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Cell Growth & Differentiation Vol. 10, 413-422, June 1999
© 1999 American Association for Cancer Research

Bovine Papillomavirus E2 Protein Activates a Complex Growth-inhibitory Program in p53-negative HT-3 Cervical Carcinoma Cells that Includes Repression of Cyclin A and cdc25A Phosphatase Genes and Accumulation of Hypophosphorylated Retinoblastoma Protein1

Lisa K. Naeger2,, 3, Edward C. Goodwin2, Eun-Seong Hwang4, Rosa Anna DeFilippis, Hui Zhang and Daniel DiMaio5

Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The bovine papillomavirus E2 protein can inhibit the proliferation of HT-3 cells, a p53-negative cervical carcinoma cell line containing integrated human papillomavirus type 30 DNA. Here, we analyzed HT-3 cells to explore the mechanism of p53-independent E2-mediated growth inhibition. Expression of the E2 protein repressed expression of the endogenous human papillomavirus type 30 E6/E7 genes. This was accompanied by hypophosphorylation and increased accumulation of p105Rb and repression of E2F1 expression. The E2 protein also caused reduced cyclin-dependent kinase (cdk) 2 activity, but this did not appear to be due to increased expression of cdk inhibitors. Rather, expression of cyclin A, which regulates cdk2 activity, and the cdc25A and cdc25B phosphatases, which are thought to activate cdk2, was significantly reduced at both the RNA and protein levels in response to E2 expression. The E2 protein reduced expression of cdc25A and cdc25B in both HT-3 and HeLa cells, but not in cells that were not growth-inhibited by the E2 protein. E2 point mutants unable to inhibit cell growth did not repress cdc25A and cdc25B expression, nor did the cell cycle inhibitors hydroxyurea and mimosine. Based on these results and the known properties of cell cycle components, we propose a model to account for E2-induced growth inhibition of cervical carcinoma cell lines.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The HPVs6 play an important role in the development of anogenital carcinomas by influencing the expression or activity of cellular proteins that regulate the cell cycle machinery. The major transforming proteins of the high-risk HPV types are the E6 and E7 proteins (for review, see Ref. 1 ). The E7 protein binds and inactivates p105Rb and other members of the Rb family of tumor suppressor proteins. Many of the growth-regulatory effects of the Rb family are mediated by the E2F family of transcription factors, which can both repress and activate the transcription of genes required for cell cycle progression (2, 3, 4, 5, 6, 7, 8, 9, 10) . Free E2F/DP heterodimers can activate transcription from promoters containing E2F sites, whereas complexes of E2F and hypophosphorylated Rb family members actively repress transcription by binding to E2F sites. Phosphorylation of Rb family members by cdks disrupts repressing complexes and promotes release of E2F family members, thereby permitting the G1-S-phase transition and transit through S phase. The HPV E6 protein binds to p53 and accelerates its ubiquitin-mediated degradation (1) . p53 modulates Rb activity because it can induce the expression of p21WAF1/CDI1, which binds to cdks and inhibits their activity (11 , 12) . cdks are also regulated by specific phosphorylation and dephosphorylation events and by association with cyclin-regulatory subunits (7) . For example, cdk2 activation around the G1-S-phase boundary requires complex formation with cyclin E and cyclin A. In addition, full activity of cdk2 requires phosphorylation at threonine 160 by CAK and dephosphorylation at residues threonine 14 and tyrosine 15 (7) . In vitro, cdc25A phosphatase can remove these inhibitory phosphates and activate cdk2 (13, 14, 15, 16, 17, 18) , but the in vivo role of cdc25A is not clear (19) .

Expression of the HPV E6 and E7 proteins is regulated by the viral E2 protein, a multifunctional, site-specific DNA-binding protein. Papillomavirus E2 proteins can repress transcription of the promoter that controls expression of the E6 and E7 genes of the high-risk HPV types (20 , 21) . Furthermore, the HPV E2 gene is often disrupted in cervical carcinomas by viral DNA integration, suggesting that loss of E2 expression may facilitate carcinogenic progression (1) . These findings prompted us to test the effect of introducing the wild-type E2 protein into cervical carcinoma cells. We and others have shown that expression of the BPV or HPV16 E2 protein exerts a significant growth-inhibitory effect in certain cell lines (22, 23, 24, 25, 26) . In our experiments, we have used a recombinant SV40-based viral vector to express the BPV E2 protein in a controlled fashion in cervical carcinoma cell lines (22 , 27) . Acute expression of the E2 protein in HeLa cells, which contain HPV18 DNA and wild-type p53 and p105Rb, caused up to a 99% inhibition of DNA synthesis and blocked entry into the S phase of the cell cycle (22) . In these cells, the E2 protein repressed the expression of the viral E6 and E7 oncogenes (22 , 23 , 25) , an effect that probably contributed to cell growth suppression by restoring the p53 and Rb function. In accord with this view, the E2 protein activated a p53/p21 growth-inhibitory pathway in HeLa cells that led to cdk inhibition and accumulation of p105Rb in its hypophosphorylated growth-inhibitory form (23 , 25 , 27) . Another group reported that transfection of the BPV or HPV18 E2 gene into HeLa cells caused apoptosis as well as growth arrest, possibly because of the higher level of E2 expression attained after transfection (25) . In those experiments, expression of a dominant negative p53 mutant blocked the growth arrest induced by the transfected E2 gene.

