mtNOS and high mitochondrial NO light on cell cycle arrest in NIH starved cells

ALIPPE Y, SERRA MP, PAGNINI C, CARRERAS MC, PODEROSO JJ.

Santiago, Chile

Congreso; I Meeting of the SFRBM- South American Group; 2009

SFRBM- South American Group

Stimulatory growth factors are required for survival and proliferation in most of cultured cell types. After serum withdrawal, inhibition of the proliferation of NIH/3T3 cells is correlated with an apoptotic response. Otherwise, increase of mtNOS (nNOS translocated to mitochondria with posttranslational modifications) and a burst of hydrogen peroxide (H2O2) have been implicated in cell arrest and apoptosis during liver and brain development and in hypothyroidism. To link these evidences, NIH/3T3 cells were deprived of serum from 0 to 72h. Serum withdrawal during 24-48 h resulted in strong arrest and minimal levels of apoptosis, as detected by flow cytometry and cell counting. After 24 hs of serum starvation, mRNA and protein nNOS expression began to increase (RT-PCR and WB). At 48-72h, increase of nNOS was followed by translocation of the enzyme to mitochondria, as demonstrated by its relative decrease in cytosol and the proportional increase in the organelles. Flow cytometry in whole cells and isolated mitochondria using the DAF-FM dye confirmed high levels of NO, partly reverted by the NOS inhibitor l-NMMA. High mitochondrial NO in deprived cells was observed by confocal microscopy and as well it was reflected by a lowering of oxygen uptake by inhibition of COX; in this context, increased NO was associated to high H2O2 released by mitochondria. The results suggest that transcriptional increase of NOS and translocation to mitochondria with high H2O2 starts signalling pathways that conduct to the arrest of NIH/3T3 cells in serum deprivation.