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J K Christman

Publications and source records attributed to J K Christman.

17 recordsLinked to original sources
PubMed1992

In situ dephosphorylation of p53 protein by calf intestinal alkaline phosphatase treatment.

The monoclonal antibody (mAb) 1801 has been reported to identify an N-terminal determinant within the tumor-suppressor protein p53 located between amino acids 32 and 79. This region contains two potential sites for serine phosphorylation at amino acids 33 and 46. Using a novel technique which dephosphorylates proteins in situ in fixed permeabilized cells, we have unmasked determinants in p53 recognized by mAb 1801, allowing additional sites of p53 protein to be detected in immunohistochemically reacted cells. This result indicates that phosphorylation at one or both sites within the determinant recognized by mAb 1801 previously blocked antibody-ligand interaction. It further suggests that in situ dephosphorylation may be of more general use in identifying antibodies which can only bind to epitopes in a particular phosphorylation state.

Alkaline Phosphatase↗
PubMed1991

Alterations in expression and methylation of specific genes in livers of rats fed a cancer promoting methyl-deficient diet.

We have reported earlier that hypomethylated DNA is rapidly induced in the livers of male Fischer rats fed an extremely methyl-deficient diet (MDD). The early effects of dietary methyl deficiency on the expression of several genes in the livers of such animals have now been investigated. Poly(A)+ RNA was isolated from the livers of rats fed MDD or a similar diet supplemented with adequate supplies of choline, methionine, folic acid and vitamin B12 (CSD) for periods ranging from 1 to 4 weeks. The levels of mRNAs for the c-myc and c-fos protooncogenes in livers of rats given either MDD or the liver carcinogen, 2-acetylaminofluorene (AAF), were compared by Northern blot analysis with those in livers of animals given control diets. Both AAF and MDD induced significant elevations in levels of mRNAs specific for these two genes. After 1 week of MDD intake, large increases in the levels of c-myc and c-fos mRNAs and a smaller increase in the levels of c-Ha-ras mRNAs were observed. In contrast, there were marked decreases in the levels of mRNAs for epidermal growth factor receptor and for epidermal growth factor. These effects on mRNA accumulation persisted and were further enhanced during a 4 week period of MDD feeding. The appearance of hypomethylated DNA in the livers of these MDD-fed rats coincided with the observed changes in levels of mRNA for these genes associated with the regulation of cell growth. Increases in levels of mRNA for c-fos, c-Ha-ras and c-myc were correlated with loss of methylation at specific sites within these genes as early as 1 week after the start of MDD feeding. These combined observations are consistent with the hypothesis that methyl-deficient diets are cancer promoting and/or carcinogenic, at least in part, because they induce hypomethylation of DNA with concomitant alterations in the regulation of gene expression.

Animals↗
PubMed1989

Rapid appearance of hypomethylated DNA in livers of rats fed cancer-promoting, methyl-deficient diets.

Prolonged intake of diets deficient in sources of methyl groups leads to development of hepatomas in rats and promotes chemical carcinogenesis in both rats and certain strains of mice. Since methylation of cytosine residues in regulatory regions can affect gene activity, several investigators have postulated that the effects of methyl-deficient diets on tumorigenesis result from the inability of cells to maintain normal patterns of DNA methylation. However, significant decreases in the 5-methylcytosine content of liver DNA have not been reported to occur until rats have consumed methyl-deficient diets for several months. To determine whether methyl-deficient diets have immediate effects on nucleic acid methylation, we assessed the degree to which hepatocyte DNA and tRNA were methylated in vivo, by measuring their ability to act as methyl acceptors in vitro. Hypomethylation of DNA and tRNA was detected within 1 week after rats were started on a diet deficient In methionine, choline, folic acid, and vitamin B12 and it persisted throughout the 4 weeks of study. A significant elevation in liver DNA synthesis occurred in parallel with increased hypomethylation of DNA. Chronic failure to fully methylate DNA that is newly synthesized in response to liver damage induced by methyl-deficient diets provides a feasible mechanism for changing patterns of DNA methylation. Our results indicate that such changes could occur rapidly enough to play a causal role in the cancer-promoting and, in some instances, cancer-inducing properties of the diet.

