Wild-type HL-60 Cells Research Articles

Phorbol diester-induced alterations in the expression of protein kinase C isozymes and their mRNAs. Analysis in wild-type and phorbol diester-resistant HL-60 cell clones

In an HL-60 cell subline (PR-17) which was greater than 100-fold resistant to the differentiating and cytostatic activities of phorbol 12-myristate 13-acetate (PMA), the protein kinase C phenotype was found to be nearly identical to that of wild-type HL-60 cells. A measurable decrease (30%) in the specific activities of crude preparations of PR-17 cell protein kinase C was observed when the enzyme was measured with histone as the phosphate acceptor substrate, but other aspects of the protein kinase C phenotype (intracellular concentrations and binding affinities of phorbol diester receptors, translocation of activated enzyme from cytosolic to particulate subcellular fractions, relative expression of the alpha and beta isozyme proteins) were equivalent in both PMA-resistant PR-17 cells and in wild-type HL-60 cells. Direct analysis of the behavior of the alpha and beta isozymes after the exposure of each cell type to 100 nM PMA for 12 h revealed that the activities and intracellular concentrations of both isozymes were downregulated to an equivalent extent in both wild-type and PMA-resistant cells. These results suggest that the cellular basis for the resistance to the effects of PMA was present "down-stream" from the activation and down-regulation of protein kinase C and was perhaps a nuclear component. Among the genes which were likely to be differentially regulated when each of the two cell lines were treated with PMA were those for the protein kinase C isozymes themselves. In wild-type HL-60 cells, the intracellular concentrations of type HL-60 cells, the intracellular concentrations of mRNA for each of the beta isozymes were increased (up to 5-fold) 48 h after the initiation of PMA treatment; further studies indicate that an activator of protein kinase C could influence the expression of HL-60 cell protein kinase C genes in an isozyme-specific manner. Comparable PMA-induced alterations in mRNA levels were not observed in PMA-resistant cells, even under conditions of significant activation and subsequent down-regulation of protein kinase C protein. Taken together, these data suggest that activation and down-regulation of the isozymes of protein kinase C may not represent absolute determinants of the PMA-induced differentiation of HL-60 cells, but that specific alterations in the levels of the mRNA for the beta isozymes of protein kinase C, or of other genes which may be regulated by the activated kinase isozymes, are important to the induction of leukemia cell differentiation by PMA.

Differentiation of HL-60 Cells into Cells with the Osteoclast Phenotype*

The osteoclast is the unique multinucleated cell that is responsible for bone degradation in both physiological and pathological circumstances. However, knowledge of the lineage of this inaccessible cell, the nature of its precursors, and the regulation of its formation and activation is limited and controversial. Here we show that the human promyelocytic cell line HL-60 has the potential, under the appropriate culture conditions, to differentiate into cells that have morphological and functional characteristics of osteoclasts, including multinucleation, presence of tartrate-resistant acid phosphatase activity, cross-reactivity with monoclonal antibodies that preferentially recognize osteoclasts, and capacity to resorb bone and respond to calcitonin. The multinucleated cells also contain high affinity receptors to calcitonin, in contrast to wild-type undifferentiated HL-60 cells. These data suggest that osteoclasts share a common precursor with hematopoietic cells. These undifferentiated and differentiated HL-60 cells should provide a unique model for study of the cell biology of human osteoclast differentiation, allowing molecular biological and biochemical studies heretofore not possible.

Differential effect of 6-ethylmercaptopurine on c-myc expression in wild-type and HGPRT-deficient HL-60 cells

