Oncogene (1997) 14, 1923 ± 1931  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00

Polyomavirus large T antigen overcomes p53 dependent growth arrest

Joanne Doherty and Robert Freund

Molecular and Cell Biology Program, University of Maryland at Baltimore and Department of Microbiology and Immunology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, Maryland 21201, USA

Polyomavirus transforms cells in culture and induces Lane, 1993; Di Leonardo et al., 1994). In the absence tumors in mice without apparent interaction with or of p53, cell cycle arrest is not seen and DNA damage inactivation of the p53 tumor suppressor protein. In this leads to the genomic instability present in many tumors report we investigate the ability of polyomavirus T (Livingston et al., 1992; Yin et al., 1992). Wild-type antigens to overcome the growth suppression function of p53 expression may sometimes induce programmed cell p53. A temperature sensitive p53 gene was introduced death, depending on the cell type and expression of into mouse embryo ®broblasts derived from a p53 null oncogenes such as myc and adenovirus E1A or mouse, resulting in expression of a protein with a mutant radiation induced DNA damage (Debbas and White, conformation at 378C and a functionally wild-type 1993; Lowe and Ruley, 1993; Lowe et al., 1993; conformation at 328C. We found that expression of p53 Hermeking and Eick, 1994). at 328C induced the cyclin-dependent kinase inhibitor The ability of p53 to activate transcription in a p21/WAF1 and arrested cell growth in the G1/G0 phase sequence speci®c manner is required for its growth- of the cell cycle. Only the under-phosphorylated form of suppressive function (Crook et al., 1994; Pietenpol et the retinoblastoma tumor suppressor protein (pRB) was al., 1994). It is thought that growth suppression is due detected in these growth arrested cells. We introduced in large part to the induction of the cyclin-dependent both polyomavirus large T (LT) and middle T (MT) kinase inhibitor, p21/WAF1 (El-Deiry et al., 1993, antigens into this cell line and showed that LT overcame 1994; Di Leonardo et al., 1994; Dulic et al., 1994). p53-dependent growth arrest, while MT did not. In cells Over-expression of p21 results in growth arrest in G1 grown at 328C, LT expession led to cell proliferation and and in its absence p53 is unable to mediate growth phosphorylation of pRB in the presence of p21. A mutant arrest (Harper et al., 1995; Deng et al., 1995). The p21 LT containing a defective pRB binding domain failed to protein binds to cyclin/cdk complexes and high overcome the growth arrest, indicating that interaction of concentrations of p21 inhibit the kinase activity of LT with RB proteins is required to override p53 function. these complexes (Gu et al., 1993; Harper et al., 1993, Although the polyomavirus T antigens do not interact 1995; Xiong et al., 1993; Zhang et al., 1994). One of with p53 directly, our results indicate that the virus, the substrates for cyclin/cdk complexes is the family of through LT, is able to interfere with the growth retinoblastoma tumor suppressor proteins (pRB, p107, suppressive activity of p53. p130) (Hinds et al., 1992; Ewen et al., 1993; Kato et al., 1993). Phosphorylation of pRB inactivates its Keywords: polyomavirus; large T antigen; p53; cell growth suppressive activity (Weinberg, 1995). Thus, it cycle arrest is thought that p53-dependent growth arrest is the result of inhibition of phosphorylation of the RB family of proteins by p21. Polyomavirus is a DNA tumor virus capable of Introduction transforming and immortalizing cells in culture and inducing tumors in mice (Tooze, 1981). The ability to The p53 tumor suppressor protein plays a critical role immortalize primary cells and transform established in suppressing cell proliferation and neoplastic cell lines is mediated by two viral antigens, large T transformation (Levine et al., 1991; Donehower and (LT) and middle T (MT) antigen. MT, a membrane Bradley, 1993; Levine, 1993). Inactivation of the p53 protein, is necessary and sucient for transformation gene frequently occurs during the development of both of cells in culture (Treisman et al., 1981; Raptis et al., mouse and human neoplasia (Mowat et al., 1985; 1985). MT induces transformation and tumorigenesis Hollstein et al., 1991). A variety of mechanisms can by its interaction with and activation of several cellular lead to such functional inactivation, including mutation proteins involved in mitogenic signal transduction. It and interaction with viral and cellular proteins (Levine associates with members of the src family of tyrosine et al., 1991; Mietz et al., 1992; Momand et al., 1992; kinases and activates their kinase activity (Courtneidge Yew and Berk, 1992). Wild-type p53 is thought to and Smith, 1983; Bolen et al., 1984). Phosphorylation suppress oncogene-mediated transformation through of MT promotes its binding to the signaling molecules its ability to induce growth arrest and/or activate phosphatidylinositol 3-kinase (PI3-kinase), shc and apoptosis (Bates and Vousden, 1996). A variety of phospholipase C (Whitman et al., 1985; Kaplan et signals, mostly associated with DNA damage, increase al., 1986; Talmage et al., 1989; Campbell et al., 1994; cellular p53 levels and elicit cell growth arrest in the G1 Dilworth et al., 1994; Su et al., 1995). LT is a nuclear phase of the cell cycle (Kastan et al., 1991; Lu and phosphoprotein that induces cellular DNA synthesis and cell division, is responsible for immortalizing cells, Correspondence: R Freund and is necessary for viral DNA synthesis (Francke and Received 19 August 1996; revised 8 January 1997; accepted 9 January Eckhart, 1973; Schlegel and Benjamin, 1978; Rassoul- 1997 zadegan et al., 1983). LT associates with pRB and this Polyoma large T overcomes p53 growth arrest J Doherty and R Freund 1924 association is necessary for the induction of cellular and M/puro cells at the permissive (328C) and non- DNA synthesis and immortalization of primary cells permissive (378C) temperatures and determined the (Dyson et al., 1990; Larose et al., 1991; Freund et al., viable cell numbers at 1, 2, 4, and 6 days (Figure 1a). 1992, 1994; Mudrak et al., 1994). Several other DNA M/tsp53 cells grew at 378C but not at 328C while M/ tumor viruses, including SV40, adenovirus, and human puro cells grew at both temperatures. The growth rates papilloma virus, encode proteins that associate with of M/puro cells at both temperatures, and M/tsp53 and inactivate both p53 and pRB (Sarnow et al., 1982; cells at 378C were similar. These results indicate that DeCaprio et al., 1988; Whyte et al., 1988; Munger et expression of the temperature sensitive p53 arrested cell al., 1989; Werness et al., 1990). Polyomavirus is unique growth at 328C but did not dramatically alter the rate among these DNA tumor viruses in that a direct of growth at 378C. interaction of its T antigens with wild-type p53 has not The proportion of cells in each phase of the cell been detected (Wang et al., 1989). cycle was determined by analysing the DNA content of In this report we investigate the ability of cells using propidium iodide staining and ¯ow polyomavirus T antigens to overcome the growth cytometry. The cell cycle distribution of M/puro and suppression function of wild-type p53. Mouse embryo M/tsp53 cells maintained at 378C and 328C for 24 h is ®broblasts derived from p53-de®cient mice were stabily shown in Figure 1b. M/tsp53 cells at 328C were