Animals↗
PubMed1989

5,5'-Dimethyloxazolidine-2,4-dione is a strong inducer of differentiation of human promyelocytic leukemia (HL-60) cells.

5,5'-Dimethyloxazolidine-2,4-dione (DMO), a weak non-metabolizable acid, is commonly utilized for determining intracellular pH. In these studies, DMO was tested as an inducer of differentiation on the basis that its uptake and subsequent dissociation might transiently raise intracellular pH and activate ion-fluxes critical for triggering maturation. After 5 days of exposure to 40 mM DMO, greater than 60% of HL-60 cells displayed phenotypic and functional changes characteristic of mature granulocytes. As with other inducers of HL-60 cell differentiation, commitment to differentiation required culture in the presence of DMO for more than 24 h, indicating that if transient effects on pH or ion-fluxes occurred, they were not sufficient to trigger this process. DMO was either weak or inactive as an inducer of murine erythroleukemia cell (FLC) differentiation. Although other weak acids and bases triggered differentiation of both HL-60 cells and FLC, the spectrum of response differed markedly between the two lines. These results suggest that: (1) a number of common buffering agents have the potential to alter cell phenotype, and (2) their effects must be evaluated for each individual cell type.

Animals↗
PubMed1988

A monoclonal antibody to a human neutrophil-specific plasma membrane antigen. Effect of the antibody on the C3bi-mediated adherence by neutrophils and expression of the antigen during myelopoiesis.

We have used mice selectively tolerized to antigens of human lymphocytes by treatment with cyclophosphamide to raise an mAb, BH2-C6, that reacts with a plasma membrane antigen specific for human neutrophils. This specificity is demonstrated by indirect immunofluorescence microscopy, cytochemical analysis of fluorescence-positive and -negative cell populations separated by flow cytometry, and by the selective, complement-mediated killing of mAb BH2-C6-treated neutrophils. Additional evidence for the neutrophil specificity of mAb BH2-C6 is shown by immunoelectron microscopy, which demonstrates a lack of reactivity with human eosinophils. Immunoblotting of SDS-PAGE-separated proteins of polymorphonuclear leukocytes with 125I-labeled BH2-C6 identifies protein with an average molecular mass of 157 kD. Binding studies show that, at saturation, neutrophils bind 214,000 molecules of 125I-BH2-C6 per cell. Addition of mAb BH2-C6 to neutrophils significantly reduces the number of C3bi-opsonized sheep erythrocytes (EIgMC3bi) bound by these cells. This reduction is partly reversed by the presence of soybean trypsin inhibitor (SBTI), indicating that at least one part of this inhibition is due to BH2-C6-stimulated secretion of a serine protease that may affect ligand binding. Cytochemical analysis of normal human bone marrow cells sorted by cytofluorimetry identifies the promyelocyte as the precursor cell that first expresses BH2-Ag on the plasma membrane. Using the leukemic cell line HL-60, we demonstrate that only inducers of granulocytic differentiation, cis-retinoic acid, and dimethyloxazolidine stimulate the expression of BH2-Ag. These results show that the expression of BH2-Ag during myelomonocytic differentiation is a property uniquely possessed by cells committed to the neutrophilic lineage.

Animals↗
PubMed1987

Murine cells carrying integrated tandem genomes of hepatitis B virus DNA transcribe RNAs from endogenous promoters on both viral strands and express middle and major viral envelope proteins.

Clone 4.10 cells were isolated as a methotrexate-resistant clone arising after cotransfection of mouse 3T3 cells with plasmid DNA containing a head-to-tail dimer of the hepatitis B virus (HBV) genome and DNA coding for methotrexate-resistant dihydrofolate reductase. The majority of methotrexate-resistant clones derived by this procedure have been found to contain multiple copies of the HBV genome, but the intact HBV dimer was rarely preserved. In contrast, 4.10 cells contained at least 40 copies of intact HBV dimer per cell. These cells produced large amounts of 22-nm hepatitis B surface antigen particles that included viral envelope proteins reactive with the pre-S2 region-specific antibody, indicating transcription and translation of the pre-S2 and S regions of the integrated viral genomes. The cells also synthesized viral e antigen, which was released into the culture medium. Characterization of polyadenylated viral RNAs transcribed from the long (minus) strand of the integrated HBV DNA demonstrated the presence of shorter-than-genome-length RNAs containing only X region sequences, shorter-than-genome-length RNAs containing both X and S region sequences, and longer-than-genome-length RNAs containing core, X, and S region sequences. Start sites for transcripts were mapped 5' to and within the pre-S region and 5' to and within the precore region at approximately the same sites as those utilized for HBV transcription during viral replication in infected livers. Polyadenylated RNA transcripts complementary to the short (plus) strand of HBV that initiated and terminated within the intact and integrated head-to-tail tandem viral genomes were also detected.

