Dispersed chromosomal localization of the proto-oncogenes transduced into the genome of Mill Hill 2 or E26 leukemia virus.

Both Mill Hill 2 and E26 retroviruses have transduced two cellular genes--c-myc and c-mil/mht (Mill Hill 2) and c-myb and c-ets (E26). We localized the genes transduced by these viruses to different chromosomes: c-myc and c-myb to relatively large chromosomes and c-mil/mht and c-ets to microchromosomes. Thus, like avian erythroblastosis virus, each of these retroviruses has transduced two cellular genes unlinked in the chicken genome.     

Oncogenes and avian development

In order to detect signs of oncogene activity and elucidate their possible role in avian ontogeny we implemented two different strategies. One was to detect either the protein product or messenger RNA in situ at various stages of development. The other was to try and disturb development with retroviruses carrying one or several oncogenes in their activated forms. Time- and tissue-specific expression of c-myc was apparently not related to particular phases of cell evolution, such as population amplification. Rather the presence of c-myc immunoreactive product at particular stages appeared to depend on cell types. c-myb and c-ets messenger RNAs were found expressed preferentially in the blood system, respectively in hemopoietic and differentiating endothelial cells. The developing embryo heart was found to be uniquely sensitive to the effect of retroviruses provided that two conditions were respected. The first was the injection of the virus or construct prior to E3.5. The second was the presence of the v-myc gene, whether alone or associated with one or several other v-onc. In such cases a large proportion (70%) of chick and all quail embryos developed multiple heart rhabdomyosarcomas within 10 days. In chickens the association of a second v-onc or of two others induced the formation of secondary tumors, whose type was determined by the nature of the other oncogene(s).(ABSTRACT TRUNCATED AT 250 WORDS)     

The cellular oncogenes c-myc, c-myb and c-erb are transcribed in defined types of avian hematopoietic cells

The possible role of normal chicken cellular sequences c-erb, c-myb and c-myc, together referred to as c-onc genes and related to the oncogenes of defective avian acute leukemia retroviruses (DLVs), was investigated by determining the accumulation of c-onc RNA in different avian cells an cell lines. Levels of c-myc and in some instances c-myb RNA are elevated in immature hematopoietic cells or cell lines from various lineages but more mature hematopoietic cells, as well as non-hematopoietic cells, contain only low levels. In contrast, the level of c-erb RNA is generally low, but high in a small number of normal bone marrow cells. The results indicate that the cellular homologues of the viral oncogenes are differentially expressed during hematopoiesis. They also indicate that the hypothesis that DLV target cells express their homologous c-onc genes might hold for c-erb, but is not valid in its simple form for c-myc and c-myb.     

Embryonic Sources of Definitive Hemopoietic Stem Cells

A review of one of the key problems of experimental hematology: the origin of hemopoietic stem cells in the development of vertebrates (amphibians, birds, and mammals). The appearance and functioning of two independent sources of hemopoietic stem cells (extra- and intraembryonic) were considered in amphibians, birds, and mammals. The contribution of each source to the formation of definitive hemopoietic tissue was analyzed. It was shown for amphibians and birds that intraembryonic organs such as the dorsolateral plate and the mesenchyme of dorsal aorta are involved in the formation of adult hemopoietic tissue, while the extraembryonic organs such as ventral islets and the yolk sac are devoid of true stem cells and provide only for the primary, transient hemopoiesis. New data have been considered concerning the previously unknown intraembryonic hemopoietic organ in mammals, a region of aorta–gonad–mesonephros arising in embryogenesis simultaneously with the yolk sac. Two extreme views on the involvement of stem cells of all these organs in the formation of definitive hemopoiesis have been considered. The data are provided on the interaction of the embryonic hemopoietic stem cells and the hemopoietic microenvironment of adult recipients.     

