Cyclin B1 is the regulatory subunit of cyclin-dependent kinase 1 (Cdk1) and is critical for the initiation of mitosis. Accumulating data indicate that the deregulation of cyclin B1 is tightly linked to neoplastic transformation. To study the phenotype and the potential preclinical relevance, we generated HeLa cell lines stably transfected with the plasmids encompassing short hairpin RNA (shRNA) targeting cyclin B1. We demonstrate that the reduction of cyclin B1 caused inhibition of proliferation by arresting cells in G2 phase and by inducing apoptosis. Cells, entering mitosis, were impaired in chromosome condensation and alignment. Importantly, HeLa cells with reduced cyclin B1 were more susceptible to the treatment of small interfering RNA targeting Polo-like kinase 1 (Plk1) and to the administration of the chemotherapeutic agent taxol. Finally, HeLa cells with reduced cyclin B1 showed inhibited tumor growth in nude mice compared to that of control cells. In summary, our data indicate that cyclin B1 is an essential molecule for tumor cell survival and aggressive proliferation, suggesting that the downregulation of cyclin B1, especially in combination with other molecular targets, might become an interesting strategy for antitumor intervention.

Background: Cyclin B1, the regulatory subunit of cyclin-dependent kinase 1 (Cdk1), is essential for the transition from G2 phase to mitosis. Cyclin B1 is very often found to be overexpressed in primary breast and cervical cancer cells as well as in cancer cell lines. Its expression is correlated with the malignancy of gynecological cancers.

The function of estrogen receptor beta (ER-b) in mammary tissue is not completely understood. While early observations were often conflicting, more recent data suggest an important role as a tumor-suppressor gene. A decrease of ER-b expression has been observed in ductal carcinoma in situ and invasive carcinoma as compared with benign mammary epithelial cells. The loss of ER-b resulted in abnormal growth of mammary epithelial cells. We have previously shown that the mRNA expression of the ER-b gene is almost totally suppressed in breast carcinomas from patients with a poor prognosis. Here we analyzed whether methylation changes in the different promoters of ER-b are responsible for the loss of expression of the gene. A methylation assay with high specificity and sensitivity was developed, and a panel of breast tissue samples (n = 175) was characterized for methylation status. In contrast to benign breast, more than two-thirds of invasive breast cancers showed a high degree of methylation. Importantly, increased methylation was also detectable in numerous premalignant lesions. By analysis of breast tumors, previously characterized by geneexpression profiling, methylation was predominantly detected in a subgroup of patients with an unfavorable prognosis, suggesting a possible prognostic value of the ER-b methylation status. We also investigated the structural characteristics of the two ER-b promoters, which were both found to be closely associated with a second, downstream, localized and opposite-oriented promoter. However, we could not detect endogenous antisense RNA transcribed from these promoters, which may be involved in epigenetic gene silencing. We also failed to induce ER-b promoter methylation by expressing siRNAs in cell lines. Interestingly, by comparing the promoter sequences of ER-b with other genes known to be epigenetically inactivated in breast cancers, we identified a sequence motif possibly involved in promoter methylation.

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Our experiments reveal that expression of enzymes from the sphingolipid metabolism has prognostic implications in breast cancer.