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J K Christman

Publications and source records attributed to J K Christman.

At least 19 recordsLinked to original sources
PubMed2001

Synthesis of oligonucleotide inhibitors of DNA (Cytosine-C5) methyltransferase containing 5-azacytosine residues at specific sites.

The incorporation of 5-azacytosine residues into DNA causes potent inhibition of DNA (Cytosine-C5) methyltransferases. The synthesis of oligodeoxyribonucleotides incorporating single or multiple 5-aza-2'-deoxycytidine residues at precise sites was undertaken to generate an array of sequences containing the reactive 5-azacytosine base as specific target sites for enzymatic methylation. Preparation of these modified oligonucleotides requires the use of 2-(p-nitrophenyl)ethyloxycarbonyl (NPEOC) groups for the protection of exocyclic amino functions. These groups are removed under mild conditions, thus avoiding conventional protocols that are detrimental to the integrity of the 5-azacytosine ring.

Base Sequence↗
PubMed2000

Single-site methylation within the p53 promoter region reduces gene expression in a reporter gene construct: possible in vivo relevance during tumorigenesis.

It is not known whether transcriptional suppression by de novo methylation occurs within the promoter region of the p53 gene during multistage tumorigenesis. To address this question, in vivo alterations in the CpG methylation within the rat p53 promoter region were evaluated in control, preneoplastic, and tumor tissue during tumor progression using the folate/methyl-deficient model of hepatocarcinogenesis. Alterations in CpG methylation were found to be site-specific and to vary depending on the stage of carcinogenesis. To further explore the effect of site-specific methylation on p53 promoter activity, reporter gene constructs were prepared containing specifically methylated sites within the p53 promoter region, and the transcriptional activity in cultured mammalian cells was determined in a transient transfection assay. Relative to the unmethylated construct as a positive control, single-site methylation at nucleotide (nt) -450, which occurs 216 nt upstream from the 85-nt minimal promoter region, suppressed promoter activity by 85%. In contrast, single-site methylation at nt -179, which occurs within the minimal essential promoter region, suppressed activity by only 20%. The p53 promoter constructs containing the singly methylated CpG site at nt -450 were then reevaluated for processive changes in methylation status 48 h after transfection, during maximum suppression of promoter activity. Restriction analysis with methylation-sensitive enzymes revealed that de novo methylation had occurred after transfection at previously unmethylated sites. These findings suggest that nt -450 may constitute a critical site for initiation of de novo methylation and processive spreading of methylation associated with transcriptional inactivation of the p53 gene. Furthermore, the results suggest a possible alternative mechanism for the silencing of the p53 gene in tumors that do not have p53 mutations.

Animals↗
PubMed1999

Mechanism of inhibition of DNA (cytosine C5)-methyltransferases by oligodeoxyribonucleotides containing 5,6-dihydro-5-azacytosine.

A key step in the predicted mechanism of enzymatic transfer of methyl groups from S-adenosyl-l-methionine (AdoMet) to cytosine residues in DNA is the transient formation of a dihydrocytosine intermediate covalently linked to cysteine in the active site of a DNA (cytosine C5)-methyltransferase (DNA C5-MTase). Crystallographic analysis of complexes formed by HhaI methyltransferase (M.HhaI), AdoMet and a target oligodeoxyribonucleotide containing 5-fluorocytosine confirmed the existence of this dihydrocytosine intermediate. Based on the premise that 5,6-dihydro-5-azacytosine (DZCyt), a cytosine analog with an sp3-hybridized carbon (CH2) at position 6 and an NH group at position 5, could mimic the non-aromatic character of the cytosine ring in this transition state, we synthesized a series of synthetic substrates for DNA C5-MTase containing DZCyt. Substitution of DZCyt for target cytosines in C-G dinucleotides of single-stranded or double-stranded oligodeoxyribonucleotide substrates led to complete inhibition of methylation by murine DNA C5-MTase. Substitution of DZCyt for the target cytosine in G-C-G-C sites in double-stranded oligodeoxyribonucleotides had a similar effect on methylation by M. HhaI. Oligodeoxyribonucleotides containing DZCyt formed a tight but reversible complex with M.HhaI, and were consistently more potent as inhibitors of DNA methylation than oligodeoxyribonucleotides identical in sequence containing 5-fluorocytosine. Crystallographic analysis of a ternary complex involving M.HhaI, S-adenosyl-l-homocysteine and a double-stranded 13-mer oligodeoxyribonucleotide containing DZCyt at the target position showed that the analog is flipped out of the DNA helix in the same manner as cytosine, 5-methylcytosine, and 5-fluorocytosine. However, no formation of a covalent bond was detected between the sulfur atom of the catalytic site nucleophile, cysteine 81, and the pyrimidine C6 carbon. These results indicate that DZCyt can occupy the active site of M.HhaI as a transition state mimic and, because of the high degree of affinity of its interaction with the enzyme, it can act as a potent inhibitor of methylation.

