Transformation of NIH3T3 fibroblasts by the c-Kit receptor tyrosine kinase: effect of receptor density and ligand-requirement

被引:24
作者
Caruana, G
Cambareri, AC
Gonda, TJ
Ashman, LK
机构
[1] Inst Med & Vet Sci, Div Haematol, Leukaemia Res Unit, Adelaide, SA 5000, Australia
[2] Inst Med & Vet Sci, Div Human Immunol, Hanson Ctr Canc Res, Adelaide, SA 5000, Australia
关键词
c-Kit; SLF; transformation; anchorage independence; receptor density;
D O I
10.1038/sj.onc.1201494
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Ectopic expression of the normal murine receptor tyrosine kinase, c-Kit, in NIH3T3 cells induced many phenotypic changes characteristic of transformation including anchorage-independent growth, focus formation and tumorigenicity in nude mice, Although transformation was largely dependent on the presence of recombinant murine Steel Factor (SLF), the ligand to the c-Kit receptor, anchorage independent growth did occur at a low frequency in the absence of added factor, and this could not be inhibited by neutralising antibodies or by SLF anti-sense mRNA, Clones from factor-independent colonies in semi-solid agar displayed a narrow range of c-Kit surface protein levels (4.3-6.4 x 10(4) receptors/cell) which was relatively high compared with the pool from which they were derived, Analysis of a larger series of random clones derived from adherent cultures expressing different levels of c-Kit demonstrated a positive correlation between SLF-dependent, anchorage-independent growth and c-Kit protein and mRNA expression levels (respectively, R-S = 0.58, P < 0.01; and R-S = 0.53, P < 0.01) with consistent colony formation observed with clones having > 2.5 x 10(4) receptors/cell, Interestingly, two of the three clones expressing the highest levels of c-Kit protein and mRNA produced few or no colonies in the presence or absence of SLF, Sequential overexpression of human c-KIT in NIH3T3 cells using a dihydrofolate reductase (DHFR)-encoding vector and gene co-amplification through methotrexate selection, which resulted in pools expressing up to 1.5 x 10(5) receptors/cell, confirmed that high receptor densities resulted in a decrease in colony numbers, Thus, analysis of clonal and selected populations has indicated that an optimal level of c-Kit is required for transformation of NIH3T3 cells in the presence of SLF, and that some ligand-independent transformation occurs.
引用
收藏
页码:179 / 190
页数:12
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