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Mechanism of genomic instability in cells infected with the high-risk human papillomaviruses.

Kadaja M, Isok-Paas H, Laos T, Ustav E, Ustav M - PLoS Pathog. (2009)

Bottom Line: These changes suggest that the integrated HPV replication intermediates are processed by the activated cellular DNA repair/recombination machinery, which results in cross-chromosomal translocations as detected by metaphase FISH.We also confirmed that the replicating HPV episomes that expressed the physiological levels of viral replication proteins could induce genomic instability in the cells with integrated HPV.It could be used as a starting point for the "onion skin"-type of DNA replication whenever the HPV plasmid exists in the same cell, which endangers the host genomic integrity during the initial integration and after the de novo infection.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.

ABSTRACT
In HPV-related cancers, the "high-risk" human papillomaviruses (HPVs) are frequently found integrated into the cellular genome. The integrated subgenomic HPV fragments express viral oncoproteins and carry an origin of DNA replication that is capable of initiating bidirectional DNA re-replication in the presence of HPV replication proteins E1 and E2, which ultimately leads to rearrangements within the locus of the integrated viral DNA. The current study indicates that the E1- and E2-dependent DNA replication from the integrated HPV origin follows the "onion skin"-type replication mode and generates a heterogeneous population of replication intermediates. These include linear, branched, open circular, and supercoiled plasmids, as identified by two-dimensional neutral-neutral gel-electrophoresis. We used immunofluorescence analysis to show that the DNA repair/recombination centers are assembled at the sites of the integrated HPV replication. These centers recruit viral and cellular replication proteins, the MRE complex, Ku70/80, ATM, Chk2, and, to some extent, ATRIP and Chk1 (S317). In addition, the synthesis of histone gammaH2AX, which is a hallmark of DNA double strand breaks, is induced, and Chk2 is activated by phosphorylation in the HPV-replicating cells. These changes suggest that the integrated HPV replication intermediates are processed by the activated cellular DNA repair/recombination machinery, which results in cross-chromosomal translocations as detected by metaphase FISH. We also confirmed that the replicating HPV episomes that expressed the physiological levels of viral replication proteins could induce genomic instability in the cells with integrated HPV. We conclude that the HPV replication origin within the host chromosome is one of the key factors that triggers the development of HPV-associated cancers. It could be used as a starting point for the "onion skin"-type of DNA replication whenever the HPV plasmid exists in the same cell, which endangers the host genomic integrity during the initial integration and after the de novo infection.

