C-myc protein expression in relation to the proliferation and differentiation of human tumor cells

Oncoproteins, the protein products of proto-oncogenes, are believed to be effector molecules for the malignant phenotype. As such, they constitute potential markers for neoplastic disease. Previous studies suggest that the c-myc oncoprotein (p64{dollar}sp{lcub}rm c-myc{rcub}{dollar}) is a marker of proliferation in normal and neoplastic cells. This study presents a detailed examination of cellular p64{dollar}sp{lcub}rm c-myc{rcub}{dollar} content, in relation to the proliferation and differentiation of human glioblastoma (A-172, U118MG and U138B) and promyelocytic leukemia (HL-60) cells.; Monoclonal antibodies were initially used in conjunction with immunofluorescence microscopy, flow cytometry (FCM) and immunoblot analysis to study p64{dollar}sp{lcub}rm c-myc{rcub}{dollar} in neoplastic and benign cultured glial cells and brain tumor tissue. A marked increase in p64{dollar}sp{lcub}rm c-myc{rcub}{dollar} content was found to be present in malignant relative to benign cells.; To further investigate p64{dollar}sp{lcub}rm c-myc{rcub}{dollar} in relation to total cellular protein (TCP) and DNA, a new dual-laser FCM technique utilizing fluorescein isothiocyanate, sulphorhodamine 101 and 4{dollar}spprime{dollar},6-diamidino-2-phenylindole was developed. The validity of this method was confirmed using immunoblot analysis and the Lowry technique following treatment of HL-60 cells with 2.5 {dollar}mu{dollar}g/ml cycloheximide. Such treatment resulted in a 50% reduction in p64{dollar}sp{lcub}rm c-myc{rcub}{dollar}, a modest reduction in TCP, and a shift of cells into the G{dollar}sb0{dollar}/G{dollar}sb1{dollar} phase of the cell cycle.; To investigate p64{dollar}sp{lcub}rm c-myc{rcub}{dollar} expression in relation to cell proliferation, serum deprivation experiments (growth in 0.5% serum for 0-96 hours) were undertaken using A-172, U118MG and HL-60 cells. In general, p64{dollar}sp{lcub}rm c-myc{rcub}{dollar}, TCP and %S phase decreased in response to serum deprivation, though variations were noted between the cell lines in terms of relative p64{dollar}sp{lcub}rm c-myc{rcub}{dollar}: TCP.; Lastly, p64{dollar}sp{lcub}rm c-myc{rcub}{dollar} content was evaluated during a sodium butyrate (SB) -induced differentiation response in relation to the morphology, glial fibrillary acidic protein (GFAP) content, TCP and cell cycle distribution of U138B cells. GFAP and TCP were noted to rise, whereas %S phase and p64{dollar}sp{lcub}rm c-myc{rcub}{dollar} fell following treatment with 2 mM SB. The effect of SB was found to be concentration-dependent and partially reversible.; These findings suggest an essential role for p64{dollar}sp{lcub}rm c-myc{rcub}{dollar} in relation to the proliferation of human glioblastoma cells. Knowledge of the regulation of p64{dollar}sp{lcub}rm c-myc{rcub}{dollar} therefore appears important for understanding the growth control mechanisms operational in neoplastic cells.