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Biomedical subjects

E Huberman

Publications and source records attributed to E Huberman.

At least 73 records · Page 4Linked to original sources
PubMed1990

Induction of cell differentiation in melanoma cells by inhibitors of IMP dehydrogenase: altered patterns of IMP dehydrogenase expression and activity.

To study the induction of differentiation in human melanoma cells, we treated 12 melanoma cell lines with mycophenolic acid and tiazofurin, inhibitors of IMP dehydrogenase (IMPDH). In all cell lines studied, both agents inhibited cell growth and increased melanin content. However, the degree of growth inhibition did not necessarily correspond to the increase in melanin content. A detailed analysis of the HO and SK-MEL-131 cell lines indicated that mycophenolic acid and tiazofurin caused a time- and dose-dependent increase in the expression of a series of other maturation markers, including formation of dendrite-like structures, tyrosinase activity, and reactivity with the CF21 monoclonal antibody. The growth inhibition and melanogenesis induced by the IMPDH inhibitors was abrogated by the addition of exogenous guanosine. No such effect was observed after treatment of the cells with phorbol 12-myristate 13-acetate or retinoic acid, two other inducers of differentiation in these cells. The mycophenolic acid- and tiazofurin-treated cells also showed an increased level of IMPDH mRNA and protein, perhaps because of compensation for the inhibitor-mediated decrease in IMPDH activity. In contrast, treatment with phorbol 12-myristate 13-acetate or retinoic acid resulted in decreased levels of IMPDH mRNA and protein. The lack of a consistent pattern of IMPDH expression in the cells treated with IMPDH inhibitors and phorbol 12-myristate 13-acetate or retinoic acid suggests that the altered expression of IMPDH is not a general requirement for the induction of cell differentiation in these cells. Our results also suggest that IMPDH inhibitors may provide a useful approach to circumvent the differentiation block in melanoma.

Biomarkers↗
PubMed1990

A protein complex expressed during terminal differentiation of monomyelocytic cells is an inhibitor of cell growth.

A protein complex (PC) composed of the MRP8 and MRP14 proteins has previously been shown to be a specific inhibitor of casein kinase I and II. This PC is expressed during the late stages of terminal differentiation induced in human promyelocytic HL-60 leukemia cells by 1 alpha,25-dihydroxyvitamin D3 and in human monocytic THP-1 leukemia cells by phorbol 12-myristate 13-acetate. This expression is associated with terminal cell differentiation because incubation of HL-60 cells with an agent or condition that causes suppression of growth but not induction of differentiation does not result in expression of the PC. At concentrations of 5-15 nM, the purified PC inhibited the growth of HL-60 cells and THP-1 cells, as well as other cell types belonging to different cell lineages. This growth inhibition was preceded by a reduction in [32P]phosphate incorporation and, at the higher PC concentrations, was associated with a reduction in [3H]thymidine, [3H]uridine, and [32S]methionine incorporation. The specific expression pattern and growth-inhibitory character of the PC suggests that the complex may have a role in suppressing cell growth during monomyelocytic terminal differentiation induced by specific chemical stimuli and during physiological and pathological events associated with monomyelocytic cell functions.

Calcium-Binding Proteins↗
PubMed1989

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.

Aged↗
PubMed1989

A protein containing the cystic fibrosis antigen is an inhibitor of protein kinases.

To investigate myeloid cell maturation, we established a panel of monoclonal antibodies that recognize myeloid cell nuclear antigens. One of these monoclonal antibodies was used to purify a specific protein complex (PC) from a human spleen. This PC, which is present at high levels in peripheral blood monocytes and granulocytes, contains a protein that is the cystic fibrosis (CF) antigen. The purified PC was shown to inhibit the activity of casein kinase I and II but not cAMP-dependent protein kinase, protein kinase C, v-abl tyrosine kinase, or insulin receptor tyrosine kinase. The observed Ki values for casein kinases I and II purified from several sources were 1 microM or less. Furthermore, the addition of the purified PC to a nuclear extract from human cells was able to prevent protein kinase-mediated stimulation of RNA polymerase activity. The unique inhibitory character of the PC and its elevated levels in monocytes and granulocytes and of the CF antigen in CF patients implies that this complex may be associated with myeloid cell functions and perhaps with the cause or consequence of the clinical manifestations of CF.

Amino Acid Sequence↗
PubMed1989

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.

Amsacrine↗
PubMed1988

Cloning and sequence analysis of the human and Chinese hamster inosine-5'-monophosphate dehydrogenase cDNAs.