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Related in: MedlinePlus

Activation of the ATM-Chk2 signaling pathway.(A–C) HeLa cells were transfected as follows: 5 µg of HPV18 E1 and 2 µg of HPV18 E2 expression plasmids (lane 1); 5 µg of HPV18 E1 expression plasmid (lane 2); 2 µg of E2 expression plasmid (lane 3); and a mock-transfection (lane 4). In every transfection, the amount of plasmid was adjusted to 10 µg with a carrier plasmid (pauxoMCF). Non-transfected HeLa cells are presented in lane 5 and HeLa cells that were treated 1 h with etoposide (50 µM) prior to the analysis in lane 6. Western blot analyses were performed at a 24 hrs time point to detect HPV18 E1 (A, panel a), HPV18 E2 (A, panel b), gamma histone H2AX (phosphorylated at S139) (A, panel c), and β-actin (A, panel d). Western blot analyses of Chk2 phosphorylated at Thr68 and Ser19 were performed after the immunoprecipitation with the anti-Chk2 antibody (B, panels a, b, c). Chk1 phosphorylated at Ser317 was detected from extracts that were immunoprecipitated with the anti-Chk1 antibody (C, panels a, b). (D) HeLa cells were transfected either with 2 µg of circular HPV18 genome, 2 µg of pBabePuro and 6 µg of carrier plasmid (lane 1), or with 2 µg of pBabePuro and 8 µg of carrier plasmid (lane 2). Untransfected cells were removed with puromycin treatment (2 µg/ml) 24–48 h posttransfection. Western blot analyses with anti-Chk2 (panel a) and anti-Chk2-Ser19 (panel b) antibodies were performed after the immunoprecipitation with the anti-Chk2 antibody at a 72 h time point. Untreated HeLa cells are shown in lane 3 and etoposide-treated HeLa cells in lane 4.
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ppat-1000397-g006: Activation of the ATM-Chk2 signaling pathway.(A–C) HeLa cells were transfected as follows: 5 µg of HPV18 E1 and 2 µg of HPV18 E2 expression plasmids (lane 1); 5 µg of HPV18 E1 expression plasmid (lane 2); 2 µg of E2 expression plasmid (lane 3); and a mock-transfection (lane 4). In every transfection, the amount of plasmid was adjusted to 10 µg with a carrier plasmid (pauxoMCF). Non-transfected HeLa cells are presented in lane 5 and HeLa cells that were treated 1 h with etoposide (50 µM) prior to the analysis in lane 6. Western blot analyses were performed at a 24 hrs time point to detect HPV18 E1 (A, panel a), HPV18 E2 (A, panel b), gamma histone H2AX (phosphorylated at S139) (A, panel c), and β-actin (A, panel d). Western blot analyses of Chk2 phosphorylated at Thr68 and Ser19 were performed after the immunoprecipitation with the anti-Chk2 antibody (B, panels a, b, c). Chk1 phosphorylated at Ser317 was detected from extracts that were immunoprecipitated with the anti-Chk1 antibody (C, panels a, b). (D) HeLa cells were transfected either with 2 µg of circular HPV18 genome, 2 µg of pBabePuro and 6 µg of carrier plasmid (lane 1), or with 2 µg of pBabePuro and 8 µg of carrier plasmid (lane 2). Untransfected cells were removed with puromycin treatment (2 µg/ml) 24–48 h posttransfection. Western blot analyses with anti-Chk2 (panel a) and anti-Chk2-Ser19 (panel b) antibodies were performed after the immunoprecipitation with the anti-Chk2 antibody at a 72 h time point. Untreated HeLa cells are shown in lane 3 and etoposide-treated HeLa cells in lane 4.

