Smad6 is a protein kinase X phosphorylation substrate and is required for HL-60 cell differentiation.

To gain insight into the function of human protein kinase X (PrKX), a signal-transduction protein required for macrophage differentiation, we identified regulatory subunit I alpha of protein kinase A, T54 and Smad6 as partners for this protein using a yeast two-hybrid interaction screen. Interactions between PrKX and these proteins were substantiated by co-immunoprecipitation. Interaction between Smad6 and PrKX was also confirmed in human myeloid HL-60 cells following their phorbol 12-myristate 13-acetate (PMA)-induced differentiation into macrophages. In vitro phosphorylation assays demonstrated that PrKX phosphorylates Smad6 at a serine residue. Mutagenesis of this site resulted in abrogation of PrKX phosphorylation. Both PrKX and Smad6 were shown to be co-localized to the nuclear compartment of HL-60 cells during their macrophage differentiation where PrKX levels are induced and Smad6 protein levels remain relatively constant while levels of serine phosphorylation of Smad6 increase. By using in vitro electrophoretic mobility shift assays and in vivo chromatin immunoprecipitation, we also demonstrate that during macrophage differentiation Smad6 displays an increased binding to the human osteopontin, Id2, and Hex gene promoters, which correlates to an observed increased expression of these genes. Finally, vector-based RNA interference experiments established that both Smad6 and PrKX proteins are required for PMA-induced cell attachment and spreading.

Amplification of the IMP dehydrogenase gene in Chinese hamster cells resistant to mycophenolic acid.

The regulation of IMP dehydrogenase (IMPDH) was analyzed in Chinese hamster V79 cell variants that exhibit different degrees of resistance to the cytotoxic effect of mycophenolic acid, a specific inhibitor of IMPDH. Western blot (immunoblot) analysis with an IMPDH antiserum revealed a 14- to 27-fold increase in the amount of enzyme in the mycophenolic acid-resistant cells. The antiserum was also used to screen for a phage containing the IMPDH cDNA sequence from a lambda gt11 expression library. Northern blot (RNA blot) analyses of total cellular and poly(A)+ RNA showed that an IMPDH cDNA probe hybridized to a 2.2-kilobase transcript, the amount of which was associated with increased resistance. Southern blotting with the probe indicated an amplification of the IMPDH gene in the mycophenolic acid-resistant cells. Our findings suggest that the acquired mycophenolic acid resistance of the V79 cell variants is associated with increases in the amount and activity of IMPDH and the number of IMPDH gene copies.

Regulation of IMP dehydrogenase gene expression by its end products, guanine nucleotides.

To study the regulation of IMP dehydrogenase (IMPDH), the rate-limiting enzyme of guanine nucleotide biosynthesis, we examined the effects of nucleosides, nucleotides, nucleotide analogs, or the IMPDH inhibitor mycophenolic acid (MPA) on the steady-state levels of IMPDH mRNA. The results indicated that IMPDH gene expression is regulated inversely by the intracellular level of guanine ribonucleotides. We have shown that treatment with guanosine increased the level of cellular guanine ribonucleotides and subsequently reduced IMPDH steady-state mRNA levels in a time- and dose-dependent manner. Conversely, MPA treatment diminished the level of guanine ribonucleotides and increased IMPDH mRNA levels. Both of these effects on the steady-state level of IMPDH mRNA could be negated by cotreatment with guanosine and MPA. The down regulation of IMPDH gene expression by guanosine or its up regulation by MPA was not due to major changes in transcriptional initiation and elongation or mRNA stability in the cytoplasm but rather was due to alterations in the levels of the IMPDH mRNA in the nucleus. These results suggest that IMPDH gene expression is regulated by a posttranscriptional, nuclear event in response to fluctuations in the intracellular level of guanine ribonucleotides.

A sensitive hepatocyte-mediated assay for the metabolism of nitrosamines to mutagens for mammalian cells.

