A Myb dependent pathway maintains Friend murine erythroleukemia cells in an immature and proliferating state

Friend murine erythroleukemia (MEL) cells are transformed erythroid precursors that are held in an immature and proliferating state but can be induced to differentiate in vivo by treatment with a variety of chemical agents such as N, N-hexamethylene bisacetamide (HMBA). To investigate the role of Myb proteins in maintaining MEL cells in an immature and proliferating state we have produced stable transfectants in the C19 MEL cell line that contain a dominant interfering Myb allele (MEnT) under the control of an inducible mouse metallothionein I promoter. When expression of MEnT protein was induced with ZnCl2, the stable transfectants differentiated with kinetics that were similar to wild type C19 MEL cells treated with HMBA, including induction of alpha-globin mRNA expression, assembly of hemoglobin and growth arrest. Expression of endogenous c-myb and c-myc was also decreased in response to MEnT. Expression of mad-1 mRNA was rapidly increased in response to expression of MEnT resulting in a shift from predominantly c-Myc/Max complexes to predominantly Mad/Max containing complexes. These results strongly suggest that C19 MEL cells are held in an immature and proliferating state by a pathway that is dependent on Myb activity.     

Induction of replicative competence ("priming") in normal liver

We have used a system of nutritional manipulation to investigate whether hepatocytes of the normal liver can be primed for replication in vivo. In this system, rats that are denied protein for 3 days undergo a burst of hepatic DNA synthesis and mitosis when they are refed amino acids, while normally fed or starved rats do not respond. To determine if hepatocytes of protein deprived (PD) rats have been "primed" for replication, we examined changes in protooncogene expression in livers of PD rats to see if they would mimic the pattern of gene expression that is induced early after partial hepatectomy. c-jun, c-myc, and p53 mRNAs were elevated in livers of PD rats, while c-fos and c-ras genes were not expressed. The administration of amino acids to PD rats stimulated hepatic DNA synthesis in a shorter period than is required after partial hepatectomy and induced p53 and c-ras expression. In culture, hepatocytes from PD rats had higher levels of c-myc mRNA, underwent morphological changes more rapidly, and reached maximum rates of DNA synthesis earlier than normal hepatocytes. In both normal and primed hepatocyte cultures, transforming growth factor alpha stimulated DNA synthesis more effectively than epidermal growth factor. We conclude that hepatocytes pass through a priming stage before they proliferate and that replicative competence without DNA synthesis can be induced in hepatocytes in the normal liver.     

max encodes a sequence-specific DNA-binding protein and is not regulated by serum growth factors.

Max and c-Myc proteins, produced in bacteria, were studied for DNA-binding activity using the electrophoretic band-shift assay (EMSA). Both Max homodimers and c-Myc-Max heterodimers selected the same sequence CA(C/T)GTG from an initial pool of 10(6) DNA molecules. From the pool of sequence-specific binding sites, the palindromic site (CACGTG) was preferentially selected over the CATGTG site using two different degenerate oligonucleotide probes. max expression is identical in myc-induced tumor cell lines relative to other cells. Furthermore, max expression is constant in both confluent and serum-stimulated A31 fibroblasts, in contrast to c-myc expression, which is barely detectable in confluent fibroblasts and induced 20-fold by serum growth factors. Based on recognition of the same DNA sequence by Max and c-Myc-Max complexes and differential expression of the two genes, we propose that Max homodimers and c-Myc-Max heterodimers may bind to a common set of cellular target genes.     

Basic mutant Max reverses a c-Myc block to differentiation

Murine erythroleukemia (MEL) cells overexpressing a transfected c-myc gene are blocked in their ability to undergo inducer-mediated differentiation, whereas overexpression of a transfected max gene mutated within the basic region (bm-max) accelerates differentiation. Based on these findings, we cotransfected MEL cells with plasmids which express human c-Myc constitutively and bm-Max in a zinc-inducible manner. Competition of endogenous proteins for binding to bm-Max can be considered negligible in cells expressing such high constitutive levels of c-Myc. Thus, this system provides a cell culture model for studying Myc:Max complex formation and its effect on erythroid differentiation. Clones expressing high levels of c-Myc and low levels of bm-Max are blocked in their ability to undergo N,N'-hexamethylene bisacetamide (HMBA)-mediated differentiation, presumably due to a preponderance of growth-promoting Myc:Max complexes. However, increased expression of bm-Max, in these clones, allows differentiation to occur by decreasing the levels of functional Myc:Max complexes. Although the exogenously expressed c-Myc and bm-Max associate in vivo, the basic region mutation in bm-Max abolishes the binding of Myc:bm-Max complexes to the specific E-box consensus sequence. We demonstrate that this sequestering of c-Myc by bm-Max reverses the c-Myc block to differentiation.     

Low molecular weight inhibitors of Myc-Max interaction and function

c-Myc is helix-loop-helix-leucine zipper (HLH-ZIP) oncoprotein that is frequently deregulated in human cancers. In order to bind DNA, regulate target gene expression, and function in a biological context, c-Myc must dimerize with another HLH-ZIP protein, Max. A large number of c-Myc target genes have been identified, and many of the encoded proteins are transforming. Such functional redundancy, however, complicates therapeutic strategies aimed at inhibiting any single target gene product. Given this consideration, we have instead attempted to identify ways by which c-Myc itself could be effectively disabled. We have used a yeast two-hybrid approach to identify low-molecular-weight compounds that inhibit c-Myc-Max association. All of the compounds prevented transactivation by c-Myc-Max heterodimers, inhibited cell cycle progression, and prevented the in vitro growth of fibroblasts in a c-Myc-dependent manner. Several of the compounds also inhibited tumor growth in vivo. These results show that the yeast two-hybrid screen is useful for identifying compounds that can be exploited in mammalian cells. More specifically, they provide a means by which structural analogs, based upon these first-generation Myc-Max inhibitors, can be developed to enhance antitumor efficacy.     

