Reversion of transformed phenotype in ovarian cancer cells by intracellular expression of anti folate receptor antibodies
Reversion of transformed phenotype in ovarian cancer cells by intracellular expression of anti folate receptor antibodies
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DOI：
10.1038/sj.gt.3301962
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发表时间：
2003-06-01
期刊：
影响因子：
5.1
通讯作者：
Canevari, S
中科院分区：
文献类型：
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作者：
Figini, M;Ferri, R;Canevari, S
α-叶酸受体（FR）在90%的非粘液性卵巢癌中选择性过表达，而在正常卵巢表面上皮（OSE）中未检测到表达。间接证据表明，FR表达与肿瘤进展相关，并影响细胞增殖。为了更好地评估FR的作用，我们开发了一种基于单链（sc）抗体（胞内抗体）的细胞内表达的方法来下调卵巢癌细胞中FR的膜表达。用抗FR胞内抗体转染IGF-I和SKOV 3卵巢癌细胞系。通过荧光激活细胞分选（FACS）、逆转录和聚合酶链反应（RT-PCR）和/或免疫印迹测试转染子和亲本细胞的FR、整联蛋白和抗FR胞内抗体表达。在液体、半固体和器官型培养物中评价细胞生长特性和粘附特性。抗FR scFv抑制FR表达从60%至99%。在生理浓度的叶酸，增殖变化直接作为FR表达的函数。FR下调伴随着软琼脂中集落形成能力降低、细胞形态变化、与层粘连蛋白或基质胶的粘附显着增强、α 6 β 4整合素表达增加两到三倍以及层粘连蛋白产生显着减少。在三维器官型培养中，抗FR胞内抗体转染的IGROV 1细胞生长为一个单一的有序层，让人想起体内正常的OSE生长。总之，抗FR胞内抗体逆转卵巢癌细胞中的转化表型，并可能提供一种有效的手段来选择性地抑制这些细胞的生长。Gene Therapy（2003）10，1018 -1025. doi：10.1038/sj.gt.3301962。
The alpha-folate receptor (FR) is selectively overexpressed in 90% of nonmucinous ovarian carcinomas, whereas no expression is detectable in normal ovarian surface epithelium (OSE). Indirect evidence suggests that FR expression is associated with tumor progression and affects cell proliferation. To evaluate better the role of FR, we developed an approach based on intracellular expression of single-chain (sc) antibodies (intrabody) to downmodulate membrane expression of FR in ovary cancer cells. IGROV-1 and SKOV3 ovarian carcinoma cell lines were transfected with an anti-FR intrabody. Transfectants and parental cells were tested for FR, integrins and anti-FR intrabody expression by fluorescence-activated cell sorting (FACS), reverse transcription and polymerase chain reaction (RT-PCR) and/or immunoblotting. Cell growth characteristics and adhesion properties were evaluated in liquid, semisolid and organotypic cultures. The anti-FR scFv inhibited FR expression from 60 to 99%. At physiological concentrations of folate, proliferation varied directly as a function of FR expression. FR downmodulation was accompanied by reduced colony-forming ability in soft agar, morphological change of the cells, significant enhanced adhesion to laminin or Matrigel, a two-to three-fold increase in alpha6beta4 integrin expression, and a marked reduction in laminin production. In three-dimensional organotypic cultures, anti-FR intrabody-transfected IGROV1 cells grew as a single-ordered layer, reminiscent of normal OSE growth in vivo. In conclusion, the anti-FR intrabody reverses the transformed phenotype in ovary cancer cells and may provide an efficient means to inhibit selectively the growth of these cells. Gene Therapy (2003) 10,1018-1025. doi:10.1038/sj.gt.3301962.