The estrogen receptor (ER) can be activated as a transcription factor either by binding of cognate estrogenic ligand or, indirectly, by a variety of other extracellular signals. As a first step towards elucidating the mechanism of ‘steroid‐independent activation’ of the ER by the epidermal growth factor (EGF), we have mapped the ER target domain and determined the signaling pathway. We show that the N‐terminal transcriptional activation function AF‐1, but not the C‐terminal AF‐2, is necessary for the EGF response. Both the EGF‐induced hyperphosphorylation and the transcriptional activation of the unliganded ER depend on a phosphorylatable serine residue at position 118. However, its phosphorylation is not sufficient and, hence, there must be other target domains or proteins which fulfill an additional requirement for EGF signaling through the ER. Using dominant‐negative Ras and MAP kinase kinase (MAPK kinase) and constitutively active MAPK kinase mutants, we show that EGF activates the ER by signaling through the MAPK pathway suggesting that MAPK directly phosphorylates the critical serine 118. Our results also imply that the steroid‐independent activation of a variety of ER mutants, which arise during the malignant progression of breast tumors, may contribute to tamoxifen resistance.

Experimental evidence has shown , both in vitro and in animal models , that neoplastic growth and subsequent metastasis formation depend on the tumor's ability to induce an angiogenic switch. This requires a change in the balance of angiogenic stimulators and inhibitors. To assess the potential role of angiogenesis factors in human thyroid tumor growth and spread , we analyzed their expression by semiquantitative RT-PCR and immunohistochemistry in normal thyroid tissues , benign lesions , and different thyroid carcinomas. Compared to normal tissues , in thyroid neoplasias we observed a consistent increase in vascular endothelial growth factor (VEGF) , VEGF-C, and angiopoietin-2 and in their tyrosine kinase receptors KDR , Flt-4 , and Tek. In particular , we report the overexpression of angiopoietin-2 and VEGF in thyroid tumor progression from a prevascular to a vascular phase. In fact , we found a strong association between tumor size and high levels of VEGF and angiopoietin-2. Furthermore , our results show an increased expression of VEGF-C in lymph node invasive thyroid tumors and , on the other hand , a decrease of thrombospondin-1 , an angioinhibitory factor , in thyroid malignancies capable of hematic spread. These results suggest that , in human thyroid tumors , angiogenesis factors seem involved in neoplastic growth and aggressiveness. Moreover , our findings are in keeping with a recent hypothesis that in the presence of VEGF , angiopoietin-2 may collaborate at the front of invading vascular sprouts , serving as an initial angiogenic signal that accompanies tumor growth. (Am J Pathol 1999Pathol , 155:1967Pathol -1976

Alteration of the FHIT (fragile histidine triad) gene occurs as an early and frequent event in lung carcinogenesis. FHIT gene transfer into lung cancer cell line H460 lacking Fhit protein expression resulted in reversion of tumorigenicity. To gain insight into the biological function of FHIT, we compared the H460 cell line with its Fhit transfectants (H460͞FHIT). A significant inhibition of cell growth was observed in H460͞FHIT cells. The analysis of apoptosis by in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling revealed a high rate of apoptosis-induced DNA strand breaks in stable clones. In situ results were confirmed by FACScan analysis that showed an apoptotic rate of 44-47% compared with a 15% level in the control H460 cells. Analysis of cell cycle-phase distribution indicated a significant G 0 ͞G 1 arrest and the presence of a sub-G 1 peak in the stable clones. No significant changes in Bcl2, BclX, and Bax protein expression level were observed in the transfected clones as compared with the control H460 cells whereas a 2-fold increase in Bak protein levels was noticed. An increased level of p21 waf protein paralleled by an up-regulation of p21 waf transcripts also was found in Fhit-expressing clones compared with the H460 cell line. No differences in p53 levels were observed in the same cells, suggesting a p53-independent effect. These data suggest that the observed growth-inhibitory effect in FHIT-reexpressing cells could be related to apoptosis and cell cycle arrest and link the tumor-suppressor activity of FHIT to its proapoptotic function.The FHIT (fragile histidine triad) gene (1), at 3p14.2, is a frequent target of deletions associated with abnormal RNA and protein expression in primary tumors and cell lines of lung, head and neck, kidney, cervix, and breast cancer (2-6). Stable FHIT-transduced clones expressing exogenous wild-type Fhit, isolated after transfection of various epithelial cell lines carrying inactivated endogenous Fhit, show reduced colonyformation efficiency in vitro and inhibition of tumor development in nude mice, indicating that FHIT acts as a tumorsuppressor gene (7). The Fhit protein is a diadenosine triphosphate (Ap 3 A) hydrolase belonging to the histidine triad superfamily (HIT) of nucleotide-binding proteins (8). Our observation that the His(96)Asn mutant, lacking hydrolytic activity, still inhibits tumor formation in vivo (7) suggests that the tumor-suppressing function of Fhit is not related to catalysis of nucleotide substrates. However, the biological mechanism of FHIT activity and the cellular pathways associated with its tumor-suppressor function are not known. Crystallographic studies suggested that Ap 3 A nucleotide binding is crucial for Fhit biological activity and that enzymesubstrate complexes may be a signaling form (9). Interestingly, it has been reported that apoptosis in human cultured cells is associated with a decrease of free Ap 3 A levels (10).To study a possible involvement of FHIT in cell growth control and apoptosis, we...

The low-a nity nerve growth factor receptor p75 NTR belongs to a membrane receptor superfamily whose members, in certain cell types, are able to transduce an apoptotic signal. To investigate the e ect of p75 NTR expression in neuroblastoma cells, we transfected the p75 NTR cDNA into SK-N-BE cells, a neuroblastoma cell line that lacks expression of both p75 NTR and TrkA. Cell clones expressing elevated levels of p75 NTR showed a high degree of cell death by apoptosis, even in serumsupplemented medium. Moreover, the level of apoptosis correlated directly with the expression level of the receptor, indicating that p75 NTR could activate the cell death program by itself. Clones expressing p75 NTR showed a dramatic increase of cell death when switched into serum-free medium; these cultures rapidly extinguished. This apoptotic e ect was greatly inhibited by NGF treatment. Our results support the hypothesis that p75 NTR , when it is not bound by NGF, may play a role in neuronal selection during embryonic development and suggest that neuroblastomas may arise from immature neuroblasts that escape programmed cell death. Therefore, the loss of p75 NTR expression in developing neural crest cells might be a primary event in the genesis of neuroblastoma.