Animals↗
PubMed1998

Transcriptional activation of functional endogenous estrogen receptor gene expression in MCF10AT cells: a model for early breast cancer.

Utilizing the MCF10AT xenograft model for progression of human proliferative breast disease, we detected expression of the endogenous estrogen receptor (ER) gene only in MCF10AneoT and cells of the MCF10AT system, all of which stably express a transfected mutated T24 Ha-ras gene. ER transcripts were undetectable in the parental MCF10A cells and in MCF10A cells transfected with normal c-Ha-ras or vector. ER transcripts expressed in MCF10AT cells contain a normal full-length ER coding region and direct synthesis of a normally sized ER protein. The protein is functional based on its ability to mediate estradiol (E2)-induced increases of transcription from both endogenous and exogenous E2-regulated genes. Transcriptional activation of the endogenous ER gene does not appear to be related to a change in methylation status of the gene since a diagnostic CpG site in exon 1 that is methylated in ER-negative breast tumors and completely unmethylated in ER-positive breast tumors is hypomethylated to the same extent in ER-negative MCF10A cells and ER-positive MCF10AT cells. E2 increased both the number and size of soft-agar colonies formed by MCF10AT3c cells, a line from a third generation MCF10AT xenograft lesion. This suggests that xenograft passage has selected for growth regulatory pathways that are E2-responsive and that identification of these pathways and their role in progression will aid in determining how E2 acts to increase risk of breast cancer.

Animals↗
PubMed1996

Characterization of the estrogen receptor transfected MCF10A breast cell line 139B6.

There has been increasing evidence which suggests that abnormal expression of the estrogen receptor (ER) protein in nonmalignant breast tissue may be important in the carcinogenic process. To examine the effects of ER expression in immortalized nonmalignant mammary epithelial cells, an expression vector containing human ER cDNA was transfected into the ER negative human breast cells, MCF10A. Characterization of a clone stably expressing ER, 139B6, provided evidence for the regulated synthesis of a functional ER capable of binding estradiol-17 beta (E2) and undergoing processing. Expression of the ER gene did not enable E2 to stimulate endogenous genes [progesterone receptor (PgR), pS2, cathepsin D and TGF alpha] which normally respond to estrogens in breast cancer cells. The ER in 139B6 cells was, however, capable of inducing expression of an ERE-regulated reporter gene, indicating its ability to interact with transcriptional machinery. Furthermore, cultures in log growth displayed a slight increase in doubling time in the presence of E2. These results indicate that ER expression alone is not sufficient to induce a transformed phenotype. Thus, the 139B6 cell line should provide a new model for determining what additional changes lead to increased growth potential in response to E2 and for exploring how E2 itself may help bring about changes leading to progression of preneoplastic breast epithelial cells.

Actins↗
PubMed1995

5-Methyl-2'-deoxycytidine in single-stranded DNA can act in cis to signal de novo DNA methylation.

Methylation of cytosine residues in DNA plays an important role in regulating gene expression during vertebrate embryonic development. Conversely, disruption of normal patterns of methylation is common in tumors and occurs early in progression of some human cancers. In vertebrates, it appears that the same DNA methyltransferase maintains preexisting patterns of methylation during DNA replication and carries out de novo methylation to create new methylation patterns. There are several indications that inherent signals in DNA structure can act in vivo to initiate or block de novo methylation in adjacent DNA regions. To identify sequences capable of enhancing de novo methylation of DNA in vitro, we designed a series of oligodeoxyribonucleotide substrates with substrate cytosine residues in different sequence contexts. We obtained evidence that some 5-methylcytosine residues in these single-stranded DNAs can stimulate de novo methylation of adjacent sites by murine DNA 5-cytosine methyltransferase as effectively as 5-methylcytosine residues in double-stranded DNA stimulate maintenance methylation. This suggests that double-stranded DNA may not be the primary natural substrate for de novo methylation and that looped single-stranded structures formed during the normal course of DNA replication or repair serve as "nucleation" sites for de novo methylation of adjacent DNA regions.

Animals↗
PubMed1995

Relationship between estrogen structure and conformational changes in estrogen receptor/DNA complexes.