transfected with a murine temperature sensitive p53 arrested predominantly in the G1/G0 phase of the cell

gene. As previously described, the temperature sensitive cycle; 74% of the cells were in G1/G0, 10% were in S,

p53 protein functions as a mutant protein at 378C and and 16% were in G2/M phases. At 378C, 48% of the

as a wild-type protein at 328C (Michalovitz et al., 1990; cells were in G1/G0, 36% were in S, and 16% were in

Martinez et al., 1991). At 378C the p53 protein G2/M. The cell cycle distributions of M/puro cells, localizes to the cytosol and is complexed with the grown at both temperatures, and M/tsp53 cells grown heat shock protein, hsp70. At 328C the protein is found at 378C were similar and is consistent with their predominantly in the nucleus, where it suppresses continuous growth depicted in Figure 1a. oncogenic transformation and reversibly arrests the The level of p53 protein expressed by M/tsp53 cells

growth of cells in the G1 phase of the cell cycle at 378C and 328C was examined by Western blotting (Michalovitz et al., 1990; Martinez et al., 1991). Here (Figure 1c). High levels of p53 were expressed at both we show that expression of the temperature sensitive 378C (lane 1) and 328C (lanes 2 ± 4). The level of p53

p53 at 328C arrests cell growth in G1 and induces decreased in cells maintained at 328C for increasing expression of p21. Additionally, we found that the time. The blot was probed with antibodies to actin to pRB in these growth arrested cells is hypophosphory- con®rm that similar amounts of protein were loaded in lated. Coexpression of each of the T antigens with p53 each lane. The decrease in the amount of p53 at 328C in these cells revealed that only the LT antigen, and over time may be due to di€erences in the half-life of not the MT antigen, overcame the growth arrest the protein. These results indicate that the temperature induced by p53. LT antigen did not interfere with the sensitive p53 is expressed in M/tsp53 cells at both p53 induced expression of p21, yet these cells continued temperatures and, as previously reported with other

to grow and contained phosphorylated forms of pRB. cell types, arrests cell growth in the G1/G0 phase of the In genetic studies, a mutant LT containing a defective cell cycle when cells were maintained at 328C RB binding domain was unable to override the p53 (Michalovitz et al., 1990; Martinez et al., 1991). dependent growth arrest, indicating that the LT-RB interaction is necessary for that function. These Polyomavirus large T antigen overcomes p53 dependent experiments support and extend the results of previous growth arrest and middle T antigen does not studies that have suggested that the RB family of proteins function downstream of p53 in the cell cycle Unlike the proteins encoded by other DNA tumor regulatory pathway (Michalovitz et al., 1991; Vousden viruses, the polyomavirus T antigens display no direct et al., 1993; Demers et al., 1994; Hickman et al., 1994; interaction with or stabilization of wild-type p53 Quartin et al., 1994; Slebos et al., 1994). protein (Wang et al., 1989). Middle T antigen, a membrane protein and major oncoprotein of the virus, causes phenotypic changes in cells indicative of neoplastic transformation by associating with a Results number of di€erent signal transducing proteins (Benjamin and Vogt, 1990). Large T antigen is a Expression of temperature sensitive p53 at 328C arrests nuclear protein known to immortalize primary cells the growth of cells in G /G phase of the cell cycle 1 0 and induce cellular DNA synthesis through its Mouse embryo ®broblasts from a p53 de®cient mouse interaction with pRB and pRB-like proteins (Dyson (Donehower et al., 1992) were transfected with a et al., 1990; Larose et al., 1991; Freund et al., 1992, temperature sensitive p53 gene. Previous reports have 1994; Mudrak et al., 1994). To determine if the shown that the protein expressed by this gene is polyoma tumor antigens interfere with p53 dependent predominantly in the mutant conformation at 378C, growth arrest, expression vectors containing large T and at 328C the protein is functionally wild-type and middle T antigen were introduced into M/tsp53 (Michalovitz et al., 1990; Martinez et al., 1991). cells and clonal cell lines expressing each of the T Growth of a cell line expressing the temperature antigens individually were isolated and studied. sensitive p53 protein, M/tsp53, was compared to a Growth characteristics of a cell line expressing the control cell line, M/puro, derived from the same large T antigen (M/tsp53/LT) and a cell line expressing parental cells but expressing only the gene conveying middle T antigen (M/tsp53/MT) were determined over drug resistance. We examined the growth of M/tsp53 a six day period at 328C. Figure 2a shows that at 328C, Polyoma large T overcomes p53 growth arrest J Doherty and R Freund 1925 M/tsp53/LT cells grew while M/tsp53/MT and M/tsp53 one seen with the growth arrested M/tsp53 cells at the cells did not grow. Analysis of the growth rates permissive temperature, with the exception of a slightly indicates that M/tsp53/LT cells grew more slowly at higher G2/M phase, which may re¯ect an additional

328C than M/tsp53 cells grew at 378C, with doubling block in the G2/M phase. M/tsp53/LT cells maintained times of 35 h and 27 h respectively. This suggests that at 328C contain 20% of the cells in S phase, 70% in although LT is able to overcome p53 mediated growth G1/G0, and 10% in G2/M phase. These results suggest arrest, it may not overcome all of the growth that M/tsp53/LT cells continue to cycle at 328C, but suppressive activities of p53. their rate of progression through the cell cycle

