Differentiation therapy for myelodysplastic syndrome.
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Azacitidine↗
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Results for “DNA Modification Methylases”
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[Expression and drug resistance of human MGMT gene in hemopoietic cells mediated by bicistronic retroviral vector].
OBJECTIVE: To increase myeloid progenitors resistance to chemotherapy and prevent myelosuppression caused by alkylating agents. METHODS: Total cellular RNA was extracted from human liver and cDNA was synthesized by superscript reverse transcriptase, a polymerase chain reaction(PCR) was conducted. We obtained a full length cDNA fragment encoding human alkyguarine-DNA-alhyltransferase(MGMT). The PCR product was cloned into pGEMT-T vector and further subcloned into G1Na retrovirus expression vector. Then the recombinant plasmid was transduced into the packaging cell lines GP+E86 and PA317 by lipofect AMINE. RESULTS: By using the medium containing BCNU for cloning selection and ping-ponging supernatant infection between ecotropic produced clone and amphotropic producer clone, we obtained high titer amphotropic PA317 producer clone with the highest titer up to 8.6-10 CFU/ml. Human hematopoietic cells were infected repeatedly with this high titer virus under stimulation of hemopoietic growth factors IL-3, IL-6 and SCF. PCR, RT-PCR, Southern blot, Western blot and MTT analyses showed that MGMT gene has integrated into the genomic DNA of human hemopoietic cells and expressed efficiently. CONCLUSIONS: This study provides a foundation for application of gene therapy to tumor clinical trial.
Alkyl and Aryl Transferases↗
Selective inhibition of DNA methyltransferase enzymes as a novel strategy for cancer treatment.
A common event in the development of human neoplasia is the loss of growth regulatory tumor suppressor functions. Methylation of 5'-CpG islands of tumor suppressor genes and elevated levels of the DNA-(cytosine-5)-methyltransferase enzymes (DNMT1, 3A and 3B) are also prevalent features of human neoplasia. However, direct evidence that elevated DNMT enzyme levels alter gene expression and influence oncogenesis has been difficult to obtain due to the lack of specific DNMT inhibitors. We have developed potent and selective antisense inhibitors of the known DNA methyltransferases. MG-98, a second-generation DNMT1-specific antisense inhibitor currently in phase II clinical trials, reactivates silenced tumor suppressor genes and inhibits the growth of cancer cells in vitro and in preclinical in vivo models. Here, we will review the discovery and development of MG-98 as a cancer therapeutic.
Clinical Trials as Topic↗
Epigenomics: genome-wide study of methylation phenomena.
Epigenetics is one of the key areas of future research that can elucidate how genomes work. It combines genetics and the environment to address complex biological systems such as the plasticity of our genome. While all nucleated human cells carry the same genome, they express different genes at different times. Much of this is governed by epigenetic changes resulting in differential methylation of our genome--or different epigenomes. Individual studies over the past decades have already established the involvement of DNA methylation in imprinting, gene regulation, chromatin structure, genome stability and disease, especially cancer. Now, in the wake of the Human Genome Project (HGP), epigenetic phenomena can be studied genome-wide and are giving rise to a new field, epigenomics. Here, we review the current and future potential of this field and introduce the pilot study towards the Human Epigenome Project (HEP).
Autoimmune Diseases↗
[Methylation and expression of the FMR1 gene].
OBJECTIVE: In this paper we present a brief review on DNA methylation, the enzymes and proteins involved in the repression complex and the importance of methylation of FMR1 gene in fragile X syndrome. DEVELOPMENT: Methylation status of control region in the genome plays a critical role in the regulation of gene expression. In susceptible genes containing CpG island in the promoter, cytosine methylation favors a repressive chromatin structure that prevents the binding of transcriptional activators to the promoter. The enzyme DNA methyltransferase transfers a methyl group from the S-adenosylmethionine to the 5 carbon of cytosine in the CG sequences. Several proteins have been described that recognize the methyl cytosine and recruit the co-repressor and the histones deacetylases. The lost of the acetyl groups produces the compacting of the chromatin. Fragile X syndrome is due, in the majority of the cases, to the expansion above a threshold of the CGG repeats in the first exon of FMR1 gene. These expansions are concomitant with the methylation of the promoter and the silencing of FMR1. CONCLUSIONS: DNA methylation is a tag that enables different phenotypic expression from an identical nucleotide sequence. Aberrant methylation is the cause of different pathologies including fragile X syndrome. The comprehension of the mechanisms by which methylation induces the silencing of the genes and the study of agents that could revert this process are important for the treatment of these diseases.
