Vincristine-resistant human laryngeal carcinoma cells demonstrate increased Rous sarcoma virus promoter activity Dragomira Majhen a,1 , Anamaria Brozovic a,1 , Tvrtko Buger a , Jelka Gabrilovac b , Maja Osmak a , Andreja Ambriović-Ristov a, ⁎ a Laboratory for Genotoxic Agents, Division of Molecular Biology, Ruđer Bošković Institute, Bijenička 54, 10000 Zagreb, Croatia b Laboratory for Experimental Haematology, Immunology and Oncology, Division of Molecular Medicine, Ruđer Bošković Institute, Bijenička 54, 10000 Zagreb, Croatia abstract article info Article history: Received 28 May 2010 Accepted 19 August 2010 Keywords: Promoter Adenovirus Vincristine Resistance Gene therapy Aims: 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. Main 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. Key findings: 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. Significance: 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. © 2010 Elsevier Inc. All rights reserved. Introduction Microtubules are cytoskeletal elements that control a wide range of cellular functions. They are important targets for cancer chemo- therapeutic drugs, such as vincristine. Vincristine is a microtubule- destabilizing antimitotic drug commonly used in the treatment of leukemias and lymphomas, as well as in combination therapies of several nonhematopoietic cancers (Duflos et al. 2002). The standard treatment of head and neck cancer is surgical resection or radical radiotherapy, or sometimes both. Compared with radiotherapy alone, concomitant chemotherapy reduces the risk of dying by 19% (Pignon et al. 2009). Recently published results of a 10-year follow-up of chemoradiotherapy for locally advanced head and neck cancer showed that patients who had undergone previous surgery did not benefit from the addition of chemotherapy to adjunctive post- operative radiotherapy. However, in patients who had not undergone surgery, treatment with two courses of simultaneous non-platinum chemoradiotherapy (vincristine, bleomycin, methotrexate and fluo- rouracil) was associated with a clear benefit that persisted after a long follow-up period (Tobias et al. 2010). One major problem with cancer chemotherapy is the emergence of drug-resistant cancer cells. The development of broad-spectrum vincristine resistance is characterized by overexpression of the transmembrane efflux pump, P-glycoprotein and/or altered β-tubulin expression (Perez 2009). Still, there is a clear need for the develop- ment of new therapies. One possibility is cancer gene therapy. The success of nonviral as well as adenoviral tumor gene therapy depends on (i) efficient delivery of their genetic material to the target cell population, i.e., vector entry, and (ii) the efficiency of the therapeutic transgene expression from a given promoter. While the entry of plasmid vectors depends on the efficiency of transfection, the entry of vectors derived from adenovirus type 5 (Ad5) depends on the presence of the primary coxsackie adenovirus receptor (CAR), which promotes attach- ment (Bergelson et al. 1997; Tomko et al. 1997), and integrins αvβ1, Life Sciences 87 (2010) 468–474 ⁎ Corresponding author. Tel.: + 385 1 4571 240; fax: + 385 1 4561 177. E-mail address: andrea@irb.hr (A. Ambriović-Ristov). 1 Equal contribution. 0024-3205/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2010.08.012 Contents lists available at ScienceDirect Life Sciences journal homepage: www.elsevier.com/locate/lifescie