WO1996032935A1 - Inhibition de l'apoptose dans des cellules exprimant le ligand de fas suite a l'activation - Google Patents

Inhibition de l'apoptose dans des cellules exprimant le ligand de fas suite a l'activation Download PDF

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WO1996032935A1
WO1996032935A1 PCT/US1996/006090 US9606090W WO9632935A1 WO 1996032935 A1 WO1996032935 A1 WO 1996032935A1 US 9606090 W US9606090 W US 9606090W WO 9632935 A1 WO9632935 A1 WO 9632935A1
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cells
fas
apoptosis
expression
fasl
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PCT/US1996/006090
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WO1996032935A9 (fr
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Reid P. Bissonnette
Richard A. Heyman
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Ligand Pharmaceuticals Incorporated
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Publication of WO1996032935A9 publication Critical patent/WO1996032935A9/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/203—Retinoic acids ; Salts thereof

Definitions

  • This invention relates to means for modulating the function of intracellular receptors using ligands therefor. More specifically, this invention relates to the use of retinoid compounds having both RXR and RAR activity to regulate the expression of Fas-Ligand (FasL) following cell activation, which can be utilized to mediate the inhibition of apoptotic cell death in certain cells, e.g., T cells.
  • Fas-Ligand Fas-Ligand
  • Abbreviations used herein include: 9-cis RA, 9-cis retinoic acid; AIDS, acquired immunodeficiency syndrome; ATRA, all-trans retinoic acid; Fas, the Fas antigen or CD95; FasL, Fas-Ligand; FCS, fetal calf (bovine) serum; FITC, fluorescein iso-thiocyanate; FL1, fluorescence channel 1; FL2, fluorescence channel 2; FL3, fluorescence channel 3; IFN- ⁇ , interferon gamma; IL2, interleukin 2; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate- buffered saline; RAR, retinoic acid receptor; RAR , ⁇ or ⁇ , retinoic acid receptor alpha, beta or gamma; RARE, retinoic acid receptor response element; RNase, ribonuclease; RXR, retinoid X receptor; RXR
  • apoptosis is defined as a form of cell death in which the cell plays an active role in its own demise, via synthesis of necessary macromolecular mediators (for example Fas and FasL) and the expenditure of energy.
  • apoptosis active cell death
  • activation-induced cell death are used interchangeably and refer specifically to the induction of apoptotic death in T-cell hybridomas and/or T cells by activation of said cells via the T-cell receptor (TCR) and by means which mimic that activation.
  • Apoptosis is distinguished from another form of cell death, i.e., necrosis, in which the cell plays a passive role, dying when membrane damage proceeds to an irreparable point of no return.
  • necrosis thus describes injury-type death, and it is characterized by swelling and rupture of plasma and organelle membranes and the loss of cytoplasmic organization, which is later followed by disruption of the nucleus. Necrotic tissues often become infiltrated by neutrophils.
  • Apoptosis is a morphologically distinct form of cell death, characterized by condensation of the chromatin and loss of nuclear structure, and the formation of plasma membrane blebs.
  • apoptosis there is often a fragmentation of genomic DNA into an oligonucleosomal ladder, a phenomenon which is usually not seen in necrotic cell death.
  • apoptosis unlike necrosis, depends upon continued RNA and protein synthesis by the dying cell, leading to the concept of cell "suicide".
  • Cells that die by apoptosis are rapidly phagocytosed and cleared from the system without an inflammatory consequence.
  • Apoptosis is functionally defined by its morphological features (especially chromatin condensation) , and this has persisted as the most reliable method of characterization.
  • DNA fragmentation occurs as it often does during active cell death, it is a useful and highly quantifiable feature (see below) ; however, apoptosis can occur in the absence of such DNA fragmentation (Tomei, L.D., et al . , Proc . Na tl . Acad . Sci . U. S . A . , 90:853-857 (1993)) . It remains possible that single-stranded DNA fragmentation (Tomei, L.D., et al. , Proc. Natl . Acad. Sci . U. S. A . , 90:853-857 (1993)) and/or cleavage into very large (approx.
  • lymphocytes e.g., B-cells, T-cells
  • antigen receptors e.g., immunoglobulin, T-cell receptor
  • activation-induced apoptosis is thought to be the mechanism of negative selection in thymocytes, peripheral deletion in mature T-cells, and loss of non-infected T-cells in patients having AIDS (e.g., Meyaard, L.
  • the induction of apoptosis in response to a receptor- transduced signal is by no means restricted to the T cell, or even hemopoietic tissue compartment.
  • An example is the induction of apoptosis in normal hepatocytes following interaction between Fas antigen expressed on the surface of the liver cells and an antibody specific for the Fas antigen (Ogasawara, J., et al . , Nature, 364:806-809 (1993) ) .
  • T-cell hybridomas, thymocytes, and T cells can be induced to undergo apoptotic cell death by activation through the T-cell receptor (TCR) (Cohen, J.J., et al . , J. Immunol . , 132:38-42 (1984) ; Shi, Y., et al . , J. Immunol . , 144:3326-3333 (1990) ; Odaka, C, et al . , J. Immunol . , 144:2096-2101 (1990) ; Ucker, D.S., et al . , J. Immunol . , 143:3461-3469 (1989)) .