A sensitive cell-mediated assay has been developed for testing mutagenesis in Chinese hamster V79 cells by carcinogenic nitrosamines. Mutations were characterized by resistance to ouabain and 6-thioguanine. Since V79 cells do not metabolize nitrosamines, mutagenesis in the V79 cells was tested in the presence of primary hepatocytes capable of metabolizing nitrosamines. The hepatocytes were isolated after collagenase and hyaluronidase digestion of liver slices. All seven liver carcinogens of the nine tested nitrosamines exhibited a mutagenic response in this cell-mediated assay. The potent liver carcinogens nitrosodimethylamine, nitrosodiethylamine, nitrosoethylmethylamine, and nitrosodipropylamine could be detected with doses as low as 1 muM. The noncarcinogenic nitrosodiphenylamine was not mutagenic. Nitrosomethoxymethylamine was the only nitrosamine that exhibited mutagenic activity in the absence of hepatocytes, and this activity was diminished in the presence of hepatocytes. It is suggested that the use of hepatocytes prepared by the slicing method for carcinogen metabolism and mutable V79 cells offers a highly sensitive assay for determining the mutagenic potential of carcinogenic nitrosamines and probably of other classes of hazardous chemicals occurring in the environment.

Stimulation of differentiated functions in human melanoma cells by tumor-promoting agents and dimethyl sulfoxide.

Treatment of cultured human HO melanoma cells with the mouse skin tumor promoter phorbol-12-myristate-13-acetate (PMA) at 5 x 10(-10) to 5 x 10(-7) M resulted in a dose-related inhibition of growth and a stimulation of differentiated functions. These included melanin synthesis and formation of dendrite-like structures. Higher doses of phorbol dibutyrate, a less potent tumor promoter, were required to produce an effect comparable to that of PMA for dendrite induction. Phorbol and two other phorbol esters, which lack tumor-promoting activity, were either inactive or elicited a poor response. In addition to morphological changes, treatment with PMA altered glucosamine incorporation into membrane gangliosides. After PMA treatment, glucosamine incorporation increased 8- to 10-fold in the GM3 ganglioside and decreased 2-fold in the GM1 ganglioside, as compared to phorbol or untreated control. Inhibition of cell growth and stimulation of melanin synthesis were also observed after treatment of the HO cells with dimethyl sulfoxide. Unlike the tumor-promoting agents, dimethyl sulfoxide did not induce the formation of dendrite-like structures in the cells. These findings indicate that HO melanoma cells can be stimulated into terminally differentiated cells after treatment with tumor-promoting agents such as phorbol diesters.

Induction of differentiation and DNA strand breakage in human HL-60 and K-562 leukemia cells by genistein.

Genistein, an in vitro inhibitor of topoisomerase II and tyrosine kinases, suppressed growth and induced differentiation in HL-205 cells, a clonal population of the human promyelocytic HL-60 leukemia cells, and in K-562-J cells, a clonal population of the human erythroid K-562 leukemia cells. Maturing HL-205 cells acquired either granulocytic or monocytic markers, namely, reactivity with the murine OKM1 monoclonal antibody, expression of nitroblue tetrazolium dye reduction, and staining for nonspecific esterase. The maturing K-562-J cells stained with benzidine, which indicates the presence of hemoglobin, an erythroid maturation marker. Although the acquisition of the maturation markers in both HL-205 and K-562-J cells was time dependent up to 6 days, the kinetics of this induction differed between the two cell types. Despite the in vitro inhibitory effect of genistein, treatment of either HL-205 or K-562-J cells with 150 micrograms/ml genistein for up to 16 h did not alter topoisomerase II activity (as determined by the unknotting assay) in their nuclear extracts. Analysis with the anti-phosphotyrosine PY-20 murine monoclonal antibody indicated that treatment of K-562-J cells with genistein decreased the reactivity of the antibody with two of the cellular proteins. However, no reactivity with the PY-20 antibody was detected in untreated or genistein-treated HL-205 cells. An early event in the HL-205 and K-562-J cells, occurring after only 1 h of treatment with 30-200 micrograms/ml genistein, was the induction of DNA damage as measured by an alkaline elution assay. This damage may be a contributing factor in the genistein-induced cell differentiation in the HL-205 and K-562-J cells.

