Glutamine Deprivation Induces Abortive S-Phase Rescued by Deoxyribonucleotides in K-Ras Transformed Fibroblasts (original) (raw)
Figure 1
Glutamine shortage induces cell proliferation arrest and S-phase accumulation in oncogenic K-ras transformed fibroblasts.
Normal (⧫) and transformed (▪) cell lines were plated at 3000 cells/cm2 in 6-well plates in normal medium. Culture medium was replaced after 24 hours with a normal medium −4 mM glutamine- (A), an intermediate medium −1 mM glutamine- (B) and a low medium −0.5 mM glutamine- (C) and the cells were collected and counted at indicate time. (D) Morphological analysis of normal cells (upper images) and transformed cells (lower images) cultured in 4 mM glutamine (left images) and 0.5 mM glutamine (right images) at 96 hours. Cells were observed at phase-contrast microscopy. Exponentially growing normal and transformed cells were incubated for increasing periods of time (24, 48, 72, 96 and 144 h) in medium containing 4 mM and 0.5 mM glutamine, then harvested, counted and subjected to FACS analysis after staining with propidium iodide (E), or pulsed 30 minutes with BrdU and then analyzed by fluorescence microscopy using an anti-BrdU specific antibody (F). Results are the mean of at least triplicate determinations with standard deviations indicated. For BrdU staining were analyzed at least 200 cells for each sample.