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D K Poluha

Publications and source records attributed to D K Poluha.

5 recordsLinked to original sources
PubMed1996

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.

Aphidicolin↗
PubMed1995

TrkA neurogenic receptor regulates differentiation of neuroblastoma cells.

We examined events associated with neuronal differentiation of neuroblastoma cell line SH-SY5Y. Treatment with nerve growth factor (NGF) and aphidicolin, an inhibitor of DNA polymerases alpha and delta, induces terminal differentiation of SH-SY5Y cells. Following 3-4 days of treatment with NGF + aphidicolin, the cells irreversibly ceased proliferation and differentiated. There was a succession of events preceding differentiation. Down-regulation of c-myc was an early event occurring after less than 1 day of treatment with NGF + aphidicolin. Upregulation of the trkA and low-affinity NGF receptors (LNGFR) occurred after 3 days of NGF + aphidicolin treatment and required treatment with both NGF and aphidicolin. To test the role of TrkA in neuroblastic differentiation, we transfected SH-SY5Y cells with a TrkA-expression plasmid. In response to NGF in the absence of aphidicolin, the TrkA-transformant line ceased proliferation and irreversibly differentiated. SH-SY5Y cells bearing a control plasmid displayed only modest, reversible differentiation and did not cease cell proliferation in response to NGF. Hence, expression of NGF receptors is upregulated during differentiation of SH-SY5Y cells, and overexpression of TrkA enhances NGF-induced differentiation.

Aphidicolin↗
PubMed1994

Retrograde axonal transport and lesion-induced upregulation of the TrkA high-affinity NGF receptor.

Long-term physiological responses of nerve growth factor (NGF) and other neurotrophins require gene regulation and likely depend on retrograde axonal transport of NGF or a signaling molecule activated by ligand-receptor interaction. The low-affinity neurotrophin receptor p75LANR is retrogradely transported, but this receptor is not sufficient for NGF-dependent cell survival or differentiation. In this study we examined the distribution and transport of the TrkA NGF receptor using two anti-peptide polyclonal antibodies and a monoclonal antibody, all of which are TrkA specific. We find that (1) in the adult rat brain TrkA-like immunoreactivity is similar with all antibodies in striatal and basal forebrain neurons, (2) TrkA is upregulated in neuronal and nonneuronal cells near the sites of injury, and (3) TrkA immunoreactivity builds up within the proximal and distal segments of transected fimbrial axons, which is consistent with its transport in the anterograde and retrograde directions. Thus, TrkA may itself be, or be a component of, the neurotrophic intraaxonal messenger by which NGF regulates gene expression in sensitive neurons.

Animals↗
PubMed1992

Neuronal differentiation triggered by blocking cell proliferation.

Treatment of the neuroblastoma cell line SHSY5Y with nerve growth factor (NGF) resulted in limited neurite extension, but proliferation continued. However, SHSY5Y cells treated with NGF and a pulse of the DNA polymerase alpha and delta inhibitor aphidicolin showed dramatic neuronal differentiation. Few differentiated cells were observed immediately following the NGF-aphidicolin treatment; however, continued treatment of the cells with NGF in the ensuing week resulted in extension of long neurites (> 400 microns). Neurite extension was not observed for cells treated with aphidicolin alone. Hence, aphidicolin and NGF act synergistically to induce differentiation of SHSY5Y cells. If maintained in NGF, the differentiated cells were stable for at least 1 month and displayed many neuronal characteristics. They were mitotically inactive, and, in contrast to control or NGF-treated cells, the differentiated cells required NGF for survival. The cells expressed multiple microtubule-associated proteins (MAP), including MAP 1A, MAP 1B, and tau. There was expression of synaptic vesicle antigens synaptophysin and SV2, but not synapsin Ia/b or synapsin IIa/b. Both hydroxyurea and thymidine, which inhibit synthesis of nucleotides, act synergistically with NGF to induce differentiation of SHSY5Y cells. Since aphidicolin, hydroxyurea, and thymidine are chemically unrelated, we conclude that these drugs enhance NGF-induced differentiation by blocking cell proliferation and not through an unrelated side effect. The model suggested by these studies is that differentiation is triggered by two simultaneous signals: NGF and cessation of cell proliferation.

Aphidicolin↗