytes), which second sample serves as an agent to be tested for its effect on the responding lymphocytes. The stimulatory lymphocyte preparation may be, if desired, subjected to mitomycin or irradiation before use, to prevent proliferation of stimulatory cells during the exposure period. By measuring the amount of substantially intact rRNA present in lymphocyte samples exposed for different times and to different levels of a stimulatory lymphocyte preparation, it is possible to correlate the amount of substantially intact rRNA in the exposed sample on the stimulatory lymphocytes. Such assays are useful for HLA matching for tissue and organ transplantation, paternity testing and the like. For example, the stimulatory lymphocyte preparation may be from a potential organ donor and the responding lymphocyte preparation may be from a potential organ recipient. The greater the proliferation of the responding lymphocytes as measured by rRNA amounts, the more likely a transplanted organ will be rejected by the recipient.
Another embodiment of a method according to the invention comprises quantitating the amount of substantially intact rRNA in a sample of eukaryotic cells exposed to a pre-determined amount of one or more compounds to be incorporated into a growth supplement. By measuring the amount of substantially intact rRNA present in populations exposed to different levels of a growth supplement, it is possible to correlate such an amount with the effect of the supplement on eukaryotic cells of the sample.
Any eukaryotic cell sample for which it is desired to quantitate the effect of a growth supplement may be used. The compound to be tested is incorporated into a culture medium and the sample is incubated for a period of time sufficient to allow the presence or absence of the compound to exert an effect upon cell proliferation or viability. rRNA amounts are then quantitated on the cell sample and are correlated to the effect of the compound.
An embodiment of a kit suitable for chemoresponse assays, comprises a detection probe, a capture substance and pre-weighed, dried, aliquots of chemotherapeutic agents in appropriate-sized, disposable vessels within a suitable packaging material. Such vessels may be microwell strips that fit into a housing with a configuration similar to a microwell plate. This configuration facilitates transfer of channel pipettor. Drug-containing microwell strips can be packaged in aluminum foil pouches or other air- and light-tight packaging material with desiccant, and sealed. The strip format allows the user flexibility in the selection of different chemotherapeutic agents to test. Suitable embodiments of such microwell strips are disclosed in, for example, U.S. Patent No. 5,242,806, issued September 7, 1993, and incorporated herein by reference.
An embodiment of a kit suitable for lymphocyte assays comprises a detection probe and a capture substance within a suitable packaging material. Such a kit may further comprise pre-weighed, dried, aliquots of antigenic or mitogenic agents in appropriate-sized, disposable vessels such as microwell strips or a microwell plate.
The invention will be further understood with reference to the following illustrative embodiments, which are purely exemplary, and should not be taken as limiting the true scope of the present invention as described in the claims.
EXAMPLE 1 Level of rRNA in Different Tumor Cell Lines An experiment was performed to determine the level of rRNA in various tumor cell lines. The experiment also evaluated the efficacy of 18S and 28S rRNA detection probes.
Oligonucleotides were synthesized on a Milligen 8700 DNA synthesizer using standard phosphoramidite chemistries. (Millipore, Marlborough, MA). The CAP215 and CAP207 capture substances contained a biotin molecule at the 5' end of the DNA. Both capture substances were synthesized using biotin phosphoramidite (Glenn Research, Sterling, VA). Unless otherwise indicated, detector oligonucleotide sequences contained an amino group at the 3' end o'f (Glenn Research, Cat. No. 20-2957-42). Amine-containing detector oligonucleotides were conjugated through the amino group to horseradish peroxidase (HRP). Oligonucleotide sequences of capture substances and detection probes are shown in Table 1.
Table 1 ,
Oligonucleotide Sequences
Seq. ID Name Sequence No.
1 BB193 ACGCACACCA CACGCG
2 BB200 GTCCACTCTC GACTGC
3 CAP207 ATTACCGCGG CTGCTGGCAC CAGACTTGCC C
4 DET212 ATTTAAAGTG GACTCATTCC
5 CAP215 AAAAAAATTA CCGCGGCTGC TGGCACCAGA CTTGCCC
6 DET216 CGGCCGTGCG TACTTAGACA
7 DET218 GTGGGTAATT TGCGCGCCT
8 DET231 GTCACCCGTG GTCACCAT
K562 and SiHa cells were grown in RPMI medium containing 1 % fetal bovine serum (FBS) and 1X Cyto-Gro 289. K562 is a line of pleuroeffusion cells derived from a 53 year old female suffering from chronic myelogenous leukemia (CML) in blast crisis. SiHa is a HPV- infected cervical carcinoma cell line. Lines were obtained from American Type Culture Collection (Rockville, Maryland).
