Vincristine-resistant human laryngeal carcinoma cells demonstrate increased Rous sarcoma virus promoter activity.
OBJECTIVE
Gene therapy is a candidate approach for treating cancer patients whose tumors have developed resistance to some drugs. Our study aims to examine possible alteration in Ad5RSVβgal-mediated transgene expression in a vincristine-resistant cells (VK2) derived from the human laryngeal carcinoma cell line HEp2, and the underlying mechanism(s) thereof.


METHODS
Adenovirus-mediated transgene expression in HEp2 and VK2 cells was measured by β-gal staining. Semiquantitative PCR was used to evaluate attachment of adenovirus to the cell surface and adenovirus internalization into cells. After transfection of cells with plasmid DNA, promoter activity was measured by semiquantitative RT-PCR.


RESULTS
We show here that VK2 cells exhibited increased Ad5RSVβgal-mediated transgene expression, despite moderately decreased Ad5RSVβgal attachment and internalization, as compared with HEp2 cells. The increased transgene expression was also observed with a virus (Ad5FbΔ639RSVβgal) that does not use the coxsackie-adenovirus receptor (CAR), suggesting that increased transgene expression is independent of CAR. Upon transfection of VK2 cells with a plasmid expressing a reporter gene under the control of the RSV promoter or a plasmid containing the complete Ad5RSVβgal genome, RSV promoter activity was 33- and 4.7-fold higher, respectively, than in HEp2 cells.


CONCLUSIONS
The increased Ad5RSVβgal-mediated transgene expression in the VK2 cells is due to the increased RSV promoter activity in VK2 cells. Our results point out that (i) drug-resistance may be accompanied with an alteration in promoter activity; (ii) the proper choice of promoter could contribute to a decrease in the vector dose required to achieve a therapeutic effect during gene therapy.
Majhen
Dragomira
D
Laboratory for Genotoxic Agents, Division of Molecular Biology, Ruđer Bošković Institute, 10000 Zagreb, Croatia.
Brozovic
Anamaria
A
Buger
Tvrtko
T
Gabrilovac
Jelka
J
Osmak
Maja
M
Ambriović-Ristov
Andreja
A
D000256:Adenoviridae / Q000235:genetics*; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D045744:Cell Line, Tumor; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D019008:Drug Resistance, Neoplasm; D015972:Gene Expression Regulation, Neoplastic; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D007822:Laryngeal Neoplasms / Q000473:pathology / Q000628:therapy*; D011401:Promoter Regions, Genetic; D011991:Receptors, Virus / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D052736:Rous sarcoma virus / Q000235:genetics*; D019076:Transgenes; D014750:Vincristine / Q000494:pharmacology
D000972:Antineoplastic Agents, Phytogenic; C568771:CLMP protein, human; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D011991:Receptors, Virus; D014750:Vincristine