A variety of compounds inhibit the growth and induce differentiation of human promyelocytic leukemia (HL-60) cells. HL-60 subclones that lack the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) can also be induced to differentiate with purine analogs. Mechanisms by which purine analogs induce differentiation offer unique possibilities for cancer chemotherapy. We have studied the effect of the purine analog 6-ethylmercaptopurine (e6MP) on the growth and induction of differentiation in both wild-type and HGPRT-deficient HL-60 cells. We have previously shown that e6MP inhibits cell growth in both wild-type and HGPRT-deficient HL-60 cells without activation through salvage pathways. In this report we evaluate the effect of e6MP on c-myc mRNA expression. c-Myc mRNA, which is amplified in HL-60 cells, has been shown to play a role in the induction of granulocytic differentiation in HL-60 cells. e6MP transiently down-regulates c-myc mRNA in wild-type cells but has no effect on c-myc mRNA expression in HGPRT-deficient HL-60 cells. Despite the differential effects of e6MP on c-myc mRNA, both wild-type and HGPRT-deficient HL-60 cells appear to engage in terminal differentiation. The morphological changes and nonspecific esterase activity induced by e6MP suggest differentiation down the monocytic pathway. However, early monocytic markers such as the rapid induction of c-fos and the stabilization of c-fms mRNA are not observed. In addition, e6MP inhibits TPA-induced monocytic/macrophage differentiation as characterized by stabilization of c-fms mRNA and cellular adherence.

Lineage directed HL-60 cell sublines as a model system for the study of early events in lineage determination of myeloid cells

Current experimental models are poorly suited to study the early biochemical and molecular events of the lineage determination process in myeloid progenitor cells. Viable lineage-committed precursors cannot be identified until after they have expressed their mature phenotype and these precursors cannot be grown to large number while lineage is committed but still immature. Recently, we have identified stable sublines of the HL-60 human leukemia cell line which differ from each other in that they selectively differentiate to either neutrophils (UR-1-4), monocyte/macrophages (MRI), eosinophils (clones 2 and 15), or mixtures of two (clones 7 and 8) or of all three lineages (UR-1-2) when stimulated to mature with butyric acid under identical conditions. Characterization of these sublines provided evidence that the expression of lineage in HL-60 cells is a multistep process and that the lineage tendencies (lineage direction) the clones exhibited when cultured with butyric acid represent a step in that process earlier than irreversible lineage commitment but later than the multipotential wild type HL-60 cells. First, treatment of these sublines with compounds that induce differentiation of HL-60 cells to specific lineages (dimethylsulfoxide, neutrophil; 1,25-(OH) 2 vitamin D 3, monocyte), generally induced differentiation to the lineage associated with that inducer rather than the butyrate-associated lineage. Second, culture of neutrophil or monocyte-directed sublines in medium of elevated pH for two months leads to the development of eosinophils. Culturing the sublines first in butyric acid for variable lengths of time and switching to either DMSO or VD3 indicated that irreversible lineage commitment develops on a time course similar to the development of the commitment to mature. Markers of monocytic and eosinophilic differentiation could not be simultaneously demonstrated in single mature cells, consistent with the phenomenon of lineage fidelity. In addition, several assays were validated that could reliably classify mature HL-60 cells to their lineage. The collection of these sublines appears to constitute a model system with well-defined behavior with respect to the early events of lineage determination that can be grown to quantities sufficient for biochemical and molecular analysis. Exploring the differences between these clones may provide a new way to examine the early events of the lineage development process in myeloid cells.

Human Myelogenous Leukemia Cell Line HL-60 Cells Resistant to Differentiation Induction by Retinoic Acid: Decreased content of glycosphingolipids and granulocytic differentiation by neolacto series gangliosides

