INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Simian virus 40 (SV40) large T antigen (TAg) has been used extensively as a model system to study cellular transformation. TAg has the ability to transform a wide variety of normal cells seemingly by affecting the functions of a small number of cellular proteins. To transform wild-type (WT) mouse embryo fibroblasts (MEFs), TAg utilizes at least three domains: the J domain, the LXCXE motif that binds to the retinoblastoma protein (pRB) family of proteins (pRB, p107, and p130), and the p53-binding domain (12, 13, 17, 20, 65, 77, 81). The J domain is a highly conserved element present in all members of the DnaJ/Hsp40 family of molecular chaperones as well as all polyomavirus T antigens (39). DnaJ proteins bind specifically to hsp70 homologues to perform various chaperone activities, including the destruction of specific proteins (reviewed in reference 63). The J domain of TAg binds to hsc70 and participates in the inactivation of pRB family members (27, 56, 60, 68, 69). The J domain and the LXCXE motif of TAg cooperate to disrupt the ability of pRB family members to repress E2F-dependent transcription and to decrease the levels of hyperphosphorylated p107 and p130 (27, 60, 64, 65, 68, 69, 77). Thus, the J domain and LXCXE motif of TAg appear to induce transformation and promote cell growth by interfering with the functions of pRB, p107, and p130. The p53-binding domain of TAg binds to the specific DNA-binding domain of p53, thereby directly interfering with the ability of p53 to activate transcription (3, 21, 51). Therefore, it is believed that TAg can transform cells primarily by interfering with p53 and the pRB family.

The original observations that TAg binds to p53 and the pRB family set into motion a large field of research that has led to a more complete understanding of the role of these tumor suppressors in the development of cancer (13, 44, 46). Not only is pRB itself mutated in a wide variety of cancers, wild-type pRB can be functionally inactivated by expression of an LXCXE-containing viral oncoprotein. pRB can also be inactivated by hyperphosphorylation as a result of overexpression of cyclin D1 or loss of expression p16^INK4a. p16^INK4a binds to cdk4 and blocks the association of cdk4 with D-type cyclins (57). Overexpression of p16^INK4a in RB^+/+ cells prevents phosphorylation and subsequent inactivation of pRB by cyclin D1-cdk4 and promotes a cell cycle arrest in G₁ (41, 48). In contrast, p16^INK4a is unable to induce a cell cycle arrest in RB^/ cells, suggesting that the pRB pathway is required for p16^INK4a-mediated cell cycle arrest (48). p16^INK4a may also affect the phosphorylation status of p107 and p130, as each of these proteins migrates as a lower-phosphorylation form in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis after p16^INK4a overexpression (74; J. Zalvide and J. A. DeCaprio, unpublished observations). Therefore, p16^INK4a may regulate all three members of the pRB family.

In addition to p16^INK4a, the INK4a locus encodes a second gene product, p19^ARF (54). While each of these proteins is encoded by a unique first exon, the second and third exons encode p19^ARF in an alternate reading frame than p16^INK4a (54). It has been demonstrated that p19^ARF binds and inactivates MDM2 (36, 52, 67, 80). Since MDM2 contributes to the degradation of p53, overexpression of p19^ARF can result in an increased amount of p53, leading to an arrest in G₁ (28, 80). Therefore, through the expression of p16^INK4a and p19^ARF, the INK4a locus apparently participates in the regulation of both the pRB and p53 pathways.

Although TAg-mediated transformation is thought to involve primarily the inactivation of pRB family members and p53, studies of mice that contain mutations in the INK4a locus would seem to indicate that the functional inactivation of pRB and p53 is not sufficient to cause cellular transformation. INK4a knockout mice lack the second and third exons of the INK4a locus and do not express p16^INK4a or p19^ARF, although they may express a truncated version of p19^ARF that may be partially active (5, 53, 58). Despite this interference with both the p53 and pRB pathways, INK4a^/ MEFs were not transformed. Although they readily became immortalized, INK4a^/ MEFs were unable to grow in soft agar and required the expression of activated H-ras to become transformed (37, 58). INK4a^/ mice do have an increased incidence of several types of tumors including lymphomas and fibrosarcomas, which presumably occur after the mice receive additional genetic mutations (58). Curiously, the ARF^/ mice that fail to express p19^ARF but retain expression of WT p16^INK4a display a similar range of tumors as the INK4a^/ mice that have lost both p19^ARF and p16^INK4a (35, 37). In addition, the ARF^/ MEFs were readily immortalized and could be transformed by activated H-ras alone similarly to the INK4a^/ MEFs (37). Therefore, at least in the mouse model, it has been difficult to observe the specific contribution of p16^INK4a to tumor suppression since the INK4a^/ and ARF^/ knockouts result in a similar phenotype.

