Arrest at the G2/M transition of the cell cycle by protein-tyrosine phosphatase inhibition: studies on a neuronal and a glial cell line.
The addition of the peroxovanadium (pV) derivatives potassium bisperoxo(1,10-phenanthroline)oxovanadate(v) (bpV[phen]) or potassium bisperoxo(pyridine-2-carboxylato) oxovanadate(v) (bpV[pic]), both of which are potent inhibitors of protein tyrosine phosphatases (PTPs) [Posner et al. (1994): J Biol Chem 269:4596-4604], to the culture medium of neuroblastoma NB 41 and glioma C6 cells resulted in a marked decrease in their proliferation rates and a progressive accumulation at the G2/M transition of the cell cycle. The effect was dependent on dose, cell type, and a pV compound employed. Mean values of the RNA-to-DNA and RNA-to-protein ratios in NB cells treated for 48 h with increased doses of bpV[phen] showed that general synthetic functions were not altered, nor did we observe oxidative damage to DNA using a sensitive DNA-nick detection assay. No changes in the expression and localization of vimentin, a component of the intermediate filament cytoskeleton, were observed by indirect immunofluorescence, showing that treatment did not disturb the cytoskeleton network. Measurements of BrdU incorporation into newly synthesized DNA showed that cells treated were not totally arrested. Furthermore, cells arrested G2/M were able to reenter the cycle rapidly after the release of inhibition. This progressive accumulation of G2/M coincided with the detection of tyrosine-phosphorylated p34cdc2 and a dramatic reduction in its kinase activity toward histone H1 by 48 h of culture. Both compounds were equally potent in inhibiting the catalytic activity of a yeast and the structurally distant mouse cdc25B in vitro, suggesting that augmented tyrosine phosphorylation of p34cdc2 derived from the in vivo inhibition of cdc25. Their equal in vitro potency contrasted with the considerably greater potency of bpV[phen] in vivo, in vivo suggesting that factors regulating the intracellular access of these compounds to cdc25 might be critical in determining in vivo specificity. In conclusion the final consequence of long-term exposure to potent and structurally defined PTP inhibitors on two highly proliferative nerve cell lines is to restrict cell growth. The corresponding hyperphosphorylation and reduced activity of p34cdc2 likely reflects the unusual sensitivity of cdc25 as an in vivo target for peroxovanadium compounds.
Faure
R
R
Département Médecine Génétique et Moléculaire, Centre Hospitalier, Université Laval, Ste-Foy, Québec, Canada.
Vincent
M
M
Dufour
M
M
Shaver
A
A
Posner
B I
BI
D016203:CDC2 Protein Kinase / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors; D002455:Cell Division; D002470:Cell Survival; D004249:DNA Damage; D016195:G2 Phase / Q000187:drug effects*; D005910:Glioma; D006657:Histones / Q000378:metabolism; D008938:Mitosis / Q000187:drug effects*; D009447:Neuroblastoma; D009457:Neuroglia / Q000166:cytology* / Q000187:drug effects; D009474:Neurons / Q000166:cytology* / Q000187:drug effects; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D019000:Phosphotyrosine / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors*; D011506:Proteins / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014638:Vanadates / Q000494:pharmacology; D014746:Vimentin / Q000032:analysis; D020687:cdc25 Phosphatases
D018797:Cell Cycle Proteins; D006657:Histones; D011506:Proteins; D014746:Vimentin; C069276:peroxovanadate; D019000:Phosphotyrosine; D014638:Vanadates; D012313:RNA; D016203:CDC2 Protein Kinase; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases; D020687:cdc25 Phosphatases