Proc. Natl. Acad. Sci. USA Vol. 89, pp. 402-406, January 1992 Cell Biology

Interferons and interleukin 6 suppress phosphorylation of the retinoblastoma protein in growth-sensitive hematopoietic cells (cytokines/growth suppression/cyclin A/c-myc)

DALIA RESNITZKY, NAVA TIEFENBRUN, HANNA BERISSI, AND ADI KIMCHI* Department of Molecular Genetics and Virology, The Weizmann Institute of Science, Rehovot 76100, Israel

Communicated by Michael Sela, September 17, 1991 (received for review July 31, 1991)

phenotype that was completely growth-resistant to the cytokine (8). The same partially resistant phenotype was also generated by depletion of protein kinase C (PKC) from sensitive cells (9). However, the latter manipulation did not interfere with the IFN-induced c-myc inhibition, suggesting that PKC might be coupled to other target genes that control growth arrest. Multiple pathways, therefore, mediate the growth inhibitory effects of a single cytokine and the IFNand IL-6-induced suppression of c-myc turned out, from these experiments, to be necessary but not sufficient by itself to block the proliferation of cycling cells at the Go/Gj phase

One approach to identify postreceptor moABSTRACT lecular events that transduce the negative-growth signals of inhibitory cytokines is to analyze the cytokine-induced modifications in the expression of cell-cycle-controlling genes. Here we report that suppression of phosphorylation of the retinoblastoma gene product (pRb) is a receptor-generated event triggered by interferons and interleukin 6 (IL-6) in hematopoietic cell lines. The conversion of pRb to the underphosphorylated forms occurs concomitantly with the decline in c-myc protein expression and both events precede the G./Gj-phase arrest induced by the cytokines. Loss of IL-6-induced c-myc responses in cells that have been stably transfected with constitutive versions of the c-myc gene abrogates the typical GO/G1-phase arrest but does not prevent the specific dephosphorylation of pRb. Conversely, depletion of protein kinase C from cells interferes with part of the interferon-induced suppression of pRb phosphorylation and relieves the G./Gj-phase cell-cycle block without affecting the extent of c-myc inhibition. None of the cytokines, including transforming growth factor , reduce the phosphorylation of pRb in S-phase-blocked cells. In contrast, the other IL-6-induced molecular responses, including the decline in c-myc mRNA levels, are not phase-specific and develop normally in S-phase-blocked cells that are depleted of the underphosphorylated functional forms of pRb. These experiments distinguish between the reduction of c-myc expression and the suppression of pRb phosphorylation, which occur independently of each other, and suggest that the development of the interferon- or IL-6-induced GO/Gl-specific arrest requires at least these two receptor-generated events.

(8, 9).

Another transcription factor with potential cell-cycleregulatory functions is the product of the retinoblastoma (Rb) gene (for review, see refs. 10 and 11). Unlike the c-myc gene, which is controlled at the transcriptional level, the Rb gene is posttranslationally regulated-the Rb protein (pRb) is functionally regulated by modifying its phosphorylation. Previous studies have shown that pRb is phosphorylated in a cell-cycle-dependent manner occurring in late G1 and S phases and disappearing as the cells leave mitosis (12-15). Moreover, growth arrest that was caused by deprivation of growth factors, high cell density, or induction of differentiation and of senescence was associated with the disappearance of the hyperphosphorylated forms of pRb (13-16). Based on this correlative information, dephosphorylation has been implicated in the functional activation of pRb. Underphosphorylated pRb forms have been shown to be tightly associated with nuclear structures (17) and to form specific complexes with a cellular target for pRb, transcription factor E2F (18). Therefore, it was of interest to study the possibility that growth-inhibitory cytokines suppress pRb phosphorylation and thus activate pRb as part of their antiproliferative activities. Experiments that addressed this problem were performed by Laiho et al. (19) who showed that TGF-p prevented the phosphorylation of pRb that takes place at the G1/S boundary in mink lung epithelial cells. However, as a result of this challenging line of experiments, it became clear that other strategies had to be used to determine whether this molecular event leads to the growth arrest or is the indirect consequence of the TGF-p3-induced change in cell-cycle distribution. In the present study, specific genetic and drug manipulations that interfere with part of the signaling pathways of IFNs and IL-6 and prevent the GO/Gj-phase arrest have been used to study the relationship between cytokines and pRb. We show that the suppression of pRb phosphorylation is a receptorgenerated event triggered by IFNs or IL-6. pRb dephosphorylation is neither dependent on nor essential for the reduction of c-myc expression and, by itself, is insufficient to cause