DNA Methylation↗
DNA methylation and demethylating drugs in myelodysplastic syndromes and secondary leukemias.
BACKGROUND AND OBJECTIVES: Methylation of DNA is a common epigenetic modification that plays an important role in the control of gene expression in mammalian cells. This process involves CpG dinucleotide sequences and is catalyzed by DNA-methyltransferase enzymes. Under physiological conditions, methylated CpG sites are only present in DNA sequences typical of bulk chromatin, where the DNA is inaccessible to transcription factors. In contrast, CpG islands of promoter regions are usually unmethylated (with few exceptions such as the genes on the inactive X-chromosome). DNA methylation abnormalities have recently emerged as the most frequent molecular changes in hematopoietic neoplasms. INFORMATION SOURCES: The authors of the present review are currently working in the field of myelodysplastic syndromes and secondary leukemias and have contributed original papers to peer-reviewed journals. The material analyzed in the present review includes articles and reviews published in journals covered by the Science Citation Index, and abstracts presented at recent international oncology and hematology meetings. STATE OF THE ART: Methylation and transcriptional status are inversely correlated, the hypermethylation of genes involved in cell-cycle control and apoptosis could have a pathogenetic role in the development of cancer. In particular, high-risk myelodysplastic syndromes (MDS) and secondary leukemias (SL) show a high prevalence of tumor-suppressor gene hypermethylation. The use of irreversible DNA methyltransferase inhibitors, such as 5-azacytidine and decitabine, appears to be a promising therapeutic option for the treatment of MDS and SL. Large clinical trials are still ongoing, but preliminary data recently published indicate for the first time that the natural history of MDS may be changed by a non-intensive treatment. CONCLUSIONS AND PERSPECTIVES: Treatment with demethylating agents, 5-azacytidine and decitabine, at present results in significantly higher response rates, improved quality of life, reduced risk of leukemic transformation, and improved survival, when compared to supportive care. Azacytidine and decitabine provide a new treatment option, and should be the treatment choice for elderly patients with high risk MDS. It is whorty in fact that azacytidine and decitabine are especially active in patients with poor prognosis MDS. The combination with histone deacetylase inhibitors may increase the efficacy of hypomethylating agents in vivo.
Antimetabolites, Antineoplastic↗
Dependence of histone modifications and gene expression on DNA hypermethylation in cancer.
We examined the relationship between aberrant DNA hypermethylation and key histone code components at a hypermethylated, silenced tumor suppressor gene promoter in human cancer. In lower eukaryotes, methylated H3-lysine 9 (methyl-H3-K9) determines DNA methylation and correlates with repressed gene transcription. Here we show that a zone of deacetylated histone H3 plus methyl-H3-K9 surrounds a hypermethylated, silenced hMLH1 promoter, which, when unmethylated and active, is embedded in methyl-H3-K4 and acetylated H3. Inhibiting DNA methyltransferases, but not histone deacetylases, leads first to promoter demethylation, second to gene reexpression, and finally to complete histone code reversal. Our findings suggest a new paradigm-DNA methylation may directly, or indirectly by inhibiting transcription, maintain key repressive elements of the histone code at a hypermethylated gene promoter in cancer.
Acetylation↗
Decitabine has promising clinical activity in chronic myeloid leukemia.
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Antineoplastic Agents↗
[Inhibitory effect on proliferation of KG1a cell line by methyltransferase inhibitors].
To explore the possibility of a new therapeutic strategy for leukemia by intervening in the DNA methylation to re-express p15 suppressor gene, methylation inhibitors, 5-Aza-2'-deoxycytidine (5-Aza-CdR) and cell differentiation agent (CDAII) were used to treat myelogenous leukemia cell line KG1a in which p15 gene expression was suppressed due to DNA hypermethylation. The biological characteristics of KG1a cells untreated or treated with the agents were investigated and analyzed using morphology, methylation specific-PCR (MSP), (3)H-labeled microassay technique, restriction endonuclease reaction, flow cytometry and immunofluorescence methods. The results indicated that both agents showed concentration-dependent and time-dependent inhibition of cell proliferation. 5-Aza-CdR and CDAII induced apoptosis and cell differentiation with G(2) and G(0)/G(1) arrest respectively. Furthermore, DNA methyltransferase activity and level of methylation in genomic DNA were decreased and p15 protein was re-expressed partially. It is concluded that it is possible to treat leukemia by intervening in the DNA methylation using methyltransferase inhibitors and it is worth to make a thorough study on mechanism of the new strategy.
