VIROLOGY

191,

754-764

(1992)

Lymphotropic

Papovavirus Transforms Hamster Ceils without the Amount or Stability of ~53

Altering

SANGSUN KANG AND WILLIAM R. FOLK’ Department

of Biochemistry,

University

of Missouri

at Columbia. Columbia, Missouri

652 12

Received May 28, 1992; accepted August 20, 1992 Expression of the early regions of several primate polyomaviruses (SV40, BKV, JCV, and LPV) in hamster cells induces transformation, manifested by the ability to grow in soft agar. Hamster cells transformed by SV40 contain complexes between the SV40 T antigen and the cellular tumor suppressor protein ~53. We detected analogous complexes between ~53 and the BKV T antigen in hamster cells transformed by the BKV early region, where the half life of p53 increased 16-fold. However, neither a LPV-transformed hamster fibroblast cell line [LPV-HE (F); K. K. Takemoto and T. Kanda, 1984, J. Viral. 50, 100-l 051 nor BHK-21 cells transformed by the LPV early region contained detectable complexes between the LPV T antigen and ~53, nor was the stability of ~53 in LPV transformed BHK-21 cells altered. Association between hamster ~53 and the LPV T antigen expressed as glutathione S-transferase fusion protein could not be detected in vitro. These data indicate that alteration of the amount or stability of ~53 is not required for transformation of hamster cells by LPV. However, as viruses such as SV40 and BKV whose T antigens bind ~53 are oncogenic in hamsters, whereas LPV is not, the alteration of ~53 amount or stability may be required for tumorigenesis. 0 1993 Academic Press, Inc.

INTRODUCTION

vine, 1979) ~105 (the Rb gene product), ~107 (a homolog of Rb) (DeCaprio eta/., 1988; Dyson eta/., 1989; Ewen et a/., 1989) DNA polymerase-a primase (Dornreiter et al., 1990; Gannon and Lane, 1987; Smale and Tjian, 1986) and ~34~~~~ (Milner et al., 1990). Many of these activities have been assigned to discrete domains. The SV40 T antigen N-terminal residues are largely responsible for complex formation with pRb, ~107 (DeCaprio et al., 1988; Dyson et al., 1989; Ewen et al., 1989), and complement mutants of ElA defective for ~300 binding (Yaciuk et al., 1991). The C-terminal region of SV40 T antigen contains the binding domain for ATP (residues 418 to 529; Bradley et a/., 1987) and p53 (residues 27 1 to 708; Manfredi and Prives, 1990). Binding of p53 by SV40 T antigen appears to be important for the immortalization of rodent and human ceils (Lin et al., 1991 b; Zhu et al., 1991) and transformation of some rodent ceils (Zhu et a/., 1992; Srinivasan et al., 1989); however, SV40 is capable of transforming many types of rodent cells without altering p53 (Clayton et a/,, 1982; Colby and Shenk, 1982; Sompayrac et a/., 1983, 1988, 1991; Srinivasan et al., 1989). p53 binding to SV40 T antigen may overcome the ability of the cell to restrict cell division and/or DNA replication (Lane and Benchimol, 1990; Levine, 1990; Levine et a/., 1991; Weinberg, 1991). Wild type p53 protein is localized in the nucleus, where it suppresses transformation byras and E 1a and consequently is considered to be a tumor suppressor

Lymphotropic papovavirus (LPV) productively infects monkey B and T cells and human B cells immortalized by Epstein-Barr Virus (EBV) (Brade et a/., 1981; Takemoto et a/., 1982; zur Hausen et a/., 1979). Transgenic mice expressing the LPV early region develop tumors of the choroid plexus and lymphoid proliferative disorders (Chen et a/., 1989). However, LPV is unlike the related SV40, BK and JC viruses in that it is not oncogenie in hamsters and mice (Takemoto and Kanda, 1984; Yoshiike and Takemoto, 1986). An explanation for the lack of oncogenicity of LPV in hamsters has not been offered and might prove to illuminate how the other polyomaviruses cause tumors. Because of their amino acid sequence similarities, it is probable that the proteins encoded by these viruses have similar functions. The SV40 T antigen has been intensively studied and serves as a paradigm (for reviews see DePamphilis and Bradley, 1986; Fanning and Knippers, 1992). It expresses multiple activities including an ATPase (Clark et al., 1981; Giacherio and Hager, 1979) and DNA helicase (Stahl eta/., 1986) and has the capacity to stimulate cellular DNA and RNA synthesis (Chou et a/., 1975; Soprano et al., 1981; Tjian and Graessmann, 1978). It has affinities for numerous cellular proteins, including AP 2 (Mitchell eta/., 1987) p53 (Lane and Crawford, 1979; Linzer and Le’ To whom reprint requests should be addressed. 0042-6822193

$5.00

Copyrtght 0 1993 by Academic Press, Inc. All rights of reproduction I” any form reserved

754

LPV T ANTIGEN

AND HAMSTER

protein (Finlay et a/., 1989; Mercer et al., 1990). Mutations in a variety of conserved regions cause it to associate with cellular stress proteins (hsp72/73) (Hinds et a/., 1987) and these complexes are important in the development of an immune response (Davidoff et al., 1992). Mutant ~53 genes are capable of immortalizing cells (Jenkins eta/., 1984) and when cotransfected with activated ras cause transformation of primary cells (Eliyahu et a/., 1984; Parada eta/., 1984). Mutant forms of p53 protein occur in the cells of a wide range of human cancers, and mice lacking p53 develop multiple spontaneous tumors, but the biochemical function(s) of ~53 is still unclear (Barteck et a/., 199 1; Donehower et a/., 1992; Lane and Benchimol, 1990; Levine et a/., 1990, 1991; Soussi et al., 1990; Weinberg, 1991). In the course of studying a hamster embryo cell line transformed by the lymphotropic papovavirus (LPV-HE (F); Takemoto and Kanda, 1984) we could find no sign of a complex between the LPV T antigen and hamster ~53, in contrast with the well-known ability of SV40 T antigen to complex with hamster ~53 (Lane and Crawford, 1979; Linzer and Levine, 1979). To more fully explore whether LPV T antigen interacts with the hamster ~53, we constructed BHK-21 cells expressing the LPV early region and for comparison, BHK-21 cells expressing the BKV early region. The capacity of BKV T antigen to bind ~53 has not been previously demonstrated, but its similarity to SV40 T antigen in the region responsible for complex formation with ~53 (amino acid residues 337-5 17; Levine et al., 199 1; Manfredi and Prives, 1990) suggests that it should. Here we report that BKV T antigen, like the SV40 T antigen, forms a complex which concomitantly stabilizes hamster ~53. However, the LPV T antigen fails to form a detectable complex with hamster ~53, and in LPV transformed cells, ~53 has a half life which is unchanged from that in untransformed cells. As BHK-21 cells expressing the LPV early region grow in soft agar, this indicates that ~53 accumulation may not be involved in the growth control of immortalized hamster cells. Furthermore, the failure of LPV T antigen to form a complex with hamster ~53 provides a possible explanation for why LPV is not tumorioenic. in contrast to SV40 and BK viruses, which are tumorigenic and whose T antigens form complexes with ~53.

