No abstract

DNA topoisomerase IIalpha is an essential enzyme for chromosome segregation during mitosis. Consistent with a cell division-specific role, the expression of the topoisomerase IIalpha gene is strongly influenced by the proliferation status of cells. The p53 protein is one of the most important regulators of cell cycle progression in mammals, with an apparent dual role in the induction of cell cycle arrest following cytotoxic insults and in the regulation of the apoptotic cell death pathway. We have analysed whether p53 plays a role in regulating expression of the human topoisomerase IIalpha gene. We show that wild-type, but not mutant, p53 is able to decrease substantially the activity of the full length topoisomerase IIalpha gene promoter. Using a series of constructs comprising various deleted or mutated versions of the promoter lacking critical cis-acting elements, we show that this p53-specific regulation of the topoisomerase IIalpha promoter is independent of all characterised transcription factor binding sites and is directed at the minimal gene promoter. We conclude that expression of wild-type p53 induces downregulation of the human topoisomerase IIalpha promoter by acting on the basal transcription machinery. These findings implicate topoisomerase II as one of the downstream targets for p53-dependent regulation of cell cycle progression in human cells.

S_mnary Topoisomerase II is a key target for several anti-cancer drugs used for breast cancer therapy, including doxorubicin, epirubicin and mitoxantrone. Two isoforms of topoisomerase II (x and /) have been described in human cells which differ in their subcellular localisation, biochemical properties and susceptibility to inhibition by anti-cancer drugs. The relative level of expression of the x and isoforms may contribute to the degree of tumour responsiveness to different chemotherapeutic agents. To assess the relationship between expression of topoisomerase II isoforms and established prognostic factors and pathological variables, 56 primary breast tumour samples were studied. The expression of the two topoisomerase II genes was apparently not co-ordinately regulated in these tissue samples. There was no relationship between any of the commonly used pathological variables [tumour size, lymph node status, S-phase fraction (SPF)] and the level of expression of topoisomerase II mRNA. However, high topoisomerase 112 gene expression was significantly associated with a high SPF (sign-rank test; P=0.01). Moreover, the ratio of mRNA levels for topoisomerase I12 and showed a stronger relationship to SPF (median ratio 0.62 for tumours with SPF 10, and 1.64 for SPF> 10; P=0.0021, sign-rankl test). As expected from previous studies, an SPF> 10 was associated with poor overall survival (P=0.01). Immunohistochemical analysis revealed that topoisomerase II was widely distributed (>90% positive tumour cells), but that topoisomerase H2 expression was less widely expressed, with a pattern of expression similar to that of the proliferation-dependent antigen recognised by Ki67. Because topoisomerase II gene expression showed a log-normal distribution, log-transformed data were used in multivariate analysis of relapse-free survivaL This showed that lymph node status and topoisomerase II mRNA expression were the only significant survival factors (P=0.001 and 0.05, respectively, with relative risks of 1.3 and 1.8). These results indicate that topoisomerase W1, but not /3, expression is dependent upon cellular proliferation status, but that the more widely expressed topoisomerase II protein may play a significant role as a target for antitumour therapy.