RNA was isolated using RNAzol B (Teltest, Friendswood, Tx) according to the manufacturers instructions. Two different preparations of K562 RNA were used, one of which had been stored frozen until use. Serial dilutions of the target RNA from the preparations were transferred to a membrane in a slot blot apparatus. Northern blots were performed as described in U.S. Patent Application No. 08/264,556, June 23, 1994, which application is incorporated herein by reference. The probes used were 32P-labeled 28S rRNA detection oligonucleotides (BB193 and BB200). The hybrids formed by these probes have melting temperatures (Tm) of about 54°C (BB193) and about 52°C (BB200).
Autoradiograms of the blots are shown in Figures 1 and 2 (using BB193 and BB200, respectively, as probes). As shown in Figures 1 and 2, approximately the same amount of rRNA is produced by each of the cell lines. Furthermore, a stored preparation of K562 cells had only a slightly smaller amount of rRNA (Columns A and B, Figures 1 and 2). However, 28S probe BB193 does not appear to be as sensitive as 28S probe BB200 (Figures 1 and 2). The difference in sensitivity is most likely due to secondary structure at hybridizing sites in the target rRNA and is likely not due to the difference in Tm of the two hybrids. Additional experiments showed that BB200 did not detect 28S rRNA in nucleic acid isolated from a human foreskin cell line.
Total RNA was isolated from six different cell lines and used as target nucleic acid in a Northern blot. The cell lines used were KG1 , SiHa, HeLa, CEM/s, HL-60 and K562 (ATCC, Rockville, MC). KG1 is an acute myelopathic leukemia line from a 59 year old male. HeLa is an HPV-18 infected cervical carcinoma cell line. CEM/s is a T-cell lymphoma blastoid cell line. HL-60 is a promyelocytotic cell line from a 36 year old female with acute promyelocytotic leukemia. Serial dilutions of the RNA were applied to a slot blot apparatus and Northern blots were performed as described above. An autoradiogram using 32P- labeled, non-amine derivatized DET231 as the probe is shown in Figure 3 and an autoradiogram using 32P-labeled, non-amine derivatized DET212 as the probe is shown in Figure 4. The results indicate that all 6 cell lines have approximately the same amount of ribosomal RNA per cell, and that probes DET231 and DET212 appear to be approximately equivalent in the amount of rRNA detected. These same probes were able to detect 18S rRNA in a human foreskin cell line. EXAMPLE 2 Ribosomal RNA Detection Assay This example teaches that the effect of a chemotherapeutic agent on a tumor cell line can be determined by measuring the amount of rRNA present after the cell line is grown in the presence of the agent.
SW480 tumor cells were grown in RPMI medium, 1 % FBS and 1 % 1X Cyto-Gro 289. SW480 is a coleorectal adenocarcinoma cell line. About 104 cells in 200 μl of medium were dispensed into wells containing bleomycin sulfate (Sigma Chemical Co. St. Louis, MO, Cat. No. B507), cis-platinum diamminedichloride (Sigma, Cat. No. P4394, also known as Platinol® or cisplatinum), and etoposide (Sigma, Cat. No. E1383, also known as VP-16). The concentration of each drug ranged from 0.01 μg to 10 μg per well, as indicated in Figures 5 and 6. Cells were incubated at 37°C for 3 days and were analyzed in duplicate at each drug concentration.
Total RNA was isolated from each well using RNAzolr B. The RNA was applied to a slot blot apparatus and the blot probed as described in Example 1 , using 32P-labeled BB200 28S rRNA detection oligonucleotide. An autoradiogram of the rRNA slot blot is shown in Figures 5 and 6.
Note that the RNA was lost in Figure 5 for lane A, slot 5 and slot 9, and that the RNA was mixed in Figure 6 for slots 12 and 13, lane A.
