Figure 6

Telomerase activity in MCF-7 monolayer and mammosphere cells. (a) Telemorase activity was determined by Telomeric Repeat Amplification Protocol (TRAP) analysis. Cell extracts were prepared from unirradiated and irradiated MCF-7 monolayer and mammospheres cells. The assay involves the addition, mediated by telomerase in the cell extract, of a number of telomeric repeats onto the 3' end of an oligonucleotide substrate, which is then subjected to amplification by PCR. When run on a 10 to 12% native polyacrylamide gel, the telomerase-extended primer appears as a ladder of 6 bp increments. The lowest band on the gel shows the 36 bp internal PCR control. (The absence of this band in the lane showing results with 10-Gy irradiated mammospheres is due to excessively high telomerase activity because amplification of the TRAP products and the internal control is semi-competitive.) The positive (+ve) control lane shows the results using a telomerase positive cell extract provided by the manufacturer of the assay kit, while a cell extract prepared from human fibroblasts (CRL 2322) served as a negative control. (b) Quantification of telomerase activity. The mean values and standard deviations were calculated from three individual determinations at each dose. The difference in activity between the monolayer and mammosphere populations at each dose were statistically significant (P < 0.05, Student's t-test).