Growth-suppressive signals are often generated by the interaction of diffusible polypeptides with their specific cell surface receptors. A major challenge in understanding the mechanisms of growth arrest is to identify the postreceptor genetic elements that transduce the signals triggered by those polypeptides (designated growth-inhibitory cytokines). One approach is based on the use of antisense cDNA expression libraries for rescuing those genes (1). Another approach includes a systematic analysis of the specific modifications that these cytokines might exert on the expression or the function of cell-cycle-controlling transcription factors. Thus, the antiproliferative effects of interferons (IFNs), transforming growth factor P (TGF-/3), and interleukin 6 (IL-6) have been correlated in certain hematopoietic and epithelial cells with the selective reduction of c-myc mRNA expression (2-7). Genetic manipulations were then performed to determine the contribution of the reduction in the expression of c-myc to the negative-growth signaling of cytokines (8). The transfection of myeloid cells with a constitutive version of c-myc that fails to be switched off by IFN or IL-6 abrogated the GO/Gl-phase arrest but was not sufficient to generate a

Abbreviations: IFN, interferon- IL-6, interleukin 6; Rb, retinoblastoma; pRb, Rb protein; pRbPkos, slow-migrating phosphorylated pRb; TGF-P, transforming growth factor (3; PKC, protein kinase C; PMA, phorbol 12-myristate 13-4cetate. *To whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 402

Cell Biology: Resnitzky ~Proc. Natl. Acad. Sci. USA 89 (1992) etet al.al. Cell Resnitzky Biology:

G0/Gj arrest in cells that express c-myc constitutively. The

reduction of c-myc expression and the suppression of pRb phosphorylation appear to be two parallel events that are triggered simultaneously and reflect the existence of more than one pathway through which these cytokines act.

MATERIALS AND METHODS Cell Lines and Culture Conditions. Ml mouse myeloblastic cells (20) were grown in Eagle's minimal essential medium (GIBCO) supplemented with 10% (vol/vol) heat-inactivated fetal calf serum (Bio-Lab, St. Paul). S6, the clone of Ml used in this study, was described in detail elsewhere (8). S6MP cells represent a pool of clones that have been stably transfected with pSV-myc-2, a plasmid containing the two coding exons of murine c-myc linked to the simian virus 40 early promoter as described (8). The Daudi Burkitt lymphoma cell line was grown in RPMI 1640 medium (GIBCO) supplemented with 10% heat-inactivated fetal calf serum (Bio-Lab). For PKC depletion, Daudi cells were exposed for 24 h prior the experiment to phorbol 12-myristate 13-acetate (PMA; 5 ng/ml; Sigma) as detailed (9). The cell-cycle distribution was determined as described (8). Immunoblot Analysis and Antibodies. Approximately 5-10 x 106 cells were lysed in extraction buffer [10 mM Tris-HCI, pH 7.2/150 mM NaCl/1% Triton X-100/0.1% SDS/1% sodium deoxycholate/5 mM EDTA/1 mM phenylmethylsulfo-

