ABSTRACT
The aims of the present study were to investigate the effect of overexpressed exogenous glutathione S-transferase π 1 (GSTP1) gene on the radiosensitivity of the HeLa human cervical cancer cell line and conduct a preliminarily investigation into the underlying mechanisms of the effect. The full-length sequence of human GSTP1 was obtained by performing a polymerase chain reaction (PCR) using primers based on the GenBank sequence of GSTP1. Subsequently, the gene was cloned into a recombinant eukaryotic expression plasmid, and the resulting construct was confirmed by restriction analysis and DNA sequencing. A HeLa cell line that was stably expressing high levels of GSTP1 was obtained through stable transfection of the constructed plasmids using lipofectamine and screening for G418 resistance, as demonstrated by reverse transcription-PCR. Using the transfected HeLa cells, a colony formation assay was conducted to detect the influence of GSTP1 overexpression on the cell radiosensitivity. Furthermore, flow cytometry was used to investigate the effect of GSTP1 overexpression on cell cycle progression, with the protein expression levels of the cell cycle regulating factor cyclin B1 detected using western blot analysis. Colony formation and G2/M phase arrest in the GSTP1-expressing cells were significantly increased compared with the control group (P<0.01). In addition, the expression of cyclin B1 was significantly reduced in the GSTP1-expressing cells. These results demonstrated that increased expression of GSTP1 inhibits radiosensitivity in HeLa cells. The mechanism underlying this effect may be associated with the ability of the GSTP1 protein to reduce cyclin B1 expression, resulting in significant G2/M phase arrest.
ABSTRACT
OBJECTIVES: To investigate the expression of P300/CBP-associated factor (PCAF) protein in intestinal type gastric cancer (ITGC); analyze the relationship between the expression of PCAF protein and the clinical pathological characteristics of patients; explore the effects of PCAF protein on biological behaviors of ITGC. RESULTS: The expression of PCAF was markedly down-regulated in GC cell lines and ITGC tissues. PCAF was able to suppress tumorigenicity of GC cells both in vitro and in vivo, including colony formation in soft agar and tumor formation in nude mice. PCAF could also inhibit GC cells entering S phase from G1 phase. Statistical analysis displayed a significant correlation in PCAF expression with the gastric wall invasion, tumor size, TNM stage, p21, pRb (P<0.001) and PCNA (P<0.01) in ITGC specimens. A reduced PCAF protein expression correlated significantly with a mutant type p53 protein expression (P<0.01). Univariate analysis indicated that the patients demonstrating the high-PCAF/wild type p53 expression have a significantly (P<0.0001) better overall survival (OS), while multivariate analysis indicated that the location, lymph node metastasis, PCAF/p53 (P<0.0001), gastric wall invasion (P=0.001) and PCNA (P=0.018) are independently significant prognostic factors for OS. METHODS: Immunohistochemistry was performed to evaluate the expression of PCAF in a large subset containing 406 ITGC samples. Eukaryotic expression plasmid pcDNA3.1/PCAF was constructed and transfected into the human gastric cancer cell line SGC-7901 and protein expression was detected by Western blot. The proliferation and cell cycle of gastric cancer cells were evaluated by MTT assay and flow cytometry. Tumor growth in nude mice was used to access the tumorigenicity of gastric cancer cells. Apoptosis cells were detected by TUNEL staining. CONCLUSION: Reduced expression of PCAF plays an important role in the development of ITGC and correlates with a poor clinical outcome.
