Growth factor receptor bound (Grb)14 is a member of the Grb7 family of src homology (SH)2 domain-containing proteins. These proteins perform both adaptor and modulatory roles in receptor tyrosine kinase (RTK) signaling, although their regulation is poorly understood. In this study, a positive correlation between Grb14 protein expression and ER alpha status in breast cancer cell lines led us to investigate regulation of Grb14 by estradiol and insulin, which synergize in the regulation of breast cancer cell proliferation. In MCF-7 cells maintained in charcoal-stripped serum, Grb14 expression was downregulated by estradiol and increased by the pure anti-estrogen ICI 182780. Under serum-free conditions, insulin enhanced Grb14 expression but this effect was repressed by estradiol when both hormones were used in combination. Using a system in which c-Myc induction drives cell cycle progression independently of estradiol, we demonstrated that Grb14 regulation was specific to estradiol treatment. Finally, we demonstrated a novel functional role for Grb14 whereby its overexpression inhibited not only insulin- but also estrogen-induced cell cycle progression. This was associated with decreased extracellular signal-regulated kinase (Erk)1/2 activation in insulin-stimulated Grb14-overexpressing cells. These data represent the first demonstration of regulation of Grb14 expression levels in response to hormonal stimuli, and are consistent with its role as a repressor of insulin signaling where it is induced as a negative feedback mechanism. A role for Grb14 is also shown in estrogen/insulin crosstalk since estradiol blocks the insulin-induced induction of this protein.
Cell cultures exposed to DNA-damaging agents such as gamma radiation respond by arresting at cell cycle checkpoints, and the p53 tumor suppressor protein is strongly implicated in this behavior. We have investigated the TP53 status and cell cycle response to ionizing radiation of a series of early passage cell lines (designated NZM1 to NZM15) previously developed from patients with metastatic melanoma. The TP53 status of each of the cell lines was determined by singlestrand conformation polymorphism and DNA sequence analysis. The majority of the lines appeared to have a wild-type TP53 gene sequence, consistent with published studies. Two lines (NZM4 and NZM7.2) were found to have an identical T→C transition mutation in nucleotide 721 (exon 7) of the coding region. NZM7.2 (mutant) and NZM7.4 (wild-type) were clonally derived from the same line (NZM7). The existence of radiation-induced cell cycle arrest in G 1 and/or G 2 M phase was determined 16 h after irradiation (6.3 Gy) by DNA staining and flow cytometric analysis. The mitotic inhibitor paclitaxel was used as a reference compound, with or without irradiation, to assess the efficiency of radiation-induced cell cycle arrest. G 1 phase arrest was associated only with the presence of the wild-type TP53 gene, but the efficiency of induced arrest varied among the cell lines and the period of G 1 phase arrest appeared to be short. A significant difference (P < 0.002) was also found between the efficiency of induction of G 2 phase arrest and the presence of wild-type TP53 gene. The results provide evidence that although the melanoma cell lines generally had an intact TP53 gene, the efficiency of p53-mediated cycle arrest might be deficient and contribute to the resistance of this tumor to treatment.
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