Most human somatic cells divide 50-60 times before senescence occurs. These cells invariably enter a state of irreversibly arrested growth. This process, termed replicative senescence, is thought to be a tumor-suppressive mechanism and an underlying cause of aging.1) The molecular mechanism underlying cellular senescence has been characterized. It is well accepted that telomeres play a major role in the process. Telomeres are the physical ends of linear eukaryotic chromosomes. They consist of hundreds to thousands of tandem repeats of the sequence TTAGGG, that are essential for stabilizing chromosomes.2) Because conventional DNA polymerase cannot replicate the very end of chromosomes, telomere length is shortened upon each DNA replication. Telomerase is the enzyme activity that elongates short telomeres. Because telomerase activity is low or not detectable in most normal human somatic cells, telomeric DNA is progressively shortened with each cell division. The shortened telomeres then signal cells to enter senescence through a DNA damage signaling pathway. Telomere length is considered as a biological clock that is capable of determining the proliferative capacity of most human somatic cells.
3,4)While telomerase is not detected or is low in most of the normal human cells, it is detected in ca. 85-90% of the immortalized or tumor cells. In humans, telomerase activity is tightly regulated by expression of the human telomerase reverse transcriptase (hTERT) gene, which appears to be the key regulator for telomerase activity. Inhibition or activation of the hTERT expression could profoundly affect the proliferative capacity of normal cells and cancers.5-7) Because telomerase is required for the sustained proliferation of most immortal cells, including cancer cells, it has become the focus of much attention as a novel and potentially highlyspecific target for the development of new anticancer chemotherapeutics. In this respect, antisense oligonucleotides or small molecular inhibitors have been identified as inhibitors to telomerase. 8) Several of them effectively inhibit telomerase in vitro and limit the proliferation of cancer cells in vivo.9) However, because a lag period is required for telomeres to be shortened to critical short length, the clinical applicability of telomerase inhibitor was questioned. For example, it takes ca. 20 additional cell divisions for telomeres of HeLa cells to be shortened to critical length.8) It is not realistic for telomerase inhibitor to be used in treating cancers. Thus, it was proposed that telomerase inhibitors should work with other chemotherapeutic agents for treating cancers.10)The use of cytotoxic agents to kill cancer cells and telomerase inhibitor to limit the proliferative potential of residual cancer cells should be a better approach in cancer chemotherapy. Interestingly, guanine-quadruplex (G-quadruplex) stabilizers such as 2,6-pyridine-dicarboxamide derivatives and 3,6,9-trisubstituted acridine compounds caused accelerated senescence in cancer cells. Molecules able to s...
Telomeres are the protective structures at the end of eukaryotic chromosomes. Telomerase is a ribonucleoprotein that contains both an RNA and a protein component for the maintenance of telomere length. Telomerase activity is detected in the majority of malignant tumors, but not in normal somatic cells, suggesting that telomerase reactivation is a crucial step in cell immortality and carcinogenesis. The mechanism of how telomerase is activated during tumorigenesis remains unclear. However, the expression of the human telomerase reverse transcriptase (hTERT) gene, which encodes the catalytic protein subunit of human telomerase, has been shown to be the major determining factor. To gain insight into the mechanisms regulating hTERT expression and to facilitate the screening of agents that affect hTERT expression, we have established cell-based systems for monitoring hTERT expression. We linked the hTERT promoter to two different reporter genes encoding green fluorescence protein (GFP) and secreted alkaline phosphatase (SEAP), respectively. These constructs were then transfected into H1299 and hTERT-BJ1 cells. Stable clones harboring these DNA constructs were isolated. In these cells, hTERT expression can be monitored through the quantification of GFP or SEAP activity on an automatic plate reader. Using these systems, we have identified several small molecule compounds that affect the expression of telomerase.
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