Inosine-5'-monophosphate dehydrogenase, a key enzyme in the regulation of guanine nucleotide biosynthesis, was purified to homogeneity; and a polyclonal antibody directed against the purified protein was used to isolate human and Chinese hamster IMP dehydrogenase cDNA clones. These clones were sequenced and found to contain an open reading frame of a protein containing 514 amino acids. A sequence of 35 amino acids obtained by analysis of the purified protein is identical to a segment of the protein sequence deduced from the IMP dehydrogenase cDNA. The molecular mass of the deduced protein is 56 kDa, which is the observed molecular mass of the purified protein and of the immunoprecipitated in vitro translation product. Comparison of the protein sequences deduced from the human and Chinese hamster cDNA clones indicates only eight amino acid differences, suggesting that IMP dehydrogenase is a highly conserved protein.

Amino Acid Sequence↗
PubMed1988

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.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

Myeloperoxidase: a myeloid cell nuclear antigen with DNA-binding properties.

An antigen histochemically localized in the nuclei and cytoplasmic granules of normal and leukemic human myeloid cells has been identified as myeloperoxidase (MPO; EC 1.11.1.7). The localization and amount of the enzyme was determined by using a murine monoclonal antibody designated H-43-5 raised against nuclear proteins derived from human promyelocytic HL-60 leukemia cells. The highest amount of nuclear MPO (3.5 micrograms per 10(6) nuclei) was found in granulocytes; less than half of this amount was detected in nuclei from HL-60 cells. Still lower levels were found in nuclei from monocytes and a series of human monomyelocytic leukemia cells. MPO from HL-60 cells was purified by immunoaffinity chromatography and fractionated into three components (forms I, II, and III) by CM-cellulose chromatography. Chromatography of these MPO forms on DNA-Sepharose columns confirmed that all three forms of MPO were tightly bound to DNA with apparent relative affinities in the order of form III greater than form II greater than form I. The affinity of MPO form III for DNA was sufficient to enable the formation and elution of DNA-MPO complexes during size-exclusion chromatography at high ionic strength and neutral pH. This form of MPO was also able to shield DNA from strand scission induced by active oxygen species generated by xanthine oxidase acting aerobically on xanthine. These data suggest that intranuclear MPO may help to protect DNA against damage resulting from oxygen radicals produced during myeloid cell maturation and function.

Antibodies, Monoclonal↗
PubMed1988

Sodium fluoride promotes morphological transformation of Syrian hamster embryo cells.

Sequential treatment of Syrian hamster embryo (SHE) cells with a chemical carcinogen followed by sodium fluoride (NaF) resulted in a higher yield of morphologically transformed cell colonies than treatment of the cells with carcinogen alone. For example, cells treated with benzo[a]pyrene (B[a]P; 3 micrograms/ml) for 3 days, then with NaF (25 micrograms/ml) for 4 days, exhibited a transformation frequency more than six times greater than that obtained by summing the transformation frequencies from cells treated with either B[a]P or NaF alone. This enhancement/promotion of cell transformation by NaF was only expressed after the cells had been pretreated with either direct-acting carcinogens or procarcinogens. Pretreatment of the cells with noncarcinogens or weakly-acting carcinogens or administration of NaF prior to treatment with the carcinogen failed to enhance the yield of transformation. Transformation was enhanced even when the NaF treatment was delayed for several days after the carcinogen treatment. However, the continued presence of NaF was necessary for maintenance of the increased level of transformation. Removal of NaF prior to termination of the assay resulted in a reversal of the transformed clonal morphologies to a normal phenotype such that the final yield of transformants was decreased, but was still greater than that observed after carcinogen treatment alone. A similar pattern for reversibility of the transformation enhancement also occurs for the widely recognized tumor promoter phorbol 12-myristate 13-acetate (PMA). Seven different SHE cell pools were tested for sensitivity to NaF promotion following carcinogen treatment. Although the response was heterogeneous, no carcinogen-treated cell pool was refractory to the NaF-induced enhancement. A second fluorocompound, sodium monofluorophosphate (NaMFP), was also found to enhance carcinogen-induced cell transformation in a manner resembling that of NaF.

Animals↗
PubMed1988

Perturbation of the cell cycle by adeno-associated virus.

Infection of nonpermissive cells with adeno-associated virus (AAV) or AAV inactivated by uv light inhibited their multiplication in culture and interfered with their transit through the cell cycle. The perturbation of the cell cycle led to the accumulation of cells in the late S and/or G2 phases. The AAV-mediated inhibition of growth was dependent upon high concentrations of input virus and the types of cells. Presenescent embryonic fibroblasts of Syrian hamster and human origin were the most sensitive cell types examined; in contrast, immortalized lines of Syrian hamster and human origin were relatively resistant. We suggest that the inhibition of cell division results from a reaction between a cellular target and the incoming AAV virion (or a component of the virion) and that parental viral gene expression is not required.

