Chen L, Waxman D J, Chen D, Kufe D W
Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Cancer Res. 1996 Mar 15;56(6):1331-40.
The cancer chemotherapeutic agent cyclophosphamide (CPA) and its isomer ifosfamide (IFA) are alkylating agent prodrugs that require metabolism by liver cytochrome P450 (P450) enzymes for antitumor activity. The therapeutic effectiveness of these oxazaphosphorines is limited by the hematopoietic, renal, and cardiac toxicity that accompanies the systemic distribution of liver-derived activated drug metabolites. Transfer of a liver cytochrome P450 gene, CYP2B1, into human breast MCF-7 cancer cells is presently shown to greatly sensitize these cells to oxazaphosphorine toxicity as a consequence of the acquired capacity for intratumoral CPA and IFA activation. Thus, CPA and IFA were highly cytotoxic to MCF-7 cells following stable transfection of CYP2B1 but exhibited no toxicity to parental tumor cells or to a beta-galactosidase-expressing MCF-7 transfectant. This cytotoxicity could be appreciably blocked by the CYP2B1 inhibitor metyrapone. Cell cycle analysis revealed that CPA arrested the CYP2B1-expressing cells, but not CYP2B1-negative cells, at G(2)-M phase. A strong bystander cytotoxicity effect that does not require direct cell-cell contact was mediated by CYP2B1-expressing MCF-7 cells on non-CYP2B1 cells. Intratumoral CYP2B1 expression conferred a distinct therapeutic advantage when treating MCF-7 tumors grown in nude mice with CPA, as revealed by a 15-20-fold greater in vivo cytotoxicity, determined by tumor excision/colony formation assay, and by the substantially enhanced antitumor activity, monitored by tumor growth delay, for CYP2B1-e xpressing MCF-7 tumors as compared to CYP2B1-negative control tumors. These enhanced therapeutic effects were obtained without any apparent increase in host toxicity. To evaluate the extent to which a CPA/P450 gene therapy strategy may be generally applicable to other tumor cell types, a replication-defective recombinant adenovirus carrying the CYP2B1 gene driven by the cytomegalovirus (CMV) promotor ad.CMV-2B1 was constructed and used to infect a panel of human tumor cell lines. Ad.CMV-2B1 infection rendered each of the cell lines highly sensitive to CPA and IFA cytotoxicity, with substantial chemosensitization seen at multiplicities of infection as low as 10. The CPA/P450 prodrug activation system may thus serve as a useful paradigm for further development of novel cancer gene therapy strategies that utilize drug susceptibility genes to significantly potentiate the antitumor activity of conventional cancer chemotherapeutic agents.
癌症化疗药物环磷酰胺（CPA）及其异构体异环磷酰胺（IFA）是烷基化剂前体药物，需要通过肝脏细胞色素P450（P450）酶进行代谢才能发挥抗肿瘤活性。这些恶唑磷的治疗效果受到肝脏衍生的活性药物代谢产物全身分布所伴随的造血、肾脏和心脏毒性的限制。目前研究表明，将肝脏细胞色素P450基因CYP2B1转入人乳腺癌MCF-7癌细胞后，由于肿瘤内CPA和IFA激活能力的获得，这些细胞对恶唑磷毒性的敏感性大大增加。因此，在稳定转染CYP2B1后，CPA和IFA对MCF-7细胞具有高度细胞毒性，但对亲本肿瘤细胞或表达β-半乳糖苷酶的MCF-7转染细胞无毒性。这种细胞毒性可被CYP2B1抑制剂甲吡酮明显阻断。细胞周期分析显示，CPA使表达CYP2B1的细胞在G(2)-M期停滞，但不使CYP2B1阴性细胞停滞。表达CYP2B1的MCF-7细胞对非CYP2B1细胞介导了一种强烈的旁观者细胞毒性效应，这种效应不需要直接的细胞间接触。当用CPA治疗裸鼠体内生长的MCF-7肿瘤时，肿瘤内CYP2B1表达赋予了明显的治疗优势，通过肿瘤切除/集落形成试验测定，体内细胞毒性提高了15 - 20倍，通过肿瘤生长延迟监测，与CYP2B1阴性对照肿瘤相比，表达CYP2B1的MCF-7肿瘤的抗肿瘤活性显著增强。在宿主毒性没有任何明显增加的情况下获得了这些增强的治疗效果。为了评估CPA/P450基因治疗策略在多大程度上可能普遍适用于其他肿瘤细胞类型，构建了一种携带由巨细胞病毒（CMV）启动子驱动的CYP2B1基因的复制缺陷型重组腺病毒ad.CMV-2B1，并用于感染一组人肿瘤细胞系。Ad.CMV-2B1感染使每个细胞系对CPA和IFA细胞毒性高度敏感，在低至10的感染复数下就观察到了显著的化学增敏作用。因此，CPA/P450前体药物激活系统可能作为一种有用的范例，用于进一步开发利用药物敏感性基因显著增强传统癌症化疗药物抗肿瘤活性的新型癌症基因治疗策略。
