We have previously shown that the human myeloid cell nuclear differentiation antigen (MNDA) is expressed at both the antigen and mRNA levels specifically in human monocytes and granulocytes and earlier stage cells in the myeloid lineage. A 200 amino acid region of the MNDA is strikingly similar t o a region in the proteins encoded by a family of interferon-inducible mouse genes, designated Mi-201, Mi-202, K-203, etc, that are not regulated in a cell-or tissue-specific fashion. However, a new member of the lfi-ZOO gene family, 03, is induced in mouse mononuclear phagocytes but not in fibroblasts by interferon. The same 200 amino acid region, duplicated in the mouse Mi-ZOO gene family, is also repeated in the recently characterized human IF1 16 gene that is constitutively expressed specifically in lymphoid cells and is induced in myeloid cells by interferon y. The 1.8-kb MNDA mRNA, which contains an interferon-stimulated response HE HUMAN MYELOID cell nuclear differentiation antigen (MNDA) is detected only in nuclei of cells of the granulocyte-monocyte lineage.' We also evaluated MNDA expression in 21 cases of human acute leukemia classified by French-American-British (FAB) Group criteria.2 Cases in the FAB M2, M3, M4, and M5 categories, in which myeloid cell maturation is evident, were all positive with variability in percentage of positive cells and in the intensity of the staining reactions. In five cases of acute myeloblastic leukemia without maturation (FAB MI), three were negative and two were positive. The four cases of acute lymphocytic leukemia were all negative. Two cases of biphenotypic acute leukemia and one case of lymphoid blast crisis of chronic granulocytic leukemia were also negative.2 These results indicated that MNDA expression is correlated with granulocyte and monocyte differentiation in cases of acute leukemia. Our MNDA amino acid and cDNA sequence a n a l y~i s~.~ provided insight into its functional significance. The strict nuclear localization of the MNDA in interphase cells is consistent with its containing sequence motifs commonly found in gene regulatory protein^.^ In addition, DNA binding activity of the MNDA was previously demonstrated using DNAprotein cross-linking.5 MNDA contains extended regions of sequence at both the DNA and protein levels similar to a class of mouse genes cluster) that are interferon-ind~cible.~.~ The mouse @-202 and @-204 genes each encode contiguous 200 amino acid (aa) regions that are highly conserved within each protein and between proteins in the family. The recently characterized mouse D3 gene, a new member of the @-ZOO gene family, contains only a single copy of the 200-aa conserved sequence and exhibits a sequence similar to the I$-204 gene outside of the 200-aa conserved sequence that is unique from other members of the @-200 gene family.' The MNDA also contains only a single copy of the 200-aa conserved sequence and more than half of the 67 residues of its NH2 terminal sequence are identical to the NH2 terminal domain of the mouse 204 protein.' In the ...

The human leukemic cell lines, K562, KG-1, and HL-60, and the blast subclones, KG-1a and HL-60 blast, were utilized to relate differences in nonhistone protein antigens to stages of myeloid cell differentiation. Chromatin proteins were separated on SDS- polyacrylamide gels, transferred electrophoretically to nitrocellulose sheets, and visualized by the peroxidase-antiperoxidase method of Sternberger. Screening with antisera raised against total and dehistonized chromatin and a nuclear extract from these cells revealed quantitative as well as qualitative differences between the cell lines. A decrease in antigen content seemed to parallel progressive stages of myeloid cell development. The results indicate that a number of chromosomal protein antigens are lost or modified during differentiation. An antigen(s) of approximately 55,000 molecular weight was found in HL-60 chromatin, but was not present in its less differentiated subclone or in the other lines representative of earlier stage cells. Upon the induction of HL-60 cells to mature to end stages with 4 microM retinoic acid, a significant increase in the mol wt 55,000 activity was seen. This antigen was detected only with antisera against HL-60 total chromatin and granulocyte nuclei, and it was found only in normal mature granulocytes and in the later stage cells of the HL-60 culture. Thus, the antigen appears to be associated with a differentiated myeloid function.

Antisera raised to dehistonized chromatin from isolated normal human granulocytes revealed the presence of chromatin-associated antigens specific for the human neutrophils that appear during late stages of myeloid cellular differentiation. Immunological specificity was demonstrated by C fixation, immunodiffusion, and immunocytochemical reactions. Chromatin prepared from both normal granulocytes and specimens of myeloid leukemia showed immunologic reactivity. Although the normal antigens were detected in a specimen of CML, the position of immunodiffusion precipitin lines was different from that obtained with normal granulocyte chromatin. In addition, chromatin prepared from the myeloid leukemic cell line HL-60 expressed only one of the three precipitin bands normally found in immunodiffusion. The immunocytochemical staining reaction was confined to the nucleus of mature neutrophils in normal peripheral blood smears. Greater than 90% of cells in peripheral blood specimens of CML showed positive immunocytochemical nuclear staining. In other types of leukaemia, the normal mature granulocyte reacted with antiserum, but the nonmyeloid leukemic cells in these specimens did not. The specificity of immunologic reactions described here suggests the usefulness of nuclear antigens as cell markers.