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• W2023228900 abstract "Article15 January 1997free access Abrogation of a mitotic checkpoint by E2 proteins from oncogenic human papillomaviruses correlates with increased turnover of the p53 tumor suppressor protein Mark G. Frattini Mark G. Frattini Department of Microbiology-Immunology, Northwestern University, 303 E. Chicago Ave, Chicago, IL, 60611 USA Search for more papers by this author Stephen D. Hurst Stephen D. Hurst Department of Microbiology-Immunology, Northwestern University, 303 E. Chicago Ave, Chicago, IL, 60611 USA Search for more papers by this author Hock B. Lim Hock B. Lim Department of Microbiology-Immunology, Northwestern University, 303 E. Chicago Ave, Chicago, IL, 60611 USA Search for more papers by this author Sathyamangalam Swaminathan Sathyamangalam Swaminathan Department of Microbiology-Immunology, Northwestern University, 303 E. Chicago Ave, Chicago, IL, 60611 USA Search for more papers by this author Laimonis A. Laimins Corresponding Author Laimonis A. Laimins Department of Microbiology-Immunology, Northwestern University, 303 E. Chicago Ave, Chicago, IL, 60611 USA Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 303 E. Chicago Ave, Chicago, IL, 60611 USA Search for more papers by this author Mark G. Frattini Mark G. Frattini Department of Microbiology-Immunology, Northwestern University, 303 E. Chicago Ave, Chicago, IL, 60611 USA Search for more papers by this author Stephen D. Hurst Stephen D. Hurst Department of Microbiology-Immunology, Northwestern University, 303 E. Chicago Ave, Chicago, IL, 60611 USA Search for more papers by this author Hock B. Lim Hock B. Lim Department of Microbiology-Immunology, Northwestern University, 303 E. Chicago Ave, Chicago, IL, 60611 USA Search for more papers by this author Sathyamangalam Swaminathan Sathyamangalam Swaminathan Department of Microbiology-Immunology, Northwestern University, 303 E. Chicago Ave, Chicago, IL, 60611 USA Search for more papers by this author Laimonis A. Laimins Corresponding Author Laimonis A. Laimins Department of Microbiology-Immunology, Northwestern University, 303 E. Chicago Ave, Chicago, IL, 60611 USA Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 303 E. Chicago Ave, Chicago, IL, 60611 USA Search for more papers by this author Author Information Mark G. Frattini1,2, Stephen D. Hurst1, Hock B. Lim1, Sathyamangalam Swaminathan1 and Laimonis A. Laimins 1,3 1Department of Microbiology-Immunology, Northwestern University, 303 E. Chicago Ave, Chicago, IL, 60611 USA 2The University of Chicago, Pritzker School of Medicine, Chicago, IL, 60637 USA 3Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 303 E. Chicago Ave, Chicago, IL, 60611 USA The EMBO Journal (1997)16:318-331https://doi.org/10.1093/emboj/16.2.318 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Human papillomavirus (HPV) E2 and E1 proteins are required for the replication of viral genomes in vivo. We have examined the effects of increasing the level of E2 on viral and cellular replication using recombinant adenoviruses. Infection of cells which maintain HPV 31 DNA episomally with E2 recombinant adenoviruses resulted in a 5-fold increase in genome copy number as well as an S phase arrest allowing for the continued replication of cellular DNA. Similar effects on cell cycle progression were seen following infection of normal human foreskin keratinocytes, the natural host cell. The DNA content of these cells increased beyond 4N indicating that multiple rounds of replication had occurred without an intervening mitotic event. In addition, increased cyclin A and E associated kinase activity was observed, while no change was detected in cyclin B associated kinase activity or in the activation state of cdc2 kinase. Interestingly, the levels of the p53 tumor suppresser protein were dramatically reduced through a post-transcriptional mechanism following infection. These data suggest a role for E2 in regulating viral and cellular replication by abrogation of a mitotic checkpoint, which is, at least in part, controlled by p53. Introduction Progression through the eukaryotic cell cycle is regulated at distinct checkpoints to insure the ultimate fidelity of DNA replication. These