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

Abstract

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 ± 6.4610 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 di erent 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, P50.01; and R S =0.53, P50.01) with consistent colony formation observed with clones having 42.5610 4 receptors/cell. Interestingly, two of the three clones expressing the highest levels of cKit 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-ampli®cation through methotrexate selection, which resulted in pools expressing up to 1.5610 5 receptors/cell, con®rmed 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.

Isoforms of c-KIT differ in activation of signalling pathways and transformation of NIH3T3 fibroblasts

“…Only cells expressing the GNNK7 isoform were tumorigenic in vivo with a latency period of approximately 42 days ( Table 1). As previously reported (Caruana et al, 1998), NIH3T3 cells expressing murine GNNK7 c-Kit also induced tumours, in this case with an average latency of 50 days. Table 1 for details) were assayed for focus formation in the presence or absence of SLF.…”

“…A representative set of experiments is shown in Table 1; in each case the results were con®rmed in at least one additional experiment. As previously described for human GNNK+ KIT expressed from pRSV009/A+ vector (Caruana et al, 1998), anchorage independent growth was observed in cells expressing both isoforms, and the yield of colonies was lower at high levels of expression. Similar to the previous study, colony formation was substantially independent of added SLF for human but not murine Kit.…”

“…Early passage NIH3T3 cells expressing murine GNNK7 cKit (as previously described; Caruana et al, 1998) were included for comparison. A representative set of experiments is shown in Table 1; in each case the results were con®rmed in at least one additional experiment.…”

“…Truncation and mutation of feline or murine c-Kit (Besmer et al, 1986;Herbst et al, 1995a), or point mutation of human c-KIT (Furitsu et al, 1993) results in transformation and tumorigenicity. Ectopic expression of normal KIT can also contribute to tumorigenicity: expression of murine or human c-KIT in immortalized ®broblasts such as NIH3T3 cells leads to transformation which is partly factor independent (Alexander et al, 1991;Caruana et al, 1998).…”

“…We now report di erences in the induction of various attributes of cell transformation in NIH3T3 cells stably expressing human c-KIT isoforms. Care has been taken to ensure that the levels of expression are comparable, since this is known to have a considerable e ect on the outcome (Caruana et al, 1998). In addition, the a nities of the isoforms for SLF have been compared and the kinetics of receptor activation and downstream signalling have been examined.…”