No abstract

EMBL accession no. X51829 cDNA clones of myeloid differentiation primary response (MyD) genes activated in the absence of protein sysnthesis in M1 myeloblastic leukemia cells induced for terminal differentiation by IL6, have been isolated (1). Here we report the full length nucleotide and deduced amino-acid sequences of MyDi 16, a novel MyD gene, which exhibits biphasic kinetics of expression, with an early peak (1 hr) and a decline followed by increased levels of steady state mRNA in terminally differentiated Ml cells (1). MyD 116 expression was observed also in murine bonemarrow, but not in non-myeloid tissues (1). MyD1 16 encodes for a 2.5 kb mRNA, having a very short 3' untranslated region with multiple AT3 motifs (underlined), implicated in mRNA destabilization (2). The MyDi 16 encoded protein (657aa) harbors 419 almost perfect 38aa long repeats (underlined), each containing a PEST region, characteristic of short lived proteins (3). The MyDi 16 protein does not contain protein secretory signals, transmembrane domains or known protein-DNA binding motifs; it contains, however, potential protein kinase phosphorylation sites (position 4, 181), amidation site (position 653), and 19 casein kinase II phosphorylation sites, including 3 in each repeat.

The proto-oncogenes c-jun, junB, junD, and c-fos recently have been shown to encode for transcription factors with a leucine zipper that mediates dimerization to constitute active transcription factors; juns were shown to dimerize with each other and with c-fos, whereasfos was shown to dimerize only with juns. Growth and differentiation of animal cells is a wellcontrolled and highly conserved process involving multiple changes in gene expression that are developmentally regulated and result in the conversion of proliferating, undifferentiated cells into nonproliferating, highly specialized cells. A profound example of this process, which continues throughout life, is the complex blood cell formation, whereby a hierarchy of hematopoietic progenitor cells in the bone marrow proliferate and differentiate along multiple, distinct cell lineages, including the proliferation and differentiation of myeloid precursor cells into mature granulocytes and macrophages (57). The establishment of in vitro culture systems for the clonal development of bone marrow cells (6,43) and the availability of Ml myeloblastic leukemia cells, which proliferate autonomously and can be induced for differentiation and loss of leukemogenicity by physiological myelopoietic factors (15,47,49), provide an excellent biological system with which to study, side by side, the molecular biology of normal blood cell development and lesions that afflict it in leukemia and upon its progression (20,23,29,30).To enhance our understanding of the regulation of normal terminal differentiation and alterations in these regulatory processes that block differentiation, leading to leukemogenicity and its progression, recently we isolated and charac-

The c-myb proto-oncogene is abundantly expressed in tissues of hematopoietic origin, and changes in endogenous c-myb genes have been implicated in both human and murine hematopoietic tumors. c-myb encodes a DNA-binding protein capable of trans-activating the c-myc promoter. Suppression of both of these proto-oncogenes was shown to occur upon induction of terminal differentiation but not upon induction of growth inhibition in myeloid leukemia cells. Myeloblastic leukemia M1 cells that can be induced for terminal differentiation with the physiological hematopoietic inducers interleukin-6 and leukemia inhibitory factor were genetically manipulated to constitutively express a c-myb transgene. By using immediate-early to late genetic and morphological markers, it was shown that continuous expression of c-myb disrupts the genetic program of myeloid differentiation at a very early stage, which precedes the block previously shown to be exerted by deregulated c-myc, thereby indicating that the c-myb block is not mediated via deregulation of c-myc. Enforced c-myb expression also prevents the loss in leukemogenicity of M1 cells normally induced by interleukin-6 or leukemia inhibitory factor. Any changes which have taken place, including induction of myeloid differentiation primary response genes, eventually are reversed. Also, it was shown that suppression of c-myb, essential for terminal differentiation, is not intrinsic to growth inhibition. Taken together, these findings show that c-myb plays a key regulatory role in myeloid differentiation and substantiate the notion that deregulated expression of c-myb can play an important role in leukemogenicity.

Interleukin-6 (IL-6) and leukemia inhibitory factor (LIF), two multifunctional cytokines, recently have been identified as physiological inducers of hematopoietic cell differentiation which also induce terminal differentiation and growth arrest of the myeloblastic leukemic M1 cell line. In this work, it is shown that c-myc exhibited a unique pattern of expression upon induction of M1 terminal differentiation by LIF or IL-6, with an early transient increase followed by a decrease to control levels by 12 h and no detectable c-myc mRNA by 1 day; in contrast, c-myb expression was rapidly suppressed, with no detectable c-myb mRNA by 12 h. Vectors containing the c-myc gene under control of the beta-actin gene promoter were transfected into M1 cells to obtain M1myc cell lines which constitutively synthesized c-myc. Deregulated and continued expression of c-myc blocked terminal differentiation induced by IL-6 or LIF at an intermediate stage in the progression from immature blasts to mature macrophages, precisely at the point in time when c-myc is normally suppressed, leading to intermediate-stage myeloid cells which continued to proliferate in the absence of c-myb expression.