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PubMed1983

DNA modification, differentiation, and transformation.

Substantial evidence has accumulated over the last 5 years that the methylation of cytosine residues in vertebrate DNA is implicated in the control of gene expression. We have used analogs of cytidine, modified in the 5 position, as specific inhibitors of DNA methylation to probe the relationship between this process and cellular differentiation. 5-Azacytidine effected marked changes in the differentiated state of cultured cells and induced the formation of biochemically differentiated muscle, fat, and chondrocytes from mouse fibroblast cell lines. Since the analog is a powerful inhibitor of DNA methylation, we suggest that this inhibition is causally related to the mechanism of phenotypic conversion. DNA extracted from cells treated with 5-azacytidine was hemimethylated and was used as an efficient acceptor of methyl groups in an in vitro reaction in the presence of eukaryotic methylases. In vitro methylation was inhibited if the substrate DNA was preincubated with a diverse range of chemical carcinogens including benzo(a)pyrene diolepoxide. Thus, chemical carcinogens may induce changes in gene expression by alteration of cellular methylation patterns. Recent experiments have also demonstrated that freshly explanted diploid fibroblasts from mice, hamsters, and humans lose substantial quantities of 5-methylcytosine during cell division and aging in culture. Taken together, these experiments suggest that the genomic distribution of 5-methylcytosine might have importance in normal differentiation and also in the aberrant gene expression found in cancer and senescence in culture.

5-Methylcytosine↗

PubMed1987

Enhanced interleukin-1 production by human monocyte cell lines following treatment with 5-azacytidine.

Recent studies suggest that DNA modification, such as methylation of specific bases or substitution of altered bases, can play an important role in the control of eukaryotic gene expression. We chose to examine the effects of two DNA modifying agents, 5-azacytidine (AZA) and 5-bromodeoxyuridine (BUdR), on interleukin-1 (IL-1) gene expression in the human monocyte cell lines THP-1 and U937.1. THP-1 produced IL-1 upon stimulation with lipopolysaccharide (LPS), whereas U937.1 did not. Following treatment with AZA, U937.1 cells could be induced to produce IL-1 beta mRNA and release IL-1, but only if a stimulus such as LPS was present. In addition, the level of IL-1 beta mRNA produced and IL-1 released by THP-1 cells after induction could be doubled by treatment with AZA. In contrast, we were unable to alter IL-1 production by treatment of cells with BUdR in the absence or presence of inducers. These results suggest that the production of IL-1 may be, in part, regulated by methylation of DNA.

Azacitidine↗

PubMed1987

Generation of macrophage variants with 5-azacytidine: selection for mannose receptor expression.

Mannose receptors are expressed only in primary macrophages. Established macrophage-derived cell lines, although apparently possessing the potential to synthesize mannose receptors, do not express them on their plasma membranes. Using the drug 5-Azacytidine, mannose receptor expression was induced in the macrophage-derived mouse cell line J774. Receptor positive cells were sorted through a fluorescent activated cell sorter (FACS) prior to cloning. Clones were isolated which continuously express mannose receptors in culture. These macrophages were able to endocytose beta-glucuronidase and phagocytose yeast particles via mannose receptors. Secretion of the lysosomal enzyme beta-hexosaminidase was also reduced in proportion to the degree of mannose receptor expression.

Animals↗

PubMed1975

Photobiological behaviour of bacteria and phages supplemented with aza-analogoues of nucleic acid bases.

The photochemical stability of anomalous nucleic acid bases of the azatype, 5-azacytosine (I), 5-azacytidine (II), 6-azacytosine (III), 6-azacytidine (IV), 6-azathymine (V), 6-azauracil (VI), and 8-aza-adenine (VII) to U. V. light of the wavelength 254 nm differs from the U. V. stability of the normal constituents. Changes of the U.V. inactivation of Escherichia coli K12 C600, E. coli B, Bacillus cereus, as well as E. coli phages gamma cb2 and gamma b2b5 supplemented with azaderivatives prior to irradiation were investigated. It was found that I, II, III, IV, and VII are more, V and VI less sensitive to U. V. light compared with corresponding natural nucleic acid bases. Their changed U. V. sensitivities are reflected in the survival curves after U. V. -irradiation in as far as azabases are incorporated into the nucleic acids in vivo. This explains the increase in U.V. sensitivity of E. coli K12 C600, E. coli B, and B. cereus supplemented with I, II, III, IV, and VII and the decrease in U.V. sensitivity of Streptococcus faecalis supplemented with V (the latter information was taken from Gunther and Prusoff 1967). The lack of any significant influence on inactivation curves of E. coli K12 C600 by V and VI, and on E. coli phages gamma cb2 and gamma c2b5 by II, is discussed in terms of too small incorporation rates. No discrimination was put forward with respect to DNA and RNA incorporation.