Protein kinase C activities with different characteristics, including substrate specificity, from two human HL-60 leukemia cell variants.

To evaluate the role of protein kinase C in the cellular maturation processes induced by phorbol diesters, we examined the biochemical activity of protein kinase C from HL-205, a cell variant from the human promyelocytic HL-60 leukemia that is susceptible to differentiation induced by phorbol 12-myristate 13-acetate, and from HL-525, an HL-60 variant that is resistant to such an induction. The activities of protein kinase C from the two cell types differed in their requirements for the cofactors Ca2+ and lipids. These enzyme activities also differed in their abilities to phosphorylate protamine and a series of four oligopeptides. We suggest that the differences in vitro in the activities of protein kinase C between HL-205 and HL-525 cells, especially in their substrate specificity, are closely related to the different phosphorylation patterns induced in vivo by phorbol 12-myristate 13-acetate in these cells. We also suggest that these differences may be responsible for the different susceptibilities of the two cell types to maturation induced by phorbol diesters.

Activation of the colon carcinogen 1,2-dimethylhydrazine in a rat colon cell-mediated mutagenesis assay.

Suspensions of rat colon epithelial cells metabolized the potent colon carcinogen, 1,2-[14C]dimethylhydrazine (DMH), into 14C-labeled, alkali-soluble volatile products, presumably CO2. The colon cell suspensions, however, were less effective than rat hepatocyte suspensions. In addition, we used a cell-mediated mutagenesis assay to test rat colon epithelial cells grown from tissue explants for their ability to metabolize DMH into products mutagenic for human P3 teratoma cells. Mutagenesis in the P3 cells was indicated by an acquired resistance to 6-thioguanine. Cocultivation of the colon cells with the P3 cells in the cell-mediated assay resulted in mutagenesis, whereas in the absence of the colon cells, no mutagenesis by DMH was observed. Similar results were obtained in a hepatocyte-mediated mutagenesis assay. Colon cells were also able to activate another carcinogen, benzo(a)pyrene, into products mutagenic for the P3 cells. Individual epithelial clonal populations isolated from the colon cultures grown from tissue explants, however, expressed different capacities to activate DMH and benzo(a)pyrene into mutagens, and a high degree of DMH activation by cells from a colon clone was not necessarily associated with a similar degree of benzo(a)pyrene activation. Our results indicate that the colon itself contains epithelial cell types capable of effectively converting DMH into mutagenic (and presumably carcinogenic) products without necessarily involving intermediary metabolism by hepatocytes as previously thought.

Alteration in glycosphingolipid pattern during phorbol-12-myristate-13-acetate-induced cell differentiation in human T-lymphoid leukemia cells.

We analyzed the patterns of glycosphingolipids (GSLs) from a line of cells derived from a clone of the human T-cell leukemia cells (CEM) that had been induced to differentiate by phorbol-12-myristate-13-acetate (PMA) into cells with a suppressor-like phenotype. We characterized the differentiation state of the cells by immunofluorescence by using anti-cell surface differentiation-specific monoclonal antibodies (OKT3, OKT4, OKT6, and OKT8). The GSLs were extracted and separated by thin-layer chromatography and the individual bands were quantitated by a dual-wavelength densitometer or by autoradiography of GSLs labeled with [14C]glucosamine and [14C]galactose. Treatment of the CEM cells with 0.16-16 nM PMA for 6 h to 6 days resulted in a dose- and time-dependent increase in the amount of two neutral GSLs [ceramide monohexoside and ceramide dihexoside] and three gangliosides [monosialoganglioside (GM3), sialosylparagloboside, and disialoganglioside (GD3)]. The increase in the neutral GSLs after PMA treatment reached its maximum at 30 h while GM3 peaked at 96 h. The increases in GM3 and sialosylparagloboside are presumably due to an increase in their synthesis levels because PMA promoted an elevated incorporation of glucosamine and galactose into these GSLs. The increase in the amount of GD3, on the other hand, is due to either a decrease in its degradation or use in other metabolic pathways because no detectable increase in glucosamine and galactose incorporation into this ganglioside could be found. Incubation of control or PMA-induced CEM cells with GM3 fractions purified from either CEM cells, human brain, or dog erythrocytes caused a reduction in cell growth and prevented the increase in reactivity of the induced cells with the OKT3 antibody. Incubation with semisynthetic ceramide dihexoside, however, prevented the decrease in reactivity with the OKT4 antibody. The observed changes in GSL patterns during PMA-induced differentiation of the CEM cells into suppressor-like cells and the inhibition of CEM cell growth by GM3 fractions suggest that the GSLs play a role in the control of cell growth and differentiation in the PMA-treated CEM cells.