Azacitidine↗
[Prokaryotic DNA methyltransferases: the structure and the mechanism of interaction with DNA].
The review considers current views on the function of DNA methyltransferases (MTases) that belong to prokaryotic type II restriction-modification systems. A commonly accepted classification of MTases is described along with their primary and tertiary structures and molecular mechanisms of their specific interaction with DNA (including methylation). MTase inhibitors are also considered. Special emphasis is placed on the flipping of the target heterocyclic base out of the double helix and on the methods employed in its analysis. Base flipping is a fundamentally new type of DNA conformational changes and is also of importance in the case of other DNA-operating enzymes. MTases show unique sequence homology, and are similar in structure of functional centers and in the mechanism of methylation. These data contribute to the understanding of the general biological significance of methylation, since prokaryotic and eukaryotic MTases are structurally and functionally similar.
Catalysis↗
Inactivation of the RASSF1A in osteosarcoma.
We investigated the expression and mutation of three isoforms of the Ras effector RASSF1 in 10 primary osteosarcomas and 6 osteosarcoma cell lines. RASSF1A was not expressed in 40% (4/10) of the primary osteosarcomas and 83.3% (5/6) of the osteosarcoma cell lines. RASSF1B and RASSF1C expression was absent in 30% (3/10) and 0% (0/10) of primary tumors, and 100% (6/6) and 0% (0/6) of osteosarcoma cell lines, respectively. Treatment of these cell lines with the DNA methylation inhibitor 5-aza-2'-deoxycytidine reactivated the transcription of RASSF1A, but not that of RASSF1B or RASSF1C. No somatic mutations were noted in RASSF1 in either the primary tumors or cell lines. Our data indicate that epigenetic inactivation of RASSF1A by hypermethylation of its promoter region is a frequent event, and may play an important role in the tumorigenesis of osteosarcomas.
Azacitidine↗
[The study of 5-Aza-2'-deoxycytidine on transcription regulation of p16/CDKN2 gene demethylation in RKO human colorectal cell line].
OBJECTIVE: To explore the transcription regulation of DNA 5'CpG island demethylation on p16/CDKN2 tumor suppressor gene and effects of growth on RKO human colorectal cancer cell line. METHODS: RKO cell line was exposed to the specific demethylating agent, 5-Aza-2'-deoxycytidine, for seventy-two hours to detect whether the silencing of p16/CDKN2 cell cycle regulatory gene could be reversed. Methylation-specific PCR (MSP), T-A cloning and sequence analysis were evaluated for methylation status. Growth speed, expression of DNA methyltransferase mRNA, p16/CDKN2 mRNA and protein were determined by MTT assay, reverse transcription polymerase chain reaction (RT-PCR), Real-time PCR, immunohistochemistry and western blot. RESULTS: (1) All cytosines in CpG dinucleotides in untreated RKO cells with 5-Aza-2'-deoxycytidine remain as C, while all cytosines in treated RKO cells have been converted to thymidine. (2) RKO cell line after treatment with three different concentration 5-Aza-2'-deoxycytidine grew slowly and double time increased to 1.49, 1.64, 1.87-times respectively. (3) The expression of RKO cell p16/CDNK2 gene mRNA treated with 5-Aza-2'-deoxycytidine increased to 4.89, 16.91, 19.97-times respectively, but the expression of DNA methyltransferase mRNA was inhibited. (4) Immunohistochemistry and western blot indicated that 5-Aza-2'-deoxycytidine could increase the p16/CDKN2 gene protein expression. CONCLUSION: DNA promoter hypermethylation is associated with p16/CDKN2 gene silence in RKO human colorectal cancer cell line. 5-Aza-2'-deoxycytidine may effectively cause demethylation and inhibit the growth of tumor cell by reactivating the gene transcription silenced by aberrant hypermethylation.
