EXPERIMENTAL THERAPEUTICS EXPLOITING FOLATE RECEPTORS

我们的长期目标是靶向表达叶酸受体（FR）的宫颈癌细胞进行基因治疗，进一步上调FR表达。这将从三个方面帮助肿瘤生长控制：首先，由于体内FR过表达与细胞增殖呈反比关系，肿瘤加倍将从4.5天显著推迟到9天；其次，转导的细胞将更容易接受包裹在叶酸系链脂质体(F)中的顺铂的摄取，顺铂通过FR进入细胞，从而预测较低的IC50和较小的全身毒性；第三，伴随FR过表达的胸苷激酶（TK）活性的增加将使这些细胞对其他无毒浓度的azidothymidine（或更昔洛韦）过敏。在实现这一目标的同时，我们建议继续我们的基础研究，以阐明FR mRNA 5'-UTR中18碱基顺式元件与46- kda -反式因子在细胞FR翻译中的相互作用（潜在的临床）意义。因此，我们将测试这种顺式因子和反式因子相互作用是培养的HeLa-IU1细胞中FR合成的关键决定因素的假设，通过将纯化的反式因子和反义寡核苷酸通过F引入顺式因子的抗血清，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。我们还将验证46- kda -反式因子cDNA（或高亲和突变体）的转导可以上调HeLa-IU1细胞中的FR，降低细胞增殖，并增加更昔洛维对tk诱导的细胞毒性的易感性。这可以通过FR基因的直接转导以外的方式发现调节FR表达的新线索。在这些研究中，我们还将启动一个项目，系统地测试一系列假设，以证实F在向宫颈癌细胞递送顺铂或逆转录病毒构建物方面的优越性，并验证更昔洛韦增强了在体外和体内过度表达FR的转导Hela-IU1细胞的细胞毒性。这将使我们能够最终验证以下假设，即通过携带hela特异性启动子驱动FR cDNA的含F逆转录病毒构建物进行基因治疗，在HeLa-IU1细胞衍生的皮下侧腹肿瘤上扩增FR将(a)减少肿瘤加倍；(b)使细胞更容易接受F +顺铂的后续摄取，从而导致更大的细胞毒性。由于TK活性的平行增加，(c)更昔洛韦的加入将导致肿瘤细胞与F +顺铂的协同细胞毒作用，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“转化”性质，我们正试图将从实验室研究中产生的令人兴奋的结果连接到临床前环境，作为将这种方法用于晚期宫颈癌妇女的前奏。

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.