Novobiocin- and phorbol-12-myristate-13-acetate-induced differentiation of human leukemia cells associated with a reduction in topoisomerase II activity.

Studies were conducted to determine the possible involvement of DNA topoisomerase II (Topo II) in the induction of differentiation in two human promyelocytic HL-60 leukemia cell variants that are either susceptible or resistant to differentiation induced by phorbol-12-myristate-13-acetate (PMA), a protein kinase C activator. The acquisition of maturation markers and changes in the activity, level, and phosphorylation of Topo II were determined after treatment with either novobiocin, a Topo II inhibitor, or PMA. Novobiocin at 50-150 microM induced differentiation in the HL-205 cells but not in the HL-525 cells, although both cell types were equally susceptible to novobiocin-evoked cytotoxicity. In both cell types, novobiocin induced similar reductions in topoisomerase I activity but different reductions in Topo II activity. Treatment with novobiocin at 150 microM for 6 h or at 2 mM for 30 min resulted in a 4-fold or higher reduction in Topo II activity in the differentiation-susceptible HL-205 cells but not in the differentiation-resistant HL-525 cells. A differential response in Topo II activity was also observed after treatment with PMA. The novobiocin-evoked decrease in Topo II activity seems to be due to an enhanced enzyme proteolysis, whereas the PMA-elicited decrease in Topo II activity is associated with an increase in Topo II phosphorylation. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine, which is an inhibitor of protein kinases, including protein kinase C, diminished the novobiocin-elicited proteolysis of Topo II and the PMA-induced Topo II phosphorylation, as well as the decrease in Topo II activity and the acquisition of differentiation markers induced by either novobiocin or PMA. These results suggest that induction of differentiation in HL-60 cells by novobiocin or PMA is associated with a reduction in Topo II activity, mediated directly or indirectly by a protein kinase(s), perhaps protein kinase C.

Alteration of the c-mos locus in "normal" tissues from humans exposed to radium.

The structure of a number of human protooncogenes of persons with internal systemic exposure to radium was analyzed by restriction enzyme digestion and Southern blotting of their DNA. Two extra c-mos EcoRI restriction fragment length bands of 5.0 and 5.5 kilobases were found in tissue DNA from six of seven such individuals. The extra c-mos bands were detected in DNA from many, but not all, of the tissues of the individuals exposed to radium. Kidney DNA, however, from three of four individuals exposed to radium contained these alterations; kidney DNA from six age-matched controls did not. The 5.0- and 5.5-kilobase bands, which were of a similar intensity, varied in their intensity with respect to that of the normal 2.5-kilobase band of the c-mos gene. The DNAs that have the polymorphic bands also appear to have a more complex c-mos methylation pattern. Our results suggest that the c-mos restriction fragment length alterations found in individuals exposed to radium were induced rather than inherited, are epigenetic in origin, and most likely result from changes in the methylation of bases surrounding the single exon of the c-mos protooncogene.

Increased inosine-5'-phosphate dehydrogenase gene expression in solid tumor tissues and tumor cell lines.