Animals↗
PubMed1985

Formation of highly stable complexes between 5-azacytosine-substituted DNA and specific non-histone nuclear proteins. Implications for 5-azacytidine-mediated effects on DNA methylation and gene expression.

Incubation of 5-azacytosine-substituted DNA ([5-aza-C]DNA) with nuclear proteins leads to the formation of highly stable DNA . protein complexes which remain intact in the presence of 1 M NaCl and/or 0.6% Sarkosyl. The proteins involved in binding double-stranded [5-aza-C]DNA in these stable complexes comprise a specific subset of non-histone nuclear proteins that includes DNA methyltransferase. Complex formation does not require S-adenosylmethionine and does not involve covalent linkage of protein to DNA or modification of 5-azacytosine residues. Non-histone nuclear proteins do not form complexes with double-stranded unsubstituted DNA that are resistant to dissociation with NaCl and Sarkosyl but are capable of forming such complexes with single-stranded DNA regardless of whether it contains 5-azacytosine residues or not. However, it can be demonstrated 1) that single-stranded regions do not account for stable binding of proteins to native [5-aza-C]DNA and 2) that many nuclear proteins which form stable complexes with single-stranded DNA are incapable of forming such complexes with double-stranded [5-aza-C]DNA. Synthesis of [5-aza-C]DNA by cells growing in the presence of either 5-azacytidine or 5-aza-2'-deoxycytidine leads to rapid loss of extractable DNA methyltransferase (Creusot, F., Acs, G., and Christman, J.K. (1982) J. Biol. Chem. 257, 2041-2048). Analogous depletion of non-histone nuclear proteins capable of forming stable complexes with [5-aza-C]DNA in vitro is observed, suggesting that the same proteins can form highly stable complexes with [5-aza-C]DNA in vitro and in vivo. Formation of stable complexes between non-histone nuclear proteins and [5-aza-C]DNA could potentially affect not only the activity of DNA methyltransferase but the action of other regulatory proteins or enzymes that interact with DNA. Such interactions could explain effects of 5-azacytidine on gene expression that cannot be directly linked to loss of methyl groups from DNA.

Animals↗
PubMed1985

Enzyme-linked immunoassay of pre-S gene-coded sequences in hepatitis B vaccines.

Pre-S gene coded domains of the hepatitis B virus (HBV) envelope protein are highly immunogenic in experimental animals and humans. Their presence in HBV and hepatitis B surface antigen (HBsAg) particles leads to production of anti-pre-S-specific antibodies during the course of HBV infection. Since antibodies specific for pre-S domains are capable of preventing the attachment of HBV to hepatocytes and are virus neutralizing, it would seem desirable to produce HBV vaccines with a standardized level of pre-S determinants to ensure their potential for eliciting the same repertoire of protective antibodies as found after recovery from natural infection. However, a test with appropriate sensitivity for detecting pre-S determinants to ensure their potential for eliciting the same repertoire of protective antibodies as found after (ELISA) for detecting pre-S determinants in vaccines containing less than or equal to 20 micrograms of HBsAg. The components of this assay are (1) antibodies to a synthetic peptide pre-S (120-145) adsorbed to polystyrene beads, and (2) beta-lactamase-labelled antibodies purified from anti-HBV serum on the basis of their affinity for a pre-S (120-174) beta-galactosidase fusion protein produced in Escherichia coli. Results of an evaluation of the pre-S content of HBV vaccines from two different commercial sources are discussed.

Amino Acid Sequence↗
PubMed1985

Expression in Escherichia coli of a cloned DNA sequence encoding the pre-S2 region of hepatitis B virus.