Animals↗
PubMed1987

Isolation, complementary DNA sequence, and regulation of rat hepatic lauric acid omega-hydroxylase (cytochrome P-450LA omega). Identification of a new cytochrome P-450 gene family.

Lauric acid omega-hydroxylase (cytochrome P-450LA omega) was purified from livers of rats that had been given the hypolipidemic drug clofibrate. Immunoblot analysis with anti-cytochrome P-450LA omega antibody revealed the presence of two clofibrate-induced cytochrome P-450 proteins in both liver and kidney. This antibody was used to screen a lambda gt11 expression library constructed from liver mRNA isolated from rats given clofibrate. The clone, pP-450LA omega, was isolated and found to contain 2062 base pairs with an open reading frame of 509 amino acids (Mr 58,222). The pP-450LA omega cDNA insert was placed in the yeast expression vector pAAH5, and the resultant plasmid expressed a protein of the same size and activity as cytochrome P-450LA omega. The mechanism of the regulation of the cytochrome P-450LA omega gene by hypolipidemic agents was examined. The rate of cytochrome P-450LA omega gene transcription was increased as early as 1 h after administration of clofibrate, and this increase was followed by an elevation of cytochrome P-450LA omega mRNA, immunochemically detectable protein, and cytochrome P-450LA omega activity. In contrast, no increase in transcriptional activity was detected after clofibrate administration for the cytochrome P-450PCN or P-450b/e genes. The cytochrome P-450LA omega has several structural features in common with other cytochrome P-450s, including a conserved cysteine-containing heme-binding fifth ligand fragment and a hydrophobic amino terminus. Overall, cytochrome P-450LA omega shared less than 35% cDNA nucleotide and amino acid similarity with cytochromes P-450c, P-450d, P-450e, and P-450PCN, indicating that it is a member of a new cytochrome P-450 gene family. Southern blot analysis indicates that this family contains two or three genes.

Amino Acid Sequence↗
PubMed1987

Cannabinoids induce incomplete maturation of cultured human leukemia cells.

Monocyte maturation markers were induced in cultured human myeloblastic ML-2 leukemia cells after treatment for 1-6 days with 0.03-30 microM delta 9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana. After a 2-day or longer treatment, 2- to 5-fold increases were found in the percentages of cells exhibiting reactivity with either the murine OKM1 monoclonal antibody or the Leu-M5 monoclonal antibody, staining positively for nonspecific esterase activity, and displaying a promonocyte morphology. The increases in these differentiation markers after treatment with 0.03-1 microM THC were dose dependent. At this dose range, THC did not cause an inhibition of cell growth. The THC-induced cell maturation was also characterized by specific changes in the patterns of newly synthesized proteins. Pronounced among these changes was an increase in the synthesis of at least 10 proteins that are found abundantly in monocytes. The THC-induced differentiation did not, however, result in cells with a highly developed mature monocyte phenotype; the THC-treated cells failed to exhibit other monocyte markers such as attachment to the surface of tissue culture dishes or morphological maturation beyond the promonocyte stage. However, treatment of these "incompletely" matured cells with either phorbol 12-myristate 13-acetate or 1 alpha,25-dihydroxycholecalciferol, which are inducers of differentiation in myeloid leukemia cells (including ML-2 cells), produced cells with a mature monocyte morphology. Two other cannabinoids, cannabidiol and cannabinol, which were more cytotoxic than THC at comparable doses, also caused an increase in the expression of maturation markers, but at doses higher than those required for THC. The ML-2 cell system described here may be a useful tool for deciphering critical biochemical events that lead to the cannabinoid-induced "incomplete" cell differentiation of ML-2 cells and other related cell types. Findings obtained from this system may have important implications for studies of cannabinoid effects on normal human bone-marrow progenitor cells.

Calcitriol↗
PubMed1987

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.

Animals↗
PubMed1987

Mutagenesis and cytotoxicity in human epithelial cells by far- and near-ultraviolet radiations: action spectra.