nyl fluoride/aprotenin (50 g&g/ml)/leupeptin (50 A&g/ml)/20 mM NaF/100 jLM vanadate]. Extracts were prepared and samples were gel-fractionated and electroblotted as described (8). For pRb detection, immunoblots were incubated for 2 h at room temperature with a 1:100 dilution of monoclonal anti-pRb antibodies (G3-245; 500 pg/ml; PharMingen, San Diego). For c-myc protein detection, immunoblots were incubated with monoclonal antibodies against a myc synthetic peptide (C59B) that corresponds to amino acids 173188 in human c-myc [1:10 dilution of hybridoma supernatant, MYCl-3C7 (21)]. In some immunoblots (see Figs. 1 and 3), the next step for pRb and c-myc detection consisted of incubating the immunoblots for 1.5 h with a 1:500 dilution of polyclonal. rabbit anti-mouse antibodies (2 mg/ml; PharMingen) followed by a 1-h incubation with iodinated protein A (Amersham; 500,000 cpm/ml). The second step for the immunoblots (see Figs. 2 and 4) was a 2-h incubation with 1:10,000 dilution of goat anti-mouse IgG heavy and light chains coupled to peroxidase (Jackson ImmunoResearch), followed by using an ECL Western blotting detection system (ECL, Amersham). To detect cyclin A protein, the immunoblots were incubated for 2 h at room temperature with a 1:500 dilution of rabbit polyclonal antibodies against cyclin A (antiserum 93; M. Pagano and G. Draetta, personal communication) and then further incubated for 1 h with protein A coupled to peroxidase (1:10,000 dilution; Amersham), followed by using an ECL Western blotting detection system. Northern Blot Analysis and DNA Probes. Total cellular RNA was isolated by a LiCl/urea procedure and analyzed on blots as described (4). Hybridization was done with 1 x 107 cpm of the following random-primer-labeled DNA probes: the 2.4-kilobase (kb) Xba I-Xho I genomic fragment of murine c-myc containing the second and third exons (22), the 0.75-kb and 1.3-kb EcoRI fragments of the murine c-myb cDNA (plasmid MM49) (23); the 0.5-kb BamHIl-Sac I fragment of the murine junB cDNA (24), and the 1. 3-kb Pst I-Pst I fragment of rat GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA (25). Cytokines. Murine IFN (a plus /3) was purchased from Lee Biomolecular Laboratories (San Diego) (2 x 108 units/mg). Human IFN-a was purified to 5 x i08 units/mg as described (2). TGF-f31 prepared from human platelets was purchased from R & D Systems (Minneapolis). Human recombinant

IL-6 purified to 2 x 107 units/mg pharm, Rehovot, Israel.

was

403

provided by Inter-

RESULTS Daudi Burkitt lymphoma cells are extremely sensitive to the antigrowth effect of IFNs (a or /3) (2, 3). The IFN-induced inhibition of cellular proliferation results from a specific block at the GO/Gl-phase of the cell cycle (2). The total pool of pRb was visualized by immunoblot analysis of whole-cell extracts using an anti-Rb monoclonal antibody. Previous work in various cell systems has shown that hyperphosphorylated pRb forms migrate more slowly in gels than under- or unphosphorylated pRb forms (12, 26). Fig. la shows that, in exponentially growing Daudi cells, a major portion of pRb appears to be the slow-migrating phosphorylated forms (designated pRbphos) and, with exposure to IFN-a, a progressive conversion to fast-migrating forms takes place. The latter forms are not represented in the nontreated culture. The time kinetics indicated that by 4 h after adding IFN-a (150 units/ ml), fast-migrating pRb forms were still hardly detected but

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FIG. 1. Immunoblot analysis of the IFN-a-induced response of pRb, c-myc, and cyclin A in Daudi Burkitt lymphoma cells. (a) Daudi Burkilt lymphoma cells (1.5 x i05 cells per ml) were treated with human IFN-a (150 units/ml). Cellular extracts were prepared when indicated and 40 g.g of protein was analyzed on immunoblots with anti-pRb and anti-c-myc antibodies. pRbPhos, slow-migrating phosphorylated pRb. (b) IFN-a treatment was as described in a except that a portion of the cell culture was pretreated for 24 h with PMA (5 ng/ml), and the drug was retained during the subsequent period of IFN treatment. Immunoblots were incubated with anti-pRb, anti-cmyc, or anti-cyclin A antibodies. +, PMA present; -, PMA absent; C, control. (a and b) For cytofluorimetric studies to determine the DNA content for cell-cycle analysis, 1 x 106 cells were analyzed prior to protein extraction. The values shown are the percent of cells that carry a DNA content of 2N (where iN is equivalent to the haploid amount of DNA).

