Pericentromeric regions are refractory to prompt repair after replication stress-induced breakage in HPV16 E6E7-expressing epithelial cells

Abstract

Chromosomal instability is the major form of genomic instability in cancer cells. Amongst various forms of chromosomal instability, pericentromeric or centromeric instability remains particularly poorly understood. In the present study, we found that pericentromeric instability, evidenced by dynamic formation of pericentromeric or centromeric rearrangements, breaks, deletions or iso-chromosomes, was a general phenomenon in human cells immortalized by expression of human papillomavirus type 16 E6 and E7 (HPV16 E6E7). In particular, for the first time, we surprisingly found a dramatic increase in the proportion of pericentromeric chromosomal aberrations relative to total aberrations in HPV16 E6E7-expressing cells 72 h after release from aphidicolin (APH)-induced replication stress, with pericentromeric chromosomal aberrations becoming the predominant type of structural aberrations (~70% of total aberrations). In contrast, pericentromeric aberrations accounted for only about 20% of total aberrations in cells at the end of APH treatment. This increase in relative proportion of pericentromeric aberrations after release from APH treatment revealed that pericentromeric breaks induced by replication stress are refractory to prompt repair in HPV16 E6E7-expressing epithelial cells. Telomerase-immortalized epithelial cells without HPV16 E6E7 expression did not exhibit such preferential pericentromeric instability after release from APH treatment. Cancer development is often associated with replication stress. Since HPV16 E6 and E7 inactivate p53 and Rb, and p53 and Rb pathway defects are common in cancer, our finding that pericentromeric regions are refractory to prompt repair after replication stress-induced breakage in HPV16 E6E7-expressing cells may shed light on mechanism of general pericentromeric instability in cancer.

Figure 1. Examples of de novo centromeric breaks.

Centromeric regions were identified by the centromeric constrictions, dark DAPI staining and pan-centromere FISH (green). Note that there is no telomere signal at the deleted sites indicated by arrows.

Figure 2. Chromatid breaks induced by APH treatment.

A: Frequencies of chromatid breaks measured at the end of APH (+) or DMSO (−) treatment. P≤0.05 for all frequencies after APH treament compared with DMSO-treated cells. B: Examples of chromatid breaks (indicated by arrows). The upper panel shows non-pericentromeric chromatid breaks in fragile sites. The lower panel shows pericentromeric chromatid breaks.

Figure 3. Chromosomal aberrations 72 h after release from APH treatment.

A: Frequencies of non-clonal chromosomal aberrations. B: Examples of pericentromeric chromosomal aberrations. Centromeric regions were identified by the centromeric constrictions, intenseDAPI staining and pan-centromere FISH (green). First panel: An example of pericentromeric chromosomal deletion. Second panel: An example of pericentromeric chromosomal breaks with both arms present. Third panel: An example of pericentromeric chromosomal translocation. Note that the joined region was at centromeric constriction region with centromere FISH signals. Lowest panel: An example of dicentrics with joined regions involving centromeric ends (Xp and 21p).

Figure 4. Chromosome aberrations after APH treatment in hTERT-immortalized cell lines without expression of HPV16 E6E7.

A: Frequencies of chromatid breaks measured at the end of APH treatment. B: Frequencies of non-clonal chromosomal aberrations measured at 72 h after removal of APH.

Figure 5. Number of large γ-H2AX foci juxtaposed with centromeres per 100 cells.

Two hundred cells were analyzed for each experimental condition. All cell lines were analyzed at PD 80. P<0.05 for the differences between HPV 16-E6E7-hTERT-immortalized cell lines and hTERT-immortalized cell lines of the same cell origins without APH treatment, or 72 h after removal of APH.

Figure 6. Immunofluorescene staining of centromeres and γ-H2AX.

Typical examples of co-immunostaning of centromeres (red) and γ-H2AX (green). DNA was stained blue. Arrows indicate the large γ-H2AX foci juxtaposed to centromeres.