The primary objective of our study is to elucidate the role of amino acid transport for fetal ontogeny, in particular, for the differentiation and development of erythroid cells. In this study, the erythroid cells will be isolated from rat fetal livers at known days of gestation. In addition, we will also use the erythroleukemic cells K562 and MEL55 as an in vitro model of erythroid cells. These cells have been used extensively for studying the changes in cell surface antigens and the expression of globin genes associated with differentiation and development.

Our studies are designed to examine the following questions: (1) Are amino acid transport proteins important in regulation of growth and differentiation of erythroid cells? (2) How is the expression of the individual transport protein regulated during differentiation? (3) What are the genetic factors that control the regulation of these proteins during differentiation and development? (4) Does the activity of a given amino acid transport protein serve as a cell surface marker representing specific stages of development? We plan to examine these questions by carrying out a series of complementary studies in a sequential manner. These studies can be grouped into three major catagories. The first involves the isolation of a homogeneous population of erythroid cells at specific stages in gestation, and developing optimal culture conditions to study their subsequent differentiation and growth. We will first identify and characterize the various amino acid transport systems which may be expressed at specific stages of development. Second, we will develop a series of studies in which we can study the regulation of amino acid transport, as induced by (a) hormones such as erythropoietin, insulin, and glucagon; (b) chemical agents such as the polyamines, DMSO and HMBA; (c) the regulatory effect of protein kinase C on both cell differentiation and amino acid transport. These studies on regulation will enable us to identify the specific transport proteins expressed at specific stages of erythroid cell differentiation and development. Third, we will attempt to isolate (a) the transport proteins and (b) the genes that are responsible for regulating the expression of these transport proteins during development, and during hormonally or chemically induced regulation at each stage of development. Protein isolation will be attempted by using photoaffinity probes or protein modifying agents. Genetic studies will involve a combination of techniques including mRNA isolation, in vitro protein synthesis, immunoprecipitation, differential hybridization and c-DNA cloning.

The underlying mechanisms that control the gene expression of the amino acid transport proteins will provide relevant contributions to clinical medicine and developmental biology.

R29DK039147

1987-08-01

1992-11-30