a

MATERIALS

AND METHODS

Construction of BKV and LPV early region expression plasmids To promote expression of the LPV early region in BHK-21 cells, we replaced the LPV enhancer region with the spleen necrosis virus LTR (Embreston et a/., 1986) by inserting the LPV early sequences between

~53

755

F

coR I

Xhol

BamHl

FIG. 1. Structures of (a) LPV T antigen expression T antigen expression plasmid.

plasmid (b) BKV

nucleotide 153 and nucleotide 2290 including the T antigen coding region (Pawlita et al., 1985) into the Kpnl site of p636/214 (a gift from Dr. M. Hannink), yielding p636/LPT (Fig. 1a). The BKV Dik strain early region (ter Schegget et al., 1985) was excised with Pvull and inserted at the Smal site of pBluescript SK(-), yielding pBlueBKT-1. A BKV early region expression plasmid (pBK-5 Neo) was constructed by the insertion of the neomycin resistance gene as anXhol fragment from pZipNeoSV(x) (Cepko et al., 1984) into the Xhol site of pBlueBKT-1. This plasmid utilizes the BKV enhancer to express T antigen (Fig. 1b). Construction

of recombinant

pGEX-2T plasmids

A LPV T antigen cDNA, generated by PCR amplification of each exon and subsequent ligation (a gift from Dr. M. Riley), was inserted into the BarnHI site of pGEX2T (Pharmacia Co.) (termed pLG73). The carboxy terminal 400 amino acids of LPV T antigen were placed in phase downstream of the glutathione S-transferase leader seauence bv cleavaae of pLG73 with Ncol and Xbal and subsequent~self-ligation, yielding pLG73DNX. The BKV T antigen exon 2 coding region (2.2 kb) was excised from pBlue BKT-1 with Ban1 and cloned at the Smal site of pBluescript SK(-), resulting in a 5.1-kb

KANG AND FOLK

756

recombinant DNA. A 2.2-kb DNA fragment containing the BKV T antigen exon 2 coding region was isolated by cleaving with BarnHI; this was cloned at the BarnHI site of pGEX-2T, yielding pGSTBK. As a result, the entire exon 2 region of BKV T antigen (amino acids 81695) was attached in phase downstream of glutathione S-transferase leader sequence. Cell culture

and DNA transfection

BHK-21 cells (ATCC CCLlO) cells were maintained in Dulbecco’s Modified Essential Medium (DMEM) containing 10% fetal calf serum (FCS). Lipofectin reagent (Bethesda Research Laboratories) was used to transfect pBK-5 Neo and p636/LPT DNA, following the methods provided by the manufacturer. Drug-resistant foci were isolated after 2 1 days in selective media containing 400 pg/ml G418 (GIBCO Co.) or 100 hg/ml Hygromycin B (Sigma Co.); to avoid studying rare transformants, three independent drug-resistant foci of pBK-5 Neo or p636/LPT DNA were recloned, expanded, and characterized. The capacity of cell lines to grow in suspension was measured by the methods described by Macpherson and Montagnier (1964). BHK-21, BHWBK, or BHWLPT cells (1 O3 or 1O4 cells per 60-mm dish) were plated in DMEM containing 10% FCS and 0.3% Bacto-Agar (Difco Lab.). Every 3 days, 0.5 ml of media containing either no drug, 400 Kg/ml G418 (GIBCO Co.), or 100 pug/ml Hygromycin B (Sigma Co.) was added. Three weeks after seeding, the colonies were counted and photographed at 100X magnification. The plates were then stained with p-iodonitro tetrazolium violet (Sigma Co.) and photographed without magnification.

supernatant were mixed for 30 min at 4” with 25 ~1 of glutathione Sepharose, which had been previously washed three times and resuspended (final concentration 1 :l [v/v]) in NETN containing 0.5% powdered milk. The glutathione Sepharose beads were then washed three times with NETN. For analysis of bound bacterial proteins, the beads were boiled in SDS sample buffer (2% SDS, 10% glycerol, 62 mM Tris [pH 6.81, SB) and loaded on SDS-7% polyacrylamide gels. Proteins were visualized by Coomassie blue staining. In vitro p53 binding assay A modification of the procedure of Kaelin eta/. (1991) was used to detect p53 binding in vitro. Nearly confluent BHK-21 and mouse NIH 3T3 cells grown in DMEM containing 10% FCS were washed two times with PBS (136 mlVl NaCI, 2.7 mn/l KCI, 10 mM Na,HPO,, 1.8 mM KH,PO, [pH 7.21) and scraped off the plate into a microfuge tube and centrifuged for 30 set at 4’. Pellets were lysed with ice-cold NETN (800 ~1) for 30 min on ice and the suspension was centrifuged for 10 min at 4”. Glutathione S-transferase T antigen fusion proteins were expressed in bacteria (20 ml culture) and recovered on glutathione Sepharose beads as described above. The beads were incubated with aliquots of cell lysates (equivalent to confluent cells in four 15-cm plates) for 1 hr at 4’ and then washed five times. The beads were then boiled in SB and bound proteins resolved on SDS-7% polyacrylamide gels. p53 was detected by Western blotting with pab240 (Gannon et a/., 1990; Yewdell et al., 1986) as described below. Detection

Glutathione

S-transferase

fusion proteins

Expression and purification of glutathione S-transferase fusion proteins were performed essentially as described by Smith and Johnson (1988). Fresh overnight cultures of Escherichia co/i DH5-cu strain (Bethesda Research Laboratories) transformed with either pGEX-2T or one of the pGEX-2T-T antigen recombinants were diluted 1: 10 in LB media containing ampicillin (100 pg/ml) and incubated for 5 hr at 37” with shaking. After 1 hr of growth, isopropyl-~-D-thiogalactopyranoside (IPTG; Sigma Co.) was added to a final concentration of 0.1 mM. For recovery of fusion proteins, glutathione Sepharose beads (Pharmacia Co.) were used. Bacteria were pelleted by centrifugation at 5000 g for 5 min at 4” and resuspended in l/20 vol NETN (20 mM Tris [pH 8.01, 100 mM NaCI, 1 ml\/l EDTA, 0.59/o Nonidet P-40). The bacteria were then disrupted by sonication at 0” and centrifuged at 10,000 g for 5 min at 4”. Aliquots (1 ml) of bacterial

of T antigens

and p53 proteins

Nearly confluent LPV-HE (F) (Takemoto and Kanda, 1984) BHK-21, BHWBK, and BHWLPT cells grown in DMEM containing 10% FCS were labeled with either [35S]methionine (200 &i/ml, Trans label, ICN Radiochemicals) in DMEM without methionine or with [32P]or-thophosphate (500 &i/ml, Amersham) in DMEM without phosphate for 2 hr at 37”. For pulse-chase labeling, cells were labeled as described above with [35S]methionine for 15 min (pulse), washed with PBS, incubated with normal DMEM containing 10% FCS and extracted immediately or collected for 0.5, 1, 3, 6, 9, 12, and 24 hr (chase). The cells were washed two times with PBS and scraped off the plate into a microfuge tube and centrifuged for 30 set at 4”. Pellets were lysed with ice-cold 800 ~1 RIPA buffer (2 mM EDTA, 150 mNI NaCI, 10 mM Na,HPO, [pH 7.21, 1% Nonidet P-40, 1% Sodium deoxycholate, 0.1% SDS, 0.1 mg/ml aprotinin, 0.174 mg/ml phenylmethylsulfonylfluoride) for 30 min on ice. The suspension was centrifuged for

