Inhibition of cell growth and telomerase activity in osteosarcoma cells by DN-hTERT

Abstract

In order to study the effects of dominant negative human telomerase reverse transcriptase (DN-hTERT) on cell growth and telomerase activity in osteosarcoma cell line MG63, MG63 cells were transfected with DN-hTERT-IRES2-EGFP9 (DN) or IRES2-EGF (I, blank vector) with lipofectamine 2000. The stably transfected cells were selected with G-418. Cell growth properties were examined under a fluorescence microscope. The hTERT mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). Telomerase activities were measured by TRAP-ELISE. The tumorigenicity was studied with tumor xenografts by subcutaneous injection of cancer cells into nude mice. The results showed that cell growth was suppressed in MG63 cells transfected with DN-hTERT. The hTERT mRNA was increased in N-hTERT transfected-MG63 cells (MG63/DN). The telomerase activity was 2.45±0.11 in MG63/DN cells, while 3.40±0.12 in the cells transfected with blank vector (MG63/I), (P<0.05); DN-hTERT-expressing clones did not form tumors in 2 weeks, but the ratio of tumorigenesis was 30 % in nude mice bearing MG63/I (P<0.01). It was concluded that DN-hTERT could specifically inhibit the cell growth and telomerase activity in MG63 cells.

Keywords

dominant negative human telomerase reverse transcriptase / MG63 / telomerase activity

References

[1] KimN. W., PiatyszekM. A., ProwseK. R., et al.. Specific association of human telomerase activity with immortal cells and cancer. Science, 1994, 266(5193): 2011-2015

[2] FengJ., FunkW. D., WangS. S., et al.. The RNA component of human telomerase. Science, 1995, 269(5228): 1236-1241

[3] MataJ. E., JoshiS. S., PalenB., et al.. A hexameric phosphorothioate oligonucleotide telomerase inhibitor arrests growth of Burkitt’s lymphoma cells in vitro and in vivo. Toxicol Appl Pharmacol, 1997, 144(1): 189-197

[4] MeyersonM., CounterC. M., EatonE. N., et al.. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell, 1997, 22;90(4): 785-795

[5] YuJ., DubeauL.. Telomerase and malignant transformation. Cancer Treat Res, 2002, 107(1): 213-228

[6] HelderM. N., WismanG. B., van der ZeeG. J.. Telomerase and telomeres: from basic biology to cancer treatment. Cancer Invest., 2002, 20(1): 82-101

[7] DavisA. J., SiuL. L.. Telomerase: Therapeutic potential in cancer. Cancer Invest, 2000, 18(2): 269-277

[8] PendinoF., TarkanyiI., DudognonC.. Telomeres and telomerase: Pharmacological targets for new anticancer strategies?. Curr Cancer Drug Targets., 2006, 6(2): 147-80

[9] HsuC. P., HsuN. Y., LeeL. W., et al.. Ets2 binding site single nucleotide polymorphism at the hTERT gene promoter—effect on telomerase expression and telomere length maintenance in non-small cell lung cancer. Eur J Cancer, 2006, 42(10): 1466-1474

[10] Batinac T, Zamolo G, Hadzisejdic I. Telomerase in anti-tumor response. Med Hypotheses. 2006 Aug 25.

[11] ChinL., ArtandiS. E., ShenQ., et al.. P53 deficiency rescues the adverse effects of telomere loss and cooperates with telomere dys2 function to accelerate carcinogenesis. Cell, 1999, 97(4): 527-538