checkpoints insure that transit into the next part of the cell cycle occurs following successful completion of the previous phase. Transition through these checkpoints is controlled by cellular proteins which detect incomplete replication or DNA damage. The cellular proteins that regulate these processes consist of complexes of a regulatory cyclin subunit and a catalytic subunit with kinase activity referred to as a cyclin-dependent kinase (CDK) (Grana and Reddy, 1995; Morgan, 1995). These cyclin–CDK complexes act to phosphorylate other proteins, such as the retinoblastoma protein, which regulate the G1, S and G2/M phases of the cell cycle. In the fission yeast, Schizosaccharomyces pombe, a single CDK (p34cdc2) regulates cell cycle progression in combination with different phase-specific cyclin proteins (Enoch and Nurse, 1991; Hayles et al., 1994; King et al., 1994; Nurse, 1994). However, in mammalian cells, a series of cyclin proteins are synthesized sequentially during the cell cycle and form active complexes with multiple CDKs (Heichman and Roberts, 1994; Grana and Reddy, 1995; Morgan, 1995; Su et al., 1995). The D-type cyclins associate with several CDK proteins in mid-G1, while cyclins E and A associate with cdk2 in late G1 and S phase, respectively. The transition from S phase into mitosis and the induction of spindle formation is controlled by complexes of cyclins A and B with the cdc2 gene product (Enoch and Nurse, 1991; King et al., 1994; Nurse, 1994). Following completion of each phase of the cell cycle, the active complexes are specifically degraded or rendered inactive in order to prevent any interference with progression. Cell cycle progression is also regulated through negative feedback on the cyclin–CDK activity by inhibitors of the CDKs which include p21, p27, p16, p18, p19, p20 and p57 (Grana and Reddy, 1995; Morgan, 1995). The transcriptional activator, p53, senses damage to DNA and, in response, activates the expression of the p21 gene product (El-Deiry et al., 1993). This increase in p21 protein level inhibits cyclin E–cdk2 and cyclin A–cdk2 activities, resulting in cell cycle arrest. Another important checkpoint exists at the end of S phase, insuring that DNA replication has been completed faithfully prior to progression into mitosis (Enoch and Nurse, 1991; Heichman and Roberts, 1994; Nurse, 1994; Su et al., 1995). The process of DNA replication is highly coordinated and dictates that each replication origin is activated once per cell cycle even though initiation occurs at different times during S phase. The cellular factors that regulate this process remain largely undefined, and examples of abrogation of this checkpoint in higher eukaryotes are rare. Several gene products have been identified recently in fission yeast which, when deleted (or overexpressed), have been shown to abrogate the normal checkpoint control mechanism linking DNA replication to mitosis. These events include destruction or inhibition of cdc2 kinase; overexpression of the rum1 protein, which is a direct inhibitor of the cyclin B–cdc2 kinase activity; deletion of the cdc13 gene product which encodes the cyclin B protein; and deletion/overexpression of the cdc18 gene product which has been shown to be directly involved in coupling S phase to cell division in fission yeast (Kelly et al., 1993; Hayles et al., 1994; Heichman and Roberts, 1994; Nurse, 1994; Su et al., 1995; Jallepalli and Kelly, 1996; Muzi-Falconi et al., 1996; Schneider et al., 1996). Information on the regulators of this mitotic checkpoint comes from cell fusion studies (Rao and Johnson, 1970). When G1-arrested nuclei are transplanted into cells in S phase, a new round of DNA synthesis is initiated, implicating a trans-acting factor in the regulation of entry into S phase. However, G2 nuclei transplanted into S phase cells do not induce re-replication (or endoreduplication) nor do they not prevent the continued replication of S phase nuclei. This indicates that nuclear localized negative regulatory signals exist to prevent re-replication. Using an in vitro replication system, Blow and Laskey (1988) demonstrated the ability to induce a second round of DNA replication without an intervening mitosis by permeabilizing Xenopus