A simple and inexpensive method to control oxygen concentrations within physiological and neoplastic ranges.

Traditional methods for regulating oxygen concentration ([O2]) in in vitro experiments over the range found in normal and tumor tissues require the use of expensive equipment to generate controlled gas atmospheres or the purchase of a range of gas cylinders with certified O2 percentages. Here we describe a simple and inexpensive enzymatic method for generating low, precise steady-state [O2] levels that are stable for several hours. This method is particularly applicable to the in vitro study of some classes of hypoxia-targeted antitumor prodrugs and bioreductively activated agents.

Regulation of the differentiation of WEHI-3B D+ leukemia cells by granulocyte colony-stimulating factor receptor.

To investigate the role of the G-CSF receptor (G-CSFR) in mediating the action of G-CSF, WEHI-3B D+ murine myelomonocytic leukemia cells were transfected with a plasmid containing the murine G-CSFR gene. Overexpression of G-CSFR in transfected clones was demonstrated by northern blotting, binding of [125I]rhG-CSF and cross-linking experiments. A high level of expression of the G-CSFR did not promote or suppress cellular proliferation or initiate differentiation; however, exposure of transfected cells to G-CSF in suspension culture caused a large percentage of the population to enter a differentiation pathway, as determined by two markers of the mature state, the ability of cells to reduce nitroblue tetrazolium (NBT) and to express the differentiation antigen Mac-1 (CD11b) on the cell surface. Thus, upon treatment with 10 ng/ml of G-CSF, 60% or more of transfected cells exhibited NBT positivity; whereas, in contrast, nontransfected cells exhibited only 6% NBT positivity in response to G-CSF. An eightfold increase in Mac-1 expression over that of the parental line was also observed in transfected cells exposed to G-CSF. The growth rate of the transfected clones was decreased by exposure to G-CSF, presumably due to terminal differentiation. The findings suggest that the predominant function of G-CSF and its receptor in WEHI-3B D+ cells is to mediate differentiation and that the level of the G-CSFR portion of the signal transduction mechanism in this malignant cell line is important for a response to the maturation inducing function of the cytokine.

Production of membrane whorls in rat liver by some inhibitors of protein synthesis.

Inhibitors of protein synthesis capable of differential effects on nascent peptide synthesis on membrane-bound and free polyribosomes were employed to investigate the structure and function of cellular membranes of liver. The formation of membranous whorls in the cytoplasm and distension of nuclear membranes were induced by inhibitors of protein synthesis (i.e., cycloheximide and emetine) which predominantly interfere with nascent peptide synthesis on membrane-bound polyribosomes in situ. Other inhibitors of protein synthesis such as puromycin and fusidic acid, which inhibit nascent peptide synthesis on both free and membrane-bound polyribosomes, and chloramphenicol, which inhibits mitochondrial protein synthesis, did not induce these alterations. Cycloheximide, puromycin, and chloramphenicol produce some common cellular lesions as reflected by similar alterations in morphology, such as swelling of mitochondria, degranulation of rough endoplasmic reticulum, and aggregation of free ribosomes. The process of whorl formation in the cytoplasm, the incorporation of [(3)H]leucine and of [(3)H]choline into endoplasmic reticulum and the total NADPH-cytochrome c reductase activity of the endoplasmic reticulum were determined. During maximum formation of membranous whorls, [(3)H]leucine incorporation into cytoplasmic membranes was inhibited, while [(3)H]choline incorporation into these structures was increased; maximum inhibition of protein synthesis and stimulation of choline incorporation into endoplasmic reticulum, however, preceded whorl formation. Cycloheximide decreased the activity of NADPH-cytochrome c reductase of rough endoplasmic reticulum, but increased NADPH-cytochrome c reductase activity of smooth endoplasmic reticulum. In addition, cycloheximide decreased the content of hemoprotein in both the microsomal and mitochondrial fractions of rat liver, and the activities of mixed function oxidase and of oxidative phosphorylation were impaired to different degrees. Succinate-stimulated microsomal oxidation was also inhibited. The possible mechanisms involved in the formation of membranous whorls, as well as their functions, are discussed.

Modulation of the terminal differentiation of human squamous carcinoma cells in vitro by all-trans-retinoic acid.