  • TCR T-cell receptor
  • This process requires macromole- cular synthesis and thus gene expression, and has been shown to be influenced by factors regulating gene transcription (Shi, Y., et al. , Science , 257:212-214 (1992) ; Bissonnette, R.P., et al . , J. Exp. Med. , 180:2413- 2418 (1994) ; Woronicz, J.D., et al. , Nature, 367:277-281 (1994) ; Liu, Z.G., et al . , Nature, 367:281-284 (1994)) .
  • Fas-Ligand Fas-Ligand
  • Both Fas and FasL molecules are induced within 4 hours of activation, and competitive inhibition of their inter ⁇ action blocks the induction of apoptosis (Brunner, T., et al. , Nature, 373:441-444 (1995)) .
  • FasL is not displayed on the cell surface, but rather is secreted, where it forms trimers and prior to interacting with the target cell expressing the Fas antigen (Dhein, J., et al. , Nature, 373:438-441 (1995)) .
  • activation-induced apoptosis in T cells proceeds via a cell-autonomous (single cell) Fas/FasL interaction.
  • Fas/FasL interaction multiple other signaling pathways are also known to be involved in regulating cell death.
  • activation-induced apoptosis is inhibited by several different agents, including the immunosuppressive drugs cyclosporine (Shi, Y., et al., Nature, 339:625-626 (1989) ; Shibuya, H., . et al., Cell , 70:57-67 (1992) ; Dowd, D.R., et al. , J. Biol . Chem .
  • T cells and other cells for example B cells
  • B cells activation-induced apoptosis in T cells and other cells
  • homeostasis e.g., the elimination of autoreactive cells or negative selection
  • immune reactivity systemically clonal deletion or peripheral tolerance
  • the ability to modulate this basic biological pathway has the potential to be of great benefit.
  • Retinoids modulate the specific expression of target genes by binding to and activating two distinct subfamilies of intracellular receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) (reviewed in Mangelsdorf, D.J., et al. , The Retinoids: Biology. Chemistry and Medicine 2nd ed. , pp. 319-349 (1994) .
  • RARs retinoic acid receptors
  • RXRs retinoid X receptors
  • retinoids examples include all- rans retinoic acid (ATRA) , 9-cis retinoic acid (9-cis RA) , and (E) -4- [2- (5,5,8, 8tetramethyl-5, 6,7, 8-tetrahydro-2 - naphthalenyl) -1-propenyl] benzoic acid (TT ⁇ PB) .
  • ATRA all- rans retinoic acid
  • 9-cis RA 9-cis retinoic acid
  • E -4- [2- (5,5,8, 8tetramethyl-5, 6,7, 8-tetrahydro-2 - naphthalenyl) -1-propenyl] benzoic acid
  • TT ⁇ PB nuclear receptor superfamily
  • RARs ⁇ ,( ⁇ and ⁇ ) and RXRs ⁇ , ⁇ and 7
  • DR direct repeat
  • All-trans retinoic acid is a high affinity ligand for RAR (a , ⁇ and 7) only, whereas 9-cis retinoic acid (9-cis RA) , the active stereoisomer of ATRA, is a high affinity ligand for both RARs and RXRs (Allegretto, E.A., et al. , J. Biol . Chem. , 268:26625-26633 (1993)) .
  • RAR/RXR heterodimers activate transcription in response to ATRA or 9-cis RA, while RXR/RXR homodimers transactivate in response to 9-cis RA.
  • 9-cis RA is 10-fold more potent than
  • ATRA in blocking activation-induced apoptosis suggests that this inhibition involves retinoic X receptors (Yang,
  • Retinoids are potent modulators of apoptosis in a number of experimental models. However in most of the experimental models where the effects of retinoids have been investigated, retinoids have been found to induce either differentiation or apoptosis, or in many cases both (for extensive reviews, see Moon, R.C., et al. , The Retinoids: Biology. Chemistry, and Medicine 2nd ed. , 573- 595 (1994) ; ibid., Hong, W.K., et al. , 597-630) .
  • retinoids have also been shown to inhibit activation-induced apoptosis in T-cell hybridomas and developing T-cells (thymocytes) (Yang, Y.L., et al. , Proc . Natl . Acad. Sci . U. S. A. , 90:6170-6174 (1993) ; Iwata, M. , et al., J. Immunol . , 149:3302-3308 (1992)) .
  • the specific pathway by which retinoids act to inhibit activation-induced apoptosis has not been previously identified or demonstrated.
  • retinoid compounds inhibit T-cell receptor mediated apoptosis in T-cell hybridomas by specifically blocking the expression of Fas-Ligand (FasL) on T cells following activation, and in particular, retinoid compounds having high affinity and activating potency for both RARs and RXRs.
  • Fas-Ligand Fas-Ligand
  • Figure 1 shows the inhibition of activation-induced apoptosis in anti-CD3 stimulated 2B4 T-cell hybridomas cells by 9-cis RA, expressed as % cells undergoing apoptosis.
  • the T-cell hybridoma 2B4 was activated in anti-CD3-coated 6-well microtiter plates for 12 hours at 37°C, in the presence or absence of 1 ⁇ M 9-cis RA.
  • the cells were collected, fixed in a 1% parafor aldehyde in phosphate-buffered saline (PBS) , assayed for DNA fragmentation by terminal deoxytransferase UTP nick end-labeling (TUNED, according to the kit manufacturer's instructions, and analyzed by flow cytometry.