Alteration of nuclear proto-oncogene expression by erythropoietin (Epo) in Epo-responsive murine cell lines

The signal transduction system of erythropoietin (Epo) and the accompanying molecular control mechanism of proliferation and differentiation of erythroid progenitors remains largely unknown. In this study, the effect of Epo on the expression of nuclear oncogenes was investigated in two murine cell lines which respond to the hormone in different ways: ELM-I-1 cells proliferate independently of Epo, but differentiate in response to the hormone, while the growth of DA-1ER cells is absolutely dependent on Epo or interleukin (IL) 3. The cell lines were stimulated with Epo or IL-3, and total RNA was extracted. Then expression of nuclear proto-oncogenes (c-myc, c-fos and c-myb) was analyzed by northern blotting. The change in c-fos expression observed during the first two h following stimulation with either stimulant were common to both cell lines; a rapid and temporary increment. Before stimulation, c-myc and c-myb were strongly expressed in both lines. No apparent change in c-myc expression was observed during the first two h of stimulation, while c-myb expression in ELM-I-1 cells was slightly reduced 1 h after stimulation with Epo but not with IL-3. Three days after stimulation with Epo, but not with IL-3, only ELM-I-1 produced hemoglobin and expressed a lower amount of c-myb mRNA. These data suggest the importance of c-fos in the early signaling system of Epo, and the involvement of c-myb in erythroid differentiation but not in proliferation.     

Deregulated c-myb disrupts interleukin-6- or leukemia inhibitory factor-induced myeloid differentiation prior to c-myc: role in leukemogenesis.

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.     

Regulated expression of the c-myb and c-myc oncogenes during erythroid differentiation

We have investigated the expression of the genes c-myb, c-myc, and alpha globin in murine erythroid cells at different stages of development, in viral-induced erythroleukemias, as well as in two mouse erythroleukemia cell lines that can be induced to terminally differentiate when exposed to dimethylsulfoxide. We find that there is a reciprocal correlation between the cell's production of messenger RNA for c-myb and globin. c-myc message shows a similar but less dramatic decrease coincident with globin RNA production. Initially with the administration of an inducing agent, dimethylsulfoxide, there is a rapid decrease of myc and myb mRNA, which is followed by signs of differentiation in the induced culture. We conclude that these oncogenes function in early maturational stages of development of these cells. In the erythroleukemic state these genes are down-regulated by forced differentiation and may play a direct role in influencing the state of differentiation of these cells.     

Cellular oncogene expression in human tumors transplantable to athymic mice

Expression of 3 cellular oncogenes among 7 ones under investigation is identified in the majority of 20 strains of human tumors, passaged in nude mice without significant specificity as far as the type of the tumor is concerned. The levels of the expression of these 3 oncogenes (c-myc, c-fos, c-ras) were higher than the ones in primary human tumors except for the human melanoma Mel-2 strain, where the expression of c-myb oncogene was identified. All the rest oncogenes (c-mos, B-lym, c-sis, c-myb) showed no expression in human tumors of the examined strains.     

Functional analysis of c-Myb protein in T-lymphocytic cell lines shows that it trans-activates the c-myc promoter.

The function of c-Myb protein was revealed by transfecting an expression vector containing the entire c-Myb protein-coding sequence into the murine CTLL-2 T-cell line. Expressions of high levels of c-Myb protein did not alter the expression of several T-cell markers, c-fos mRNA expression, responses to interleukin-2, and growth characteristics of these cells. Interestingly, expression of the c-myc gene was drastically increased in this clone. Further, the c-myb expression plasmid, but not a frameshift mutant of c-myb, enhanced the expression of a hybrid construct of c-myc promoter linked to a reporter gene by 8- to 14-fold. These results demonstrate a role of c-Myb protein in c-myc gene expression.     

IL-2 modulation of murine T-cell oncogene expression

C-myb, a cellular oncogene associated with normal thymic development, was found to be highly expressed in four interleukin 2 (IL-2)-independent T-cell lines, but not in two of three IL-2-dependent T-cell lines. The IL-2-dependent lines, HT2 and CTLL-2, were found to have low levels of c-myb mRNA in the presence of IL-2. However, short-term IL-2 depletion resulted in at least fivefold increases in c-myb message. Add-back of IL-2 after 30 hr IL-2 depletion of CTLL-2 cells resulted in return to baseline low-level c-myb mRNA. Expression of the oncogenes myc, bas, raf, and abl as well as the T-cell genes Thy-1 and CT beta did not parallel that of c-myb. These studies indicate that removal of a growth factor can result in increased levels of a specific cellular oncogene and that two nuclear protooncogenes (c-myb and c-myc) are expressed differentially during cell growth. These results may help to explain aspects of intrathymic T-cell differentiation where there is very high c-myb expression in the face of limiting amounts of growth factors such as IL-2.