Although the experiments summarized above suggested that activation of the p53 pathway played an important role in E2-induced growth inhibition, E2 expression also induced substantial growth inhibition in HT-3 cells, a cervical carcinoma cell line that contains a dominant negative p53 gene and a mutant p105Rb allele (22 , 28, 29, 30) . HT-3 cells were initially classified as HPV DNA negative (31 , 32) , suggesting that E2-induced growth inhibition in HT-3 cells may proceed in a HPV-independent fashion that does not involve restoration of tumor suppressor activity. However, recent re-evaluation of HT-3 cells revealed that they harbor HPV30 DNA, which has also been detected in some primary anogenital carcinomas (33 , 34) .7

Here, we have characterized HT-3 cells to identify potential p53-independent growth-inhibitory signals mobilized by acute expression of the BPV E2 protein. Expression of the E2 protein in HT-3 cells caused repression of HPV30 E6/E7 expression and accumulation of hypophosphorylated p105Rb. Although cdk activity was reduced in HT-3 cells expressing the E2 protein, cdk inhibitors were not induced in these cells. Rather, E2 expression caused dramatic inhibition of the expression of cyclin A and the cdk-activating phosphatases cdc25A and cdc25B. On the basis of these results and known cell cycle-regulatory circuits, we propose that E2 expression mobilizes multiple growth-inhibitory signals that converge on Rb family members in both p53-positive and p53-negative cervical carcinoma cell lines.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Growth Inhibition by the BPV E2 Gene.
The BPV E2 gene was delivered acutely into the HT-3 human cervical carcinoma cell line by infection with a BPV/SV40 recombinant virus (22) . This method introduced the E2 gene into the vast majority of HT-3 cells, enabling us to carry out physiological and biochemical analysis of the infected cells. [3H]Thymidine incorporation assays performed in parallel with the experiments reported here documented that the E2 protein typically caused a 70–85% inhibition of DNA synthesis in HT-3 cells within 60 h of infection, and the arrested cells displayed predominantly G0-G1 DNA content as assessed by fluorescence-activated cell sorting (Ref. 22 ; data not shown). A mutant virus containing a nonsense mutation in the E2 gene caused no significant growth inhibition and was used as a negative control.

Repression of HPV30 E6/E7 Expression by the BPV E2 Protein.
To determine whether the BPV E2 protein repressed expression of the endogenous HPV30 DNA in HT-3 cells, RNA was isolated from cells 57 h after mock infection, infection with a virus expressing the wild-type E2 protein, or infection with viruses expressing a panel of transactivation-defective E2 mutants with single amino acid substitutions in the E2 transactivation domain (35) . The RNA was subjected to Northern blot analysis with a probe consisting of the E6/E7 region of HPV30 DNA (Fig. 1)Citation . Mock-infected cells and cells infected with the E2 amber mutant contained a major E6/E7 RNA that migrated at approximately 2200 bases. Expression of the wild-type E2 protein caused a dramatic, dose-dependent reduction in the level of E6/E7 RNA. At a high MOI, the level of E6/E7 RNA was reduced more than 30-fold. In contrast, a variety of transactivation-defective E2 point mutants that were defective for inducing growth arrest in HT-3 cells were also markedly impaired in their ability to repress E6/E7 expression. The two mutants F87S and E105G, which retained low-level residual transactivation and growth-inhibitory activity (35) , also retained a partial ability to repress E6/E7 expression. These results demonstrated that HT-3 cells expressed HPV30 E6/E7 RNA and that E2 expression caused profound repression of this expression. Furthermore, the ability of various E2 mutants to repress E6/E7 expression displayed an absolute correlation with their growth-inhibitory activity.



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Fig. 1. HPV30 E6/E7 repression by the E2 protein. RNA was purified from mock-infected HT-3 cells or cells infected with a virus expressing the wild-type (wt) E2 gene at a MOI of 5 or 20 or the indicated E2 mutants at a MOI of 20. HPV30 E6/E7 RNA was detected by Northern blotting. E2amber expresses a mutant E2 gene containing a premature translation termination codon. For the other mutants, the first letter and the number in the mutant designation identifies the wild-type amino acid at that position in the E2 protein, and the last letter identifies the substituted amino acid (35) . +, 5–6-fold decrease in DNA synthesis; +/-, 3-fold decrease; -, <2-fold decrease compared to mock-infected cells.

 
Effects of E2 Expression on p105Rb and E2F1 Expression.
Because HT-3 cells do not contain wild-type p53 and therefore cannot mount a p53-mediated growth-inhibitory response (Refs. 29 and 30 ; see below), we examined the status of p105Rb, the prototype of the other tumor suppressor family targeted by the high-risk HPV oncogenes. HT-3 cells contain a mutant form of the Rb1 gene, which is predicted to result in the synthesis of a truncated version of p105Rb (30) . A wild-type Rb1 cDNA was also cloned from HT-3 cells, a finding that was initially interpreted as indicating heterogeneity of the initial cell population (30) . We analyzed the expression of p105Rb and its state of phosphorylation in HT-3 cells by immunoblotting with an anti-p105Rb antibody (Fig. 2A)Citation . Extracts prepared from HT-3 cells contained a rapidly migrating species (designated {Delta}Rb), which is not expressed by HeLa cells and was presumably the truncated form of p105Rb encoded by the mutant allele. The other protein species recognized by the p105Rb antibody in mock-infected HT-3 cells comigrated with the forms present in mock-infected HeLa cells: (a) a discrete band that corresponded to hypophosphorylated full-length wild-type p105Rb (designated Rb); and (b) a more slowly migrating smear that corresponded to hyperphosphorylated p105Rb (designated ppRb). Phosphatase treatment confirmed that the slowly migrating smear was phosphorylated p105Rb (data not shown). This same pattern was observed in 10 of 10 independently derived single cell clones of HT-3 cells (data not shown), indicating that HT-3 cells are heterozygous for wild-type and mutant alleles of the Rb1 gene.



