hTERT mRNA expression correlates with telomerase activity in human breast cancer.

AIMS: Telomerase is a ribonucleoprotein enzyme that synthesises telomeres after cell division and maintains chromosomal length and stability thus leading to cellular immortalisation. hTERT (human telomerase reverse transcriptase) gene seems to be the rate-limiting determinant of telomerase reactivation. hTERT mRNA expression was reported to correlate with telomerase activity in cell lines and some human tumours. However the correlation between telomerase activity and hTERT mRNA expression has not been previously examined in human breast cancer. The present study aims to quantitatively measure the expression of hTERT mRNA and telomerase activity in human breast cancer and examine the relationship between these parameters. Furthermore the associations with other parameters including estrogen receptor (ER) and progesterone receptor (PgR) status, DNA ploidy, and S-phase fraction (SPF) are also examined. METHODS: RNA was extracted from 18 breast carcinomas and hTERT mRNA expressions were estimated by reverse transcriptase-PCR (RT-PCR) and Taqman methodology. These tumours had already been analysed for ER and PgR status using ligand-binding assays and had had their DNA ploidy and S-phase fractions measured by flow cytometry. Telomerase activity had already been determined by using a modified telomeric repeat and amplification protocol (TRAP) assay. RESULTS: The expression of hTERT mRNA in the breast tumours ranged between 1.3 and 2.7 x 10(7) copy numbers per micro g of cellular RNA (the median value was 2.7x10(5) and the mean was 3.1 x 10(6)). Telomerase activity was between 0 and 246 units of Total Protein Generated (TPG), where one unit of TPG was equal to 600 molecules, of telomerase substrate primers extended by at least three telomeric repeats. The median level of TPG was 60 units and the mean level was 81 units). Telomerase activity was found to significantly correlate with hTERT expression (r(s)=0.51112, P=0.0302). There was no significant correlation between hTERT and other parameters. CONCLUSION: hTERT mRNA expression significantly correlates with telomerase activity in human breast cancer. This is consistent with the hypothesis that hTERT is the catalytic and rate-limiting determinant subunit of the enzyme.

hTERT expression in human breast cancer and non-cancerous breast tissue: correlation with tumour stage and c-Myc expression.

Telomerase is a ribonucleoprotein enzyme that synthesises telomeres after cell division and maintains chromosomal length and stability thus leading to cellular immortalisation. hTERT (human telomerase reverse transcriptase) gene is the rate-limiting determinant of telomerase reactivation. The present study aims to quantitatively measure the expression of hTERT mRNA in human breast cancer, adjacent non-cancerous tissue (ANCT) and benign breast lesions, examine the association between hTERT and the clinicopathological characteristics of the cancer specimens and to explore the relationship between c-Myc and hTERT expressions. RNA was extracted from 49 breast carcinomas, 46 matched ANCT, and eight fibroadenomas. hTERT and c-Myc mRNA expressions were estimated by reverse transcriptase-PCR (RT-PCR) and Taqman methodology. hTERT mRNA was present in all of the cancerous and most of ANCT specimens with levels being much higher in the cancerous tissue than in ANCT. The ratio of hTERT mRNA in tumour to that in ANCT was 2011 (95% confidence interval 373-10,853, P < 0.0001). There was no significant association between tumour hTERT expression and patient's age, tumour size, grade, nodal metastasis, estrogen receptor (ER) positivity, lymphovascular (LVI) or c-Myc expression. However, there was a weak but significant negative correlation between hTERT expression and progesterone receptor (PR) status (p = 0.04) in tumours. hTERT mRNA expression was also significantly higher in carcinomas (median = 2.61 x 10(6)) than in fibroadenomas (median = 424).We conclude that hTERT mRNA expression is significantly higher in human breast cancer than in non-cancerous breast tissue suggesting that hTERT has a potential role in breast cancer diagnosis. The hTERT mRNA levels in tumour do not seem to be associated with the patient's age or advanced tumour stage. Furthermore, hTERT mRNA expression does not correlate with c-Myc mRNA expression in breast cancer.

Stimulation of aromatase activity in breast fibroblasts by tumor necrosis factor alpha.

The conversion of androstenedione to estrone, the reaction mediated by the aromatase enzyme complex, may make an important contribution to the synthesis of estrogens in breast tissues. In the present study, the effect of the cytokine. TNF alpha, on aromatase activity was examined in breast fibroblasts derived from normal and malignant breast tissue. TNF alpha (2.5-10.0 ng/ml), in the presence of stripped fetal calf serum and dexamethasone, significantly stimulated fibroblast aromatase activity in a dose-dependent manner. IL-1 and IL-6 also stimulated fibroblast aromatase activity, but no marked synergism between TNF alpha and IL-1 or IL-6 was detected. Using a specific radioimmunoassay, significant concentrations of TNF alpha were detected in samples of breast cyst fluid and breast tumor cytosol, which had previously been shown to stimulate aromatase activity, but not in conditioned medium from breast tumor-derived fibroblasts. As TNF alpha may be preferentially expressed and produced in the adipose tissue component of the breast, this cytokine may have an important role in regulating estrogen synthesis in normal and malignant breast tissues.