Epigenetic modification is involved in aberrant expression of class III beta-tubulin, TUBB3, in ovarian cancer cells.

Aberrant expression of class III beta-tubulin, TUBB3, has been reported to be one of the important mechanisms responsible for taxane resistance in diverse human malignancies. We investigated aberrant TUBB3 expression and its epigenetic modification in 66 primary tumors and 3 cell lines (OVCAR-3, JHOC-5 and JHOC-8) of ovarian cancers. Overexpression of TUBB3 protein was observed in 56 (85%) of the 66 ovarian cancers, and was significantly associated with aggressive tumor behavior (advanced stage, presence of ascites, suboptimal cytoreduction at surgery and presence of lymph node metastasis) (P<0.05). Responses to treatment with a demethylating agent (5-aza-2'-deoxycytidine, 5-Aza-CdR) and a histone deacetylase inhibitor (4-phenylbutyric acid, PBA) differed among the ovarian cancer cell lines. In 2 cell lines with weak expression of TUBB3 protein (OVCAR-3 and JHOC-8), TUBB3 induction was independently induced by treatment with 5-Aza-CdR (JHOC-8) or PBA (OVCAR-3), while neither agent markedly altered TUBB3 mRNA/protein expression in a strongly TUBB3-expressing cell line (JHOC-5). A CpG island within intron 1 was hypermethylated in 1 cell line (JHOC-8) that expressed TUBB3 weakly and required 5-Aza-CdR treatment for gene expression. A CpG island of another cell line showing faint expression of TUBB3 protein (OVCAR-3), in which a significant gain of TUBB3 expression was induced by treatment with PBA but not with 5-Aza-CdR, was hypomethylated, similarly to a cell line (JHOC-5) showing constitutive expression of TUBB3. We evaluated methylation status in this region in 14 primary tumors using methylation-specific PCR, but there was no significant relationship with TUBB3 immunoreactivity. These findings suggest that aberrant expression of TUBB3 protein might be associated with aggressive behavior of ovarian cancers, and that epigenetic modulation (DNA methylation and chromatin acetylation) might be partly involved in TUBB3 expression.

Downregulation of miR-138 is associated with overexpression of human telomerase reverse transcriptase protein in human anaplastic thyroid carcinoma cell lines.

Alterations of several microRNA (miRNA) have been linked to cancer development and its biology. To search for unique miRNA that might play a role in the development of anaplastic thyroid carcinoma (ATC), we examined the expression of multiple miRNA and their functional effects on target genes in human thyroid carcinoma cell lines. We quantitatively evaluated the expression of multiple miRNA in 10 ATC and five papillary thyroid carcinoma (PTC) cell lines, as well as primary tumors from 11 thyroid carcinoma patients (three ATC and eight PTC), using the stem-loop-mediated reverse transcription real-time polymerase chain reaction method. We also examined the target gene specificity of unique miRNA that showed differences in expression between ATC and PTC cell lines. One miRNA, miR-138, was significantly downregulated in ATC cell lines in comparison with PTC (P < 0.01). Eleven miRNA (including miR-138) potentially targeting the human telomerase reverse transcriptase (hTERT) gene were totally downregulated in both ATC and PTC cell lines in comparison with normal thyroid tissues. A tendency for an inverse correlation between miR-138 and hTERT protein expression was observed in the thyroid cancer cell lines, although this failed to reach significance (r = -0.392, P = 0.148). We demonstrated that overexpression of miR-138 induced a reduction in hTERT protein expression, and confirmed target specificity between miR-138 and the hTERT 3'-untranslated region by luciferase reporter assay. These results suggest that loss of miR-138 expression may partially contribute to the gain of hTERT protein expression in ATC, and that further multiple miRNA targeting hTERT mRNA might be involved in the development of thyroid carcinoma.

Expression of human telomerase reverse transcriptase and correlation with telomerase activity in placentas with and without intrauterine growth retardation.

BACKGROUND: Human telomerase reverse transcriptase has been found in telomerase-positive tumor tissues, but not in telomerase-negative nonmalignant somatic cells. METHODS: Thirty-two first-trimester chorionic villi specimens (Group A), 33 second- and third-trimester placenta specimens without asymmetric intrauterine growth retardation (Group B) and 13 specimens of placenta tissue from cases with intrauterine growth retardation (Group C) were examined for telomerase activity and expression of human telomerase reverse transcriptase by reverse transcription polymerase chain reaction and quantitative reverse transcription polymerase chain reaction. RESULTS: Telomerase activity was detected in 29 of the 32 specimens (90.6%) in Group A, in 20 of the 33 specimens (60.6%) in Group B and none of the 13 specimens (0.0%) in Group C. Human telomerase reverse transcriptase was identified in all 32 specimens of Group A (100%), all 33 specimens of Group B (100%) and 2 of the 13 specimens in Group C (15.4%) by nested reverse transcription polymerase chain reaction. Copy numbers of human telomerase reverse transcriptase were 202.3 +/- 73.0 (n=32), 8.8 +/- 2.9 (n=33) and 0 (n=13) in Groups A, B, and C, respectively. Significant differences were observed between Groups A and B, Groups A and C, and Groups B and C (p <0.01, p < 0.01, and p < 0.01, respectively). CONCLUSIONS: Our findings indicate that human telomerase reverse transcriptase expression is the rate-limiting determinant of telomerase activity in chorionic villi during the first trimester. Telomerase activity was not detected in placentas with intrauterine growth retardation, whereas human telomerase reverse transcriptase was expressed in some placentas with intrauterine growth retardation.