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by 8 h they had reached 30-40% of the pRb population as determined by densitometric tracing. The ratio became biased in favor of the fast-migrating underphosphorylated forms of pRb 16 and 24 h after IFN treatment and reached =100% conversion to the rapidly migrating pRb form at 48 h (Fig. la). Next, in light of our report (2) that IFN-a reduces the transcription rate of c-myc in Daudi cells, we used the same cell lysates for testing the levels of c-myc protein on immunoblots. IFN-a reduced c-myc protein levels beginning between 4 and 8 h and by 24 h the signal was no longer detected (Fig. la). Samples from these cultures were also assayed for the IFN-a-mediated changes in cell-cycle distribution before their extraction. Fig. la shows that a 48-h IFN-a treatment caused 80%o of the cells to accumulate in the Go/Gj resting phase of the cell cycle. After an 8-h IFN treatment, the cell-cycle distribution was identical to that of control nontreated cells but after a 16-h treatment the distribution started to change. To further explore the relationship between IFN-a and the posttranslational modification of pRb, we depleted Daudi cells of PKC by prolonged exposure to PMA (9). Treatment of Daudi cells with PMA (5 ng/ml) eliminated >90% of the PKC protein and enzyme activity without causing any other detectable side effects (9). It generated a partially resistant phenotype, in which cells were no longer blocked at Go/G1 by IFN-a, without losing many of the IFN-a-induced molecular responses, so that eventually the cells stopped proliferating in the presence of IFN in a non-cell-cycle-specific manner (9). We examined the effect of PKC depletion on the IFNinduced pRb response. Pretreatment with PMA partially interfered with the IFN-mediated change in the phosphorylated state of pRb (Fig. lb). By 24 h only 35% of the pRb appeared in the rapidly migrating underphosphorylated forms in PKC-desensitized cells compared to 60%o of the pRb in IFN-a-treated naive cells as determined by densitometric tracing. At 48 h, although almost 100% of the pRb population was converted into underphosphorylated forms in the naive cells, in the PKC-desensitized cells, only 50% of the pRb population was converted in response to IFN-a (Fig. lb). This partial abrogation of pRb responses was repeated in two additional experiments. It represents one of the few molecular responses to IFN that is blocked by the PMA treatment. Another molecular response that was abrogated in the PMApretreated cells is the IFN-a-mediated reduction in cyclin A protein levels. Whereas the level of cyclin A protein sharply declined in the naive cells 24 and 48 h after IFN-a treatment, this reduction was completely abrogated in the PKCdesensitized cells. In contrast, and as expected from our report (9), depletion of PKC did not interfere with c-myc responses to IFN-a and the extent of the IFN-a-induced c-myc reduction was identical in naive and PMA-pretreated cells. The cell-cycle data confirm that, as shown (9) PKC depletion interfered with the ability of IFN-a to arrest cells efficiently in Go/G1 phase. The relaxed regulation of pRb and cyclin A in the PKC-desensitized cells could contribute to the loss of the IFN-mediated Go/G1 arrest in spite of the continuous c-myc responses. The Ml myeloblastic cell line (20) represents another intriguing system to study the various molecular pathways that block cell-cycle progression at Go/Gj phase. The Go/G1 arrest can be induced in these cells by three cytokines, IFNs (a and ,B), TGF-13, and IL-6 (refs. 8 and 27; see also Fig. 2). The IL-6-induced growth arrest develops in these cells as they differentiate morphologically and functionally to monocytes (8, 28). Fig. 2 illustrates that treatment of Ml cells (S6 clone) (8) with IFN, TGF-,B, or IL-6 induces the appearance of underphosphorylated forms of pRb that become the major forms by 48 h. These underphosphorylated forms of pRb are absent in the exponentially growing nontreated cells. IL-6 converts almost 100%o of the pRb population to underphos-

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Biology: Resnitzky et al.

proliferating in a non-cell-cycle-specific manner. We show here that in spite of the loss of Go/Gj arrest (Fig. 3), the IL-6-induced pRb response is not affected by the transfected sequences. The comparison between the S6 and S6MP (a pool of myc-transfected clones) shows an identical pattern of pRb migration on gels 24 h after IL-6 treatment and only minor differences at 48 h where the dominant underphosphorylated forms migrate in a slightly more diffused pattern in the myc-transfected cells. This almost identical pattern of pRb responses occurs in the absence of changes in cell-cycle distribution and stands in sharp contrast to the failure of IL-6 to down-regulate the expression of the c-myc protein from the exogenous gene. We conclude from the data in Fig. 3 that most if not all of the IL-6-induced conversion of pRb to the underphosphorylated forms is the result of receptorgenerated events that occur even if cell cycle responses have been interrupted. In addition, these data indicate that the constitutive levels of the c-myc protein in the transfected clones do not prevent the pRb response to IL-6, and the latter is not sufficient by itself to cause a Go/Gj block. One or more of the three cytokines could activate a non-cell-cycle-dependent pRb phosphatase and thus lead to the reduction in the phosphorylated state of pRb. To test this possibility, we generated a system where the responses to IFN, TGF-p, and IL-6 develop under conditions where all the pRb molecules are "locked" in the hyperphosphorylated form and not exposed to the M-phase phosphatase (29). Those conditions were obtained by blocking cells in S phase with hydroxyurea. As shown in Fig. 4a, after a 48-h treatment with 0.15 mM hydroxyurea, the cycling Ml cells accumulated in S phase and contained only the most slowly migrating hyperphosphorylated forms of pRb. Cells exposed to IFN, TGF-p8, or IL-6 during the last 24 h of hydroxyurea treatment showed no detectable effect on the migration of these pRb forms, whereas the naive cells showed the typical 24-h shift in electrophoretic mobility (Fig. 4a). The lack of any detectable shift in pRb migration in S-phase-blocked cells excludes the simple possibility that one of these cytokines activates a specific phosphatase that recognizes pRb in addition to the M-phase-specific phosphatase (29). Instead, the cytokines could inhibit the activity of cell-cycle-dependent pRb kinase(s) or increase the activity of a phase-specific phosphatase.

