Identification of the signal pathways modulated by expression of p210$\rm\sp{bcr-abl}$ and the inhibition of p210$\rm\sp{bcr-abl}$ transforming activity by polypeptides interacting with p210$\rm\sp{bcr-abl}$ in murine myeloid 32D cells

The Bcr-Abl fusion oncogene which resulted from a balanced reciprocal translocation between chromosome 9 and 22, t(9;22)(q11, q34), encodes a 210 KD elevated tyrosine specific protein kinase that is found in more than 95 percent of chronic myelogenous leukemia patients (CML). Increase of level of phosphorylation of tyrosine is observed on cell cycle regulatory proteins in cells overexpressing the Bcr-Abl oncogene, which activates multiple signaling pathways. In addition, distinct signals are required for transforming susceptible fibroblast and hematopoietic cells, and the minimal signals essential for transforming hematopoietic cells are yet to be defined. In the present study, we first established a tetracycline repressible p210$\rm\sp{bcr-abl}$ expression system in a murine myeloid cell line 32D c13, which depends on IL3 to grow in the presence of tetracycline and proliferate independent of IL3 in the absence of tetracycline. Interestingly, one of these sublines does not form tumors in athymic nude mice suggesting that these cells may not be completely transformed. These cells also exhibit a dose-dependent growth and expression of p210$\rm\sp{bcr-abl}$ at varying concentrations of tetracycline in the culture. However, p210$\rm\sp{bcr-abl}$ rescues IL3 deprivation induced apoptosis in a non-dose dependent fashion. DNA genotoxic damage induced by gamma-irradiation activates c-Abl tyrosine kinase, the cellular homologue of p210$\rm\sp{bcr-abl},$ and leads to activation of p38 MAP kinase in the cells. However, in the presence of p210$\rm\sp{bcr-abl}$ the irradiation failed to activate the p38 MAP kinase as examined by an antibody against phosphorylated p38 MAP kinase. Similarly, an altered tyrosine phosphorylation of the JAK1-STAT1 pathways was identified in cells constitutively overexpressing p210$\rm\sp{bcr-abl}.$ This may provided a molecular mechanism for altered therapeutic response of CML patients to IFN-$\alpha.$^ Bcr-Abl oncoprotein has multiple functional domains which have been identified by the work of others. The Bcr tetramerization domain, which may function to stabilize the association of the Bcr-Abl with actin filaments in p210$\rm\sp{bcr-abl}$ susceptible cells, are essential for transforming both fibroblast and hematopoietic cells. We designed a transcription unit encoding first 160 amino acids polypeptide of Bcr protein to test if this polypeptide can inhibit the transforming activity of the p210$\rm\sp{bcr-abl}$ oncoprotein in the 32D c13 cells. When this vector was transfected transiently along with the p210$\rm\sp{bcr-abl}$ expression vector, it can block the transforming activity of p210$\rm\sp{bcr-abl}.$ On the other hand, the retinoblastoma tumor suppressor protein (Rb), a naturally occurring negative regulator of the c-Abl kinase, the cellular homologue of Bcr-Abl oncoprotein, binds to and inhibits the c-Abl kinase in a cell cycle dependent manner. A polypeptide obtained from the carboxyl terminal end of the retinoblastoma tumor suppressor protein, in which the nuclear localization signal was mutated, was used to inhibit the kinase activity of the p210$\rm\sp{bcr-abl}$ in the cytoplasm. This polypeptide, called Rb MC-box, and its wild type form, Rb C-box, when overexpressed in the 32D cells are mainly localized in the cytoplasm. Cotransfection of a plasmid transcription unit coding for this polypeptide and the gene for the p210$\rm\sp{bcr-abl}$ resulted in reduced plating efficiency of p210$\rm\sp{bcr-abl}$ transfected IL3 independent 32D cells. Together, these results may lead to a molecular approach to therapy of CML and an in vitro assay system to identify new targets to which an inhibitory polypeptide transcription unit may be directed. ^

Health care policy analysis of Medicaid's early and periodic screening, diagnosis, and treatment (EPSDT) benefit in Colorado

The purpose of this thesis is to identify "best practice" recommendations for successful implementation of the EPSDT outreach program at Memorial Health System's Hospital for Children in Colorado Springs through a policy analysis of Medicaid EPSDT services in Colorado. A successful program at Memorial will increase education and awareness of EPSDT services, enrollment, and access to and utilization of health care services for eligible children. Methodology utilized in this study included questionnaires designed for the EPSDT contract administrator and outreach coordinators/workers; analysis of current federal and state policies; and studies conducted at the federal and state level, and by various advocacy groups. The need for this analysis of EPSDT came about in part through an awareness of increasingly high numbers of children in poverty and who are uninsured. Though the percentage of children living in poverty in Colorado is slightly below the national average (see Table 2), according to data analyzed by The Annie E. Casey Foundation, the percentage of children (0-18) living in poverty in Colorado increased from 10% in 2000 to 16% in 2006, a dramatic increase of 60% surpassed by only one other state in the nation (The Annie E. Casey Foundation, 2008). By comparison, the U.S. percentage of children in poverty during the same time frame rose from 17% to 18% (The Annie E. Casey Foundation, 2008). What kind of health care services are available to this vulnerable and growing group of Coloradans, and what are the barriers that affect their enrollment in, access to and utilization of these health care services? Barriers identified included difficulty with the application process; system and process issues; a lack of providers; and a lack of awareness and knowledge of EPSDT. Fiscal restraints and legislation at the federal and state level are also barriers to increasing enrollment and access to services. Outreach services are a critical component of providing EPSDT services, and there were several recommendations regarding outreach and case management that will benefit the program in the future. Through this analysis and identification of a broad range of barriers, a clearer picture emerged of current challenges within the EPSDT program as well as a broad range of strategies and recommendations to address these challenges. Through increased education and advocacy for EPSDT and the services it encompasses; stronger collaboration and cooperation between all groups involved, including providing a Medical Home for all eligible children; and new legislation putting more money and focus on comprehensive health care for low-income uninsured children; enrollment, access to and utilization of developmentally appropriate and quality health care services can be achieved. ^