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Fig. 2. Effect of the E2 protein on p105Rb expression and phosphorylation. A, left panel, whole cell extracts from mock-infected HeLa or HT-3 cells and cells infected with a virus expressing the wild-type E2 protein or the indicated E2 mutants were prepared, electrophoresed, and immunoblotted with an antibody against p105Rb. The hyperphosphorylated form of full-length p105Rb is indicated by the designation ppRb, the hypophosphorylated form is indicated by Rb, and the truncated form of p105Rb found in HT-3 cells is indicated by {Delta}Rb. Right panel, total cellular RNA was prepared from mock-infected HT-3 cells or cells infected with a virus expressing the wild-type E2 protein (E2) or the amber mutant (E2amber). Rb1 RNA was detected by Northern blotting. B, extracts from mock-infected HT-3 cells or cells infected with virus expressing the wild-type E2 protein or the indicated mutants were immunoprecipitated with an E2 monoclonal antibody, electrophoresed, and blotted with the same antibody. The full-length E2 protein is indicated.

 
Expression of the E2 protein had no effect on the amount or migration of the deleted version of p105Rb protein. In contrast, E2 expression converted the slowly migrating hyperphosphorylated form of full-length p105Rb into the hypophosphorylated form, and the overall levels of p105Rb were increased (Fig. 2ACitation , left panel). Northern blot analysis demonstrated that expression of the E2 gene had no significant effect on the level of Rb1 RNA in HT-3 cells (Fig. 2ACitation , right panel). Increased accumulation of hypophosphorylated p105Rb was not observed in HT-3 cells infected with viruses expressing the E2 nonsense mutant or either of two representative growth inhibition-defective E2 mutants with amino acid substitutions in the transactivation (E39G) or DNA binding (K339M) domain. Western blot analysis confirmed that these mutant E2 proteins accumulated in infected HT-3 cells (Fig. 2B)Citation . Thus, expression of the wild-type but not defective E2 proteins caused marked accumulation of the hypophosphorylated, growth-inhibitory form of p105Rb in HT-3 cells.

Expression of E2F1 RNA and protein is markedly inhibited in HeLa cells expressing the BPV E2 protein (27) . We proposed that this was due to titration of E2F family members by hypophosphorylated p105Rb, leading to repression of the E2F1 gene. Experiments were carried out to test whether the E2 protein also inhibited E2F1 expression in HT-3 cells. As shown in Fig. 3Citation , expression of the wild-type E2 protein caused a significant reduction in the amount of E2F1 and E2F1 RNA, whereas a DNA binding-defective E2 mutant that does not cause growth inhibition did not affect E2F1 expression. Therefore, E2 expression and the accumulation of hypophosphorylated p105Rb were accompanied by repression of E2F1 expression in HT-3 cells as well as in HeLa cells. Furthermore, expression of E2F-responsive genes including B-myb (data not shown) and cyclin A (see below) was also inhibited by E2 expression. Thus, the E2 protein appeared to activate the Rb growth-inhibitory pathway in HT-3 cells.



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Fig. 3. Effect of the E2 protein on E2F1 expression. Left panel, HT-3 cells were mock-infected or infected with a virus expressing the wild-type E2 protein. Whole cell extracts from these cells were analyzed by immunoblotting using an anti-E2F1 antibody. Right panel, total RNA was isolated from mock-infected HT-3 cells and cells infected with viruses expressing the wild-type E2 protein or the E2 DNA binding mutant C340R. RNA was examined by Northern blotting by using a labeled E2F1 cDNA fragment as a probe.

 
In vitro cdk Activity in HT-3 Cells Expressing the E2 Protein.
To test whether the E2-induced reduction in hypophosphorylated p105Rb in HT-3 cells was due to reduced cdk activity, kinase complexes were immunoprecipitated from cell extracts by using antibody recognizing either cdk2, cyclin E, or cyclin A and assayed for their ability to phosphorylate the exogenous substrate histone H1 in vitro. Matched nonimmune antibodies were used as controls. As shown in Fig. 4Citation , expression of the E2 protein caused a marked reduction in the kinase activity of total cdk2 complexes and of cyclin A complexes compared to complexes isolated from mock-infected cells or cells expressing a mutant E2 protein. The extent of inhibition of cdk2 activity was approximately 5-fold, as quantitated by PhosphorImager. In contrast, the E2 protein did not exert a significant effect on cyclin E-associated kinase activity. We next carried out experiments to explore the basis for the reduced cdk activity in HT-3 cells expressing the E2 protein.



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Fig. 4. cdk activity in HT-3 cells expressing the E2 protein. Whole cell extracts were prepared from mock-infected, E2 virus-infected, or E2 amber virus-infected HT-3 cells. After immunoprecipitation of active kinase complexes using anti-cdk2, anti-cyclin A, or cyclin E antibody or immunoprecipitation with the appropriate nonimmune control antibody, in vitro cdk activity was measured using histone H1 as a substrate. Phosphorylated histone H1 is shown.

 
Expression of cdk Inhibitors.
Acute introduction of the E2 gene into HeLa cells caused a dramatic transcriptional induction of the gene encoding the cdk inhibitor p21 (27) . To determine whether p21 was induced in cells that do not express wild-type p53, extracts were prepared from metabolically labeled HT-3 cells, and p21 protein levels were examined by immunoprecipitation with an anti-p21 antibody. There was no increase in p21 protein levels upon E2 expression in HT-3 cells as compared to either mock-infected or mutant E2-infected cells, whereas E2 expression in HeLa cells processed in parallel resulted in a dramatic induction of p21 (Fig. 5A)Citation . Northern blotting demonstrated that p21 RNA levels were not affected by E2 expression in HT-3 cells (data not shown). Thus, p21 induction did not contribute to growth inhibition in these cells. The lack of p21 induction in p53-negative HT-3 cells further suggested that E2-mediated induction of p21 in HeLa cells was dependent on p53 function.



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Fig. 5. Expression of cdk inhibitors in response to the E2 protein. A, p21 levels were determined by immunoprecipitation from metabolically labeled cells after mock infection or infection with viruses expressing the wild-type or amber mutant E2 gene, as indicated. Left panel, HT-3 cells; right panel, HeLa cells. B, soluble cdk-inhibitory activity was measured by determining the ability of cellular extracts to inhibit the histone H1 kinase activity of active cdk2/cyclin E complexes assembled in vitro. The indicated amount of protein (µg) from E2-infected (+) or uninfected (-) HeLa or HT-3 cell extracts was added to the cdk2/cyclin E complexes. Phosphorylated histone H1 is shown.