We analysed the cell cycle distribution of T antigen decreases in the G1/G0 phase compared to growth at expressing cell lines maintained at 378C and 328C for 378C. 24 h (Figure 2b). M/tsp53/MT cells at 328C contained A Western blot of extracts from each cell line approximately 66% of the cells in the G1/G0 phase of revealed that p53 and the appropriate T antigen were the cell cycle, 8% in S, and approximately 26% in G2/ expressed at both temperatures (Figure 2c). As noted M. There was no indication that MT induced apoptosis earlier, the amount of p53 is reduced in all cell lines under these conditions as determined by FACS grown at 328C compared to those grown at 378C. At analysis. This cell cycle distribution is similar to the 328C, the amount of p53 expressed in the growth

a

a

b b

c 1234 c 12345678

— p53 — p53 —LT —MT — Actin

0 24h 72h 144h — Actin Figure 1 Cells expressing temperature sensitive p53 are growth arrested in the G1/G0 phase of the cell cycle at 328C. (a) Growth Figure 2 Polyomavirus large T antigen overcomes p53 growth curve of M/tsp53 and M/puro cells at 378C and 328C. Cells were arrest and middle T antigen does not. (a) Growth curves of M/ plated at 26104 per ml and incubated at the indicated tsp53/LT, M/tsp53/MT, M/tsp53 cells at 328C and M/tsp53 cells at temperatures for 24, 48, 96, 144 h. Viable cell number was 378C. Cell growth was determined as described in Figure 1a. (b) determined by counting cells that excluded trypan blue. Triplicate Cell cycle analysis of M/tsp53/LT and M/tsp53/MT cells after samples were counted and averaged for each time point. (b) Cell incubation at 378C and 328C for 24 h. Cells were stained with cycle analysis of M/puro and M/tsp53 cells after incubation at propidium iodide and cell cycle distribution was analysed by ¯ow 378C and 328C for 24 h. The cells were stained with propidium cytometry as described in Figure 1b. The average of three iodide and DNA content determined by ¯ow cytometry. experiments is shown. (c) Western blot of cell extract from M/ Distribution of cells at di€erent phases of the cell cycle was puro (lanes 1 and 2), M/tsp53 (lanes 3 and 4), M/tsp53/LT (lanes 5 analysed using Verity's ModFit LT software. The cell cycle and 6), and M/tsp53/MT (lanes 7 and 8) grown at 378C (odd lanes) analysis is an average of three separate experiments. (c) Western and 328C (even lanes). The top panel shows the blot probed with blot of protein extract from M/tsp53 cells grown at 378C (lane 1), an anti-p53 antibody (Pap240), the middle panel shows the blot and moved to 328C for 24 h (lane 2), 72 h (lane 3), and 144 h probed with an anti-T antigen antibody (F4) that recognizes both (lane 4). The blot was probed with an anti-p53 speci®c antibody large (LT) and middle T (MT) antigens, and the bottom panel (Pab240) (top) and an anti-actin antibody (I-19) (bottom) shows the blot re-probed with an antibody to actin (I-19) Polyoma large T overcomes p53 growth arrest J Doherty and R Freund 1926 arrested cells, M/tsp53 (lane 4) and M/tsp53/MT (lane (Dyson et al., 1990). Genetic studies have indicated 8), is similar to the amount of p53 expressed by M/ that these interactions are responsible for the ability of tsp53/LT cells (lane 6) when normalized to the amount LT antigen to immortalize primary cell and induce of actin in each sample (bottom panel). cellular DNA synthesis (Larose et al., 1991; Freund et Several independent cell lines were tested to avoid al., 1992, 1994; Mudrak et al., 1994). Other functions the possibility that results were due to single clonal of the protein, such as induction of viral DNA variants. A total of nine large T-expressing clones and synthesis, are mediated by separate domains of LT six middle T-expressing clones were examined, and (Cowie et al., 1986; Gjorup et al., 1994). We have each displayed similar growth characteristics and cell expressed a mutant large T antigen in M/tsp53 cells cycle distributions as M/tsp53/LT and M/tsp53/MT that is unable to bind to pRB, to determine if (not shown). These results demonstrate that expression association of LT with the RB proteins is required to of LT antigen overcomes p53 dependent growth arrest, overcome p53 dependent growth arrest. The growth while expression of middle T antigen is unable to properties of these cells, designated M/tsp53/LT7Rb, override the inhibition of cell growth induced by p53. along with M/tsp53, and M/tsp53/LT, were assayed for 6 days (Figure 3a). M/tsp53/LT7Rb cells and M/tsp53 cells failed to grow at 328C while M/tsp53/LT cells Association of large T antigen with RB proteins is grew at 328C and M/tsp53 cells grew at 378C. At 328C necessary to overcome p53 dependent growth arrest the cell cycle distribution of M/tsp53/LT7Rb cells Polyomavirus large T antigen has been shown to (Figure 3b) was similar to the growth arrested M/ associate with the retinoblastoma tumor suppressor tsp53 cells (Figure 1b) and M/tsp53/MT cells (Figure protein (pRB) and pRB-like proteins (p107 and p130) 2b). A Western blot analysis of extract from M/tsp53/ LT7Rb cells revealed that p53 and mutant large T antigen were expressed at both 378C and 328C (Figure a 3c). Nine independent clonal cell lines expressing mutant large T antigen were tested and all displayed growth arrest when incubated at 328C (not shown). The ability of the mutant LT expressed in M/tsp53/ LT7Rb cells to support replication of polyomavirus DNA was tested to ensure that activities other than those associated with RB binding of the mutated protein were still functional. A plasmid containing polyomavirus origin of replication was transfected into M/tsp53/LT, M/tsp53/LT7Rb and M/tsp53, and cells were incubated at 378C and 328C. DNA was isolated by Hirt extraction and replication detected by Southern blot analysis. Figure 4 shows that at 378C both M/ tsp53/LT and M/tsp53/LT7Rb cells supported replica- tion of the plasmid DNA containing the viral origin of replication, while at 328C only M/tsp53/LT cells b