LPV T ANTIGEN

10 min at 4’. The supernatant was incubated with 50 ~1 of SV40 T antigen monoclonal antibody pab416 (Harlow et al., 1981) or 10 ~1 of hamster polyclonal antibody against SV40 (Flow Laboratories) or LPV T antigen (developed from LPV T antigen fusion protein expressed in E. co/i; unpublished data) for 1 hr on ice and then incubated with 10 ~1 of protein G Sepharose 50% (v/v) with agitation at 4“ for 2 hr. The immunoprecipite was collected by centrifugation in a microfuge for 3 min at 4’ and then washed five times with 800 ~1 of RIPA buffer. The resulting pellet was suspended in 25 ~1 of SB, boiled for 5 min, and centrifuged for 5 min. Proteins were resolved on SDS-7% polyacrylamide gels and detected by autoradiography. To determine the stability T antigen or ~53, the radioactivity from the gel bands was measured with a Phospholmager (Molecular Dynamics Co.). For Western blots, the unlabeled total cell lysate was immunoprecipitated as described above and proteins were fractionated on SDS-7% polyacrylamide gels. Transfer of proteins from gel to nitrocellulose was performed as described by Burnette (1981). An ImmunoBio Assay Kit (Bio-Rad Co.) was used as recommended by the manufacturer. A 1:50 dilution of the supernatant of the hybridoma cell culture pab240 was used to detect ~53. RESULTS LPV T antigen does not form a stable complex with hamster ~53 The LPV-HE (F) ceil lines arose after infection of hamster embryo cells with LPV viral DNA (Takemoto and Kanda, 1984). lmmunoprecipitation of LPV-HE (F) cell [32P] proteins with a hamster LPV T antigen polyclonal antibody detected LPV T antigen (84 kd); however, a band corresponding to hamster p53 was not observed (Fig. 2a, lane 2; also see Fig. 4 of Takemoto and Kanda, 1984). A protein migrating close to the expected position of p53 was observed, but was not ~53, as it was also immunoprecipitated by pab416 (Harlow eta/., 1981) (Fig. 2a, lane l), which does not recognize LPV T antigen or p53 (unpublished data). As an alternative means of detecting ~53, unlabeled cell proteins were immunoprecipitated with a hamster LPV T antigen polyclonal antibody and then a Western blot was probed with pab240, which recognizes denatured hamster and mouse ~53. This confirmed that hamster p53 was not immunoprecipitated by the LPV T antigen polyclonal antibody (Fig. 2b, lane 2). These results contrast with the well-known capacity of SV40 T antigen to bind p53 (Lane and Crawford, 1979; Linzer and Levine, 1979) and with a recent report demonstrating that LPV T antigen forms a weak

AND HAMSTER

p53

757

complex with mouse p53 (Symonds et al., 1991). Because LPV-HE (F) cells have been in culture for some time, we could not rule out the possibility that mutation or deletion of the p53 gene had occurred in this cell line, preventing p53 from being expressed in a functional form. To resolve this question, we introduced into the pseudodiploid BHK-2 1 hamster cell line a vector encoding LPV T antigen, p636/LPT (Fig. la), and determined whether hamster p53 forms a complex with LPV T antigen. For comparison, we introduced into BHK-21 cells a vector expressing the BKV T antigen, pBK-5 Neo (Fig. 1b), which is closely related to the SV40 T antigen. Analysis of the cells expressing BKV T antigen (BHWBK) revealed that a 55-kDa phosphoprotein was coimmunoprecipitated with BKV T antigen (88 kDa) by pab416, a SV40 T antigen monoclonal antibody that also reacts with BKV T antigen (Fig. 2c, lane 3). The identity of this phosphoprotein was confirmed to be hamster p53 by Western blot analysis with pab240 (Gannon and Lane, 1987; Yewdell et al., 1986) which recognizes the denatured hamster p53 (Fig. 2b, lane 3, and Fig. 2d, lane 1). In contrast, although BHK21 cells transfected with p636/LPT (Fig. 1a) expressed LPV T antigen (BHK/LPT), coimmunoprecipitation of p53 with LPVT antigen was not observed (Fig. 2c, lane 2, Fig. 2d, lane 2). To ensure that this was characteristic of these viruses, three independent drug resistant foci of each type of transformant were tested with same results (data not shown). In summary, these results show that LPV T antigen does not form a detectable complex with hamster p53 in either LPV-HE (F) or BHWLPT cells. However, hamster p53 is capable of complexing with BKV T antigen. Thus, the lack of a complex between LPV T antigen and hamster p53 is attributable to a property of the LPV T antigen. Properties of hamster cells transformed early region

by the LPV

BHK-21 cells do not grow in soft agar, but are one step removed from being malignant (Bouck and di Mayorca, 1982; di Mayorca eta/., 1973). Conversion to malignancy and the capacity to induce angiogenesis are highly correlated with anchorage independence in this cell line (Bouck et al., 1986; Rastinejad et a/., 1989). To better understand the role of p53 in these transformants, we compared the properties of BHW LPT and BHWBK to the parental cell line BHK-21. The cellular morphology of each line is shown in Fig. 3a. Confluent BHK-21 cells exhibited a parallel, nonoverlapping alignment characteristic of nontransformed cells, whereas confluent cells expressing BKV or LPV early regions grew randomly and were highly refractile