The effect of estrogen structure on the conformation of the complex formed with estrogen receptor (ER) and the consensus estrogen response element (EREc) has been examined with gel mobility shift assay. Proteins in MCF-7 cell extracts formed three distinct complexes with ERE. Only the slowest moving complex contained ER as indicated by binding with anti-ER antibodies H222 and D547. This ER-ERE complex displayed increased electrophoretic mobility when formed in the presence of estradiol (E2) and bound radiolabeled 16 alpha-iodoestradiol. The antiestrogen ICI 164,384 decreased the mobility of the ER-ERE complex and blocked the effect of E2. The results reported here indicate that the position and location of hydroxyl groups on the estratriene nucleus is an important factor in determining the mobility of ER-EREc (or a variant ERE) in gel shift assays. The ability of E2 analogs to cause conformational changes detectable as altered mobility was not directly related either to their binding affinity for ER or to their ability to activate E2 responsive genes. Although several dihydroxyestrogens (estradiol-16 alpha, 1- and 2-hydroxyestratrien-17 beta-ol) caused an increase in the mobility of the ER-EREc, other ligands (estradiol-17 alpha, 4-hydroxyestratriene-17 beta-ol, 3-hydroxy estratriene, estratrien-17 beta-ol and 5-androsten-3 beta, 17 beta-diol) with a capacity for activating at least some E2 responsive genes in MCF-7 cells had little or no effect. On the basis of these and previously published results, it can be concluded that specific structure features of estrogens are responsible for conformational changes of ER-ERE complexes detectable in gel-shift assays. Furthermore, the identified structural characteristics of the ligand which are required for gel-shift are not the same as those previously reported to be essential for stimulation of transcriptional activity of ER.

Base Sequence↗
PubMed1995

Moderate folate deficiency does not cause global hypomethylation of hepatic and colonic DNA or c-myc-specific hypomethylation of colonic DNA in rats.

Global and gene-specific DNA hypomethylation is considered to be an important early epigenetic event in several human neoplasms. A growing body of evidence has suggested that DNA methylation can be altered by dietary manipulation of methyl group donors. This study investigated whether moderate depletion of folate, a dietary component needed for the synthesis of methyl groups, would cause decreased hepatic and colonic S-adenosylmethionine concentrations, and thereby lead to global and/or protooncogene-specific DNA hypomethylation. Weanling rats were fed an amino acid-defined diet containing either 0 or 8 mg folate/kg diet for 15 or 24 wk. Significantly lower systemic, hepatic and colonic folate concentrations were observed in the moderately folate-depleted rats than in controls at both 15 and 24 wk (P < 0.005). Although hepatic S-adenosylmethionine was significantly lower in the moderately folate-depleted rats than in controls at the two time points (P < 0.03), colonic S-adenosylmethionine concentrations were not significantly different between the two groups at either time point. No significant differences between the folate-depleted and control animals could be detected with regard to global DNA methylation in the liver or colonic mucosa. Furthermore, c-myc protooncogene-specific DNA methylation in the colonic mucosa was not significantly different between these two groups of animals. These results indicate that moderate folate depletion does not cause a significant reduction in global DNA methylation in liver or colonic mucosa or in c-myc-specific colonic mucosal DNA methylation in this rat model.

Animals↗
PubMed1994

Regulation of CD9 expression during 12-O-tetradecanoyl-phorbol-13-acetate- induced differentiation of human myeloid leukemia (HL-60) cells.

CD9 antigen is a member of the tetra spans superfamily of proteins which are expressed on the surface of a variety of hematopoietic and epithelial cell types. CD9 appears to play a role in platelet activation and to enhance sensitivity of cells to diphtheria toxin through its association with the diphtheria toxin receptor. Although several studies indicate that treatment of specific hematopoietic cells with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), induces CD9 expression, the mechanisms by which CD9 expression is regulated have not been elucidated. Here, we provide evidence that, in HL-60 cells, increases in the level of CD9 protein occur in parallel with TPA-induced differentiation. More than 80% of HL-60 cells exposed to 17 nM TPA become CD9 positive within 24 h. CD9 mRNA levels increase within 8-10 h after starting TPA treatment. Activation of CD9 transcription occurs during the same time period. Both transcriptional activation and accumulation of CD9 mRNA require protein synthesis. However, once CD9 mRNA has accumulated, inhibition of protein synthesis has no effect on its level or rate of turnover. These results suggest that CD9 expression in TPA-treated HL-60 cells is regulated at the transcriptional level and that activation of transcription occurs subsequent to the production of proteins induced as an immediate-early response to TPA. Since CD9 expression is not induced in HL-60TR cells, which respond to TPA but are resistant to its differentiating effects, the results also indicate that CD9 expression may serve as a marker for monocyte/macrophage differentiation of HL-60 cells.

Antigens, CD↗
PubMed1993

Regulation of the expression of galactoside-binding lectin during human monocytic differentiation.