  • Cells undergoing apoptosis with accompanying DNA fragmentation incorporate label (UTP-digoxigenin/digoxigenin-FITC) and can be detected by flow cytometry as a peak shifted to the right of the non-apoptotic cells, indicating an increase in DNA fragmentation and apoptosis.
  • the data shown represents the percentage of apoptotic cells (%TdT positive) in each sample (inset) .
  • Figure 2 shows the ability of 9-cis RA to inhibit the expression of FasL functional activity, expressed as % DNA fragmentation induced in Fas * target cells.
  • FasL functional activity was measured as the cytotoxic activity of 2B4 cells against Fas" target cells.
  • L1210 and L1210.fas target cells were labeled with [ 3 H] -TdR, then plated with either 2B4 cells, at the ratios indicated, into 96-well plates coated with anti-CD3 antibody.
  • the cultures were incubated in the presence or absence of 9-cis RA for 10 hours at 37°C, extracted, and the radioactivity retained on glass fiber mats was counted in a scintillation counter. The results shown are expressed as % DNA fragmentation relative to control wells (absence of 2B4 effector cells) and represent the mean of 6 replicates with standard deviation (bars) .
  • D unstimulated effector cells (2B4) o anti-CD3-stimulated, ⁇ unstimulated effector cells plus 1 ⁇ M 9-cis RA, • anti-CD3 stimulated effector cells plus 1 ⁇ M 9-cis RA.
  • Figure 3 demonstrates that the ability of 9-cis RA to inhibit the expression of FasL functional activity is specifically via modulation of FasL expression and not by interference with the ability of the Fas antigen to transduce a death signal.
  • this is shown where the induction of the death signal is mediated by the interaction between Fas and FasL directly, and is expressed as the % DNA fragmentation induced in Fas+ target cells.
  • 2B4 cells were plated at time 0 into 96-well plates coated with anti-CD3 antibodies, in the presence or absence of 9-cis RA. The L1210 and L1210.fas target cells were not added at this time.
  • T hybridoma cells were then cultured for 6 hours in the presence of the activating antibody to allow the expression of FasL, at which time the [ 3 H] -TdR-labeled L1210 and L1210.fas target cells (as in Figure 1) were added.
  • All plates were cultured a further 6 hours and extracted as described above in Figure 1. The results shown are expressed as % DNA fragmentation relative to control wells (absence of effector cells) and represent the mean of 6 replicates with standard deviation (bars) .
  • Figure 4 demonstrates that the ability of 9-cis RA to inhibit the expression of FasL functional activity is specifically via the modulation of FasL expression and not by interfering with the ability of the Fas antigen to transduce a death signal.
  • this is shown where the induction of the death signal is mediated by a monoclonal antibody specific for Fas, expressed as the % DNA fragmentation induced in Fas '* targets cells. Fas * Jurkat cells were labeled with
  • [ 3 H] -TdR then plated with into 96-well plates together with anti-Human Fas at the indicated concentration and 1 ⁇ M 9-cis RA for 10 hours at 37°C.
  • the results shown are expressed as % DNA fragmentation relative to control wells (absence of effector cells) and represent the mean of 6 replicates with standard deviation (bars) . Symbols: D Untreated control cultures, ⁇ cells plus 1 ⁇ M 9-cis RA.
  • Figure 5 shows the ability of 9-cis RA to modulate the expression of FasL at the mRNA level, detected qualitatively by the reverse transcription of polyA mRNA and specific oligodeoxynucleotide pri er/Taq DNA polymerase amplification of FasL mRNA (RT/PCR) .
  • RT/PCR analysis was performed on total RNA obtained from 2B4 cells activated by anti-CD3 in the presence or absence of 1 ⁇ M 9-cis RA.
  • RNA was reverse transcribed using an oligo dT primer and the resulting cDNA phenol :chloroform extracted.
  • PCR amplification was performed on 10-fold serially diluted cDNA using the primer pairs designed for each mRNA.
  • the gel was dried, exposed to X-ray film, and the resulting autoradiograph analyzed by densitometry scan.
  • the data shown in the graph represents the amount of FasL mRNA expression normalized to the g-actin in each sample and is expressed as fold induction FasL signal relative to unactivated 2B4 controls.
  • Figure 7 shows the correlation between the ability of 9-cis RA to block FasL expression at the mRNA level and the ability of 9-cis RA to prevent the expression of FasL protein, detected on the cell surface by flow cytometry.
  • 2B4 cells were incubated for 6 hours at 37°C on 6-well plates coated with anti-CD3, in the presence or absence of 1 ⁇ M 9-cis RA.
  • the cells were stained with the chimeric protein FasFc, which comprises the N-terminal portion of Fas, minus the transmembrane and cytoplasmic regions, with the Fc portion of human IgGl. FasFc binds to FasL, which is then detected by staining with anti-human Fc/biotin, and streptavidin PerCP.
  • Figure 8 shows the structures of the receptor- selective retinoid compounds used to demonstrate the bifunctional (RAR and RXR) receptor requirements for the inhibition of FasL expression by retinoids.