KANG AND FOLK

758

a

b

116

-

84

-

58

-

-

84

-

58

-

36

116

+

LPV T

Hamster P53

e

Hamster P53

36 -

-

FIG. 2. lmmunoprecipitation and Western blot of LPV-HE (F), BHWBK, BHWLPT, and BHK-21 cell lines. (a) LPV-HE (F) cell [32P]proteins were immunoprecipitated with a polyclonal hamster LPV T antigen antibody or with pab416. Proteins were resolved on SDS-7% polyacrylamide gels and detected by autoradiography. Molecular sizes (kiloDaltons) are given at the left and the position of LPV T antigen at the right. (b) Proteins from unlabeled BHK-21, LPV-HE (F), or BHWBK cell lines were immunoprecipitated with antibodies as indicated above. A Western blot was performed with pab240. The position of hamster p53 is indicated at the right. (c) BHKIBK, BHWLPT, or BHK-21 cells were labeled with [32P]orthophosphate and proteins were immunoprecipitated with antibodies as indicated above. Proteins were resolved on SDS-7% polyacrylamide gels and detected by autoradiography. The position of LPV (90 kDa), BKV (88 kDa) T antigen or hamster p53 (55 kDa) are marked at the right. (d) Proteins from unlabeled BHWBK, BHK/LPT, or BHK-21 cell lines were immunoprecipitated with antibodies as indicated above. A Western blot was performed with pab240 which detects hamster and mouse ~53. The position of hamster p53 is indicated.

and rounded. As expected from these morphologies, BHWBK and BHWLPT cell lines formed foci in soft agar (Fig. 3b and 3~). This was verified for the cell lines from the three independent drug-resistant foci. Although the colony sizes of the BHWLPT cell lines were slightly smaller than those of the BHWBK cell lines, the plating efficiency of each was similar (- 17%) (Table 1).

P53 stability

is unchanged

in LPV-transformed

cells

The SV40 T antigen, adenovirus El B protein, and human papillomavirus E6 proteins each alter the stability of murine ~53. SV40 T antigen and adenovirus El B protein help stabilize it (Lane and Benchimol, 1990; Levine et al., 1991) but the papillomavirus E6 protein causes its degradation (Scheffner et a/., 1990). As we could detect no p53 in the LPV-transformed BHK-21 cells, it clearly does not accumulate, as for instance, in the BKV-transformed BHK-21 cells. However, it might

be rapidly degraded (as for instance, in murine cells transformed by papilloma virus). Consequently, we compared the half-life of p53 in the parental BHK-21 and in its derivatives. Cell were labeled with [“5S]methionine for 15 min and either lysed immediately or incubated with unlabeled methionine for 0.5, 1, 3, 6, 9, 12, or 24 hr. Cell extracts were prepared and immunoprecipitated with hamster SV40 tumor sera (Lane and Crawford, 1979; Linzer et a/., 1979). We resorted to using this sera, as we knew of no monoclonal antibodies directed against hamster p53 in its native state. This analysis indicated that the half life of p53 in BHK21 cells was approximately 30 min, while that of p53 in the BHWBK cells was approximately 8 hr, a 16-fold increase (Fig. 4a), which is very likely due to its formation of a complex with BKV T antigen. In contrast, we observed that the half life of p53 in BHK/LPV cells was approximately 30 min, the same as that in the parental BHK-21 cells; this indicates that the stability of p53 is unaffected by the expression of LPV T antigen.

LPV T ANTIGEN

AND HAMSTER

~53

BHK-21

BHK/BK

BHKILF’T

BHK/BK

BHK/BK

BHK/LPT

BHK/LPT

FIG. 3. Growth of BHK-21, BHWBK or BHWLPT cell lines in soft agar. (a) Each cell line (1000 cells) was plated on 0.3941agar in 60.mm dishes. (b) Three weeks after seeding, the colonies were photographed at 100X magnification. (c) The plates were stained with piodonitro tetrazolium violet and photographed without magnification.

ln vitro association of T antigen glutathione transferase fusion proteins and p53

S-

As a further test of the ability of these T antigens to associate with ~53, we compared the binding of the respective glutathione S-transferase BKV or LPV T antigen fusion proteins to hamster or mouse p53 in vitro. In place of the intact T antigen proteins, it was convenient to use the carboxyl terminal domains homologous to the region of SV40 T antigen containing the binding site for p53 (pGSTBK; BKV T antigen residues 81-695aa and pLG73DNX; LPV T antigen residues 299-697aa). These expression vectors produced the expected fusion proteins in E, co/i, as well as degrada-

tion products (data not shown). Cell lysates were prepared from unlabeled BHK-21 and mouse NIH 3T3 cells. Aliquots of the lysates were incubated with glutathione beads containing either the glutathione S-transferase leader sequence or the T antigen fusion proteins. Subsequently, these beads were repeatedly washed and bound proteins were eluted and resolved by SDS-polyacrylamide gel electrophoresis. Then, a Western blot was performed with pab240 which recognizes denatured hamster ~53. As a positive control, immunoprecipitations were performed with hamster SV40 tumor sera which recognizes native ~53. The data showed that BKV T antigen fusion proteins bound both hamster and mouse ~53. LPV T antigen fusion

KANG AND FOLK

760 TABLE 1 ANCHORAGE-INDEPENDENTGROWTHOF CELLS

Conversion of cells from a normal to a neoplastic state requires multiple genetic changes. BHK-21 cells are already immortalized and require only a single mu-

MicrocoloniesB Cell line

1

2

BHK-21 BHWBK BHWLPT

7+7 TMTCb TMTC

8+8 164 f 18 179 + 19

’ 1O4 cells (column 1) or 1O3 cells (column 2) were seeded in each of four plates. Three weeks later, colonies in each plate were counted. b TMTC, too many to count.

a

120,

p>j

+ --o-ci; 8 2 *

Despite similarities in genome organization and in DNA sequence, LPV differs from other primate polyomaviruses by its lack of oncogenicity in hamsters and mice. SV40 is highly oncogenic and BKV and JCV are moderately or weakly oncogenic, but LPV has never been found to induce tumors in hamsters and mice, despite repeated attempts (Takemoto and Kanda, 1984; Yoshiike and Takemoto, 1986). Our results might provide an explanation for this lack of tumorigenicity. We could not detect a stable complex between LPV T antigen and hamster p53 in three independent isolates of BHK-21 cells expressing the LPV early region. The failure of LPV T antigen to complex with hamster p53 is not solely a property of BHK-21 hamster cells (Fig. 2c and 2d), because LPV-HE (F), hamster embryo fibroblast cells transformed by LPV, also display no evidence of LPV T antigen-p53 complexes (Fig. 2a and 2b, also see Fig. 4 of Takemoto and Kanda, 1984). In addition, in vitro binding assays demonstrate a lack of complex formation between LPV T antigen and hamster p53 (Fig. 4b).

BHK-21 BHK/LF’T BHK/BK

60 40

-H

IO

protein did not bind hamster p53 and bound mouse p53 only weakly (Fig. 4b). Binding of the LPV T antigen to mouse p53 confirms the observation of Symonds et al. (1991). The low affinity of LPV T antigen for p53 is likely to be attributable to its primary sequence. Lin and Simmons (1991 a) reported that met-401 and asp-402 are key residues in SV40 T antigen (3g7L L P K US V V 405) for stable binding to monkey, human, and mouse p53 (Lin and Simmons, 1991 b). BKV T antigen has identical residues (3QQL L P Q M D S V I 457); however, the sequence of LPV T antigen (482L L D N ND V F 44*) is significantly different in this region (as well as in other regions).

