a.In the following experiments, the role of telomerase in the growth of human ca
ID: 192035 • Letter: A
Question
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?
Cell A Cell B Control Wild type MutantControl Wild type Mutant b· Telomere length in these transfected cells was examined by Southern blot analysis of total genomic DNA digested with a restriction enzyme and probed with a telomere-specific DNA sequence. What do you conclude about the lengths of the telomeres in cells A and B after transfection with the wild-type or mutant h mechanism do cells A and B maintain their telomere lengths? TERT? By what Cell A Control Wild type Mutant Cell B Control Wild type Mutant c. The proliferation of transfected cells A and B was assayed by measuring the number of cells versus time in culture. What do you conclude about the effect of the mutant hTERT on cell proliferation in transfected cells A Cell A Cell B 20 4o Time (hr 20 0 Time (hr Wild type d. Do the results of the three assays support or refute the proposed role of telomerase in long-term survival of cells in culture? What is the value of comparing transfection of cells A with that of cells B? e. Tumorigenic cells, which can replicate continuously in culture, can cause tumors when injected into mice that lack a functional immune system (nude mice). From the above assays, what would you predict would happen if wild-type or mutant hTERT transfected cells A were injected into nude mice?Explanation / Answer
A)
Telomerase reverse transcriptase (hTERT in humans) is a catalytic subunit of the enzyme telomerase, which, together with the telomerase RNA component (TERC), comprises the most important unit of the telomerase complex. These observations indicate that telomere maintenance is essential to the proliferation of tumor cells. We show here that expression of a mutant catalytic subunit of human telomerase results in complete inhibition of telomerase activity, reduction in telomere length and death of tumor cells.
In mutanted hTERT, expressing an hTERT protein that is catalytically actiive unable to elongate telomeres . These results imply that expression of mutant telomerase activity in transfected cells is lethal, and that lethality is dependent upon expression of a biologically active enzyme.
Deletion type of mutation would represent this.
B)
Cells B, telomerase negative cells that maintain telomeres by the ALT pathway can be converted to a telomerase-positive phenotype by transfection of appropriate components of the enzyme. Cell B is example of ALT lines which retain expression of hTER, we were able to restore enzymatic activity by reintroduction of only the catalytic subunit of telomerase, hTERT.
The dynamics of telomeres in ALT cells, a pattern of erosion and rapid increase in the length of telomeric DNA tracts, has suggested that recombination may similarly be responsible for the ALT mechanism.
Reintroduction of wild-type hTER in that subset of ALT lines that lack it, does not restore enzymatic activity. hTERT alone is insufficient to convert to telomerase-positive those ALT cells that fail to express hTER, and in this case reintroduction of both subunits is necessary and sufficient for acquisition of this phenotype.
Ans C)
Expression of mutant telomerase in VA13/hTERT-HA cells had no effect on cell growth or viability.
The effect of telomerase enzymes on cell growth and viability was measured directly on the polyclonal populations immediately after their rescue from selection. While control populations transfected with wt hTR or the empty vector grew with comparable rates and reached, populations expressing mutant telomerases were significantly retarded in their growth and attained lower than controls over the same period of time. Their colony forming abilitywas also reduced relative to controls.
Ans e)
The enzyme is reactivated when somatic cells are immortalized in vitro or during tumorigenesis; inhibition of telomerase in these cells results in cell death, demonstrating a requirement for telomere maintenance to sustain indefinite cell proliferation
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