nuclei with detergents. These studies suggested the existence of ‘licensing factors’ which allow for replication and prohibit replication in their absence. Candidate genes, the minichromosome maintenance (MCM) proteins, have been identified in yeast, but none has been identified yet in mammalian cells (Romanowski and Madine, 1996). Genetic studies in yeast have identified cyclins, namely Clb5 and Clb6, as being important for faithful completion of S phase, and mutant alleles of cdc2 and cyclin B have been isolated which lead to re-replication (Schwob and Nasmyth, 1993; Hayles et al., 1994). Abrogation of replication control has also been demonstrated in Drosophila melanogaster (Sauer et al., 1995). This occurs as part of the normal process of terminal differentiation and results in polytene chromosomes or polyploid cells. Therefore, several mechanisms exist which allow for the abrogation of the normal cell cycle checkpoint at the end of S phase. In contrast to studies in yeast and Drosophila, little is known about the mammalian factors regulating the mitotic checkpoint and controlling the fidelity of DNA replication. A single conditional mutant Chinese hamster ovary cell line has been isolated which can accumulate up to 32N DNA at the restrictive temperature (Handeli and Weintraub, 1992). The gene product responsible, however, has not been isolated, and the mechanism remains undefined. Based on studies of fibroblasts derived from p53 knockout mice, a role has recently been suggested for this tumor suppressor protein in regulating the mitotic checkpoint (Cross et al., 1995). Upon passage in culture, these p53-deficient cells rapidly become aneuploid. In addition, treatment of these cells with the mitotic spindle inhibitor, nocodazole, resulted in an S phase arrest with continued chromosomal replication. Viral systems have often provided useful insights into the biochemistry of eukaryotic DNA replication as well as in cell cycle control. Viral proteins target cell cycle progression to facilitate their own replicative cycle. Prominent examples are the targeting of Rb family members by the simian virus 40 (SV40) large T antigen, adenovirus E1A and the E7 protein of high risk human papillomaviruses (HPVs), which results in the constitutive activation of the E2F family of transcription factors (Nevins, 1992). HPVs, in particular, provide a unique example of viral targeting of cell cycle progression. HPVs infect the basal cells of stratified epithelia and establish their genomes as low copy viral episomes (Laimins, 1993; Howley, 1996). In these infected cells, viral episomes replicate in synchrony with cellular chromosomes. As infected cells migrate from the basal layer and undergo differentiation, lytic viral replication and late gene expression are induced (Bedell et al., 1991; Meyers et al., 1992; Laimins, 1993; Frattini et al., 1996; Howley, 1996). In normal epithelia, differentiating suprabasal cells exit the cell cycle after leaving the basal layer. In contrast, in HPV-infected epithelia which are actively producing virus, viral proteins arrest suprabasal cells at the G1–S boundary until they reach terminal differentiation, at which point cells are induced to re-enter S phase (Laimins, 1993; Howley, 1996). The viral replication proteins, E1 and E2, are both necessary and sufficient to activate viral replication in transient transfection assays, while the E7 oncoprotein, in the absence of other viral proteins, has been shown to activate cellular replication in a stratified epithelium (Ustav and Stenlund, 1991; Blanton et al., 1992; Chiang et al., 1992; DelVecchio et al., 1992; Frattini and Laimins, 1994a; Cheng et al., 1995). The E1 protein functions as the viral initiator protein, and the E2 protein, while having the structure of a classical transcriptional activator, has been shown to be involved in replication through heteromeric complex formation with E1 (Androphy et al., 1987; Dostatni et al., 1988; Mohr et al., 1990; Blitz and Laimins, 1991; Lambert, 1991; Yang et al., 1991; Hegde et al., 1992; Seo et al., 1993; Thorner et al., 1993; Frattini and Laimins, 1994b; Li and Botchan, 1994). Previous studies from our laboratory used recombinant amphotrophic retroviruses expressing the