Signal Transduction Through the EGF Receptor Transfected in IL-3-Dependent Hematopoietic Cells

Abstract

An expression vector for the epidermal growth factor (EGF) receptor was introduced into the 32D myeloid cell line, which is devoid of EGF receptors and absolutely dependent on interleukin-3 (IL-3) for its proliferation and survival. Expression of the EGF receptor conferred the ability to utilize EGF for transduction of a mitogenic signal. When the transfected cells were propagated in EGF, they exhibited a more mature myeloid phenotype than was observed under conditions of IL-3-directed growth. Moreover, exposure to EGF led to a rapid stimulation of phosphoinositide metabolism, while IL-3 had no detectable effect on phosphoinositide turnover either in control or EGF receptor-transfected 32D cells. Although the transfected cells exhibited high levels of functional EGF receptors, they remained nontumorigenic. In contrast, transfection of v-erbB, an amino-terminal truncated form of the EGF receptor with constitutive tyrosine kinase activity, not only abrogated the IL-3 growth factor requirement of 32D cells, but caused them to become tumorigenic in nude mice. These results show that a naïve hematopoietic cell expresses all of the intracellular components of the EGF-signaling pathway necessary to evoke a mitogenic response and sustain continuous proliferation.

Epidermal growth factor (EGF) suppresses staurosporine-induced apoptosis by inducing mcl-1 via the mitogen-activated protein kinase pathway

“…This idea may be strengthened by other groups' observations. For example, Kulik et al, (1997) and Pierce et al, (1988) also found that EGF prevented apoptosis only in EGF receptor-overexpressing Rat-1 ®broblasts or 32D hematopietic cells. In addition, the numbers of EGF receptor were more than 10 5 per cell in those cells which EGF has been reported as a survival factor (Merlo et al, 1995;Yao and Cooper, 1995).…”

cbl-b inhibits epidermal growth factor receptor signaling

“…The data presented here show that cbl-b, but not ccbl, inhibits EGFR induced growth in 32D/EGFR cells ( Figure 5). These cells are absolutely dependent on growth factor (either EGF or IL-3) and rapidly undergo apoptosis in the absence of exogenous growth factor (Greenberger et al, 1983;Ihle et al, 1981Ihle et al, , 1982Pierce et al, 1988;Prystowsky et al, 1982). The inhibition of growth in the cbl-b clones is not associated with any cell cycle arrest but it is associated with a dramatic increase in the fraction of cells undergoing apoptosis (Figure 6).…”

“…32D cells do not normally express any endogenous EGFR (or other members of the EGFR family) and do not grow in EGF (Alimandi et al, 1997). In contrast, 32D cells overexpressing the EGFR (32D/EGFR) can be grown in the presence of either IL-3 or EGF (Pierce et al, 1988). These 32D/EGFR cells, used in our experiments, were maintained in Figure 3 cbl-b and c-cbl associate with Grb2.…”

“…WEHI 3B conditioned media containing IL-3 was produced by culturing the WEHI 3B cells to a high density and then harvesting the supernatants. 32D/EGFR cells were transfected by electroporation as previously described (Pierce et al, 1988) and stable clones were selected and maintained in growth media supplemented with 750 mg/ml G418 (GIBCO ± BRL). To assess the growth in di erent conditions, cells were pelleted, resuspended in RPMI 1640 containing 15% FCS, 1% PenStrep, but no growth factor.…”

Interleukin 3 and phorbol ester stimulate tyrosine phosphorylation of overlapping substrate proteins

“…In addition Pierce et al [30] have introduced the gene encoding the epidermal growth factor receptor into IL3-dependent cells and established EGF-dependent cell growth. Their work shows that tyrosine kinase activity can substitute for IL3, which suggests that the mechanism of IL3 biological activity involves amplification of tyrosine phosphorylation of a select group of proteins.…”