Abstract:Binding of extracellular ligands to epidermal growth factor receptors (EGFR) activate signal transduction pathways associated with cell proliferation, and these events are inhibited by monoclonal antibodies against EGFR. Since efficient DNA repair in actively growing cells may require growth factor signaling, it was of interest to explore any linkage between EGFR-mediated signaling and DNA-dependent protein kinase (DNA-PK), an enzyme believed to be involved in repairing double strand breaks and V(D)J recombina… Show more
“…DNA damage is modulated by metabolic DNA repair processes. Interestingly, Bandyopadhyay et al (1998) have shown that EGFR-mediated signalling may control the activity of DNA-PK, an enzyme directly involved in DNA damage repair; more For each combination sequence of ZD1839 and radiation, the dose-effect curve of the combination was compared with the dose-effect curves of ZD1839 alone and radiation alone. Radiation dose ranged from 0.5 to 10 Gy.…”
Elevated levels of epidermal growth factor receptor in head and neck cancer have been extensively reported, and are correlated with poor prognosis. The combination of cisplatin and 5-fluorouracil is a standard treatment regimen for head and neck cancer, with radiation representing another therapeutic option. Six head and neck cancer cell lines were used to study the cytotoxic effects of combining ZD1839 ('Iressa'), a new selective epidermal growth factor receptor tyrosine kinase inhibitor, and radiation. Two of the cell lines were also used to study the combination of ZD1839 and cisplatin/5-fluorouracil. Cytotoxic effects were assessed by the MTT test. The results indicated that ZD1839 applied before radiation gave the best effects (P=0.002); an effect that was strongest in those p53-mutated cell lines that express the highest epidermal growth factor receptor levels. The effects of ZD1839 with cisplatin and/or 5-fluorouracil were sequence dependent (P50
“…DNA damage is modulated by metabolic DNA repair processes. Interestingly, Bandyopadhyay et al (1998) have shown that EGFR-mediated signalling may control the activity of DNA-PK, an enzyme directly involved in DNA damage repair; more For each combination sequence of ZD1839 and radiation, the dose-effect curve of the combination was compared with the dose-effect curves of ZD1839 alone and radiation alone. Radiation dose ranged from 0.5 to 10 Gy.…”
Elevated levels of epidermal growth factor receptor in head and neck cancer have been extensively reported, and are correlated with poor prognosis. The combination of cisplatin and 5-fluorouracil is a standard treatment regimen for head and neck cancer, with radiation representing another therapeutic option. Six head and neck cancer cell lines were used to study the cytotoxic effects of combining ZD1839 ('Iressa'), a new selective epidermal growth factor receptor tyrosine kinase inhibitor, and radiation. Two of the cell lines were also used to study the combination of ZD1839 and cisplatin/5-fluorouracil. Cytotoxic effects were assessed by the MTT test. The results indicated that ZD1839 applied before radiation gave the best effects (P=0.002); an effect that was strongest in those p53-mutated cell lines that express the highest epidermal growth factor receptor levels. The effects of ZD1839 with cisplatin and/or 5-fluorouracil were sequence dependent (P50
“…The lysates were centrifuged in an Eppendorf centrifuge at 48C for 15 min. Cell lysates containing equal amounts of protein were resolved on a 10% SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with the appropriate antibodies, using an ECL method or alkaline phosphatase-based color reaction method (Bandyopadhyay et al, 1998).…”
Section: Cell Extracts Immunoblotting and Immunoprecipitationmentioning
The mechanisms through which heregulin (HRG) regulates the progression of breast cancer cells to a more invasive phenotype are currently unknown. Recently we have shown that HRG treatment of breast cancer cells leads to the formation of lamellipodia/ ®lopodia, and increased cell migration and invasiveness through the phosphatidylinositol 3-kinase (PI-3 kinase).Since the process of cell migration must involve changes in adhesion, we explored the potential HRG regulation of paxillin, a major cytoskeletal phosphoprotein of focal adhesion. We report that HRG stimulation of noninvasive breast cancer cells resulted in stimulation of p38 mitogen-activated protein kinase (p38 MAPK ), extracellular signal-regulated kinases (ERK) and PI-3K, and a concurrent unexpected increase in the level of paxillin phosphorylation on serine residue which was sensitive to protein-phosphatase 2b but not to protein tyrosine phosphatase 1. In addition, HRG triggered a rapid redistribution of paxillin to the perinuclear regions from the tyrosine-phosphorylated focal adhesions, and increased cell scattering. There was no e ect of HRG on the state of phosphorylation and localization of focal adhesion kinase. The HRG-induced increase in serine phosphorylation of paxillin and cell scattering were selectively inhibited by a speci®c inhibitor of p38 MAPK or a dominant-negative p38 MAPK mutant, but not by inhibitors of p42/44 MAPK or PI-3 kinase pathways. For the ®rst time our results have shown that HRG, a potent migratory growth factor stimulates serine phosphorylation of paxillin. These ®ndings suggest a role of p38 MAPKdependent signal transduction pathway(s) in serine phosphorylation and disassembly of the paxillin from the focal complexes during HRG-induced cell shape alterations and motility.
“…Cellular localization of Bax and p53 was determined using indirect immuno¯uorescence (Martin et al, 1987) with some modi®cations (Bandyopadhyay et al, 1998). Brie¯y, cells grown on glass coverslips were ®xed in methanol at 7208C for 10 min.…”
Section: Immuno¯uorescence and Confocal Studiesmentioning
Homeostasis in colonic epithelial cells is regulated by the balance between proliferative activity and cell loss by apoptosis. Because epithelial cells at the apex of colonic crypts undergo apoptosis and proliferative activity is usually restricted to the base of the crypts, it has been proposed that the limited availability of growth factorsignals at the upper portions of the crypts may trigger apoptosis. In the present studies, we investigate the mechanism of apoptosis mediated by growth factor deprivation in colorectal carcinoma cells by delineating the possible involvement of Bax and its subcellular localization. We report that inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase activity and downregulation of EGFR by anti-EGFR mAb 225 induces apoptosis in human colorectal carcinoma DiFi and FET cells. Induction of apoptosis was preceded by enhanced expression of newly synthesized Bax protein, and required protein synthesis. In the mAb 225-treated cells, Bax was redistributed from the cytosol to the nucleus and subsequently, to the nuclear membranes. The observed induction of Bax expression by mAb 225 was not associated with p53 induction. However, mAb 225 treatment also triggered relocalization of p53 from the cytosol to a nuclear membrane-bound form. Induction of Bax and its redistribution to the nucleus of DiFi cells during apoptosis was also demonstrated in response to butyrate, a physiological relevant molecule in colonic epithelial cells as it is the principal short-chain fatty acid produced by bacterial fermentation of dietary ®ber in colonic epithelium. Using immuno¯uorescence and confocal microscopy, we observed that Bax is predominantly localized in the cytosol, but during apoptosis it is localized both inside and along the nuclear membrane. Taken together, these ®ndings suggest that apoptosis induced by growth factor-deprivation or butyrate may involve the subcellular redistribution of Bax in human colorectal carcinoma cells.
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