E2 gene product of the oncogenic papillomavirus, HPV 31, to infect monolayer cultures of a human keratinocyte cell line (CIN 612-9E) which stably maintains HPV 31 episomes at ∼50 copies per cell. Following infection by the E2-expressing retrovirus, these cells exhibited a 5- to 9-fold increase in viral copy number which was not seen following infection with retroviruses expressing other HPV gene products (Frattini and Laimins, 1994b). The nuclei of these E2-expressing cells were significantly enlarged, and the cells appeared growth arrested. Small cells quickly grew out of the large cell colonies and, since a period of drug selection was required, these smaller cells constituted the population which was analyzed for viral copy number. The changes in cell morphology observed at early time points suggested that increased levels of E2 might have an immediate effect on cell cycle progression. This is consistent with reports that high level E2 expression is toxic to cells, and cell lines which stably overexpress E2 have been difficult to isolate (M.G.Frattini and L.A.Laimins, unpublished observation). In this study, we investigated the effects of the E2 protein on cell cycle progression using an adenovirus-mediated expression system. High titer recombinant adenoviruses expressing the E2 gene product were constructed and, following infection of either immortalized or normal human keratinocytes, we observed that infected cells arrested in S phase and continued to replicate cellular DNA. This loss of replication control correlated with an increased turnover of the p53 tumor suppressor gene product, consistent with a role for this protein in control of the mitotic checkpoint in mammalian cells. Results Construction of recombinant adenoviruses In order to study the immediate effects of increasing the levels of the papillomavirus replication protein, E2, on cellular proliferation and HPV copy number, we constructed a high titer recombinant adenovirus expressing the HPV 31 E2 gene product (Figure 1). The HPV 31 E2 gene was cloned under the control of a cytomegalovirus (CMV) promoter into a plasmid which contained 452 bp of the 5′ end of the adenovirus genome. In this construct, the adenovirus E1A gene was replaced by the CMV promoter and the E2 gene. A fragment containing the 5′ end of the adenovirus genome and the E2 expression cassette was then ligated to purified restricted recombinant adenovirus genomes which lacked both the 452 bp 5′ end and the E1A coding sequences. The ligation products were then transfected into 293 cells, which express the E1A gene products. The resultant viruses were plaque purified, and a single recombinant E2-expressing adenovirus (rAdE2-1) was used in the majority of the following studies, although identical effects were also seen with the initial heterogeneous viral stock. A similar methodology was used to construct a recombinant adenovirus expressing the HPV 31 E1 gene product. Figure 1.Diagram of the recombinant adenoviruses used in these studies. Recombinant adenoviruses expressing the HPV 31 E1 and E2 proteins and a control virus expressing the adenovirus E4 ORF 6/7 protein were generated as described in Materials and methods. These recombinant viruses were constructed by replacing the E1A coding sequences of the adenovirus type 5 mutant, dl309, with the indicated CMV expression cassette. The control CMV recombinant virus replaces both the adenovirus E1A and E1B coding sequences with an empty CMV expression cassette as indicated. Adenovirus coding sequences are shown and ML represents the major late promoter. Download figure Download PowerPoint rAdE2-1 expresses active E2 protein To demonstrate that the rAdE2-1 virus was expressing active E2 protein, we used a functional assay for E2 DNA binding. A squamous cell carcinoma cell line, SCC-13, was infected with a high multiplicity of rAdE2-1. Nuclear extracts were then prepared at various time points following infection and subjected to an electromobility shift assay (EMSA) using a 32P-labeled DNA probe which contained two E2 binding sites. As shown in Figure 2, E2 DNA binding was first detected at 12 h post-infection and increased steadily throughout the length of the time course. This pattern of E2 protein activity