Mentions: The data presented above clearly indicate that the DNA double-strand break repair machinery is recruited to the replication foci of integrated HPV. We further studied the activation status of the DNA DSB repair systems. HeLa cells were transfected with the HPV18 E1 and E2 expression vectors (Figure 6A–6C, lane 1), the HPV18 E1 expression vector (Figure 6A–6C, lane 2), the HPV18 E2 expression vector (Figure 6A–6C, lane 3), or carrier DNA (Figure 6A–6C, lane 4). Untransfected HeLa cells (Figure 6A–6C, lane 5) and HeLa cells that were treated for 1 hour with etoposide (Figure 6A–6C, lane 6) were used as controls. The transfected cells were first analyzed for E1 and E2 expression (Figure 6A, panels a and b, respectively) 24 hrs post-transfection using normalized western blot analysis. DNA double-strand break repair originating from diverse causes in eukaryotic cells are accompanied by the upregulation of phosphorylated γH2AX protein (at serine 139) at the sites of DSBs in chromosomal DNA. This phosphorylated form of γH2AX is rapidly formed in cells that are treated with ionizing radiation (IR) and also during V(D)J and class-switch recombination and apoptosis. Since γH2AX appears within minutes after IR, the production of the phosphorylated form of γH2AX is considered to be a sensitive and selective signal for the existence of DNA double-strand breaks. Indeed, treatment of the HeLa cells with etoposide, which generates DNA DSBs, considerably elevates the formation of the phosphorylated form of the γH2AX in these cells (Figure 6A, panel c, lane 6). In addition, we detected a considerable increase of the phosphorylated form of γH2AX when the E1 and E2 proteins were co-transfected into HeLa cells (Figure 6A, panel c, lane 1). This indicates that the cellular response to the DNA DSBs that are generated by the replication of the integrated HPV DNA is clearly activated. We further analyzed the activation status of Chk2 at the same time point by using IP-western blot analysis with phosphorylation specific antibodies (Figure 6B). HeLa cells, which were transfected in a manner similar to the procedure that was used in Figure 6A, were lysed and subjected to immunoprecipitation with the anti-Chk2 antibody. The immunoprecipitated protein samples were further analyzed with phosphorylation-specific antibodies targeted against the Chk2 phosphopeptides that contain Thr68 or Ser19. These sites are part of a cluster of S/TQ phosphorylation sites that are recognized by PIKKs (PI3 kinase-like kinases) such as ATM and ATR [55]. It is known that all S/TQ sites in the N terminus of Chk2 are individually sufficient to activate the protein [56]. As expected, strong phosphorylation of Chk2 at Thr68 and Ser19 were detected in the case of etoposide–treated cells (Figure 6B, lane 6). In addition, a modest activation of Chk2 can be observed in the cells that were transfected with E1 expression vector alone (Figure 6B, lane 2). However, this effect was considerably enhanced when E1 and E2-dependent replication was initiated in HeLa cells (Figure 6B, lane 1). Interestingly, Ser19 is phosphorylated exclusively in response to DSBs in an ATM- and Nbs1-dependent but ATR-independent manner [57]. We conclude that E1 protein expression can, to some extent, activate the Chk2 kinase, which is further activated by the replication of the integrated HPV. Similar IP-western blot analysis of Chk1 activation in these cells showed a very weak elevation of the phosphorylation at Ser317 in the E1-transfected cells, which was not enhanced by the replication of integrated HPV and, by no means, was comparable to the effect of the etoposide-treatment of the cells (Figure 6C, compare lanes 1 and 2 to lane 6). We can only speculate why Chk1 and Chk2 are slightly activated in response the E1 expression. It could be either direct interactions with the components of the DNA repair pathways or an unspecific binding and unwinding of the cellular DNA.


Mechanism of genomic instability in cells infected with the high-risk human papillomaviruses.

Kadaja M, Isok-Paas H, Laos T, Ustav E, Ustav M - PLoS Pathog. (2009)

Activation of the ATM-Chk2 signaling pathway.(A–C) HeLa cells were transfected as follows: 5 µg of HPV18 E1 and 2 µg of HPV18 E2 expression plasmids (lane 1); 5 µg of HPV18 E1 expression plasmid (lane 2); 2 µg of E2 expression plasmid (lane 3); and a mock-transfection (lane 4). In every transfection, the amount of plasmid was adjusted to 10 µg with a carrier plasmid (pauxoMCF). Non-transfected HeLa cells are presented in lane 5 and HeLa cells that were treated 1 h with etoposide (50 µM) prior to the analysis in lane 6. Western blot analyses were performed at a 24 hrs time point to detect HPV18 E1 (A, panel a), HPV18 E2 (A, panel b), gamma histone H2AX (phosphorylated at S139) (A, panel c), and β-actin (A, panel d). Western blot analyses of Chk2 phosphorylated at Thr68 and Ser19 were performed after the immunoprecipitation with the anti-Chk2 antibody (B, panels a, b, c). Chk1 phosphorylated at Ser317 was detected from extracts that were immunoprecipitated with the anti-Chk1 antibody (C, panels a, b). (D) HeLa cells were transfected either with 2 µg of circular HPV18 genome, 2 µg of pBabePuro and 6 µg of carrier plasmid (lane 1), or with 2 µg of pBabePuro and 8 µg of carrier plasmid (lane 2). Untransfected cells were removed with puromycin treatment (2 µg/ml) 24–48 h posttransfection. Western blot analyses with anti-Chk2 (panel a) and anti-Chk2-Ser19 (panel b) antibodies were performed after the immunoprecipitation with the anti-Chk2 antibody at a 72 h time point. Untreated HeLa cells are shown in lane 3 and etoposide-treated HeLa cells in lane 4.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2666264&req=5

