In the early 90s while new techniques were being discovered to handle cancerous cells. Some assays were published mentioning the activity of telomerase. TELOMERASE is an enzyme found in cancers that, by re-forming the telomeres at the ends of chromosomes, prevents the shortening that usually limits the number of replications, thus allowing cancer growth. In laboratories experiments are made for multiple purposes.
What the Assay has to state:In this assay, an aliquot of the test lysate is incubated with a mixture containing a synthetic telomere-like oligonucleotide, which, in the presence of telomerase activity, will be elongated by a number of hexameric repeats. In a conventional polymerase chain reaction, the mixture of telomerase products, which differ in size by a multiplicand of 6 bases, is amplified and separated on polyacrylamide gels. In case of telomerase activity, a typical 6-bp incremental ladder is visualized.
Numerous modifications of the original protocol have been made in order to improve reliability, linearity and sensitivity, to exclude false-positive results due to primer-dimer formation, to quantify enzyme levels, and to exclude false negativity due to the presence of Taq polymerase inhibitors. We perform quick, safe and effective detection of nonlabeled amplicons using the DNA stain ethidium bromide, and a CCD camera-coupled software package. Assays offered by Del Doc 1000 Molecular Analyst, BioRad Laboratories GmbH,
Telomerase activity was detected in 20/22 (91%) of primary mesotheliomas, and all tumor-derived mesothelioma cell lines. Levels of enzyme activity were semiquantitatively graded using lysate dilution series, and different levels were observed in the tumors of different histological subtypes. Telomerase activity could not be detected in the 6 normal mesothelial cell cultures or in 2 mesotheliomas. Unexpectedly, both benign solitary fibrous tumors showed strong telomerase activity.
The finding of telomerase activity in over 90% of mesotheliomas is in agreement with the reported activity in a variety of other cancer types and is consistent with the hypothesis that telomerase activation may be a feature of carcinogenesis in mesothelioma as perhaps in many other cancers. Carcinogenesis is recognized as a multistep process resulting from the accumulation of sequential genetic alterations in a cell. The very long latency period that is characteristic of mesothelioma suggests that multiple cumulative genetic, cytotoxic and proliferative events occur during the tumorigenic conversion of the progenitor cell. Of the known changes that we and others investigated in mesothelioma, the most frequent are in the p16 and NF2 genes .
According to the telomere-senescence model, such genetically altered premalignant cells finally may be eliminated before they ever can develop a full-blown malignant phenotype. Conversely, the presence of telomerase activity in mesothelioma may indicate that these cells possibly can overcome the restraint of their finite lifespan and are no longer hampered in their clonal evolution towards a more malignant phenotype. We did not detect telomerase activity in normal mesothelial cells. There is evidence that mesothelioma originates from surface mesothelial cells rather than from a ‘multipotential’ subserosal stem cell, although this has not been established beyond all doubt. Assuming the former scenario is correct, the absence of detectable telomerase activity in mesothelial cells, in contrast to the presence of activity in mesotheliomas, could fit with the interpretation that most adult tumors develop from telomerase-negative precursors after telomerase reactivation. In contrast, others may interpret these results as evidence for the abovementioned stem cell origin of mesothelioma, arguing that these telomerase-positive tumors indeed arise from telomerase-positive stem cells without the need for a reactivation step.
Importantly, we neither detected telomerase in proliferating nor in resting mesothelial cells. This result conceivally argues against a model in which telomerase is regarded merely as a proliferation marker.Indeed, extensive TRAP analyses and hTERC in situ hybridization studies showed a similar (but not identical!) distribution pattern of telomerase activity and/or hTERC RNA and the Ki67 (Mib-1) proliferation marker. Other cell types, that scored telomerase negative in vivo, appeared competent to express telomerase when subjected to a sufficient proliferative stimulus in vitro. Therefore, it is currently only valid to apply the classic reactivation model to a particular organ system when the cell of origin is effectively telomerase negative, not just in its in vivo state but also when subject to ‘excessive’ growth stimulation. This controversial issue was subject of a more detailed study, in which we determined telomerase activity and hTERT mRNA expression in primary melanocyte cultures. Here too, highly proliferating melanocyte cultures, showing nuclear staining for Ki67 and for KiS2 in 65% and in 35% of the cells, respectively, lacked telomerase activity and hTERT transcripts. KiS2 binds to an epitope that is present during the entire cell cycle, with exception of the rate-limiting G1 phase, thereby being a more accurate marker of the actively proliferating cell fraction than Ki67. The detected strong telomerase activity in both benign solitary fibrous tumors could reflect their hypothesized origin from telomerase-positive, CD34-positive fibroblastic stem cells or their unpredictable clinical behavior. Similarly, Umbricht et al found 19% of benign follicular tumors of the thyroid with detectable telomerase activity and argued that some histologically benign lesions may be precursor lesions of follicular carcinomas. Our observation can also indicate that the application of telomerase activity as a marker for malignancy of serosal lesions needs further validation.
It is obvious that larger series need to be studied, which should also include unequivocal benign, mesothelium-derived tumors, e.g., adenomatoid tumors. The ‘classic model’ states that telomerase up-regulation is forced by critical telomere erosion beyond the point where cell multiplication normally stops. An in vitro situation is seen during continuous culture of SV40 large T antigen-transformed, telomerase-negative human cells, which eventually undergo ‘crisis’, the condition in which cellular chromosomes are characterized by ultrashort telomeres and that coincides with telomerase activation. At present it is not known whether SV40-like viruses, for which DNA sequences have been found by us and others in some mesotheliomas, are responsible for an in vivo equivalent of crisis during mesothelioma carcinogenesis. The ‘co-selection hypothesis’ states that telomerase is indirectly reactivated as part of a ‘package’ of changes in gene expression that occurs after some other genetic event. It remains speculative whether asbestos-induced DNA damage represents such a genetic event. Similarly, others have discussed that telomerase activation is a mere side effect of dedifferentiation. We observed different levels of enzyme activity in the tumors of varying grades of differentiation. Because of the limited study size, we did not perform statistical analysis to find any correlation between the level of telomerase activity in the tissue lysates and histologic subtype. Anyway, statistical results may be inaccurate because of weaknesses in the TRAP assay. Indeed, heterogeneity of tumor differentiation within a tissue section as well as morphology is lost during the in vitro biochemical assay. Further, the TRAP assay requires access to fresh or frozen samples, indicating the need for alternatives that can detect telomerase activity at the cellular level in archived materials. Such alternatives will be extremely helpful to study the telomerase status in archived samples of serosal precursor lesions. It is important to understand any cell activity to see how it reacts to other elements. Medicines and treatment methods highly depend on these lab reports. Any wrong theory or experiment can cause deaths and other complications. Human life is the most precious thing in this world. Where there are health issues there is no compromise.