correlated with the level of E2 gene expression as determined by Western blot analyses at similar times post-infection (data not shown). Figure 2.The recombinant adenovirus, rAdE2-1, expresses active E2 protein. SCC-13 cells were infected, and nuclear extracts prepared at the indicated time points (in hours) post-infection as described in Materials and methods. For the EMSA analysis, 7.5 μg of extract was mixed with a 32P-labeled DNA probe containing two E2 binding sites. FP represents free probe, and the plus sign (+) represents 70 ng of a purified GST–E2 fusion protein containing the carboxy-terminal DNA binding domain, a positive control for E2 DNA binding. Protein–DNA complexes were visualized by autoradiography. Download figure Download PowerPoint Infection of CIN 612-9E cells with rAdE2-1 results in an increase in viral copy number and an accumulation of cells with greater than 4N DNA content We next examined if, as we previously observed using recombinant retroviruses (Frattini and Laimins, 1994b), increasing the level of E2 protein in cells which stably maintain HPV genomes as episomes (CIN 612-9E) would result in an increase in viral genomic copy number. Monolayer cultures of CIN 612-9E cells were infected with a control AdE4 open reading frame (ORF) 6/7-expressing recombinant adenovirus (AdE4) or recombinant HPV 31 E1- or E2-expressing adenoviruses, and viral copy number was determined by Southern analyses (Figure 3). While infection with AdE4- or E1-expressing viruses had minimal effect on HPV 31 copy number, infection by rAdE2-1 alone increased the copy number ∼5-fold, and infection by both E1- and E2-expressing viruses increased copy number ∼20-fold, as quantitated by phosphoimager analysis. Cells infected with rAdE2-1, but not the other recombinant viruses, exhibited enlarged nuclei similar to what we observed previously following infection with E2-expressing recombinant retroviruses (data not shown). To examine if E2 had an effect on cell cycle progression, rAdE2-1-infected cells were fixed with ethanol at 48 h post-infection, stained with propidium iodide and examined by fluorescence-activated cell sorter (FACS) analyses. As seen in Figure 4, the majority of cells in mock- or AdE4 control-infected cells were in G0/G1. In contrast, the majority of cells infected with the E2 recombinant adenovirus were consistent with cells in S or G2/M phases of the cell cycle. In addition, a significant number of these rAdE2-1-infected cells had DNA contents >4N (Figure 4). Figure 3.Infection of CIN 612-9E cells with recombinant adenoviruses expressing the E2 protein results in an increase in viral DNA copy number. Recombinant adenoviruses expressing the AdE4, E1 or E2 proteins were used to infect CIN612-9E cells as described in Materials and methods. Total genomic DNA was isolated at 6, 24 and 48 h post-infection, and 10 μg of total cellular DNA was examined by Southern analyses using a 32P-labeled HPV 31-specific DNA probe from the non-coding region. Hybridizing species were visualized by autoradiography. Lanes 1, 2 and 3 represent 1000, 250 and 50 copies per cell of linearized HPV 31 DNA, respectively. Lane 4 is 10 μg of normal human foreskin keratinocyte DNA. Molecular weight standards are indicated in kb. I, II and III indicate monomeric supercoiled, nicked and linearized viral genomes, respectively. The asterisk (*) represents multimeric concatenated genomic species. The fold increase in viral genome copy number was quantitated using a phosphoimager and normalized to the values obtained for the copy number controls. Download figure Download PowerPoint Figure 4.Infection of CIN 612-9E cells with recombinant adenoviruses expressing the E2 protein results in an accumulation of cells with >4N DNA content. Recombinant adenoviruses expressing the AdE4 or E2 proteins were used to infect CIN612-9E cells and, at 48 h post-infection, cells were fixed, stained with propidium iodide and subjected to FACS analyses as described in Materials and methods. Mock represents uninfected cells; 2N and 4N indicate cells in G0/G1 and G2/M phases of the cell cycle, respectively. Download figure Download PowerPoint rAdE2-1-infected NHK cells contain greater than 4N DNA content and display an aberrant