ppat-1000397-g006: Activation of the ATM-Chk2 signaling pathway.(A–C) HeLa cells were transfected as follows: 5 µg of HPV18 E1 and 2 µg of HPV18 E2 expression plasmids (lane 1); 5 µg of HPV18 E1 expression plasmid (lane 2); 2 µg of E2 expression plasmid (lane 3); and a mock-transfection (lane 4). In every transfection, the amount of plasmid was adjusted to 10 µg with a carrier plasmid (pauxoMCF). Non-transfected HeLa cells are presented in lane 5 and HeLa cells that were treated 1 h with etoposide (50 µM) prior to the analysis in lane 6. Western blot analyses were performed at a 24 hrs time point to detect HPV18 E1 (A, panel a), HPV18 E2 (A, panel b), gamma histone H2AX (phosphorylated at S139) (A, panel c), and β-actin (A, panel d). Western blot analyses of Chk2 phosphorylated at Thr68 and Ser19 were performed after the immunoprecipitation with the anti-Chk2 antibody (B, panels a, b, c). Chk1 phosphorylated at Ser317 was detected from extracts that were immunoprecipitated with the anti-Chk1 antibody (C, panels a, b). (D) HeLa cells were transfected either with 2 µg of circular HPV18 genome, 2 µg of pBabePuro and 6 µg of carrier plasmid (lane 1), or with 2 µg of pBabePuro and 8 µg of carrier plasmid (lane 2). Untransfected cells were removed with puromycin treatment (2 µg/ml) 24–48 h posttransfection. Western blot analyses with anti-Chk2 (panel a) and anti-Chk2-Ser19 (panel b) antibodies were performed after the immunoprecipitation with the anti-Chk2 antibody at a 72 h time point. Untreated HeLa cells are shown in lane 3 and etoposide-treated HeLa cells in lane 4.
Mentions: The data presented above clearly indicate that the DNA double-strand break repair machinery is recruited to the replication foci of integrated HPV. We further studied the activation status of the DNA DSB repair systems. HeLa cells were transfected with the HPV18 E1 and E2 expression vectors (Figure 6A–6C, lane 1), the HPV18 E1 expression vector (Figure 6A–6C, lane 2), the HPV18 E2 expression vector (Figure 6A–6C, lane 3), or carrier DNA (Figure 6A–6C, lane 4). Untransfected HeLa cells (Figure 6A–6C, lane 5) and HeLa cells that were treated for 1 hour with etoposide (Figure 6A–6C, lane 6) were used as controls. The transfected cells were first analyzed for E1 and E2 expression (Figure 6A, panels a and b, respectively) 24 hrs post-transfection using normalized western blot analysis. DNA double-strand break repair originating from diverse causes in eukaryotic cells are accompanied by the upregulation of phosphorylated γH2AX protein (at serine 139) at the sites of DSBs in chromosomal DNA. This phosphorylated form of γH2AX is rapidly formed in cells that are treated with ionizing radiation (IR) and also during V(D)J and class-switch recombination and apoptosis. Since γH2AX appears within minutes after IR, the production of the phosphorylated form of γH2AX is considered to be a sensitive and selective signal for the existence of DNA double-strand breaks. Indeed, treatment of the HeLa cells with etoposide, which generates DNA DSBs, considerably elevates the formation of the phosphorylated form of the γH2AX in these cells (Figure 6A, panel c, lane 6). In addition, we detected a considerable increase of the phosphorylated form of γH2AX when the E1 and E2 proteins were co-transfected into HeLa cells (Figure 6A, panel c, lane 1). This indicates that the cellular response to the DNA DSBs that are generated by the replication of the integrated HPV DNA is clearly activated. We further analyzed the activation status of Chk2 at the same time point by using IP-western blot analysis with phosphorylation specific antibodies (Figure 6B). HeLa cells, which were transfected in a manner similar to the procedure that was used in Figure 6A, were lysed and subjected to immunoprecipitation with the anti-Chk2 antibody. The immunoprecipitated protein samples were further analyzed with phosphorylation-specific antibodies targeted against the Chk2 phosphopeptides that contain Thr68 or Ser19. These sites are part of a cluster of S/TQ phosphorylation sites that are recognized by PIKKs (PI3 kinase-like kinases) such as ATM and ATR [55]. It is known that all S/TQ sites in the N terminus of Chk2 are individually sufficient to activate the protein [56]. As expected, strong phosphorylation of Chk2 at Thr68 and Ser19 were detected in the case of etoposide–treated cells (Figure 6B, lane 6). In addition, a modest activation of Chk2 can be observed in the cells that were transfected with E1 expression vector alone (Figure 6B, lane 2). However, this effect was considerably enhanced when E1 and E2-dependent replication was initiated in HeLa cells (Figure 6B, lane 1). Interestingly, Ser19 is phosphorylated exclusively in response to DSBs in an ATM- and Nbs1-dependent but ATR-independent manner [57]. We conclude that E1 protein expression can, to some extent, activate the Chk2 kinase, which is further activated by the replication of the integrated HPV. Similar IP-western blot analysis of Chk1 activation in these cells showed a very weak elevation of the phosphorylation at Ser317 in the E1-transfected cells, which was not enhanced by the replication of integrated HPV and, by no means, was comparable to the effect of the etoposide-treatment of the cells (Figure 6C, compare lanes 1 and 2 to lane 6). We can only speculate why Chk1 and Chk2 are slightly activated in response the E1 expression. It could be either direct interactions with the components of the DNA repair pathways or an unspecific binding and unwinding of the cellular DNA.

