*************Answer PART E only *************** Analyze the Data 8-1: Telomerase

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*************Answer PART E only ***************

Analyze the Data 8-1: Telomerase in Cancer Cells

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?

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 hTERT? By what mechanism do cells A and B maintain their telomere lengths?

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 and B?

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?

*************ANSWER PART E FIRST***************

Explanation / Answer

If wild or mutant hTERt cells were injected into nude mice, cell proliferation increases leading to cancer.

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