Adenine↗

PubMed1978

Photochemistry and photobiology of 5-azacytidine: effect of repair on photostabilization of Escherichia coli.

The photochemical stability of the anomalous nucleic acid base 5-azacytidine (z5Cyd) on irradiation at 254 nm is by about one order of magnitude less than that of cytidine (Cyd). Contrary to the photochemical behaviour, incorporation of z5Cyd into the nucleic acids of E. coli strains SR 20 (uvr+ rec+), SR 74 (uvr+ rec-) and SR 22 (uvr- rec+) produced a higher resistance to UV light. Only the SR 73 (uvr- rec-) strain was shown to have an increased UV sensitivity. This latter finding is in accord with the photochemical properties of z5Cyd. The results led to the conclusion that excision and recombination repair processes contribute to the observable protective effect. The fact that inhibition of excission repair by caffeine or proflavine of the mutant uvr+ rec- changes protection into sensitization supports this idea.

Acriflavine↗

PubMed1975

Kinetics and mechanisms of degradation of the antileukemic agent 5-azacytidine in aqueous solutions.

The hydrolytic degradation of 5-azacytidine was studied spectrophotometrically as a function of pH, temperature, and buffer concentration. Loss of drug followed apparent first-order kinetics in the pH region below 3. At pH less than 1,5-azacytosine and 5-azauracil were detected; at higher pH values, drug was lost to products which were essentially nonchromophoric if examined in acidic solutions. The apparent first-order rate constants associated with formation of 5-azacytosine and 5-azauracil from 5-azacytidine are reported. Above pH 2.6, first-order plots for drug degradation are biphasic. Apparent first-order rate constants and coefficients for the biexponential equation are given as a function of pH and buffer concentration. A reaction mechanism consistent with the data is discussed together with problems associated with defining the stability of the drug in aqueous solutions. At 50 degrees, the drug exhibited maximum stability at pH 6.5 in dilute phosphate buffer. Similar solutions were stored at 30 degrees to estimate their useful shelflife. Within 80 min, 6 times 10(-4) M solutions of 5-azacytidine decreased to 90% of original potency based on assumptions related to the proposed mechanisms.

Azacitidine↗

PubMed1979

5-Azacytidine hydrolysis kinetics measured by high-pressure liquid chromatography and 13C-NMR spectroscopy.

Hydrolysis of 5-azacytidine, an experimental anticancer drug, in aqueous buffers was measured using a high-pressure liquid chromatographic (HPLC) procedure and a 13C-NMR method. The former utilized a 17.5-micron Aminex A-6 strong cation-exchanger column eluted with 0.4 M, pH 4.6 ammonium formate buffer at a flow rate of 0.4 ml/min. The hydrolysis sequence as well as the existence of a labile intermediate, N-formylguanylribosylurea, was unequivocally established using 6-13C-5-azacytidine and NMR spectral techniques. A reversible ring opening step to the N-formylguanylribosylurea with an equilibrium constant of 0.58 +/- 0.03 between pH 5.6 and 8.5, followed by an irreversible formation of guanylribosylurea, was found by HPLC. The data confirm previous assumptions on the hydrolytic kinetics. The pH dependency of hydrolysis was examined, and the hydrolysis profile gave a normal V shape with the most stable pH at 7.0. Rather stable intravenous dosage forms can be formulated.

Azacitidine↗

PubMed1979

Stabilization of 5-azacytidine by nucleophilic addition of bisulfite ion.

