EXPERIMENTAL THERAPEUTICS EXPLOITING FOLATE RECEPTORS

利用叶酸受体的实验治疗

• 批准号: 6129304
• 负责人: Asok Antony
• 金额: $ 20.13万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-16 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6129304
• 关键词: HeLa cells; Retroviridae; antisense nucleic acid; apoptosis; athymic mouse; cervix neoplasms; cis platinum compound; complementary DNA; drug interactions; folate; ganciclovir; gene therapy; genetic promoter element; human papillomavirus; immunoglobulin G; liposomes; neoplasm /cancer therapy; nonhuman therapy evaluation; oligonucleotides; receptor expression; transcription factor; vitamin receptor; zidovudine

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.

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。 这将从三个方面帮助控制肿瘤生长：首先，由于FR过表达与体内细胞增殖之间存在反比关系，肿瘤倍增将从4.5天显着延迟至9天；其次，转导的细胞将更容易接受封装在叶酸束缚脂质体 (F ) 中的顺铂，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 过表达而增加的胸苷激酶 (TK) 活性将使这些细胞对原本无毒浓度的叠氮胸苷（或更昔洛韦）过敏。 在追求这一目标的同时，我们建议继续进行基础研究，以阐明 FR mRNA 5'-UTR 中的 18 个碱基顺式元件与 FR mRNA 中的 46-kDa 反式因子相互作用的（潜在临床）意义。 FR 在细胞中的翻译。 因此，我们将通过将纯化的反式因子的抗血清和顺式元件的反义寡核苷酸引入细胞中来检验这一假设，即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通过 F 并确定淬灭 FR（在 FR 表达对于细胞活力至关重要的情况下）是否会引发叶酸缺乏的细胞死亡。 我们还将测试以下假设：正义 46-kDa 反式因子 cDNA（或更高亲和力突变体）的转导可以上调 HeLa-IU1 细胞中的 FR、减少细胞增殖并增加对 TK 诱导的细胞毒性的敏感性。更昔洛韦。 这可以识别通过直接转导 FR 基因以外的方式调节 FR 表达的新线索。在这些研究期间，我们还将启动一项计划，系统地测试一系列假设，以确认 F 将顺铂或逆转录病毒构建体递送至宫颈癌细胞的优越性，并验证更昔洛韦增强转导的 Hela-IU1 细胞的细胞毒性，在荷瘤小鼠体内和体外过度表达 FR。 这将使我们能够最终检验以下假设：通过使用含有 F 的逆转录病毒构建体（带有 HeLa 特异性启动子驱动的有义 FR cDNA）进行基因治疗，在 HeLa-IU1 细胞衍生的皮下肿瘤上扩增 FR 将（a）减少肿瘤加倍； (b) 使细胞更容易接受随后摄取的F+顺铂，从而导致更大的细胞毒性。 并且由于TK活性的平行增加，(c)更昔洛韦的添加将导致与F+顺铂对肿瘤细胞产生协同细胞毒作用，从而降低其IC50和对正常组织的毒性。 这种方法具有创新性，因为其固有的“转化”性质，我们试图将实验室研究产生的令人兴奋的结果与临床前环境联系起来，作为将这种方法用于晚期宫颈癌女性的前奏。