Giunciuglio D, Culty M, Fassina G, Masiello L, Melchiori A, Paglialunga G, Arand G, Ciardiello F, Basolo F, Thompson E W
Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy.
Int J Cancer. 1995 Dec 11;63(6):815-22. doi: 10.1002/ijc.2910630612.
Infection with erbB-2 (E) of Ha-ras (H) oncogene-transfected cells has been previously shown to cooperatively induce anchorage-independent growth of the MCF10A human mammary epithelial cell line in vitro, but not to induce nude mouse tumorigenicity. Here we show that oncogene-transformed MCF10A are able to halt in the lungs of nude mice, a sign of organ colonization potential. We have therefore studied the transformants for in vitro migratory and invasive properties known to correlate with the metastatic potential of human mammary carcinoma cells in nude mice. MCF10A transfected with Ha-ras, infected with a recombinant retroviral vector containing the human c-erB-2 proto-oncogene (MCF10A-HE cells), show a higher invasive index than either the single transfectant (MCF10A-H) or MCF10A-erB-2(MCF10A-E) cells in the Boyden chamber chemotaxis and chemoinvasion assays. The MCF10A-HE cells also adopted an invasive stellate growth pattern when plated or embedded in Matrigel, in contrast to the spherical colonies formed by the single transformants MCF10A-H, MCF10A-E, and the parental cells. Dot-blot analysis of gelatinase A and TIMP-2 mRNA levels revealed increasing gelatinase A mRNA levels (HE > E > H > MCF10A) and reduced TIMP-2 expression in both single and double transformants. Furthermore, MCF10A-HE cells show more MMP-2 activity than parental MCF10A cells or the single transformants. CD44 analysis revealed differential isoform banding for the MCF10A-HE cells compared to parental cells, MCF10A-H and MCF10A-E, accompanied by increased binding of hyaluronan by the double transformants. Our results indicate that erB-2 and Ha-ras co-expression can induce a more aggressive phenotype in vitro, representative of the malignancy of mammary carcinomas.
先前研究表明，Ha-ras（H）致癌基因转染细胞感染erbB-2（E）可协同诱导MCF10A人乳腺上皮细胞系在体外进行不依赖贴壁的生长，但不会诱导裸鼠致瘤性。在此我们发现，致癌基因转化的MCF10A细胞能够在裸鼠肺部停滞，这是具有器官定植潜力的标志。因此，我们研究了这些转化细胞的体外迁移和侵袭特性，已知这些特性与人类乳腺癌细胞在裸鼠中的转移潜力相关。用Ha-ras转染并感染含人c-erB-2原癌基因的重组逆转录病毒载体的MCF10A（MCF10A-HE细胞），在Boyden小室趋化和化学侵袭试验中，其侵袭指数高于单一转染细胞（MCF10A-H）或MCF10A-erB-2（MCF10A-E）细胞。与单一转化体MCF10A-H、MCF10A-E以及亲代细胞形成的球形集落相比，当MCF10A-HE细胞接种或包埋在基质胶中时，它们还呈现出侵袭性的星状生长模式。对明胶酶A和TIMP-2 mRNA水平的斑点印迹分析显示，单一和双重转化体中明胶酶A mRNA水平均升高（HE>E>H>MCF10A），而TIMP-2表达降低。此外，MCF10A-HE细胞比亲代MCF10A细胞或单一转化体表现出更强的MMP-2活性。CD44分析显示，与亲代细胞、MCF10A-H和MCF10A-E相比，MCF10A-HE细胞的异构体条带不同，双重转化体与透明质酸的结合增加。我们的结果表明，erB-2和Ha-ras共表达可在体外诱导更具侵袭性的表型，这代表了乳腺癌的恶性程度。
