Telomerase is a reverse transcriptase ribonucleoprotein that is able to generate and maintain the length of telomeres (DNA sequences that make up the ends of chromosomes). The RNA component of telomerase (hTR) serves as the template for elongation of telomeric DNA. The catalytic protein subunit of telomerase (hTERT, 127kDa in humans) contains the activity of a DNA polymerase and has a conserved amino acid motif also found and highly conserved in other reverse transcriptases(2,7). Telomerase elongates the 3’ ends of preexisting telomeres by reverse transcribing the template region of its RNA component, generating DNA sequences that consist of tandem repeats of (TTAGGG)(1,4,7,9). By doing so, it addresses the “end-replication problem” (1,11) of cells in that results in a loss or shortening of terminal telomeric sequences during normal replication of chromosomes. The shortening of the telomeric sequences is due to DNA polymerase’s inability to fully replicate the 3’ end of the lagging strand of linear DNA. In the absence of telomerase, telomeres progressively shorten every cell cycle causing a cell to eventually undergo senescence. Thus the length of telomeres acts as a molecular clock(8) that dictates what will be the Hayflick limit or the maximum number of times a cell can replicate (1,3).
Figure 1-Detection of telomerase reverse transcriptase expression in cells using RT-PCR. A) A total of100 ng of RNA isolated from the indicated cell lines was subjected to RT-PCR using a primer set that spanned regions of exons 8 and 9 of telomerase reverse transcriptase cDNA (Accession # NM_003219). This primer set amplified a158bp product from telomerase reverse transcriptase cDNA, indicated by the left arrow. B) To control for the amount of RNA used in each sample, RTPCR for B-actin (Accession # NG_002724) was performed using a primer set that amplified a 444 bp product, indicated by the right arrow
Figure 2-Sequencing results for the amplified product of Telomerase Reverse transcriptase (hTERT). The nucleotide sequence of the amplified products (shown in fig.2A) was blasted against NCBI’s nucleotide databases. The results of the alignment confirm that the amplified 158 bp product corresponds to sequences in the variant 1 mRNA of hTERT (Accession Number NM_003219). The N’s in the above sequence alignment indicate regions on the sequencing gel that were unreadable
In this study, the expression of telomerase reverse transcriptase or hTERT was compared between a normal immortalized breast cell line (MCF10A), a noninvasive tumor breast cell line (MCF7) and an invasive tumor breast cell line (HTB26) through the use of RT-PCR. The detection of telomerase reverse transcriptase (hTERT) mRNA and not hTR mRNA (the RNA component of telomerase) is used to identify cells with telomerase activity. It has been shown that all human cells express hTR (1) but only telomerase activity has been detected in cells that expressed hTR and hTERT. Cells that lack expression of hTERT (the catalytic subunit of telomerase) also lack telomerase activity.
The results of the RT-PCR for this study revealed that all three cell lines contained detectable levels of telomerase reverse transcriptase mRNA. Other reports have also shown that immortalized and cancerous cell lines contain a higher level of telomerase activity than normal cell lines (which do not have the ability to continuously divide). Compared to the MCF10A band resulting from RT-PCR, the expression of hTERT mRNA seems to be more prominent in the two cancer breast cell lines(MCF7 and HTB26), indicating that there is a higher amount of telomerase activity in those cell lines than in the normal immortalized breast cell line. Like previous reports mentioned above (12), these results reveal that the amount of telomerase activity is an indication of the level of malignancy of a cancerous cell.
From the RT-PCR results, one can predict by looking at the intensity of the bands that MCF7 and HTB26 cell lines are more malignant than the MCF10A cell line, since they express higher amounts of hTERT mRNA. The MCF10A cell line contained detectable levels of hTERT mRNA was because it was immortalized. This does not mean that MCF10A is cancerous (because it is still contact inhibited and will not form tumors if injected in an organism) but just that it can now continuously divide, maintaining its telomeres through telomerase activity. If it were not immortalized, no detectable levels of telomerase mRNA would have been expressed. MCF7 and HTB26 cell lines, on the other had are not contact inhibited and will form tumors in an organism. These results show that detection of telomerase activity can be a useful method for identifying cancerous cells and even their degree of malignancy when compared to normal cells.
Currently a more sensitive assay is being used for the detection of telomerase activity called the TRAP assay (or Telomerase repeat amplification protocol assay), which can detect as few a one to ten telomerase positive cells (12). But an assay for telomerase alone will not be an efficient method for detecting all cancerous cells since it has been reported that some cancer cell lines do not express telomerase activity. These cancer cells maintain their telomeres through an alternative method that is currently under investigation. It is recommended that an assay for the detection of telomerase activity should be used in addition to other methods that can identify other possible markers of cancerous cells. This will ensure the efficient detection of all cancerous cells, including the ones that do not express telomerase activity.
I would like to thank Jingson Qiu, Brian Fox and my fellow classmates for their helpful discussions. I am thankful to Dr. Raj Kandpal for providing me with the cells used in this project. Dr. Rubin, thank you for providing us with the opportunity to put into practice the knowledge we have learned in class and for pushing us to do our best.
|This document was last modified 01/31/2006.|
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