ABSTRACT
PURPOSE: Genes involved in liver cancer cell growth have been identified using an antisense library of large circular (LC-) genomic DNA of a recombinant M13 phage. MATERIALS AND METHODS: A subtracted cDNA library was constructed by combining procedures of suppression subtractive hybridization (SSH) and unidirectional cloning of the subtracted cDNA into an M13 phagemid vector. Utilizing the life cycle of M13 bacteriophages, LC-antisense molecules derived from 1, 200 random cDNA clones selected by size were prepared from the culture supernatant of bacterial transformants. The antisense molecules were arrayed for transfection on 96-well plates preseeded with HepG2. RESULTS: When examined for growth inhibition after antisense transfection, 153 out of 1, 200 LC-antisense molecules showed varying degrees of growth inhibitory effect to HepG2 cells. Sequence comparison of the 153 clones identified 58 unique genes. The observations were further extended by other cell-based assays. CONCLUSION: These results suggest that the LC-antisense library offers potential for unique high-throughput screening to find genes involved in a specific biological function, and may prove to be an effective target validation system for gene-based drug discovery.
Subject(s)
Bacteriophage M13 , Bacteriophages , Clone Cells , Cloning, Organism , DNA , DNA, Complementary , Drug Discovery , Gene Library , Hep G2 Cells , Life Cycle Stages , Liver Neoplasms , Liver , Mass Screening , TransfectionABSTRACT
PURPOSE: In the present study, ribbon antisense to the hTR RNA, a component of the telomerase complex, was employed to inhibit telomerase activity and cancer cell growth. MATERIALS AND METHODS: Ribbon antisense molecules to the human hTR gene (hTR-RiAS) were constructed and complexed with a short modified peptide and cationic liposomes to improve the cellular uptake of the antisense molecules. The DPL complexes containing hTR-RiAS were transfected into target cancer cells. Various assays were performed to confirm the effects of the hTR-RiAS on the gene expression and cell proliferation. RESULTS: When cancer cells were treated with hTR-RiAS, the cellular level of hTR mRNA was reduced by more than 95%, as shown by RT-PCR. Further, the telomerase activity was also affected by the antisense treatment. In contrast, both mismatched and scrambled oligonucleotides failed to reduce the levels of hTR mRNA and telomerase activity. When checked for cancer cell viability, hTR- RiAS inhibited cell growth by more than 70%, in a very rapid manner. The reduced cell viability was found to be due to apoptosis of cancer cells. CONCLUSION: These results show that hTR-RiAS is a powerful anticancer reagent, with the potential for broad efficacy to diverse malignant tumors.
Subject(s)
Humans , Apoptosis , Cell Proliferation , Cell Survival , Gene Expression , Liposomes , Oligonucleotides , RNA , RNA, Messenger , Telomerase , TransfectionABSTRACT
PURPOSE: IL-10, one of the potent Th2 cytokine, has strong anti-inflammatory reaction and immunosuppression action by stabilization of polarized Th2 cell gene expression and blocking of function of antigen presenting cells. The aims of this study were to investigate the immunosuppressive effect of recombinant adenovirus mediated rat IL-10 (Ad:ratIL-10) in mouse to rat skin graft. METHODS: The transgene expression of the recombinant adenovirus was confirmed by X-gal staining of Ad:LacZ infected skin graft. The skin graft was done between mouse and rat after 1 hour infection of donated skin with Ad:ratIL-10, which was already been produced in our Institute for Medical Science. Checked gene expression in grafted skin by in situ RT-PCR and systemic blood by ELISA on day 1, 3, 5 and 7. Immunosuppressive effect of the Ad:ratIL-10 was evaluated by graft survival and compared with control group which was infected by saline. RESULTS: Transgene expression of recombinant adenovirus was peak on third day of skin graft and became disappeared on day 5 and 7. The same expression was confirmed by in situ RT-PCR of Ad:ratIL-10 infected skin graft. The systemic blood level of infected Ad:ratIL-10 checked by ELISA was undetectable but their expression checked in culture cell line of HeLa cell was 275 ng/mL on day 5. Mean grafted skin survival was 6.0 +/- 0.7 days in Ad:ratIL-10 group but statistically indistinguishable to control group of infection with saline (5.6 +/- 0.6 days, p>0.05). CONCLUSION: In summary, the Ad:ratIL-10 alone, infected in grafted skin, was peak on day 3 after graft but showed no immunosuppressive effect on mouse-to-rat skin graft.
Subject(s)
Animals , Humans , Mice , Rats , Adenoviridae , Antigen-Presenting Cells , Cell Line , Enzyme-Linked Immunosorbent Assay , Gene Expression , Graft Survival , HeLa Cells , Immunosuppression Therapy , Interleukin-10 , Skin , Th2 Cells , Transgenes , TransplantsABSTRACT
BACKGROUND: Aberrant expression of c-myb gene is often detected in transformed leukemic cells. Inhibition of c-myb expression by antisense oligos was shown to inhibit growth of normal as well as leukemic cells. C-myb antisense oligo for inhibition of tumor cell growth was, however, not decisive enough to be an effective anti-cancer agent. Thus, we set out to devise a systematic approach to find effective target sites for c-myb antisense oligos and to compare cellular uptake of antisense oligos complexed with different liposomes. METHODS: A computer simulation program for RNA secondary structures was employed to choose 8 potential target sites free of secondary structures along the entire c-myb mRNA sequence. Linear phosphorothioate-capped antisense oligos complementary to the selected target sites were synthesized and delivered into HL-60 and K562 cancer cell lines as liposomes complexes. RESULTS: Three of the 8 target sites were found to be relatively effective for reducing c-myb message. The three oliogs, MIJ-4, -17 and -18 were able to reduce c-myb message by more than 70% and suppressed tumor cell growth by about 70%. When three different cationic liposomes were used to facilitate the cellular uptake of antisense c-myb oligos, distinct liposome formulations were found to be comparably effective for reduction of c-myb message and inhibition of tumor cell growth. CONCLUSION: These results show that simulation of RNA secondary structure can be used to search effective target sites for antisense oligos and oligo uptake can be significantly enhanced by liposomes. However, cellular uptake of antisense oligos by liposomes needs further improvement.
Subject(s)
Cell Line , Computer Simulation , Genes, myb , Liposomes , RNA , RNA, MessengerABSTRACT
PURPOSE: Aberrant expression of the c-myb gene is often detected in transformed leukemic cells. Inhibition of c-myb expression by antisense oligos could be an effective way to abort rapid growth of leukemic cells. Developing stable antisense oligos combined with enhanced delivery into cells would be of great use in developing an effective anti-cancer molecular agent. MATERIALS AND METHODS: Selection of target sites was carried out by employing computer simulation of mRNA secondary structures. Multiple antisense oligo sequences were adjoined and AS-oligos were then covalently closed to evade exonuclease activities. C-myb antisense oligos with a novel structure were complexed with cationic liposomes and used to treat HL-60 leukemic cells. RESULTS: We developed covalently closed antisense oligos which harbor four adjoined antisense sequences. The c-myb antisense oligos were found to be exceptionally stable and effective in specifically ablating c-myb mRNA. The antisense oligos were able to inhibit growth of leukemic cell line (HL-60) by about 80%. Antisense effect was more pronounced when the cells were treated twice with the antisense oligos at lower concentrations. CONCLUSION: The novel covalently closed antisense oligo (CMAS-oligos) was found to be effective and exceptionally stable, Growth of HL-60 was significantly inhibited, showing a rational way to develop an effective molecular anti-cancer agent.