A DNA sequence encoding the entire pre-S2 region (amino acids 120-174; serotype ayw) of human hepatitis B virus envelope protein has been inserted into the lacZ gene of the plasmid pSKS105 yielding a recombinant, pWS3. Lac+ colonies of the Escherichia coli M182 delta (lacIOPZYA), isolated after transformation with pWS3, produced a pre-S2 peptide-beta-galactosidase fusion protein. This fusion protein, which comprised as much as 3% of the total bacterial protein, was purified to greater than 90% homogeneity by affinity chromatography on p-aminophenyl-beta-D-thiogalactoside-Sepharose. It is immunoprecipitable with rabbit antibodies to a synthetic peptide corresponding to amino acids 120-145 of the pre-S2 region of serotype adw [pre-S(120-145)] or with antibodies to hepatitis B virus. pre-S(120-145) completely blocked the binding of either antibody to the pre-S2 peptide-beta-galactosidase fusion protein. These results indicate that there are antigenic determinants on the fusion protein that are closely related to, if not identical to, determinants on synthetic pre-S(120-145) and on pre-S2 sequences of native hepatitis B virus particles. Thus, bacteria transformed with pWS3 can provide an abundant source of pre-S2-beta-galactosidase fusion protein, which may prove useful either as a diagnostic reagent possessing marker enzyme activity suitable for ELISA tests or as an immunogen with potential to contribute to active prophylaxis of hepatitis B.

Amino Acid Sequence↗
PubMed1979

The effect of 12-O-tetradecanoyl-phorbol 13-acetate on the ribonuclease activity of circulating human lymphocytes.

12-O-Tetradecanoyl-phorbol 13-acetate is a very effective tumor promotor and inflammatory agent and can act as a mitogen for a subset of T lymphocytes. We report here that even short exposure of lymphocytes to 12-O-tetradecanoyl-phorbol 13-acetate changes the balance between the levels of neutral ribonuclease and ribonuclease inhibitor. The most dramatic change occurs in a B-lymphocyte-enriched population. We find that most, if not all, of the neutral ribonuclease activity in circulating lymphocytes is associated with this population and that this activity is lost with exposure to 12-O-tetradecanoyl-phorbol 13-acetate. Both 12-O-tetradecanoyl-phorbol 13-acetate and phytohaemagglutinin increase the level of ribonuclease inhibitor in T cells. However, phytohaemagglutinin has no effect on the ribonuclease or inhibitor level of the B-cell-enriched population.

B-Lymphocytes↗
PubMed1978

Effect of L-ethionine on macromolecular synthesis in mitogen-stimulated lymphocytes.

2--4 mM L-ethionine completely inhibits DNA synthesis in phytohaemagglutinin- or concanavalin A-stimulated lymphocytes even though it does not prevent the morphological changes characteristic of blast formation. Evidence is presented which indicates that complete commitment to DNA synthesis as well as a substantial increase in the rates of RNA and protein synthesis can occur in the presence of ethionine. Ethionine, however, does inhibit methylation of tRNA and prevents mitogen-induced increase in the activity of histone-modifying enzymes. All of these effects of exposure to ethionine are completely reversible. Removal of ethionine after 24 h or more of exposure results in a rapid, synchronous wave of DNA synthesis, an increase in the rate of methylation of RNA and an increase in activity of histone-modifying enzymes.

Acetyltransferases↗
PubMed1978

Specificity of response in hamster cells induced to produce plasminogen activator by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate.

A phorbol ester promoting agent, 12-O-tetradecanoylphorbol-13-acetate, enhances plasminogen activator production in hamster cell lines that synthesize plasminogen activator immunologically identical with plasminogen activator of normal hamster lung cells. 12-O-Tetradecanoylphorbol-13-acetate exhibits a high degree of specificity in the type of plasminogen activator evoked, which is always of the lung form. No enhancement of plasminogen activator production was detected in hamster cell lines synthesizing plasminogen activator of different antigenic form, although in the presence of 12-O-tetradecanoylphorbol-13-acetate, cells of one such line initiated synthesis of plasminogen activator of the lung form.

Animals↗
PubMed1977

Correlation between hypomethylation of DNA and expression of globin genes in Friend erythroleukemia cells.

