Differential promoter methylation may be a key molecular mechanism in regulating BubR1 expression in cancer cells

The BubR1 mitotic-checkpoint protein monitors proper attachment of microtubules to kinetochores, and links regulation of chromosome-spindle attachment to mitotic-checkpoint signaling. Thus, disruption of BubR1 activity results in a loss of checkpoint control, chromosomal instability caused by a premature anaphase, and/or the early onset of tumorigenesis. The mechanisms by which deregulation and/or abnormalities of BubR1 expression operate, however, remain to be elucidated. In this study, we demonstrate that levels of BubR1 expression are significantly increased by demethylation. Bisulfite sequencing analysis revealed that the methylation status of two CpG sites in the essential BubR1 promoter appear to be associated with BubR1 expression levels. Associations of MBD2 and HDAC1 with the BubR1 promoter were significantly relieved by addition of 5-aza-2'-deoxycytidine, an irreversible DNA methyltransferase inhibitor. However, genomic DNA isolated from 31 patients with colorectal carcinomas exhibited a +84A/G polymorphic change in approximately 60% of patients, but this polymorphism had no effect on promoter activity. Our findings indicate that differential regulation of BubR1 expression is associated with changes in BubR1 promoter hypermethylation patterns, but not with promoter polymorphisms, thus providing a novel insight into the molecular regulation of BubR1 expression in human cancer cells.

Plasma TGF-beta1 as a Tumor Marker in Breast Cancer Patients / 대한암학회지

PURPOSE: Transforming Growth Factor-beta1(TGF-beta1) is the most potent inhibitor of the progression of normal mammary epithelial cells through the cell cycle. However, advanced breast cancers are mostly refractory to TGF-beta mediated growth inhibition and produce large amounts of TGF-beta, which may enhance tumor cell invasion and metastasis by its effects on extracellular matrix. Yet, little is known about the association of TGF-beta1 with progression of malignant disease in vivo. In this study, we evaluated the preoperative and postoperative plama level of TGF- in breast cancer and analyzed the utility of plasma TGF-beta1 as possible tumor marker. MATERIALS AND METHODS: ELISA(enzyme-linked immunosorbent assay) was used to measure plasma TGF-beta1 level in 45 newly diagnosed breast cancer patients and in 15 normal healthy people, and the results were compared with clinicopathologic characteristics. RESULTS: The mean plasma TGF-beta1 levels were 1.73+/-0.47 ng/ml in normal people and 5.05+/-1.41 ng/ml in breast cancer patiens. In 37 operated patients, the preoperative plasma TGF-beta1 level was 6.34+/-1.34 ng/ml and decreased to 4.48+/-1.07 ng/ml in patients with follow-up after surgery and 4.74+/-0.79 ng/ml in patients with chemotherapy. However, there was no significant correlation between plasma TGF-beta1 level and known prognostic factors including tumor size, LN involvement, tumor grade, hormone receptor status, and pathology. CONCLUSION: These findings suggest that the plasma TGF-g level can be a tumor marker in breast cancer patients and the association with progression of breast cancer will be explored in future studies.

Effect on Malignant Phenotype of Gastric Cancer Cell Line after p53 Gene Transduction / 대한암학회지

PURPOSE: To evaluate the effect of wild type p53 gene transduction on the malignant phenotypes for metastasis in gastric cancer, we compared the biological phenpotypes of gastric cancer cell lines based on p53 gene status. Then, after retrovirus-mediated wild-type p53 gene transduction, we compared those phenotypes among parent YCC-3 cell line, vector transduced YCC-3v cell line and a clone of YCC-3C3. MATERIAL AND METHODS: Four human gastric cancer celi lines were used; YCC-l(mutant), YCC-2(wild), YCC-3(mutant) and AGS(wild). DNAs of the cell lines were analyzed to evaluate the mobility shift with PCR-SSCP. Tumorigenecity and proliferation were evaluated by soft agar assay and proliferation assay. Migratory capacity was measured by adhesion assay and Boyden chamber assay. p53 protein expression was measured by Western blot analysis and VEGF, WAF-1 were measured by ELISA assay. Angiogenic activity was measured by cross-feeding assay and cell cycle analysis was performed by flowcytometry. In vivo tumorigenicity was measured by xenograft in nude mice. RESULTS: YCC-3 cell line with mutant p53 gene expressed all the phenotypes for the metastasis such as tumorigenicity, migration and angiogenesis. In a stable clone of YCC-3C3, no differences were found in proliferation, cell cycle and WAP-1 expression when compared to those of the control YCC-3v and parent YCC-3 cell line, even if increased p53 protein production was found by Western blot analysis. However, both in vitro and in vivo tumorigenicity were decreased in a stably transduced YCC-3C3 clone. The adhesive capacity was also decreased in YCC-3C3 clone whereas the endothelial cell growth stimulatory effect and VEGF production showed no difference compared to those of the YCC-3v cell line. CONCLUSION: Wild-type p53 gene transduction in gastric cancer cell line decreased tumorigenicity which resulted from decreased colony forming activity and adhesive capacity but not formed changes of angiogenic activity. This suggested the possible application of anti- metastasis strategy with p53 gene therapy in gastric cancer.