Given that disruption of the INK4a locus was not sufficient to induce transformation of MEFs, the pRB and p53 pathways may retain some of their tumor suppressor activity in INK4a^/ cells. Indeed, it has been noted that p53 can be activated in the ARF^/ MEFs by ionizing radiation but not by adenovirus E1A (14, 37). It is thus possible that p53 may retain the ability to act as a tumor suppressor under some conditions in the ARF^/ and INK4a^/ mice. Alternatively, there may be tumor suppressor pathways other than pRB and p53 that retain activity in the INK4a^/ or ARF^/ MEFs. We wished to determine whether the loss or functional inactivation of tumor suppressor genes that are targeted by TAg would abrogate the need for the J domain, the LXCXE motif, or the p53-binding domain in TAg-mediated transformation. Using this approach, it had been previously demonstrated that the LXCXE motif was required to transform RB^/ MEFs, suggesting that p107 and p130 were also targeted by the LXCXE motif (11, 76). To identify additional pathways that may be targeted by SV40 TAg, we generated WT, RB^/, ARF^/, and INK4a^/ MEFs that stably expressed WT or selected mutants of TAg. We observed that TAg could fully transform MEFs from each of these genetic backgrounds. Notably, the LXCXE motif was absolutely required for transformation of WT and RB^/ MEFs but was dispensable for anchorage-independent growth of ARF^/ and INK4a^/ MEFs, suggesting that the pRB pathway was functionally inactivated by loss of p19^ARF. However, pRB family members were expressed normally, and their ability to repress E2F activity was intact in the ARF^/ and INK4a^/ MEFs expressing the LXCXE mutants of TAg. These results suggest the possibility that TAg's LXCXE motif may functionally inactivate other growth suppressors in addition to the pRB family and that p19^ARF may have activity beyond the regulation of MDM2 and p53.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Plasmids. The SV40 large TAg cDNA expression plasmids pSG5-T, pSG5-H42Q, pSG5-K1, pSG5-C105G, and pSG5-PVU-1 have been previously described (69, 76). pSG5-dl434-444 was cloned by substituting the PflMI-PstI fragment from dl434-444 containing the mutant p53-binding site into pSG5-T (40). pSG5-HQ-K1 was similarly cloned by substituting the PflMI-PstI fragment of pSG5-K1 into pSG5-HQ. pSG5-T1-135 and pSG5-T1-350 were generated by PCR amplification of a TAg cDNA with the appropriate primers. DNA sequencing confirmed the identity of all constructs.

Cells. INK4a^/ MEFs and ARF^/ MEFs have been previously described (37, 58). RB^/ and WT MEFs were prepared from 13.5-day embryos as previously described (32, 76). All MEFs were cultured in Dulbecco modified Eagle medium (DMEM) containing 10% Fetal Clone Serum I (HyClone) and penicillin-streptomycin. MEFs were cotransfected with pEpuro and fivefold excess SV40 TAg-encoding plasmid by the calcium phosphate precipitation method or Fugene 6 (Boehringer Mannheim). After 16 h of exposure to the plasmid DNA, cells were refed with complete medium and grown for 24 h before splitting into medium containing puromycin (2 µg/ml). Before reaching confluence, colonies were pooled and expanded in three 100-mm-diameter plates, at which point they were considered to be established. Cells of no more than three passages after this stage were used for transformation studies and were always passaged at subconfluence.