After culturing SW480 cells in the presence of 10 μg of VP16, there is little or no detectable rRNA (Figure 6, slots 9 and 10). This result indicates that SW480 cells have little or no substantially intact rRNA after exposure to VP16. In contrast, SW480 cells have easily detectable amounts of rRNA when the cells are cultured in the presence of 10 μg of bleomycin sulfate (Figure 5, slots 9 and 10). Even in the same cell line cultured in the presence of 1.0 μg of bleomycin sulfate (compare Figure 6, slots 15 and 16 to Figure 5, slots 1 1 and 2). These results demonstrate that, on a weight basis, VP16 is more toxic than bleomycin to SW480 cells. A companion plate using the same cell line was grown under the same conditions and the same concentrations of chemotherapeutic drugs. The cells on the companion plate then were pulsed with tritiated thymidine (3H TdR, 1 μC\ per well) for 16 hours as described in U.S. Patent 5,242,806, incorporated herein by reference. Acid-precipitable radioactivity was determined using a scintillation counter; the results are plotted in Figure 7 as a percentage of the radioactivity in no-drug control wells. A comparison of Figures 5 and 6 (rRNA assay) and Figure 7 (3H TdR assay) indicates that the same relative cytotoxicity of the 3 agents was found in both assays (etoposide > cis-platinum > bleomycin sulfate). These results show that the amount of ribosomal RNA present in cells after exposure to a chemotherapeutic agent can be used to determine the effect of an agent on such cells.
EXAMPLE 3 Evaluation of Capture and Detection Oligonucleotides Different capture oligonucleotides and detection oligonucleotides were tested in an automated microwell plate assay, using MOLT-4 cells that were cultured as described in Example 2.
About 104 cells in each well were lysed in 50 μl of 0.3% SDS, 5 mM vanadyl ribonucleoside complex (VRC) for 5 minutes at 50°C. Four picomoles of the indicated capture substance and 1 picomole of the indicated detector probe in 50 μl of 2X hybridization buffer (10% PEG- 8000, 1 % BSA, 10X SSC, 0.2% polyviπylpyrrolidone) were then added to each well. Lysed cells and hybridization solution were gently mixed by tapping the corner of the microwell plate and the mixtures incubated at the indicated temperature for 30 minutes.
Each mixture was transferred to a streptavidin-coated well and incubated at room temperature for 20 minutes. Wells then were rinsed five times with wash buffer (2X SSC, 0.05% Tween-20 and 0.01 % thimerosal). Two drops of 0.04%, 3,3',5,5'-tetramethyl benzidine and 0.02% hydrogen peroxide were added to each well and the mixture was incubated at room temperature for 15 minutes, avoiding light. After the 15 minute period, 100 μl of 1 M H3PO4 was added to each well. The absorbance at 450 nm (A450) of each well was measured and recorded.
Streptavidin-coated microwell plates were prepared by incubating in
10 μg/ml streptavidin, 50 mM sodium borate, pH 9.0 for 4 hours at
37°C. The plates were covered during this incubation. The plates were washed 5X with 0.05% Tween-20®, 10 mM Tris, pH 7.4 and 0.01 % thimerosal, packed in a foil lined pouch with desiccant and stored at 4°C.
The results for various combinations of capture substance and detection probes are shown in Table 2. The hybridization temperature that resulted in the highest absorbance for each capture/detection pair varied somewhat, but was generally about 50°C. Table 2 shows that, for any given detection probe, the use of CAP215 as capture substance resulted in a higher absorbance value compared to the absorbance when CAP207 was used as the capture substance.
Probes DET212, DET216, DET218 and DET231 , were tested again as described above using a hybridization temperature of 50°C. The results are shown in Table 3. The two detector probes that had the highest absorbance were DET231 and DET212. DET218 also produced a high absorbance but the background absorbance was high with this Table 2.
A^ at Different Hvbridization Temperatures
Capture/ 23°C 40°C 50° 60°C 50°C Detection Pair (No Cells)
207/231 0.O47 1.086 2.862 2.362 0.037
215/231 0.619 1.157 3.619 3.735 0.278
207/212 0.092 1.388 3.072 2.234 0.038
215/212 0.306 1.737 4.000 3.594 0.132
207/218 0.140 0.810 1.750 1.657 0.062
215/218 3.594 4.000 4.000 3.800 3.891
207/216 0.138 0.898 2.245 2.495 0.102
215/216 1.749 1 .973 3.978 3.794 2.031
Table 3.
A__n of Capture/Detection Combinations
Figure imgf000033_0001
EXAMPLE 4 Effect of Combining Detector Oligonucleotides
DET231 and DET212 were tested as detector probes separately and mixed together. The experiment was carried out as described in Example 3, using 5 pmoles per well of CAP2 5 as the capture substance and 1 to 4 pmoles of each probe. The hybridization temperature was 50°C. The data (Table 4) showed that when the amount of added RNA corresponded to a relatively high cell concentration (104 cells or higher) and both DET231 and DET212 were used, the A450 was approximately the sum of the absorbance values for each probe separately. At RNA amounts corresponding to lower cell numbers, (103 cells or less), the absorbance value when both DET231 and DET212 were used was greater than the sum of the absorbance values for each probe used separately. These results indicate that using DET231 and DET212 together may increase the sensitivity of the assay at low cell concentrations. Table 4.