Inosine-5'-phosphate (IMP) dehydrogenase, a regulatory enzyme of guanine nucleotide biosynthesis, may play a role in cell proliferation and malignancy. To assess this role we examined IMP dehydrogenase expression in a series of human solid tumor tissues and tumor cell lines in comparison with their normal counterparts. Increased IMP dehydrogenase gene expression was observed in brain tumors relative to normal brain tissue and in sarcoma cells relative to normal fibroblasts. Similarly, in several B- and T-lymphoid leukemia cell lines, elevated levels of IMP dehydrogenase mRNA and cellular enzyme were observed in comparison with the levels in peripheral blood lymphocytes. These results are consistent with an association between increased IMP dehydrogenase expression and either enhanced cell proliferation or malignant transformation.

Double minute chromatin bodies and other chromosome alterations in human myeloid HL-60 leukemia cells susceptible or resistant to induction of differentiation by phorbol-12-myristate-13-acetate.

An analysis of the chromosomal karyotype of the human promyelocytic HL-60 leukemia cell line and of a number of its sublines that exhibit varying degrees of resistance to induction of differentiation by phorbol-12-myristate-13-acetate was conducted. The HL-60 cell line and the derived sublines contained two consistent marker chromosomes [9p- and t(10;13)], which suggested that they have a common and possibly clonal origin. HL-60 cells that are susceptible to phorbol-12-myristate-13-acetate-induced cell differentiation contained double minute chromatin bodies. The sublines with different degrees of resistance showed a corresponding sequential reduction of double minute chromatin bodies in metaphase cells. This loss of double minute chromatin bodies was not associated with an appearance of homogeneously staining chromosomal regions. Resistant and susceptible HL-60 cells differed also in a number of other chromosomal alterations, including gains or losses involving chromosomes 5, 8, 11, 13, 16, and 17. Thus, it is suggested that acquisition of resistance to phorbol-12-myristate-13-acetate-induced cell differentiation in the HL-60 cells may involve one or more of the above chromosomal changes.

Maintenance of adult rat hepatocytes on C3H/10T1/2 cells.

A procedure is described for maintaining primary cultures of adult rat hepatocytes on a layer of irradiated C3H/10T1/2 cells. These hepatocytes were capable of metabolizing the liver carcinogen N-2-acetylaminofluorene to water-soluble products and after 14 days in culture could still metabolize approximately 70% of the Day 1 level. Hepatocytes maintained on the C3H/10T1/2 cells were inducible for the liver-specific enzyme tyrosine aminotransferase, and exhibited approximately a 4-fold induction by hydrocortisone during a 10-day culture period. Morphologically, these hepatocytes retained many characteristics of hepatocytes in vivo. By contrast, hepatocytes maintained on plastic lost both N-2-acetylaminofluorene-metabolizing ability and tyrosine aminotransferase activity by Day 5. This was presumably due to degeneration of the hepatocytes and an overgrowth by fibroblasts. The maintenance of morphologically and biochemically functional hepatocytes in culture on feeder cells may provide a valuable approach for studying drug metabolism and liver cell transformation in vitro.

Alterations in lipid metabolism induced by 12-O-tetradecanoylphorbol-13-acetate in differentiating human myeloid leukemia cells.

Several aspects of lipid metabolism were evaluated in differentiating human myeloid leukemia (HL-60) cells after treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Modifications accompanying the phorbol ester-induced differentiation include an increase in the incorporation of acetate into free fatty acids and neutral lipids, an increase in the amount of neutral glycerolipids, and a selective incorporation of long-chain fatty alcohols into triacylglycerols and ether-linked alkyldiacylglycerols. Additionally, an enhanced stimulation of phospholipid metabolism, as measured by the incorporation of 32P and labeled precursors of the polar head groups, could be detected within 4 hr after treatment of cells with the tumor promoter. 4-O-Methyltetradecanoylphorbol-13-acetate, an analog with poor tumor-promoting activity, failed to elicit any activity on phospholipid metabolism.

Expression of maturation-specific nuclear antigens in differentiating human myeloid leukemia cells.