We have recently reported that neolacto series gangliosides (NeuAc-nLc) are increased during granulocytic differentiation of human myelogenous leukemia cell line HL-60 cells induced by retinoic acid and that HL-60 cells are differentiated into mature granulocytes when the cells are cultivated with NeuAc-nLc (Nojiri, H., Kitagawa, S., Nakamura, M., Kirito, K., Enomoto, Y., and Saito, M. (1988) J. Biol. Chem. 263, 7443-7446). In contrast to these wild-type-HL-60 cells, HL-60 cells resistant to differentiation induction by retinoic acid showed a markedly decreased content of gangliosides, especially NeuAc-nLc, and did not show any increase in the content of gangliosides when cultivated with retinoic acid. Neutral glycosphingolipids, the precursors of gangliosides, were not accumulated in these resistant cells. When retinoic acid-resistant HL-60 cells were cultivated in the presence of NeuAc-nLc, the cells were found to be differentiated into mature granulocytes on morphological and functional criteria. The differentiation of cells was dependent on the concentration of gangliosides and was accompanied by inhibition of cell growth. Wild-type HL-60 cells differentiated by NeuAc-nLc showed the changes in ganglioside composition, which were similar to those in wild-type HL-60 cells differentiated by retinoic acid; among the gangliosides changed, 2----3 sialylparagloboside and 2----3 sialylnorhexaosylceramide were increased. These findings suggest (a) that the synthesis of particular NeuAc-nLe molecules is an important step for retinoic acid-induced granulocytic differentiation and this step could be bypassed or replaced by exogenous NeuAc-nLc, and (b) that the defective synthesis of particular NeuAc-nLc molecules is responsible for the failure of differentiation induction in retinoic acid-resistant HL-60 cells by retinoic acid.

Variable regulation of sensitivity to retinoic acid-induced differentiation in wild-type and retinoic acid-resistant HL-60 cells.

The initial cell association and metabolic conversion of retinoic acid (RA) by HL-60 cells in serum-free, transferrin/insulin-supplemented, RPMI 1640 medium was greater than or equal to 10-fold greater than in RPMI 1640 medium containing 10% fetal bovine serum (FBS). This was paralleled under the serum-free conditions by 10-fold greater sensitivity to RA-induced differentiation, which was partially reversed by the addition of purified bovine serum albumin to the same concentration present in 10% FBS. In serum-free HL-1 medium, HL-60 cell sensitivity to RA-induced differentiation was approximately 250-fold less than in serum-free RPMI 1640 medium but, in this comparison, there was little difference in RA cell association or metabolism. A greater than 200-fold RA-resistant HL-60 subline had RA cell-association and metabolism rates similar to those of wild-type cells under all culture conditions. No significant qualitative differences in the high performance liquid chromatography elution patterns of polar metabolites were observed under any circumstances. These results indicate that inherent cellular properties, not associated with gross differences in RA uptake or metabolism, primarily determined the relative sensitivity or insensitivity of HL-60 cells to RA-induced differentiation but that RA responsiveness was markedly regulated by extracellular factors, one of which, serum albumin, appeared to act by decreasing the initial cell association and metabolism of RA, whereas other, as yet unidentified exogenous factors, may have acted independently of these functions.

Regulation of gene expression of myeloperoxidase during myeloid differentiation.

Myeloperoxidase (MPO) is a major heme enzyme involved in inflammatory responses of polymorphonuclear leukocytes. Using cDNA and intron specific probes for MPO we studied the regulation of MPO expression during myeloid differentiation of the promyelocytic HL-60 leukemia cell line. Mature MPO mRNA species of 3.3, 2.8 and 1.6 kb and heterogeneous nuclear (hn) RNA of greater than 8 and approximately 4 kb were observed in wildtype HL-60 cells. Induction of differentiation of the cells towards either granulocytes or macrophages resulted in a profound decrease (greater than 95%) in the concentration of MPO mRNA levels, showing that gene expression of MPO mRNA is closely linked to the stage of development of myeloid cells. Studies using normal and leukemic hematopoietic cells confirmed these findings and showed that myeloblasts and promyelocytes contain MPO mRNA. Rate of transcription of MPO was measured by a nuclear run-on assay in wild-type and day 3- and day -4 differentiated HL-60 cells and was nearly the same in all three. In contrast, rate of transcription of c-myc in the same nuclei became almost undetectable with induction of differentiation. Overall transcription decreased by 60% and 80% on day 3 and 4 of differentiation, respectively, compared to wild-type cells. Stability of mature MPO mRNA was also measured and found to be the same in wild-type and differentiated HL-60. Half-life of MPO hnRNA was less than or equal to 30 min in wild-type HL-60; nevertheless, this hnRNA was easily detectable 3 days after induction of differentiation of these cells. Taken together, the results show that decreased expression of MPO mRNA with differentiation occurs in part post-transcriptionally, possibly due to a failure in RNA processing. In addition, as overall transcription decreases during differentiation, MPO transcription is concomitantly reduced. This indicates that transcriptional and post-transcriptional mechanisms cooperate in the control of MPO gene expression.