A_4y. at Different Cell Concentrations
RNA (μg) Equivalent 231 & 212 231 212 Cell No.
2.0 10 3.933 3.681 3.839
0.2 104 1.592 0.785 0.734
0.02 10a 0.358 0.099 0.160
0.002 10* 0.280 0.44 0.055
0.0002 101 0.268 0.075 0.053 none 0 0.279 0.046 0.064 noneD 0 0.039 0.036 0.035 EXAMPLE 5. Effect of Various Additions to Hvbridization Buffer The effect of adding various components to the hybridization buffer was investigated. Chemoresponse assays were carried out as described in Example 3 except that 0.2 μg total RNA isolated from SW480 cells was used instead of cultured cells. CAP215 (5 picomoles per well) was used as the capture substance and DET212/DET231 (4 picomoles each per well) were used as the probe. The hybridization temperature was 50° C. Each of the additional components was prepared as a stock solution in 0.1 % PVP, 5X SSC and added to the hybridization solution of Example 3 prior to addition of hybridization solution to the wells.
The results (Tables 5 and 6) indicate that the addition of glycerol (10-50%), sorbitol (10-50%), Tween-20® (1-5%) or 3-[(3- cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate
(CHAPSO, 0.5-2.5%) did not significantly increase the A450. However, the addition of 5% polyethylene glycol 8000 or the addition of 0.5% bovine serum albumin increased the absorbance values compared to the standard hybridization buffer.
Page missing at the time of publication
EXAMPLE 6 Assay of a Clinical Tumor Specimen Using rRNA Detection
A biopsy of an ovarian cancer (Sample No. 92018S) was obtained from a patient. The specimen was minced and resuspended in RPMI medium containing 1 % FBS and 1X Cyto-Gro 289.
Foil packaged microwell drug strips were allowed to reach room temperature before opening the package in a laminar flow hood. The microwell strips, containing pre-determined amounts of the indicated drugs, were removed from their foil pouches, placed in a plate frame and snapped firmly into place. A strip label at the bottom of the frame was coded according to the particular drug present in that strip.
After placing all drug and control strips in plate frames, 100 μl of media were pipetted into each microwell. Wells containing the lowest drug concentration were mixed first, followed by wells containing increasingly higher concentrations. Pipet tips were changed before each medium addition. A homogeneous mixture was obtained by allowing the drugs to solubilize for five minutes and by repeatedly aspirating the mixture.
The drugs used in this experiment were doxorubicin hydrochloride (also known as Adriamycin®), mitomycin-C, 5-fluorouracil, L-Pharm, bleomycin sulfate, cis-platinum and VP16. The amounts used were 0, 0.01 , 0.1 and 1.0 μg per well.
Tumor cells (about 104 cells) in 100 μl of medium were then transferred into each well in a laminar flow hood using sterile techniques. Transfer was facilitated by the use of a multi-channel pipettor and V-shaped reservoir (Costar, Cambridge, MA). The dispensing procedure was completed in less than one minute to ensure an even distribution of cells. The vessels were covered and incubated at 37°C, 5% C02 for 3 days in a fully humidified incubator. At the end of the incubation period, the cells were centrifuged, the supernatant was discarded and 50 μl of lysing buffer (0.3% SDS and 5 mM vanadyl ribonucleoside complex, VRC) were added to each well of the incubation plate. The cells were incubated in the lysing solution for about 5 minutes at about room temperature. About 50 μl of the hybridization solution of Example 3, containing 4 picomoles each of horseradish peroxidase-conjugated DET231 and DET212 and 4 picomoles of CAP215 were added to each well. The lysed cells and hybridization solution were gently mixed by tapping the corner of the microwell plate and incubated at 50°C for 30 minutes.
The lysed cell/hybridization mixtures was transferred to corresponding streptavidin-coated wells of an assay plate, the assay plate was rinsed three times with wash buffer (2X SSC, 0.05% Tween- 20® and 0.01 % thimerosal) and two drops (about 100 μl) of 0.04% 3,3*,5,5'-tetramethyl benzidine and 0.02% hydrogen peroxide were added to each well. The plate was incubated at room temperature for 15 minutes, avoiding light. After the 15 minute period, about 100 μl of 1 M H3PO4 was added to each well. The A450 of each well was measured and recorded. The results are shown in Table 7.
The results indicate that this ovarian tumor is most sensitive to doxorubicin hydrochloride and 5-fluorouracil, and is least sensitive to cis-platinum. The chemoresponse of this tumor to the tested drugs would be reported to the clinician responsible for treatment of this patient for ovarian cancer. Table 7.