Western blotting and antibodies. Cells were lysed in high-salt EBC (50 mM Tris-HCl [pH 8.0], 300 mM NaCl, 0.5% Nonidet P-40) containing aprotinin (10 µg/ml), leupeptin (10 µg/ml), 0.1 mM phenylmethylsulfonyl fluoride, 4 mM sodium fluoride, and 0.1 mM sodium orthovanadate. Lysates were cleared by centrifugation at 14,000 × g, and protein concentration was determined by the Bradford assay (Bio-Rad); 100 µg of each sample was separated in SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked for 1 h with 5% nonfat dry milk and 1% goat serum in TBS-T (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% Tween 20), before overnight incubation with the primary antibody in TBS-T containing 1% bovine serum albumin at 4°C. The following antibodies were used in this study: anti-TAg polyclonal antibodies (PAb) 101 and 419 (26) (American Type Culture Collection), anti-RB PAb 245 (Pharmingen), anti-p107 C-18 (Santa Cruz Biotechnology), anti-p130 C-20 (Santa Cruz), anti-p53 FL-393 (Santa Cruz), anti-p15^INK4b Ab-3 (Neomarkers), anti-p16^INK4a M-156 (Santa Cruz), and anti-p21^CIP1 C-19 (Santa Cruz). The anti-p19^ARF rabbit PAb was previously described (37). Detection of proteins was performed with the appropriate horseradish peroxidase-conjugated secondary rabbit or goat antibody (Pierce) at a 1:5,000 dilution in TBS-T containing 2.5% milk and 0.5% goat serum. Immunoblots were developed using enhanced chemiluminescence (Pierce) according to the manufacturer's protocol.

Transformation assays. Soft agar assays were performed in 35-mm-diameter dishes coated with 2 ml of DMEM containing 10% fetal bovine serum (HyClone) and 0.6% agarose (Gibco-BRL). Cells were seeded on top of this layer at a density of 5 × 10⁴ cells per plate in DMEM containing 10% fetal bovine serum and 0.3% agarose. Soft agar colony formation was evaluated 6 weeks after the initial plating. To determine growth to high density, cells were seeded at low density (5 × 10⁴ per 60-mm-diameter dish) and fed every 3 days with DMEM containing 10% fetal bovine serum. Triplicate plates of cells were counted every 2 or 3 days.

Promoter reporter assays. WT and ARF^/ cell lines were plated at a density of 10⁵ cells per 35-mm-diameter dish. Cells were transfected the following day with Fugene 6 and 0.5 µg of the appropriate luciferase reporter construct and pCMX--Gal (23). At 48 h after transfection, cells were washed twice in phosphate-buffered saline and assayed for -galactosidase and luciferase activities as previously described (31).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Immortalization of MEFs by SV40 TAg. It has been reported that TAg-mediated transformation of WT MEFs requires the inactivation of the pRB family by the LXCXE motif and J domain and inactivation of p53 function through the p53-binding domain (10, 76, 81). It is possible that these transforming domains of TAg have other functions or affect additional cellular targets. To determine whether these TAg domains contributed to transformation in cells that had undergone inactivation of the pRB and p53 pathways, we used selected knockout mouse strains. INK4a^/ MEFs contain a deletion of the second and third exons of the INK4a locus (58). This prevents the expression of both p16^INK4a and p19^ARF, effectively disrupting both the p53 and the pRB pathways. ARF^/ MEFs selectively delete the first exon of the p19^ARF protein and retain an intact p16^INK4a (37). RB^/ MEFs lose pRB but express the pRB-related proteins p107 and p130 (76).

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View larger version (18K): [in this window] [in a new window]   FIG. 1.   SV40 TAg constructs. The J domain is contained within the first 82 residues and includes the conserved HPD (residues 42 to 44). The point substitution mutant H42Q inactivates the J domain function of TAg (6, 68). The LXCXE motif is present in residues 103 to 107. The K1, C105G, and PVU-1 mutations disrupt binding to all pRB family members (76). The HQ-K1 mutant contains inactivating mutations in the J domain and LXCXE motif. The dl434-444 mutant disrupts the ability of TAg to bind to p53 (40). T1-350 contains the first 350 amino acids of TAg. T1-135 contains the first 131 amino acids of TAg plus 4 additional residues (79). All TAg constructs were expressed as cDNAs in a pSG5 plasmid under an SV40 promoter.

