Autonomous expression of c-myc in BC3H1 cells partially inhibits but does not prevent myogenic differentiation.

Myogenic differentiation is obligatorily coupled to withdrawal of myoblasts from the cell cycle and is inhibited by specific polypeptide growth factors. To investigate the potential involvement of c-myc in the control of myogenesis, the BC3H1 muscle cell line was stably transfected with a simian virus 40 promoter:c-myc chimeric gene. In quiescent cells in 0.5% serum, the exogenous c-myc gene was expressed at a level more than threefold greater than the level of endogenous c-myc in undifferentiated, proliferating cells of the parental line in 20% serum. The transfected myc gene partially inhibited the expression of both muscle creatine kinase and the nicotinic acetylcholine receptor, but was not sufficient to prevent the induction of these muscle differentiation products upon mitogen withdrawal.     

Modulation of c-myc expression in the HL-60 cell line

A decrease in the expression of the myc proto-oncogene of HL-60 cells has been reported as an accompaniment of myeloid differentiation induced by either dimethylsulfoxide or retinoic acid. We report herein that several inhibitors of poly(ADP-ribose)-polymerase induced myeloid differentiation in HL-60 cultures. Studies on the expression of the c-myc gene in total cell RNA populations indicate that expression of this gene is inversely correlated with the state of differentiation, either myeloid or monocytic, of the cultured cells independent of the inducer and the rate of cell proliferation.     

Independent variations of myc amplification, inducibility, maturation, and proliferation states in HL60

We have investigated the possible relationship between c-myc gene activity and other variable traits in HL60. In a panel of variant lines, a good correlation was observed between myc gene copy number and the level of myc mRNA. There was no correlation between myc amplification or expression and the resistance of the lines to induction of terminal neutrophilic or monocytic differentiation. Therefore, myc mRNA level does not appear to determine the ability of the variant HL60 lines to respond to inducers of differentiation. Flow cytometric analyses of the expression of a differentiation antigen (AGF 4.36) revealed stable negative and positive subpopulations in growing HL60. myc amplification, expression, and inducibility were identical in these subpopulations, suggesting that variation of these traits in HL60 sublines and variants is not due to maturation state differences. myc gene copy number was also identical in transferrin receptor positive (proliferating) and negative (resting) populations. These data contradict the notion that myc amplification has been important in determining the in vitro biological properties of HL60.     

c-myc and c-fos expression in differentiating mouse primary keratinocytes.

Expression of the myc and fos genes has been monitored in mouse primary keratinocytes after induction of terminal differentiation by calcium or tetradecanoylphorbol acetate (TPA). myc RNA levels in growing cells are very high and remain elevated even at late times after calcium-induced differentiation. Thus, keratinocytes provide the first example of normal primary cells with persistent c-myc expression irrespective of their proliferative or differentiated state. fos expression is also relatively unaffected by addition of calcium. In contrast to calcium, TPA-induced differentiation is accompanied by dramatic changes in proto-oncogene expression: marked c-fos induction and considerable although transient decrease in c-myc expression. These effects might be important for the keratinocyte response to TPA: TPA treatment of a keratinocyte cell line (RBK) resistant to this substance has no effect on c-myc expression and leads only to minimal c-fos induction. In these cells full fos induction can still be triggered by addition of fresh medium. Thus, the fos gene in normal keratinocytes is inducible through at least two independent mechanisms, only one of which has been lost during derivation of the TPA-resistant cell line.     

Contrasting expression patterns of three members of the myc family of protooncogenes in the developing and adult mouse kidney

The myc family of protooncogenes encode similar but distinct nuclear proteins. Since N-myc, c-myc, and L-myc have been found to be expressed in the newborn kidney, we studied their expression during murine kidney development. By organ culture studies and in situ hybridization of tissue sections, we found that each of the three members of the myc gene family shows a remarkably distinct expression pattern during kidney development. It is known that mesenchymal stem cells of the embryonic kidney convert into epithelium if properly induced. We demonstrate the N-myc expression increases during the first 24 h of in vitro culture as an early response to induction. Moreover, the upregulation was transient and expression levels were already low during the first stages of overt epithelial cell polarization. In contrast, neither c-myc nor L-myc were upregulated by induction of epithelial differentiation. c-myc was expressed in the uninduced mesenchyme but subsequently became restricted to the newly formed epithelium and was not expressed in the surrounding loose mesenchyme. At onset of terminal differentiation c-myc expression was turned off also from the epithelial tubules. We conclude that N-myc is a marker for induction and early epithelial differentiation states. That the undifferentiated mesenchyme, unlike stromal cells of later developmental stages, express c-myc demonstrates that the undifferentiated mesenchymal stem cells are distinct from the stromal cells. The most astonishing finding, however, was the high level of L-myc mRNA in the ureter, ureter-derived renal pelvis, papilla, and collecting ducts. In the ureter, expression increased, rather than decreased, with advancing maturation and was highest in adult tissue. Our results suggest that each of the three members of the myc gene family are involved in quite disparate differentiation processes, even within one tissue.     