DISCUSSION

SLaalllLy

Time (hr)

20

30

chase (hr)

BHK-21

BHK-21/LPT

0

1

3

6

9

12

24

BHK-21/BK

b

BHK-21

FL3T3 Q

%

Hamster 1153 I$ Mouse

p53

FIG. 4. Detection of p53 in viva and in vitro. (a) BHK-21, BHKIBK, and BHKILPT cell lines were pulse-chased with [35S]methionine as described under Materials and Methods. Polyclonal hamster SV40 T antigen antibody was used to immunoprecipitate hamster p53 and resolved on SDS-7% polyacrylamide gels. The amount of p53 in the gel bands was measured by Phospholmager analysis. (b) Unlabeled BHK-21 or FL3T3 cell lysates were incubated with glutathione Sepharose bound with pGEX-2T, pGSTBK, or pLG73DNX or immunoprecipitated with polyclonal hamster SV40 T antigen antibody. Proteins were resolved on a SDS-7% polyacrylamide gel and visualized by Western blotting with pab240. The position of the hamster and the mouse p53 are indicated.

LPV T ANTIGEN

tational event to grow in suspension, which is the last of the progressive stages leading to tumorigenicity (Bouck and Mayorca, 1982; di Mayorca et al., 1973; Jarett and Macpherson, 1968; Stoler and Bouck, 1985). Transformed BHK-21 cells which grow in suspension are highly tumorigenic (di Mayorca et al., 1973; Jarett and Macpherson, 1968; Macpherson and Montaignier, 1964) as are the LPV HE (F) cells (Takemoto and Kanda, 1984). Expreksion of the LPV T antigen is capable of inducing BHK-21 cells to grow in suspension, but our results indicate that p53 is unlikely to be involved in this process. Instead, another activity of LPV T antigen must be responsible. The Rb protein is a likely candidate for causing the transformed phenotype (Huang et al., 1988) or alternatively another yet undetected activity of T antigen may be responsible (Manfredi and Prives, 1990; Zhu et a/., 1992). Studies with mutants of SV40 implicate the N-terminal domain of its T antigen, which includes the binding domains for Rb and DNA polymerase CYand the activity capable of complementing El A mutants defective for ~300 binding, as being important in the transformation of some rodent cells (Clayton et al., 1982; Colby and Shenk, 1982; DeCaprio et al., 1988; Ewen et al., 1989; Sompayrac et al., 1983, 1988, 1991; Yoshiike and Takemoto, 1986). LPV T antigen is capable of binding to human Rb in vitro (Dyson et al., 1990) and we have found it to be capable of binding to hamster proteins the size of Rb in vitro and in viva (our unpublished observation). Studies of other transformed Syrian hamster cells provide evidence for tumor suppressor genes which block anchorage independence and responsiveness to paracrine and autocrine stimuli (Koi et a/., 1989) and genetic evidence suggests that one or more of these may be encoded in human cells by chromosome 1 (Stoler and Bouck, 1985). Additionally, human chromosome 1 induces cellular senescence in immortalized Syrian hamster cells (Sugawara et a/., 1990) and it is noteworthy that genetic changes in chromosome 1 have been detected in a variety of human cancers (Ghose et al., 1990; Leister et al., 1990; Weinberg, 1991). These multiple lines of evidence point to the presence on human chromosome 1 of genes or regulators of genes important in the control for the immortalization and progression of a hamster cell to a tumorigenie state. It seems likely that LPV T antigen interacts with the genes or gene products affecting these events. Might this occur through complex formation with the Rb protein? Although the Rb gene is located on human chromosome 13 (Cavenee et al., 1983) its effects are pleiotropic because of its affinity for multiple cellular proteins including the transcription factor E2F (Bagchi et a/., 1991; Chellapan et a/., 1991; Chittenden

AND HAMSTER

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et a/., 1991; Huang et al., 1991; Kaelin et al., 1991); thus, targets for Rb (or E2F) action might be located on human chromosome 1. Why is LPV incapable of forming tumors in hamsters? Our data suggests that it might be for the inability of LPV T antigen to bind ~53, and consequently, through the failure to efficiently immortalize hamster cells. The BHK-21 cells we have used in these experiments are already immortalized, and in the instance of other hamster cells transformed by LPV (Takemoto and Kanda, 1984) they may also have been spontaneously immortalized, as transformation occurred after a long time in passage. lmmof-talization (or suppression of senescence) is required for tumor formation by virus-transformed fibroblasts (O’Brien et al., 1986). It has been suggested that rodent cells are immortalized through the continued expression of SV40 T antigen only after bypassing the crisis stage (M 1) (Radna et al., 1989; Wright et al., 1989), and then they can be transformed and become tumorigenic. In contrast, immortalization of normal human cells has been suggested to occur as a result of bypassing two immortality stages Ml and M2 (Goldstein, 1990; Wright et al., 1989). Human fibroblasts transformed by SV40 are not tumorigenie, as binding of p53 by SV40 T antigen suffices to bypass only the crisis stage (Ml) of primary human cells in culture (Lin and Simmons, 1991a) and other mechanisms are required for full immortalization (Resnick-Silverman et al., 1991). That LPV T antigen weakly forms a complex with the mouse p53 might also explain its lack of tumorigenicity in mice (Yoshiike and Takemoto, 1986). However, expression of the LPV early region induces choroid plexus and lymphoid tumors in mice (Chen eta/., 1989; Symonds et al., 1991) suggesting that chronic expression .(or a very high level) of LPV T antigen in certain cells might lead to sufficient inactivation of p53 function to cause immortalization. These reports also indicate that the LPV enhancer does not strictly restrict viral gene expression to lymphoid cells of primates (Mosthaf et al., 1985) which could be argued to be an alternative reason for its lack of tumorigenicity in hamsters and mice. Why does LPV T antigen have a low affinity for ~53, while other primate polyomavirus T antigens (such as SV40, BK, JC virus) bind p53? Perhaps it is because in lymphoid cells (a target cell for LPV), the cell cycle is subject to different regulation than occurs with fibroblasts. The amount of p53 in lymphocytes is very low (Milner, 1984), and the variety of cell cycle mediators modulating lymphocyte activation and subsequent replication may not function through p53 activation/inactivation. Consequently, LPV may have adapted its T antigen to modify another pathway(s), so as to better repli-

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cate in lymphoid cells. A search for the connection between the LPV T antigen and these other pathways might prove illuminating and help to explain the basis for immortalization of lymphoid cells in human tumors. It is noteworthy that although p53 mutation in solid human tumors occurs very frequently (Barteck et a/., 1991), the incidence of p53 mutation in human lymphoid malignancies is quite low (Gaidano et a/., 199 1). ACKNOWLEDGMENTS We thank Sarah Scanlon, John Lednicky, Mark Martin, and Michael Riley for discussions and advice. We also thank E. Gurney, J. Butel, S. Tevethia, and E. Harlow for antibodies and K. Yoshiike, M. Pawlita, and M. Hannink for DNAs. This work was supported by grants from the National Cancer Institute (CA 45033 and CA 38538) and by the University of Missouri-Columbia. Note added in proof. Expression of very high levels of LPV T antigen in hamster embryo cells may lead to formation of a complex with p53 or a related protein (von Hoyningen-Huene et a/., Virology 190, 155-167).