 
In addition to p21, cells have the potential to express a variety of proteins that inhibit cdk2 activity. To carry out a broader search for soluble cdk-inhibitory activity in E2-infected HT-3 cells, we tested the ability of cell extracts to inhibit cdk2 kinase complexes. Active kinase complexes were first assembled in vitro from human cyclin E and HA-tagged human cdk2 expressed in insect cells. Extracts from mock-infected or E2 virus-infected HeLa or HT-3 cells were then added to these complexes as a source of putative cdk inhibitor. HA-tagged cdk2 was immunoprecipitated with anti-HA antibody, and cdk2 kinase activity of the immunoprecipitate was assayed in vitro (Fig. 5B)Citation . As little as 50 µg of extract from E2-infected HeLa cells caused a significant decrease in kinase activity in comparison to the activity of complexes treated with extract from mock-infected HeLa cells. This reduction in kinase activity was presumably due to p21 that was induced in HeLa cells by E2 expression. In contrast, cdk2 kinase activity was not affected by up to 200 µg of extract prepared from E2 virus-infected HT-3 cells. Taken together, these results suggest that cdk inhibitors were not responsible for reduced cdk2 activity in HT-3 cells.

Cyclin Expression and CAK Activity.
To determine whether decreased levels of cyclin/cdk complex components contributed to the decrease in cdk2 activity in HT-3 cells, the expression of cdk2, cyclin E, and cyclin A was analyzed by immunoblotting. Upon expression of the wild-type E2 protein, there was no change in the level of cdk2 relative to mock-infected or E2 mutant virus-infected cells, and there was an approximately 2-fold increase in the level of cyclin E (Fig. 6Citation , left panel; data not shown). Because cyclin E-associated cdk kinase activity did not show a corresponding increase (Fig. 4)Citation , the specific activity of cyclin E complexes appears slightly reduced in cells expressing the E2 protein. In contrast, the level of cyclin A and cyclin A RNA was significantly reduced in E2-infected HT-3 cells compared to mock-infected and E2 mutant virus-infected cells (Fig. 6Citation , left panel). PhosphorImager analysis indicated that the E2-induced reduction in cyclin A RNA was approximately 5-fold. Because p105Rb appears to be a substrate of cyclin/cdk2 complexes (5 , 36 , 37) , reduced cyclin A expression and the consequent decreased concentration of cyclin A/cdk2 complexes are likely to contribute to the reduced cdk2 activity and reduced p105Rb phosphorylation in HT-3 cells expressing the E2 protein.



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Fig. 6. Cyclin expression and CAK activity. Left panel, equal amounts of whole cell extracts from mock-infected, E2 virus-infected, or mutant E2 amber-infected HT-3 cells were electrophoresed, and cyclin E (top) and cyclin A (middle) were detected by immunoblotting. In the bottom panel, RNA was analyzed by Northern analysis using a cyclin A cDNA fragment as a probe. Right panel, CAK was immunoprecipitated from the indicated amount of extracted protein (in µg) from uninfected (-) or E2-infected (+) HT-3 cells and incubated with inactive cdk2/cyclin E complexes. cdk was immunoprecipitated, and kinase activity was measured using histone H1 as a substrate.

 
For cyclin/cdk2 complexes to be enzymatically active, cdk2 must be phosphorylated by CAK at the threonine at position 160 (7) . Therefore, we examined E2-expressing cells for the ability of cellular CAK to stimulate the kinase activity of cdk2 expressed from baculoviruses. First, an antibody was used to immunoprecipitate CAK from mock-infected or E2 virus-infected HT-3 cells. Lysates of insect cells infected separately with baculoviruses expressing cyclin E and cdk2 were mixed and incubated with the immunoprecipitated CAK in the presence of ATP. To determine whether CAK in the immunoprecipitates activated cdk2, the cdk2 complexes were immunoprecipitated with anti-cdk2 antibody and assayed for kinase activity in vitro (Fig. 6Citation , right panel). When no CAK was added to the baculovirus lysates, minimal cdk2 kinase activity was detected. However, CAK immunoprecipitated from both mock-infected and E2 virus-infected HT-3 cell extracts activated cdk2 complexes to a similar extent. These results indicated that CAK activity in HT-3 cells was not affected by E2 expression.

cdc25A and cdc25B Phosphatase Expression in E2-infected Cervical Carcinoma Cells.
We also examined the level of the phosphatases cdc25A and cdc25B, which can remove inhibitory phosphate groups from cdk2 in vitro (13) . Immunoblot analysis revealed a marked reduction in the levels of cdc25A and cdc25B in extracts of HT-3 cells expressing the E2 protein as compared to mock-infected or mutant E2 virus-infected cells (Fig. 7)Citation . Northern blot analysis showed that cdc25A and cdc25B RNA levels were also markedly reduced in E2-infected HT-3 cells (Fig. 8ACitation ; data not shown). To determine whether E2 expression affected cdc25A expression in other cell lines, we also analyzed HeLa cells, CV1 monkey kidney epithelial cells, and A431 cells, a HPV-negative human vulvar carcinoma cell line (Fig. 8ACitation ; data not shown). As was the case in HT-3 cells, expression of HeLa cell cdc25A and cdc25A RNA was dramatically repressed by E2 expression. In contrast, E2 expression did not affect cdc25A RNA expression in A431 and CV1 cells, two cell lines not subject to E2-induced growth arrest (22) . In addition, immunoblotting demonstrated that the E2 protein repressed the expression of the cdc25A and cdc25B proteins in HeLa cells as well (Fig. 7)Citation . These results suggest that reduced cdc25A expression may also contribute to reduced cdk2 activity and reduced p105Rb phosphorylation in cells undergoing E2-induced growth inhibition.