The widely distributed hL-31 (CBP35, epsilon BP, mL-34, L-29, Mac-2) is a Ca(2+)-independent galactoside-binding lectin which functions as a receptor on mammalian cells for glycoproteins containing poly-N-acetyllactosamine side chains. Little is known about the regulation of its expression. The human promyelocytic leukemia cell line, HL-60, was used to determine whether expression of hL-31 (Mac-2) correlated with macrophage/monocyte differentiation. Nondifferentiated HL-60 cells and HL-60 cells grown in the presence of 1.24 microM retinoic acid expressed only trace amounts of hL-31. In contrast, both hL-31 transcripts and protein were detected at 8 h after addition of 17 nM 12-O-tetradecanoylphorbol-13-acetate and reached maximal levels at 24 h. Addition of actinomycin D along with 12-O-tetradecanoylphorbol-13-acetate blocked accumulation of hL-31 mRNA. In contrast, addition of actinomycin D to 12-O-tetradecanoylphorbol-13-acetate-treated HL-60 cells that had already accumulated high levels of hL-31 mRNA did not cause significant reduction in RNA levels until 6-8 h had elapsed. Since increased hL-31 expression was not associated with an increase in transcriptional activity of the hL-31 gene, these results suggest that hL-31 expression is regulated at the posttranscriptional level, at least in part, by stabilization of its mRNA. The results also indicate that the processes leading to increased hL-31 expression in HL-60 cells may be specific to differentiation along the monocyte/macrophage pathway.

Actins↗
PubMed1993

Effects of estradiol-17 beta analogues on activation of estrogen response element regulated chloramphenicol acetyltransferase expression.

These experiments were designed to examine the effect of structural modifications to the estradiol-17 beta (E2) molecule on the estrogen response element (ERE) dependent activation of the thymidine kinase (tk) promoter. Estrogen receptor (ER) positive MCF-7 cells were transfected with plasmids containing one or two vitellogenin EREs inserted upstream of the tk promoter in p(-37)tk. Transient expression of the CAT gene in these constructs was measured after cells had been maintained for 36-42 h in the presence of E2 or an E2 analogue. E2 induced CAT expression at levels as low as 10(-13) M, with maximum induction at 10(-11) M. CAT activity decreased at higher concentrations of E2. Estratriene, which has low affinity for ER, was active only at micromolar concentrations. 3-Hydroxyestratriene displayed maximal activity at 10(-9) M, with higher levels being less active. Still higher concentrations (10(-7) M) of estratrien-17 beta-ol were required to induce maximum CAT activity. All positional and conformational alterations in the D-ring hydroxyl group of E2 yielded active ligands. Movement of the phenolic hydroxyl group of E2 to other positions on the A-ring produced dihydroxyestrogens with varied capacities to activate CAT (2-hydroxyestratrien-17 beta-ol produced maximum CAT activation at 10(-11) M; 1-hydroxyestratrien-17 beta-ol required a 10(-8) M concentration for maximum activity; 4-hydroxyestratrien-17 beta-ol gave maximum CAT activation at 10(-6) M). Only those androstanediols or 5-androstenediols with a 3 beta-hydroxyl group were capable of activating CAT expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1993

Reversibility of changes in nucleic acid methylation and gene expression induced in rat liver by severe dietary methyl deficiency.

As we have reported previously, both DNA and tRNA become hypomethylated in livers of rats fed a cancer promoting, methyl-deficient diet (MDD) for as short a period as one week. Within the same period, activities of tRNA and DNA methyltransferases (MTases) increase and levels of mRNAs for several genes believed to have roles in growth regulation are altered. These diet-induced changes in nucleic acid methylation and gene expression increased in extent when MDD was fed continuously for four weeks. We also observed hypomethylation of specific CCGG sites within several genes for which mRNA levels were increased. These included c-myc, c-fos and c-Ha-ras. To investigate the reversibility of such diet-induced alterations in methylation and gene expression, animals were fed MDD for four weeks, after which a diet supplemented with adequate sources of methyl groups (CSD) was fed for 1-3 weeks. One to two weeks after the restoration of an adequate diet, the overall extent of methylation of tRNA and DNA from livers of these rats did not differ from that of tRNA and DNA from livers of age matched animals continually maintained on CSD. At the same time, activities of MTases in the liver dropped to normal values. Levels of mRNAs for all genes studied returned to control levels within three weeks after ending MDD feeding, although at different rates. In contrast, MDD-induced hypomethylation of some HpaII sites in c-myc, c-fos and c-Ha-ras genes persisted after 3 weeks refeeding of an adequate diet. These results, which demonstrate that most of the effects of MDD on the parameters we have studied occur rapidly and are essentially reversible, are consistent with the role of MDDs as promoters of hepatocarcinogenesis. However, the finding that unmethylated sites persist in genes that play a role in growth regulation suggests a mechanism by which intermittent or long term exposure to MDDs could result in heritable phenotypic changes in some hepatocytes that lead to hyperplasia and tumorigenesis.

Amino Acid Sequence↗
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↗