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Role of the LXCXE motif in anchorage-independent growth. To determine the ability of TAg or mutant derivatives to confer anchorage-independent growth, the MEF cell lines were plated in soft agar. Growth was evaluated visually, and colonies containing eight or more cells were scored as transformants (Fig. 2A). WT TAg expression in WT and RB^/ MEFs was capable of inducing growth in soft agar in approximately 40 to 50% of cells seeded. In WT and RB^/ MEFs, the J domain mutant H42Q transformed almost as well as WT TAg, with nearly the same number and size of colonies, indicating that the J domain was not required for anchorage-independent growth (68). In contrast, TAgs containing mutations in the LXCXE motif were unable to transform WT or RB^/ MEFs (68, 76). Thus, an intact LXCXE motif but not the J domain was necessary for the transformation of WT and RB^/ MEFs. Similar results were observed in p107^/, p130^/, and p107/p130^/ MEFs (Zalvide and DeCaprio, unpublished). In each of these genetic backgrounds, the LXCXE mutants failed to induce growth in soft agar, while both WT TAg and the J domain mutant of TAg induced soft agar growth in more than 40% of the cells seeded (data not shown). The truncated T1-135, lacking the p53-binding domain, was able to induce soft agar growth of RB^/ MEFs only slightly more efficiently than the full-length LXCXE mutants, suggesting that the p53-binding domain of TAg may contribute to anchorage-independent growth of RB^/ MEFs.

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View larger version (35K): [in this window] [in a new window]   FIG. 2.   Ability of TAg to form soft agar colonies in WT, RB/, INK4a/, and ARF/ MEFs. (A) TAg-immortalized MEFs from the indicated cultures were plated in soft agar and allowed to grow for 6 weeks. Colonies containing eight or more cells were scored as positive and indicated as percentage of cells seeded. Nonimmortalized WT and RB/ MEFs from the fifth passage (P5) were used as controls. Error bars indicate standard deviation. (B) Soft agar colony formation in an independent transformation of ARF/ MEFs. (C) Western blot of TAg expression in ARF/ MEFs. Equivalent amount of lysates prepared from each of the indicated MEFs were separated in an SDS-7.5% polyacrylamide gel and blotted with an anti-TAg monoclonal antibody. Sizes are indicated in kilodaltons.

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Growth to high density requires the J domain and the LXCXE motif. The TAg-transformed phenotype is also reflected in the ability of adherent cells to grow to high density on plastic surfaces. TAg-transformed cells can grow to a higher density than nontransformed cells. To overcome a density arrest, cells must overcome signals for growth arrest due to contact inhibition as well as growth factor depletion. TAg induces high-density growth in cultured cells (76). To determine the domains of TAg that participate in inducing high-density growth, the growth rate of TAg-immortalized MEF cell lines was determined. Cells were plated on a 10-cm-diameter plate at a low density and refed with medium containing 10% serum every 3 days. At various intervals, three replica plates of each transformant were harvested and counted. This growth experiment was repeated at least twice, and the results of a typical experiment are shown in Fig. 3.

View larger version (26K): [in this window] [in a new window]   FIG. 3.   Ability of TAg-transformed MEFs to grow to high density. Fifty thousand WT, INK4a/, or ARF/ MEFs stably expressing the indicated TAg constructs were seeded on replica 100-mm-diameter plates and fed with complete medium containing 10% serum every 2 to 3 days. Triplicate plates were harvested at the indicated day after seeding, and cells were counted. Error bars indicate standard deviation.

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Expression and activity of pRB in ARF^/ MEFs. The soft agar experiments shown in Fig. 2A and B suggest that while the LXCXE motif was required for TAg-dependent anchorage-independent growth in WT and RB^/ MEFs, it was dispensable in INK4a^/ and ARF^/ MEFs. This suggests that the loss of p19^ARF may have resulted in a partial inactivation of the pRB growth suppression pathway in addition to the p53 pathway. To explore this possibility, we examined the pRB and p53 pathways in WT and ARF^/ MEFs.

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View larger version (31K): [in this window] [in a new window]   FIG. 4.   Western blot for p19ARF, p16INK4a, and p15INK4b in WT and ARF/ MEFs expressing various TAg constructs. Equivalent amounts of lysates prepared from WT and ARF/ MEFs stably expressing the indicated TAg constructs were separated in SDS-polyacrylamide gels and blotted with specific antibodies.

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View larger version (67K): [in this window] [in a new window]   FIG. 5.   Expression of pRB family members, p53, and p21 in WT and ARF/ MEFs. (A) Western blot for pRB, p107, and p130 in WT and ARF/ MEFs expressing various TAg constructs. (B) Western blot for p53 and p21 in WT and ARF/ MEFs expressing various TAg constructs.