The hydroxyurea experiment also provided a powerful tool measure the possible dependence of the other molecular responses to the cytokines, especially the reduction of c-myc expression, on the presence of underphosphorylated forms of pRb. In Fig. 4b, total RNA was extracted from naive and hydroxyurea-blocked cells that were cultured with and without IL-6. The Northern blot analysis shows that c-myc mRNA levels were efficiently reduced by IL-6 in S-phaseblocked cells, whereas immunoblot analysis (Fig. 4b Inset) shows no detectable effect on the migration of pRb in S-phase-blocked cells. Two other molecular responses to IL-6, suppression of the c-myb gene and induction of junB mRNA expression, were normal in S-phase-blocked cells (Fig. 4b). Thus the hydroxyurea treatment did not destroy the functional cell surface receptors to IL-6 and permitted many molecular responses that, unlike the pRb response, were not phase-specific. Thus, suppression of c-myc transcription could develop under those conditions in the absence of any detectable functional hypophosphorylated forms of pRb. to

DISCUSSION In this work we show that three growth-inhibitory cytokines are capable of interfering with pRb phosphorylation in

growth-sensitive hematopoietic cells. By analyzing myctransfected clones that have lost cell-cycle-specific responses to IL-6, due to the genetic manipulation, we prove that this suppression of phosphorylation is a receptor-generated event

Proc. Natl. Acad. Sci. USA 89 (1992)

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FIG. 4. S-phase-blocked cells fail to reduce pRb phosphorylation in response to IFN, TGF-,8, and IL-6 but continue to reduce c-myc mRNA expression. (a) M1-S6 cells were seeded at 2 x 10 cells per ml in the presence (+) and absence (-) of 0.15 mM hydroxyurea. After 24 h, fresh hydroxyurea and one of the three cytokines IFN, TGF-,8, or IL-6 were added to the cultures. Twenty-four hours later, proteins were extracted and analyzed on immunoblots for pRb expression. Cell-cycle distribution was analyzed in parallel and the percent of cells in S phase was calculated. (b) M1-S6 cells were seeded at 2 x 105 cells per ml with (+) or without (-) 0.15 mM hydroxyurea. Twenty-four hours later, IL-6 was added (250 units/ ml) with fresh hydroxyurea and, after an additional 24 h, proteins and total RNA were extracted. The Northern blot of the total RNA was hybridized sequentially with the following DNA probes: c-myc, c-myb,junB, and GAPDH. (Inset) Immunoblot of extracted proteins with anti-pRb antibodies. +, Compound added; -, compound not added.

that continues to take place even when the cell-cycle distribution does not change. Several lines of evidence suggest that one of the putative pRb kinases is p34cdc2 or a closely related kinase (30-32). The timing of pRb phosphorylation in synchronized cells (29) suggests that p34cdc2 or related kinase must function either in a complex with cyclin A (33) or with one of the earlier G1 cyclins (34-36). Each of the cytokines could inhibit the synthesis, reduce stability, or induce posttranslational inhibitory modifications of one of the putative subunits that generate the active cdc2 kinase. Previous work (37) with synchronized mink lung epithelial cells reported that TGFP-mediated rapid inhibitory effects on p34cdc2 phosphorylation and H1 histone kinase activity, provided that the cytokine was added before the Gj/S phase boundary. Similar IFN-a-mediated interactions have been detected in the Daudi cell system (38). However, the contribution of the inhibition of p34cdc2 phosphorylation to the suppression of pRb phosphorylation is still a matter of speculation. Also it is not clear whether the reduction by the cytokines of cyclin A protein levels, shown above, may have any role in the reduction of pRb phosphorylation. Genetic manipulation with constitutive versions of the cyclin A gene could determine the contribution of cyclin A inhibition to pRb dephosphorylation

