The association between telomerase activity and expression of its RNA component (hTR) in breast cancer patients: the importance of DNase treatment

Abstract

Telomerase is a ribonucleoprotein enzyme that compensates for the telomere length shortening which occurs during the cell cycle. Telomerase activity has been detected in most tumours but not in somatic cells. However, hTR; the RNA component of telomerase; has been reported to be universally expressed in both cancerous and non-cancerous tissues. Tumour samples from 50 patients with primary invasive breast cancer were collected. The TRAP assay was used to detect telomerase activity. RT-PCR on cDNA and DNased cDNA samples and control groups was used to detect the expression of hTR, GAPDH and PGM1 genes. Seventy-two percent of samples showed telomerase activity. DNA contamination was detected in 36 (72%) of RNA samples. Without performing DNase treatment, 49 (98%) of all samples showed hTR expression, but with the application of this strategy, hTR expression decreased from 98% to 64%. A significant association (p < 0.001) between hTR expression and telomerase activity was observed. Among the 32 hTR positive samples, 30 had telomerase activity and among the 18 hTR negative samples, telomerase activity was observed in 6 cases. Thus the application of this strategy could provide an applicable tool to use instead of the TRAP assay thus facilitating telomerase research in cancer genetic investigations.

Figure 1

Telomerase activity detected by TRAP assay method. C.-: A negative control group including a telomerase negative sample, a denatured protein sample and a mixture without protein extract.

Figure 2

Electrophoregram for GAPDH and hTR PCR products. A: cDNA samples. B: Mixture of RNA and materials required for cDNA synthesis excluding Reverse Transcriptase enzyme, determining the probable DNA contamination. C: DNase treated cDNA. D: Mixture of DNased RNA and required for cDNA synthesis excluding Reverse Transcriptase enzyme, determining the accuracy of DNase treatment. All of samples had GAPDH expression but lanes 7 and 8 did not have expression of this gene (in RNA-RT samples) and so did not have DNA contamination. Lanes 1–3 had positive expression of hTR. Lanes 4–6 did not have expression on DNased treated samples and so were negative but if we had not used DNase treatment, the expression was determined and this was caused by DNA contamination. Lane 7 did not have DNA contamination and the band seen in PCR for cDNA sample does not belong to DNA contamination. Lane 8 did not have any DNA contamination and hTR expression, too. C.-: Negative control including any cDNA or DNA template. C. +: Positive control (a template with positive expression of gene) to reach the accuracy of PCR. M: DNA size marker.