REFERENCES BAGCHI, S., WEINMANN, R.. and RAYCHAUHURI,P. (1991). The retinoblastoma protein copurifies with E2F-I, an El A-regulated inhibitor of the transcription factor E2F. Cell 65, 1063-l 072. BARTECK, J., BARTKOVA, J., VOJTESEK, B., STASCOVA, Z., LUKAS, J., REITHAR, A., KOVARIK,J., MIDGLY, C. A., GANNON, J. V., and LANE, D. P. (1991). Aberrent expression of the p53 oncoprotein is a common feature of a wide spectrum of human malignancies. Oncogene 6, 1699-l 703. BOUCK, N. P., and DI MAYORCA, G. (1982). Chemical carcinogens transform BHK cells by inducing a recessive mutation. MO/. Cell. Biol. 2, 97-l 05. BOUCK, N. P., STOLER, A., and POLVERINI, P. J. (1986). Coordinate control of anchorage independence, actin cytoskeleton, and angiogenesis by human chromosome 1 in hamster-human hybrids. CancerRes. 46, 5101-5105. BRADE, L., VOGL, W., GISSMANN, L., and ZUR HAUSEN, H. (198 1). Propagation of B-lymphotropic papovavirus (LPV) in human B-lymphoma cells and characterization of its DNA. Virology 114, 228-235. BRADLEY, M. K., SMITH, T. F., L~;~HRoP, R. H., LIVINGSTON,D. M., and WEBSTER,T. A. (1987). Consensus topography in the ATP binding site of the simian virus 40 and polyoma virus large tumor antigens. Proc. Nat/. Acad. Sci. USA 84, 4026-4030. BURNETTE,W. N. (1981). “Western blotting”: Electrophoretic transfer of proteins from sodium dodecylsulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112, 195-203. CAVENEE, W. K., DRYJA, T. P., PHILLIPS, R. A., BENEDICT. W. F., GODBOUT, R., GALLIE, B. L., MURPHREE,A. L., STRONG, L. C., and WHITE, R. L. (1983). Expression of recessive alleles by chromosomal mechanism in retinoblastoma. Nature (London) 305, 779-784. CEPKO. C. L., ROBERTS,B. E., and MULLIGAN, R. C. (1984). Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell 37, 1053-l 062. CHELLAPPAN, S. P., HIEBERT, S., MUDRYL, M.. HOROWI~, J. M., and NEVINS, J. R. (1991). The E2F transcription factor is a cellular target for the Rb protein. Cell 65, 1053-l 061. CHEN, J., NEILSON, K., and VAN DYKE, T. (1989). Lymphotropic papo-

vavirus early region is specifically regulated in transgenic mice and efficiently induces neoplasia. /. Viral. 63, 2204-2214. CHITTENDEN,T., LIVINGSTON,D. M., and KAELIN,W. G.. JR. (1991). The T/ElA-binding domain of the retinoblastoma product can interact selectively with a sequence-specific DNA-binding protein. Ce//65, 1072-l 082. CHOU, J. Y.. and MARTIN, R. G. (1975). DNA infectivity and the induction of host DNA synthesis with temperature-sensitive mutants of simian virus 40.1. Viral. 15, 145-l 50. CLARK, R., LANE, D. P., and TJIAN, R. (1981). Use of monoclonal antibodies as probes of simian virus 40 T antigen ATPase activity. J. Biol. Chem. 256, 11854-l 1859. CLAYTON, C. E., MURPHY, D., LOVEIT, D., and RIGBY, P. W., JR. (1982). A fragment of the SV40 large T antigen genes transforms. Nature (London) 299, 56-61. COLBY, W. W., and SHENK, T. (1982). Fragments of the simian virus 40 transforming gene facilitate transformation of rat embryo cells. Proc. Nat/. Acad. Sci. USA 79, 5189-5 193. DAVIDOFF, A. M., IGLEHART,J. D., and MARKS, J. R. (1992). Immune response to p53 is dependent upon p53/HSP70 complexes in breast cancers. Proc. Nat/. Acad. Sci. USA 89, 3439-3442. DECAPRIO, J. A., LUDLOW,J. W.. FEGGE,J., SHEW, J.-Y.. HUANG, C.-M., LEE, W.-H., MARSILLIO, E., PAUCHA, E., and LIVINGSTON, D. M. (1988). SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell 54, 275-283. DEPAMPHILIS, M. L., and BRADLEY, M. K. (1986). Replication of SV40 and polyoma virus chromosomes, p. 99-246. /n “The Papovaviridae” (N. P. Salzman. Ed.), Vol. 1. Plenum, New York. DI MAYORCA, G., GREENEMIT, M., TRAUTHEN,T., SOLLER,A., and GIORDANO, R. (1973). Malignant transformation of BHK-2 1 clone 13 cell in vitro by nitrosamines-A conditional state. Proc. Nat/. Acad. Sci. USA 70, 46-49. DONEHOWER,L.. HARVEY, M., SLAGLE. B. L.. MCARTHER, M. J., MONTGOMERY,C. A., JR., BUTEL, J. S., and BRADLEY,A. (1992). Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumors. Nature (London) 356, 2 15-22 1. DORNREITER,I., Hess. A., ARTHUR, A. K., and FANNING, E. (1990). SV40 T antigen binds directly to the large subunit of purified DNA polymerase alpha. EMBOI. 9, 3329-3336. DYSON, N., BUCHKOVICH, K., WHME, P., and HARLOW, E. (1989). The cellular 107K protein that binds to adenovirus EIA also associates with the large T antigens of SV40 and JC virus. Cell 58, 249-255. DYSON, N., BERNARDS,R.. FRIEND, S. H., GOODING, L. R., HASSEL,J. A.. MAJOR. E. O., PIPAS, J. M., VANDYKE, T., and HARLOW, E. (1990). Large T antigens of many polyomaviruses are able to form complexes with the retinoblastoma protein. J. Viral. 64, 1353-l 356. ELIYAHU, D.. RX, A., GRUSS, P., GIVOL, D., and OREN, M. (1984). Participation of p53 cellular tumor antigen in transformation of normal embryonic cells. Nature (London) 312, 646-649. EMBRESTON,J. E., and TEMIN. H. M. (1986). Pseudotyped retroviral vectors reveal restrictions to reticuloendotheliosisvirus replication in rat cells. /. Viral. 60, 662-668. EWEN, M. E.. LUDLOW, J. W., MARSILIO, E., DECAPRIO, J. A., MILLIKAN, R. C., CHENG, S. H., PAIJCHA, E., and LIVINGSTON,D. M. (1989). An N-terminal transformation-governing sequence of SV40 large T antigen contributes to the binding of both pl 1 ORband a second cellular protein, ~120. Cell 58, 257-267. FANNING, E., and KNIPPERS,R. (1992). Structure and function of SV40 large tumor antigen. Annu. Rev. Biochem. 61, 55-86. FINLAY, C. A., HINDS, P. W., and LEVINE,A. 1. (1989). The p53 protooncogene can act as a suppressor of transformation. Cell 57, 1083-l 093. GAIDANO, G., BALLERINI,P., GONG, J. Z., INGHIRAMI,G.. NERI, A., NEW-

