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.     

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.     

Flow cytometric correlation of the c-myc oncoprotein and cell cycle kinetics of HL60 leukaemia during induced maturation with cytosine arabinoside and dimethylsulphoxide

The c-myc oncogene codes for a DNA binding protein that functions in a cell cycle-related manner. A useful model for studying the relationship of c-myc expression with cell cycle kinetics is the HL60 cell line. HL60 cells constitutively express high levels of c-myc mRNA; however, the level can be down-regulated as the cells are induced to differentiate. We have developed a flow cytometric assay for correlating c-myc oncoprotein levels with DNA content. C-myc oncoprotein levels were additionally correlated with c-myc mRNA levels as determined by slot blot hybridization. Dimethylsulphoxide (DMSO) and cytosine arabinoside were used to induce granulocytic and monocytic maturation respectively. Treatment of HL60 cells with DMSO leads to an increase in the per cent of cells in G1/G0 and a decrease in mean c-myc mRNA and oncoprotein levels. The cells with G1 DNA content show the greatest decrease in c-myc protein. ARA-c treatment of HL60 cells leads to a slowing and an accumulation of cells in S phase with a moderate decrease in mean mRNA and only a slight decrease in mean c-myc protein levels. These data support the hypothesis that c-myc is involved in the switch from G1 to G0.     

Temporal relationships between induced changes in c-myc mRNA abundance, proliferation, and differentiation in HL60 cells

Changes in the relative abundances of c-myc mRNA have been related to changes in other parameters of differentiation (histochemical, clonogenic) during the course of the differentiation of HL60 cells to monocytes/macrophages or to granulocytes. Induction of differentiation to monocytes/macrophages was marked by a rapid rate of appearance of committed cells (80 to 90% in 24 hours) and a concomitant rapid loss of c-myc mRNA. Induction of granulocytic differentiation resulted in a much slower rate of appearance of committed cells (50% in 48 hours), and a much faster rate of loss of c-myc mRNA (tenfold in 1 hour). These data are consistent with there being a direct link between down-regulation of the expression of c-myc and the onset of proliferation arrest and monocytic differentiation, but show there is no such association of c-myc mRNA abundance and proliferation or differentiation during the maturation of HL60 granulocytes.     

Isolation and characterization of the human cellular myc gene product

Antibodies against the product of the human cellular myc gene (c-myc) were prepared against a bacterially expressed human c-myc protein by inserting the ClaI/BclI fragment of the human c-myc DNA clone in an expression vector derived from pPLc24. These antibodies cross-react with viral-coded myc (v-myc) proteins from MC29 and OK10 viruses. Furthermore, IgGs specific for synthetic peptides, corresponding to the 12 carboxy-terminal amino acids of the human c-myc gene and 16 internal amino acids, were isolated. By use of the various myc-specific antisera or IgGs, a protein of Mr 64 000 was detected in several human tumor cell lines including Colo320, small cell cancer of the lung (417d), HL60, Raji, and HeLa. This protein is larger than the corresponding v-myc or chicken c-myc proteins from avian virus transformed cells or avian bursa lymphoma cells (RP9), both of which are proteins of Mr 55 000. The human c-myc protein is located in the nucleus of Colo320 cells, exhibits a half-life of about 15 min, and is expressed at significantly lower levels than the viral protein. The human c-myc protein was enriched about 3000-fold from Colo320 cells using c-myc-specific IgG coupled to Sepharose beads. The protein binds to double-stranded DNA in vitro, a reaction that can be inhibited to more than 90% by c-myc specific IgG.     

Flow cytometric correlation of the c-myc oncoprotein and cell cycle kinetics of HL60 leukaemia during induced maturation with cytosine arabinoside and dimethylsulphoxide

Abstract The c-myc oncogene codes for a DNA binding protein that functions in a cell cycle-related manner. A useful model for studying the relationship of c-myc expression with cell cycle kinetics is the HL60 cell line. HL60 cells constitutively express high levels of c-myc mRNA; however, the level can be down-regulated as the cells are induced to differentiate. We have developed a flow cytometric assay for correlating c-myc oncoprotein levels with DNA content. C-myc oncoprotein levels were additionally correlated with c-myc mRNA levels as determined by slot blot hybridization. Dimethylsulphoxide (DMSO) and cytosine arabinoside were used to induce granulocytic and monocytic maturation respectively. Treatment of HL60 cells with DMSO leads to an increase in the per cent of cells in G₁/G₀ and a decrease in mean c-myc mRNA and oncoprotein levels. The cells with G₁ DNA content show the greatest decrease in c-myc protein. ARA-c treatment of HL60 cells leads to a slowing and an accumulation of cells in S phase with a moderate decrease in mean mRNA and only a slight decrease in mean c-myc protein levels. These data support the hypothesis that c-myc is involved in the switch from G₁ to G₀.     

Overall changes in chromatin sensitivity to DNase I during differentiation

The DNase I sensitivity of total chromatin was studied in fixed cells and nuclei isolated from proliferating and terminally differentiated cells, by measuring the incorporation of labelled nucleotides into DNase-sensitive sites, and electrophoresis of DNA isolated from DNase-treated nuclei. The unfixed nuclei were sensitive to digestion at around 10 micrograms/ml, the fixed cells at 30 ng/ml DNase I concentration. Proliferating Rauscher leukemia cells were more digestible than normal spleen cells. The DNase I sensitivity of the human HL60 leukemia line decreased upon DMSO-induced differentiation but still exceeded the digestibility of nuclei from normal human peripheral blood. A novel flow-cytometric technique was developed to study DNase sensitivity at the cell level. It confirmed the relative resistance of differentiated cells to DNase I and ruled out the possibility that this could be due to an altered distribution of cell cycle phases. The overall DNase I sensitivity of chromatin was compared with the sensitivity of the c-myc gene and the myc-associated hypersensitive sites. The latter sites were detected at 1 microgram/ml DNase I in HL60 nuclei. They disappeared partially upon DMSO-induced differentiation. At 10 micrograms/ml, myc was degraded in both growing and differentiating HL60, but not in HPB cells. These data suggest that a progressive condensation of the chromatin occurs during terminal differentiation which gradually involves specific genes that need to be inactivated.