Biomedical subjects
Publications and source records attributed to J K Christman.
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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.
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.
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.
Long-term cultures of hepatocytes were established from livers of human fetuses obtained by abortion at 18 to 23 wk of gestation. Cells obtained by collagenase dissociation of liver were maintained in defined serum-free medium on a substratum of positively charged plastic. Under these conditions, the cells divide and form a confluent monolayer. After multiple passages over a period of 3 mo., the cells retained an epithelioid morphology and continued to synthesize and secrete albumin.
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.
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.
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HL-60TR, a tetraploid variant of the human promyeloid cell line HL-60, was obtained by culturing HL-60 cells for one week with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) at 400 pM. HL-60TR cells have lost the ability to develop several common markers of maturation in response to compounds that induce monocytoid or myelocytoid differentiation of HL-60 cells. In addition, they release a factor which inhibits induction of the same markers in HL-60 cells. Medium conditioned by HL-60TR cells also inhibits colony formation by normal mouse bone marrow cells. These properties have been maintained by HL-60TR cells through more than one year of constant subculture in the absence of TPA, a finding which suggests the possibility that TPA may promote tumor formation not only through direct effects on the phenotype of initiated cells but also through induction of continued production of factors that affect differentiation of normal stem cells.
One of the most readily quantitated indices of myeloid maturation in HL-60 cells is their ability to respond to the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate with increased respiratory burst activity. HL-60 cells exposed to the antileukemic drug, 5-azacytidine (3 to 5 microM) for 24 hr and subsequently cultured in its absence for 2 to 3 days develop an enhanced ability to respond to 12-O-tetradecanoylphorbol-13-acetate with increased respiratory burst activity detectable as an increase both in hexose monophosphate shunt activity and in the proportion of the population producing superoxide anion. 5-Azacytidine treatment also causes marked inhibition of DNA methyltransferase, and thus DNA synthesized by HL-60 cells during the 24-hr period of analogue treatment is essentially devoid of methylated cytosine residues. This suggests, as does our previous finding that a general inhibitor of transmethylation reactions, L-ethionine, can induce differentiation of HL-60 cells, that changes in gene expression triggered by these compounds may be linked to their ability to alter patterns of DNA methylation. Since at least 50% of HL-60 cells capable of forming colonies in soft agar after a 24-hr exposure to 5-azacytidine yield progeny that mature (i.e., produce superoxide anion in response to 12-O-tetradecanoylphorbol-13-acetate) 2 weeks after 5-azacytidine treatment, the results also indicate that the changes induced in HL-60 cells by limited exposure to 5-azacytidine are heritable and can influence gene expression many generations after treatment has been terminated.
Treatment of Friend erythroleukemia cells with the antileukemic drugs 5-azacytidine and 5-aza-2'-deoxycytidine leads to rapid, time-dependent, and dose-dependent decrease of DNA methyltransferase activity and synthesis of markedly undermethylated DNA. Since this DNA is at least partially methylated in vivo and serves as an excellent substrate for methylation in vitro, hypomethylation of DNA in analog-treated cells appears to result from the loss of DNA methyltransferase, rather than from an inherent inability of 5-azacytosine- substituted DNA to serve as a methyl acceptor. Inhibition of DNA synthesis blocks the loss of DNA methyltransferase activity while inhibitors of RNA synthesis do not, suggesting that the analogs must be incorporated into DNA to mediate their effect on the enzyme, and that minor substitution of 5-azacytosine for cytosine in DNA (approximately 0.3%) suffices to inactivate more than 95% of the enzyme in the cell. Several lines of evidence link changes in the pattern of DNA modification with differentiation. In this regard, it is significant that 5-azacytidine and 5-aza-2'-deoxycytidine act as weak inducers of erythroid differentiation of Friend erythroleukemia cells in the same concentration range where they affect DNA methyltransferase activity. For differentiation to proceed, the cells must be washed free of the drugs. Less than 24 h later, normal levels of DNA methyltransferase activity are restored and within 48 h, DNA isolated from the cells is not detectably undermethylated. This may in part explain why 5-azacytidine and 5-aza-2'-deoxycytidine induce differentiation in less than 15% of the population despite their initial profound effect on DNA methylation.