Autocrine beta-related interferon controls c-myc suppression and growth arrest during hematopoietic cell differentiation

Different hematopoietic cells produce minute amounts of beta-related interferon (IFN) following induction of differentiation by chemical or natural inducers. The endogenous IFN binds to type I cell surface receptors and modulates gene expression in the producer cells. We show that self-induction of two members of the IFN-induced gene family differs in the dose response sensitivity and the prolonged kinetics of mRNA accumulation from the response to exogenous IFN-beta 1. Production and response to endogenous IFN are also detected when bone marrow precursor cells differentiate to macrophages after exposure to colony stimulating factor 1. In M1 myeloid cells induced to differentiate by lung-conditioned medium, addition of antibodies against IFN-beta partially abrogates the reduction of c-myc mRNA and the loss in cell proliferative activity, which both occur during differentiation. The endogenous IFN therefore functions as an autocrine growth inhibitor that participates in controlling c-myc suppression and the specific G0/G1 arrest during terminal differentiation of hematopoietic cells.     

Efficient retroviral transfer of a mouse c-myc construct into HL60

We introduced an LTR-driven mouse c-myc second and third exon, Tn5Neo gene construct into the inducible human leukemia line HL60 using an amphotropic retroviral vector system. Over 90% of the cells became neo-resistant and the transfected myc gene was transcribed in several neomycin resistant clones. Making use of the simultaneous presence of the different myc genes in the same cell, we compared expression of the corresponding mRNAs after differentiation and their decay mechanisms.     

Nuclear colocalization of cellular and viral myc proteins with HSP70 in myc-overexpressing cells.

The c-myc oncogene and its viral counterpart v-myc encode phosphoproteins which have been located within cell nuclei, excluding nucleoli. We have expressed the c-myc gene under the simian virus 40 early promoter and studied the distribution of its protein product in transient expression assays in COS, HeLa, and 293 cells. We found three distinct patterns of c-myc immunofluorescence in the transfected cells: one-third of the c-myc-positive cells displayed a diffuse nuclear distribution, and in two-thirds of the cells the c-myc fluorescence was accumulated either in small amorphous or in large multilobed phase-dense nuclear structures. Unexpectedly, these structures also stained for the HSP70 heat shock protein in both heat-shocked and untreated cells. Our results indicate that both transient and stable overexpression of either the c-myc or v-myc protein induces translocation of the endogenous HSP70 protein from the cytoplasm to the nucleus, where it becomes sequestered in structures containing the myc protein. Interestingly, the closely related N-myc protein does not stimulate substantial nuclear expression of the HSP70 protein. Studies with chimeric myc proteins revealed that polypeptide sequences encoded by the second exon of c-myc are involved in colocalization with HSP70.     

c-myc inhibition of MyoD and myogenin-initiated myogenic differentiation.

In vertebrate development, a prominent feature of several cell lineages is the coupling of cell cycle regulation with terminal differentiation. We have investigated the basis of this relationship in the skeletal muscle lineage by studying the effects of the proliferation-associated regulator, c-myc, on the differentiation of MyoD-initiated myoblasts. Transient cotransfection assays in NIH 3T3 cells using MyoD and c-myc expression vectors demonstrated c-myc suppression of MyoD-initiated differentiation. A stable cell system was also developed in which MyoD expression was constitutive, while myc levels could be elevated conditionally. Induction of this conditional c-myc suppressed myogenesis effectively, even in the presence of MyoD. c-myc suppression also prevented up-regulation of a relative of MyoD, myogenin, which is normally expressed at the onset of differentiation in all muscle cell lines examined and may be essential for differentiation. Additional experiments tested whether failure to differentiate in the presence of myc could be overcome by providing myogenin ectopically. Cotransfection of c-myc with myogenin, MyoD, or a mixture of myogenin and MyoD showed that neither myogenin alone nor myogenin plus MyoD together could bypass the c-myc block. The effects of c-myc were further dissected by showing that c-myc can inhibit differentiation independently of Id, a negative regulator of muscle differentiation. These results lead us to propose that c-myc and Id constitute independent negative regulators of muscle differentiation, while myogenin and any of the other three related myogenic factors (MyoD, Myf-5, and MRF4/herculin/Myf-6) act as positive regulators.     

