Jounaidi Youssef, Waxman David J
Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, Massachusetts 02215, USA.
Cancer Res. 2004 Jan 1;64(1):292-303. doi: 10.1158/0008-5472.can-03-1798.
Cytochrome P450 (CYP) gene transfer sensitizes tumor xenografts to anticancer prodrugs such as cyclophosphamide (CPA) without a detectable increase in host toxicity. Optimal prodrug activation is achieved when a suitable P450 gene (e.g., human CYP2B6) is delivered in combination with NADPH-cytochrome P450 reductase (P450R), which encodes the flavoenzyme P450 reductase. We sought to improve this gene therapy by coordinated delivery and expression of P450 and P450R on a single bicistronic vector using an internal ribosomal entry site (IRES) sequence. Retrovirus encoding a CYP2B6-IRES-P450R expression cassette was shown to induce strong P450-dependent CPA cytotoxicity in a population of infected 9L gliosarcoma cells. Adeno-P450, a replication-defective, E1/E3 region-deleted adenovirus engineered to express CYP2B6-IRES-P450R, induced intracellular CPA 4-hydroxylation, and CPA cytotoxicity, in a broad range of human cancer cell lines. However, limited Adeno-P450 gene transfer and CPA chemosensitization was seen with certain human tumor cells, notably PC-3 prostate and HT-29 colon cancer cells. Remarkable improvements could be obtained by coinfecting the tumor cells with Adeno-P450 in combination with Onyx-017, an E1b-55k gene-deleted adenovirus that selectively replicates in p53 pathway-deficient cells. Substantial increases in gene expression were observed during the early stages of viral infection, reflecting an apparent coamplification of the Adeno-P450 genome, followed by enhanced viral spread at later stages, as demonstrated in cultured tumor cells, and in A549 and PC-3 solid tumor xenografts grown in scid mice. This combination of the replication-defective Adeno-P450 with a replication-conditional and tumor cell-targeted helper adenovirus dramatically improved the low gene transfer observed with some human tumor cell lines and correspondingly increased tumor cell-catalyzed CPA 4-hydroxylation, CPA cytotoxicity, and in vivo antitumor activity in a PC-3 tumor xenograft model. The use of tumor-selective, replicating adenovirus to promote the spread of replication-defective gene therapy vectors, such as Adeno-P450, substantially increases the therapeutic potential of adenoviral delivery systems, and should lead to increased activity and enhanced tumor selectivity of cytochrome P450 and other gene-directed enzyme prodrug therapies.
细胞色素P450（CYP）基因转移可使肿瘤异种移植对环磷酰胺（CPA）等抗癌前体药物敏感，而宿主毒性未见明显增加。当合适的P450基因（如人CYP2B6）与NADPH-细胞色素P450还原酶（P450R）联合递送时，可实现最佳的前体药物激活，P450R编码黄素酶P450还原酶。我们试图通过使用内部核糖体进入位点（IRES）序列在单个双顺反子载体上协调递送和表达P450和P450R来改进这种基因治疗。编码CYP2B6-IRES-P450R表达盒的逆转录病毒在一群受感染的9L胶质肉瘤细胞中显示出诱导强烈的P450依赖性CPA细胞毒性。腺病毒-P450是一种复制缺陷型、E1/E3区域缺失的腺病毒，经工程改造可表达CYP2B6-IRES-P450R，在多种人类癌细胞系中诱导细胞内CPA 4-羟基化和CPA细胞毒性。然而，在某些人类肿瘤细胞中，尤其是PC-3前列腺癌细胞和HT-29结肠癌细胞中，腺病毒-P450基因转移和CPA化学增敏作用有限。通过将腺病毒-P450与Onyx-017（一种E1b-55k基因缺失的腺病毒，可在p53通路缺陷的细胞中选择性复制）共同感染肿瘤细胞，可获得显著改善。在病毒感染的早期阶段观察到基因表达大幅增加，这反映了腺病毒-P450基因组的明显共扩增，随后在后期阶段病毒传播增强，这在培养的肿瘤细胞以及在严重联合免疫缺陷（scid）小鼠中生长的A549和PC-3实体瘤异种移植中得到了证实。这种复制缺陷型腺病毒-P450与复制条件性和肿瘤细胞靶向辅助腺病毒的组合显著改善了在某些人类肿瘤细胞系中观察到的低基因转移，并相应增加了肿瘤细胞催化的CPA 4-羟基化、CPA细胞毒性以及在PC-3肿瘤异种移植模型中的体内抗肿瘤活性。使用肿瘤选择性复制腺病毒来促进复制缺陷型基因治疗载体（如腺病毒-P450）的传播，可大幅提高腺病毒递送系统的治疗潜力，并应导致细胞色素P450和其他基因导向酶前体药物疗法的活性增加和肿瘤选择性增强。