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View larger version (20K): [in this window] [in a new window]   FIG. 6.   E2F and p53 promoter reporter activity in TAg-transformed WT and ARF/ MEFs. The reporter constructs (A) 3xE2F-Luc and pCMV--Gal were cotransfected into the indicated stable TAg-transformed MEFs. Luciferase activity was normalized to -galactosidase activity and expressed as relative luminometer units. The experiment was performed in triplicate; error bars indicate standard deviation. (B) Cyclin G promoter-luciferase reporter (left) and p21 promoter-luciferase promoter (right) activities were determined as described above.

Expression and activity of p53 in ARF^/ MEFs. SV40 TAg has a well-described ability to bind to p53 and to increase its level of expression at least in part through decreasing the turnover rate (70). As shown in Fig. 5B (top panel), p53 expression was extremely low in the pEpuro ARF^/ MEFs (lane 8) as well as in early-passage cultures of WT MEFs (lane 1). In contrast, WT and ARF^/ MEFs expressing WT TAg (lanes 2 and 9), LXCXE mutants (lanes 4 to 6 and 11 to 13), or J domain mutants (lanes 3, 7, 10, and 14), which are capable of binding to p53, had significantly increased levels of p53. This observation suggests that TAg could stabilize p53 both in WT cells and in cells that have lost p19^ARF. In contrast, the expression of p53-binding mutants dl434-444 (lane 15), T1-350 (lane 16), and T1-135 (lane 17) did not result in increased steady-state levels of p53.

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	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this report, the contribution of the J domain, LXCXE motif, and p53-binding domain of TAg to transformation of WT, RB^/, INK4a^/, and ARF^/ MEFs was examined. The goal was to determine whether the loss or functional inactivation of certain tumor suppressor genes would reduce the requirement for these domains in TAg transformation. WT SV40 TAg was able to induce growth of MEFs derived from each of these genetic backgrounds to high density and in an anchorage-independent manner. In WT and RB^/ MEFs, TAg's p53-binding domain was required for efficient immortalization, the LXCXE motif was needed for anchorage-independent and high-density growth, and the J domain was required for high-density growth. In contrast, TAg had different requirements for transformation of MEFs that did not express p16^INK4a or p19^ARF. The p53-binding domain of TAg was not required for immortalization, high-density growth, or soft agar growth of INK4a^/ or ARF^/ MEFs. These results were not unexpected given that the loss of p19^ARF expression has been shown to perturb the p53 growth suppression pathway, thereby reducing the requirement for p53 inactivation by TAg binding. The LXCXE motif and J domain were required for high-density growth of INK4a^/ MEFs, suggesting that the pRB family was functionally intact despite the loss of p16^INK4a expression. The most unexpected observation was that the LXCXE motif was not required for anchorage-independent growth of either INK4a^/ or ARF^/ MEFs. This latter result would seem to suggest that the growth suppression functions of the pRB family were disrupted in ARF^/ and INK4a^/ MEFs during anchorage-independent growth. Notably, the ARF^/ MEFs which expressed p16^INK4a but not p19^ARF behaved similarly as INK4a^/ MEFs in response to TAg. Both cell types were induced to grow in soft agar when expressing either p53-binding domain or LXCXE motif mutants of TAg. Since p19^ARF was reported to regulate the p53 pathway but not the pRB pathway, the LXCXE mutants were not expected to induce anchorage-independent growth in ARF^/ MEFs that expressed p16^INK4a.

It is unlikely that INK4a^/ and ARF^/ MEFs expressing the LXCXE mutant TAg constructs have acquired additional specific genetic modifications that enabled them to grow in soft agar. All assays were performed with pools of transfected cells to reduce the possibility of clonal variation. Furthermore, similar transformation results were obtained with several independently performed transfections. INK4a^/ or ARF^/ MEFs could be readily immortalized in the absence of TAg, as evidenced by the efficient ability to select for colonies in the presence of the puromycin resistance gene alone. However, INK4a^/ or ARF^/ MEFs expressing the puromycin resistance gene alone were not transformed, as determined by their inability to grow in soft agar or to high density. Expression of TAg conferred an additional transforming activity that permitted growth under these more stringent conditions. These observations are consistent with the demonstration that expression of activated H-ras induced transformation of ARF^/ and INK4a^/ MEFs (37, 58).