Antimetabolites, Antineoplastic↗
Decitabine: development of a DNA methyltransferase inhibitor for hematological malignancies.
Decitabine (DAC) is a small molecule nucleotide analog that is incorporated into DNA and traps human DNA methyltransferases. Although initially developed as a cytotoxic agent, low-dose DAC is enjoying a revival as a specific inhibitor of hypermethylation in cancer. DAC has activity in several hematological diseases, especially myelodysplastic syndrome, chronic myelogenous leukemia and acute myeloid leukemia. Clinical and preclinical advances are presented in this review.
Animals↗
Antineoplastic action of 5-aza-2'-deoxycytidine (Dacogen) and depsipeptide on Raji lymphoma cells.
Epigenetic alterations, such as aberrant DNA methylation and histone deacetylation, can silence genes that suppress leukemogenesis. The objective of our study was to investigate the in vitro antineoplastic and gene re-activation activity of 5-aza-2'-deoxycytidine (5AZA), a potent inhibitor of DNA methylation, and depsipeptide (depsi), an inhibitor of histone deacetylase, on Raji lymphoma cells. The combination of 5AZA with depsi produced a significantly greater inhibition of growth and colony formation than either agent alone. Using RT-PCR, we observed that combination also produced a synergistic activation of E-cadherin, a gene that is silenced by aberrant DNA methylation in Raji cells. This latter interaction indicates that there is cross-talk between DNA methylation and histone modifications in chromatin for E-cadherin in this cell line. 5AZA and depsi may be an interesting drug combination to investigate in patients with lymphoma.
Antimetabolites, Antineoplastic↗
[Effect of 5-aza-2'-deoxycytidine on cell of high-risk patients with myelodysplastic syndrome in vitro].
To investigate the effect of 5-aza-2'-deoxycytidine (5-Aza-CdR) on cell of high-risk patients with myelodysplastic syndrome (MDS) in vitro, the growth inhibition of MUTZ-1 cell induced by 5-Aza-CdR was detected by MTT method; apoptosis was detected by morphological observation and translocation of phosphatidylserine (PS) was examined by flow cytometry assay; the expressions of P15INK4B, DNA methyltransferases (DNMT)(1), DNMT(3A) and DNMT(3B) gene on mRNA level were detected by RT-PCR; methylation of p15INK4B gene in MUTZ-1 cells was detected by PCR using methylation specific primer (MSP). The results showed that 5-Aza-CdR inhibited the growth of MUTZ-1 cells. The IC(50) values of 24, 48 and 72 hours were 6.75, 2.82 and 5.45 mmol/L respectively. Characteristic changes of apoptosis emerged in MUTZ-1 cells after being exposed to 5-Aza-CdR in the different concentration from 0.8 mmol/L to 3.2 mmol/L, and the positive cells of annexin V on the membrane of MUTZ-1 cells were analyzed by flow cytometry. 5-Aza-CdR could activate the p15INK4B gene expression in MUTZ-1 cells by demethylation of the p15INK4B gene in a dose-dependent manner after the cells were treated for 48 hours. Furthermore, 5-Aza-CdR could significantly down-regulate the expressions of DNA methyltransferase genes DNMT(3A) at mRNA level in a dose dependent manner. However, it had no effects on DNMT(1) gene and DNMT(3B) gene. It is concluded that 5-Aza-CdR can inhibit the growth of MUTZ-1 cells and induce the apoptosis of these cells within the range of concentration from 0.8 mmol/L to 3.2 mmol/L, which may be one of the mechanisms of antitumor effects of 5-Aza-CdR. The drug can activate the expression of p15INK4B gene in MUTZ-1 cells by demethylation of the p15INK4B gene through inhibiting the expression of DNMT(3A) gene. It may be the mechanism of 5-Aza-CdR in the treatments of MDS.
Azacitidine↗
Effects of DNA methylation inhibitor on down-regulation of ribonuclease inhibitor expression in cancer cell lines.
