Immortalization of Human Fibroblasts by Sv4olarge T Antigen Results In

[CANCER RESEARCH55, 4651-4657, October 15, 19951 Immortalization of Human Fibroblasts by SV4O Large T Antigen Results in the Reduction of Cyclin Dl Expression and Subunit Association with Proliferating Cell Nuclear Antigen and Wafi' Scott R. Peterson,2Donna M. Gadhois, E. Morton Bradbury, and Paul M. Kraemer Ljfe Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 [S. R. P., D. M. G., E. M. B., P. M. K.], and Department of Biological Chemistr,, University of California at Davis, Davis, Cal@fomia95616 [E. M. B.]

ABSTRACT the accumulation of mutations to genes that change basic cell cycle regulatory networks (3). Expression of large T antigen in human Protein complexes containing cydins and cyclin-dependent protein Id diploid fibroblasts extends the normal life span of the host cell and nases (cdks) have been shown to be rearranged in both spontaneous and causes chromosomal instability, resulting in a high frequency of viral tumor antigen-transformed cells. We have examined G1- and S phase cydlln/cdk complexes as a function of the neoplastic progression of genetic aberrations and ploidy changes (4, 5). These direct effects of human diploid fibroblasts transfected with the SV4O large T antigen. We large T antigen may be mediated via its association with the tumor fmd that the expression of cydlin Dl and its association with proliferating suppressor proteins p53 and Rb (6). The life span of these cells is cell nuclear antigen (PCNA) and Wafi remain unchanged in precrisis finite, however, and they enter into a crisis period when the prolifer human fibroblasts transfected with SV4O large T antigen. However, in ative capacity of the population is exhausted and a majority of the these same cells the association of cdk4 with cydin Dl, PCNA, and Wall cells die (7). A small percentage of cells (1 in 10@)expressing large T is disrupted. Upon immortalization, cydlin Dl protein expression is de antigen escape crisis and become immortal (8). The rarity of large creased, and binding of both PCNA and Wail with the remaining cyclin T-antigen-induced immortalization suggests that it has a genetic basis Dl is reduced. In contrast, large T antigen increased the expression of cydlinA and cyclin E proteins in both precrisis and immortal cells and did and requires specific mutations to the host genome that work in not reduce the binding of PCNA or Wall to either cdk2 or cyclin A combination with the biochemical properties of large T antigen itself. proteins. These results show that large T-antigen expression in human Continued passage of large T-antigen immortalized cells can pro fibroblasts selectively uncouples cydin Dl from cdk4, and subsequent duce cell populations that display many of the characteristics of immortalization of these cells results in additional changes to the cydin tumor-derived cell lines, including loss of contact inhibition, the D1-dependent cell cycle regulatory pathways. ability to grow under low serum conditions, as well as the capacity to induce tumors in nude mice (9). In addition, protein kinase-mediated 1 phase checkpoints that are present in normal human fibroblasts are INTRODUCTION abrogated in human fibroblasts fully transformed by large T antigen Transformation of human cells to the neoplastic state is a multistep (10, 11). One of the G1 kinase control points affected in the large process driven by the accumulation of mutations to both tumor sup T-antigen-transformed cells temporally overlaps with the expression pressor genes and proto-oncogenes. Much of what we know about of the cyclin Dl protein (11). Expression of cyclin Dl is required for cancer cells comes from the study of cell lines derived from solid G1 phase progression in nontransformed cells, but is apparently dis tumors. These cells exhibit a variety of phenotypic changes when pensable for progression in many transformed cells, including large grown in culture, including the loss of contact inhibition and anchor T-antigen-transformed human fibroblasts (12). Indeed, many tumor age dependence and the ability to grow under low-serum conditions. cell lines show reduced expression of cyclin Dl protein when corn Whereas normal human cells have a limited proliferative capacity and pared to normal human diploid fibroblasts (12â€”16).This reduction in irreversibly withdraw from the cell cycle over time, most cells derived cyclin Dl protein levels has been correlated with inhibition of the from human cancers are immortal. Immortalization is thought to be a retinoblastoma (Rb) gene product by viral tumor antigen binding or critical step in the transformation process because it allows for con mutational inactivation (13â€”15,17). In addition to the alteration in tinued evolution and selection of more advanced neoplastic traits cyclin Dl expression, protein complexes containing cyclin Dl, the within the tumor cell population. Insight into the processes leading to PCNA,3 and Wafi protein (also Cipi, p21, Sdil, and cap20) (18â€”23) the immortalization of cancer cells is confounded by the difficulty in are disrupted in human fibroblasts transformed by the SV4O large T delineating the biochemical changes that cause immortalization from antigen (18). Similar changes were also observed in human papilloma alterations that are responsible for other neoplastic characteristics. virus-transformed HeLa cells and in immortal Li-Fraumeni fibro Because of the rarity of spontaneous immortalization of normal hu blasts, which have mutations in both p53 alleles (18). In normal cells, man cells in culture, model systems that utilize viral tumor antigens to these factors associate to form quaternary complexes (24), but the drive cellular transformation have been developed to study this proc functional significance of their association is not entirely clear. It has ess. These include the E6 and E7 viral oncoproteins of the human been proposed that the dissociation of these cyclin complexes in the papilloma virus 16 (1) and the SV4O large tumor antigen (reviewed in transformed cells results in deregulated cdk kinase activities (18). This Ref. 2). idea is supported by the observation that the catalytic activities of the Viral oncoproteins, including the SV4O large tumor antigen, en cdk2 and cdk4 kinase complexes are regulated by the Wafi protein hance the rate at which cells become immortal through a process that (21, 24). The inhibition of cdk activity by Wafi is proposed to be an requires the inactivation of host tumor suppressor gene products and important component of the p53-dependent G1 arrest that is induced in response to DNA damage (25, 26). Received 4/7/95; accepted 8/16/95. The observation that both the expression and composition of cyclin/ The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with cdk complexes is altered in both spontaneous and viral tumor antigen 18 U.S.C. Section 1734 solely to indicate this fact. transformed cell lines suggests that the appearance of these changes 1 This work was supported by Department of Energy Research Grant KPO4-02. 2 To whom correspondence should be addressed at Mail Stop M880, Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545. 3 The abbreviation used is: PCNA, proliferating cell nuclear antigen. 4651