Effects of Interleukin-2 Transduction into the Human Hepatoma Cell Lines Using Retroviral Vector / 대한암학회지

PURPOSE: We compared the differences between parent hepatoma cell lines and interleukin-2 (IL-2) transduced hepatoma cell lines using N2A/IL-2 and LNC/IL-2 retrovirus with regards to in vitro sensitivity to peripheral blood monocytes and in vivo tumorigenic activity. MATERIALS AND METHODS: Retroviral vector and producer cell line were constructed and IL-2 gene was transduced into the human hepatoma cell lines (SK-Hep1, Hep-G2, Hep-3B). IL-2 secretion after IL-2 transduction was measured by ELISA. MTT assay for in vitro sensitivity to peripheral blood monocytes was performed and the tumorigenic activity was observed in BALB/c mice and nude mice. RESULTS: IL-2 secretion was 186 pg/10 degrees C cells/24 hrs in SK-Hep1 cell line and was 147 pg/10 (6) cells/24 hrs in Hep-3B cell line with N2A/IL-2 retroviral vector and was 55,000 pg/10 (6) cells/24 hrs with LNC/IL-2 retroviral vector. In vitro sensitivity to peripheral blood monocytes was increased by 163.8~254% in IL-2 transduced hepatoma cell lines (Hep -3B/N2A/IL-2, Hep-G2/N2A/IL-2) compared to those of the parent cell lines. The tumorigenicity was observed in 1 of 3 BALB/c mice and all 3 nude mice. Simultaneous injection of 1 X 10 (7) cells of the parent cell line (Hep-3B) into the right flank and IL-2 transduced cell line (Hep-3B/LNC/IL-2) into the left flank of the three BALB/c mice and of 5 X 10 (5) cells for the three nude mice resulted in a complete regression of the IL-2 modified tumor cell line (Hep-3B/LNC/IL-2) in 3 weeks and the parent cell line (Hep-3B) in 5 weeks. But, after the injection of 1.5 X 10 (7) cells for other five nude mice, the tumor of the IL-2 transduced hepatoma cell line (Hep-3B/LNC/IL-2) was gradually disappeared, and the tumor of the parent hepatoma cell line (Hep-3B) was initially decreased and then gradually regrew 20 days later. CONCLUSION: IL-2 transduced hepatoma cell lines secreting IL-2 became more sensitive to peripheral blood monocytes and resulted in the increased antigenicity to the tumors formed by IL-2 transduced hepatoma cell line and parent cell line, and finally resulted in the regression of the tumors in experimental animals.

A Study of Retrovirus-mediated p53 Gene Transduction Into Human Gastric Cancer Cell Lines / 대한암학회지

PURPOSE: The development of new therapeutic modalities such as gene therapy, which still requires further investigation, is clearly important to improve the prognosis of gastric cancer. This study was conducted to evaluate the effect on the growth and the tumorigenicity of retrovirus-mediated p53 gene transduction into gastric cancer cells. MATERIALS AND METHODS: Human gastric cancer cell lines were cultured and their DNAs were analyzed to evaluate the p53 status with PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) and DNA sequencing. Retroviral supernatants were obtained from each producer cell line, PA317/LNCX and PA317/LNC/p53, after construction of retroviral vector LNC/p53 containing human p53 cDNA and producer cell line PA 317/ LNC/p53. To investigate the effect of retrovirus-mediated p53 gene transduction in human gastric cancer cell lines, the in vitro growth rates and in vivo tumorigenicities of the N-87 cell line having mutant p53 and the YCC-S-2 cell line having wild-type p53 were compared before and after infection with LNC/p53 retrovirus. RESULTS: The following results were obtained: 1) The growth inhibition of N-87 cells after p53 transduction was signficant when compared to that of the parent N-87 cells. The growth of the p53 transduced YCC-S-2 cells and the parent YCC-S-2 cells was not different. 2) In nude mice, the growth of tumors formed by N-87 cells was modestly inhibited after retrovirus-mediated wild-type p53 gene transduction. However, the growth of tumors formed by YCC-S-2 cells was not inhibited by retrovirus-mediated p53 gene transduction. 3) The expression rate of p53 protein after p53-containing retroviral infection in the KATO-III cell lines, which have no p53 gene, was dose-dependent on the m.o.i. of retrovirus, although it was not more than 15% with the m.o.i. of 100 upon immunohistochemical analysis. CONCLUSION: The growth inhibition by retrovirus-mediated p53 transduction in human gastric cancer cells was significant in a gastric cancer cell line having mutant p53 in vitro, and the growth of tumor masses formed by a gastric cancer cell line having mutant p53 was modestly inhibited after p53 transduction using retroviral vector in nude mice, although it was not statistically significant. Only modest inhibition of tumor growth using retrovirus-mediated p53 gene transduction in vivo is most likely to be due to low transduction efficiency.