Yu D C, Sakamoto G T, Henderson D R
Calydon, Inc., Sunnyvale, California 94089, USA.
Cancer Res. 1999 Apr 1;59(7):1498-504.
Human glandular kallikrein (hK2) and prostate-specific antigen (PSA) are related members of the human kallikrein gene family. The genes for hK2 and PSA are expressed predominately in the prostate, are transcriptionally up-regulated by androgens, and share 78% homology. Previously, one functional androgen response element was identified within the proximal promoter (-324 to +33 relative to the cap site) of the hK2 gene. To detect additional upstream regulatory elements, the 12.3 kbp between the PSA gene and 5' to the hK2 gene were amplified by PCR and linked to a promoterless firefly luciferase reporter gene. Transient transfection experiments showed an androgen-dependent enhancer, located between -3.4 and -5.2 kb upstream of the transcription start site of the hK2 gene. This hK2 enhancer increased luciferase expression 100-fold in the presence of the testosterone analogue R1881. The hK2 enhancer contains an androgen response element that lost activity when mutated. The hK2 enhancer/promoter demonstrated activity in PSA(+) LNCaP cells whereas the enhancer/promoter was inactive in PSA(-) 293, A549, HBL100, HUH-7, LoVo, MCF-7, OVCAR-3, and PC-3 cells. Insertion of the hK2 enhancer/promoter into adenovirus to drive the E1A genes of adenovirus type 5 (Ad5) created an attenuated replication competent adenovirus variant Calydon virus (CV) 763, which replicates similarly to wild-type adenovirus in prostate tumor cells but is attenuated in nonprostate tumor cells. In addition, CV764, an adenovirus variant containing the previously cloned prostate-specific enhancer (to drive the Ad5 E1A genes) and the hK2 enhancer/promoter (to drive the Ad5 E1B genes) was constructed. CV764 is significantly attenuated and has a high therapeutic index with a cell specificity of 10,000:1 for PSA(+) LNCaP cells, compared to ovarian cancer OVCAR-3 cells and SK-OV-3 cells and PA-1 cells. CV764 is also highly attenuated in primary human microvascular endothelial cells.
人腺激肽释放酶（hK2）和前列腺特异性抗原（PSA）是人类激肽释放酶基因家族的相关成员。hK2和PSA基因主要在前列腺中表达，受雄激素转录上调，且具有78%的同源性。此前，在hK2基因近端启动子（相对于帽位点为-324至+33）内鉴定出一个功能性雄激素反应元件。为了检测其他上游调控元件，通过PCR扩增了PSA基因与hK2基因5'端之间的12.3 kbp，并将其连接到无启动子的萤火虫荧光素酶报告基因上。瞬时转染实验显示，在hK2基因转录起始位点上游-3.4至-5.2 kb之间存在一个雄激素依赖性增强子。在睾酮类似物R1881存在的情况下，该hK2增强子使荧光素酶表达增加了100倍。hK2增强子包含一个雄激素反应元件，该元件在突变时失去活性。hK2增强子/启动子在PSA(+)的LNCaP细胞中表现出活性，而在PSA(-)的293、A549、HBL100、HUH-7、LoVo、MCF-7、OVCAR-3和PC-3细胞中无活性。将hK2增强子/启动子插入腺病毒以驱动5型腺病毒（Ad5）的E1A基因，产生了一种减毒的具有复制能力的腺病毒变体Calydon病毒（CV）763，其在前列腺肿瘤细胞中的复制方式与野生型腺病毒相似，但在非前列腺肿瘤细胞中减毒。此外，构建了腺病毒变体CV764，其包含先前克隆的前列腺特异性增强子（用于驱动Ad5 E1A基因）和hK2增强子/启动子（用于驱动Ad5 E1B基因）。与卵巢癌OVCAR-（此处原文可能有误，推测为OVCAR-3）细胞、SK-OV-3细胞和PA-1细胞相比，CV764显著减毒，对PSA(+)的LNCaP细胞具有10000:1的细胞特异性治疗指数。CV764在原代人微血管内皮细胞中也高度减毒。