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Fig. 7. Expression of cdc25A and cdc25B. Equal amounts of whole cell extracts of mock-infected cells or cells infected with wild-type or amber mutant E2 virus were immunoblotted to detect cdc25A (left panels) or immunoprecipitated with anti-cdc25B antibodies followed by immunoblotting to detect cdc25B (right panels). Arrows, cdc25A and cdc25B. Similar results were obtained when a different antiserum was used to detect cdc25A by the combined immunoprecipitation/immunoblot protocol. The top two panels show results obtained with HT-3 cells; the bottom panels show results obtained with HeLa cells.

 


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Fig. 8. Expression of cdc25A RNA. A, RNA was prepared from HT-3, HeLa, CV1, or A431 cells that were mock-infected or infected with a virus expressing the wild-type E2 protein (E2) or a DNA binding defective mutant E2 protein (C340R). After electrophoresis under denaturing conditions and transfer to a filter, the cdc25A message was detected by hybridization to a cdc25A cDNA clone. B, RNA was purified from mock-infected HT-3 cells or cells infected with wild-type or transactivation-defective E2 mutant virus and assayed as described in A. Two different preparations of mock-infected cells were used. The other lanes are labeled as described in the Fig. 1Citation legend.

 
To identify the functions of the E2 protein necessary for cdc25A repression and to further examine the correlation between repression and growth arrest in HT-3 and HeLa cells, we tested the effects of E2 mutants defective for inducing growth inhibition (35) . Fig. 8BCitation shows that cdc25A RNA levels were repressed in a dose-dependent fashion by infection with the virus expressing the wild-type E2 protein in HT-3 cells, whereas little if any repression was caused by the defective mutants. Similar results were obtained in E2-infected HeLa cells (data not shown). Thus, intact transactivation and DNA binding functions of the E2 protein were required for cdc25A repression. Furthermore, there was an absolute correlation between the inability of the various E2 mutants to inhibit cell growth and to repress the expression of cdc25A.

We also used chemical cell cycle inhibitors to test whether the decrease in cdc25A expression was an indirect consequence of cell cycle arrest rather than a specific effect of the E2 protein. RNA was prepared from HT-3 cells treated with the G1 inhibitor mimosine or the S-phase inhibitor hydroxyurea, under conditions that caused a >90% reduction in DNA synthesis. Northern blotting revealed that drug treatment did not cause a decrease in cdc25A RNA levels, whereas, as expected, there was a decrease in cyclin A RNA, which is known to be expressed in a cell cycle-dependent fashion (data not shown). Although the mechanism of growth inhibition by these chemical inhibitors presumably is unrelated to the mechanism of E2-induced inhibition, these results nevertheless established that growth inhibition of HT-3 cells does not inevitably result in cdc25A repression.


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
We are using the BPV E2 protein to characterize the status of cell cycle regulatory pathways in human cervical carcinoma cell lines and to identify cellular components that regulate proliferation of these cells. Our results indicate that the E2 protein can mobilize dormant growth-regulatory programs in HPV-positive cervical carcinoma cells, resulting in growth inhibition. Presumably, these malignant cells can tolerate the existence of these growth-inhibitory programs because they are normally neutralized by expression of the endogenous HPV E6 and E7 proteins. HT-3 cells contain HPV30 DNA, and the E2 protein represses HPV E6/E7 expression in these cells, as it does in HPV18-containing HeLa cells. Therefore, the growth-inhibitory effects of the E2 protein in cervical carcinoma cell lines may be solely a consequence of E6/E7 repression. Although we have not carried out an exhaustive survey of various cell lines, we have not identified HPV-negative cells that are growth-inhibited by infection with the SV40-based virus expressing the BPV E2 protein. However, we have not ruled out the possibility that the E2 protein exerts HPV-independent effects in some cells. In fact, Frattini et al. (24) have reported that expression of the HPV31b E2 protein from an adenovirus vector causes an S-phase arrest in both a HPV-positive cervical carcinoma cell line and in normal human keratinocytes.

As is the case for the high-risk genital HPV types, HPV16 and HPV18, the HPV30 regulatory region contains two E2 binding sites in close proximity to the promoter driving E6/E7 expression (33) . It is likely that the E2 protein represses HPV30 E6/E7 expression by binding to these sites, which are required for E2-induced repression of HPV16 and HPV18 E6/E7 expression (38 , 39) . Our results demonstrate that HPV30 is an additional HPV type subject to repression by the BPV E2 protein and provide a second example of a HPV genome in which the transactivation and DNA binding activities of the E2 protein are required for repression of E6/E7 expression.

We reported previously that the p53 growth-inhibitory pathway is activated by the E2 protein in HeLa cells, resulting in p21 induction that contributes to cdk inhibition and activation of the Rb growth-inhibitory pathway (27) . The presence of only transactivation-defective p53 in HT-3 cells suggested that the E2 protein could elicit p53-independent growth-inhibitory signals, and our demonstration that p21 was not induced in HT-3 cells provided biochemical evidence in support of this notion. In contrast, these cells express an apparently wild-type p105Rb protein, which accumulated in increased amounts in the growth-inhibitory hypophosphorylated form in response to E2 expression. Furthermore, the expression of some E2F-responsive genes, including E2F1 itself and cyclin A, was repressed. Thus, the Rb pathway was activated by the E2 protein in HT-3 cells, evidently generating the p53-independent growth-inhibitory signal in these cells. Growth inhibition is more profound in HeLa cells than in HT-3 cells (22) , perhaps reflecting the activation of both the p53 and the Rb pathways in HeLa cells and of only the Rb pathway in HT-3 cells.