  • Figure 9 demonstrates the retinoid receptor activation selectivity of the receptor-selective compounds illustrated in Figure 8, expressed as the fold induction of retinoic acid receptor (RARE) -driven and retinoid X receptor (RXRE) -driven gene expression.
  • Ligand was added to CV-1 cells transiently co-transfected with receptor 11 expression vectors (RAR or RXR) , together with either of the two reporter constructs containing response elements for RAR (RARE, DRl-tk-Luc) or RXR (RXRE, DR5-tk-Luc) . Transfection efficiency was monitored by co-transfection with a b-galactosidase expression vector.
  • the data is expressed as fold induction relative to controls without ligand, and were normalized relative to the b-galactosidase signal. Symbols: ⁇ TTNPB, ⁇ LG100268, • 9-cis RA.
  • Figure 10 shows the relative ability of the receptor-selective compounds to inhibit activation-induced apoptosis, expressed as % apoptotic cells.
  • 2B4 cells were cultured in anti-CD3 coated 6-well microtiter plates in the presence or absence of the indicated compounds, added at a concentration of l ⁇ M. After 12 hours the cells were collected and assayed for DNA fragmentation by TUNEL, with propidium iodide counterstaining.
  • FL3 PI, DNA content
  • FL1 FITC, TdT positive
  • apoptotic cells appear low and to the right (TdT positive and hypodiploid) .
  • the numbers (inset) shown represent the percentage of apoptotic cells in each sample using the region marker shown in the dot plots.
  • Figure 11 shows the relative ability of the receptor-selective compounds to inhibit the expression of the functional activity of FasL on 2B4 effector cells, expressed as the % DNA fragmentation induced in Fas* targets cells.
  • the experimental procedures used here are the same as those described for Figure 2.
  • [ 3 H] -TdR-labeled L1210 and L1210.fas target cells were exposed to anti-CD3 activated 2B4 cells at the ratios indicated in the presence or absence of 1 ⁇ M ligand.
  • the data shown are the results obtained from an effector (2B4) to target
  • FIG. 12 shows the effect of adding increasing concentrations of the indicated retinoid compounds on the induction of apoptosis in 2B4 cells following activation. The figure demonstrates that the requirement for both RAR- and RXR-selective compounds for effective inhibition of FasL expression is not simply due to the presence of more retinoid, but to bifunctional activation of both retinoid receptor (RAR, RXR) pathways, expressed as % DNA fragmentation induced in activated 2B4 cells.
  • RAR retinoid receptor
  • FasL Functional expression was determined by the ability of activated 2B4 cells to express FasL-mediated cytotoxicity on Fas+ L1210 cells, as described for Figure 2. Symbols: ⁇ unstimulated controls, +/- retinoid, ⁇ anti-CD3- activated, +/- retinoid.
  • Figure 13 demonstrates a) the requirement for both RAR- and RXR-selective compounds for effective inhibition of FasL expression at the mRNA level, and b) the specificity of that inhibition, by demonstrating the lack of similar inhibition of Fas expression, expressed as fold induction of Fas and FasL mRNA relative to controls. Fas and FasL mRNAs were quantitated by RNase protection as described in Figure 3.
  • the T hybridoma cells were activated for 2 hours in 6-well plates coated with anti-CD3, the RNA extracted for RNase protection. Retinoids were added at 1 ⁇ M each.
  • Figure 14 shows the effect of retinoids on the production of the cytokine interleukin-2 (IL2) by activated 2B4 cells, expressed as the incorporation of [ 3 H] -TdR label by IL2-responsive CTLL-2 cells, and as units of IL2 produced (in parentheses) .
  • 2B4 cells were cultured 16 hours in 96-well plates coated with anti-CD3 antibody, with the indicated retinoids added at 1 ⁇ M. The culture supernatants were collected, centrifuged, and IL2 unit concentrations determined using the IL2-dependent cell line CTLL-2 and a recombinant IL2 standard.
  • CTLL-2 cells were cultured with the supernatants for 16 hours and pulsed 6 hours with 5 ⁇ Ci/ml [ 3 H]-TdR. Units of IL2 per ml were calculated using a standard curve and linear regression analysis. The data shown represents the mean of 6 determinations (DPM) with standard deviation. Symbols: O unstimulated controls, ⁇ anti-CD3 activated, o activated plus TTNPB, • activated plus LG100268, ⁇ activated plus 9-cis RA, + standard.
  • Activation-induced apoptosis in T-cell hybridomas occurs via multiple signaling pathways, but requires and is dependent upon the expression of two molecules, Fas (CD95) and Fas-Ligand (FasL) , which interact to transduce the apoptotic signal (Ju, S.T., et al. , Nature, 373:444-448 (1995) ; Brunner, T., et al . , Na ture, 373:441-444 (1995) ; Dhein, J. , et al . , Nature, 373:438-441 (1995)) .
  • Fas CD95
  • FasL Fas-Ligand
  • retinoids such as 9-cis retinoic acid (9-cis RA) specifically inhibit expression of FasL following activation, and by that specific pathway block activation-induced apoptosis.
  • retinoids which have high affinity and activation potency for both RXRs and RARs (pan-agonists) are most effective in blocking FasL expression and thus activation-induced apoptosis, indicating that this inhibition involves retinoid X receptors.
  • RXRs 9-cis retinoic acid
  • RARs pan-agonists
  • mice T-cell hybridoma line 2B4 was used and has been described in detail elsewhere.