A malignant human cell line (SqCC/Y1) derived from a squamous carcinoma of the buccal mucosa is described. It formed a stratified cellular structure with ultrastructural characteristics of a fully differentiated stratified squamous epithelium when cultured in equal parts of Dulbecco's modified Eagle medium and Ham's medium F12, supplemented only with insulin, transferrin, and selenium. After 14 days in culture in this defined medium, 30% of the cells became keratinized (insoluble in detergent), and 75% of the cells were capable of being induced to form cornified cell envelopes. Involucrin, the precursor protein of the cornified cell envelope, could be detected by immunofluorescence only in suprabasal cells. Treatment of SqCC/Y1 cultures with 5 X 10(-8) M all-trans-retinoic acid (RA) completely inhibited stratification and markedly increased cell desquamation. In the presence of RA, less than 10% of the cells became keratinized, and only 15-20% of the cells acquired envelope-forming competence. The fraction of colony-forming cells in RA-treated cultures was tenfold higher than in fully mature cultures. Thus RA appears to be an effective inhibitor of terminal differentiation of SqCC/Y1 cells.

Interference with tumor cell-induced degradation of endothelial matrix on the antimetastatic action of nafazatrom.

The antithrombotic compound nafazatrom was evaluated in several in vivo and in vitro assays to elucidate the mechanism of its antimetastatic activity. C57BL/6 mice bearing B16 amelanotic subcutaneous tumors treated with 100 mg nafazatrom/kg/day exhibited a sixfold reduction in metastatic pulmonary lesions compared to lesion numbers in controls. The reduction in metastatic lesions was not accompanied by changes in primary tumor growth, and up to 1 microgram nafazatrom/ml did not inhibit tumor cell proliferation in vitro. Treatment of C57BL/6 mice with nafazatrom prior to iv inoculation of tumor cells failed to inhibit lung colony formation. In vitro exposure of exponentially growing B16 amelanotic cells to nafazatrom (1 microgram/ml for 72 hr) in culture did not change their ability to adhere to endothelial cell monolayers. B16 amelanotic cells degraded the matrix material of bovine endothelial cell monolayers; a heparin sulfate proteoglycan appeared to be the predominant matrix component released by these tumor cells, as judged by resistance to chondroitin ABC lyase and sensitivity to heparitinase and pronase degradation. Nafazatrom (1 microgram/ml for 72 hr) inhibited the solubilization of matrix components by approximately 60%. Tumor cell degradation of matrix components is an important event in the pathogenesis of metastasis. Thus the interference with this process appears to provide an explanation for the inhibition of malignant cell dissemination in vivo by nafazatrom.

Distribution of porfiromycin in EMT6 solid tumors and normal tissues of BALB/c mice.

The distribution of porfiromycin was studied in BALB/c mice bearing EMT6 mammary tumors. The levels of 3H in blood and most tissues peaked approximately 15 minutes after intraperitoneal injection of [3H]porfiromycin. The levels of radioactivity present in most of the tissues and in the tumors were similar at 4 hours and 24 hours after administration. Most of the normal tissues showed uniform, low grain densities when analyzed by autoradiography; the liver and the small intestine had the highest labeling densities. Only kidney, bladder, and tumor showed differential distributions of grains from [3H]porfiromycin. In the kidney, higher grain counts were found in cortex than in medullary regions; grains were uniformly distributed within each region. In the bladder, the highest labeling densities were found in regions near the lumen. Tumor regions that had some necrotic features or regions of necrosis that included some viable cells showed higher labeling intensities than healthy-looking tumor regions, probably because the abnormal microenvironments in these regions led to increased rates of activation of porfiromycin to electrophilic species. These findings show that porfiromycin can reach and be activated in tumor regions containing cells resistant to many chemotherapeutic agents and to x rays. The results also support the concept that agents such as porfiromycin can target cells in specific microenvironmental subpopulations of solid tumors.

Glycosaminoglycan production and distribution in cloned B16 murine melanoma cell lines exhibiting different lung colony-forming efficiencies.

