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 bisperox o(1,10-phenanthroline)oxo-vanadate(v) (bpV[phen]) or potassium bispero xo(pyridine-2-carboxylato)oxovanadate(v) (bpV[pic]), both of which are potent inhibitors of protein tyrosine phosphatases (PTPs) [Posner et al. (1994): 1 Biol Chem 269:4596-4604], to the culture medium of neuro blastoma NB 41 and glioma C6 cells resulted in a marked decrease in th eir proliferation rates and a progressive accumulation at the G2/M tra nsition of the cell cycle. The effect was dependent on dose, cell type , and the 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, n or did we observe oxidative damage to DNA using a sensitive DNA-nick d etection assay. No changes in the expression and localization of vimen tin, a component of the intermediate filament cytoskeleton, were obser ved by indirect immunofluorescence, showing that treatment did not dis turb the cytoskeleton network. Measurements of BrdU incorporation into newly synthesized DNA showed that cells treated were not totally arre sted. Furthermore, cells arrested at G2/M were able to reenter the cyc le rapidly after the release of inhibition. This progressive accumulat ion at G2/M coincided with the detection of tyrosine-phosphorylated p3 4(cdc2) and a dramatic reduction in its kinase activity toward histone H1 by 48 h of culture. Both compounds were equally potent in inhibiti ng the catalytic activity of a yeast and the structurally distant mous e cdc25B in vitro, suggesting that the augmented tyrosine phosphorylat ion of p34(cdc2) derived from the in vivo inhibition of cdc25. Their e qual in vitro potency contrasted with the considerably greater potency of bpV[phen] in vivo, suggesting that factors regulating the intracel lular access of these compounds to cdc25 might be critical in determin ing in vivo specificity. In conclusion the final consequence of long-t erm 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 p34(cdc2) l ikely reflects the unusual sensitivity of cdc25 as an in vivo target f or peroxovanadium compounds. (C) 1995 Wiley-Liss, Inc.