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1995 American Association for Cancer Research. SV4O TAG IMMORTALIZATION AND ALTERED CYCLIN Dl may coincide with important steps in the transformation process. cyclin A (Ab-1), anti-Rb (Ab-4), anti-SV4O large T antigen (Ab-2), and However, the role these changes play in contributing to the acquisition anti-Rb mAb (Ab-1) and anti-5V40 large T-antigen (Ab-2)-conjugated agarose of neoplastic phenotypes is not known because they have been ana beads were obtained from Oncogene Science. Monoclonal and polyclonal lyzed by comparing normal cells to fully transformed cell lines. antibodies anti-cyclin E (C-19), polyclonal anti-cyclin A (C-22), cdk2 (M2), Whether these changes are a direct consequence of viral tumor antigen and cdk4 (C-22), and anti-p53 mAb conjugated agarose beads (DO-i) were obtained from Santa Cruz Biotechnology. Polyclonal anti-cdk4 antibodies expression or occur as a consequence of subsequent genetic changes were obtained from Upstate Biotechnology. induced during the transformation process has not been determined. To address these questions, we have investigated the changes in the expression and subunit association of G1- and S-phase cyclin and cdk RESULTS complexes in human cell populations representing intermediate stages in the SV4O large T-antigen transformation pathway. We find that the Reduction of Cyclin Dl Expression and Dissociation of Cydlin expression of cyclin Dl is reduced in immortalized, but not in pre Dl Complexes in SV4O Large T-Antigen-immortalized Human crisis cells expressing large T antigen, and that upon immortalization, Fibroblasts. In human fibroblasts transformed by the SV4O large T the association of both PCNA and Wafi with the remaining cyclin Dl antigen, the composition of complexes containing cyclin Dl, cdk4, protein is inhibited. In contrast, the association of cdk4 with Wall, PCNA, and Wall is altered (18). We were interested in determining PCNA, and cyclin Dl is disrupted in both precrisis and immortal cells whether these changes are a direct consequence of large T expression expressing SV4O large T antigen and is accompanied by increased or occur as a result of subsequent changes to the cells as they become levels of cyclin A and E protein. Surprisingly, large T antigen did not increasingly neoplastic. To test this, we measured the association of influence the steady-state level of Wafi protein, which was found in PCNA with cyclin Dl in cell populations representing various stages association with both cdk2 and cyclin A in both the precrisis and in the SV4O large T-antigen transformation pathway. Whole cell newly immortalized cells. These data demonstrate that in human lysates were prepared from exponentially growing cultures of the fibroblasts, the association of cyclin Dl with cdk4 is directly altered human diploid fibroblast line HSF 43 and from large T-antigen by SV4O large T-antigen expression and that the genetic changes transfected cell lines; these included precrisis (2PC), newly immor associated with large T-induced immortalization of these cells may be talized (2N1), high passage (2HP), and tumorigenic (2Tl) cell types. responsible for additional changes in cyclin Dl-regulated biochemical Each of these cells were derived from the CT1O 2C lineage, which pathways. was obtained by transfection of the large T-antigen gene into the HSF43 fibroblasts (9). Cyclin Dl protein complexes were recovered from these extracts by MATERIALS AND METHODS immunoprecipitation by using a polyclonal anti-cyclin Dl antibody. Cell Culture. The human diploid fibroblastcell line HSF43 (passages The association of PCNA with cyclin Dl was measured by immuno 9â€”i1)was cultured in cx-MEMsupplemented with 10% heat-inactivated calf blot analysis of these immunoprecipitation reactions by using a mAb serum, penicillin, and streptomycin in a humidified incubator at 37Â°Cwitha against PCNA (Fig. 1A). We detected PCNA in cyclin Dl immuno 5% CO2atmosphere.SV4O largeT-antigen-expressingcells from the CF10 2C precipitation reactions performed by using extracts of normal and CT1O 12 lineages were cultured under identical conditions. These cells (HSF43) and precrisis cells (2PC). We did not detect PCNA under express the SV4O large T antigen under control of a Rous sarcoma virus identical conditions by using extracts prepared from newly immortal promoter (9). Precrisis cells from both lineages have undergone approximately ized (2N1), high passage (2HP), or tumorigenic derivatives (2T1). 75 cumulative population doublings and represent a mixed population of PCNA was not detected in immunoprecipitation reactions performed cells. The newly immortalized cells are clonally derived from the precrisis in parallel using control rabbit serum. Samples of the same immuno populations. precipitation reactions were also probed with an anti-cyclin Dl mAb Cell Extract Preparation. Whole cell lysates were prepared by suspend ing cells in lysis buffer [50 m@i Tris-HC1 (pH 7.9), 150 mM NaCl, .5% NP4O, (Fig. 1B). Similar amounts of cyclin Dl protein were recovered from 50 mM Tris-HC1 (jH 7.9), 20 mM EDTA, 20 @.tg/ml phenylmethylsulfonyl the HSF43 and 2PC reactions. In contrast, little cyclin Dl protein was fluoride, 1 p@g/ml aprotinin, 1 @g/mlleupeptin, 1 p.g/ml pepstatin A, 10 @Wml detected in the 2N1 reaction. Similar results were also obtained by soybean trypsin inhibitor, 20 mM sodium fluoride, 20 mr@if3-glycerophosphate, immunoblot analysis of whole cell lysates (data not shown). To test 0.1 mMsodium orthovanadate, and 1 mMDTT on ice for 30 min. Lysates were whether the reduced expression of cyclin Dl in these cells was then forced through a 22-gauge needle and centrifuged at 40,000 X g for 20 indicative of a general repression of 01 cyclins in the immortal cells, mm at 4Â°C.Proteinconcentration of the lysates was determined by Bradford we examined the level of cyclin E protein. Immunoprecipitation analysis and adjusted to 1 mg/mI with lysis buffer. reactions were performed with extracts prepared from the HSF43, Immunoprecipitations and Immunoblotting. Immunoprecipitationswere 2PC, and 2NI cells by using an anti-cyclin E polyclonal antibody or performed by incubating 1 ml of cell lysate with 1â€”5@xgofantibody and 30 p3 of a 1:1 slurry of protein A or protein G agarose beads (Pierce Biochemicais) an anti-cyclin A antibody as a control. Detection of cyclin E protein at 4Â°C for 60 mm. The resultant immunocomplexes were pelleted by micro was performed by immunoblot analysis of these reactions by using an centrifugation and washed 3 times with 1 ml of ice cold lysis buffer. Washed anti-cyclin E mAb. A band migrating with an electrophoretic mobility complexes were then boiled in 100 p3 of SDS-PAGE loading buffer and characteristic of cyclin E protein was detected in all three extracts analyzed by gel electrophoresis as described in the figure legends. Proteins (Fig. 1C) but was absent in the control immunoprecipitation reaction. were transferred to nitrocellulose by using a semi-dry apparatus, blocked for 30 In contrast to the cyclin Dl results, cyclin E protein was found to be mm with a solution of 5% Carnation nonfat milk in 1X TTBS [10 mM more abundant in both precrisis and newly immortalized cell extracts. Tris-HC1 (pH 7.4), 150 mM NaCl, and 0.1% Tween-20] and incubated with To determine whether the reduction in cyclin Dl protein expression 0.1â€”[email protected]/mlprimaryantibody at 4Â°Cfor1â€”16h.Blots were then washed 3 was limited to the CT1O 2C lineage of cells, a similar set of experi times with 250 ml of 1 x TTBS for 10 mm (each wash) and incubated with a ments was performed by using a second set of precrisis and newly horseradish peroxidase-conjugated secondary antibody (Amersham) at a dilu tion of 1:5000 in 1X @FBSfor 2 h at room temperature. The antibody wash immortalized cell lines from the CT1O 12 lineage (9). Inununopre step was then repeated, and immunoreactive blotted proteins were visualized cipitation reactions were performed by using extracts prepared from by chemiluminescent detection by using ECL reagents (Amersham). HSF43, Cr10 12 precrisis (12PC), and newly immortalized (12NI) Antibodies. Polyclonal and monoclonal (G124-326) anti-cyclin Dl anti cells under conditions identical to those used with the CT1O 2C cell bodies were obtained from PharMingen. The mAbs anti-PCNA (Ab-1), anti extracts. For these experiments, we also detected a decrease in the 4652

