TAILIEUCHUNG - Báo cáo khoa học: The involvement of human ribonucleases H1 and H2 in the variation of response of cells to antisense phosphorothioate oligonucleotides

We have analyzed the response of a number of human cell lines to treatment with antisense oligodeoxynucleotides (ODNs) directed against RNA polymerase II, replication protein A, and Ha-ras. ODN-delivery to the cells was liposome-mediated or via electroporation, which resulted in di erent intracellular locations of the ODNs. The ODN-mediated target mRNA reduction varied consider-ably between the cell lines. | Eur. J. Biochem. 269 583-592 2002 FEBS 2002 The involvement of human ribonucleases H1 and H2 in the variation of response of cells to antisense phosphorothioate oligonucleotides Anneloor L. M. A. ten Asbroek Marjon van Groenigen Marleen Nooij and Frank Baas Neurozintuigen Laboratory Academic Medical Center Amsterdam the Netherlands We have analyzed the response of a number of human cell lines to treatment with antisense oligodeoxynucleotides ODNs directed against RNA polymerase II replication protein A and Ha-ras. ODN-delivery to the cells was liposome-mediated or via electroporation which resulted in different intracellular locations of the ODNs. The ODN-mediated target mRNA reduction varied considerably between the cell lines. In view of the essential role of RNase H activity in this response RNase H was analyzed. The mRNA levels of RNase H1 and RNase H2 varied considerably in the cell lines examined in this study. The intracellular localization of the enzymes assayed by green-fluorescent protein fusions showed that RNase H1 was present throughout the whole cell for all cell types analyzed whereas RNase H2 was restricted to the nucleus in all cells except the prostate cancer line 15PC3 that expressed the protein throughout the cell. Whole cell extracts of the cell lines yielded similar RNase H cleavage activity in an in vitro liquid assay in contrast to the efficacy of the ODNs in vivo. Overexpression of RNase H2 did not affect the response to ODNs in vivo. Our data imply that in vivo RNase H activity is not only due to the activity assayed in vitro but also to an intrinsic property of the cells. RNase H1 is not likely to be a major player in the antisense ODN-mediated degradation of target mRNAs. RNase H2 is involved in the activity assayed in vitro. The presence of cell-type specific factors affecting the activity and localization of RNase H2 is strongly suggested. Keywords ribonuclease RNase H human antisense phosphorothioate. Ribonucleases H RNases H are .

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