5-Azacytidine (I) stability was increased approximately 10-fold over its stability in water or lactated Ringer injection by the addition of excess sodium bisulfite and the maintenance of pH approximately 2.5. The increased stability in the presence of bisulfite at pH 2.5 was attributed to the addition of bisulfite across the 5-6 protonated imine bond of I, which prevented the hydrolytic attack on this labile double bond. However, above pH 4, bisulfite increased I degradation. At higher pH, the compound was no longer protonated and bisulfite did not form the stable addition product. The addition compound quickly decomposed above pH 6 to give back the parent compound and, thus, acted as a I prodrug. The intact drug remaining was assayed by high-pressure liquid chromatography (HPLC), and the reversibility of the bisulfite-I addition product above pH 6 was demonstrated by UV spectrophotometry and HPLC. The potential utility of the bisulfite-I addition product as a I prodrug in parenteral and possible oral dosage forms is discussed.

Azacitidine↗

PubMed1981

High-performance liquid chromatographic analysis of chemical stability of 5-aza-2'-deoxycytidine.

The chemical stability of 5-aza-2'-deoxycytidine (I) in acidic, neutral, and alkaline solutions was analyzed by high-performance liquid chromatography. In alkaline solution, I underwent rapid reversible decomposition to N-(formylamidino)-N'-beta-D-2-deoxyribofuranosylurea (II), which decomposed irreversibly to form 1-beta-D-2'-deoxyribofuranosyl-3-guanylurea (III). The pseudo-first-order rate constants for this reaction were determined. The decomposition of I in alkaline solution was identical to that reported previously for the related analog, 5-aza-cytidine. However, in neutral solution (or water), there was a marked difference in the decomposition of I and 5-azacytidine. The same decomposition products were formed from 5-azacytidine in neutral solution as in alkaline solution. However, in neutral solution, I decomposed to II and three unknown compounds that were chromophoric at 254 nm. Compound I was most stable when stored in neutral solution at low temperature.

Azacitidine↗

PubMed2003

Inactivation of helicase-like transcription factor by promoter hypermethylation in human gastric cancer.

Helicase-like transcription factor (HLTF), a member of the SWI/SNF (mating type switching/sucrose nonfermenting) chromatin-remodeling complex, is recently found to be inactivated by promoter hypermethylation in human colorectal cancer. However, the role of this putative tumor suppressor gene in other tumors has not been determined. We evaluated the role of HLTF promoter hypermethylation in gastric cancer. Expression of HLTF was examined by reverse-transcription (RT)-polymerase chain reaction (PCR), and promoter hypermethylation in HLTF was determined by methylation-specific PCR. Bisulfite DNA sequencing was performed to determine the detailed methylation profiles of the promoter region. HLTF expression was lost in two of five gastric cell lines and in 13 (28%) of 46 primary gastric cancers. Accordingly, promoter hypermethylation was detected in the two cell lines and in nine of 13 gastric cancer samples. Of the ten normal gastric specimens and ten paired adjacent nonneoplastic tissues, methylation was detected in only one adjacent nonneoplastic tissue. Bisulfite DNA sequencing of the promoter region of HLTF showed that the CpG island was densely methylated in cell lines and cancer samples; this also appeared to correlate with expression level. Treatment of gastric cell lines that lacked HLTF expression with the demethylating agent 5-azacytidine (5-azaDC) restored HLTF expression. These results suggest that HLTF promoter hypermethylation is frequently demonstrated in human gastric cancer, and inactivation of HLTF or the chromatin-remodeling complex may play a crucial role in gastric carcinogenesis.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Epigenetic inactivation of TSLC1 gene in nasopharyngeal carcinoma.

