Myelopoiesis, the process of cell division and maturation whose end-product includes the mature circulating granulocyte, requires the orderly expression of a large number of genes. The myeloid (granulocytic) leukemias are malignancies in which the mechanisms controlling this process are disrupted, with the resultant accumulation of cells unable to progress beyond an abnormal, immature stage. The work proposed in this application is directed toward elucidating some of the control mechanisms operative during normal and leukemic myelopoiesis. To achieve this goal, factors affecting the expression of the genes for human neutrophil elastase, cathepsin G and lactoferrin will be studied in detail. These genes provide the basis for these studies because 1) they are only expressed during specific stages of myeloid maturation; 2) their expression is blocked or aberrant in cells leukemically arrested at the myeloblast or promyelocyte stage; and 3) normal expression of the genes appears to be correlated with the synthesis of normal primary (azurophilic) and secondary neutrophil granules. The methodology used in these studies will require the use of cloned cDNA probes for these genes. These will be isolated from a myeloid cell cDNA library which has already yielded transcripts of the lactoferrin gene. Screening of the library for elastase and cathepsin G clones will be accomplished with synthetic oligonucleotides designed on the basis of amino acid sequence data. The cloned cDNA's will be used to examine RNA preparations from normal and maturationally arrested granulocytes and myeloid precursors for evidence of gene expression. Isolated DNA, chromatin and whole nuclei will be studied with the cDNA probes to determine whether certain epigenetic factors known to influence gene expression play a role in regulating the activity of these genes. These studies will include determination of the nuclease sensitivity of the chromosomal genes, their relationship to the nuclear matrix and their methylation state in early, late, and terminally differentiated states of myeloid maturation. The results obtained in normal cells will be compared with observations on cells blocked by leukemic maturation arrest. Through these experiments I will test the hypothesis that an understanding of the regulation of developmentally modulated genes in normal and leukemic cells will provide important information about factors responsible for the creation and maintenance of the malignant state.
| Srivastava, C H; Rado, T A; Bauerle, D et al. (1991) Regulation of human bone marrow lactoferrin and myeloperoxidase gene expression by tumor necrosis factor-alpha. J Immunol 146:1014-9 |
| Broxmeyer, H E; Mantel, C; Gentile, P et al. (1991) Actions of H-subunit ferritin and lactoferrin as suppressor molecules of myelopoiesis in vitro and in vivo. Curr Stud Hematol Blood Transfus :178-81 |
| Han, J A; Lu, C M; Brown, G B et al. (1991) Direct amplification of a single dissected chromosomal segment by polymerase chain reaction: a human brain sodium channel gene is on chromosome 2q22-q23. Proc Natl Acad Sci U S A 88:335-9 |
| Krauss, R D; Rado, T A (1989) Current approaches to the molecular and physiological basis of cystic fibrosis. Am J Med Sci 298:334-41 |