406

Proc. Natl. Acad Sci. USA 89 (1992)

Cell Biology: Resnitzky et al.

and growth inhibition. The PKC depletion data illustrate that the IFN-a-mediated reduction in cyclin A levels and part of the suppression of pRb phosphorylation are dependent on active PKC. Since many of the other molecular responses to IFN are not coupled to PKC (9), this system provides another tool to investigate the relationship between cyclin A and cytokine-induced pRb responses. Another point raised in this work relates to the distinction between the suppression of c-myc and the dephosphorylation of pRb in hematopoietic cells that are exposed to IFNs and IL-6. The IL-6-induced dephosphorylation of pRb does not depend on c-myc reduction, as shown by the analysis of myc-transfected clones. Moreover, the hydroxyurea experiment suggests the existence of a mechanism that reduces c-myc expression in the absence of underphosphorylated pRb forms characteristic of the cytokine response. Pietenpol et al. (39) showed that TGF-,f-induced suppression of c-myc transcription in keratinocytes could be abrogated by the wellstudied transforming proteins of three DNA tumor viruses and suggested that pRb may be a likely candidate for mediating the negative effects on c-myc expression (39). Several molecular mechanisms capable of suppressing c-myc transcription might exist in mammalian cells and could be differently used in each system depending on the type of the cytokine and the genetic background of the target cells. The defined mapping of the cis-regulatory sequences responsible for the negative responses of the c-myc gene in each system should clarify this point. However, it is clear that, in the system studied in this work, the modifications of pRb and of c-myc that are induced by IFN and IL-6 do not form a simple single cascade of events. Other promoters have been shown to be trans-repressed or activated by pRb in cotransfection experiments, including the c-fos (40) and the TGF-f31 (41) promoters. The function of pRb has been associated with its ability to form complexes with transcription factor E2F (18, 42-44). The potential target genes whose expression might be up- or down-modulated through the cytokine-generated underphosphorylated forms of pRb should be studied. In conclusion, this work establishes that IFNs and IL-6 actively interfere with the phosphorylation of pRb in some growth-sensitive hematopoietic cells. From the analysis of the myc-transfected cells, it is clear that this molecular event, which is supposed to activate the growth-suppressive effects of the protein, is not sufficient by itself to block cell-cycle progression at the Go/Gj phase and must function simultaneously with the reduction of c-myc expression. The clear dissociation between pRb dephosphorylation and the IL-6mediated c-myc inhibition shown here in the hydroxyurea experiment suggests the existence of mechanisms that suppress c-myc transcription independent of the presence of dephosphorylated pRb. Thus the two molecular events might lie on parallel pathways, which further supports the concept that a single cytokine activates more than one pathway, each being necessary but not sufficient by itself to cause the Go/Gj arrest. We thank G. Draetta for the antibodies against cyclin A, G. Evan for the anti-c-myc antibodies, and Interpharm Inc. for the gift of recombinant IL-6. This work was supported by grants from Minerva and the Laub Foundation. 1. Deiss, L. P. & Kimchi, A. (1991) Science 252, 117-120. 2. Einat, M., Resnitzky, D. & Kimchi, A. (1985) Nature (London) 313, 597-600. 3. Knight, E. J., Anton, E. D., Fahey, D., Friedland, B. K. & Jonak, G. J. (1985) Proc. Nati. Acad. Sci. USA 82, 1151-1154. 4. Yarden, A. & Kimchi, A. (1986) Science 234, 1419-1421. 5. Kimchi, A., Resnitzky, D., Ber, R. & Gat, G. (1988) Mol. Cell. Biol. 8, 2828-2836. 6. Pietenpol, J. A., Holt, J. T., Stein, R. W. & Moses, H. L. (1990) Proc. Natl. Acad. Sci. USA 87, 3758-3762. 7. Coffey, R. J., Bascom, C. C., Sipes, N. J., Graves-Deal, R.,

8. 9. 10. 11. 12.

13. 14. 15.

16. 17. 18. 19.

20. 21. 22. 23. 24. 25.

26. 27. 28.

29. 30. 31.

32. 33. 34. 35. 36. 37. 38. 39. 40.

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