Deletion of 11q23 is a common genetic aberration in nasopharyngeal carcinoma (NPC). Multiple candidate tumor suppressor genes (TSG) were mapped to this region but few of them were investigated in NPC. TSLC1 (tumor suppressor in lung cancer) is recently reported to be a putative TSG on 11q23. This gene was found to be inactivated by promoter hypermethylation in non-small cell lung carcinoma (NSCLC), liver cancer, and breast cancer. To study the role of TSLC1 gene in NPC tumorigenesis, we screened for mutations and aberrant methylation of TSLC1 gene in 5 NPC cell lines, 3 NPC xenografts, and 38 primary NPC cases. No somatic mutations of TSLC1 were detected in the NPC samples, but a 9-bp (CCACCACCA) deletion in exon 8 was found in a primary NPC and its corresponding blood sample. Bisulfite sequencing revealed aberrant methylation of TSLC1 promoter in four NPC cell lines. Loss of TSLC1 gene expression was found in two cell lines (HK-1 and CNE-2) with dense methylation. Expression of this gene was restored in these cell lines after treatment with demethylating agent 5-aza-2'-deoxycytidine. Our results showed that silencing of TSLC1 gene expression in NPC was associated with promoter hypermethylation. Promoter hypermethylation of TSLC1 gene was further illustrated in 34.2% (13/38) of primary NPCs. No aberrant promoter methylation was found in any of the four investigated normal nasopharyngeal epithelia. Frequent epigenetic inactivation of TSLC1 gene in NPC suggested that this gene is one of the target tumor suppressor genes of this endemic cancer.

Adult↗

PubMed2004

CHFR-associated early G2/M checkpoint defects in breast cancer cells.

Cell division is a highly regulated process. Checkpoints can halt cell-cycle progression due to adverse conditions such as misalignment of chromosomes to prevent missegregation. The search for new regulators of the cell cycle revealed the mitotic checkpoint gene CHFR (checkpoint with forkhead-associated and ring finger). CHFR coordinates an early mitotic phase by delaying chromosome condensation in response to a mitotic stress. Because aneuploidy and chromosome instability are common in malignant breast tumors, we screened 24 breast cancer cell lines for CHFR expression and demonstrated that 50% (12 of 24) of breast cancer cell lines had low CHFR levels. Expression of CHFR was reactivated with the demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) in two low-CHFR-expressing cell lines. Eleven of these 12 (92%) low-CHFR-expressing cell lines had an unusually high number of condensed chromosomes and high mitotic indices in response to nocodazole treatment. Transfection of CHFR in one of these cancer cell lines lowered the mitotic index after nocodazole treatment. In conclusion, our data suggested that low CHFR expression associated with high mitotic indices in response to nocodazole treatment were common in the breast cancer cell lines studied. Additional flow cytometry studies and analysis of a protein that interacts with CHFR in vitro, polo-like kinase 1 (PLK1), suggests that this CHFR-associated early G(2)/M checkpoint is complex, involving additional, as yet unidentified, proteins. Further analysis of CHFR in breast cancer cells will be important for understanding the complex mechanisms leading to aneuploidy and chromosomal instability observed in breast cancer.

Azacitidine↗

PubMed2004

Expression of the p16INK4a gene and methylation pattern of CpG sites in the promoter region in rat tumor cell lines.

Loss of p16(INK4a) protein expression has frequently been related to DNA methylation in association with gene silencing. Although the methylation status of exon1alpha for p16(INK4a) involvement in various cancers has been extensively analyzed, it has been pointed out that some inconsistencies existed in its relationship to gene silencing of p16(INK4a). In this study, we focused on the expression and methylation status in the regions of nt -478 to -201, containing a putative TATA box (nt -401 to -396), and nt -233 to 26, both in a recently cloned 5' upstream region of rat p16(INK4a). We showed that rat lung adenocarcinoma RLCNR did not express the p16(INK4a) gene, whereas rat osteosarcoma COS1NR and malignant fibrous histiocytoma MFH1NR both expressed it at levels similar to normal fibroblasts, even though the region of nt -233 to 26 was hypermethylated in COS1NR rather than RLCNR. In contrast, the CpG islands near the putative TATA box region were consistently methylated in RLCNR, but not in COS1NR and MFH1NR, as well as in normal fibroblasts. Treatment with 5-aza 2'-deoxycytidine induced expression of p16(INK4a) gene in RLCNR after 48 h, but no changes were observed in COS1NR and MFH1NR. The results indicated that methylation of CpG islands near a TATA box region played a critical role for gene silencing of the rat p16(INK4a) gene, rather than that of other regions.

Adenocarcinoma↗

PubMed2001

Loss of mismatch repair activity in simian virus 40 large T antigen-immortalized BPH-1 human prostatic epithelial cell line.

