Limits...
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.

Show MeSH

Related in: MedlinePlus

Re-replication of the integrated HPV induces the instability of chromosome structure.SiHa cells were transfected with 10 µg of HPV18 E1 and 5 µg of E2 expression plasmids. Single cell subcloning was performed 72 h after transfection [30]. SiHa cells and the subclones with novel restriction patterns were analyzed by FISH. (A) FISH analysis of the interphase nuclei (left panel) and metaphase chromosome spreads (the middle and the right panels) of SiHa cells. (B) Interphase FISH (left panel) and metaphase FISH (the middle and the right panels) analyses of the subclone with cross-chromosomal translocation. The integrated HPV16 is visible as green (Alexa Fluor 488), and the subtelomeric region of the chromosome 13 is visible as red (Texas Red). The translocation of 13q is indicated with the white arrowhead.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2666264&req=5

ppat-1000397-g007: Re-replication of the integrated HPV induces the instability of chromosome structure.SiHa cells were transfected with 10 µg of HPV18 E1 and 5 µg of E2 expression plasmids. Single cell subcloning was performed 72 h after transfection [30]. SiHa cells and the subclones with novel restriction patterns were analyzed by FISH. (A) FISH analysis of the interphase nuclei (left panel) and metaphase chromosome spreads (the middle and the right panels) of SiHa cells. (B) Interphase FISH (left panel) and metaphase FISH (the middle and the right panels) analyses of the subclone with cross-chromosomal translocation. The integrated HPV16 is visible as green (Alexa Fluor 488), and the subtelomeric region of the chromosome 13 is visible as red (Texas Red). The translocation of 13q is indicated with the white arrowhead.

Mentions: Cervical carcinogenesis is associated with the acquisition of structural and numerical chromosomal abnormalities after the integration of HPV into the host cell genome [26],[34]. One potential reason is believed to be the increased levels of the HPV E6 and E7 proteins, which is caused by the disruption of E2 transcriptional repressor expression [23],[31]. However, there is also an alternative mechanism that might cause the chromosomal rearrangements, which involves the over-amplification of the integrated HPV combined with the cellular attempt to fix the resulting DSBs by HR and NHEJ. We previously demonstrated, by restriction analysis, that the local region of the integrated HPV DNA changes dramatically upon in situ re-replication of the integrated HPV [30]. In this previous work, SiHa cells were transfected with the HPV16 E1 and E2 expression plasmids, the resulting transfected cells were single cell subcloned, and changes in the HPV16 restriction pattern, which could represent either an internal rearrangement or a reintegration at a novel site, were examined. The subclones with altered restriction pattern was further investigated in the current study by metaphase FISH in order to detect possible chromosome alterations that are associated with the replication of the integrated HPV. We used the tyramide-enhanced FISH method for the detection of HPV16 sequence in combination with a subtelomeric probe specific for chromosome 13 (CytoCell). The results demonstrate that there are two chromosome 13's within the SiHa cells and that both carry the HPV sequence (Figure 7A, one nucleus in interphase, and two metaphase chromosome spreads are presented). The HPV16 DNA was labeled with Alexa Fluor 488 and was visible as green dots, while the chromosome 13 subtelomeric regions were labeled with Texas Red and were visible as red dots. More importantly, the de novo cross-chromosomal translocation of the HPV16 genome along with the entire q-arm of chromosome 13 could be detected in one of the subclones, where the DNA replication of integrated HPV had been initiated (Figure 7B, one nucleus in interphase and two metaphase chromosome spreads are presented). As a result, there is third 13q arm in the haploid genome of this subclone. Immunofluorescence analyses of the subclone showed three replication foci as compared to the two foci that we exclusively detected in SiHa cells (data not shown). Over one hundred metaphase spreads of SiHa cells were analyzed and no type of heterogenity in our cell population was detected with regard to the HPV integration site.


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)

Re-replication of the integrated HPV induces the instability of chromosome structure.SiHa cells were transfected with 10 µg of HPV18 E1 and 5 µg of E2 expression plasmids. Single cell subcloning was performed 72 h after transfection [30]. SiHa cells and the subclones with novel restriction patterns were analyzed by FISH. (A) FISH analysis of the interphase nuclei (left panel) and metaphase chromosome spreads (the middle and the right panels) of SiHa cells. (B) Interphase FISH (left panel) and metaphase FISH (the middle and the right panels) analyses of the subclone with cross-chromosomal translocation. The integrated HPV16 is visible as green (Alexa Fluor 488), and the subtelomeric region of the chromosome 13 is visible as red (Texas Red). The translocation of 13q is indicated with the white arrowhead.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000397-g007: Re-replication of the integrated HPV induces the instability of chromosome structure.SiHa cells were transfected with 10 µg of HPV18 E1 and 5 µg of E2 expression plasmids. Single cell subcloning was performed 72 h after transfection [30]. SiHa cells and the subclones with novel restriction patterns were analyzed by FISH. (A) FISH analysis of the interphase nuclei (left panel) and metaphase chromosome spreads (the middle and the right panels) of SiHa cells. (B) Interphase FISH (left panel) and metaphase FISH (the middle and the right panels) analyses of the subclone with cross-chromosomal translocation. The integrated HPV16 is visible as green (Alexa Fluor 488), and the subtelomeric region of the chromosome 13 is visible as red (Texas Red). The translocation of 13q is indicated with the white arrowhead.
Mentions: Cervical carcinogenesis is associated with the acquisition of structural and numerical chromosomal abnormalities after the integration of HPV into the host cell genome [26],[34]. One potential reason is believed to be the increased levels of the HPV E6 and E7 proteins, which is caused by the disruption of E2 transcriptional repressor expression [23],[31]. However, there is also an alternative mechanism that might cause the chromosomal rearrangements, which involves the over-amplification of the integrated HPV combined with the cellular attempt to fix the resulting DSBs by HR and NHEJ. We previously demonstrated, by restriction analysis, that the local region of the integrated HPV DNA changes dramatically upon in situ re-replication of the integrated HPV [30]. In this previous work, SiHa cells were transfected with the HPV16 E1 and E2 expression plasmids, the resulting transfected cells were single cell subcloned, and changes in the HPV16 restriction pattern, which could represent either an internal rearrangement or a reintegration at a novel site, were examined. The subclones with altered restriction pattern was further investigated in the current study by metaphase FISH in order to detect possible chromosome alterations that are associated with the replication of the integrated HPV. We used the tyramide-enhanced FISH method for the detection of HPV16 sequence in combination with a subtelomeric probe specific for chromosome 13 (CytoCell). The results demonstrate that there are two chromosome 13's within the SiHa cells and that both carry the HPV sequence (Figure 7A, one nucleus in interphase, and two metaphase chromosome spreads are presented). The HPV16 DNA was labeled with Alexa Fluor 488 and was visible as green dots, while the chromosome 13 subtelomeric regions were labeled with Texas Red and were visible as red dots. More importantly, the de novo cross-chromosomal translocation of the HPV16 genome along with the entire q-arm of chromosome 13 could be detected in one of the subclones, where the DNA replication of integrated HPV had been initiated (Figure 7B, one nucleus in interphase and two metaphase chromosome spreads are presented). As a result, there is third 13q arm in the haploid genome of this subclone. Immunofluorescence analyses of the subclone showed three replication foci as compared to the two foci that we exclusively detected in SiHa cells (data not shown). Over one hundred metaphase spreads of SiHa cells were analyzed and no type of heterogenity in our cell population was detected with regard to the HPV integration site.

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