Sensitivity of an Ovarian Tumor to Chemotherapeutic Aoents
cis- 5-
Cone. No drug Doxoru¬ Beomy- plati¬ L pharm Mitomy- No drug Fluorou- VP16 No drug] bicin cin num cin-C racil
0 0.109a 0.129 0.893 0.140 0.833 0.121 0.133a 0.342 0.100 0.057a
0 0.107a 0.088 0.692 0.253 0.488 0.162 1.314b 0.281 1.377 .051b
1.0 2.760 0.1 12 1.689 1.987 1.605 0.597 1.559b 0.418 1.310 0.051 b
1.0 2.776 0.151 1.593 1.867 1.397 0.360 1.449b 0.326 1.884 0.047b
0.1 2.846b 1.045 2.176 2.833 1.136 1.393 2.015b 1.068 1.737 0.050
0.1 2.609 1.050 2.422 3.014 1.653 1.872 1.927 1.164 1.794 0.049b
0.01 2.249b 2.558 2.586 2.423 2.528 1.680 2.065b 1.929 1.571 0.050b
0.01 3.209 2.395 2.095 3.280 2.546 2.447 2.437b 2.217 2.487 0.053b "No Cell Control b+ Cell Control
EXAMPLE 7
Optimization of Capture and Detection Oligonucleotide Concentrations
The relationship between A450 and detection probe concentration was examined at a constant capture substance concentration and constant cell number. The cells were obtained from a primary ovarian cancer (Specimen 920069). The specimen was minced and trypsinized, cultured overnight in RPMI-1640, 1 % FBS and frozen in liquid nitrogen.
Aliquots of frozen cells were thawed and allowed to recover at 37°C overnight in RPMI-1640 plus 1 X Cyto-Gro 289. Cells were added to a
96 well microwell plate at 104 cells per microwell and the plates cultured for 4 days at 37° C, 5% CO2 in RPMI-1640, 1 % FBS and 1 X
Cyto-Gro 289 in a fully humidified incubator.
At the end of the culture period, the cells were centrifuged, the supernatant was discarded and 50 μl of lysing buffer (0.3% SDS and 5 mM VRC) were added to each well of the incubation plate. The cells were incubated in the lysing solution for about 5 minutes at room temperature. Hybridization solution (50μl), containing 5 picomoles of
CAP215 and 0.25,0.5,1 ,2 or 4 pmoles/well each of DET231 and DET212 was added to each well. The mixtures were hybridized at 50°C for 45 minutes.
During the hybridization, streptavidin-coated microwell plates were blocked with 300 μl of blocking buffer (5X SSC, 5X Denhardt's solution, 1 % BSA, 0.1 % Tween-20 and 0.2 ng/ml sheared salmon sperm DNA). The blocking buffer was removed before transferring the hybridization mixture.
After hybridization, each mixture was transferred to a streptavidin- coated assay well plate and incubated for 45 minutes at 50° C. The assay plates then were rinsed four times with wash buffer (2X SSC, 0.05% Tween-20). Two drops (about 100 μl) of 0.04% 3,3', 5,5'- tetramethyl benzidine and 0.02% hydrogen peroxide were then added to each well and the mixture incubated at room temperature for 15 minutes, avoiding light. After the 15 minute period, about 50 μl of 1 M H3PO4 was added to each well. The A450 of each well was measured and recorded. The results are shown in Figure 8.
EXAMPLE 8 Correlation Between Cell Number and A-^ The relationship between the number of tumor cells and A450 was examined for 2 different tumor cell lines. SW480 cells were aliquoted into microwells at 400, 2,000, 10,000 and 50,000 cells per well. HTB158 cells were aliquoted into microwells at 300, 600, 1 ,200, 2,500, 5,000, 10,000, 20,000 and 40,000 cells per well. rRNA amounts were measured as described in Example 7, using 2.5 pmoles of CAP215 and 1 pmole of DET212. The results for the SW480 line are shown in Figure 9. The results for the HTB158 line are shown in Figure 10. EXAMPLE 9 Comparison of Chemoresponse Assay Methods This example compares an rRNA assay to a 3H TdR assay and a [3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay in measuring the effect of a chemotherapeutic drug upon eukaryotic cell viability and/or cell proliferation.