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1995 American Association for Cancer Research. SV4O TAG IMMORTALIZATION AND ALTERED CYCLIN Dl A Fig. 1. Reduced expression of cyclin Dl protein in human diploidfibroblastsimmortalizedbySV4OlargeI antigen.A, cbs HSF43 2PC 2PM 2HP 211 proteincomplexeswereimmunopredpitatedfromcellextractsby â€˜pAntIbody Dl Dl C Dl Dl C Dl Dl C Dl Dl C Dl Dl C usinga polyclonalantibodyspecificforcyclinDl (DI) or by Volume @4O40 20 40 40 204040 20 4040 20 4040 using rabbit control serum (C). Extracts were prepared from human foresldn fibroblasts (HSF 43) and from the following SV4O large T-expressing cell lines: CT1O 2C precrisis (2PC), 32k0sâ€” .@. PCNA newly immOrtalized (2N1), high passage (2HP), and tumorigenic (2T1). The indicated amounts (p3) of immunoprecipitated pro 27kDs teins were resolved by 10% SDS-PAGE and analyzed by immu noblotting with a mAb directed against PCNA. Arrow, position of the M, 36,000 PCNA protein. (B) Immunoprecipitalion reactions were performed and analyzed by using the same extracts and conditionsdescribed in A with the exception that samples were immunoblotted by using a mAb directed against cyclin Dl. Ar B C row, position of the M, 36,000 cycin Dl protem C, immunopre @@ cipitationreactionswereperformedwiththesameextractsana Cbs N$F43 2PC 2t1 Cue @! lyzed in B by using either a cydin E or A polyclonalantibodyas IPAndbody Dl C Dl C Dl C IPAnhibody C E E E a control. tmmunoprecipitated proteins were resolved by 10% $0kDs SDS-PAGEandanalyzedbyimmunoblottingwitha mAbdi 32kDeâ€” . @@@cycD1 reeled against cyclin E. Arrow, position of the cydlin E protein. @@@ 49 kOsâ€” eyeE The position of prestained molecular weight markers is indicated 27kDs inkilodaltons(kDa).