Characterization of a relationship between the T-lymphocyte derived differentiation inducing factor (DIF) and lymphotoxin: a common receptor system for DIF, lymphotoxin and tumor necrosis factor downregulated by phorbol diesters.

Here we describe results which show that recombinant lymphotoxin (rLT), like the T-lymphocyte derived differentiation inducing factor (DIF), inhibited the clonogenic growth of some myeloid leukemia cell lines by concentrations of 1 to 30 pmol/l. Wild type HL-60 cells were resistant at these concentrations but responded with differentiation into monocyte-like cells at higher concentrations. An antigenic relationship between DIF and LT was indicated because a neutralizing monoclonal anti-LT antibody bound to and neutralized both differentiation and growth inhibitory effects of DIF. An activity, which cochromatographed with DIF during all purification steps, competed with binding of both rLT and recombinant tumor necrosis factor (rTNF) to HL-60 cells. By use of radioiodinated ligand, 2100 binding sites for rLT were detected on HL-60 cells with a Kd of 330 pmol/l. At 37 degrees C bound ligand was transferred to lysosomes, followed by degradation. rTNF and rLT were shown to compete for binding sites on HL-60 cells. Receptors for both rLT and rTNF were downregulated by activators of protein kinase C such as phorbol diester or diacylglycerol; the number of cell surface receptors decreased while the Kd remained unchanged. Our observations demonstrate a functional and antigenic relationship between DIF and LT and indicate that TNF, LT and DIF share binding sites on myeloid leukemia cells that are downregulated by activation of protein kinase-C.

5′-Deoxy-5′-methylthioadenosine phosphorylase—IV: Biological activity of 2-fluoroadenine-substituted 5′-deoxy-5′ -methylthioadenosine analogs

5′-Deoxy-5′-methylthioadenosine phosphorylase (MTAPase) phosphorolyzes 5'-deoxy-5'-methylthioadenosine (MTA) generated during polyamine biosynthesis to adenine and 5-methyl-thioribose-1-phosphate. Two doubly-substituted, 2-fluoroadenine-containing analogs of MTA, 5'-deoxy-2-fluoroadenosine (5'-dFAdo) and 5′-deoxy-5′-iodo-2-fluoroadenosine (5′-IFAdo), were synthesized and studied as substrates of MTAPase: their reaction with this enzyme resulted in the liberation of the cytotoxic base, 2-fluoroadenine, as well as potentially cytotoxic analogs of 5-methylribose-1-phosphate. The activities of these MTA analogs were compared to that of the singly-substituted analog, 5'-deoxy-5'-methylthio-2-fluoroadenosine (5′-MTFAdo). The cytotoxic action of these MTA analogs depended primarily on their conversion to 2-fluoroadenine-containing nucleotides, as a cell line that contains both MTAPase and adenine phosphoribosyltransferase (APRT) activity (HL-60 human promyelocytic leukemia) readily converted these MTA analogs to 2-fluoroadenine-containing nucleotides (especially 2-fluoroadenosine triphosphate) and was highly sensitive to the growth-inhibitory effects of all three compounds ( ic 50 values in the 10 −8 M range), whereas cell lines lacking MTAPase (CCRF-CEM human T-cell leukemia) or APRT (HL-60/ aprt 1cells) did not form analog nucleotides and were relatively insensitive to these compounds ( ic 50 values in the 10 −5 M range). The doubly-substituted analogs were not more growth inhibitory than 5'-MTFAdo in wild type HL-60 cells as the potent effects of 2-fluoroadenine may mask the activity of the 5-methylthioribose-1-phosphate analogs generated in the reaction of these compounds with MTAPase. 5'-dFAdo and 5'-IFAdo also were irreversible inhibitors of idS-adenosylhomocysteine hydrolase, which may explain in part the weak but observable growth inhibitory action of these compounds against MTAPase-deficient cell lines.

