Sticky anaphase aberrations after G2-phase arrest of gamma-irradiated human skin fibroblasts: TP53 independence of formation and TP53 dependence of consequences.

We have studied the impact of TP53 status on the extent and nature of chromosome damage seen in human skin fibroblasts after gamma irradiation beyond the G1-phase checkpoint but prior to the G2-phase checkpoint. Mitotic cells were examined in the absence and presence of treatment with nocodazole and the yield of aberrations was scored as a function of time postirradiation. The results revealed substantially greater damage in the absence of nocodazole, indicating that damage was being masked in its presence. While metaphase aberrations were seen exclusively in the presence of nocodazole, anaphase aberrations were seen principally in its absence. Furthermore, these were mostly of an unseparated, or "sticky", type that showed separation of the chromatids in the centromeric region, indicating normal degradation of cohesin, with retention of adhesion further out on the chromatid arms. Using postirradiation BrdU labeling and the absence of nocodazole, we were able to identify mitotic figures up to the third postirradiation mitosis. Analysis of the data revealed that in cells wild-type for TP53 the aberrant anaphases were lost after the first postirradiation mitosis, although they were still found in gradually decreasing amounts into the second and third postirradiation mitoses in E6-expressing cells. The data indicate that the formation of these sticky anaphases is independent of TP53 status, an observation that is consistent with the TP53 independence of transient G2-phase arrest. However, the consequences of the formation of these lesions appear to be very different. In the case of cells wild-type for TP53 this is chronic G1-phase arrest, while in E6 cells it is anaphase catastrophe.

Complexity of the mechanisms of initiation and maintenance of DNA damage-induced G2-phase arrest and subsequent G1-phase arrest: TP53-dependent and TP53-independent roles.

Through a detailed study of cell cycle progression, protein expression, and kinase activity in gamma-irradiated synchronized cultures of human skin fibroblasts, distinct mechanisms of initiation and maintenance of G2-phase and subsequent G1-phase arrests have been elucidated. Normal and E6-expressing fibroblasts were used to examine the role of TP53 in these processes. While G2 arrest is correlated with decreased cyclin B1/CDC2 kinase activity, the mechanisms associated with initiation and maintenance of the arrest are quite different. Initiation of the transient arrest is TP53-independent and is due to inhibitory phosphorylation of CDC2 at Tyr15. Maintenance of the G2 arrest is dependent on TP53 and is due to decreased levels of cyclin B1 mRNA and a corresponding decline in cyclin B1 protein level. After transiently arresting in G2 phase, normal cells chronically arrest in the subsequent G1 phase while E6-expressing cells continue to cycle. The initiation of this TP53-dependent G1-phase arrest occurs despite the presence of substantial levels of cyclin D1/CDK4 and cyclin E/CDK2 kinase activities, hyperphosphoryated RB, and active E2F1. CDKN1A (also known as p21(WAF1/CIP1)) levels remain elevated during this period. Furthermore, CDKN1A-dependent inhibition of PCNA activity does not appear to be the mechanism for this early G1 arrest. Thus the inhibition of entry of irradiated cells into S phase does not appear to be related to DNA-bound PCNA complexed to CDKN1A. The mechanism of chronic G1 arrest involves the down-regulation of specific proteins with a resultant loss of cyclin E/CDK2 kinase activity.

Localization of deletion to a 300 Kb interval of chromosome 11q13 in cervical cancer.

Previous molecular genetic studies on HeLa cell (a cervical cancer cell line) derived non-tumorigenic and tumorigenic hybrids have localized a tumor suppressor gene to the long arm of chromosome 11. Analysis of cervical cancer cell lines using chromosome 11 specific probes showed deletion and translocation of 11q13 sequences in five out of eight cell lines. Fluorescence in situ hybridization (FISH), using 11q13 specific probes, has shown interstitial deletion of 11q13 sequences in the HeLa cells. In order to determine whether 11q13 deletions occur in primary cervical tumors, we analysed 36 tumors using 20 different microsatellite and RFLP markers. Semi automated fluorescein based allelotyping was performed to identify loss of heterozygosity (LOH) in tumors. The results showed allelic loss in 17 (47%) tumors. Three different regions of loss, one near MEN1, the second near D11S913, and the third near INT2 locus were observed. The smallest region of deletion overlap at the D11S913 locus was localized to a 300 Kb distance between D11S4908 and D11S5023. Fluorescence in situ hybridization (FISH), using 11q13 specific cosmid and BAC (bacterial artificial chromosome) probes, confirmed allelic deletion in the tumors. PCR analysis further identified homozygous deletion of 11q13 sequences in a primary tumor, in HeLa cells and in two HeLa cell derived tumorigenic hybrid cell lines. The homozygous deletion in the cell lines was mapped to a 5.7 kb sequence of 11q13. We hypothesize therefore that a putative cervical cancer tumor suppressor gene exists within the 300 kb of chromosome 11q13.