Chromosomal instability is correlated with telomere erosion and inactivation of G2 checkpoint function in human fibroblasts expressing human papillomavirus type 16 E6 oncoprotein

Abstract

Cell cycle checkpoints and tumor suppressor gene functions appear to be required for the maintenance of a stable genome in proliferating cells. In this study chromosomal destabilization was monitored in relation to telomere structure, lifespan control and G2 checkpoint function. Replicative senescence was inactivated in secondary cultures of human skin ®broblasts by expressing the human papillomavirus type 16 (HPV-16) E6 oncoprotein to inactivate p53. Chromosome aberrations were enumerated during in vitro aging of isogenic control (F5neo) and HPV-16E6-expressing (F5E6) ®broblasts. We found that structural and numerical aberrations in chromosomes were signi®cantly increased in F5E6 cells during aging in vitro and¯uorescence in situ hybridization (FISH) analysis using chromosome-speci®c probes demonstrated the occurrence of rearrangements involving chromosome 4 and 6 in genetically unstable F5E6 cells. Flow cytometry and karyotypic analyses revealed increased polyploidy and aneuploidy in F5E6 cells only at passages 416, although these cells displayed defective mitotic spindle checkpoint function associated with inactivation of p53 at passages 5 and 16. G2 checkpoint function was con®rmed to be gradually but progressively inactivated during in vitro aging of E6-expressing cells. Aging of F5neo ®broblasts was documented during in vitro passaging by induction of a senescence-associated marker, pH 6.0 lysosomal b-galactosidase. F5E6 cells displayed extension of in vitro lifespan and did not induce b-galactosidase at high passage. Erosion of telomeres during in vitro aging of telomerase-negative F5neo cells was demonstrated by Southern hybridization and by quantitative FISH analysis on an individual cell level. Telomeric signals diminished continuously as F5neo cells aged in vitro being reduced by 80% near the time of replicative senescence. Telomeric signals detected by FISH also decreased continuously during aging of telomerasenegative F5E6 cells, but telomeres appeared to be stabilized at passage 34 when telomerase was expressed. Chromosomal instability in E6-expressing cells was correlated (P50.05) with both loss of telomeric signals and inactivation of G2 checkpoint function. The results suggest that chromosomal stability depends upon a complex interaction among the systems of telomere length maintenance and cell cycle checkpoints.

Telomere erosion and chromosomal instability in cells expressing the HPV oncogene 16E6

“…In those same mice, a strong correlation between telomere erosion and the number of anaphase bridges was found (Rudolph et al, 2001). Correlation between chromosomal instability and telomere erosion was demonstrated in human fibroblasts expressing HPV 16E6 (Filatov et al, 1998). In human keratinocyte clones expressing the HPV oncogenes 16E6 and 16E7, the frequency of anaphase bridges is dependent on the level of telomerase activity.…”

Degradation of ATM-Independent Decatenation Checkpoint Function in Human Cells is Secondary to Inactivation of p53 and Correlated with Chromosomal Destabilization

“…42 Although the E6-expressing cells are susceptible to endoreduplication when stressed with the spindle poisons colcemid and nocodazole 80,87 they spontaneously acquire polyploidy only after in vitro aging associated with attenuation and inactivation of DNA damage-responsive G2 checkpoint function. 42,80 This result suggested that reduced G2 checkpoint function in E6-expressing cells might produce stress on the mitotic spindle and thereby stimulate endoreduplication. The results described above suggested a model that explains how G2 checkpoint function limits stress on the mitotic spindle (Fig.…”

“…84 The inactivation of G2 checkpoint function that accompanies cellular aging in the HPV16E6-expressing fibroblasts appears to be a response to chromatid damage associated with telomere erosion and formation of unstable dicentric chromosomes. 80,85 Cells lacking G2 checkpoint function gain a growth advantage by being able to progress into mitosis with damaged chromatids. Induction of telomerase before E6-expressing cells acquired chromosomal instability preserved G2 checkpoint function and stabilized chromosomes ( Table 2).…”

“…The role of p53 in ICRF-193-induced G2 delay was tested using diploid fibroblast strains that had been transduced with HPV16E6 to inactivate p53. 42,80 As shown in Figure 4A, NHF4-neo fibroblasts displayed inhibition and recovery of mitosis 2-6 h after 0.5 Gy of IR. NHF4-E6 fibroblasts expressing HPV16E6 to inactivate p53 also displayed inhibition and recovery of mitosis after γ-irradiation when at low population doubling levels (PDL<20).…”

Regulation of the G2/M transition by p53

“…The number of IMR-90 normal lung ®broblasts entering mitosis after exposure to ionizing radiation was much higher after expression of HPV-E6, suggesting that p53 is required for G2 arrest . In contrast the expression of HPV-E6 in human foreskin ®broblasts did not a ect G2 arrest in response to ionizing radiation initially (Filatov et al, 1998;Kaufmann et al, 1997;Passalaris et al, 1999;Paules et al, 1995). However, as early as 12 population doublings after the expression of HPV-E6 many cells entered mitosis after exposure to ionizing radiation (Kaufmann et al, 1997), and the G2 arrest in response to DNA damage was completely lost upon more extensive in vitro propagation (Filatov et al, 1998;Kaufmann et al, 1997).…”