Growth in soft agar measures the ability of fibroblasts to acquire anchorage-independent growth. Anchorage-independent growth has been considered a stringent in vitro method for predicting the tumor formation potential of TAg-transformed fibroblasts (55). However, it has been reported that the ability of TAg-transformed cells to form tumors in nude mice does not require an intact LXCXE motif (71). To date, there has been an absolute requirement for an intact LXCXE motif in anchorage-independent growth of TAg-transformed WT MEFs in soft agar (10, 11, 71, 76). Here, we observed that the LXCXE motif was not required for soft agar growth of INK4a^/ and ARF^/ MEFs.

Notably, expression of HQ-K1, a TAg containing inactivating mutations in both the LXCXE motif and J domain, induced INK4a^/ or ARF^/ MEFs to grow in soft agar. Despite loss of the J domain and LXCXE motif, HQ-K1 could bind to and stabilize p53. Transformation by this and other LXCXE mutants would seem to indicate that binding to p53 conferred a growth advantage and that p53 function was not completely eliminated by the loss of p19^ARF. Consistent with this possibility, p53 appeared to be functional in the ARF^/ MEFs that express TAg with mutations in the p53-binding domain, as demonstrated by the increased activity of the p21^CIP and cyclin G promoters (Fig. 6B) as well as by the increased expression of p21^CIP (Fig. 5B). This potential for activation of p53 in ARF^/ MEFs did not seem to pose a barrier to immortalization or to the ability of the p53-binding mutant TAgs to induce growth in soft agar or to high density.

The ability of adherent cells to grow to high density may reflect the cell's response to limiting amounts of growth factors and nutrients as the cell number increases. An intact J domain and LXCXE motif were both required for TAg to induce growth to high density in WT, RB^/, ARF^/, and INK4a^/ MEFs (Fig. 3 and reference 68). The contribution of the J domain to high-density growth of these MEFs is consistent with previous work that suggests that the J domain cooperates with the LXCXE motif to inactivate pRB, p107, and p130 (27, 60, 68). Further evidence for the J domain activity includes the reduction in phosphorylation of p107 and p130 in WT and ARF^/ MEFs expressing TAg (Fig. 5B). We have previously reported that J domain mutant constructs were as effective as WT TAg in promoting the high-density growth of p130^/ p107^/ double-knockout MEFs, suggesting that the J domain specifically contributed to the functional inactivation of p107 and p130 (68). The observation that the J domain and the LXCXE motif were required for overcoming density arrest of INK4a^/ and ARF^/ MEFs suggests that the pRB family including p107 and p130 was functionally intact in adherent INK4a^/ and ARF^/ MEFs. Therefore, loss of p16^INK4a or p19^ARF expression did not perturb the pRB growth suppression activity in the density arrest assay. It will be interesting to determine whether TAgs containing J domain or LXCXE motif mutants will have the ability to induce high-density or soft agar growth in MEFs homozygously deficient in Rb, p107, and p130.

The J domain cooperates with the LXCXE motif to inactivate pRB function in a variety of assays including dissociation of pRB-E2F complexes and overriding pRB repression of E2F-dependent promoters (68, 77). Recent reports suggest that the LXCXE motif of SV40 and polyomavirus large T antigens may also inhibit certain pRB functions independently of the J domain. For example, WT TAg and a J domain mutant (H42Q) but not an LXCXE mutant (K1) could override a p53-induced growth arrest (25). The LXCXE motif but not the J domain was required for polyomavirus large T antigen-induced apoptosis of C2C12 myoblasts upon serum withdrawal (61). In this report, we observed that an intact LXCXE motif but not the J domain was required for activation of the 3xE2F promoter reporter in WT and ARF^/ MEFs (Fig. 6A). In addition, an intact J domain was not required for inducing soft agar growth of MEFs derived from any of the genetic backgrounds studied. These distinctions may reflect the ability of TAg to perturb different growth-suppressing functions of the pRB family. Alternatively, the LXCXE motif may target other cellular proteins that do not require the contribution of the J domain.

