In culture, normal human cells undergo a finite number of cell divisions until t

Question

In culture, normal human cells undergo a finite number of cell divisions until they no longer proliferate; they then enter a state known as replicative senescence. The inability to maintain normal telomere length is thought to play an important role in this process. Telomerase is a ribonucleoprotein complex that regenerates the ends of telomeres lost during each round of DNA replication. Human telomerase consists of a template containing an RNA subunit and a catalytic protein subunit known as human telomerase reverse transcriptase (hTERT). Most normal cells do not express telomerase; most cancer cells do express telomerase. Thus telomerase is proposed to play a key role in the transformation of cells from a normal to a malignant state.

a.         In the following experiments, the role of telomerase in the growth of human cancer cells was investigated (see W. C. Hahn et al., 1999, Nature Medicine 5:1164–1170). Immortal, telomerase-positive cells (cells A) and immortal, telomerase-negative cells (cells B) were transfected with a plasmid expressing either a wild-type or a mutated hTERT. Telomerase activity in cell extracts was measured by the telomeric repeat amplification protocol (TRAP) assay, a PCR-based assay that measures the addition of telomere repeat units onto a DNA fragment. A six-base-pair ladder pattern is typically seen. Control indicates transfection of cells with just the “empty” plasmid expression vector that does not express any protein. Wild type and mutant indicate transfection with a plasmid vector expressing a wild-type hTERT or the mutated hTERT, respectively. What do you conclude about the effect of the mutant hTERT on telomerase activity in the transfected cells? What type of mutation would this represent?

Explanation / Answer

The effect on telomerase activity in both Cell A and Cell B shows that, the hTERT gene has only expressed in all Wild type allels in the gel run shown above. This states that the expression of the hTERT gene in the Mutant type is not shown not only because it is not expressed or the mutation has not occurred and there is no sign of malignancy in them, There could be other reasons such as lack of improper promoter complex or the quantity of cells being tranfected taken could be less.

Telomeres protect chromosome ends from fusion and from being recognized as sites of DNA damage. Dysfunctional telomeres, arising by critical shortening of telomeres in normal somatic cells during progressive cell divisions, elicit DNA damage responses that trigger cellular senescence. Cells that gain oncogenic changes bypass senescence and continue to divide until multiple critically shortened telomeres initiate crisis (a period of complete replicative senescence, chromosome end-to-end fusions, and extensive apoptosis). During anaphase, the sister chromatids are drawn apart owing to movement towards opposite poles, resulting in the formation of uneven derivative chromosomes, leading to genomic instability. The period of crisis results in extensive cell death.

Recent observations of two highly recurrent mutations at two sites within the core promoter region of hTERT suggest one possible mechanism for the activation of telomerase in cancer cells. These mutations, which occur at 124 bp and 146 bp upstream from the ATG start site, are CT transitions and on chromosome 5, and each mutation generates an identical 11 bp nucleotide stretch (5-CCCCTTCCGGG-3) containing a consensus binding motif (GGA(A/T)) for ETS transcription factors that can function as transcriptional repressor, activator or both to regulate telomerase expression. However, the molecular mechanisms of telomerase activation by ETS are not clearly understood. It has recently been reported that epidermal growth factor (EGF)-mediated activation of telomerase activity in lung cancer is associated with direct binding of ETS-2 to the hTERT promoter. The recurrent hTERT promoter mutations were first reported as germline mutations from a family of melanoma patients and were later seen through genome sequencing of sporadic melanoma and a number of cell lines across numerous cancer types and were associated with increased hTERT promoter activity.

Related Questions

Navigate

• Browse (All)
• Browse I
• Subjects

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.