Study On Inhibition Of HTR, Telomere Repeats And C-myc Gene Activity For Supression Of Lung Cancer Cell Proliferation Using Antisense Phosphorothioate Oligonucleotides

Objectives: Telomerase is a ribonucleoprotein that replicates and adds repeating units of TTAGGG to the end of chromosomes. It participates in the maintenance of telomere length after cell division. Almost immortal cells and 90% of tumor cell lines and tissues express more telomerase activity than normal somatic cells and tissues which have no detectable telomerase activity. Telomerase undertakes reverse transcription task. The RNA component of human telomerase, termed hTR, is discribed. the template region of hTR encompasses 11 nucleotides (5' CUAACCCUAAC 3'), complements to 3'end of the human telomere sequence and lengthens telomere. This suggests that it may be a novel target for gene therapy of tumor using antisense oligodeoxynucleotides.Deregulation C-myc , a proto-oncogen , may induce upset of DNA replication and mRNA transcription, which leads to cell cycle imbalance and cellular oncogenesis.Antisense oligonucleotides are able to penetrate cell membrane, which recognize and complement to cellular target gene by the specificity of Watson-Crick base pairing to inhibit DNA replication, mRNA transcription and cell metabolism.We measured telomerase activity hi 8 Lung tumor cell lines and transfected antisense phosphorothiate oligodeoxynucleotides (anti-PS-ODN) of hTR, anti PS-ODN of Telomere DNA and anti PS-ODN of C-myc as therapeutic agents into target cell LTEP-a-2 which express more tolomerase activity to investigate the effect of inhibition of telomerase activity and tumor cell growth. Methods: 1. Cell culture including GLC-82. LTEP-a-2. SPC-a-1 PA-a-1. SCLC. PG-49. 801- D and LTEP-78. 2. Telomerase activity assay hi human Lung tumor cell lines using Telomerase PCR ELISA .3. Oligonuleotides sythesisincluding Antisense PS-ODNs of hTR , Telomere DNA and C-myc Oligonucleotides were transfected with Clonfectin into LTEP-a-2 cell for 72hr. 4. Measurement of Telomerase activity, SDH. activity and cell growth, usingTelomerase PCR ELISA.. MTT optical absobance test and cell count. Results: 1. Eight cell lines showed high expression of telomerase activity. 2. Concentrations of 5uM anti-PS-ODNs of hTR. Telomere DNA and C-myc specifically reduced the telomerase activity by 25.74-34.87% for 72hr after the initial treatment. lOuM of anti-PS-ODN6 of hTR and Anti C-myc PS-ODNI2 had the ability to inhibit the telomerase activity by 49.44% and 62.7% 3. PS-ODNs of anti-hTR and anti-telomere DNA were able to reduce SDH activity by 19.4-28%.C-myc PS-ODN reduced the SDH activity by 42.38% at 5uM. lOuM of three PS-ODNs of hTR and PS-ODN9 of anti-telomere DNA reduced enzyme activity by 25.71-33.87 %. lOuM of Anti C-myc PS-ODN12 inhibit SDH activity by 57.78%?4. The concentration of 5jaM PS-ODNs of anti-hTR and Telomere DNA had the ability to inhibit cell growth by 14.77-28.13%. the inhibitory rate of 10|^M PS-ODNs of hTR were 34-36.92% and PS-ODNI2 of anti-C-myc can reduce cell growth by 52.94%. 5. The result indicated that 20uM of random sequence of PS-ODN9 failed to inhibit Telomerase, SDH activity and LTEP-a-2 cell growth specially.Conclusions: Human lung tumor cell lines expressed high activity of telomerase. PS-ODNs of anti-hTR and Telomere DNA had the ability to inhibit telomerase activity and reduced LTEP-a-2 cell growth. Anti C-myc PS-ODNI2 resulted in cell growth inhibitory activity by regulating cell cycle and the reduction was concentration dependent.