As mentioned earlier, the most unexpected observation was that the LXCXE mutants of TAg could induce anchorage-independent growth of ARF^/ MEFs. There are several models to explain how the LXCXE mutant TAgs were able to confer anchorage-independent growth of the ARF^/ and INK4a^/ MEFs. One possibility is that p19^ARF is involved in the regulation of both p53 and pRB and that loss of p19^ARF led to at least partial deregulation of both pathways. If this were true, it could explain why ARF^/ mice display the same tumor types as INK4a^/ mice. Several reports suggest that p19^ARF may regulate pRB function. For example, the expression of an antisense construct to p19^ARF could overcome a growth arrest induced by p16^INK4a whereas the expression of an antisense construct to p16^INK4a was unable to overcome a growth arrest induced by p19^ARF, suggesting that p19^ARF has functions that overlap with those of p16^INK4a (8). In addition, overexpression of p19^ARF was able to induce a growth arrest in p53^/ cells but not in p53^/ cells that overexpressed E2F-1, again suggesting that p19^ARF may be able to suppress growth through regulation of pRB as well as p53 (8). In addition, MDM2 may be directly involved in the regulation of pRB by binding to the C-terminal region of pRB (73). Alternatively, MDM2 may promote the rapid degradation of E2F-1 in cells that lack p19^ARF (4). Under such conditions, it is likely that pRB would be at least partially unable to act as a tumor suppressor gene, since pRB binding to E2F-1 is required for transcriptional repression of E2F-dependent promoters and tumor suppression (22, 75). However, our data do not support such a scenario since we were unable to detect any differences in E2F activity between WT and ARF^/ MEFs transformed by LXCXE mutants of TAg. Furthermore, the steady-state levels of E2F-1 appeared similar between the WT and ARF^/ cells as determined by Western blotting (data not shown). We have not extensively tested the ability of the pRB family to bind and inactivate other members of the E2F family in the TAg-transformed MEFs. It is possible that the activities of one or more members of the E2F family are diminished in ARF^/ MEFs. Alternatively, it is possible that the LXCXE region of TAg targets an additional growth or tumor-suppressing protein whose activity may be lacking or diminished in ARF^/ MEFs.

The ability of the LXCXE mutant constructs to induce anchorage-independent growth in ARF^/ MEFs may also reflect a novel transforming function of TAg. This additional activity could overcome the growth inhibition induced by the pRB family. p300 is an especially interesting candidate for this additional activity. p300 was originally cloned as an adenovirus E1A-associated protein (18), and both p300 and the highly related CREB-binding protein (CBP) can be functionally inactivated by E1A (1, 49). E1A binds directly to p300 and CBP through a domain distinct from its pRB family-binding motif. E1A can stimulate entry into the cell cycle from quiescence using either the p300/CBP-binding region or the pRB-binding LXCXE motif (66, 78). Thus, E1A binding to p300/CBP can circumvent at least some of the growth-inhibitory functions of pRB. The mechanism of the E1A/p300 growth-promoting activity, however, is not completely understood. The effect of p300 binding to TAg also remains unknown; however, it is possible that this permits TAg to overcome the growth-suppressing function of the pRB family in ARF^/ MEFs (2, 45). The p53-binding domain of TAg can also bind to MDM2 (29). Thus, TAg could perturb the function of several growth-regulatory factors, including p53, MDM2, p300/CBP, and pRB.

In addition to the three TAg domains described here, it is possible that TAg has additional domains that contribute to transformation. The N terminus of TAg is known to bind to other cellular proteins in addition to the pRB family, most notably a 185-kDa protein that is as yet uncharacterized (42). In addition, there has been some suggestion that the DNA-binding domain of TAg (residues 131 to 280) may contribute to growth promotion and transformation. The DNA-binding region is involved in transcriptional transactivation of many cellular and viral promoters that may promote cellular growth. For example, a point substitution mutation in the DNA-binding domain reduced the ability of TAg to induce DNA synthesis in quiescent cells (15, 34). In our experiments, ARF^/ MEFs expressing T1-135 grew to half the density of those expressing full-length TAg, T1-350, or dl434-444 (Fig. 3). This may indicate that the DNA-binding region (residues 131 to 280) or perhaps the zinc finger (residues 302 to 320) can contribute to TAg-mediated transformation, though the nature and extent of this transforming activity are as yet uncharacterized.

In conclusion, these experiments have demonstrated that mutations in either the p53-binding domain or the LXCXE motif did not diminish the ability of TAg to confer anchorage-independent growth of INK4a^/ and ARF^/ MEFs. These results support the notion that the N-terminal and C-terminal regions of TAg have transforming functions beyond the disruption of the pRB and p53 tumor suppressor pathways. Furthermore, these results suggest that p19^ARF may have tumor suppression functions in addition to control of the p53 tumor suppressor gene.