Three cloned B16 murine melanoma cell lines were characterized with respect to their ability to a) detach from a plastic tissue culture substratum, b) form experimental pulmonary metastases, and c) produce and process glycosaminoglycans (GAGS). All three of the cell lines formed pulmonary metastases to different extents. Chondroitin sulfate and heparin-heparan sulfate were the major GAGS produced by all of the clones. Although the major compositional analyses of the cell lines were similar, some differences were apparent. The clone never selected for lung colony-forming efficiency and with the lowest metastatic potential (B16YL1) exhibited a reduced cell-associated glycosaminoglycan (GAG) matrix, shed the highest proportion of labeled GAGS into the medium, and was the most easily removed from the tissue culture substratum. In contrast, the other two cell lines (B16YM1 and B16YH1), derived from cultures selected for lung colony-forming efficiency, exhibited GAG-enriched cellular coats and were more resistant to EDTA-induced detachment from the plastic substratum. Both the B16YL1 and B16YM1 clones, which exhibited the lowest lung colony-forming efficiencies, shed an unsulfated chondroitin species into the extracellular fraction that was not evident in the most metastatic clone B16YH1. Despite these differences, cellular GAG matrix development and detachment from the plastic substratum showed only a partial correlation with lung colony-forming efficiency, presumably expressing the complexity of this biological phenomenon. Nonetheless, the findings suggest that GAGS may play an important role at several steps along the cascade of events leading to the successful dissemination of malignant cells.

Therapeutic attack of hypoxic cells of solid tumors: presidential address.

Hypoxic cells of solid tumors are relatively resistant to therapeutic assault. Studies have demonstrated that oxygen-deficient tumor cells exist in an environment conducive to reductive reactions making hypoxic cells particularly sensitive to bioreductive alkylating agents. Mitomycin C, the prototype bioreductive alkylating agent available for clinical use, is capable of preferentially killing oxygen-deficient cells both in vitro and in vivo. This phenomenon is at least in part the result of differences in the uptake and metabolism of mitomycin C by hypoxic and oxygenated tumor cells, with the ultimate critical lesion being the cross-linking of DNA by the mitomycin antibiotic. The combination of mitomycin C with X-irradiation, to attack hypoxic and oxygenated tumor cell populations, respectively, has led to enhanced antitumor effects in mice bearing solid tumor implants and in patients with cancer of the head and neck. More efficacious kill of hypoxic tumor cells may be possible by the use of dicoumarol in combination with mitomycin or by the use of the related antibiotic porfiromycin. The findings support the use of an agent with specificity for hypoxic tumor cells in potentially curative regimens for solid tumors.

Regulation by epidermal growth factor of human squamous cell carcinoma plasminogen activator-mediated proteolysis of extracellular matrix.

The interaction of epidermal growth factor (EGF) with specific cell surface receptors initiates biochemical events in target cells which result in cellular proliferation and differentiation. In this report the regulation of extracellular-associated plasminogen activator (PA) production by EGF in human squamous cell carcinomas and its influence on tumor cell-mediated degradation of extracellular matrix (ECM) is described. The studies utilized the vulvar carcinoma cell line A431, which possesses an unusually large number of EGF receptors (EGF-Rs), and two A431 EGF-R expression variants (A5 and A7), which contain up to 20-fold fewer cell surface EGF-Rs. EGF enhanced the production of urokinase (u) PA activity by two- to threefold in A431 tumor cells, in a concentration-dependent manner, following a 24-h treatment, as determined by substrate hydrolysis assays, while no changes in tissue-type PA occurred. In contrast, A5 and A7 tumor cells failed to demonstrate such a response. Time course studies of the EGF-mediated induction of uPA activity in A431 tumor cells indicated that within 8 h after exposure to EGF, a twofold increase above basal untreated control levels was observed using the substrate hydrolysis assay. EGF increased the steady state levels of uPA mRNA threefold in A431 tumor cells following a 24-h treatment, while in contrast, no such response was observed in EGF-R variant tumor cells. In accord with an EGF enhancement of uPA mRNA levels in A431 tumor cells, a similar increase of two- to threefold in the de novo synthesis of [35S]methionine-radiolabeled uPA was observed by immunoprecipitation following EGF treatment, while no measurable increase was observed in the EGF-R tumor variants. A431 tumor cells progressively degraded [3H]glucosamine-radiolabeled bovine corneal subendothelial ECM in the presence of EGF, resulting in 8.7-, 4.3-, and 1.7-fold increases above untreated control values, after a 48-h exposure to 100, 10, and 1 ng/ml of EGF, respectively. In contrast, A5 and A7 tumor cells did not demonstrate an increase in ECM degradation in the presence of EGF, even though these tumor cells possessed the ability to degrade ECM in the absence of the growth factor. The observed increase in ECM degradation mediated by EGF in A431 tumor cells was dependent upon the presence of plasminogen and could be inhibited by an anticatalytic uPA monoclonal antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytotoxicity and DNA lesions produced by mitomycin C and porfiromycin in hypoxic and aerobic EMT6 and Chinese hamster ovary cells.