123

37C ¨

c M/tsp53/LT–Rb

— p53 32C —LT ¨

37C 32C Figure 3 Large T-pRB interaction is required to overcome p53 Figure 4 Large T antigen mutant defective in pRB binding dependent growth arrest. (a) Growth of M/tsp53/LT, M/tsp53/ retains viral DNA replication capability. A plasmid containing LT7Rb, and M/tsp53 cells at 328C and M/tsp53 cells at 378C. M/ the polyomavirus origin of replication was transfected into M/ tsp53/LT7Rb cells express a mutant LT antigen containing a tsp53 (lane 1), M/tsp53/LT (lane 2), and M/tsp53/LT7Rb (lane 3) single amino acid change in the pRB binding domain that cells using the DEAE dextran method. Cells were incubated at disrupts the LT-pRB interaction. Cell growth was determined as 378C and 328C for 48 h and plasmid DNA was extracted by the described in Figure 1a. (b) Cell cycle analysis of M/tsp53/LT7Rb Hirt procedure. To remove unreplicated plasmid DNA, the cells grown at 378C and 328C for 24 h. The cell cycle distribution sample was digested with DpnI, which recognizes and cuts was determined by ¯ow cytometry, as described in Figure 1b. The sequences methylated by E. coli methylase. Remaining DNA, average of three experiments is shown. (c) Western blot of cell which has been replicated in eucaryotic cells, was linearized by extract from M/tsp53/LT7Rb cells incubated at 378C (lane 1) and EcoRI and detected by Southern blot analysis. Migration of 328C for 24 h (lane 2). The top panel shows the blot probed with replicated DNA is indicated by an arrow. M/tsp53/LT and M/ an anti-p53 antibody (Pab240) and the bottom panel shows the tsp53/LT7Rb cells support viral replication at 378C while only M/ blot probed with an anti-T antigen antibody (F4) tsp53/LT cells support viral replication at 328C Polyoma large T overcomes p53 growth arrest J Doherty and R Freund 1927 demonstrated viral DNA replication. Cells not expres- the Western blot, M/tsp53, M/tsp53/LT and M/tsp53/ sing large T antigen, M/tsp53, were unable to replicate MT cells maintained at 328C expressed approximately the plasmid at either temperature, demonstrating that equal amounts of p21. M/tsp53/LT7Rb cells appear to LT is necessary for viral replication. The vector express less p21 at 328C (lane 10) although the levels of without the viral origin of replication was not actin suggest that less protein was loaded. These results replicated in any of the cell lines (not shown), indicate that the temperature sensitive p53 is able to indicating that replication is dependent on the induce expression of p21, and further supports the presence of the viral origin. These results clearly assumption that p53 is functionally wild-type at the indicate that the mutant large T antigen expressed in permissive temperature of 328C. Additionally, this M/tsp53/LT7Rb cells retains the ability to support viral indicates that large T antigen does not interfere with DNA replication. Interestingly, the results also suggest expression of p21 and is able to overcome p53 that viral replication requires both cell growth and LT, dependent growth arrest in the presence of high levels since M/tsp53/LT cells supported viral DNA replica- of p21. tion at 328C, while the growth arrested M/tsp53/LT7Rb cells did not. Hyperphosphorylated pRB is detected in cells expressing p53 and large T antigen Large T antigen does not interfere with p53 The underphosphorylated form of pRB is thought to transcriptional activation of p21 negatively regulate progress through the cell cycle by The ability of p53 to induce growth arrest is correlated sequestering and inactivating the transcription factor with its ability to function as a sequence speci®c E2F (Chellappan et al., 1991; Hiebert et al., 1992; transcriptional activator (Crook et al., 1994; Pietenpol Flemington et al., 1993). Cell cycle progression is et al., 1994). One of the genes induced in response to associated with phosphorylation of pRB and is p53 expression is the cyclin-dependent kinase inhibitor believed to cause the release and activation of E2F p21/WAF-1 (p21) (El-Deiry et al., 1993). p21 inhibition (Buchkovich et al., 1989; Chen et al., 1989; DeCaprio of cyclin-dependent kinases has been shown to interfere et al., 1989). This suggests that p53 mediates growth with progression of the cell cycle, resulting in growth arrest by inducing p21 and inhibiting the cyclin- arrest at the G1/G0 phase (Dulic et al., 1994; Harper et dependent kinases that phosphorylate pRB. Polyoma- al., 1995). We examined expression of p21 in all of the virus large T antigen, as well as other viral proteins cell lines at both 378C and 328C. Figure 5 shows that such as SV40 large T antigen, HPV E7, and adenovirus only cells that expressed the temperature sensitive p53 E1A, associate with underphosphorylated pRB and and were cultured at 328C expressed p21. M/puro cells, cause the release of transcriptionally active E2F which do not express p53, failed to express p21 at (Dyson et al., 1989; Ludlow et al., 1989; Chellappan either temperatures (lanes 1 and 2) while M/tsp53 cells et al., 1992; Freund et al., 1992). To explore the expressed p21 at 328C (lane 4) but not at 378C (lane 3). possible connection between the p53 induced expres- Similarly, at 328C, M/tsp53/LT cells (lane 6) and M/ sion of p21 and the ability of large T antigen to tsp53/MT cells (lane 8) expressed p21. As indicated by overcome growth arrest, we investigated the phosphorylation state of pRB in various cell lines. Cells were incubated at 378C and 328C and the level of pRB phosphorylation was assessed by its migration on SDS ± PAGE and visualized by Western blot analysis –Rb (Figure 5). M/puro cells at 378C (lane 1) and at 328C (lane 2) contained slowly migrating, hyperphosphory- lated forms of pRB. M/tsp53 cells contained hyperpho-

M/puro M/tsp53 M/tsp53/LT M/tsp53/MT M/tsp53/LT sphorylated pRB at 378C (lane 3), but only fast migrating, hypophosphorylated pRB at 328C (lane 4). — Rb-P At 328C M/tsp53/MT and M/tsp53/LT7Rb contained —Rb only hypophosphorylated pRB (lanes 8 and 10), while M/tsp53/LT cells contained both the hypo- and hyper- phosphorylated forms of pRB (lane 6). These results

demonstrate that growth arrest in the G1/G0 phase of — p21 the cell cycle correlates with the appearance of hypophosphorylated pRB and the absence of hyper- phosphorylated pRB. In addition, it indicates that a — Actin population of pRB is phosphorylated in cells expres- sing LT, p53, and p21 at 328C. This suggests that large 12345678910 T antigen expression overrides the p21 inhibition of Figure 5 The e€ect of p53 and the T antigens on the kinase activity or activates another pRB kinase. phosphorylation status of the retinoblastoma protein (pRB) and on expression of the cyclin-dependent kinase inhibitor p21/ WAF1. Cells were grown at 378C (odd lanes) and at 328C (even lanes). Cell extract from M/puro cells (lanes 1 and 2), M/ Discussion tsp53 cells (lanes 3 and 4), M/tsp53/LT cells (lanes 5 and 6), M/ tsp53/MT cells (lanes 7 and 8), and M/tsp53/LT7Rb cells (lanes 9 The experiments reported here explore the interaction and 10) was analysed by Western blotting. Top panel shows a blot probed with pRB antibody (pAB245), middle panel shows a between polyomavirus T antigens and the p53 tumor blot probed with anti-p21 (C-19) and bottom panel shows the p21 suppressor protein. Unlike several other DNA tumor blot re-probed with anti-actin antibody (I-19) viruses, polyomavirus does not encode proteins that Polyoma large T overcomes p53 growth arrest J Doherty and R Freund 1928 directly inactivate the p53 protein. We have shown that overcomes p53 growth arrest, possibly by inducing an the polyomavirus large T antigen (LT) interferes with inhibitor of p21 (Hermeking et al., 1995). the growth arrest function of p53. In addition, we have p53 growth suppression was not completely reversed found that association of LT with RB proteins is by expression of LT in this system. At 328C, M/tsp53/ essential in overcoming p53 dependent growth arrest, LT cells grew more slowly than cells expressing mutant further supporting the evidence that the RB family of p53 or those not expressing p53, indicating that in the proteins are components of the p53 growth suppression presence of LT p53 was still able to suppresss cell pathway. growth, but not block it completely. This may be due This and other studies have shown that over- to the non-physiological levels of p53 expressed in expression of wild-type p53 results in growth arrest these cells. The high levels of p53 may prevent LT from