  • the 2B4 cell line expresses the T-cell receptor-CD3 (TCR-CD3) surface receptor complex and undergoes activation-induced apoptosis when exposed to anti-TCR antibodies (Shi, Y., et al., Nature, 339:625-626 (1989) ; Mercep, M. , et al. , J. Immunol . , 142:4085-4092 (1989)) .
  • TCR-CD3 T-cell receptor-CD3
  • the pan-RAR/RXR agonist 9-cis retinoic acid (9-cis RA) was synthesized at Ligand Pharmaceuticals. The synthesis of 9-cis RA has been described in detail elsewhere (Boehm, M.F., et al., J. Med. Chem., 37:2930-2941 (1994)) . 9-cis RA was solvated to 1 mM in DMSO:ethanol.
  • 2B4 cells used in the assay were maintained in log phase growth. To activate the cells to undergo apoptosis, the cells were incubated in the presence of a monoclonal anti-CD3 antibody (clone 145.2C11)
  • the B cell hybridoma clone 145.2C11 secreting a monoclonal hamster anti-mouse CD3 antibody was obtained from the ATCC.
  • 145.2C11 culture supernatants were clarified and the antibody was affinity purified using protein A-Sepharose chromatography (Pharmacia, Piscataway, ⁇ J) .
  • Tissue culture plates used in the activation-induced cell death experiments were pre-coated with the above purified antibody, which had been diluted in to 0.5 ⁇ g/ml in a Tris-HCl buffer, (0.05 M pH 9.0) .
  • the plates/dishes were coated for 2 hours @ 37°C, following which they were stored at 4°C for a period of no more than 2 weeks before use.
  • the plates/dishes were washed extensively with a phosphate-buffered saline solution (PBS) to remove unbound antibody.
  • PBS phosphate-buffered saline solution
  • the cells were seeded in to plates, together with retinoid, for a period of not less than 8 hours.
  • the experiments were allowed to proceed for a period of 16 hours before assays of apoptosis would be performed.
  • activated but otherwise untreated 2B4 cells undergo apoptosis in excess of 50%.
  • FITC-labeled anti-digoxygenin antibody to detect the amount of UTP-label incorporated and thus the amount of DNA fragmentation present.
  • the cells were then counterstained with propidium iodide for DNA content, and analyzed by flow cytometry on a FACScan using
  • Macintosh-based CELLQuestTM software (Becton-Dickinson, San Jose, CA) .
  • Figure 1 The data is depicted by a histogram, where the area under the histogram at any one point along the x
  • (horizontal) axis represents the number of cells (y-axis) having a certain amount of fluorescence (x-axis) , which here is direct quantitation of DNA fragmentation and thus apoptosis.
  • 9-cis RA effectively inhibited activation-induced apoptosis in anti-CD3 stimulated 2B4 (from 77.0% to 28.6%) cells.
  • concentration of 9-cis RA required to produce a 50% inhibition of this response was 150 nM (data not shown) .
  • Similar results were obtained using other methods (not shown) , and these observations are consistent with those reported by others (Yang, Y.L., et al. , Proc . Natl . Acad. Sci . U. S. A. , 90:6170-6174 (1993)) , demonstrating that the RAR/RXR pan-agonist 9-cis RA is a potent modulator of activation-induced apoptosis.
  • Example 2 The inhibition of apoptosis in activated T-cell hybridomas via the negative regulation, bv 9-cis RA, of Fas-Ligand expression:
  • FasL Functional activity was determined by the ability of cells expressing FasL to induce apoptosis in the Fas * L1210 target cell line transfected to constitutively express mouse Fas (L1210.fas, (Rouvier, E., et al., J. Exp . Med . , 177:195-200 (1993))) .
  • L1210.fas L1210 target cell line transfected to constitutively express mouse Fas
  • DNA fragmentation was determined as follows: L1210 and L1210.fas cells were labeled for 2 hours at 37°C with 5 ⁇ Ci/ml [ ** __] -Thymidine deoxyribonuleotide ( [ 3 H] -TdR) in RPMI 1640/10% FCS. The cells (50xl0 3 per well) were washed twice in warm HBSS, resuspended in IMDM/10% FCS, and seeded into 96-well plates uncoated or coated anti-CD3 antibody.
  • L1210 wild type and Fas+ L1210.fas cells were labeled for 2 hours at 37°C with 5 ⁇ Ci/ml t 3 H] -TdR in RPMI 1640/10% FCS. The cells were washed twice in warm HBSS and resuspended in IMDM/10% FCS. The target cells (25 x 10 3 per well) were added to 96-well plates containing T hybridoma cells which had been seeded into uncoated or anti-CD3 coated wells at densities yielding the indicated final ratio of effector to target cells.
  • L1210 T cell leukemia cells normally express neither FasL nor CD3, and only barely detectable levels of Fas, and are therefore resistant to FasL-induced cell death.
  • L1210 cells transfected with mouse fas (L1210.fas) cells express high levels of Fas constitutively and are sensitive to the induction of apoptosis either by cells expressing FasL, or anti-Fas antibody. Therefore, they can be used as target cells to detect FasL expression when mixed with 2B4 effector cells activated via the TCR to express FasL (and Fas) .