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3T3 cells containing hepatitis B virus DNA sequences can be efficiently selected by exposure to methotrexate after cotransfection with cloned viral DNA and DNA coding for a methotrexate-resistant dihydrofolate reductase. More than 75% of methotrexate-resistant cells isolated after cotransfection with a head-to-tail tandem of the hepatitis B virus genome synthesized viral surface antigen. The antigen was released into the culture medium in the form of 22-nm particles with buoyant density of 1.20 g/ml. No other virally coded proteins were detected in the cells or the culture medium. Application of selective pressure by increasing the concentration of methotrexate resulted in an amplification of viral DNA sequences and a concomitant increase in the rate of synthesis and release of hepatitis B surface antigen. The ability to produce large amounts of surface antigen appears to be a stable trait and has been maintained in these cultures through more than 30 passages.
Chromatin fragments released from intact Friend erythroleukemia cell nuclei during limited incubation with micrococcal nuclease, DNase II or DNase I were analyzed to determine the distribution of DNA methyltransferase in chromatin. The enzyme was released in a free form when internucleosomal DNA was digested with micrococcal nuclease but was found associated with Mg++-precipitable polynucleosomes after DNase II digestion. Less than 25% of the enzyme was released from nuclei incubated with DNase I under conditions where transcriptionally active chromatin should have been completely digested. These results indicated that the bulk of DNA methyltransferase was bound to "linker" DNA in condensed regions of chromatin. Preferential rebinding of free enzyme to linker DNA was also demonstrated in vitro. The possibility that chromatin proteins play a role in regulating access of DNA methyltransferase to specific sites in DNA is discussed in light of these findings.
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DNA from mammalian cells has been shown to contain significant amounts of 5-methyl cytosine resulting from enzymatic transfer of methyl groups from s-adenosylmethionine to cytosine residues in the DNA polymer. The function of this modification is not known. We have found that DNA synthesized during chemically induced differentiation of friend erythroleukemia cells is hypomethylated, as measured by its ability to accept methyl groups transferred by homologous DNA methyltransferases in vitro. The extent of hypomethylation detected by this sensitive method is small, a decrease of less than 1.6 percent in 5-methylcytosine content. Hypomethylated DNA can be isolated from friend erythroleukemia cells grown in the presence of dimethyl sulfoxide, butyrate, hexamethylene-bis- acetamide, pentamethylene-bis acetamide, and ethionine. However, hypomethylated DNA is found only under conditions where differentiation is actually induced. DNA isolated from cells of a dimethyl sulfoxide- resistant subclone grown in the presence of that agent is not hypomethylated, although DNA of these cells becomes hypomethylated after growth in the presence of inducers that can trigger their differentiation. We also find that the DNA of friend erythroleukemia cells does not become hypomethylated when the cells are exposed to inducing agents in the presence of substances that inhibit differentiation. These results suggest a close link between genome modification by methylation and differentiation of friend erythroleukemia cells.
The induction of aryl hydrocarbon hydroxylases in lymphocytes is dependent on their activation. The tumor-promoting phorbol esters which induce blast formation and DNA synthesis in lymphocytes enable polycyclic aromatic hydrocarbons to induce aryl hydrocarbon hydroxylases. Melittin, the major constituent of bee venom, acts synergistically with these phorbol esters in enhancing both lymphocyte activation and hydroxylase synthesis. Since aryl hydrocarbon hydroxylases convert procarcinogens to carcinogens these results suggest that tumor promotion by phorbol esters may be associated with their ability to affect the induction of these enzymes. This hypothesis is supported by the finding that phorbol and phorbol esters which lack tumor-promoting activity fail to enhance induction of aryl hydrocarbon hydroxylases in lymphocytes. However mezerein, although rather ineffective in promoting tumors, activates lymphocytes and permits polycyclic aromatic hydrocarbons to induce their hydroxylases effectively.