Hyposialylation of differentiation-inducer-resistant HL-60 cells

The total sialic acid concent of retinoic acid (RA)-resistant or 6- thioguanine (6TG)-resistant HL-60 cells was more than tenfold lower and of dimethylsulfoxide (DMSO)-resistant HL-60 cells was approximately twofold lower than that of parental, wild-type (wt) HL-60 cells. Neuraminidase-inaccessible, ie residual cell-associated sialic acid after neuraminidase treatment, was four- to twelvefold lower in the three differentiation-inducer-resistant sublines than in the parent line. Neuraminidase treatment of 125I-labeled surface membrane glycoproteins (SMGs) from wt HL-60 cells converted the two-dimensional gel electrophoretic pattern to one having features in common with RA- and 6TG-resistant cells. However, neuraminidase treatment did not alter the sensitivity of wt HL-60 cells to differentiation induction by RA, hypoxanthine (purine base), or DMSO. These results indicate that differences in peripheral, neuraminidase-accessible sialic acids are important determinants of the gel electrophoretic mobility of the SMGs of the HL-60 line and sublines but are not likely related to the differentiation-resistance mechanism. Further studies are required to determine if hyposialylation of cryptic, neuraminidase-inaccessible sites has functional significance.

Hyposialylation of Differentiation-Inducer-Resistant HL-60 Cells

The total sialic acid concent of retinoic acid (RA)-resistant or 6-thioguanine (6TG)-resistant HL-60 cells was more than tenfold lower and of dimethylsulfoxide (DMSO)-resistant HL-60 cells was approximately twofold lower than that of parental, wild-type (wt) HL-60 cells. Neuraminidase-inaccessible, ie residual cell-associated sialic acid after neuraminidase treatment, was four- to twelvefold lower in the three differentiation-inducer-resistant sublines than in the parent line. Neuraminidase treatment of 125I-labeled surface membrane glycoproteins (SMGs) from wt HL-60 cells converted the two-dimensional gel electrophoretic pattern to one having features in common with RA- and 6TG-resistant cells. However, neuraminidase treatment did not alter the sensitivity of wt HL-60 cells to differentiation induction by RA, hypoxanthine (purine base), or DMSO. These results indicate that differences in peripheral, neuraminidase-accessible sialic acids are important determinants of the gel electrophoretic mobility of the SMGs of the HL-60 line and sublines but are not likely related to the differentiation-resistance mechanism. Further studies are required to determine if hyposialylation of cryptic, neuraminidase-inaccessible sites has functional significance.

T lymphocyte-derived differentiation-inducing factor inhibits proliferation of leukemic and normal hemopoietic cells

A differentiation-inducing factor (DIF) for the promyelocytic HL-60 cell line is constitutively produced by the malignant T lymphocyte line HUT-102. DIF was highly purified from HUT-102-conditioned media by means of diethylaminoethanol (DEAE)-chromatography, gel chromatography, and high-resolution, ion-exchange chromatography on a MonoQ column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition to inducing differentiation of wild-type HL-60 cells, resulting in secondary inhibition of growth, DIF, at a tenfold lower concentration, inhibited the growth of some clones of the monoblastic U-937 cell line as well as that of subclones of HL-60. The latter effect was most likely a primary growth inhibition and not secondary to differentiation; 50% inhibition of clonogenic growth in agar was seen at approximately 1.0 pmol/L of DIF. In addition, the clonogenic growth of fresh leukemia cells from 10 of 12 patients with acute myeloid leukemia (AML) was inhibited with 50% inhibition at approximately 10 pmol/L of DIF. The growth of normal granulocyte-macrophage colonies was inhibited at a similar concentration, whereas early erythroid colonies were much more resistant. DIF and interferon-gamma (gamma-IFN) were shown to be separate molecules inasmuch as a neutralizing antibody for gamma-IFN did not abolish the DIF effect. The differentiation effect on wild-type HL-60 and the proliferation inhibitory effect on leukemic and normal myeloid cells cochromatographed through all purification steps suggest that both activities are exhibited by identical polypeptides. DIF may have a role in regulating normal hemopoiesis. The growth inhibitory effect of DIF and the ability to induce differentiation of some leukemia cells may suggest a clinical utility in the treatment of leukemia.