of cells in the G1/G0 phase of the cell cycle completely overcoming the suppression of growth. (Michalovitz et al., 1990; Martinez et al., 1991; Lin et Alternatively, p53 may mediate growth arrest through al., 1992; Di Leonardo et al., 1994). Growth arrest a combination of growth suppressive mechanisms and coincides with expression of the cyclin dependent LT interferes with only a subset of these mechanisms. kinase inhibitor, p21, and accumulation of underpho- Further work is needed to distinguish these possibi- sphorylated retinoblastoma tumor suppressor protein lities; however the results indicate that p53 mediated (pRB). This supports the premise that p53 dependent growth arrest is abrogated by LT. growth arrest results from expression of p21, which in In addition, we have shown that the major turn inhibits the phosphorylation of pRB. Unpho- oncoprotein of polyomavirus, middle T antigen sphorylated pRB and other RB family members (p107 (MT), does not overcome p53 dependent growth and p130) remain associated with the family of E2F arrest nor induce p53 dependent apoptosis. MT transcription factors, inhibiting their ability to promote transforms cells in culture through activation of

transcription of genes necessary for the G1 to S phase several mitogenic signaling pathways (Benjamin and transition (Chellappan et al., 1991; Hiebert et al., 1992; Vogt, 1990). Through the association of MT antigen Flemington et al., 1993). with pp60c-src and the resulting activation of its Polyomavirus large T antigen associates with and is tyrosine kinase activity, MT associates with PI 3- thought to inactivate the pRB proteins (Dyson et al., kinase, shc, and phospholipase C (Bolen et al., 1984; 1990; Larose et al., 1991; Freund et al., 1992). This Whitman et al., 1985; Kaplan et al., 1986; Talmage et protein-protein interaction releases E2F, which allows al., 1989; Campbell et al., 1994; Dilworth et al., 1994; its subsequent transcriptional activity (Mudrak et al., Su et al., 1995). Studies have shown that middle T 1994). Our results have shown that the association of antigen induces expression of the early response RB proteins with polyomavirus LT prevents p53 from genes, myc, fos and jun (Zullo et al., 1987; arresting cell growth. Expression of a mutant LT Schonthal et al., 1992). Several experimental systems (LT7Rb), which contains a single amino acid change in have suggested that growth stimulation signals the RB binding domain and is unable to bind RB concurrent with p53 growth arrest trigger an proteins, does not overcome the growth arrest. These apoptotic response (Debbas and White, 1993; results con®rm and extend previous studies with other Hermeking and Eick, 1994; Lowe and Ruley, 1993; DNA tumor viruses that indicated an involvement of Wu and Levine, 1994). The inability of middle T to RB proteins in p53 mediated growth arrest (Michalo- induce apoptosis in the presence of high levels of p53 vitz et al., 1991; Vousden et al., 1993; Demers et al., may be due to a variety of factors. The MEF cells 1994; Hickman et al., 1994; Quartin et al., 1994; Slebos may not express certain gene products that are et al., 1994). These studies showed that expression of necessary to support an apoptotic response, or MT an SV40 large T antigen, which contains an RB may induce expression of survival or anti-apoptotic binding domain but does not associate with p53, and genes. Recent studies have suggested a requirement expression of HPV E7 and adenovirus E1A, which also for the PI 3-kinase signaling pathway in preventing associate with pRB, overcomes the growth arrest apoptosis (Yao and Cooper, 1995). Our results induced by p53. suggest, however, that activation of c-src tyrosine Our results have shown that in the presence of LT, kinase by middle T antigen, which deregulates p53 induced p21 expression and cells contained both signaling pathways and induces early response genes,

hypo- and hyper-phosphorylated forms of pRB. Large is insucient to overcome the p53 mediated G1/G0 T antigen did not interfere with the ability of p53 to cell cycle block. transcriptionally activate p21, or with the expression of In summary, we have shown that polyomavirus large the p21 protein. However, a population of pRB T antigen overcomes p53 induced growth arrest. It became phosphorylated even when high levels of p21 accomplishes this in part by its association with and were expressed, indicating the presence of an active inactivation of the RB family of proteins. The data pRB kinase. This result suggests that LT interferes also suggest that LT antigen expression may overcome with p21 activity or induces a p21-resistant pRB inhibition of cyclin dependent kinase activity, which kinase. LT may interact directly with p21, or may results in phosphorylation of pRB. Further studies are induce another protein(s) that inactivates p21. Thus, needed to determine if large T antigen inactivates the LT antigen not only inactivates the RB proteins but CDK inhibitor, p21, or induces an RB kinase. Our also allows their phosphorylation in the presence of results indicate that polyomavirus has evolved an p21. Both of these activities may be required to indirect mechanism to overcome growth suppression overcome p53 dependent growth arrest. Recently it by targeting the downstream e€ector molecules of p53. has been reported that adenovirus E1A interacts with These novel molecular mechanisms may be functioning and inactivates the CDK inhibitor p27KIP1 (Mal et al., in non-virally induced cancers that retain wild-type p53 1996). Additionally it has been reported that c-myc expression. Polyoma large T overcomes p53 growth arrest J Doherty and R Freund 1929 Materials and methods Western blotting Cell extract was prepared by lysing cells in NP40 extraction Construction and maintenance of cell lines bu€er (0.137 M NaCl, 0.02 M Tris-HCl pH 9.0, 0.001 M