Action spectra were determined for cell killing and mutation by monochromatic ultraviolet and visible radiations (254-434 nm) in cultured human epithelial P3 cells. Cell killing was more efficient following radiation at the shorter wavelengths (254-434 nm) than at longer wavelengths (365-434 nm). At 254 nm, for example, a fluence of 11 Jm-2 gave 37% cell survival, while at 365 nm, 17 X 10(5) Jm-2 gave equivalent survival. At 434 nm little killing was observed with fluences up to 3 X 10(6) Jm-2. Mutant induction, determined at the hypoxanthine-guanine phosphoribosyltransferase locus, was caused by radiation at 254, 313, and 365 nm. There was no mutant induction at 334 nm although this wavelength was highly cytotoxic. Mutagenesis was not induced by 434 nm radiation, either. There was a weak response at 405 nm; the mutant frequencies were only slightly increased above background levels. For the mutagenic wavelengths, log-log plots of the mutation frequency against fluence showed linear regressions with positive slopes of 2.5, consistent with data from a previous study using Escherichia coli. The data points of the action spectra for lethality and mutagenesis were similar to the spectrum for DNA damage at wavelengths shorter than 313 nm, whereas at longer wavelengths the lethality spectrum had a shoulder, and the mutagenesis spectrum had a secondary peak at 365 nm. No correlation was observed for the P3 cells between the spectra for cell killing and mutagenesis caused by wavelengths longer than 313 nm and the induction of DNA breakage or the formation of DNA-to-protein covalent bonds in these cells.

Cell Line↗
PubMed1986

c-fos expression is neither sufficient nor obligatory for differentiation of monomyelocytes to macrophages.

The c-fos gene is rapidly and transiently expressed when human U-937 and HL-60 leukemia cells are induced to differentiate to macrophages. We show that the expression of c-fos is controlled primarily at the transcriptional level. c-fos mRNA is very labile, with a half-life of less than 30 min. Superinduction of c-fos in the presence of cycloheximide occurs primarily because of stabilization of c-fos mRNA. When U-937 cells are serum-stimulated or treated with diacylglycerol, a c-kinase agonist, c-fos is transiently expressed to high levels; however, the cells fail to differentiate to macrophages. Furthermore, HL-60 cell variants resistant to TPA can be induced to differentiate to macrophages in the absence of detectable c-fos expression.

Calcitriol↗
PubMed1986

An interlaboratory comparison of transformation in Syrian hamster embryo cells with model and coded chemicals.

Three independent laboratories tested eight "model" and five coded chemicals in the Syrian hamster embryo clonal transformation assay system to establish the intra- and interlaboratory reproducibility of the system and to identify sources of variability. When a common cell pool and the same lot of fetal calf serum were used, the three laboratories obtained consensus on the activity of eight model chemicals: five chemicals (benzo(a)pyrene, 7,12-dimethylbenz(a)anthracene, N-methyl-N'-nitro-N-nitrosoguanidine, nitroquinoline-N-oxide, and lead chromate) induced morphological transformation without exogenous metabolic activation and three (N-2-fluorenylacetamide, pyrene, and anthracene) produced no transformation response. Five coded chemicals (2,6-dichloro p-phenylenediamine, 4,4'-oxydianiline, cinnamyl anthranilate, dichlorvos, and reserpine), representative of environmental chemical classes, but not necessarily strong carcinogens, produced more equivocal responses in this interlaboratory study. Thus, while the assay can be used to distinguish between transforming and nontransforming chemicals in some cases, the intrinsic limitations in low transformation frequency and in achieving any dose-response results are major constraints to the use of this system in a routine testing program at the present time. Efforts to increase the transformation frequency or to amplify the expression of the transformed phenotype constitute some of the approaches which should be explored in order to overcome these limitations.

Animals↗
PubMed1986

Inhibition of metabolic cooperation in Chinese hamster V79 cells by 1 alpha, 25-dihydroxycholecalciferol, the active metabolite of vitamin D3.

The biologically active metabolite of vitamin D3, 1 alpha, 25-dihydroxycholecalciferol (1,25-(OH)2D3), inhibited the metabolic cooperation between cocultivated Chinese hamster V79 cells that are either susceptible or resistant to the cytotoxic effect of 6-thioguanine (TG). This inhibition was characterized by an increased survival of TG-resistant cells when they are cocultured with susceptible cells in the presence of TG. TG-resistant cells in the absence of susceptible cells and after treatment with TG yielded a cloning efficiency of about 90%. Cocultivation of these cells with the susceptible cells reduced this cloning efficiency to about 30%. At 0.5 microgram/ml, 1,25-(OH)2D3 restored the cloning efficiency of the TG-resistant cells in the cocultures to 62%. Phorbol 12-myristate 13-acetate (PMA), a known inhibitor of metabolic cooperation, at 0.5 microgram/ml restored the cloning efficiency to a similar extent. Vitamin D3, 24,25-dihydroxycholecalciferol (24,25-(OH)2D3, and phorbol were ineffective in inhibiting this metabolic cooperation. 1,25-(OH)2D3, unlike PMA, did not inhibit the binding of [3H]phorbol 12,13-dibutyrate (PDBu) to its cellular receptors in either the TG-susceptible or resistant cells. These results indicate that 1,25-(OH)2D3 resembles tumor-promoting agents such as PMA in its capacity to inhibit metabolic cooperation, but this capacity is not mediated through phorbol diester receptors.

Animals↗