The expression of three myeloid-specific nuclear antigens was studied by indirect immunofluorescence with murine monoclonal antibodies in human myeloid (HL-60, ML-2, KG-1, and B-II) leukemia cells treated with chemical inducers of cell differentiation. Treatment of the promyelocytic HL-60 cells with dimethyl sulfoxide or 1,25-dihydroxyvitamin D3 induced the cells to acquire a phenotype that resembled that of granulocytes and monocytes-macrophages, respectively. These phenotypes were characterized by changes in cell growth, cell morphology, expression of specific cell surface antigens, and activities of lysozyme and nonspecific esterase enzymes. Induction of these differentiation markers in the HL-60 cells was associated with induction of the myeloid-specific nuclear antigens. The ML-2 cells, which are arrested at the myeloblast-promyelocyte stage, were also susceptible to the induction of cell differentiation and to changes in the expression of the nuclear antigens, but the degree of susceptibility was less than in the HL-60 cells. The less-differentiated KG-1 and B-II myeloid cells were either not responsive or responded only in a limited degree to the induction of cell differentiation or to changes in the expression of the nuclear antigens. We suggest that the reactivity of cells with monoclonal antibodies to specific nuclear antigens can be used as a maturational marker in cell differentiation studies. Furthermore, nuclear antigens expressed early in cellular differentiation may provide information about changes in regulatory elements in normal and malignant cells.

Tumor necrosis factor gene expression is mediated by protein kinase C following activation by ionizing radiation.

Tumor necrosis factor (TNF) production following X-irradiation has been implicated in the biological response to ionizing radiation. Protein kinase C (PKC) is suggested to participate in TNF transcriptional induction and X-ray-mediated gene expression. We therefore studied radiation-mediated TNF expression in HL-60 cells with diminished PKC activity produced by either pretreatment with protein kinase inhibitors or prolonged 12-O-tetradecanoylphorbol-13-acetate treatment. Both treatments resulted in attenuation of radiation-mediated TNF induction. Consistent with these results, we found no detectable induction of TNF expression following X-irradiation in the HL-60 variant deficient in PKC-mediated signal transduction. The rapid activation of PKC following gamma-irradiation was established using an in vitro assay measuring phosphorylation of a PKC specific substrate. A 4.5-fold increase in PKC activity occurred 15 to 30 s following irradiation, which declined to baseline at 60 s. Two-dimensional gel electrophoresis of phosphoproteins extracted from irradiated cells demonstrated in vivo phosphorylation of the PKC specific substrate Mr 80,000 protein at 45 s following X-irradiation. These findings indicate that signal transduction via the PKC pathway is required for the induction of TNF gene expression by ionizing radiation.

Ectopic expression of 2-5A synthetase in myeloid cells induces growth arrest and facilitates the appearance of a myeloid differentiation marker.

Two variants of the human myeloid leukemia cell line HL-60 were used to study the possible involvement of the IFN-induced protein 2-5A synthetase in cell growth arrest and differentiation. The two variants, HL-205 and HL-525, are equally susceptible to differentiation to the granulocyte lineage by exposure to DMSO, but only HL-205 cells acquire the macrophage phenotype following exposure to phorbol esters. The kinetics of 2-5A synthetase activity was established in both variants exposed to either DMSO or phorbol 12-myristate 13-acetate. With DMSO treatment, 2-5A synthetase activity was markedly induced in both variants, although with slightly different kinetics. With phorbol 12-myristate 13-acetate treatment, 2-5A enzymatic activity increased only in HL-205; no activity was detected up to 96 h after treatment in HL-525. The induction of 2-5A synthetase activity is apparently alpha/beta-IFN dependent, because only antibodies directed against a mixture of alpha- and beta-IFN completely abolished the increase in activity detected during differentiation of HL-205 cells. To directly establish the role of 2-5A synthetase in differentiation, HL-205 cells were transfected with an expression vector harboring the cDNA for the 43-kDa isoform of murine 2-5A synthetase fused to the inducible metallothionein promoter. Two clones, clone 6, which yielded a low level of 2-5A synthetase activity in response to ZnCl2 (which activates the promoter), and clone 7, which was a high responder, were further analyzed and compared with the control clone, neo. Reductions in the rates of cell growth and thymidine incorporation were observed with both clone 6 and clone 7 cells exposed to ZnCl2; clone 7 was more responsive. In addition, the level of c-myc-specific RNA transcript was greatly reduced in ZnCl2 or beta-IFN-treated clone 7 cells, whereas the neo cells responded similarly only after beta-IFN treatment. Treatment of clone-neo cells with beta-IFN resulted in conversion of pRb protein from the phosphorylated to the underphosphorylated form within 24 h; ZnCl2 had no effect, even after 72 h. In contrast, the accumulation of the underphosphorylated form of pRb was observed in clone 7 cells treated either with beta-IFN or ZnCl2. Finally, a significant increase in nitro blue tetrazolium-positive cells, an indication of differentiation, was evident with ZnCl2-treated clone 6 and clone 7 cells; no such increase was observed with clone-neo cells under similar conditions. We conclude that ectopic expression of 2-5A synthetase in HL-205 cells results in cell growth arrest and facilitates the appearance of a myeloid differentiation marker.

