Chung I, Schwartz P E, Crystal R G, Pizzorno G, Leavitt J, Deisseroth A B
Yale University School of Medicine, New Haven, Connecticut 06520-8032, USA.
Cancer Gene Ther. 1999 Mar-Apr;6(2):99-106. doi: 10.1038/sj.cgt.7700017.
The objective of this study was to develop an adenoviral vector system that would generate a pattern of expression of exogenous therapeutic genes appropriate for the treatment of ovarian cancer. For this purpose, we have generated a replication-deficient recombinant adenoviral vector, AdLPLacZ, which contains the human L-plastin (LP) promoter (LP-P) driving the Escherichia coli LacZ gene. LP is constitutively expressed at high levels in malignant epithelial cells but is not expressed in normal tissues, except at low levels in mature hematopoietic cells. Because adenoviral vectors infect early hematopoietic multilineage precursor cells only poorly or not at all, this vector would be of use in the peritoneal cavity and in vitro for marrow purging. We first analyzed the expression of the LacZ reporter gene in ovarian and breast cancer cell lines, normal fibroblasts, and leukemia cell lines using the adenoviral vector in which the LacZ gene is governed by the LP-P promoter (AdLPLacZ) or in which the LacZ gene is governed by the cytomegalovirus (CMV) promoter (AdCMVLacZ). We found equivalent and high levels of expression of beta-galactosidase (beta-gal) by AdLPLacZ and AdCMVLacZ vectors in the breast or ovarian cancer cell lines as well as in a fibrosarcoma cell line, indicating that the adenoviral vectors infected these cells and expressed their transgenes equally with the LP and CMV promoters. Expression of the LacZ gene with the CMV vector but not with the LP-P vector was observed in experiments with normal fibroblasts, indicating that the vectors infected the cells, but that the LP-P was not active within them. In hematopoietic cells such as U937 cells, no measurable beta-gal activity was detected in cells infected either by AdLPLacZ or by AdCMVLacZ, indicating that the adenoviral vectors were not infecting the cells. Although beta-gal activity was observed in fresh ascitic ovarian cancer cells after infection with adenoviral vectors containing CMV or the LP promoters, beta-gal activity was detected in a portion of a biopsy of normal peritoneum when the tissues were exposed to the AdCMVLacZ vector, but not when tissues were exposed to the AdLPLacZ vector. These results suggest that the transcription of therapeutic genes in cells infected by the AdLP vectors would be restricted to LP expression-positive ovarian carcinoma cells but would not be seen in the normal mesothelial cells of the peritoneal cavity. This possibility implies that adenoviral vectors carrying therapeutic genes driven by the LP-P would be of use for the intracavitary treatment ovarian cancer.
本研究的目的是开发一种腺病毒载体系统，该系统能够产生适合治疗卵巢癌的外源治疗基因表达模式。为此，我们构建了一种复制缺陷型重组腺病毒载体AdLPLacZ，其包含驱动大肠杆菌LacZ基因的人L-肌动蛋白（LP）启动子（LP-P）。LP在恶性上皮细胞中持续高水平表达，但在正常组织中不表达，仅在成熟造血细胞中有低水平表达。由于腺病毒载体对早期造血多谱系前体细胞的感染能力很差或根本无法感染，因此该载体可用于腹腔内以及体外骨髓净化。我们首先使用LacZ基因由LP-P启动子调控的腺病毒载体（AdLPLacZ）或LacZ基因由巨细胞病毒（CMV）启动子调控的腺病毒载体（AdCMVLacZ），分析了LacZ报告基因在卵巢和乳腺癌细胞系、正常成纤维细胞以及白血病细胞系中的表达情况。我们发现AdLPLacZ和AdCMVLacZ载体在乳腺癌或卵巢癌细胞系以及纤维肉瘤细胞系中β-半乳糖苷酶（β-gal）的表达水平相当且较高，这表明腺病毒载体能够感染这些细胞，并通过LP和CMV启动子同等程度地表达其转基因。在正常成纤维细胞实验中，观察到CMV载体可表达LacZ基因，而LP-P载体则不能，这表明载体能够感染细胞，但LP-P在细胞内不具有活性。在造血细胞如U937细胞中，无论是AdLPLacZ还是AdCMVLacZ感染的细胞均未检测到可测量的β-gal活性，这表明腺病毒载体未感染这些细胞。尽管在用含有CMV或LP启动子的腺病毒载体感染新鲜腹水卵巢癌细胞后观察到了β-gal活性，但当正常腹膜组织暴露于AdCMVLacZ载体时，在部分活检组织中检测到了β-gal活性，而暴露于AdLPLacZ载体时则未检测到。这些结果表明，AdLP载体感染的细胞中治疗基因的转录将局限于LP表达阳性的卵巢癌细胞，而在腹腔正常间皮细胞中则不会出现。这种可能性意味着携带由LP-P驱动的治疗基因的腺病毒载体可用于卵巢癌的腔内治疗。