Studies on the kinetics of expression of cell cycle dependent proto-oncogenes during mitogen-induced liver cell proliferation

The present study was undertaken to determine the kinetics of DNA synthesis and expression of cell cycle dependent proto-oncogenes in response to two types of cell proliferative stimuli in male Wistar rat liver. The peak of DNA synthesis was approximately 24 h after a compensatory cell proliferative stimulus induced by 2/3 partial hepatectomy and approximately 36 h following a mitogenic stimulus obtained with a single dose of lead nitrate (10 micromol/100 g body wt, through femoral vein). Even though both proliferative stimuli induced the expression of c-fos, c-myc and c-Ha-ras, the extent of the increase in c-fos expression was 4- to 5-fold less in mitogen-induced cell proliferation. In addition, while the expression of c-myc, following partial hepatectomy returned to basal level by 4 h, the induced expression of c-myc persisted for up to 40 h during the lead nitrate-induced liver cell proliferation.     

Epidermal growth factor and proliferation in rat hepatocytes in primary culture isolated at different times after partial hepatectomy

Primary hepatocyte cultures have been prepared from normal adult rat liver and from rat liver at 4, 8, 12, 24, and 48 h following partial hepatectomy (removal of 70% of the liver). Cells were maintained in minimal essential medium alone or supplemented with hormones. Comparing DNA synthesis in normal adult rat hepatocytes with DNA synthesis in hepatocytes isolated from regenerating livers, we found with minimal essential medium alone little DNA synthesis in normal adult rat hepatocytes and in hepatocytes isolated 4, 8, or 12 h after 70% hepatectomy. In hepatocytes isolated 24 h after partial hepatectomy, however, the incorporation of [3H]thymidine was 3 times the rate of normal hepatocytes. The addition of insulin to minimal essential medium had minimal effect on DNA synthesis in all hepatocytes. Addition of epidermal growth factor alone or in combination with insulin resulted in a dramatic increase in DNA synthesis in hepatocytes from regenerating rat liver. Increased incorporation was detectable as early as 4 h after partial hepatectomy and reached a maximum at 24 h after the operation. Results obtained with [3H]thymidine incorporation were confirmed by autoradiography and by direct DNA determinations in hepatocyte cultures. Epidermal growth factor binding to the hepatocytes was determined and agreed with previously reported binding studies. Binding of epidermal growth factor in hepatocytes isolated at 4 h after partial hepatectomy was the same as in normal hepatocytes but was undetectable in hepatocytes isolated from rats at 12 and 24 h after partial hepatectomy.     

Expression of c-raf-1 and A-raf-1 during regeneration of rat liver following surgical partial hepatectomy

The major objective of this study was to investigate the expression of members of the raf family of proto-oncogenes during rat liver regeneration. The steady-state level of expression of both c-raf-1 and A-raf-1 increased three- to fivefold 18-24 h following partial hepatectomy, and it returned to basal levels by 72 h. Expression of c-myc and Ha-ras mRNA was increased at 3 and 18-24 h, respectively, confirming previous reports. Increased steady-state levels of c-raf-1, A-raf-1, and Ha-ras mRNA were also detected in hepatocytes isolated from rat liver 24 h after partial hepatectomy. Thus, elevated expression of the raf genes closely correlated with that of Ha-ras, beginning at 12 h and reaching maximal levels during the first peak of DNA synthesis following partial hepatectomy.     

Differential expression of myc, max and RB1 genes in human gliomas and glioma cell lines.

Deregulated expression of myc proto-oncogenes is implicated in several human neoplasias. We analysed the expression of c-myc, N-myc, L-myc, max and RB1 mRNAs in a panel of human gliomas and glioma cell lines and compared the findings with normal neural cells. The max and RB1 genes were included in the study because their protein products can interact with the Myc proteins, being thus putative modulators of Myc activity. Several gliomas contained c/L-myc mRNAs at levels higher than those in fetal brain, L-myc predominantly in grade II/III and c-myc in grade III gliomas. High-level N-myc expression was detected. In one small-cell glioblastoma and lower levels in five other gliomas. In contrast, glioma cell lines totally lacked N/L-myc expression. The in situ hybridisations revealed mutually exclusive topographic distribution of myc and glial fibrillary acidic protein (GFAP) mRNAs, and a lack of correlation between myc expression and proliferative activity, max and RB1 mRNAs were detected in most tumours and cell lines. The glioma cells displayed interesting alternative splicing patterns of max mRNAs encoding Max proteins which either suppress (Max) or augment (delta Max) the transforming activity of Myc. We conclude that (1) glioma cells in vivo may coexpress several myc genes, thus resembling fetal neural cells; but (2) cultured glioma cells expression only c-myc; (3) myc, max and RB1 are regulated independently in glioma cells; and (4) alternative processing of max mRNA in some glioma cells results in delta Max encoding mRNAs not seen in normal fetal brain.