EXPERIMENTAL THERAPEUTICS EXPLOITING FOLATE RECEPTORS

我们的长期目标是针对表达叶酸受体(FR)的宫颈癌细胞进行基因治疗，以进一步上调FR的表达。这将在3个方面帮助肿瘤生长控制：首先，由于FR过表达与体内细胞增殖之间存在负相关关系，肿瘤细胞的倍增将从4.5天大幅推迟至9天；其次，转导细胞将更容易接受通过FR进入细胞的叶酸锚定脂质体(F)包裹的顺铂的摄取，从而预测较低的IC50，且全身毒性较小；第三，与FR过度表达相伴随的胸苷激酶(TK)活性增加将使这些细胞对其他无毒浓度的齐多夫定(或更昔洛韦)更敏感。在追求这一目标的同时，我们建议继续我们的基础研究，以阐明FR mRNA 5‘端非编码区18碱基顺式元件与46 kDa反式因子在细胞内翻译FR中的相互作用(潜在的临床)意义。因此，我们将通过F将纯化的反式因子的抗血清和顺式元件的反义寡核苷酸导入细胞，并确定FR猝灭(在FR表达对细胞生存至关重要的条件下)是否会引发叶酸缺乏的细胞死亡，以检验这种顺式元件和反式因子的相互作用是培养的HeLa-IU1细胞中FR合成的关键决定因素的假设。我们还将验证这样的假设，即转导正义46 kDa反式因子cDNA(或更高亲和力突变体)可以上调HeLa-IU1细胞中的FR，降低细胞增殖，并增加对TK诱导的细胞毒性的敏感性。这可能通过直接转导FR基因以外的其他方式来识别调节FR表达的新的线索。在这些研究中，我们还将启动一项计划，系统地测试一系列假设，以确认F向宫颈癌细胞运送顺铂或逆转录病毒载体的优越性，并验证更昔洛韦在体外和体内增强体内过表达FR的转导Hela-IU1细胞的细胞毒作用。这将使我们能够最终检验这样的假设，即通过基因治疗HeLa-IU1细胞衍生的皮下侧翼肿瘤的FR扩增，带有HeLa特异性启动子驱动的正义FR cDNAF的逆转录病毒载体将：(A)减少肿瘤倍增；(B)使细胞更容易接受随后的F+顺铂摄取，从而产生更大的细胞毒性。由于TK活性的平行增加，(C)更昔洛韦的加入将导致F+顺铂对肿瘤细胞的协同细胞毒作用，从而降低其IC50和对正常组织的毒性。这种方法是创新的，因为它固有的“翻译”性质，其中我们试图将从试验台研究产生的令人兴奋的结果连接到临床前环境，作为对晚期宫颈癌妇女使用这种方法的前奏。

Our long-term goal is to target folate receptor (FR)-expressing cervical carcinoma cells for gene therapy to further up-regulate FR expression. This will assist tumor growth Control in 3 ways: first, because of an inverse relationship between FR overexpression and cell proliferation in vivo, tumor doubling will be dramatically delayed from 4.5 to 9 days; second, transduced cells will be more receptive to the uptake of cisplatin that is encapsulated in folate-tethered liposomes (F ) which enter cells via FR, thereby predicting a lower IC50 with less systemic toxicity; and third, the increase in thymidine kinase (TK) activity which accompanies FR overexpression will render these cells hypersensitive to otherwise nontoxic concentrations of azidothymidine (or ganciclovir). While pursuing this goal, we propose to continue our basic studies to elucidate the (potential clinical) significance of the interaction of the 18-base cis-element in the 5'-UTR of FR mRNA and 46-kDa-trans-factor in the translation of FR in cells. So we will test the hypothesis that this cis-element and trans-factor interaction is a critical determinant for the synthesis of FR in cultured HeLa-IU1 cells by introducing antisera to the purified trans-factor and antisense oligonucleotides to the cis-element into cells via F and determining if quenching FR (under conditions when FR expression is essential for the viability of the cell) will trigger a folate-deficient cell death. We will also test the hypothesis that transduction of the sense 46-kDa-trans-factor cDNA (or higher-affinity mutant) can up-regulate FR in HeLa-IU1 cells, decrease cell proliferation, and increase susceptibility to TK-induced cytotoxicity by ganciclovir. This could identify new leads to modulate FR expression by means other than through direct transduction of FR genes. During these studies, we will also initiate a program to systematically test a series of hypotheses to confirm the superiority of F to deliver either cisplatin or retroviral constructs to cervical carcinoma cells, and to verify that ganciclovir augments the cytotoxicity of transduced Hela-IU1 cells that overexpress FR in vitro and in vivo in tumor-bearing mice. This will place us in a position to eventually test the hypothesis that amplification of FR on HeLa-IU1 cell-derived subcutaneous flank tumors by gene therapy with F containing retroviral constructs bearing HeLa-specific promoter-driven sense FR cDNA will (a) reduce tumor doubling; (b) render cells more receptive to subsequent uptake of F +cisplatin resulting in greater cytotoxicity. And because of the parallel increase in TK activity, (c) the addition of ganciclovir will lead to synergistic cytotoxic effects to tumor cells with F +cisplatin thereby reducing its IC50 and toxicity to normal tissues. This approach is innovative because of its inherent 'translational' nature wherein we are attempting to bridge exciting results that have arisen from bench research to the preclinical setting as a prelude to using this approach for women with advanced cervical cancer.