There appear to be multiple mechanisms that account for the activation of the Rb pathway in HT-3 cells expressing the E2 protein. First, E2-induced repression of E7 expression releases active p105Rb from the E7 protein/p105Rb complex (3) . Second, E2 expression also leads to a higher steady-state level of p105Rb. This effect is posttranscriptional and may also be due to E7 repression, because the E7 protein is known to accelerate the degradation of Rb family members in a variety of cell types, including keratinocytes (40, 41, 42) . Finally, reduced cdk2 activity ensures that the p105Rb in these cells is maintained in the hypophosphorylated, growth-inhibitory state.

The E2 protein did not induce p21 or other soluble cdk2-inhibitory activity in these cells, and there was little or no effect on cdk2 expression or CAK activity. In contrast, the E2 protein repressed expression of positive regulators of cdk activity, cyclin A, cdc25A, and cdc25B. The repression of these genes in HT-3 cells indicated that repression did not require wild-type p53 function. The cyclin A gene is normally transcribed in a cell cycle-dependent fashion (36 , 43 , 44) , and the human cyclin A promoter contains multiple E2F binding sites (as well as a CHR site found in some E2F-repressible genes) and can be regulated by ectopic E2F1 expression (45, 46, 47) . Furthermore, HPV16 E7 stimulates cyclin A expression, an effect that is dependent on E2F sites in the cyclin A promoter (46) . Therefore, E2-induced repression of E7 and the resulting perturbations in p105Rb and E2F signaling may be the proximal cause of cyclin A repression in this system. On the other hand, the decreased cyclin A RNA in cells treated with the chemical cell cycle inhibitors is also consistent with cyclin A repression being a more indirect consequence of the growth arrested state. In contrast, expression of the cyclin E promoter, which contains E2F binding sites but not the CHR site, is induced by the E2 protein, thus emphasizing the complexity of gene regulation by E2F family members (47, 48, 49) . Unlike cyclin A or cyclin E, cdc25A levels do not vary markedly with the cell cycle in HeLa cells (16) . Furthermore, expression of cdc25A RNA was not reduced by mimosine or hydroxyurea at concentrations sufficient to cause substantial growth inhibition, demonstrating that E2-mediated repression of cdc25A was not an inevitable consequence of growth inhibition. There is a consensus E2 binding site and multiple E2F binding sites in the 5' flanking region of the human cdc25A gene (50) ,8 which might be involved in transcriptional control. The inability of the E2 protein to repress cdc25A expression in non-HPV cell lines suggests that the E2 binding site is not sufficient to mediate E2 repression, and it has recently been reported that transforming growth factor ß-induced repression of the cdc25A promoter is mediated by altered E2F regulation (50) . Experiments are underway to explore the mechanism of E2-mediated repression of the cdc25A gene.

Repression of cyclin A and cdc25A may play a role in E2-induced growth arrest by reducing cdk2 activity, thus maintaining hypophosphorylated p105Rb. Although cyclin A plays important roles during S phase and mitosis, it is also involved in entry into S phase (7) . Inhibition of cyclin A expression or function can inhibit the G1 to S-phase transition in a variety of cell types, including HeLa cells, whereas ectopic expression of cyclin A can accelerate entry into S phase and overcome a p105Rb-induced block to cell proliferation (5 , 36 , 37 , 51, 52, 53) . These effects are probably mediated by alterations of p105Rb phosphorylation, because cyclin A/cdk2 complexes can phosphorylate Rb family members in vitro, promote Rb phosphorylation in vivo, and relieve Rb-mediated repression of an E2F-responsive promoter (5 , 7 , 54) . Similarly, in several settings, inhibition of cdc25A function or expression results in growth inhibition. For example, inhibition of cdc25A by antibody microinjection can induce G1-S-phase growth arrest in HeLa cells (16 , 55) . In addition, repression of cdc25A expression appears to play a role in the inhibitory effects of IFN-{alpha} on the proliferation of Burkitt’s lymphoma cells and of transforming growth factorß on the proliferation of human mammary epithelial cells (56 , 57) . Conversely, ectopic expression of cdc25A can accelerate the activation of cyclin A-associated cdk activity and S-phase entry (19) . However, recent results suggest that the role of cdc25A in promoting cell cycle progression is not restricted to cdk2 dephosphorylation (19) . We are currently testing the role of cdc25A repression in E2-mediated growth inhibition by attempting to express cdc25A constitutively in the face of E2 expression.

Taken together, our results suggest the following model to explain growth inhibition by the E2 protein (Fig. 9)Citation . E2-induced elimination of HPV E7 results in increased accumulation of active Rb family members, which leads to repression of certain E2F-responsive genes required for cell cycle progression, including E2F1 itself (58) and cyclin A. This results in decreased cdk2 activity and reduced phosphorylation of Rb family members, thus reinforcing the Rb growth-inhibitory signal. In addition, the E2 protein represses cdc25A and cdc25B expression, which may also contribute to cdk inhibition and Rb hypophosphorylation. Thus, the initial Rb growth-inhibitory signal elicited by E7 repression is amplified by positive feedback loops that maintain Rb family members in the hypophosphorylated state. In cells containing an intact p53 pathway, repression of HPV E6 expression by the E2 protein induces p21 expression, which also contributes to cdk inhibition and Rb activation. In this setting, E7 repression may also remove inhibitory constraints on p21 action (59) .



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Fig. 9. Model of E2-induced growth inhibition in cervical carcinoma cell lines. It is not known whether repression of cdc25A expression is a direct effect of the E2 protein binding to the cdc25A promoter or an indirect effect mediated by E2F family members. In p53-positive cells, E6 repression results in the induction of p21 and inhibition of cdk2 activity.