Solid neoplasms may contain deficient or poorly functional vascular beds, a property that leads to the formation of hypoxic tumor cells, which form a therapeutically resistant cell population within the tumor that is difficult to eradicate by ionizing irradiation and most existing chemotherapeutic agents. As an approach to the therapeutic attack of hypoxic cells, we have measured the cytotoxicity and DNA lesions produced by the bioreductive alkylating agents mitomycin C and porfiromycin, two structurally similar antibiotics, in oxygen-deficient and aerobic cells. Mitomycin C and porfiromycin were preferentially cytotoxic to hypoxic EMT6 cells in culture, with porfiromycin producing a greater differential kill of hypoxic EMT6 cells relative to their oxygenated counterparts than did mitomycin C. Chinese hamster ovary cells were more resistant to these quinone antibiotics; although in this cell line, porfiromycin was significantly more cytotoxic to hypoxic cells than to aerobic cells, and the degree of oxygenation did not affect the toxicity of mitomycin C. Alkaline elution methodology was utilized to study the formation of DNA single-strand breaks and DNA interstrand cross-links produced by mitomycin C and porfiromycin in both EMT6 and Chinese hamster ovary cells. A negligible quantity of DNA single-strand breaks and DNA interstrand cross-links were produced in hypoxic and aerobic Chinese hamster ovary cells by exposure to mitomycin C or porfiromycin, a finding consistent with the considerably lower sensitivity of this cell line to these agents. In EMT6 tumor cells, no single-strand breaks appeared to be produced by these antitumor antibiotics under both hypoxic and aerobic conditions; however, a significant number of DNA interstrand cross-links were formed in this cell line following drug treatment, with substantially more DNA interstrand cross-linking being produced under hypoxic conditions. Mitomycin C and porfiromycin caused the same amount of cross-linking under conditions of oxygen deficiency; however, mitomycin C produced considerably more DNA cross-linking than did porfiromycin in oxygenated cells. DNA interstrand cross-links were observed in hypoxic EMT6 cells throughout a 24-h period following removal of mitomycin C and porfiromycin, with a decrease in DNA interstrand cross-links observed at 24 h. An increase in DNA interstrand cross-links occurred in aerobic EMT6 cells treated with mitomycin C and porfiromycin at 6 h after drug removal, with a decrease in these lesions being observed by 24 h, suggesting that the rate of formation of the cross-links may be slower and the removal of cross-links more rapid under aerobic conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of growth and differentiation of human keratinocytes by type beta transforming growth factor and epidermal growth factor.

The role of type beta transforming growth factor (TGF beta) and epidermal growth factor (EGF) as regulators of the growth and differentiation of cultured human neonatal epidermal cells and squamous carcinoma cells was investigated in postconfluent cultures. Neither cell proliferation nor DNA synthesis was affected by treatment with TGF beta alone; however, EGF significantly stimulated cell growth, and this process was specifically antagonized by TGF beta. In addition, TGF beta inhibited the maturation of human foreskin-derived epidermal cells, as measured by their competence to synthesize involucrin and to form cornified cell envelopes, in a dose-dependent manner. Although treatment with EGF did not affect the maturation of human foreskin-derived epidermal cells, the combination of a low concentration of TGF beta with EGF resulted in significant enhancement of the maturation of these normal keratinocytes. Growth of three of four squamous carcinomas in the presence of EGF was not inhibited by TGF beta. In addition, all four carcinomas were either totally or partially resistant to the induction of maturation by the combination of TGF beta and EGF. This resistance of squamous carcinomas to TGF beta was paralleled by an increased sensitivity to the antikeratinizing effects of EGF. Thus, TGF beta inhibited the mitogenic stimulation of keratinocytes by EGF and induces cell maturation.

Inhibition of the synthesis of glycoproteins and induction of the differentiation of HL-60 promyelocytic leukemia cells by 6-methylmercaptopurine ribonucleoside.