Tumor cells were obtained from a human lung cancer cell line, A549 (ATCC, Rockville, MD). Cells were washed with RPMI, 1 % FBS and resuspended in RPMI, 1 % FBS, IX Cyto-Gro 289™ The cells were plated at about 1.5 X 103 cells per well in BC-ECM coated microwell plates and cultured in the presence of chemotherapeutic drugs for 4 days as described in Example 6. BC-ECM coated microwell plates were prepared as described in U.S. Patent 5,242,806, issued Sept. 7, 1993 and incorporated herein by reference. The drugs used in the experiment were: Adriamycin®, bleomycin sulfate, Platinol® and mitomycin-C at concentrations from about 0.01 μg/ml to about 10 μg/ml. After culturing the cells, an rRNA assay was carried out as described in Example 7, using 5 pmoles CAP215 and 1 pmole of DET212.
In parallel, A549 cells were exposed to chemotherapeutic drugs as described above in two separate plates. For one plate, an MTT assay was carried out by adding 25 μl of MTT solution to each well. MTT solution contained 5 mg/ml MTT in phosphate buffered saline (PBS), stored at -20°C. After incubating for 4 hours at 37°C, the blue formazan product was dissolved in DMSO and the absorbance at 570 nm was measured. For the second plate, a 3H TdR assay was carried out as described in U.S. Patent 5,242,806, issued September 7, 1993, incorporated herein by reference. The 3H TdR (specific activity 6.7 //Ci/ml) was added at 1 Ci/well. Comparisons of the 3 methods for each chemotherapeutic drug are shown in Figures 11 to 14, in which the A450 is presented as a percentage of a control well containing no drug. The data indicate that the A549 line is most sensitive to mitomycin-C and least sensitive to bleomycin sulfate. The dose-response pattern for A549 cells to each agent was similar for each method, except for the MTT assay with Platinol®. This assay predicted a higher Platinol® sensitivity than did the rRNA or 3H TdR assays. The results show that an rRNA assay is suitable for measuring chemosensitivity of a tumor. EXAMPLE 10
Comparison of Chemoresponse Assay Method This example compares an rRNA assay to a 3H TdR assay and an MTT assay in measuring the effect of a chemotherapeutic drug upon cell viability and/or cell proliferation of a clinical tumor specimen. Tumor cells were obtained from a primary ovarian cancer. The specimen was minced, trypsinized, counted, and incubated overnight in 1 % DMEM. Cells were plated and cultured in the presence of chemotherapeutic drugs for 4 days as described in Example 9. The drugs used in the experiment were: Adriamycin®, bleomycin sulfate, cis- diammine[1 ,1-cyclobutane-dicarboxylato] platinum (also known as carboplatin), paclitaxel from Taxus brevifolia (also known as taxol) and 5-fluorouracil. Carboplatin and taxol were obtained from Sigma or Bristol-Myers.
After culturing the cells, rRNA, MTT and 3H TdR assays were carried out as described in Example 9, except that 3H uridine (specific activity >20 //Ci/ml, 1 /Ci/well) was used instead of 3H TdR in the 5- Fluorouracil assay. The results are shown in Figures 15 to 19. The data indicate that this ovarian cancer is most sensitive to Adriamycin®, and EXAMPLE 11 Effect of a Growth Stimulator on Tumor Cells This example teaches that the effect of a growth stimulating agent upon eukaryotic cell viability or proliferation can be determined by measuring the amount of substantially intact rRNA present after exposure to the agent. Cells from the MCF-7 breast cancer cell line were used in this experiment. After washing and resuspending cells as described in Example 9, 5 X 103 cells were transferred to 2 wells of a 96 well microwell plate and incubated in RPMI- 1640, 1 % FBS, IX Cyto- Gro289 or in RPMI-1640, 1 % FBS without Cyto-Gro289 for 3 days at 37°C, 5% C0 in a fully humidified incubator. rRNA amounts in the wells were measured as described in Example 9, using 5 pmoles of CAP215 and 1 pmole of DET212. The A450 for cells grown in the presence of Cyto-Gro 289 was 1.4, whereas the A450 for cells grown in the absence of Cyto-Gro 289 was 0.7.
The foregoing detailed description has been provided for a better understanding of the invention only and no unnecessary limitation should be understood therefrom as some modifications will be apparent to those skilled in the art without deviating from the spirit and scope of the appended claims.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i)APPLICANT: DADE INTERNATIONAL INC.
