Ambriović-Ristov Andreja, Gabrilovac Jelka, Cimbora-Zovko Tamara, Osmak Maja
Laboratory for Genotoxic Agents, Division of Molecular Biology, Ruder Bosković Institute, Zagreb, Croatia.
Int J Cancer. 2004 Jul 10;110(5):660-7. doi: 10.1002/ijc.20176.
In our study, we investigated molecular mechanisms of increased adenoviral transduction efficacy in cisplatin-resistant human laryngeal carcinoma cells CA3ST as compared to parental cells HEp2. Using reverse transcription-PCR, the genes potentially implicated in adenoviral entry were screened. In cisplatin-resistant cells, only upregulation of alphavbeta3 integrin was detected, which was additionally confirmed by flow cytometry. Moderately increased expression of CAR was determined in cisplatin-resistant CA3ST cells using flow cytometry and measurement of wild-type adenovirus Ad5CMVbetagal attachment. In order to test the implication of alphavbeta3 integrin in transduction efficacy, 6 HEp2-derived alphavbeta3-expressing clones with graded expression of alphavbeta3 were isolated. To a certain degree of density, expression of alphavbeta3 positively correlated with Ad5CMVbetagal transduction efficacy (i.e., increased viral transduction), suggesting a role of alphavbeta3 in transduction efficacy. However, HEp2 clones with the highest alphavbeta3) expression were negatively correlated with transduction efficacy (i.e., decreased viral transduction). This was shown to be associated with downregulation of alphavbeta5 integrin, also involved in viral transduction, in clones with the highest alphavbeta3 expression. The implication of CAR in increased adenoviral transduction efficacy in cisplatin resistant CA3ST cells was further assessed by transduction experiments using adenoviral mutant Ad5FbDelta639 whose entry is only to a very small extent dependent on the presence of CAR. Indeed, Ad5FbDelta639 infected 2.5-fold more, in comparison to wild-type adenovirus, which infected 5-fold more efficiently resistant CA3ST cells than parental HEp2 cells, indicating that increased expression of CAR contributes to increased efficacy of adenoviral transduction. Thus, the data presented provide evidence that both alphavbeta3 integrin and CAR are involved in increased adenoviral transduction efficacy in cisplatin resistant CA3ST cells. These findings may have significant implications in human gene therapy using adenoviruses, especially in patients after unsuccessful cisplatin treatment.
在我们的研究中，我们调查了与亲代细胞HEp2相比，顺铂耐药的人喉癌细胞CA3ST中腺病毒转导效率提高的分子机制。使用逆转录聚合酶链反应筛选可能与腺病毒进入有关的基因。在顺铂耐药细胞中，仅检测到αvβ3整合素上调，流式细胞术进一步证实了这一点。使用流式细胞术和顺铂耐药CA3ST细胞中野生型腺病毒Ad5CMVβgal附着的测量，确定了CAR的表达适度增加。为了测试αvβ3整合素在转导效率中的作用，分离了6个具有不同αvβ3表达水平的HEp2来源的αvβ3表达克隆。在一定密度下，αvβ3的表达与Ad5CMVβgal转导效率呈正相关（即病毒转导增加），表明αvβ3在转导效率中起作用。然而，αvβ3表达最高的HEp2克隆与转导效率呈负相关（即病毒转导降低）。这表明在αvβ3表达最高的克隆中，也参与病毒转导的αvβ5整合素下调。通过使用腺病毒突变体Ad5FbDelta639进行转导实验，进一步评估了CAR在顺铂耐药CA3ST细胞中腺病毒转导效率增加中的作用，其进入仅在很小程度上依赖于CAR的存在。事实上，与野生型腺病毒相比，Ad5FbDelta639的感染率高出2.5倍，野生型腺病毒感染顺铂耐药CA3ST细胞的效率比亲代HEp2细胞高出5倍，表明CAR表达增加有助于提高腺病毒转导效率。因此，所呈现的数据提供了证据，表明αvβ3整合素和CAR都参与了顺铂耐药CA3ST细胞中腺病毒转导效率的提高。这些发现可能对使用腺病毒的人类基因治疗具有重要意义，特别是在顺铂治疗失败的患者中。