HL-60 promyelocytic leukemia cells were induced to differentiate along the granulocytic pathway by exposure to 6-methylmercaptopurine ribonucleoside (6-MMPR). The interference with cellular replication and the induction of terminal maturation by 6-MMPR appeared to be a consequence of the inhibition of purine nucleotide biosynthesis de novo, since the simultaneous exposure of HL-60 cells to 6-MMPR and adenine completely prevented cellular differentiation, as measured by both nitro-blue tetrazolium reduction and the phagocytosis of latex beads, and partially prevented growth inhibition. The induction of HL-60 leukemia cell maturation by 6-MMPR was preceded by a marked reduction in the incorporation of [3H]mannose into glycoproteins and into the dolichol-oligosaccharide precursors of N-linked glycoprotein biosynthesis. Simultaneous exposure of HL-60 cells to 6-MMPR and adenine completely prevented the reduction in [3H]mannose incorporation into glycoproteins produced by the purine nucleoside antimetabolite. These findings suggest that the utilization of mannose for glycoprotein biosynthesis may be a component of the mechanism by which 6-MMPR causes the induction of the terminal differentiation of HL-60 leukemia cells.

Reductive activation of mitomycin C by NADH:cytochrome b5 reductase.

Mitomycin C requires bioreduction in order to exert its cytotoxic action. Activation of mitomycin C to an electrophile was equally supported by NADPH and NADH in EMT6 tumor cell sonicates under hypoxia. No alkylation was observed under aerobic conditions. Purified NADH:cytochrome b5 reductase catalyzed the reduction of mitomycin C with a Km of 23 microM at pH 6.6. At pH 7.6, the rate of enzymatic reduction of mitomycin C was 61% of that at pH 6.6. NADH:cytochrome b5 reductase catalyzed the activation of mitomycin C to alkylating metabolites under both hypoxic and aerobic conditions, with alkylation being 1.5 times greater in hypoxia. Dicumarol at 100 microM inhibited the NADH:cytochrome b5 reductase-catalyzed reduction of mitomycin C by 24% and by 57% at 300 microM. The degree of inhibition of the enzyme by dicumarol was the same at both pH 6.6 and 7.6. NADH:cytochrome b5 reductase exhibited a small but measurable NADH-oxidase activity, which was unaffected by 300 microM dicumarol. These findings demonstrate that (a) NADH:cytochrome b5 reductase can metabolically activate mitomycin C and (b) dicumarol is capable of inhibiting this enzymatic activity.

Biochemical characterization of tyrosine kinase and phosphotyrosine phosphatase activities of HL-60 leukemia cells.

The cellular phosphotyrosine content of the HL-60 promyelocytic leukemia markedly decreased during the induced granulocytic and monocytic maturation of these cells. This occurs in the face of major increases in tyrosine kinase and protein phosphotyrosine phosphatase activities (D. A. Frank and A. C. Sartorelli, Biochem. Biophys. Res. Commun., 140: 440-447, 1986). In the present work, these two activities were characterized in the particulate fraction of HL-60 cells, since both enzymes are membrane bound. The tyrosine kinase activity utilized ATP as a phosphate donor, although GTP and other nucleotides were competitive with ATP. The enzyme was temperature sensitive, had a pH optimum of 6.5, and required Mg2+ or Mn2+ for activity, with additional stimulation of activity being produced by Zn2+. Agents such as epidermal growth factor and insulin, which stimulate other tyrosine kinase enzymes, were without effect on the tyrosine kinase activity of HL-60 cells. Enzyme activity was stimulated, however, by non-ionic detergents and was inhibited by quercetin. The protein phosphotyrosine phosphatase activity was paralleled by that of p-nitrophenyl phosphatase, was inhibited by VO3-4, Zn2+ and F-, and was maximally active at a pH of 7 to 8. The characteristics of the tyrosine kinase and the protein phosphotyrosine phosphatase activities were distinct from those of other known proteins of these classes. Tyrosine kinase activity was predominantly located on the plasma membrane, while the protein phosphotyrosine phosphatase activity was concentrated on internal membranes. The activities of both enzymes present on the plasma membrane appeared to exist on the cytoplasmic face of this membrane. Further characterization of the activities of these enzyme systems and their contribution to the regulation of tyrosine phosphorylation would appear to be important to an understanding of the control of cellular proliferation and differentiation.

Alterations in tyrosine phosphorylation during the granulocytic maturation of HL-60 leukemia cells.