 
The effects elicited by the E2 protein in cervical carcinoma cell lines may reflect events occurring during the virus life cycle. Vegetative papillomavirus DNA replication and assembly of virus particles normally take place in terminally differentiated keratinizing epithelia. Removing the growth-stimulatory effects of cdc25A and cdc25B may help drive the cells from a proliferative state toward a more differentiated state compatible with viral replication. Alternatively, viruses often encode proteins that inhibit apoptosis, which might otherwise impair virus replication (60) . In some systems, p21 has been shown to exert an antiapoptotic effect (61, 62, 63) , whereas cdc25A displays proapoptotic activity (64) . Thus, by inducing p21 in p53-positive cells and repressing cdc25A, the E2 protein may interfere with apoptosis. Unlike the antiapoptotic proteins expressed by other viruses that directly interfere with the apoptotic machinery, the E2 protein modulates the expression of cellular genes that regulate apoptosis. Although other laboratories have reported that expression of papillomavirus E2 proteins can stimulate apoptosis under some circumstances (24, 25, 26) , the BPV E2 protein does not induce apoptosis with the expression vectors and host cells used here.9

Analysis of the cell cycle targets of viral oncogenes has provided important new insights into cell cycle control. Analysis of the interaction of viral growth-inhibitory proteins such as papillomavirus E2 proteins and the cell cycle machinery can also be expected to provide new insights into virus-cell interactions and cell growth regulation. In addition, elucidating the mechanism of E2-mediated repression of the cdc25A gene may suggest new approaches for the treatment of malignancies characterized by overexpression of this gene (65 , 66) .


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cells and Virus Stocks.
HT-3 cells were maintained in McCoy’s 5a media containing 15% FBS, and HeLa cells were maintained in DMEM containing 10% FBS. Recombinant BPV/SV40 virus stocks were prepared essentially as described previously (35) , with the modification that infected cell pellets were resuspended in 10 mM Tris-1 mM EDTA (pH 8.8) and freeze-thawed (between -70°C and 37°C) three times, and cell debris was removed by centrifugation. Viral stocks were titered on CMT4 cells in eight-well chamber slides (Nunc-177399) infected with serial dilutions of virus in DMEM containing 2% FBS supplemented with 100 µM ZnCl2 and 1 µM CdSO4 to induce large T antigen expression (35) , followed by indirect immunofluorescence of the SV40 VP1 protein. CMT4 cells were plated at 104 cells/well and infected the next day with dilutions of virus. After 20–24 h, cells were fixed for 5 min with 4% formaldehyde (Formaldefresh; Fisher), washed four times with PBS, treated with 0.2% Triton X-100 and 0.1 M glycine in PBS for 5 min, and washed twice in PBS. Primary anti-VP1 mouse monoclonal antibody supernatant (Pab597 from E. Harlow, Boston, MA) diluted 1:20 in PBS was added to the cells and incubated for 1 h at 37°C. Cells were washed with PBS three to four times, and then a 1:200 dilution of goat antimouse H+L fluorescein secondary antibody (Jackson Laboratories, West Grove, PA) was added and incubated for 1 h at 37°C. 4',6-Diamidino-2-phenylindole stain was added to secondary antibody to compare the total number of cells to the number of VP1-positive cells. Cells were washed in PBS three to four times and mounted with a cover slide using Gel Mount (Biomeda). 4',6-Diamidino-2-phenylindole-stained cells and VP1-stained cells were counted by fluorescence microscopy. An approximate titer was calculated assuming a Poisson distribution of infected cells. Viral stocks used in each experiment were titered in parallel to minimize differences caused by assay variability.

Equivalent amounts of Pava1 or Pava5'B{Delta}S, BPV/SV40 recombinant viruses expressing the wild-type BPV E2 protein (22 , 35) , Pava-E2am1, a virus containing an amber nonsense mutation in the 5' portion of the E2 gene, and isogenic viruses expressing various E2 mutants containing missense mutations (35) were used to infect HT-3 or HeLa cells at MOIs of 20–100. Cell growth inhibition was assayed by measuring [3H]thymidine incorporation into acid-insoluble material 48–57 h after infection, as described previously (22) .

RNA Analysis.
Total RNA was prepared from cells 48 or 57 h after infection by using the Trizol reagent according to the manufacturer’s instructions (Life Technologies, Inc., Bethesda, MD). RNA (10 µg) was subjected to agarose gel electrophoresis in the presence of formaldehyde, UV cross-linked to Nytran (Schleicher & Schuell) membranes, and hybridized with random primed cDNA probes for cdc25A or cdc25B [both obtained from K. Galaktionov and D. Beach, (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and Baylor College of Medicine, Houston, TX)], E2F1 (obtained from J. Nevins, Duke University, Durham, NC), cyclin A (obtained from R. Baserga, Jefferson Medical College, Philadelphia, PA), or p105Rb, or with nucleotides 100–751 of HPV30 DNA containing the E6 gene and part of the E7 gene (obtained from E-M. deVilliers, Deutsches Krebsforschungzentrom, Heidelberg, Germany). Sequential hybridizations were performed after stripping the previous probe from the membrane according to the manufacturer’s instructions. RNA levels were normalized by either ethidium bromide staining of ribosomal RNA bands or by probing for {gamma}-actin followed by quantitation on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Immunoprecipitation, Immunoblotting, and cdk2 Kinase Assay.
For immunoprecipitation of p21, infected HT-3 and HeLa cells were starved in media minus cysteine and methionine for 30 min and then labeled with 200 µCi of [35S]cysteine and methionine translabel (ICN) for 3 h. Cells were lysed in EBC buffer [50 mM Tris-HCl (pH 8.0), 120 mM NaCl, and 0.5% NP40], and equal amounts of trichloroacetic acid-precipitable counts were subjected to immunoprecipitation with an anti-p21 antibody (PharMingen, San Diego, CA).