BACKGROUND & OBJECTIVE: Human ribonuclease inhibitor (RI) could effectively block angiogenin-induced angiogenesis, and inhibit growth of transplant solid tumors in animals. However, its exact molecular mechanism of antitumor has not been totally ascertained. Many tumor suppressor genes occur loss of expression by aberrant methylation in promoter region. Demethylation, treated with methylation inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR),can restore the gene expression. To further explore functions of RI, and investigate relationship between RI and tumorigenesis, the study was designed to explore effects of 5-Aza-CdR on expression of RI in cancer cell lines. METHODS: Human breast cancer cell line MCF-7, human gastric cancer cell line BGC-823, human prostate cancer cell line DU-145, and human colon cancer cell line HT-29 were treated with 5-Aza-CdR. Expression of RI was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, immunofluorescence, and immunocytochemistry. RESULTS: Expression of RI significantly elevated by 5-Aza-CdR at both mRNA and protein level in MCF-7, BGC-823, and DU-145 cells (P< 0.01). Compared with control cells, mRNA levels of RI in MCF-7, BGC-823, and DU-145 cells treated with 5-Aza-CdR were increased by percentages of 37.2%, 46.0%, and 32.4%, respectively; protein levels of RI were increased by percentages of 26.4%, 20.9%, and 24.4%, respectively; but no obvious change observed in HT-29 cells. CONCLUSION: RI gene may be involved in tumorigenesis of gastric, prostate, and breast cancer.
Azacitidine↗
[Growth and gene expression of leukemia cell after treated with methylation inhibitor 5-aza-2'-deoxycytidine].
OBJECTIVE: To investigate the mechanism of demethylation therapy of leukemia by 5-aza-2'-deoxycytidine (5-aza-CdR). METHODS: By using MTT test, NBT reduction reaction and DNA agarose gel electrophoresis, changes in proliferation, differentiation and apoptosis were observed in K562, HL-60 and fresh leukemia cells after treated with 5-aza-CdR. The mRNA expressions of DNMTs, p15, p53 and bcl-2 were measured by RT-PCR. The status of p15(INK4B) gene methylation was examined by methylation-specific PCR (MSP-PCR). RESULTS: The growth inhibition of K562, HL-60 and fresh leukemia cells displayed a dose and time-dependent manner after treated by 5-aza-CdR. The differentiation-inducing ability on HL-60 cells was obvious at 0.5 micromol/L of 5-aza-CdR. The up-regulation of p15 mRNA and p53 mRNA expression and down-regulation of bcl-2 mRNA expression were obvious as compared with the control, but the DNMTs expression was not significantly different from the control. The methylation status of p15 gene in fresh leukemia cells decreased gradually with increasing concentration of 5-aza-CdR. CONCLUSION: The proliferation of leukemia cells was obviously inhibited by 5-aza-CdR, its mechanism maybe related to the up-regulation of p15 and p53 genes and down-regulation of bcl-2 gene. The decrease of p15 gene methylation was associated with the competitive inhibition of 5-aza-CdR.
Antimetabolites, Antineoplastic↗
The mouse DNA methyltransferase 5'-region. A unique housekeeping gene promoter.
We have cloned and characterized 5'-flanking sequences of the DNA methyltransferase (MeTase) gene. DNA MeTase gene transcription is initiated at a few discrete sites: 343 and 90 base pairs upstream of the translation initiation site as determined by RNase protection and primer extension assays. The promoter sequences that regulate expression of DNA MeTase, as defined by chloramphenicol acetyltransferase assays, reside between position -171 and the transcription start site. The promoter of DNA MeTase does not contain TATAA or CAAT boxes and is unusual because it does not contain the CG-rich elements characteristic of TATAA-less housekeeping genes. The 5'-flanking region of DNA MeTase contains AP-1, AP-2 and glucocorticoid response elements, suggesting possible regulation by cellular signal transduction pathways. The base composition of the DNA MeTase promoter is markedly different from that of other housekeeping genes. Whereas most housekeeping genes are characterized by CG-rich areas in their 5'-flanking regions, the TG dinucleotide is over-represented in DNA MeTase 5'-flanking sequences, including a perfect tandem repeat of T/G between positions -685 and -650. DNA methylation patterns play an important role in the developmental regulation of gene expression in vertebrates. DNA MeTase activity is probably regulated to maintain this pattern of methylation. We suggest that the DNA MeTase promoter represents a new class of housekeeping gene promoters that was designed to ensure high fidelity regulation of gene expression.
3T3 Cells↗