Granulocytic maturation of HL-60 promyelocytic leukemia cells induced by dimethylsulfoxide has been shown to produce a decrease in cellular protein phosphotyrosine residues and increases in both tyrosine kinase and protein phosphotyrosine phosphatase activities (D. A. Frank and A. C. Sartorelli, Biochem. Biophys. Res. Commun., 140: 440-447, 1986). These changes have been shown to not be restricted to dimethylsulfoxide-induced differentiation, since similar changes occur in HL-60 cells initiated with retinoic acid and in HL-60 sublines resistant to dimethylsulfoxide-induced differentiation treated with the retinoid. These regulatory events are not directly coupled to growth arrest, which accompanies terminal maturation, since the anthracycline antibiotics aclacinomycin A and marcellomycin, which induce HL-60 differentiation, cause these changes in phosphotyrosine metabolism, while Adriamycin, at a level which produces an equivalent degree of growth inhibition but does not initiate the maturation of HL-60 cells, does not. Furthermore, an HL-60 subline deficient in hypoxanthine-guanine phosphoribosyltransferase, which differentiates in the presence of 6-thioguanine, produced a decrease in phosphotyrosine residues and increases in tyrosine kinase and phosphotyrosine phosphatase activities in response to the purine antimetabolite, while the parental HL-60 line, in which 6-thioguanine inhibits cellular proliferation but does not induce maturation, does not exhibit these changes. Finally, similar alterations in phosphotyrosine regulation were exhibited during anthracycline-induced differentiation of the murine myelomonocytic leukemia cell line WEHI-3B D+, supporting the concept that the phenomena measured represent a general response to inducers of the granulocytic differentiation of leukemia cells.

Dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells in the absence of cytokinesis.

Friend erythroleukemia cells grown in culture and induced to differentiate along the erythroid developmental pathway by dimethyl sulfoxide (DMSO) were used as a model system to investigate the requirement for cellular replication to express a differentiated erythroid phenotype. That cytokinesis is not essential for DMSO-induced erythroid differentiation as measured by the synthesis and accumulation of hemoglobin was shown by experiments using cytochalasin B. In these studies, hemoglobin was found to accumulate in Friend cells treated simultaneously with DMSO and cytochalasin B; such treatment caused cells to become enlarged and multinucleated due to inhibition of cytokinesis by cytochalasin B. In contrast, exposure of cells to cytochalasin B for at least 48 hr prior to DMSO caused significant inhibition of erythroid differentiation. The findings support the concept that cellular division and, thereby the production of new cellular types are not required for gene activation and the expression of an erythroid phenotype. These effects of cytochalasin B on DMSO-induced differentiation of Friend leukemia cells also suggest plasma membrane-cytoskeleton involvement in the initiation of the erythroid maturation process in this system.

Effects of 6-thioguanine on macromolecular events in regenerating rat liver.

Regenerating rat liver was used as a semisynchronous system in which to investigate the effects of 6-thioguanine on biochemical processes occurring in discrete phases of the cell cycle. 6-Thioguanine inhibited the first wave of DNA biosynthesis in regenerating rat liver. This effect appeared to be the result of a decrease, caused by 6-thioguanine, in the induction of several enzyme activities (i.e., thymidine kinase, deoxycytidylate deaminase, cytidine diphosphate reductase, and DNA polymerase) necessary for the initiation of DNA replication in regenerating liver. There was a fairly short period during which 6-thioguanine could be given to rats to accomplish the inhibition of the appearance of the induced activities of these enzymes; this period corresponded to the time just before enzyme induction. The inhibition of the induced synthesis of this group of enzymes occurred in the presence of an intact translational apparatus and intact polysomes and in the absence of interference with the incorporation of radioactive leucine and tyrosine into total protein of liver. Synthesis of polyadenylate-containing RNA was depressed in 6-thioguanine-treated rats, whereas the synthesis of polyadenylate-lacking RNA was unaffected. It is suggested that the inhibition of the synthesis of polyadenylate-containing RNA by 6-thioguanine is at least in part responsible for the observed decrease in induced enzyme activities and the resulting interference with DNA replication.

Inhibition of glycoprotein biosynthesis by the inducers of HL-60 cell differentiation, aclacinomycin A and marcellomycin.