Bottom Line: These changes suggest that the integrated HPV replication intermediates are processed by the activated cellular DNA repair/recombination machinery, which results in cross-chromosomal translocations as detected by metaphase FISH.We also confirmed that the replicating HPV episomes that expressed the physiological levels of viral replication proteins could induce genomic instability in the cells with integrated HPV.It could be used as a starting point for the "onion skin"-type of DNA replication whenever the HPV plasmid exists in the same cell, which endangers the host genomic integrity during the initial integration and after the de novo infection.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.

ABSTRACT
In HPV-related cancers, the "high-risk" human papillomaviruses (HPVs) are frequently found integrated into the cellular genome. The integrated subgenomic HPV fragments express viral oncoproteins and carry an origin of DNA replication that is capable of initiating bidirectional DNA re-replication in the presence of HPV replication proteins E1 and E2, which ultimately leads to rearrangements within the locus of the integrated viral DNA. The current study indicates that the E1- and E2-dependent DNA replication from the integrated HPV origin follows the "onion skin"-type replication mode and generates a heterogeneous population of replication intermediates. These include linear, branched, open circular, and supercoiled plasmids, as identified by two-dimensional neutral-neutral gel-electrophoresis. We used immunofluorescence analysis to show that the DNA repair/recombination centers are assembled at the sites of the integrated HPV replication. These centers recruit viral and cellular replication proteins, the MRE complex, Ku70/80, ATM, Chk2, and, to some extent, ATRIP and Chk1 (S317). In addition, the synthesis of histone gammaH2AX, which is a hallmark of DNA double strand breaks, is induced, and Chk2 is activated by phosphorylation in the HPV-replicating cells. These changes suggest that the integrated HPV replication intermediates are processed by the activated cellular DNA repair/recombination machinery, which results in cross-chromosomal translocations as detected by metaphase FISH. We also confirmed that the replicating HPV episomes that expressed the physiological levels of viral replication proteins could induce genomic instability in the cells with integrated HPV. We conclude that the HPV replication origin within the host chromosome is one of the key factors that triggers the development of HPV-associated cancers. It could be used as a starting point for the "onion skin"-type of DNA replication whenever the HPV plasmid exists in the same cell, which endangers the host genomic integrity during the initial integration and after the de novo infection.

Show MeSH
Related in: MedlinePlus