Control of macrophage cell differentiation in human promyelocytic HL-60 leukemia cells by 1,25-dihydroxyvitamin D3 and phorbol-12-myristate-13-acetate.

Human promyelocytic leukemia cells (HL-60) were induced to differentiate into macrophage-like cells in a dose (3 X 10(-10) to 10(-7) M) and time (1 to 6 days)-dependent manner by 1,25-dihydroxyvitamin D3 and the tumor promoter, phorbol-12-myristate-13-acetate. Differentiation was determined by an increase in the percentage of morphologically mature cells, in lysozyme and nonspecific esterase activities, and in reactivity with the murine OKM1 monoclonal antibody. Two HL-60 cell variants, designated as R-80 and B-II, were also examined. R-80 cells, which are resistant to induction of cell differentiation by phorbol-12-myristate-13-acetate, also exhibited resistance, although to a lesser degree, to induction of cell differentiation by 1,25-dihydroxyvitamin D3. The resistance to the action of the two compounds is presumably not due to similar binding sites for the two inducers, since 1,25-dihydroxyvitamin D3 was unable to compete for the phorbol diester binding sites as measured by [3H]phorbol-12,13-dibutyrate binding. B-II cells were resistant to induction of cell differentiation by 1,25-dihydroxyvitamin D3, phorbol-12-myristate-13-acetate, retinoic acid, and dimethyl sulfoxide. Two-dimensional electrophoretic analysis of HL-60 cell protein patterns indicated that treatment of the HL-60 cells with 1,25-dihydroxyvitamin D3, phorbol-12-myristate-13-acetate, retinoic acid, and dimethyl sulfoxide caused the cells to express various monocyte-macrophage and granulocyte marker proteins. None of the inducers caused a protein pattern identical to that of peripheral monocytes or granulocytes in the HL-60 cells, but the protein pattern of the HL-60 cells treated with 1,25-dihydroxyvitamin D3 was the closest to that of peripheral blood monocytes. These results indicate that 1,25-dihydroxyvitamin D3 induces in the HL-60 cells a phenotype that resembles, but is not identical to, that of peripheral monocytes-macrophages.

gamma-Glutamyl transpeptidase and malignant transformation of cultured liver cells.

The relationship between gamma-glutamyl transpeptidase (GGT)and malignant cell transformation was analyzed in malignant and nonmalignant culture epithelial cell lines derived from rat livers and fibroblastic cell types derived from hamsters and mice. GGT activity was prominent (25 to 90% of cells) in 3 of 5 malignant epithelial liver cell lines. None of the 9 fibroblastic or 4 nonmalignant epithelial cell lines exhibited GGT activity. Our results suggest that by use of GGT activity we can detect in cultured liver cells a significant fraction of the spontaneously or chemically induced malignant cells. Thus, in conjunction with other markers, this marker may help in identifying tumorigenic cells in liver epithelial cultures.