cell/nuclear size Since the cells used in the previous experiments were from an immortalized cell line (CIN 612-9E) which express HPV gene products, it was not clear whether the effects on cell cycle progression we observed were the result of overexpression of E2 alone or a synergistic effect of E2 with other papillomaviral proteins. Studies were initiated, therefore, using normal human foreskin keratinocytes (NHK), the natural host cell for HPV infection. As seen in Figure 5A, a similar switch in the proportion of cells in G0/G1 to those in S and G2/M was observed following infection of NHK cells with rAdE2-1. No such change was seen in cells infected with the control CMV adenovirus, and similar results were observed with the recombinant AdE4 virus (data not shown). The appearance of cells with >4N DNA content was most pronounced at 66 h post-infection, at which time we also observed the appearance of cells with <2N DNA content, indicative of apoptotic cells in which the DNA was being degraded (Darzynkiewicz et al., 1992). Figure 5B shows a representative phase contrast micrograph of control CMV- and rAdE2-1-infected NHK cells at 22 and 66 h post-infection. The photograph demonstrates that as early as 22 h post-infection large cells with enlarged nuclei can be detected specifically in the rAdE2-1-infected cells. At 66 h post-infection, the control CMV-infected cells approached confluency with no change in cell/nuclear size. In contrast, the average size of the rAdE2-1-infected population continued to increase with the appearance of smaller refractile cells. The presence of small cells in the rAdE2-1 infections most likely represents the apoptotic population detected in the FACS analysis in Figure 5A. Figure 5.Infection of NHK cells with recombinant adenoviruses expressing the E2 protein results in an accumulation of cells with >4N DNA content and aberrant cell/nuclear size. (A) CMV and rAdE2-1 recombinant adenoviruses were used to infect NHK cells and, at the indicated time points post-infection, cells were fixed, stained and subjected to FACS analyses as described in Materials and methods. 2N and 4N indicate cells in G0/G1 and G2/M phases of the cell cycle, respectively. The peak of cells with <2N DNA content at 66 h post-infection with the rAdE2-1 virus represents an apoptotic cell population. (B) Phase contrast microscopy of CMV (upper left quadrant, lower left quadrant) and rAdE2-1 (upper right quadrant, lower right quadrant) infected NHK cells at 22 (upper left and right quadrants) and 66 h (lower left and right quadrants) post-infection. Download figure Download PowerPoint The majority of rAdE2-1-infected NHK cells are in S phase and re-replicate cellular DNA In order to confirm that the cells with >4N DNA content were synthesizing DNA; mock-, AdE4- and rAdE2-1-infected NHK cells were labeled for 1 h at 40 h post-infection with bromodeoxyuridine (BrdU), fixed and stained. A two-dimensional FACS analysis of these cells (Figure 6) demonstrated that a majority of the mock- and AdE4-infected cells were in G0/G1 (60.4 and 61.5%, respectively) while the majority of the rAdE2-1-infected cells were in S phase (62.5%). In addition, many of the rAdE2-1-infected cells in S phase had DNA contents >4N. We therefore conclude that infection of normal human keratinocytes with recombinant adenoviruses expressing the E2 protein induces a specific accumulation in S phase resulting in re-replication of cellular DNA. Figure 6.Two-dimensional FACS analyses demonstrate that >60% of the cells infected with rAdE2-1 are actively synthesizing DNA. Recombinant adenoviruses expressing the AdE4 or E2 proteins were used to infect NHK cells. At 40 h post-infection, cells were pulsed for 1 h with BrdU and subsequently fixed, stained and subjected to FACS analyses as described in Materials and methods. The NHK panel represents uninfected cells. The upper left and right quadrants correspond to cells actively synthesizing DNA, as determined by BrdU incorporation, while cells in the lower left and right quadrants represent cells in G0/G1 and G2/M, respectively. Download figure Download PowerPoint rAdE2-1-infected NHK cells are arrested in S phase This accumulation in S phase could either be the result of cells