LPV T ANTIGEN COMB, E. W.. MAGRATH, I. T.. KNOWLES, D. M., and DALLA-FAVERA, R. (1991). p53 mutation in human lymphoid malignancies: Association with Burkitt lymphoma and chronic lymphocytic leukemia. Proc. Natl. Acad. Sci. USA 88, 5413-5417. GANNON, J. V., and LANE, D. P. (1987). p53 and DNA polymerase (Y compete for binding to SV40 T antigen. Nature (London) 329, 456-458. GANNON, J. V., GREAVES,R., IGGO, R., and LANE, D. P. (1990). Activating mutations in p53 produce a common conformational effect. A monoclonal antibody specific for the mutant form. EAJBO /. 9, 1595-l 602. GHOSE. T.. LEE, C. L. Y., FERNANDEZ, L. A., LEE, S. H. S., RAMAN, R., and COLP, P. (1990). Role of 1q trisomy in tumorigentcity, growth, and metastasis of human leukemic B-cell clones in nude mice. Cancer Res. 50, 3737-3742. GIACHERIO, D., and HAGER, L. P. (1979). A poly (dT)-stimulated ATPase activity associated with SV40 large T antigen. /. Biol. Chem. 254, 8113-8116. GOLDSTEIN,S. (1990). Replicative senescence: The human fibroblast comes of age. Science 249, 1129-l 133. HARLOW, E., CRAWFORD, L. V., PIM, D. C., and WILLIAMSON, N. M. (1981). Monoclonal antibodles specific for simian virus 40 tumor antigens. /. Viral. 39, 861-869. HINDS, P. W., FINLAY, C. A., FREY, A. B., and LEVINE, A. J. (1987). Immunological evidence for the association of p53 with a heat shock protein, hsc70, in p53-plus-ras transforming cell lines. Mol. Ce//. Biol. 7, 2863-2869. HUANG, H.-J. S., YEE, J.-K., SHEW, J.-Y., CHEN, P.-L., BOOKSTEIN, R., FRIEDMANN, T., LEE, E. Y.-H. P.. and LEE, W.-H. (1988). Suppression of the neoplastic phenotype by replacement of the RB gene in human cancer cells. Science 242, 1563-l 566. HUANG, S., LEE, W.-H., and LEE, Y.-H. P. (1991). Acellularprotein that competes with SV40 T antigen for binding to the retinoblastoma gene product. Nature (London) 350, 160-l 62. JARRET, O., and MACPHERSON, I. (1968). The basis for the tumorigenicity of BHK-21 cells. In?. /. Cancer 3, 657-662. JENKINS,J. R., RUDGE, K., and CURRIE. G. A. (1984). Cellular immortalization by a cDNA clone encoding transformation-associated phosphoprotein ~53. Nature (London) 312, 651-654. KAELIN, W. G. J., PALMS. D. C., DECAPRIO, J. A., KAYE. F. J., and LIVINGSTON,D. M. (1991). Identification of cellular proteins that can interact specificallywlth the T/ElA-binding region of the retinoblastoma gene product. Cell 64, 521-532. KOI, M.. AFSHARI, C. A., ANNAB, L. A., and BARRET, J. C. (1989). Role of a tumor-suppressor gene in the negative control of anchorageindependent growth of Syrian hamster cells. Proc. Nat/. Acad. SC;. USA 86, 8773-8777. LANE, D. P., and CRAWFORD,L. V. (1979). Tantigen is bound to a host protein in SV40-transformed cells. Nature (London) 278, 261263. LANE, D. P., and BENCHIMOL, S. (1990). ~53: Oncogene or anti-oncogene? Genes Dev. 4, l-8. LEISTER,I., WEITH, A., BRUDERLEI,S., CZIPLUCH, C., KANGWANPONG,D., SCHLAG, P., and SCHWAB, M. (1990). Human colorectal cancer: High frequency of deletion at chromosome 1~35. CancerRes. 60, 7232-7235. LEVINE, A. J. (1990). The p53 protein and its interactions with the oncogene products of the small DNA tumor viruses. Virology 177, 419-426. LEVINE, A. J., MOMAND, J., and FINLAY, C. A. (1991). The ~53 tumor suppressor gene. Nature (London) 351, 453-456. LIN, J.-Y., and SIMMONS, D. T. (1991a). Stable T-p53 complexes are not required for replication of simian virus 40 in culture or for en-