A cell line expressing a temperature sensitive p53 protein MgCl2,0.001MCaCl2, 10% glycerol, 1% NP40, 0.01 mg/ (M/tsp53) was generated by transfecting a temperature ml aprotinin, 0.005 mg/ml leupeptin, 0.1 mM sodium sensitive p53 expression plasmid (pLTRp53cGval135) orthovanadate, 0.1 mg/ml PMSF). Cell debris was re- (Eliyahu et al., 1985; Michalovitz et al., 1990) and a moved by centrifugation and protein quanti®ed by puromycin selectable marker plasmid (pBabepuro) (Mor- Bradford Dye Assay (BioRad) according to the manufac- genstern and Land, 1990) into primary mouse embryo turer's directions. Equal amounts of total protein were ®broblasts (MEF) derived from p53-de®cient mice (Harvey separated on SDS polyacrylamide gels and electrophor- et al., 1993). MEF cells were transfected with the etically transferred to nitrocellulose. Blots were probed puromycin selectable marker plasmid alone as a control with primary antibodies and appropriate secondary (M/puro). Transfections were carried out using the calcium antibodies, washed as described (Harlow and Lane, phosphate method and transformants were selected using 1988), and antigen was detected by Amersham's ECL 1.25 mg/ml puromycin (Sambrook et al., 1989). Single antibody detection kit. p53 was detected on Western blots colonies were picked, expanded, and screened for p53 with a mouse monoclonal antibody that recognizes both expression by Western blotting. A single cell line was mutant and wild-type p53 (pAb240, Oncogene Science); chosen and used to generate all other cell lines. actin was detected with I-19 (Santa Cruz Biotechnology); T Cell lines expressing each of the polyoma T antigens along antigen expression was detected with F4 (Oncogene with the temperature sensitive p53 were created by infecting Science), a mouse monoclonal antibody that recognizes M/tsp53 cells with defective murine retrovirus containing the the amino terminus of polyomavirus large, middle and cloned T antigen gene (Cepko et al., 1984; Cherington et al., small T antigens (Pallas et al., 1986); pRB was detected 1986). The culture medium of cloned psi-2 cells containing using the mouse monoclonal antibody, pAB245 (PharMin- retrovirus was ®ltered, supplemented with 8 mg/ml polybrene, gen); and p21 expression was detected with C-19 (Santa and used to infect M/tsp53 cells. Infected cells were selected Cruz Biotechnology), a rabbit polyclonal antiserum. in medium containing geneticin (G418) at 1 mg/ml for approximately 2 weeks and single colonies were picked, Replication assays expanded and assayed for T antigen expression. Cell lines expressing large T antigen (M/tsp53/LT), middle T antigen A plasmid containing the polyomavirus origin of replica- (M/tsp53/MT) and a mutant large T antigen (M/tsp53/ tion (Pori) was used to assay for large T antigen dependent LT7Rb) were generated. LT7Rb contains a cysteine to glycine replication of viral DNA. The polyomavirus DNA change at amino acid 144, which has been previously shown fragment from Bcl1 (nuc. 5021) to BstX1 (nuc. 173) to disrupt the LT:pRB association (Freund et al., 1992). encompassing the origin of replication (Tooze, 1981) was All cell lines were routinely cultured in Dulbecco's cloned into the pBS vector (Stratagene). 1 mgofPoriwas modi®ed Eagle medium (DMEM) supplemented with transfected into M/tsp53, M/tsp53/LT, and M/tsp53/LT7Rb 0.375% sodium bicarbonate, 100 units/ml penicillin, 100 mg/ cells using the DEAE dextran method (Sambrook et al., ml streptomycin, and 10% calf serum in a 5% CO2 incubator 1989). After transfection the cells were incubated at 378C at 378C. Puromycin resistant cells were maintained in 1 mg/ml or 328C for 48 h and DNA was isolated by Hirt extraction puromycin, and cells resistant to both puromycin and G418 (Hirt, 1967). To remove unreplicated plasmid DNA, were maintained in 1 mg/ml puromycin and 50 mg/ml G418. samples were digested with DpnI, which recognizes and To express p53 in a wild-type conformation, cells containing cuts sequences that have been methylated by the bacterial the temperature sensitive p53 gene were incubated at 328C. enzyme, Dam methylase. DNA replicated in eucaryotic cells is not digested by DpnI. The remaining DNA was linearized with EcoRI and analysed by Southern blotting Growth assays as described (Sambrook et al., 1989). Digoxigenin labeled Cells were plated at 26104 per ml in a 24 well plate and Pori was used as a probe and detected with anti- incubated at 378C for 24 h. Plates were moved to 328Cand digoxigenin antibody conjugated to chemiluminescence viable cell number was determined after incubation for alkaline phosphatase (Boehringer Mannheim). 24 h, 48 h, 96 h and 144 h. Cells were trypsinized and viable cell number was determined by counting cells that excluded trypan blue. The average number from three wells at each time point was recorded.

Acknowledgements Flow cytometry The authors wish to thank L Donehower for the mouse 56105 cells were plated in 10 cm tissue culture plates and embryo ®broblasts isolated from the p53-de®cient mouse. incubated at 378C for 24 h. Plates were transferred to 328C We also thank A Levine for the temperature sensitive p53 for 24 h and processed for propidium iodide staining as expression plasmid, pLTRp53cGval, and D Talmage for described (Gray and Cono, 1979). Brie¯y, cells were ®xed the puromycin selectable marker expression plasmid, in 80% ethanol, permeabilized with 0.5% Tween 20 and pBabepuro. This work was supported by grants from stained with 10 mg/ml propidium iodide in 100 units/ml American Cancer Society/Maryland Division, Inc. and

RNAase and 0.1% NaN3 foratleast2hat48C. A Becton- National Cancer Institute (CA63111) to RF. JD was Dickinson FACscan and CELLQuest software were used supported in part by the Molecular and Cell Biology to collect the data and Verity's ModFit LT software was Program and the Department of Microbiology and used to analyse the data. Immunology.