(ii)TITLE OF INVENTION: EUKARYOTIC CELL ASSAY USING RIBOSOMAL RNA DETECTION
(iii)NUMBER OF SEQUENCES: 8
(iv)CORRESPONDENCE ADDRESS:
(A)ADDRESSEE: Dade International Inc. (B)STREET: 1717 Deerfield Road (C)CITY: Deerfield (D)STATE: Illinois (E)COUNTRY: USA (F)ZIP: 60015-0778
(v)COMPUTER READABLE FORM:
(A)MEDIUM TYPE: Floppy disk
(B)COMPUTER: IBM PC compatible
(C)OPERATING SYSTEM: PC-DOS/MS-DOS
(D)SOFTWARE: PatentIn Release #1.0, Version 1.25
(vi)CURRENT APPLICATION DATA: (A)APPLICATION NUMBER: (B)FILING DATE: November 8, 1995 (C)CLASSIFICATION:
(vii)PRIOR APPLICATION DATA:
(A)APPLICATION NUMBER: 08/336,730 (B)FILING DATE: 11/9/94
(viii)ATTORNEY/AGENT INFORMATION: (A)NAME: WINSTON, Lois K. (B)REGISTRATION NUMBER: 39,074 (C)REFERENCE/DOCKET NUMBER:BA1-4711
(ix)TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 708/267-5364 (B)TELEFAX: 708/267-5376 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARCTERISTICS: (A)LENGTH: 16 (B)TYPE: nucleic acid (C)STRANDEDNESS: single stranded (D)TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: ACGCACACCA CACGCG 16
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 16 (B)TYPE: nucleic acid (C)STRANDEDNESS: single stranded (D)TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GTCCACTCTC GACTGC 16
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 31 (B)TYPE: nucleic acid (C)STRANDEDNESS: single stranded (D)TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATTACCGCGG CTGCTGGCAC CAGACTTGCC C 31
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 20 (B)TYPE: nucleic acid (C)STRANDEDNESS: single stranded (D)TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ATTTAAAGTG GACTCATTCC 20
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 37 (B)TYPE: nucleic acid (C)STRANDEDNESS: single stranded (D)TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: AAAAAAATTA CCGCGGCTGC TGGCACCAGA CTTGCCC 37
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 20 (B)TYPE: nucleic acid (C)STRANDEDNESS: single stranded (D)TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CGGCCGTGCG TACTTAGACA 20
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 19 (B)TYPE: nucleic acid (C)STRANDEDNESS: single stranded (D)TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GTGGGTAATT TGCGCGCCT 19
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 18 (B)TYPE: nucleic acid (C)STRANDEDNESS: single stranded (D)TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GTCACCCGTG GTCACCAT 18

Claims

WHAT IS CLAIMED IS:
1. A method for determining the effect of an agent on eukaryotic cell viability or proliferation, comprising: (a) quantitating the amount of ribosomal RNA in a sample of eukaryotic cells exposed to said agent, said quantitating occurring by specific hybridization of a detection probe to said ribosomal RNA; and (b) correlating said amount with said effect.
2. The method of claim 1 , wherein said probe comprises a nucleic acid hybridizing to 18S ribosomal RNA.
3. The method of claim 2, wherein said nucleic acid comprises a sequence selected from the group consisting of: SEQ. ID NO: 4, SEQ. ID NO: 6, SEQ. ID NO: 7, SEQ. ID NO: 8 and a sequence that hybridizes to a sequence complementary to the sequence of SEQ. ID NO: 4, SEQ. ID NO: 6, SEQ. ID NO: 7 or SEQ. ID NO: 8.
4. The method of claim 1 , wherein said probe comprises a nucleic acid hybridizing to 28S ribosomal RNA.
5. The method of claim 4, wherein said nucleic acid comprises a sequence selected from the group consisting of: SEQ. ID NO: 1 , SEQ. ID NO: 2, and a sequence that hybridizes to a sequence complementary to the sequence of SEQ. ID NO: 1 or SEQ. ID NO: 2.
6. The method of claim 1 wherein said probe further comprises a label coupled to said nucleic acid.
7. The method of claim 6, wherein said label is selected from the group consisting of horseradish peroxidase, alkaline phosphate, β- galactosidase and ATPase.
8. The method of claim 1 , wherein said quantitating step comprises the steps of:
(i) capturing said ribosomal RNA by hybridizing a capture substance to said ribosomal RNA, said capture substance effective for binding to an immobilizing compound on a solid phase; and
(ii) immobilizing said captured ribosomal RNA by binding of said capture substance to said immobilizing compound on said solid phase.
9. The method of claim 8, wherein said capture substance comprises a nucleic acid hybridizing to 18S ribosomal RNA.
10. The method of claim 9, wherein said capture substance comprises a nucleotide sequence selected from the group consisting of SEQ. ID NO: 5, SEQ. ID NO: 3 and a sequence that hybridizes to a sequence complementary to the sequence of SEQ. ID NO: 3 or SEQ. ID NO: 5.