expression of cyclin Dl protein in the newly immortalized extracts noprecipitation reactions using an anti-Wafi mAb showed Waf 1 (Fig. 2A) but to a lesser degree than what was observed for the 2N1 associates with cyclin Dl complexes isolated from extracts prepared extracts. In agreement with the results using the CT1O 2C cells, from HSF43 cells and both CT1O 2C (2PC) and CT1O 12 (12PC) coimmunoprecipitation of PCNA with cyclin Dl was observed in precrisis cell lines (Fig. 4A). Coimmunoprecipitating Wafi was not extracts prepared from 12PC cells but not in the extracts prepared detected in either of the newly immortalized cell extracts. The asso from the 12N1 cells (Fig. 2B). These results were not due to a decrease ciation of Wafi with cyclin Dl in the 2N1 extracts was also reduced in PCNA expression in the immortalized cells because immunoblot under conditions that took into account the reduced expression of analysis detected equal amounts of PCNA in both the precrisis and cyclin Dl in these cells (Fig. 4B). Immunoblot analysis of cell extracts newly immortalized cell extracts (Fig. 2C). revealed that this result is not due to a reduction in Wafi protein levels Because the limited expression of cyclin Dl protein in the newly in the 2N1 cells (Fig. 4C). immortalized cells could by itself account for the limited recovery of Decreased expression of cyclin Dl has been reported previously to coimmunoprecipitating PCNA, we reassayed under conditions where occur in cells transformed by large T antigen, adenovirus E1A, and in equal amounts of cyclin Dl protein were detected in immunoprecipi several tumor-derived cell lines (12, 15, 17). In these reports, this tation reactions from both the HSF43 and 2N1 cell extracts (Fig. 3A). effect was correlated with the inactivation of the Rb gene product by We found that there was still a reduction in the amount of PCNA tumor antigen binding or mutational inactivation. However, in our protein coimmunoprecipitating with cyclin Dl from the 2NI cell experiments changes in cyclin Dl protein levels are only evident in extract (Fig. 3B). Similar results were obtained by using the CT1O 12 the cells immortalized by large T antigen. To ensure that this differ NI cells (data not shown). ence was not caused by differential expression of large T antigen in In normal human fibroblasts, the cyclin-dependent kinase regula the precrisis and immortal cell lines, a mAb specific for large T tory factor Wafi is found in association with various cyclin/cdk antigen was used to probe immobilized proteins from 2PC, 2NI, complexes in combination with PCNA (18, 23, 27). To determine if 12PC, and 12N1 cells (Fig. 5A). Similar amounts of large T antigen Wall also dissociates from cyclin Dl immunocomplexes as a function were detected in extracts prepared from both lineage's of precrisis and of immortalization, we measured the amount of Wafl coimmunopre newly immortalized cells. However, we did note that the large T cipitating with cyclin Dl. Immunoblot analysis of cyclin Dl immu antigen present in the 12PC cells appears to consist of two protein