References

Bates S and Vousden KH. (1996). Curr. Opin. Gen. Dev., 6, Benjamin T and Vogt PK. (1990). Virology, Fields. B (ed.) 12 ± 19. Raven Press: New York, pp. 347 ± 367. Polyoma large T overcomes p53 growth arrest J Doherty and R Freund 1930 Bolen JB, Thiele CJ, Israel MA, Yonemoto W, Lipsich LA Gjorup OV, Rose PE, Holman PS, Brockus BJ and and Brugge JS. (1984). Cell, 38, 767 ± 777. Scha€hausen BS. (1994). Proc. Natl. Acad. Sci. USA, 91, Buchkovich K, Du€y LA and Harlow E. (1989). Cell, 58, 12125 ± 12129. 1097 ± 1105. Gray JW and Cono P. (1979). Methods in Enzymology, CampbellKS,OgrisE,BurkeB,SuW,AugerKR,Druker Volume 58, Cell Culture. Jakoby W B and Pastan I H. BJ, Scha€hausen BS, Roberts TM and Pallas DC. (1994). (eds.) Academic Press, Inc.: San Diego, pp. 233 ± 247. Proc. Natl. Acad. Sci. USA, 91, 6344 ± 6348. Gu Y, Turck CW and Morgan DO. (1993). Nature, 366, Cepko CL, Roberts BE and Mulligan RC. (1984). Cell, 37, 707 ± 710. 1053 ± 1062. Harlow E and Lane D. (1988). Antibodies: a Laboratory Chellappan S, Kraus VB, Kroger B, Munger K, Howley PM, Manual. Cold Spring Harbor Laboratory Press: Cold Phelps WC and Nevins JR. (1992). Proc. Natl. Acad. Sci. Spring Harbor. USA, 89, 4549 ± 4553. Harper JW, Adami GR, Wei N, Keyomarsi K and Elledge Chellappan SP, Hiebert S, Mudryj M, Horowitz JM and SJ. (1993). Cell, 75, 805 ± 816. Nevins JR. (1991). Cell, 65, 1053 ± 1061. Harper JW, Elledge SJ, Keyomaarsi K, Dynlacht B, Tsai L, Chen P, Scully P, Shew J, Wang JYJ and Lee W. (1989). Cell, Zhang P, Dobrowolski S, Bai C, Connell-Crowley L, 58, 1193 ± 1198. Swindell E, Fox MP and Wei N. (1995). Mol. Biol. Cell, 6, Cherington V, Morgan B, Spiegelman BM and Roberts TM. 387 ± 400. (1986). Proc. Natl. Acad. Sci. USA, 83, 4307 ± 4311. Harvey M, Sands AT, Weiss RS, Hegi ME, Wiseman RW, Courtneidge SA and Smith AE. (1983). Nature, 303, 435 ± Pantazis P, Giovanella BC, Tainsky MA, Bradley A and 439. Donehower LA. (1993). Oncogene, 8, 2457 ± 2467. Cowie A, de Villiers J and Kamen R. (1986). Mol. Cell. Biol., Hermeking H and Eick D. (1994). Science, 265, 2091 ± 2093. 6, 4344 ± 4352. Hermeking H, Funk JO, Reichert M, Ellwart JW and Eick Crook T, Marston NJ, Sara EA and Vousden KH. (1994). D. (1995). Oncogene, 11, 1409 ± 1415. Cell, 79, 817 ± 827. Hickman ES, Picksley SM and Vousden KH. (1994). Debbas M and White E. (1993). Genes & Dev., 7, 546 ± 554. Oncogene, 9, 2177 ± 2181. DeCaprio JA, Ludlow JW, Figge J, Shew JY, Huang CM, Hiebert SW, Chellappan SP, Horowitz JM and Nevins JR. Lee WH, Marsillio E, Paucha E and Livingston DM. (1992). Genes & Dev., 6, 177 ± 185. (1988). Cell, 54, 275 ± 283. Hinds PW, Mittnacht S, Dulic V, Arnold A, Reed SI and DeCaprio JA, Ludlow JW, Lynch D, Furukawa Y, Grin J, Weinberg RA. (1992). Cell, 70, 993 ± 1006. Piwnica-Worms H, Huang C and Livingston DM. (1989). Hirt B. (1967). J. Mol. Biol., 26, 365 ± 369. Cell, 58, 1085 ± 1095. Hollstein M, Sidransky D, Vogelstein B and Harris CC. Demers GW, Foster SA, Halbert CL and Galloway DA. (1991). Science, 75, 839 ± 841. (1994). Proc. Natl. Acad. Sci. USA, 91, 4382 ± 4386. Kaplan DR, Whitman M, Scha€hausen BS, Raptis L, Deng C, Zhang P, Harper JW, Elledge SJ and Leder P. Garcea RL, Pallas D, Roberts TM and Cantley L. (1995). Cell, 82, 675 ± 684. (1986). Proc. Natl. Acad. Sci. USA, 83, 3624 ± 3628. Di Leonardo A, Linke SP, Clarkin K and Wahl GM. (1994). Kastan MB, Onyekwere O, Didransky D, Vogelstein B and Genes & Dev., 8, 2540 ± 2551. Graig RW. (1991). Cancer Res., 51, 6304 ± 6311. Dilworth SM, Brewster CEP, Jones MD, Lanfrancone L, Kato J, Matsushime H, Hiebert SW, Ewen ME and Sherr CJ. Pelicci G and Pelicci PG. (1994). Nature, 367, 87 ± 90. (1993). Genes & Dev., 7, 331 ± 342. Donehower LA, Harvey M, Slagle BL, McArthur MJ, Larose A, Dyson N, Sullivan M, Harlow E and Bastin M. Montgomery CA Jr, Butel JS and Bradley A. (1992). (1991). J. Virol., 65, 2308 ± 2313. Nature, 356, 215 ± 221. Levine AJ, Momand J and Finlay CA. (1991). Nature, 351, Donehower LA and Bradley A. (1993). Biochim. Biophys. 453 ± 456. Acta., 1155, 181 ± 205. Levine AJ. (1993). Annu. Rev. Biochem., 62, 623 ± 651. Dulic V, Kaufmann WK, Wilson SJ, Tlsty TD, Lees E, Lin D, Shields MT, Ullrich SJ, Appella E and Mercer WE. Harper JW, Elledge SJ and Reed Sl. (1994). Cell, 76, (1992). Proc. Natl. Acad. Sci. USA, 89, 9210 ± 9214. 