T Lymphocyte-Derived Differentiation-Inducing Factor Inhibits Proliferation of Leukemic and Normal Hemopoietic Cells

Abstract A differentiation-inducing factor (DIF) for the promyelocytic HL-60 cell line is constitutively produced by the malignant T lymphocyte line HUT-102. DIF was highly purified from HUT-102-conditioned media by means of diethylaminoethanol (DEAE)-chromatography, gel chromatography, and high-resolution, ion-exchange chromatography on a MonoQ column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition to inducing differentiation of wild-type HL-60 cells, resulting in secondary inhibition of growth, DIF, at a tenfold lower concentration, inhibited the growth of some clones of the monoblastic U- 937 cell line as well as that of subclones of HL-60. The latter effect was most likely a primary growth inhibition and not secondary to differentiation; 50% inhibition of clonogenic growth in agar was seen at approximately 1.0 pmol/L of DIF. In addition, the clonogenic growth of fresh leukemia cells from 10 of 12 patients with acute myeloid leukemia (AML) was inhibited with 50% inhibition at approximately 10 pmol/L of DIF. The growth of normal granulocyte-macrophage colonies was inhibited at a similar concentration, whereas early erythroid colonies were much more resistant. DIF and interferon-gamma (gamma-IFN) were shown to be separate molecules inasmuch as a neutralizing antibody for gamma-IFN did not abolish the DIF effect. The differentiation effect on wild-type HL-60 and the proliferation inhibitory effect on leukemic and normal myeloid cells cochromatographed through all purification steps suggest that both activities are exhibited by identical polypeptides. DIF may have a role in regulating normal hemopoiesis. The growth inhibitory effect of DIF and the ability to induce differentiation of some leukemia cells may suggest a clinical utility in the treatment of leukemia.

Surface Membrane Glycoproteins of Wild-Type and Differentiation-Inducer-Resistant HL-60 Cells

AbstractSurface membrane glycoproteins (SMGs) of cells from the parental wild-type HL-60 cell line and from three sublines variably cross-resistant to the granulocyte differentiation-inducing effects of retinoic acid (RA), dimethylsulfoxide (DMSO), and certain purine bases (6-thioguanine [6TG] or hypoxanthine) were studied by one-dimensional and two-dimensional gel electrophoresis. After both oligosaccharide (periodate/borotritiide) and peptide (1,3,4,6-tetrachloro-3α, 6α-diphenylglycouril) ectolabeling procedures, striking common changes were noted in the gel electrophoretic patterns of the SMGs from the RA- and 6TG-resistant sublines compared to those from the wild-type HL-60 line or the DMSO-resistant subline. Most prominently, this included the presence in the RA- and 6TG-resistant cells of an apparent high molecular weight acidic glycoprotein(s) (mol wt, 200 to 285 kilodaltons [kD]; isoelectric point range [pI], 4.5 to 6.0) not observed in the wild-type or DMSO-resistant cells and, conversely, the presence of a lower molecular weight glycoprotein(s) (mol wt, 120 to 165 kD; pl, 4.2 to 5.9) in the wild-type and DMSO-resistant cells, which was absent or much reduced in the RA- and 6TG-resistant cells. These acidic SMGs did not change as a function of the induction of granulocyte differentiation. However, some other more basic SMGs varied as a function of granulocyte differentiation in both the wild-type and differentiation inducer-resistant sublines, including the loss of the transferrin receptor and the gain of a mol wt 55-to 60-kD neutrophil-associated protein. In the context of previously reported information, these results indicate (1) that the overall pattern of SMG changes in the RA- and 6TG-resistant cells closely resembles that associated with multidrug (pleiotropic) resistance to cytotoxic agents in a variety of mammalian cells; (2) that the RA/6TG resistance-associated SMG changes are not granulocyte differentiation stage-specific; and (3) that either the RA/6TG resistance-associated SMG changes are not related to the resistance mechanism or they are involved in the resistance/cross-resistance mechanism(s) for RA/purine bases but not for DMSO.