Simian virus 40 large T antigen (SVLTAg) has been used to immortalize cells; however, the mechanism leading to immortalization is still unclear. We hypothesize that DNA mismatch repair (MMR) activity is important during SVLTAg-induced immortalization. To test this hypothesis, we used the SVLTAg-immortalized cell line BPH-1 derived from human benign prostate epithelial cells to analyze MMR activity and the expression of MMR genes (hMLH1, hPMS1, hPMS2, hMSH2, hMSH3, and hMSH6). The results demonstrated that BPH-1 cells were deficient in repairing G:T, A:C, and G:G mispairs in bacteriophage M13mp2. Reverse-transcription polymerase chain reaction experiments indicated MMR genes (hMSH3, hMSH6, and hPMS1) were expressed at a low level in BPH-1 cells. In contrast, all six MMR genes were expressed in human benign prostate hyperplasia tissues. Downregulation of hMSH3, hMSH6, and hPMS1 genes is not a result of the hypermethylation mechanism because demethylation with 5-aza-2'-deoxycytidine did not restore expression of these genes. Although the hMLH1 gene is expressed in BPH-1 cells, western blotting and exon analyses demonstrated that hMLH1 was mutated and/or deleted in BPH-1 cells.

Adaptor Proteins, Signal Transducing↗

PubMed2001

CpG methylation of promoter region inactivates E-cadherin gene in renal cell carcinoma.

CpG methylation in the promoter region has been shown to be important in the regulation of genes implicated in malignant transformation. The present study was designed to test the hypothesis that CpG methylation of the promoter region of the E-cadherin gene may inactivate its expression in renal cell carcinoma. To test this hypothesis, five kidney cancer cell lines and 34 microdissected renal cell carcinoma samples were analyzed for gene and protein expression by reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. CpG methylation in the promoter regions of the E-cadherin gene was analyzed by the sodium bisulfite genome sequencing technique. Our results show that all normal renal tissue expressed the E-cadherin gene and protein. Of the renal cancer tissues analyzed, 67% (23 of 34) lacked E-cadherin expression, with an associated increase in methylation, compared with normal tissue. E-cadherin gene promoter was methylated in all renal cancer cell lines and was accompanied by a loss of E-cadherin gene and protein expression. The treatment of renal cancer cell lines with the demethylating agent 5-aza-2'-deoxycytidine restored E-cadherin mRNA expression in all renal cancer cell lines. This is the first report that shows inactivation of the E-cadherin gene and protein in renal cell carcinoma through CpG hypermethylation in the promoter region of this gene. The results of these experiments may contribute to an understanding of the role of E-cadherin inactivation in renal cell carcinoma.

Azacitidine↗

PubMed2001

Hypermethylation of the tumor necrosis factor receptor superfamily 6 (APT1, Fas, CD95/Apo-1) gene promoter at rel/nuclear factor kappaB sites in prostatic carcinoma.

DNA hypermethylation of CpG-rich promoter sequences is associated with tumor suppressor gene inactivation in many human cancers, notably in carcinoma of the prostate and the urinary bladder. Recently, the mouse homologue of the tumor necrosis factor receptor superfamily 6 (TNFRSF6) gene was reported to be inactivated by DNA methylation in various cell types. The Fas (CD95, Apo-1) protein encoded by the TNFRSF6 gene is an important mediator of apoptosis, which also is downregulated in different types of human carcinoma. We therefore investigated the methylation of the TNFRSF6 promoter in prostatic and bladder carcinomas and cell lines. In a restriction enzyme polymerase chain reaction assay, four of 32 prostatic carcinomas and three of 15 advanced bladder carcinomas showed evidence of hypermethylation at the rel/nuclear factor kappaB (NFkappaB) binding sites essential for promoter activity. The DU145 cell line derived from a metastasis of a prostate carcinoma also displayed hypermethylation in this assay, which was confirmed by bisulfite sequencing. Treatment of DU145 cells with the methylation inhibitor deoxyazacytidine slightly increased Fas protein expression, as detected by flow cytometry analysis. In vitro methylation of the TNFRSF6 promoter at the rel/NFkappaB sites completely abolished its activity. Thus, although the TNFRSF6 gene can be inactivated efficiently by DNA methylation, hypermethylation occurs neither frequently nor extensively in human carcinomas and appears to play a limited role in downregulation of Fas expression.