arrested in S phase or, alternatively, a distinct population of rapidly cycling cells. To distinguish between these possibilities, control and rAdE2-1-infected NHK cells were treated with nocodazole, a mitotic spindle inhibitor, at 24 h post-infection. At 44 h post-infection, the cells were fixed, stained with propidium iodide and examined by FACS analyses. As shown in Figure 7, mock- or AdE4-infected NHK cells treated with nocodazole were arrested in G2 and exhibited the expected cell cycle distribution. In contrast, the distribution of rAdE2-1-infected cells, including those cells with >4N DNA content, remained unchanged following treatment with nocodazole. It appears that the cells infected by rAdE2-1 were not cycling rapidly, but were arrested in S phase and continued to replicate cellular DNA. These cells failed to progress into mitosis and were, therefore, unaffected by treatment with nocodazole. Figure 7.rAdE2-1-infected NHK cells are arrested in S phase. Recombinant adenoviruses expressing the AdE4 or E2 proteins were used to infect NHK cells. At 24 h post-infection, cells were treated with (+) or without (−) the mitotic spindle inhibitor, nocodazole, for 20 h. At 44 h post-infection, cells were fixed, stained and subjected to FACS analyses as described in Materials and methods. NHK represents uninfected cells; 2N and 4N indicate cells in G0/G1 and G2/M phases of the cell cycle, respectively. Download figure Download PowerPoint rAdE2-1-infected NHK cells contain increased cyclin A- and E-associated histone H1 kinase activity, but the activation state of cdc2 kinase remains unchanged The mechanism by which HPV E2 proteins induced an S phase arrest most likely involved the altered activity or expression of cellular proteins which regulate passage through the cell cycle. We therefore investigated the levels and activities of cellular proteins which regulate transit through the G1 and S phases. The level of cyclin A, E and B kinase activity was examined by associated histone H1 kinase activity of immunoprecipitated protein complexes. At 40 h post-infection, NHK cell extracts were prepared, and cyclin proteins immunoprecipitated with the corresponding antibodies. The immunoprecipitated complexes were then incubated with [γ-32P]ATP and histone H1, and the levels of phosphorylation examined following SDS–PAGE. As shown in Figure 8A, increased levels of kinase activity associated with cyclins A and E were observed specifically in rAdE2-1-infected cells, while the level of kinase activity associated with cyclin B remained relatively unchanged. The presence of equivalent amounts of cyclin B kinase activity in control and rAdE2-1-infected cells is in agreement with approximately equal distribution of cells in G2/M as shown in Figure 6. The increase in cyclin A and E kinase activity in rAdE2-1-infected cells is consistent with the majority of these infected cells being arrested in S phase and not progressing into mitosis. Figure 8.Infection of NHK cells with rAdE2-1 results in increased cyclin A- and E-associated histone H1 kinase activity, but no change in the activation state of cdc2 kinase. Recombinant adenoviruses expressing the AdE4 (A) or E2 (E2) proteins were used to infect NHK cells. (M) represents uninfected cells. At 40 h post-infection, total cell extracts were prepared as described in Materials and methods. (A) Immunoprecipitation and associated histone H1 kinase activity assay. Equal amounts of protein extract were immunoprecipitated with the indicated antibody and subsequently incubated with [γ-32P]ATP and histone H1. Following SDS–PAGE, phosphorylated histone H1 was visualized by autoradiography. (B) Cdc2 immunoprecipitation and anti-phosphotyrosine Western blot analyses. Equal amounts of protein extract were immunoprecipitated with a cdc2 antibody and, following SDS–PAGE, the amount of cdc2 containing phosphorylated tyrosine residues was visualized by Western analyses" @default.
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• W2023228900 title "Abrogation of a mitotic checkpoint by E2 proteins from oncogenic human papillomaviruses correlates with increased turnover of the p53 tumor suppressor protein" @default.
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