The G₂ Checkpoint Is Maintained by Redundant Pathways

Abstract

While p53 activity is critical for a DNA damage-induced G(1) checkpoint, its role in the G(2) checkpoint has not been compelling because cells lacking p53 retain the ability to arrest in G(2) following DNA damage. Comparison between normal human foreskin fibroblasts (HFFs) and HFFs in which p53 was eliminated by transduction with human papillomavirus type 16 E6 showed that treatment with adriamycin initiated arrest in G(2) with active cyclin B/CDC2 kinase, regardless of p53 status. Both E6-transduced HFFs and control (LXSN)-transduced cells maintained a prolonged arrest in G(2); however cells with functional p53 extinguished cyclin B-associated kinase activity. Down regulation was mediated by p53-dependent transcriptional repression of the CDC2 and cyclin B promoters. In contrast, cells lacking p53 showed a prolonged G(2) arrest despite high levels of cyclin B/CDC2 kinase activity, at least some of which translocated into the nucleus. Furthermore, the G(2) checkpoint became attenuated as p53-deficient cells aged in culture. Thus, at late passage, E6-transduced HFFs entered mitosis following DNA damage, whereas the age-matched parental HFFs sustained a G(2) arrest. These results indicate that normal cells have p53-independent pathways to maintain DNA damage-induced G(2) arrest, which may be augmented by p53-dependent functions, and that cells lacking p53 are at greater risk of losing the pathway that protects against aneuploidy.

p21-Mediated Nuclear Retention of Cyclin B1-Cdk1 in Response to Genotoxic Stress

“…Centrosomal staining with an anti-PS133 signal together with an elevated Mpm2 signal in drugtreated E6 cells led us to conclude that these cells were not truly arrested in G2 phase but were in fact undergoing early mitotic events ( Figure 3D). This is in agreement with the observation that in drug-arrested E6 cells, cyclin B1-associated kinase activity, albeit weak in comparison with mitotic cells, is significantly higher than that of cyclin B1 complexes in G2-arrested wild-type NHFs (Dulic et al, 1998;Passalaris et al, 1999;Baus et al, 2003).…”

“…Centrosomal staining with an anti-PS133 signal together with an elevated Mpm2 signal in drugtreated E6 cells led us to conclude that these cells were not truly arrested in G2 phase but were in fact undergoing early mitotic events ( Figure 3D). This is in agreement with the observation that in drug-arrested E6 cells, cyclin B1-associated kinase activity, albeit weak in comparison with mitotic cells, is significantly higher than that of cyclin B1 complexes in G2-arrested wild-type NHFs (Dulic et al, 1998;Passalaris et al, 1999;Baus et al, 2003).To eliminate the possible contribution of other p53 (or E6) targets, we studied cyclin B1 localization in response to DNA damage (ICRF-193 and ␥-irradiation) in asynchronously growing p21 null fibroblasts (Wei et al, 2001). Unlike their wild-type counterparts (LF1), p21 Ϫ/Ϫ NHF failed to block pRb phosphorylation and efficiently inactivate cyclin B1-associated kinase activity in the presence of ICRF-193 (Figure 4, A and B), thus confirming our earlier observation in E6 cells (Baus et al, 2003).…”

“…We have recently shown that this could be due to its ability to block the Cdk-dependent phosphorylation of pocket proteins (Baus et al, 2003). Interestingly, in spite of its strong association with cyclin A-Cdk1 (Baus et al, 2003), data suggest that cyclin B1-Cdk1 complexes are not an important target of p21 (Winters et al, 1998;Passalaris et al, 1999;Flatt et al, 2000;Smits et al, 2000;Deming et al, 2001).…”

Hsp90-inhibitor geldanamycin abrogates G2 arrest in p53-negative leukemia cell lines through the depletion of Chk1

“…As previously reported in p53-positive cells after g-radiation (Bunz et al, 1998), p21 should directly block the cyclin B1-Cdc2 activity even after the depletion of Chk1 in ML-1 cells. The G 2 checkpoint of ML-1 cells should thus consist of p53-dependent and p53-independent pathways (Passalaris et al, 1999), and GM abrogates only the latter Geldanamycin-induced Chk1 depletion abrogates G 2 arrest K Sugimoto et al pathway(s). In contrast, intra-S phase arrest elicited by cytarabine or gemcitabine seems to be dependent almost solely on Chk1 even in ML-1 cells, and therefore readily abrogated by GM (Arlander et al, 2003;Mesa et al, 2005).…”

Distinctive patterns of Her‐2/neu, c‐myc, and cyclin D1 gene amplification by fluorescence in situ hybridization in primary human breast cancers

“…The findings in the present study that genetic instability, as exemplified by Her-2/neu amplification, c-myc amplification, and gross aneuploidy, occurs almost exclusively in nonlobular breast tumors, draw attention to the role that p53 dysfunction might play in the development of structural and/or numerical chromosomal abnormalities in non-lobular breast cancers. Studies in experimental systems have demonstrated a relationship between the loss of wild type p53 function and the development of structural chromosomal abnormalities (49) including gene amplification (50,51), loss of pre-, intra-, and post-mitotic checkpoint control (52,53), endoreduplication (52), unequal chromosome segregation at mitosis due to centrosome abnormalities (54) and the development of tetraploidy and gross aneuploidy (49,50). The finding that excess mdm2 also leads to centrosome hyperamplification and to numerical chromosomal instability (55) lends further support to a causal relationship between loss of wild type p53 function and chromosomal instability.…”