  • Fas antigen When the Fas antigen interacts with Fas-Ligand, either in solution or on the surface of the same or other cells, that interaction between these two molecules results in the transduction of one-way death signal into the Fas-expressing cell.
  • Fas-Ligand a Fas-Ligand that interacts with Fas-Ligand, either in solution or on the surface of the same or other cells, that interaction between these two molecules results in the transduction of one-way death signal into the Fas-expressing cell.
  • Jurkat cells express Fas, and can be induced to undergo apoptosis by either FasL or by monoclonal antibodies specific for Fas. Thus the interaction between Fas and antibody binding Fas can be thought to mimic the interaction between Fas and FasL.
  • Figure 4 shows that the result of that interaction between Fas and antibody specific for Fas causes apoptosis, and is not blocked by 9-cis RA. The data confirms that 9-cis RA blocks not Fas signal transduction or the interaction between Fas and FasL if FasL is already expressed, but rather prevents the expression of FasL.
  • RA mRNAs expressed following activation were detected by Reverse transcriptase-Polymerase Chain reaction (RT/PCR) .
  • Total RNA was obtained from cells, activated as described in detail in examples 1-3, by acid-guanidium thiocyanate-phenol extraction (Xie, W.Q., et al. , Biotechniques, 11:324-327 (1991)) .
  • Reverse transcriptase synthesis of cDNAs was performed using oligo dT primers and a commercially obtained kit (cDNA Cycle Kit, Invitrogen, San Diego, CA) .
  • the resulting cDNA:RNA hybrids were phenol :chloroform extracted, precipitated, and serially diluted for PCR amplification with Taq polymerase (Gibco/BRL, Grand Island, NY) and the following primer pairs: GAPDH, sense (5' GTGAAGGTCGGAGTCAACG) and antisense (5' TGAAGACGCCAGTGGACTC) ; nur77a, sense (5' GGAACACCAGCAACGAGC) and antisense (5' CATCTGGAGGCTGCTTGG) ; FasL, sense (5' TTCTCTGGAGCAGTCAGCGT) and antisense (5' TAAGGACCACTCCATGGACC) .
  • the products of the PCR reaction were resolved on 1.5% agarose in TAE (40 mM Tris acetate, pH 8.5, 2 mM EDTA) gels, stained with ethidium bromide and photographed.
  • the developed films were scanned and the bands representing the resolved PCR products corresponding to the indicated mRNAs were analyzed using a PDI (Huntington Station, NY) scanner and Quantity OneTM software.
  • FasL expression in 2B4 cells as measured by RT/PCR was induced as a result of anti-CD3 stimulation. This expression was inhibited by the presence of 9-cis RA.
  • ⁇ ur77a expression was induced following activation. However, it was not altered by the addition of 9-cis RA to the cultures.
  • RNA used in this example was prepared as described for Example 4. However, in this example, the very sensitive and quantitative technique of RNase protection was used to determine the levels of FasL mRNA, following activation, in the presence of 9-cis RA.
  • FasL mRNA expression was determined by RNase protection using RT/PCR-generated DNA probe templates corresponding to the sequences of murine FasL and human g-actin, which has 100% homology with the corresponding murine g-actin sequence.
  • RNA obtained from activated 2B4 cells was 22 reverse transcribed and amplified with primer pairs s p e c i f i c f o r F a s L ( s e n s e 5 ' GGCCGAATTCAGATGGAAGGAGGTCTGTGA and antisense 5' GGCCAAGCTTAACGGCCTCTGTGAGGTAGT) .
  • cDNAs possessed convenient EcoRI and Hindlll linker ends, which were used for insertion into pGEM4Z (Promega) .
  • a similar procedure was employed to construct the control g-actin probe (sense 5' TTGATGCTGCAGGTCACCAACTGGGACGACATG and antisense 5' AACCCTAAGCTTCGCAGCTCGTTGTAGAAGG) .
  • the probes were sequenced to ensure identity with target sequences.
  • a- 32 P-UTP-labeled RNA transcripts were prepared with a kit (Ambion, MAXIscriptTM) using T7 RNA polymerase .
  • RNase protections were performed on total RNA samples according to the kit (RPA IITM, Ambion) manufacturer's instructions. The samples were resolved on 8 M urea/6% polyacrylamide sequencing gels, the gels were dried and exposed to Kodak Biomax ARTM film. The developed films were scanned and analyzed using a PDI (Huntington Station, NY) scanner and Quantity OneTM software. Measured by RNase protection (Figure 6) , FasL expression in 2B4 cells was induced greater than 50-fold following anti-CD3 stimulation, and this expression was profoundly inhibited by the presence of 9-cis RA (to 20% of control) .
  • FasL a chimeric Fas-Fc protein recognizing FasL expressed on cells following activation was utilized
  • Fas-Fc chimeric protein was produced in a baculovirus expression system (Crowe, P.D., et al . , J. Immunol .
  • FasFc expression vector The details of the construction of the FasFc expression vector have been described elsewhere (Brunner, T., et al . , Nature, 373:441-444 (1995)) . Briefly, the Fas-Fc insert was constructed from the cD ⁇ A encoding the extracellular domain of Fas, ligated to the cD ⁇ A encoding the hinge, CH2 and CH3 domains of human IgGl (Brunner, T., et al . , Nature, 373:441-444 (1995)) .