1013 ± 1023. Livingston LR, White A, Sprouse J, Livanos E, Jacks T and Dyson N, Howley PM, Munger K and Harlow E. (1989). Tlsty T. (1992) Cell, 70, 923 ± 935. Science, 243, 934 ± 936. Lowe SW and Ruley HE. (1993). Genes & Dev., 7, 535 ± 545. Dyson NR, Bernards R, Friend SH, Gooding LR, Hassell Lowe SW, Schmitt EM, Smith SW, Osborne BA and Jacks T. JA, Major EO, Pipas JM, VanDyke T and Harlow E. (1993). Nature, 362, 847 ± 852. (1990). J. Virol., 64, 1353 ± 1356. Lu X and Lane DP. (1993). Cell, 75, 765 ± 778. El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons Ludlow JW, DeCaprio JA, Huang CM, Lee WH, Paucha E R, Trent JM, Lin D, Mercer WE, Kinzler KW and and Livingston DM. (1989). Cell, 56, 57 ± 65. Vogelstein B. (1993). Cell, 75, 817 ± 825. Mal A, Poon RYC, Howe PH, Toyoshima H, Hunter T and El-Deiry WS, Harper JW, O'Connor PM, Velculescu VE, Harter ML. (1996). Nature, 380, 262 ± 265. Canman CE, Jackman J, Pietenpol JA, Burrell M, Hill Martinez J, Georgo€ I and Levine A. (1991). Genes & Dev. 5, DE, Wang Y, Wiman KG, Mercer WE, Kastan MB, Kohn 151 ± 159. KW, Elledge SJ, Kinzler KW and Vogelstein B. (1994). Michalovitz D, Halevy O and Oren M. (1990). Cell, 62, 671 ± Cancer Res., 54, 1169 ± 1174. 680. Eliyahu D, Michalovitz D and Oren M. (1985). Nature, 316, Michalovitz DM, Yehiely F, Gottlieb E and Oren M. (1991). 158 ± 160. J. Virol., 65, 4160 ± 4168. EwenME,SlussHK,SherrCJ,MatsushimeH,KatoJand Mietz JA, Unger T, Huibregtse JM and Howley PM. (1992). Livingston DM. (1993). Cell, 73, 487 ± 497. EMBO J., 11, 5013 ± 5020. Flemington EK, Speck SH and Kaelin WG Jr. (1993). Proc. Momand J, Zambetti GP, Olson DC, George D and Levine Natl. Acad. Sci. USA, 90, 6914 ± 6918. AJ. (1992). Cell, 69, 1237 ± 1245. Francke B and Eckhart W. (1973). Virology, 55, 127 ± 135. Morgenstern JP and Land H. (1990). Nuc. Acids. Res., 18, Freund R, Bronson RT and Benjamin TL. (1992). Oncogene, 3587 ± 3596. 7, 1979 ± 1987. Mowat M, Cheng A, Kimura N, Bernstein A and Benchimol Freund R, Bauer PH, Crissman HA, Bradbury EM and S. (1985). Nature, 314, 633 ± 636. Benjamin TL. (1994). J. Virol., 68, 7227 ± 7234. Polyoma large T overcomes p53 growth arrest J Doherty and R Freund 1931 Mudrak I, Ogris E, Rotheneder H and Wintersberger E. Tooze J. (1981). DNA Tumor Viruses. Cold Spring Harbor (1994). Mol. Cell. Biol., 14, 1886 ± 1892. Laboratory: Cold Spring Harbor. Munger K, Werness BA, Dyson N, Phelps WC, Harlow E Treisman R, Novak U, Favaloro J and Kamen R. (1981). and Howley PW. (1989). EMBO J., 8, 4099 ± 4105. Nature, 292, 595 ± 600. Pallas DC, Schley C, Mahoney M, Harlow E, Scha€hausen Vousden KH, Vojtesek B, Fisher C and Lane D. (1993). BS and Roberts TM. (1986). J. Virol., 60, 1075 ± 1084. Oncogene, 8, 1697 ± 1702. Pietenpol JA, Tokino T, Thiagaligam S, El-Deiry W, Kinzler Wang EH, Friedman PN and Prives C. (1989). Cell, 57, 379 ± KW and Vogelstein B. (1994). Proc. Natl. Acad. Sci. USA, 392. 91, 1998 ± 2002. Weinberg RA. (1995). Cell, 81, 323 ± 330. Quartin RS, Cole CN, Pipas JM and Levine AJ. (1994). J. Werness BA, Levine AJ and Howley PM. (1990). Science, Virol., 68, 1334 ± 1341. 248, 76 ± 79. Raptis L, Lamfrom H and Benjamin TL. (1985). Mol. Cell. Whitman M, Kaplan DR, Scha€hausen BS, Cantley L and Biol., 5, 2476 ± 2485. Roberts TM. (1985). Nature, 315, 239 ± 242. Rassoulzadegan M, Naghashfar Z, Cowie A, Grisoni M, Whyte P, Buchkovich KJ, Horowitz JM, Friend SH, Kamen R and Cuzin F. (1983). Proc. Natl. Acad. Sci. Raybuck M, Weinberg RA and Harlow E. (1988). USA, 80, 4345 ± 4358. Nature, 334, 124 ± 129. Sambrook J, Fritsch EF and Maniatis T. (1989). Molecular Wu X and Levine AJ. (1994). Proc. Natl. Acad. Sci. USA, 91, Cloning, a laboratory manual. Cold Spring Harbor 3602 ± 3606. Laboratory Press: Cold Spring Harbor. XiongY,HannonGJ,ZhangH,CassoD,KobayashiRand Sarnow P, Ho YS, Williams J and Levine AJ. (1982). Cell, 28, Beach D. (1993). Nature, 366, 701 ± 704. 387 ± 394. Yao R and Cooper GM. (1995). Science, 267, 2003 ± 2006. Schlegel R and Benjamin T. (1978). Cell, 14, 587 ± 599. Yew PR and Berk AJ. (1992). Nature, 357, 82 ± 85. Schonthal A, Srinivas S and Eckhart W. (1992). Proc. Natl. Yin Y, Tainsky MA, Bischo€ FZ, Strong LC and Wahl GM. Acad. Sci. USA, 89, 4972 ± 4976. (1992). Cell, 70, 937 ± 948. Slebos RJC, Lee MH, Plunkett BS, Kessis TD, Williams BO, Zhang H, Hannon GJ and Beach D. (1994). Genes & Dev., 8, Jacks T, Hedrick L, Kastan MB and Cho KR. (1994). 1750 ± 1758. Proc. Natl. Acad. Sci. USA, 91, 5320 ± 5324. Zullo J, Stiles CD and Garcea RL. (1987). Proc. Natl. Acad. Su W, Liu W, Scha€hausen BS and Roberts TM. (1995). J. Sci. USA, 84, 1210 ± 1214. Biol. Chem., 2705, 12331 ± 12334. Talmage DA, Freund R, Young AT, Dahl J, Dawe CJ and Benjamin TL. (1989). Cell, 59, 55 ± 64.