Childhood neuroblastomas (NBs) are very chemosensitive tumors with a high proportion of all grades responding well to standard chemotherapy regimens. Unfortunately, most children presenting with either advanced disseminated disease or tumors with an amplified N-myc gene, ultimately relapse with a tumor which is then drug resistant. Since NB tumors respond in a similar manner in response to treatment with a variety of different drugs, the Principal Investigator hypothesizes that NB cells are chemosensitive owing to their susceptibility to undergo apoptosis and in the rare cell which survives therapy there is an alteration, either transient or stable, in the apoptotic pathway allowing cell survival and tumor repopulation. In order to test this hypothesis the Applicant proposes to evaluate the contribution of wild-type p53 (wtp53) and downstream proteins in dictating the low apoptotic threshold of this tumor type. She will determine whether p53-dependent apoptosis occurs in NB cells and contributes to their chemosensitive phenotype, both in NB cell lines in vitro (Aim one), and human tumor xenografts in vivo (Aim 4). Although amplified N-myc is a poor prognostic indicator for NB tumors, over-expression together with a wtp53 gene is thought to be pro-apoptotic. These pro-apoptotic factors appear to be balanced by high level expression of the anti-apoptotic protein, Bcl-2, in NB cells allowing maintenance of cell growth. However, the high expression of Bcl-2 is an observation inconsistent with the chemosensitive phenotype of NBs and therefore, this application will also investigate whether Bcl-2 expression is negatively regulated by wtp53 protein and how Bcl-2 family members dimerize in response to exposure to chemotherapeutic agents (Aim 2). Once it has been determined how these factors contribute to NB chemosensitivity she will examine changes in NB cells which may result in an altered apoptotic threshold and a chemoresistant phenotype. The Applicant proposes to investigate the function and relationship of p53 and the Bcl-2 family members in both colonies of cells which survive a drug dose capable of killing 99.9 percentof the population (Aim3) and in xenograft tumor nodules which regrow from previously undetectable minimal residual disease (Aim 4). Expansion of these cells or xenografts will allow determination of stability of the resistant phenotype. The studies proposed will assist in understanding mechanisms by which NB develop pleiotropic resistance to agents having different mechanisms of action and allow insight into new approaches to treat advanced NB.