  • Tn5Bl-4 cells were infected with a baculovirus transfer vector containing the Fas-Fc cD ⁇ A, and the Fas-Fc protein was protein-G purified from cells grown in serum-free medium (Brunner, T., et al . , Nature, 373:441-444 (1995)) .
  • Fas-Fc 15 ⁇ g of purified Fas-Fc was added as above to the cells suspended in PBS/0.1% sodium azide/l% FCS/normal mouse IgG and incubated for 30 minutes on ice.
  • the cells were washed once in PBS/0.1% sodium azide/l% FCS, incubated 30 minutes with anti-human IgG-biotin, washed, and stained with Streptavidin TRICOLOR (Caltag, San Francisco, CA) or Streptavidin-PerCP (Becton-Dickinson, San Jose, CA) for 30 minutes on ice.
  • Streptavidin TRICOLOR Caltag, San Francisco, CA
  • Streptavidin-PerCP Becton-Dickinson, San Jose, CA
  • retinoid-receptor selective retinoids to determine the bifunctional Nature of the inhibition of FasL expression by 9-cis RA.
  • Retinoids are known to mediate their effects through two separate classes of retinoid receptors; the RARs and the RXRs.
  • RAR/RXR heterodimeric complexes regulate gene transcription by binding to retinoic acid response elements (RAREs) (Glass, C.K., Endocr. Rev., 15:391-407 (1994)) .
  • RAREs retinoic acid response elements
  • RXRs will form homodimers and activate transcription by binding to retinoid X response elements (RXREs) .
  • the relative selectivity of the compounds synthesized are routinely determined using retinoid receptor binding assays and co-transfection assays designed to detect the activation of the retinoid receptor's ability to regulate gene transcription.
  • Ligand competition-binding assays were performed using receptors prepared with a baculovirus expression system.
  • Receptors were obtained from lysis-extracts (Allegretto, E.A., et al., J. Biol . Chem. , 268:26625-26633 (1993)) of SF21 cells infected with baculovirus transfer vectors expressing hRAR , ⁇ and 7 or hRXR a , ⁇ and 7. The receptor extracts were incubated for 2 hours at 0°C with
  • the co-transfection assay to determine ligand activation of receptor transcriptional activity has been described in detail elsewhere (Heyman, R.A. , et al . , Cell, 68:397-406 (1992)) .
  • the retinoid receptor expression vectors used in this example were pRS-hRAR 7 (human RAR 7) (Giguere, V., et al . , Nature, 330:624-629 (1987)) and pRS-hRXR a, (human RXR a) (Mangelsdorf, D.J., et al. , Nature, 345:224-229 (1990)) .
  • the RAR reporter construct used contains two copies of the TRE-palindromic response element inserted into the basal reporter construct ⁇ MTV-Luciferase.
  • the RXR reporter construct TK-CRBPII-Luc contains one copy of the DR1 response element from CRBPII (cellular retinol binding protein II) , linked to the herpes simplex virus thymidine kinase (tk) minimal promoter upstream of the luciferase gene.
  • CRBPII cellular retinol binding protein II
  • CV-1 cells were seeded into 96-well plates and transiently co-transfected using the calcium phosphate method with 10 ng of either receptor-expression vector, 50 ng of reporter plasmid, and 50 ng of pRS-/3-GAL
  • Table 1 shows the comparative abilities (Kd) of the RAR-selective retinoid agonist, TTNPB, the RXR-selective LG100268 and the RAR/RXR pan-agonist 9-cis RA to bind the different baculovirus-expressed retinoid receptors.
  • Kd the comparative abilities of the RAR-selective retinoid agonist
  • TTNPB the RXR-selective LG100268
  • RAR/RXR pan-agonist 9-cis RA The structures of these compounds are shown in Figure 8.
  • LG100268 binds strongly to RXR ⁇ , ⁇ and 7, but not to RAR CK, ⁇ and 7.
  • TTNPB binds with high affinity to RAR ⁇ , ⁇ and 7, but not at all to RXR a, ⁇ and 7.
  • the pan-agonist 9-cis RA binds with high affinity to both RARs and RXRs in this assay.
  • the RAR-selective retinoid TTNPB effectively activated RAR-dependent transcription but not RXR-dependent transcriptional responses.
  • the RXR-selective LG100268 activated only an RXR-dependent response.
  • 2B4 cells were cultured in anti-CD3 coated 6-well microtiter plates in the presence or absence of the indicated compounds . After 12 hours the cells were collected and assayed for DNA fragmentation by TUNEL, with propidium iodide counterstaining. Propidium iodide counterstaining is widely used to facilitate the quantitation of the cell's DNA content, and is very useful as a measure of DNA fragmentation. Cells which have undergone DNA fragmentation contain less than normal amounts of DNA (are said to be hypodiploid) due the loss of the soluble DNA fragments, which can been observed by flow cytometry as a shift to the left (reduction) in 28 fluorescence intensity.
  • the RXR-selective compound LG100268 was found to be significantly less potent than 9-cis RA.
  • the numbers (inset) shown represent the percentage of apoptotic cells in each sample using the region marker shown in the dot plots.
  • the RAR-selective compound TTNPB was much less effective than the pan agonist 9-cis RA.