A B Ip Antibody a-cyclln Dl IP Antibody u-cyclin Dl

C.,). 43 I2PC 12P11 Cills 43 12PC l2NI Fig. 2. Reduced expression and subunit association of cyclin Dl with PCNA in CF1O 12 newly immortalized cells. A, im munoprecipitation reactions were performed with polyclonal @ cyc Dl PCNA cyclin Dl antibody as described for Fig. IA by using extracts prepared from exponentially growing cultures of HSF43 (43) cells and Cr10 12 precrisis (J2PC) and CTIO 12 newly im mortalized (12N1) cells. Immunoprecipitated proteins were re solved by 10% SDS-PAGE and analyzed as described for Fig. 1A. Arrow, position of cyclin Dl. B, reactions identical to those used in A were resolved by SDS-PAGE and probed with a mAb C directed against PCNA. Arrow, position of PCNA. C, samples 2 12 from protein extracts used in A and B were boiled in SDS-PAGE loading buffer, resolved by 10% SDS-PAGE, transferred to CsIls 43 PC NI PC NI nitrocellulose, and immunoblotted by using a mAb directed against PCNA. Arrow, position of PCNA. Left, position of prestained molecular weight markers in thousands. @@@@ 32 â€” â€” [email protected]

27â€”

4653

A B munoprecipitation reactions performed with HSF43 cell extracts (Fig. IP Antibody u.cyclln Dl IP Antibody u.cyclln Dl 6B). Analysis of cdk4 immunoprecipitation reactions from the same Cbs 43 Cells 43 2N1 set of extracts showed Wafi was associated with cdk4 complexes 49â€” isolated from HSF43 cell extracts (Fig. 6C) but this association was not found in cdk4 immunocomplexes isolated from either of the @ 32 â€” [__] CYC Dl @.PCNA 27â€” precrisis or newly immortalized cell extracts. These results were not due to repressed cdk4 expression because immunoblot analysis of Fig. 3. Binding of PCNA to cyclin Dl is disrupted in SV4O large T-antigen-immor these extracts showed the amount of cdk4 protein in extracts prepared talized cells. A, proteins were immunoprecipitated from normal human diploid fibroblasts from both the precrisis and newly immortalized cells was similar to (HSF 43) and C'l'lO 2C newly immortalized (2N1) cell extracts by using rabbit polyclonal antibodies directed against cyclin Dl . Immunoprecipitated proteins were resolved by 10% that found in the HSF43 cell extracts (Fig. 6D). SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with a mAb Association of PCNA and Wall with Cydlin A and cdk2 Is Not directed against cyclin Dl. Four times more material was loaded for the 2Nl sample. Disrupted by SV4O Large T Antigen. To test whether the disruption Arrow, position of cyclin Dl. B, samples identical to those used in A were analyzed by immunoblotting with a mAb directed against PCNA. Arrow, position of PCNA. Left, of cyclin Dl and cdk4 complexes by large T-antigen expression is a position of prestained molecular weight markers in thousands. general effect on cyclin-dependent protein kinase complexes or is unique for the cyclin Dl pathway, we measured the association of PCNA and Wafi with both cyclin A and cdk2. Cyclin A immune populations that are distinguished by different electrophoretic mobil complexes were recovered from HSF43 cell extracts and from extracts ity's. It has been reported previously that phosphorylation of large T prepared from both lineage's of precrisis and newly immortalized antigen can alter its electrophoretic mobility in SDS-PAGE gels (28), cells by using a polyclonal anti-cyclin A antibody. In contrast to the suggesting that the relative phosphorylation state of large T antigen results obtained for the association of cyclin Dl with PCNA, cyclin may vary between the 12PC and 12N1 cells. We also examined the A/PCNA complexes were enriched in both the precrisis and newly expression of Rb protein in these cells and found that the amount of immortalized cell extracts (Fig. 7A). This result agrees with a previous Rb protein and its phosphorylation state did not vary within each cell report which showed an increased abundance of cyclin AIPCNA lineage (Fig. SB). The only striking difference we found was that the complexes in the fully transformed, tumorigenic derivative CT1O 2C amount of Rb protein immunoprecipitated from all the large T Ti (18). The amount of Wafi protein associated with cyclin A antigen-expressing cell extracts was lower than that recovered from immunocomplexes was also greater in cells expressing large T antigen the HSF43 cells. In particular, the abundance of the low mobility, (Fig. 7B). These results may be a consequence of the increased hyperphosphorylated Rb 3pecies was reduced in both the precrisis and expression of cyclin A in the precrisis and newly immortalized cells newly immortalized cells. This may be due to decreased affinity of the (Fig. 7C). immunoprecipitating antibody for the large T-antigen-complexed Rb The association of both PCNA (Fig. 8A) and Wafi (Fig. 8B) with protein. the G1-S kinase cdk2 was not significantly altered in any of the large The association of both PCNA and Wafl with various cyclin T-expressing cells. dependent protein kinase complexes has been suggested previously to correlate with functional p53 (18). In our experiments, PCNA is DISCUSSION associated with cyclin Dl complexes isolated from extracts prepared from the large T-antigen expressing precrisis cells but not from The data we present in this report shows that included among the extracts made from the newly immortalized cells. To test whether this changes associated with SV4O large T-antigen-induced cellular im was due to differences in p53 expression between the precrisis and mortalization is the alteration of the expression and subunit associa newly immortalized cells, we measured the p53 protein extracts from both of these cells by immunoblotting with an anti-p53 mAb (Fig. A SC). Similar amounts of p53 were found in each precrisis and newly IP Antibody a-.cycllnDl immortalized cell population. In addition, the amount of large T Cells 43 2PC 2N1 4312PC12N1 antigen bound to p53 in each of the precrisis and newly immortalized 32â€” cell extracts was equivalent (Fig. SD). The difference in the abun 27â€” @ .-.â€”â€”â€”.â€”â€” ______Wet-i dance of p53 in the large T-antigen-producing cells is likely due to the 18 â€” association of large T antigen with p53 protein. Dissociation of cdk4 Complexes Occurs as a Direct Conse quence of Large T-Antigen Expression in Human Fibroblasts. The cyclin-dependent protein kinase cdk4 can be isolated as part of a B C multiprotein complex that includes cyclin Dl, PCNA, and Wafi/Cip Ip AntIbOdy a-cycftn Di Cells 43 2PC 211 (18, 23, 27). In viral tumor antigen-transformed cells, these complexes Cbs 43 2N1 32â€” @ are disrupted and cdk4 associates with the putative tumor suppressor 32â€” 27â€” @@@ protein p16 (18, 29). On the basis of our observation that cyclin Dl 27 â€” â€˜.0@Wall @@ dissociates from PCNA and Wafl as a function of large T-antigen Wall 18 @ 18â€” induced immortalization, we predicted that the association of cdk4 with cyclin Dl, PCNA, and Wafi would follow the same trend. To Fig. 4. Binding of Wafi to cyclin Dl is disrupted in large T-antigen immortalized fibroblasts. A, immunoprecipitation reactions were performed as described in Fig. IA by test this, we immunoprecipitated cdk4 complexes from extracts pre using anti-cyclin DI polyclonal serum. Immunoprecipitated proteins were resolved by pared from HSF43 cells and from both of the lineages of CflO 2C and 12.5% SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with a cr10 12 precrisis and newly immortalized cells by using a polyclonal mAb directed against Wafi. Arrow, position of WafI protein. B, cyclin Dl immunopre cipitation samples described in Fig. 3A were resolved by 12.5% SDS-PAGE, transferred anti-cdk4 antibody. Cyclin Dl coimmunoprecipitated with cdk4 corn to nitrocellulose, and analyzed by immunoblotting with a mAb directed against Wafi. plexes isolated from extracts prepared from HSF43 cells but not from Arrow, position of Wafl. C, immunoblot analysis of Wafi was performed as described in Fig. 2C with the exception that cellular protein extracts were resolved by 12.5% SDS cell extracts prepared from the precrisis or newly immortalized cells PAGEandanalyzedbyimmunoblottingwithananti-WafimAb.Arrow,positionofWafi (Fig. 6A). Similarly, PCNA was only recovered from anti-cdk4 im protein. Left, position of prestained molecular weight markers in thousands. 4654