Recombinant human interferon sensitizes resistant myeloid leukemic cells to induction of terminal differentiation

Recombinant human leukocyte interferon (IFN-αA) inhibits growth of the human promyelocytic leukemic cell line HL-60 without inducing these cells to differentiate terminally. When IFN-αA is combined with agents capable of inducing differentiation in HL-60 cells, such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), cis or trans retinoic acid (RA) or dimethylsulfoxide (DMSO), growth suppression and induction of differentiation are dramatically increased. By growing HL-60 cells in increasing concentrations of TPA, RA, or DMSO, a series of sublines have been developed which are resistant to the usual growth inhibition and induction of differentiation seen when wild type HL-60 cells are exposed to these agents. Treatment of these resistant HL-60 cells with the combination of IFN-αA and the appropriate inducer results, however, in a synergistic suppression in cell growth and a concomitant induction of terminal differentiation. The ability of interferon to interact synergistically with agents which promote leukemic cell maturation may represent a novel means of reducing resistant leukemic cell populations.

Characterization of differentiation-inducer-resistant HL-60 cells

Sub-lines of the cultured human promyelocytic leukemia cell line HL-60 were individually selected for their ability to sustain exponential growth in the presence of 3 structurallyunrelated inducers of granulocytic differentiation — retinoic acid (RA), dimethylsulfoxide (DMSO), and 6-thioguanine (6TG). Selections were made by step-wise augmentation to final drug concentrations of 10 −3mM RA, 169mM (1.2%) DMSO and 0.12mM (20 μg ml −1) 6TG. In addition to growth resistance, cells in each sub-line displayed variable cytodifferentiation resistance to each of the 3 selective agents, which was quantitated as the ratio of the concentration of drug required to induce differentiation in 50% of the cells in each resistant sub-line versus comparably-passaged wild-type HL-60 cells. The levels of resistance/cross-resistance were as follows: RA-resistant (res) sub-line > 2700-fold to RA, 1.3-fold to DMSO and > 1.5-fold to hypoxanthine (HXN; the noncytotoxic purine base inducer analogue of 6TG); DMSO-res sub-line 2.5-fold to DMSO, 137-fold to RA and > 1.5-fold to HXN; and 6TG-res sub-line > 1.5-fold to HXN, 9-fold to RA and 1.6-fold to DMSO. These sub-lines were not cross-resistant to sodium butyrate (NaBut), a monocyte inducer, or to 12- O-tetradecanoylphorbol 13-acetate (TPA), a macrophage inducer. HL-60 sub-lines selected by exposure to a single high concentration of 5-bromo-2′-deoxyuridine (BUdR; 3.3 × 10 −2mM) or oubain (Ou; 5 × 10 −3mM) were not or were slightly cross-resistant to either granulocyte or monocyte inducers. Although some variations in line/sub-line phenotype were observed, this was minor compared to the quantitative variations in response to individual inducing agents. The RA-res and 6TG-res sub-lines contained numerous double minute chromosomes (indicators of amplified genes) which were either absent or present in much smaller numbers in the parental wild-type cells or in the other drug-resistant sub-lines. There was little change or a decrease in the amplification level of the known amplified oncogene c- myc in the various drug-resistant sub-lines compared to wild-type HL-60 cells. These results (a) confirm that the neutrophilic granulocytic and monocytic/macrophagic differentiation programs in HL-60 cells are mechanistically different and separable; (b) suggest that both agent-specific and common quantitative alterations contribute to the mechanism(s) for resistance to granulocyte differentiation; and (c) suggest that the latter quantitative defects could be related to amplification of genes other than c- myc.