AND HAMSTER

~53

763

hanced phosphorylation of T antigen and ~53.1. Viral. 65, 20662072. LIN, J.-Y., and SIMMONS, D. T. (1991 b). The ability of IargeT antigen to complex with p53 is necessary for the increased life span and partial transformation of human cells by simian virus 40. /. Viral. 65,6447-6453. LINZER, D. I. H., and LEVINE, A. J. (1979). Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40 transformed cells and uninfected embryo carcinoma cells. Cell 17, 43-52. MACPHERSON, I., and MONTAGNIER, L. (1964). Agar suspension culture for the selective assay of cells transformed by polyoma virus. Virology 23, 291-294. MANFREDI, J. J., and PRIVES,C. (1990). Binding of p53 and pl05-RB is not sufficient for oncogenic transformation by a hybrid polyomavirus-simian virus 40 large T antigen. 1. Viral. 64, 5250-5259. MERCER,W. E., AMIN, M., SAUVE, G. J., APPELLA. E., ULLRICH. S. J., and ROMANO, J. W. (1990). Wild type human p53 is antiproliferative in SV40-transformed hamster cells. Oncogene 5, 973-980. MITCHELL, P. J., WANG, C., and TIJIAN, R. (1987). Positive and negative regulation of transcription in vitro: Enhancer-binding protein AP-2 is inhibited by SV40 T antigen. Cell 50, 847-861. MILNER, 1. (1984). Different forms of p53 detected by monoclonal antibodies in non-dividing and dividing lymphocytes. Nature (London) 310,143-145. MILNER, J., COOK, A., and MASON, J. (1990). p53 is associated with ~34~~~’ in transformed cells. EMBO 1. 9, 2885-2889. MOSTHAF, L., PAWLITA. M., and GRUSS, P. (1985). A viral enhancer element specifically active in human haematopoietic cells. Nature (London) 315, 597-660. O’BRIEN, W., STENMAN, G., and SANGER. R. (1986). Suppresston of tumor growth by senescence in virally transformed human fibroblasts. Proc. Nat/. Acad. Sci. USA 83, 8659-8663. PARADA, L. F., LAND, H.. WEINBERG,R. A., WOLF, D., and ROTTER,V. (1984). Cooperation between gene encoding p53 tumor antigen and ras In cellular transformation. Nature (London) 312, 649-651. PAWLITA, M., CLAD, A., and ZUR HAUSEN, H. (1985). Complete DNA sequence of lymphotropic papovavirus: Prototype of a new species of the polyomavirus genus. virology 143, 196-21 1. RADNA, R. L., CATON, Y., JHA, K. K., KAPLAN, P., LI, G., TRAGANOS, F.. and OZER. H. L. (1989). Growth of immortal simian virus 40&A transformed human fibroblasts IS temperature dependent. Mol. Cell. Viol. 9, 3093-3096. RASTINEJAD,F., POLVERINI,P. J., and BOUCK, N. P. (1989). Regulation of the activity of a new inhibitor of angiogenesis by a cancer suppressor gene. Cell 56, 345-355. RESNICK-SILVERMAN,L., PANG, Z., LI, G., JHA, K. K., and OZER, H. L. (1991). Retinoblastoma protein and simian virus 40.dependent immortalization of human fibroblasts. /. Viral. 65, 2845-2852. SCHEFFNER.M., WERNESS,B. A., HUIBREGTSE,J. M., LEVINE,A. J., and HOWLEY, P. M. (1990). The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of ~53. Ce// 63, 1129-l 136. SMALE, S. T.. and TJIAN, R. (1986). T-antigen-DNA polymerase 01 complex implicated in simian virus 40 DNA replication. MO/. Ce//. Biol. 6, 4077-4087. SMITH, D. B., and JOHNSON, K. S. (1988). Single step purification of polypeptides expressed in Escherichia colias fusions with glutathtone S-transferase. Gene 67, 31-40. SOMPAYRAC. L., and DANA, K. J. (1991). The amino-terminal 147 amino acids of SV40 large T antigen transform secondary rat embryo fibroblasts. Virology 181, 412-415. SOMPAYRAC,L. M., and DANNA, K. J. (1988). A new SV40 mutant that encodes a small fragment of T antigen transforms established rat and mouse cells. Virology 163, 39 l-396.

764

KANG AND FOLK

SOMPAYRAC, L. M., GURNEY, E. G., and DANNA, K. J. (1983). Stabilization of the 53,000 dalton nonviral tumor antigen is not required for transformation by simian virus 40. Mol. Cell. Biol. 3, 290-296. SOPRANO, K. J., JONAK, G., GALANTI, N., FLOROS, J., and BASERGA,R. (1981). Identification of an SV40 DNA sequence related to the activation of silent rRNA gene in human mouse hybrid cells. Virology 109, 127-l 36. Sousa, T., CARON DE FROMENTEL,C., and MAY, P. (1990). Structural aspects of the p53 protein in relation to gene evolution. Oncogene 5,945-952. SRINIVASAN,A., PEDEN, K. W. C.. and PIPAS, J. M. (1989). The large tumor antigen of simian virus 40 encodes at least two distinct transforming functions. J. Viral. 6, 5459-5463. STAHL, H., DROGE, P., and KNIPPERS,R. (1986). DNA helicase activity of SV40 large tumor antigen. EMBO /. 5, 1939-l 944. STOLER,A., and BOUCK, N. P. (I 985). Identification of single chromosome in the normal human genome essential for suppression of hamster cell transformation. froc. Nat/. Acad. Sci. USA 82, 570574. SUGAWARA,O., OSHIMURA, M., KOI, M., ANNAB, L. A., and BARRET, J. C. (1990). Induction of cellular senescence in immortalized cells by human chromosome 1. Science 247, 707-710. SYMONDS, H., CHEN, J., and VAN DYKE. T. (1991). Complex formation between the lymphotropic papovavirus large tumor antigen and the tumor suppressor protein ~53.1. Viral. 65, 5417-5424. TAKEMOTO, K. K., and KANDA, T. (1984). Lymphotropic papovavirus transformation of hamster embryo cells. J. Viral. 50, 100-l 05. TAKEMOTO, K. K., FURUNO, A., KATO, K., and YOSHIIKE, K. (1982). Biological and biochemical studies of African green monkey lymphotropic papovavirus. J. Viral. 42, 502-509. TER SCHEGGET,J., SOL, C. J. A., BAAN, E. W., VAN DERNOORDAA, J., and

VAN ORMONDT, H. (1985). Naturally occurring BK virus variants (JL and Dik) with deletions in the putative early enhancer-promoter sequences. J. Viral. 53, 302-305. TJIAN, R., and GRAESSMANN,A. (1978). Biological activity of purified simian virus 40 T antigen. Proc. Nat/. Acad. Sci. USA 75, 12701283. WEINBERG, R. A. (1991). Tumor suppressor genes. Science 254, 1138-1153. WRIGHT, W. E., PEREIRA-SMITH,0. M., and SHAY, J. W. (1989). Reversible cellular senescence: Implications for immortalization of normal human diploid fibroblasts. Mol. Cell. Biol. 9, 3088-3092. YACIUK, P.. CARTER. M. C.. PIPAS,J. M., and MOWN, E. (1991). Simian virus 40 large T antigen expresses a biological activity complementary to the p300-associated transforming function of the adenovirus El A gene products. Mol. Cell. Biol. 11, 21 16-2 124. YEWDELL, J. W., GANNON, J. V., and ONE, D. P. (1986). Monoclonal antibody analysis of p53 expression in normal and transformed cells. 1. Viral. 59, 444-452. YOSHIIKE, K., and TAKEMOTO, K. K. (1986). Studies with BK virus and monkey lymphotropic papovavirus. In “The Papovaviridae” (N. P. Salzman, Ed.), Vol. 1, pp. 295-326. Plenum, New York. ZHU, J., RICE, P. W., GORSCH, L., ABATE, M., and COLE, C. N. (1991). The ability of SV40 large T antigen to immortalize primary mouse embryo fibroblasts cosegregates with its ability to bind ~53. J. Viral. 65, 6872-6880. ZHU, J., RICE, P. W., GORSCH, L., ABATE, M., and COLE, C. N. (1992). Transformation of a continuous rat embryo fibroblast cell line requires three separate domains of simian virus 40 large T antigen. f. Viral. 66, 2780-2791. ZUR HAUSEN, H., and GISSMANN, L. (1979). Lymphotropic papovavirus isolated from African green monkey and human cells. Med. Microho/. Immunol. 167, 137-l 53.

Lymphotropic papovavirus transforms hamster cells without altering the amount or stability of p53.

Expression of the early regions of several primate polyomaviruses (SV40, BKV, JCV, and LPV) in hamster cells induces transformation, manifested by the...
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