A B 2 12 Ip Antibody a-Rb Cells 43 PC NI PC NI 2 12 Cells 43 PC NI PC NI 112â€” @ 79.5 â€” â€” â€” â€” @.Large-I 117â€” @ 105â€” @- â€” 80â€”

C D 2 12 IP AntIbody a-p53

Cells 43 PC NI PC NI 2 12 Cells 43 PC NI PC NI 79.5â€” @;@â€˜r@ -. @@ 105â€” ;@ @ . Large-I @ 49â€” @@-p53 @T @:@:.

Fig. 5. Immunoblot analysis of large T-antigen and Rb protein expression. A, protein extracts prepared from exponentially growing cultures of HSF43 (43) cells, CTI() 2C precrisis (2PC), and newly immortalized (2N1) cells, and CF1O12 precrisis(J2PC) and newly immortalized (/2N1) cells were boiled in SDS-PAGE loadingbuffer, resolved by 8% SDS-PAGE. transferred to nitrocellulose, and immunoblotted by using a mAb directed against SV4O large T antigen. Five ,.@gof total protein were run for each sample. Arrow, position of large T antigen.B,immunoprecipitationreactionswereperformedbyusinganti-RbproteinmAbbeadsandthecellextractsdescribedinA.Immunoprecipitatedproteinswereresolvedby 6.5%SDS-PAGE,transferredtonitrocellulose,andanalyzedby immunoblottingwitha mAbdirectedagainstthe Rb protein.Arrows,positionsof the hypophosphorylatedand hyperphosphorylated forms of the Rb protein. C, 20 p@gof total protein from HSF43 cell lysates and 2 @xgfromC110 2C and CTIO 12 precrisis and newly immortalized cell lysates were resolved by 8% SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with a mAb anti-p53. Arrow, position of p53 protein. D, immunoprecipitation reactions were performed as described in B by using anti-p53 agarose beads. Immunoprecipitated proteins were resolved by 8% SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with a mAb directed against SV4O large T antigen. Arrow, position of large T protein. Left, position of prestained molecular weight markers in thousands.

