Overexpression of Aurora-A kinase promotes tumor cell proliferation and inhibits apoptosis in esophageal squamous cell carcinoma cell line.

Aurora-A kinase, a serine/threonine protein kinase, is a potential oncogene. Amplification and overexpression of Aurora-A have been found in several types of human tumors, including esophageal squamous cell carcinoma (ESCC). It has been demonstrated that cells overexpressing Aurora-A are more resistant to cisplatin-induced apoptosis. However, the molecular mechanisms mediating these effects remain largely unknown. In this report, we showed that overexpression of Aurora-A through stable transfection of pEGFP-Aurora-A in human ESCC KYSE150 cells significantly promoted cell proliferation and inhibited cisplatin- or UV irradiation-induced apoptosis. Cleavages of caspase-3 and poly (ADP-ribose) polymerase (PARP) in Aurora-A overexpressing cells were substantially reduced after cisplatin or UV treatment. Furthermore, we found that silencing of endogenous Aurora-A kinase with siRNA substantially enhanced sensitivity to cisplatin- or UV-induced apoptosis in human ESCC EC9706 cells. In parallel, overexpression of Aurora-A potently upregulated the expression of Bcl-2. Moreover, the knockdown of Bcl-2 by siRNA abrogated the Aurora-A's effect on inhibiting apoptosis. Taken together, these data provide evidence that Aurora-A overexpression promoting cell proliferation and inhibiting apoptosis, suggesting a novel mechanism that is closely related to malignant phenotype and anti-cancer drugs resistance of ESCC cells.

[Gadd45 mediated G2/M cell cycle arrest induced by BRCA1].

BACKGROUND & OBJECTIVE: It is considered that tumor suppressor gene BRCA1 is an important factor in the regulation of cell cycle checkpoint, but the molecular mechanism by which BRCA1 regulates cell cycle G(2)/M arrest is less known. The objective of this study was to investigate the effects of Gadd45 on the BRCA1-induced cell growth suppression. METHODS: BRCA1 induction of Gadd45 protein was analyzed using Western-blot assay following cells transfection with BRCA1 expression vector and cell sorting. Activation of the Gadd45 promoter by BRCA1 was determined by CAT assay. Effect of antisense Gadd45 on the BRCA1-induced cell cycle G(2)-M arrest was examined by flow cytometry analysis. And the effects of antisense Gadd45 on BRCA1-mediated growth suppression in HeLa and HCT116 cell lines was determined by colony formation assay. RESULTS: Gadd45 protein was highly induced after expression of BRCA1. BRCA1 strongly activated the Gadd45 promoter. Antisense Gadd45 substantially abrogated BRCA1-activated cell cycle G(2)-M arrest and BRCA1-induced cell growth suppression on HeLa and HCT116 cell lines. CONCLUSION: Gadd45 is a BRCA1-regulated downstream gene and mediates the role of BRCA1 in the control of cell cycle G(2)/M arrest and growth suppression.

Telomerase activity in cervical intraepithelial neoplasia.

BACKGROUND: It was reported that telomerase expression is closely associated with cellular immortality and cancer. This study was designed to investigate the relationship between telomerase expression and the carcinogenesis of cervical cancer, the possible use of telomerase as a marker of cervical intraepithelial neoplasia (CIN) progression or regression, and the natural history of CIN. METHODS: Telomeric repeat amplification protocol (TRAP) assay was used to measure telomerase activity in cervical scrapings and biopsy samples obtained from 105 cases affected with various cervical conditions, including chronic cervicitis (n = 20), CIN (n = 64, 16 cases of CIN I, 20 cases of CIN II, and 28 cases of CIN III), and invasive squamous cell carcinoma (n = 21). RESULTS: In exfoliated cell samples, telomerase activity was detected in 5 of 20 (25.0%) cases of cervicitis, 10 of 16 (62.5%) cases of CIN I, 11 of 20 (55.0%) cases of CIN II, 23 of 28 (82.1%) cases of CIN III, and 13 of 21 (61.9%) cases of carcinoma. In cervical biopsy samples, telomerase activity was detected in 6 of 20 (30.0%) cases of cervicitis, 8 of 16 (50.0%) cases of CIN I, 9 of 20 (45.0%) cases of CIN II, 27 of 28 (96.4%) cases of CIN III, and 20 of 21 (95.2%) cases of carcinoma. Telomerase activation was significantly higher in CIN samples than in cervicitis samples. Telomerase activity was detected at similar frequency in samples from cervical scrapings and cervical biopsies. CONCLUSION: These results seem to suggest that telomerase expression may be associated with carcinogenesis of the cervix. TRAP assay of cervical scraping samples could be used to monitor and predict the development of CIN in clinical practice.

[Suppression of HCT116 growth of cells by Gadd45].

OBJECTIVE: To study the mechanism of suppression of growth of HCT116 human colon carcinoma cells by Gadd45 gene. METHODS: HCT116 human colon carcinoma cells were transfected with pTRE-Gadd45 vector so as to establish Gadd45-inducible cell line that was cultured in medium with tetracycline. Then tetracycline was withdrawn. The number of cell clones was counted. Flow cytometry was used to detect the percentages of cells at the G1, S, and G2-M phases. TUNEL technique was used to detect the apoptosis of cells. Western blotting was used to analyze the expression of Gadd45 protein, and apoptosis-related proteins: poly-ADP-ribose polymerase (PARP), and caspase 3 protein. RESULTS: Gadd45 protein was not expressed in the HCT116 cells cultured in the medium with tetracycline, however, it was expressed with a gradually increased level in the cells cultured in the medium from which tetracycline was withdrawn, The clone formation rate of HCT116 cells was 100% in the medium with tetracycline, however, was only 14.2% in the medium with tetracycline withdrawal, with a suppression rate of more than 85%. The percentage of cells in G(2)-M phase was significantly increased in the cells cultured in the medium with tetracycline withdrawal. 24-36 hours after the withdrawal of tetracycline, PARP and caspase 3 protein were activated remarkably. CONCLUSION: High expression of Gadd45 inhibits the growth of HCT116 cells, through inducing G2-M arrest and activating apoptotic pathway.