For immunoblotting, cells were lysed in modified EBC buffer [50 mM Tris-HCl (pH 7.5), 120 mM NaCl, 2 mM EDTA, 0.4% NP40, 1 mM NaF, 0.1 mM sodium orthovanadate, 5 µg/ml leupeptin, and 5 µg/ml aprotinin] 48 h after infection, and total protein concentration was determined by BCA Protein Assay reagent (Pierce, Rockford, IL) and processed as described previously (27) . The following primary antibodies were used for immunoblotting: (a) anti-cdk2 (SC-163-G; Upstate Biotechnology Inc., Lake Placid, NY); (b) anti-cyclin E antibody (06-134; Upstate Biotechnology Inc.); (c) anti-p21 antibody (65951A, PharMingen); (d) anti-p105Rb antibody (14001A; PharMingen); (e) anti-cdc25A antibody (SC-7389, Santa Cruz Biotechnology); or (f) anti-cyclin A polyclonal antibody. Blots were then incubated with donkey antirabbit or antimouse IgG conjugated to horseradish peroxidase secondary antibody or protein A-conjugated horseradish peroxidase secondary antibody (Jackson Laboratories), and bands were visualized by enhanced chemiluminescence (Amersham). E2F1 was detected as described previously (27) with polyclonal anti-E2F1 antiserum obtained from J. Nevins. cdc25B protein was detected by first immunoprecipitating with anti-cdc25B antibody (obtained from K. Galaktionov and D. Beach) and then immunoblotting with the same antibody. The mutant and wild-type BPV E2 proteins were detected by first using the monoclonal antibody B202 to immunoprecipitate the E2 protein from 250 µg of HT-3 cell extract and then immunoblotting with the same antibody as described previously (35) .

To measure cyclin/cdk2 kinase activity, infected HT-3 cells were lysed in modified EBC buffer 48 h after infection. Total protein (50 µg) was immunoprecipitated with polyclonal anti-cyclin A antibody, anti-cyclin E antibody (Santa Cruz Biotechnology; HE111), or anti-cdk2 antibody (Santa Cruz Biotechnology; 163-G) for 2 h, and histone H1 kinase activity was measured as described previously (27) . Nonimmune, species-matched antibodies were used as negative controls.

Assay for Soluble cdk-inhibitory Activity.
The presence of endogenous cdk inhibitors in human cells was assayed by testing the ability of cell extracts to inhibit the kinase activity of active cdk2/cyclin E complexes assembled in vitro according to Koff et al. (67) , with modifications. Lysates of insect cells infected separately with baculoviruses encoding human cdk2, tagged with a HA epitope (HA-cdk2), and human cyclin E were incubated together at 30°C for 30 min in the presence of 20 µM ATP to allow complex formation and activation by insect cell CAK. E2 virus-infected or mock-infected HeLa or HT-3 cells were lysed in modified EBC buffer, and 50, 100, or 200 µg of extract were depleted of endogenous human CAK by immunoprecipitation with anti-CAK antibody and depleted of ATP by treatment with hexokinase. These treated extracts were incubated with the HA-cdk2/cyclin E complexes for 30 min at 30°C. The complexes were immunoprecipitated with the anti-HA monoclonal antibody 12CA5 and assayed for kinase activity in vitro.

Assay for CAK Activity.
Infected HT-3 cells were lysed in modified EBC buffer, and 100 or 250 µg of total protein lysate were precleared with protein A-Sepharose beads and then immunoprecipitated with anti-CAK antibody. Immune complexes were washed sequentially with modified EBC buffer and kinase assay buffer. Baculovirus lysates containing cdk2 and cyclin E prepared separately, as described previously (68) , were added to the washed CAK immunoprecipitate and incubated in extract reaction buffer [10 mM MgCl2, 50 mM Tris (pH 7.5), 0.5 mM DTT, 40 mM creatine phosphate, 50 µg/ml creatine kinase, and 1 mM ATP] for 30 min at 30°C. The supernatant was removed from the beads and immunoprecipitated with anti-cdk2 antibody, and then kinase assays were performed as described above.

Experiments with Chemical Inhibitors.
Exponentially growing HT-3 cells were treated with 2 mM hydroxyurea (Sigma) or 200 µM L-mimosine (Sigma) for 49 or 36 h, respectively, to induce growth arrest. DNA synthesis assays and RNA extractions were performed in parallel, and the RNA was transfered to a membrane and sequentially probed with radiolabeled cdc25A and cyclin A cDNAs. Under these conditions, hydroxyurea resulted in an approximately 91% inhibition of DNA synthesis, and mimosine resulted in an approximately 98% inhibition.


    Acknowledgments
 
We thank Konstantin Galaktionov and David Beach for anti-cdc25A and cdc25B antibodies and cDNA clones, Renato Baserga for the cyclin A cDNA clone, Joseph Nevins for the E2F1 cDNA and antibody, Ethel-Michelle deVilliers for HPV30 DNA and communication of results before publication, and Ed Harlow for anti-VP1 antibodies.


    Footnotes
 
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 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from the National Cancer Institute (CA16038) and the American Cancer Society. L. K. N. was supported by postdoctoral fellowships from the American Cancer Society and the Anna Fuller Fund for Cancer Research. E. C. G. was supported by a NIH postdoctoral training grant and the Anna Fuller Fund for Cancer Research. R. A. D. was supported by a predoctoral training grant from the NIH. Back

2 L. K. N. and E. C. G. contributed equally to this work. Back

3 Present address: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404. Back

4 Present address: Department of Life Science, University of Seoul, 90 Jeonnogdong Dongdaemoongu, Seoul, Korea 130-743. Back

5 To whom requests for reprints should be addressed, at Department of Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510. Phone: (203) 785-2684; Fax: (203) 785-7023; E-mail: daniel.dimaio{at}yale.edu Back

6 The abbreviations used are: HPV, human papillomavirus; cdk, cyclin-dependent kinase; BPV, bovine papilloma virus; CAK, cdk-activating kinase; HA, hemagglutinin; FBS, fetal bovine serum; MOI, multiplicity of infection. Back

7 E-M. de Villiers, personal communication. Back

8 K. Galaktionov and K. Helin, personal communication. Back

9 L. K. Naegar, V. Reddy, and D. DiMaio, unpublished observations. Back

Received for publication 11/24/98. Revision received 3/ 9/99. Accepted for publication 4/15/99.


    References
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 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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