This report identifies L-ethionine as an inducer of differentiation in murine erythroleukemia cells. When Friend erythroleukemia cells are grown in the presence of 4mM L-ethionine, globin mRNA accumulates and in 4-5 days, 25-30% of the cells in the culture contain hemoglobin. Incubation of the cells with bromodeoxyuridine prevents both ethionine-induced accumulation of globin mRNA and erythroide differentiation. At the concentration where L-ethionine acts as an inducer of FL cell differentiation it inhibits methylation of DNA and tRNA in vivo but does not prevent macromolecular synthesis or cell division. To establish whether a link existed between inhibition of a specific methyltransferase and activation of globin synthesis in FL cells, we examined the degree of hypomethylation of DNA and tRNA from FL cells induced to differentiate with dimethylsulfoxide and butyrate. In contrast to the tRNA from ethionine-treated cells, tRNA from cells induced by butyrate or Me2SO cannot be methylated in vitro using homologous enzymes. DNA isolated from cells exposed to any of the three inducers, however, was significantly hypomethylated when compared with DNA from uninduced cells. These data suggest that methylation of DNA may play a role in the regulation of gene expression.

Cell Differentiation↗
PubMed1976

The reovirus replicative cycle.

Soon after entry into their host cells ssRNA viruses form doule-stranded replicative intermediates. Most RNA viruses exist only transiently in this double-stranded form. Double-strand formation occurs irrespective of whether the genome is of the same or complementary polarity to viral mRNA. In contrast, reoviruses contain dsRNAs from the outset. Single, and not double, strands are the intracellular intermediates through which genetic information is transferred from parental to progeny reoviruses. Double strands are the repositories in which the information is stored. We emphasize this distinction because it permits dsRNA-containing viruses to replicate conservatively, a mode of replication that is not shared by any other viruses. One important consequence of the conservative mode of replication is that cellular enzymes never gain access to the reovirus genome but only to its ssRNA precursors.

Binding Sites↗
PubMed1975

Immunological analysis of plasminogen activators from normal and transformed hamster cells. Evidence that the plasminogen activators produced by SV40 virus-transformed hamster embryo cells and normal hamster lung cells are antigenically identical.

Rabbits were immunized against the plasminogen activator released by SV4- virus-transformed hamster embryo cells. The resulting antiplasminogen activator immunoglobulin (APA-IgG) inhibited the enzymatic activity of the plasminogen activator produced by SV40-transformed hamster cells, and the plasmin-catalyzed release of these cells from the tissue culture dish. APA-IgG was not cytotoxic for these cells even in the presence of complement and did not inhibit their release of plasminogen activator. APA-IgG formed a single precipitin line in immunodiffusion plates using highly purified plasminogen activator as antigen. APA-IgG inhibited the plasminogen activator produced by newborn hamster lung cells and by an established diploid line (DON) of hamster lung cells, but did not inhibit plasminogen activators produced by normal or transformed hamster kidney cells or by cells of other species (mouse and human). We derive three major conclusions from these data: (a) There are several immunologically distinguishable forms (isozymes) of plasminogen activators in normal hamster tissues. (b) The plasminogen activators produced by normal hamster lung cells and by SV40 virus-transformed hamster embryo cells share antigenic determinants and are presumably the same isozyme. (c) The plasminogen activators produced by different hamster tumor cells do not share antigenic determinants and are presumably different isozymes.

Animals↗
PubMed1975

Correlated suppression by 5-bromodeoxyuridine of tumorigenicity and plasminogen activator in mouse melanoma cells.

The decrease of tumorigenicity by mouse melanoma clone B559 after growth in the presence of 5-bromodeoxyuridine (BrdU) has been correlated with a decrease in detectable cellular plasminogen activator. Reduction of both activities occurs after one to two cell divisions in the presence of this thymidine analog and is virtually complete within three to four cell cycles. These changes are fully reversible; four to five cell divisions in the absence of BrdU are sufficient to allow both tumorigenicity and plasminogen activator levels to return to normal. These results support the hypotheses that (a) the expression of a cellular plasminogen activator is closely associated with the transformation of normal to malignant cells and that (b) the suppression of tumorigenicity by BrdU reflects the capacity of this base analog to inhibit the expression of specialized functions which accompany the malignant state.

Animals↗