tion of the G1 regulatory protein cyclin Dl . These changes are distinct from subsequent genetic changes associated with the acquisition of from the direct phenotypic effects of large T-antigen expression, neoplastic phenotypes. which include the disruption of cdk4 kinase complexes and increased The disruption of quaternary complexes composed of cyclin Dl, expression of both cyclin A and E proteins. These results both cdk4, PCNA, and Wafi was previously proposed to result from the compliment and extend previous work by others demonstrating sub- inactivation of the p53 protein by tumor antigen binding or mutational unit rearrangements of cyclin-dependent kinase complexes in human inactivation (18). Our data are not consistent with this hypothesis fibroblasts transformed by large T antigen (18). In particular, by because cyclin Dl is associated with both PCNA and Wafi in the measuring these parameters at both early and advanced steps in the large T-antigen-expressing precrisis cells. This suggests that p53 large T-antigen transformation pathway, we have been able to differ- inactivation by large T antigen binding does not directly induce the entiate changes brought about directly by large T-antigen expression dissociation of these complexes. However, we do note that expression

A P Antibody a-cdk4 IP Antibody a-cdk4

CellsB432PC 2NI4312PC12NII Cells 43 2PC 2NI 43 12PC12NI @@@ :@:@DlD32â€” -*PCNA 27â€”

C Ip Antibody a-cdk4 2 12

Cells 43 2PC 2NI 4312PC12N1 Cells 43 PC NI PC Ni 32 â€” @@@ 27 â€” 32 â€” cdk4

@@ â€”, Waf.1 27â€” Fig. 6. Binding of cdk4 to cyclin Dl and PCNA is disrupted in both precrisis and newly immortalized cells expressing SV4Olarge T antigen. A, immunoprecipitation reactions were performed by using a polyclonal anti-cdk4 antibody and extracts prepared from exponentially growing cultures of HSF43 (43) cells, CTIO 2C precrisis (2PC), and newly immortalized (2N1) cells and CT1O 12 precrisis (J2PC) and newly immortalized (12N1) cells. Immunoprecipitated proteins were resolved by 10% SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with a mAb directed against cyclin Dl. Arrow, position of cyclin Dl protein. B, samples from the same immunoprecipitation reactions analyzed in A were resolved by 10% SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with a mAb directed against PCNA. Arrow, position of PCNA protein. C, immunopre cipitation reaction samples prepared as described in A were resolved by 12.5% SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with a mAb directed against Waft. Arrow, position of Wafi protein. D, immunoblot analysis was performed as described in Fig. 2C and analyzed by immunoblotting with anti-cdk4 polyclonal serum. Arrow, position of cdk4 protein. Left, position of prestained molecular weight markers in thousands. 4655

A B

Ip Antibody a-cyclin A IPAntibody a-cyclin A 2 12 2 12 Cells 43 PC NI PC NI Cells 43 PC NI PC NI

45â€” 27â€”

@@ . â€” .@. PCNA 31 â€” Wall 18 â€”

C 2 12 Cells 43 PC NI PC NI

80â€”

@@ 49 â€” @.cycA

Fig. 7. Analysis of PCNA and Wafi association with cyclin A complexes in precrisis and newly immortalized cells. A, immunoprecipitation reactions were performed by using anti-cyclin A polyclonal antibody and extracts prepared from HSF43 (43), 2PC, 2N!, J2PC, and 12N1 cells. Immunoprecipitated proteins were resolved by 12.5% SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with a mAb directed against PCNA. Arrow, position of PCNA protein. B, the same samples analyzed in A were immunoblotted with an anti-WafI mAb. Arrow, position of Wafl protein. C, immunoblot analysis of cell lysates used in A and B was performed as described in Fig. 2C with the exception that cell extract proteins were resolved by 10% SDS-PAGE and analyzed by immunoblotting with an anti-cyclin A mAb. Arrow, position of cyclin A protein. Left, position of prestained molecular weight markers in thousands.