  • RARs and RXRs can form functional heterodimers which can be activated by either TTNPB or 9-cis RA, and since TTNPB alone was insufficient, these data suggested that the engagement of both RAR/RXR heterodimer and RXR/RXR homodimer receptor pathways was important in mediating the inhibition.
  • the two receptor-selective ligands were added together. Shown in Figure 10, the combination of the RAR- and RXR selective retinoids together was as effective as 9-cis RA, inhibiting anti-CD3-induced apoptosis in 2B4 cells.
  • retinoid-receptor selective retinoids to demonstrate the requirement for both RAR- and RXR-selective ligands for the inhibition of FasL functional activity by 9-cis RA.
  • TNPB TNPB
  • LG100268 RXR-selective
  • retinoids do not block the expression of Fas mRNA, as detected by RNase protection, which correlates well with flow cytometric data showing no significant change in Fas surface protein levels (not shown) .
  • T-cell hybridomas activated via the TCR produce IL2 (Achier, D.S., et al . , J. Immunol . , 143:3461-3469 (1989) ; Ashwell, J.D., et al. , J. Exp . Med. , 165:173-194 (1987) ; Mercep, M. , et al . , J. I-nmunol. , 142:4085-4092 (1989) ; Mercep, M. , et al. , J. Immunol., 140:324-335
  • lymphokine is often used as an indicator that activation has taken indeed place (Shi, Y. , et al . , Science, 257:212-214 (1992) ;
  • tissue culture super ⁇ natants were obtained from 2B4 cells which had been stimulated to undergo apoptosis in the presence or absence of 9-cis RA, as well as the two receptor-selective ligands TTNPB and LG100268.
  • the collected supernatants were titrated into cultures of the IL2-dependent cell line CTLL-2, which had been starved of IL2 for 4 hours prior to the addition of the IL2-containing supernatants.
  • the cells were cultured 16 hours and then pulsed for 6 hours with 5 ⁇ Ci/ml of [ 3 H] -TdR.
  • the labeled DNA was harvested and the amount of label incorporated determined. Units of IL2 per ml were calculated using a standard curve obtained with recombinant IL2 and linear regression analysis.
  • Activation-induced apoptosis in normal T lymphocytes is known to occur under various conditions in vivo . Stimulation of immature thymocytes via the T-cell receptor results in apoptosis and this is an important mechanism of negative selection during T-cell development.
  • Another form of activation-induced activation-induced apoptosis occurs in mature T-cells in vivo and in vi tro, following expansion of antigen-specific T-cells, and this homeostatic process has been called peripheral deletion. This process is mediated by Fas/FasL interaction (Russell, J.H., et al., Proc . Na tl . Acad . Sci . U. S . A .
  • Activation-induced apoptosis in T-cells also occurs under pathological conditions, and it has been implicated as a mechanism for the loss of both CD4+ and CD8+ T cells in AIDS.
  • Fas and FasL have been implicated.
  • One example of such a condition is the disappearance of hepatocytes during chronic Hepatitis C infections. It has been shown hepatocytes express Fas in both normal (Ogasawara, J., et al . , Nature 364:806-809
  • lymphocytes infiltrating the liver of such patients express FasL (Mita, E., et al . , Bioche ⁇ i. Biophys . Res . Commun . , 204:468-474 (1994)) .
  • FasL The implication here is that T-cells express FasL as a result of chronic stimulation due to persistent infection, thus interact with Fas expressed on hepatocytes to induce apoptosis in the liver cells, thus causing the massive destruction of liver cells associated with this disease.
  • FasL Another example of the potential role for FasL and thus its regulation in disease can be found in several inflammatory conditions where there are skin lesions which have been characterized by apoptosis in keratinocytes.
  • the common factors associated with these conditions are 33 lesions, the expression of both Fas and the cell:cell adhesion molecule ICAM-1 on keratinocytes associated with these lesions, and a lymphoid component in the pathology of the condition.
  • These include but are not restricted to lesions caused by Lichenoid drug eruption, Herpes zoster virus (varicella zoster virus) , Erythema Multiforme, and Contact dermatitis (Sayama, K. , et al . , J. Invest . Derma tol .
  • IFN-g Interferon-gamma
  • FasL lymphoid
  • FasL lymphoid
  • the selective blocking of the expression of Fas-Ligand following activation by use of the retinoid compounds of the type discussed herein can be expected to be useful in inhibiting T-cell receptor-induced Fas/FasL-mediated apoptosis and its effects in patients.

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Abstract

Procédé d'induction de l'expression de ligands de FAS par des cellules de mammifères après activation à l'aide de composés de rétinoïde et plus particulièrement de composés de rétinoïde activant à la fois les récepteurs du rétinoïde X et de l'acide rétinoïque. L'acide 9-cis rétinoïque et les composés associés constituent des exemples de ces composés de rétinoïde. Ce procédé permet d'obtenir un moyen d'inhiber l'apoptose dans des cellules exprimant des ligands de FAS après activation.
PCT/US1996/006090 1995-04-21 1996-04-17 Inhibition de l'apoptose dans des cellules exprimant le ligand de fas suite a l'activation WO1996032935A1 (fr)

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US5998654A (en) * 1997-07-25 1999-12-07 Ligand Pharmaceuticals Incorporated Retinoic acid receptor antagonist compounds and methods

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