Wojciech Poluha

The cyclin-dependent kinase inhibitor p21 (WAF1) is required for survival of differentiating neuroblastoma cells.

We are employing recent advances in the understanding of the cell cycle to study the inverse relationship between proliferation and neuronal differentiation. Nerve growth factor and aphidicolin, an inhibitor of DNA polymerases, synergistically induce neuronal differentiation of SH-SY5Y neuroblastoma cells and the expression of p21WAF1, an inhibitor of cyclin-dependent kinases. The differentiated cells continue to express p21WAF1, even after removal of aphidicolin from the culture medium. The p21WAF1 protein coimmunoprecipitates with cyclin E and inhibits cyclin E-associated protein kinase activity. Each of three antisense oligonucleotides complementary to p21WAF1 mRNA partially blocks expression of p21WAF1 and promotes programmed cell death. These data indicate that p21WAF1 expression is required for survival of these differentiating neuroblastoma cells. Thus, the problem of neuronal differentiation can now be understood in the context of negative regulators of the cell cycle.

Cell-cycle arrest in TrkA-expressing NIH3T3 cells involves nitric oxide synthase.

We have examined nerve growth factor (NGF)‐triggered signaling in two NIH3T3 cell lines exogenously expressing the NGF receptor, TrkA. TRK1 cells cease to proliferate and extend long processes in response to NGF, while E25 cells continue to proliferate in the presence of NGF. These two cell lines express similar levels of TrkA and respond to NGF with rapid elevation of mitogen‐activated protein kinase (MAPK) activity. MAPK activation is slightly more sustained for E25 cells than for TRK1 cells, although sustained activation of MAPK has been suggested to cause cell‐cycle arrest. As judged by NADPH‐diaphorase staining, nitric oxide synthase (NOS) activity is increased in TRK1 cells upon exposure to NGF. In contrast, diaphorase staining in E25 cells is unaffected by NGF treatment. Immunocytochemistry shows that levels of the brain NOS (bNOS) isoform are increased in TRK1, but not E25, cells exposed to NGF. Furthermore, Western blots show that NGF elevated cyclin‐dependent kinase inhibitor, p21WAF1, in TRK1 cells only. NGF‐induced p21WAF1 expression, cell‐cycle arrest and process extension are abolished by N‐nitro‐L‐arginine methyl ester (L‐NAME), a competitive inhibitor of NOS. The inactive enantiomer, D‐NAME, did not inhibit these responses. Furthermore, even though E25 cells do not respond to NGF or nitric oxide donors, they do undergo a morphological change in response to NGF plus a nitric oxide donor. Therefore, NOS and p21WAF1 are induced only in the TrkA‐expressing NIH3T3 cell line that undergoes cell‐cycle arrest and morphological changes in response to NGF. These results demonstrate that sustained activation of MAPK is not the sole determining factor for NGF‐induced cell‐cycle arrest and implicate NO in the cascade of events leading to NGF‐induced morphological changes and cell‐cycle arrest. J. Cell. Biochem. 81:193–204, 2001.