Interleukin-6- and leukemia inhibitory factor-induced terminal differentiation of myeloid leukemia cells is blocked at an intermediate stage by constitutive c-myc.

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.     

Probability that the commitment of murine erythroleukemia cell differentiation is determined by the c-myc level

During the commitment of mouse erythroleukemia cell differentiation, c-myc mRNA levels change dramatically. To examine the involvement of c-myc in the commitment of these cells, we have introduced the rat c-myc gene driven by inducible, heterologous (human metallothionein IIA) gene promoter into murine erythroleukemia cells and we have examined the ability of the transformed cells to undergo commitment to terminal differentiation. The induction of the exogenous c-myc gene expression inhibited the commitment of these cells. Time-dependent inhibition of the commitment was observed with the addition of zinc at an appropriate time after the induction with dimethyl sulfoxide. The result clearly indicated that late decline, not early decline, is required for the commitment. By examining the transformants expressing the exogenous c-myc mRNA at different levels, and the induction of the exogenous c-myc mRNA by varying the concentration of zinc, we demonstrated that the commitment may be determined by a stoichiometric amount of c-myc in the defined period. The data also suggest that the probability value for the commitment process occurring in a stochastic manner is well-correlated with the amount of c-myc mRNA.     

Contrasting roles for c-Myc and L-Myc in the regulation of cellular growth and differentiation in vivo.

Although myc family genes are differentially expressed during development, their expression frequently overlaps, suggesting that they may serve both distinct and common biological functions. In addition, alterations in their expression occur at major developmental transitions in many cell lineages. For example, during mouse lens maturation, the growth arrest and differentiation of epithelial cells into lens fiber cells is associated with a decrease in L- and c-myc expression and a reciprocal rise in N-myc levels. To determine whether the down-regulation of L- and c-myc are required for mitotic arrest and/or completion of differentiation and whether these genes have distinct or similar activities in the same cell type, we have studied the consequences of forced L- and c-myc expression in the lens fiber cell compartment using the alpha A-crystallin promoter in transgenic mice (alpha A/L-myc and alpha A/c-myc mice). With respect to morphological and molecular differentiation, alpha A/L-myc lenses were characterized by a severely disorganized lens fiber cell compartment and a significant decrease in the expression of a late-stage differentiation marker (MIP26); in contrast, differentiation appeared to be unaffected in alpha A/c-myc mice. Furthermore, an analysis of proliferation indicated that while alpha A/L-myc fiber cells withdrew properly from the cell cycle, inappropriate cell cycle progression occurred in the lens fiber cell compartment of alpha A/c-myc mice. These observations indicate that continued late-stage expression of L-myc affected differentiation processes directly, rather than indirectly through deregulated growth control, whereas constitutive c-myc expression inhibited proliferative arrest, but did not appear to disturb differentiation. As a direct corollary, our data indicate that L-Myc and c-Myc are involved in distinct physiological processes in the same cell type.     

Immunologic competence of B cells subjected to constitutive c-myc oncogene expression in immunoglobulin heavy chain enhancer myc transgenic mice

B cell activation is associated with a marked transient rise in expression of the c-myc proto-onco-gene. A unique opportunity to examine the effects of constitutive c-myc expression upon B cell function is provided by transgenic mice in which the c-myc oncogene is regulated by the enhancer (E mu) from the immunoglobulin locus (E mu-myc mice). We have examined the immunologic competence of B cells from E mu-myc mice both in vitro and in vivo. Upon stimulation, many E mu-myc B cells can proliferate to form clones most of which contain antibody-forming cells. However, the frequency of responsive B cells from E mu-myc donors is only about 30% that of B cells from normal littermates. Thus, enforced myc expression is not sufficient to block the differentiation of all B cells, but a much larger fraction of the immunoglobulin-bearing cells from E mu-myc mice are incompetent. Upon immunization, E mu-myc mice mounted specific antibody responses, although some responses were delayed. Isotype switching can occur, since we observed hemolytic plaques of both IgM and IgG type and detected specific antibody of both classes in the serum. Moreover, the serum from nonimmunized E mu-myc mice contained normal levels of both IgM and IgG. Thus constitutive expression of the c-myc gene appears to retard B cell differentiation, but does not grossly impair immunologic function in the intact animal.