of large T antigen does appear to affect the dissociation of cdk4 from protein expression. Only the large T-antigen-immortalized cells, de cyclin Dl, PCNA, and Wafi. This effect appears to be specific rived from these same precrisis cell populations, showed a reduction because association of cdk2 with PCNA or Wafi was not similarly in cyclin D 1 protein expression. One interpretation of our data is that affected. The disruption of the cdk4 complexes occurs in parallel with the loss of functional Rb protein by tumor antigen binding or muta the increased expression of cyclin A and E proteins, both of which tional inactivation may predispose cells to a particular immortaliza regulate late 01 and S progression (30â€”33).Induction of cyclin A tion pathway that ultimately results in the inhibition of cyclin Dl protein by viral tumor antigens has been described previously (13, 34, protein expression. It is also possible that in the precrisis cells, cyclin 35), and increased expression of both cyclin A and E has been found Dl expression may be activated through Rb-independent mechanisms in breast cancer cells (36â€”38). Overexpression of these proteins, that mask the direct effects of large T antigen on the Rb-dependent which are normally expressed subsequent to the appearance of both cyclin Dl transcriptional pathway. Silencing of these Rb-independent cyclin Dl and cdk4 proteins, may be responsible for the effect on cdk4 pathways on immortalization could then result in the observed reduc complex equilibrium, perhaps via the induction of a regulatory cas tion in cyclin Dl expression in the newly immortalized cells. cade that feeds back to suppress cdk4 activity. One possible mecha In addition to the down-regulation of cyclin Dl, we also find that nism for this could involve the induction of the cdk4 inhibitor protein the proportion of cyclin Dl protein bound to both PCNA and Waf 1/ p16, which is expressed at higher levels in tumor virus-transformed Cip in the newly immortalized cells is reduced. The combined effect cells and cells lacking a functional Rb protein (39) and inhibits the of reducing cyclin Dl expression and the inhibition of its association association of cdk4 with cyclin Dl (29). with cdk4, PCNA, and Wafi suggest that the large T-antigen-immor There is now a large body of evidence that links the deregulation of talized cells have developed the capacity to proliferate independently cyclin Dl protein expression to a variety of neoplastic cell types. The of cyclin Di-regulated pathways. This hypothesis is supported by the observed reduction of cyclin Dl protein expression in viral oncopro observation that normal human and rodent cells electroporated with tein-transformed cells has been proposed to be a direct result of Rb cyclin Dl antibodies arrest in G1 (15, 40), whereas transformed cells inactivation (13â€”15,17). However, our observation that the precrisis that suppress cyclin Dl expression, including those transformed by cells express cyclin Dl protein at levels comparable to the parental viral tumor antigens, are unaffected by the same treatment (15). human fibroblasts suggests that the association of SV4O large T Bypassing the requirement for cyclin Dl expression for G@traverse antigen with the Rb protein by itself is not sufficient to alter cyclin Dl may allow for subsequent changes that reduce the expression of cyclin

A B IPAntibody a-Cdk2 IPAntibody a-cdk2 2C 12 2C 12 Cills 43 PC NI PC NI Cells 43 PC NI PC NI

49 â€” 27â€” @@@@ 32 â€” @.p@NA @ 18â€” @-Wall

Fig. 8. Association ofcdk2 with both PCNA and WafI is not affected by SV4O large T-antigen expression in precrisis and newly immortalized cells. A, immunoprecipitation reactions were performed as described in Fig. 7A by using an anti-cdk2 polyclonal antibody. Samples were analyzed as described in Fig. 7A by using anti-PCNA mAb. Arrow, position of PCNA protein. B. immunoprecipitation reactions were performed and analyzed as described in A with the exception that they were immunoblotted using an anti-Wafi mAb. Arrow, position of Wafi protein. Left, position of prestained molecular weight markers is indicated in thousands.

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Dl protein. This may allow the cells to further circumvent cell cycle 17. Muller, H., Lukas, J., Schneider, A., Warthoe, P., Bartek, J., and Eilers, M. Cyclin Dl checkpoints mediated through cyclin Dl. An example of this may be expression is regulated by the retinoblastoma protein. Proc. NatI. Acad. Sci. USA, 91: 2945â€”2949,1994. the recent work showing that cyclin Dl may act to prevent DNA 18. Xiong, Y., Zhang, H., and Beach, D. Subunit rearrangement of the cyclin dependent synthesis and serve as a part of a cell cycle checkpoint in normal kinases is associated with cellular transformation. Genes & Dcv., 7: 1572â€”1583, 1993. human fibroblasts by inhibiting the activity of PCNA in S phase (41). 19. El-Diery, W. S., Tokino, T., Velculescu, V. E., Levy. D. B., Parsons, R., Trent, J. M., Additionally, the expression of cyclin Dl protein is enhanced in Lin, D., Mercer, W. E., Kinzler, K. W., and Vogelstein, B. WAFI. a potential senescent cells, leading to the accumulation of inactive cdk complexes mediator of p53 tumor suppression. Cell, 75: 817â€”825, 1993. 20. Gu, Y., Turck, C. W., and Morgan, D. 0. Inhibition of CDK2 activity in vivo by an (42, 43). Taken together, these data suggest that in addition to its role associated 20K regulatory subunit. Nature (Lond.), 366: 707â€”710,1993. as an activator of G1 progression, cyclin Dl may also serve under 21. Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., and Elledge, S. J. The p21 some circumstances to inhibit cell cycle progression. Thus, immor Cdk-interacting protein Cipi is a potent inhibitor of Gl cyclin-dependent kinases. Cell,75:805â€”816,1993. talization of human fibroblasts by large T antigen may be linked to the 22. Noda, A., Ning, Y., Venable, S. F., Pereira-Smith, 0. M., and Smith, J. R. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1995 American Association for Cancer Research. Immortalization of Human Fibroblasts by SV40 Large T Antigen Results in the Reduction of Cyclin D1 Expression and Subunit Association with Proliferating Cell Nuclear Antigen and Wafl

Scott R. Peterson, Donna M. Gadbois, E. Morton Bradbury, et al.

Cancer Res 1995;55:4651-4657.

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