Genomic Modifications Occur during Maturation in Myeloid Cells

Objective: A chromosomal translocation plays an important role on the leukemia-generation from the normal hematopoietic cells. Various kinds of translocation-patterns and -partners have been reported; however, a typical translocation has been reported like AML-1 and MTG8 in acute myelogenous leukemia (AML) (M2), and PML and RARα in AML (M3). This implies that some common mechanism exists on the chromosomal double strand breaks and the generation of translocations. We make a hypothesis that, when normal immature cells come into a maturation process, a specific event may occur on DNA level to induce irreversible changes toward cell-death. And a target region for the irreversible change may be the near site to chromosomal translocations. We compared the digested fragment-size for restriction-endonucleases using southern blotting among hematopoietic cell lines, normal bone marrow immature progenitor cells and normal blood mature neutrophils.

Materials and Methods: Bone marrows and bloods were obtained from informed healthy persons, and mononuclear cells were prepared after Ficoll-Paque sedimentation method, and neutrophil-rich fractions were prepared with density gravity centrifugation and hypotonic RBC lyses method. Various kinds of human cell lines, cultured in DMEM with 10% FCS, were also prepared. High molecular weight DNA was extracted from these cells, and they were digested with several kinds of restriction endonucleases. After electrophoresis, the digested DNA was transferred to a nitrocellulose membrane, and southern blot-hybridization was examined. The probe used was AML-1, MTG-8, PML, and RARα, in which the 5′ and 3′ breakpoint regions observed in chromosomal translocations were selected, respectively. Results and Discussion: No changes on the blotted fragment-size were observed among in the cultured cell lines, bone marrow immature mononuclear cells, and blood mononuclear cells; however, when 5′-PML probe was hybridized after DNA was digested with HindIII, the observed blot size was changed between normal immature myeloid progenitor cells and normal blood mature neutrophilic granulocytes. This change was not due to the personal polymorphism. These observations indicate that when myeloid cells become mature to neutrophilic granulocytes, some change occurs at DNA level. PML works on the maintenance of nucleobody for cell division, and it is very important to determine whether PML gene is maintained in neutrophilic granulocytes. We now prepare a genomic library from blood neutrophils to isolate the target clone, and determine DNA sequence near at the HindIII site to identify the modification of DNA during maturation of myeloid cells.