Aged↗

PubMed2005

Histone modification in the TGFbetaRII gene promoter and its significance for responsiveness to HDAC inhibitor in lung cancer cell lines.

We previously reported silencing of the TGF-beta type II receptor gene (TGFbetaRII), involving histone deacetylation, instead of DNA methylation (DNA-Me). Because different histone modifications may play crucial roles in the epigenetic alterations, we further studied links with silencing of the TGFbetaRII gene promoter in six lung cancer cell lines. ChIP assays demonstrated three chromatin patterns for this gene silencing (Pattern I: histone H3 acetylation (H3-Ac)(+/-)/histone H3 lysine 4 methylation (H3K4-Me)(+)/DNA-Me(-), Pattern II; H3-Ac(-)/H3K4-Me(+/-)/DNA-Me(-), and Pattern III; H3-Ac(-)/H3K4-Me(-)/DNA-Me(+)), indicating possible progressive alterations with H3K4-Me alteration. With exposure to a histone deacetylase inhibitor (HDAC-I), trichostatin A, cell lines with the pattern II demonstrated strong and persistent induction of TGFbetaRII expression, while those with the pattern III showed only weak or no induction. ACC-LC-91 cell line, one of the pattern II examples demonstrated strong and continuous induction of H3K4-Me similar to TGFbetaRII expression. In contrast, ACC-LC-176 with the pattern III showed only weak and transient induction of H3K4-Me, similar to TGFbetaRII expression. Treatment with 5-aza-2'-deoxycytidine (5aza-dC) in addition to HDAC-I resulted in strong and continuous induction of TGFbetaRII expression and H3K4-Me in ACC-LC-176, although 5aza-dC alone was without such effects. In ACC-LC-91, both H3-Ac and H3K4-Me were promptly and simultaneously induced by HDAC-I, and similarly inhibited by wortmannin, a PI3K family inhibitor, together with TGFbetaRII induction. These findings suggested progressive alterations of chromatin configuration including H3K4-Me alteration in TGFbetaRII gene silencing. A possible involvement of a wortmannin-sensitive kinase in histone modification was also suggested.

Acetylation↗

PubMed1990

Factors influencing the expression of endogenous retrovirus-like sequences in Rat 6 cells.

Previous studies from this laboratory have demonstrated increased levels of expression of endogenous rat leukemia virus (RaLV) and 30S retrovirus-like sequences in liver and colon tumors induced by chemical carcinogens in rats. During the process of normal liver regeneration, the levels of RaLV RNA were dramatically increased, whereas the levels of 30S RNA did not change. The present study examined several factors that might influence the expression of these sequences in the Rat 6 embryo fibroblast cell line. Rat 6 cells in either log-phase or confluent cultures were treated with cycloheximide, the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), or the activated carcinogen benzo[a]pyrene diol epoxide (BPDE) for various periods of time up to 48 h. Northern blot analyses of total RNA indicated that cells in untreated cultures in log phase had higher levels of RaLV and 30S RNAs than did confluent cells. Within 10 h cycloheximide (2 or 10 micrograms/mL) markedly increased the levels of RaLV and 30S RNAs in both log-phase and confluent cells. BPDE (100 ng/mL) induced a marked increase in the levels of RaLV RNA at 4 to 10 h, which returned to the basal level by 24 h in the log-phase cells; but no significant change was seen in the confluent cells. The level of 30S RNA also increased moderately in the BPDE-treated log-phase cells and was maximal at 24 h; but no change was seen in confluent cells. Treatment with TPA (100 ng/mL) induced no significant increase in either RaLV or 30S RNA levels in the log-phase or confluent cells. The exposure of Rat 6 cells to 5-azacytidine (3 microM for 24 h) led to a marked increase in the levels of both RaLV and 30S RNAs, which persisted during at least 15 subsequent passages in the absence of the drug. Thus, inhibition of protein synthesis, DNA damage, or hypomethylation can increase the expression of certain endogenous retrovirus-like sequences, but an activator of protein kinase C does not.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