Selection of HSV-1 TK gene-transfected murine mammary carcinoma cells resistant to (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and ganciclovir (GCV).
We evaluated the molecular mechanism of resistance in herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene-transfected murine mammary carcinoma (FM3ATK-/HSV-1 TK+) cells, that were selected for resistance against (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and ganciclovir (GCV) by prolonged exposure of the cell cultures to dose-escalating concentrations of these compounds. Drug-resistant FM3ATK-/HSV-1 TK+ cells showed marked differences in their sensitivity spectrum to a series of antiherpetic nucleoside analogues. BVDU-resistant FM3ATK-/HSV-1 TK+ cells displayed the same sensitivity profile as wild-type FM3A/0 cells. In contrast, GCV-resistant FM3ATK-/HSV-1 TK+ cells were still sensitive to BVDU, (E)5-(2-iodovinyl)-2'-deoxyuridine (IVDU) and (E)-5-(2bromovinyl)-2'-deoxycytidine (BVDC), a typical feature of FM3A TK cells lacking cytosolic TK. Southern blot and PCR analysis revealed that HSV-1 TK genes were not deleted from the genome of the drug-resistant FM3ATK-/HSV-1 TK+ cells. However, the TK genes in drug-resistant FM3ATK-/HSV-1 TK+ cells were shown to be heavily methylated. Accordingly, RT-PCR demonstrated the complete abrogation of TK mRNA production resulting in a complete loss of TK enzyme activity in drug-resistant FM3A TK-/HSV-1 TK+ cells.
Degrève
B
B
Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium.
De Clercq
E
E
Balzarini
J
J
D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D015139:Blotting, Southern; D001973:Bromodeoxyuridine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D004279:DNA, Viral / Q000032:analysis; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005260:Female; D015774:Ganciclovir / Q000737:chemistry / Q000494:pharmacology*; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D008325:Mammary Neoplasms, Experimental / Q000235:genetics*; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013937:Thymidine Kinase / Q000032:analysis / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
D000998:Antiviral Agents; D004279:DNA, Viral; C020235:brivudine; D013937:Thymidine Kinase; D001973:Bromodeoxyuridine; D015774:Ganciclovir