11. The method of claim 9, wherein said capture substance further comprises a binding component coupled to said capture substance nucleic acid, said binding component effective for binding to said immobilizing compound.
12. The method of claim 1 , wherein said agent comprises a
13. The method of claim 1 , wherein said agent comprises an antigenic or mitogenic compound.
14. The method of claim 1 , wherein said agent comprises a population of lymphocytes.
15. A kit for determining the effect of an agent on eukaryotic cell viability or proliferation, comprising: a) packaging material; and b) a detection probe accompanying said packaging material, said probe effective for quantitating ribosomal RNA in a sample of eukaryotic cells exposed to said agent.
16. The kit of claim 15, wherein said probe comprises a nucleic acid effective for hybridization to 18S ribosomal RNA.
17. The kit of claim 16, wherein said nucleic acid comprises a nucleotide sequence selected from the group consisting of: SEQ. ID NO: 4, SEQ. ID NO: 6, SEQ. ID NO: 7, SEQ. ID NO: 8 and a sequence that hybridizes to a sequence complementary to the sequence of SEQ. ID NO: 4, SEQ. ID NO: 6, SEQ. ID NO: 7 or SEQ. ID NO: 8.
18. The kit of claim 15, wherein said probe comprises a nucleic acid effective for hybridization to 28S ribosomal RNA.
19. The kit of claim 18, wherein said nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ. ID NO: 1 , SEQ. ID NO: 2, and a sequence that hybridizes to a sequence complementary to the sequence of SEQ. ID NO: 1 or SEQ. ID NO: 2.
20. The kit of claim 15, wherein said probe further comprises a label coupled to said nucleic acid.
21. The kit of claim 20, wherein said label is selected from the group consisting of horseradish peroxidase, alkaline phosphate, β- galactosidase and ATPase.
22. The kit of claim 15, further comprising a capture substance accompanying said packaging material, said capture substance effective for binding to an immobilizing compound on a solid phase.
23. The kit of claim 22, wherein said capture substance comprises a nucleic acid effective for hybridization to 18S ribosomal RNA.
24. The kit of claim 23, wherein said capture substance nucleic acid comprises a sequence selected from the group consisting of SEQ. ID NO: 5, SEQ. ID NO: 3 and a sequence that hybridizes to a sequence complementary to the sequence of SEQ. ID NO: 3 or SEQ. ID NO: 5.
25. The kit of claim 15, further comprising a plurality of vessels accompanying said packaging material.
26. The kit of claim 25, wherein a selected number of said vessels comprise a solid phase.
27. The kit of claim 25, wherein a selected number of said vessels contain a pre-determined amount of a chemotherapeutic drug.
28. The kit of claim 25, wherein a selected number of said vessels contain a pre-determined amount of an antigenic or mitogenic compound.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994002636A1 (en) * 1992-07-28 1994-02-03 Hitachi Chemical Company Ltd. Gene detection system

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994002636A1 (en) * 1992-07-28 1994-02-03 Hitachi Chemical Company Ltd. Gene detection system

Non-Patent Citations (5)

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Title
CANCER CHEMOTHER PHARMACOL, 1989, 25 (1) P37-44, GERMANY, WEST, XP002001929 GREENHALGH DA ET AL: "Effects of 5-fluorouracil on cytotoxicity and RNA metabolism in human colonic carcinoma cells." *
CANCER RES. (1992), 52(1), 11-16 CODEN: CNREA8;ISSN: 0008-5472, XP002001931 FERRARI, SERGIO ET AL: "Abundance of the primary transcript and its processed product of growth-related genes in normal and leukemic cells during proliferation and differentiation" *
CYTOMETRY (1993), 14(2), 136-43 CODEN: CYTODQ;ISSN: 0196-4763, XP002001933 WALLNER, GUENTER ET AL: "Optimizing fluorescent in situ hybridization with rRNA targeted oligonucleotide probes for flow cytometric identification of microorganisms" *
HISTOCHEMISTRY (1991), 96(1), 73-81 CODEN: HCMYAL;ISSN: 0301-5564, XP002001930 PAJOR, L. ET AL: "Flow cytometric measurement of rRNA levels detected by fluorescent in situ hybridization in differentiating K-562 cells" *
PROC. NATL. ACAD. SCI. U. S. A. (1981), 78(2), 727-31 CODEN: PNASA6;ISSN: 0027-8424, XP002001932 GRUMMT, INGRID: "Specific transcription of mouse ribosomal DNA in a cell-free system that mimics control in vivo" *

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