The trisaccharide-containing anthracyclines aclacinomycin A (ACM) and marcellomycin (MCM) have been shown by our laboratory to be potent inducers of HL-60 leukemia cell maturation, while the monosaccharide-containing anthracyclines Adriamycin and pyrromycin were inactive and significantly less potent, respectively, as initiators of the differentiation of these malignant cells. We have now observed that ACM and MCM are potent inhibitors of both total glycoprotein synthesis and the formation of lipid-linked oligosaccharide intermediates in intact HL-60 cells. This inhibitory activity was both concentration and time dependent and was maximal after 12 hr exposure to 30 nM ACM or MCM, conditions under which both cell growth and total protein synthesis were maintained at levels equal to those of untreated cells. In contrast, exposure of HL-60 cells to pyrromycin or Adriamycin, even at cytotoxic concentrations, did not result in specific decreases in the synthesis of glycoproteins containing asparagine-linked oligosaccharides. Maximum inhibition of the formation of lipid-linked intermediates by ACM or MCM preceded the loss of cell surface transferrin-binding activity (maximal at 24 hr), which in turn preceded the minimal exposure time necessary for significant induction of differentiation by either of the active anthracyclines (36 hr). These findings demonstrate a new biochemical site of action for ACM and MCM and suggest that this activity is involved in the induction of terminal differentiation of HL-60 leukemia cells by these antitumor agents.

Structure-activity relationships for the induction of differentiation of HL-60 human acute promyelocytic leukemia cells by anthracyclines.

Marcellomycin, a newly developed anthracycline antibiotic with antineoplastic activity, was tested as an inducer of differentiation of the human promyelocytic leukemia cell line HL-60 in vitro. The percentage of cells reducing nitro blue tetrazolium, an indication of a stimulus-induced respiratory burst typical of mature phagocytes, was used as a functional measure of the extent of differentiation. Marcellomycin was a potent inducer of maturation, with 95% of the cells expressing a terminally differentiated state after 10 days of exposure to a concentration of 40 nM anthracycline. Cells exposed to marcellomycin exhibited a 35-fold increase in their total superoxide anion-generating capacity, an 80% increase in acid phosphatase activity, and a loss of myeloperoxidase and chloroacetate esterase activities. In addition, marcellomycin-treated cells stained negatively for alpha-naphthyl acetate esterase. These findings provided evidence for the granulocytic nature of the mature cells. In contrast, marcellomycin was not an effective inducer of differentiation of Friend murine erythroleukemia cells. Studies on the relationship between structure and the ability to induce differentiation of HL-60 leukemia cells demonstrated that removal from the marcellomycin molecule of the terminal 2-deoxyfucose (musettamycin) or its substitution by cinerulose (aclacinomycin A) did not alter differentiation-inducing capacity. However, removal of the carbomethoxy group from the C-10 position of marcellomycin substantially reduced its potency as an initiator of maturation, and removal of the two terminal 2-deoxyfucose moieties (pyrromycin) decreased both potency and the maximal percentage of differentiated cells produced in the population. The monosaccharide anthracyclines Adriamycin and carminomycin were completely inactive as inducers of HL-60 leukemia cell maturation. The results suggest that certain anthracyclines would be reasonable candidate drugs to use in a clinical trial aimed at reducing the leukemic stem cell burden through maturation rather than through cytodestruction.

Biochemical and cytotoxic actions of 3,6-dihydroxy-4,5-dimethylphthalaldehyde in sarcoma 180 cells.

The replication of Sarcoma 180 cells in culture was inhibited by 3,6-dihydroxy-4,5-dimethylphthalaldehyde (HMPA). The inhibition of growth caused by HMPA was evident after treatment of cells with drug for only 15 min. This exposure period caused decreased in (a) cloning efficiency, (b) transport and/or phosphorylation of [3H]thymidine and [3H]uridine, (c) incorporation of radioactive nucleosides into acid-insoluble material, and (d) incorporation of [3H]leucine into protein. Examination of the cytotoxicities of the model compounds 2,3,5,6-tetramethyl-1,4-dihydroquinone (durohydroquinone) and o-phthalaldehyde indicated that the dialdehyde portion of the molecule was responsible for the cytocidal effects of HMPA. The ratio of adenosine triphosphate to adenosine diphosphate in the acid-soluble fraction of Sarcoma 180 cells incubated in vitro with HMPA for 45 min was reduced in a concentration-dependent manner. The reduction in the ATP pool size produced by HMPA contrasts with the action of the periodate oxidation product of cytidine dialdehyde, which has been reported to increase the intracellular concentration of adenosine triphosphate.