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Cytidine (CR) deaminase is a key enzyme in the catabolism of cytosine nucleoside analogues, since their deamination results in a loss of their pharmacological activity. In this report we have investigated the importance of CR deaminase with respect to the antineoplastic action of inhibitors of DNA methylation, 5-aza-2'-deoxycytidine (5-AZA-CdR) and zebularine. Zebularine has a dual mechanism of action, since it can also inhibit CR deaminase. The objective of our study was to investigate the importance of zebularine as an inhibitor of CR deaminase with respect to the antineoplastic action of 5-AZA-CdR. Using an in vitro clonogenic assay, we investigated the antineoplastic action of 5-AZA-CdR and zebularine, alone and in combination on wild type 3T3 murine fibroblasts and corresponding V5 cells transduced with CR deaminase gene to express a very high level of CR deaminase activity. The V5 cells were much less sensitive to 5-AZA-CdR than the wild type 3T3 cells. The addition of zebularine significantly enhanced the antineoplastic action of 5-AZA-CdR on V5 cells, but not 3T3 cells. Enzymatic analysis on CR deaminase purified from the V5 cells showed that zebularine is a competitive inhibitor of the deamination of 5-AZA-CdR. These in vitro observations are in accord with our in vivo study in mice with L1210 leukemia, which showed that zebularine increased the antileukemic activity of 5-AZA-CdR. Pharmacokinetic analysis also showed that zebularine increased the plasma level of 5-AZA-CdR during an i.v. infusion in mice. Our results indicate that the major mechanism by which zebularine enhances the antineoplastic action of 5-AZA-CdR is by inhibition of CR deaminase. These findings provide a rationale to investigate 5-AZA-CdR in combination with zebularine in patients with advanced leukemia. [ABSTRACT FROM AUTHOR]

Background: The inactivation of tumor suppressor genes (TSGs) by aberrant DNA methylation plays an important role in the development of malignancy. Since this epigenetic change is reversible, it is a potential target for chemotherapeutic intervention using an inhibitor of DNA methylation, such as 5-aza-2'-deoxycytidine (DAC). Although clinical studies show that DAC has activity against hematological malignancies, the optimal dose-schedule of this epigenetic agent still needs to be established.Methods: Clonogenic assays were performed on leukemic and tumor cell lines to evaluate the in vitro antineoplastic activity of DAC. The reactivation of TSGs and inhibition of DNA methylation by DAC were investigated by reverse transcriptase-PCR and Line-1 assays. The in vivo antineoplastic activity of DAC administered as an i.v. infusion was evaluated in mice with murine L1210 leukemia by measurement of survival time, and in mice bearing murine EMT6 mammary tumor by excision of tumor after chemotherapy for an in vitro clonogenic assay.Results: Increasing the DAC concentration and duration of exposure produced a greater loss of clonogenicity for both human leukemic and tumor cell lines. The reactivation of the TSGs (p57KIP2 in HL-60 leukemic cells and p16CDKN2A in Calu-6 lung carcinoma cells) and the inhibition of global DNA methylation in HL-60 leukemic cells increased with DAC concentration. In mice with L1210 leukemia and in mice bearing EMT6 tumors, the antineoplastic action of DAC also increased with the dose. The plasma level of DAC that produced a very potent antineoplastic effect in mice with leukemia or solid tumors was > 200 ng/ml (> 1 microM).Conclusion: We have shown that intensification of the DAC dose markedly increased its antineoplastic activity in mouse models of cancer. Our data also show that there is a good correlation between the concentrations of DAC that reduce in vitro clonogenicity, reactivate TSGs and inhibit DNA methylation. These results suggest that the antineoplastic action of DAC is related to its epigenetic action. Our observations provide a strong rationale to perform clinical trials using dose intensification of DAC to maximize the chemotherapeutic potential of this epigenetic agent in patients with cancer. [ABSTRACT FROM AUTHOR]

Epigenetic gene silencing by DNA methylation and histone methylation by EZH2 play an important role in the development of acute myeloid leukemia (AML). EZH2 catalyzes the trimethylation of histone H3-lysine 27-trimethylated (H3K27me3). These epigenetic alterations silence the expression of the genes that suppress leukemogenesis. Reversal of this gene silencing by 5-aza-2'-deoxycytidine (5-Aza-CdR), an inhibitor of DNA methylation, and by 3-deazaneplanocin-A (DZNep), an inhibitor of EZH2, results in synergistic gene reactivation and antileukemic interaction. The objective of this study is to determine if the addition of another epigenetic agent could further enhance the antileukemic action of these inhibitors of DNA and histone methylation. Vitamin C (Vit C) is reported to enhance the antineoplastic action of 5-Aza-CdR on AML cells. The mechanism responsible for this action of Vit C is due to its function as a cofactor of alpha-ketoglutarate-dependent dioxygenases (α-KGDD). The enhancement by Vit C of the catalytic activity of α-KGDD of the ten eleven translocation (TET) pathway, as well as of the Jumonji C histone demethylases (JHDMs), is shown to result in demethylation of DNA and histones, leading to reactivation of tumor suppressor genes and an antineoplastic effect. This action of Vit C has the potential to complement the antileukemic action of 5-Aza-CdR and DZNep. We observe that Vit C remarkably increases the antineoplastic activity of 5-Aza-CdR and DZNep against myeloid leukemic cells. An important step to bring this novel epigenetic therapy to clinical trial in patients with AML is the determination of its optimal dose schedule.

Most patients with acute myeloid leukemia (AML) have a poor prognosis. Curative therapy of AML requires the complete eradication of the leukemic stem cells (LSCs). One aspect of LSCs that is poorly understood is their low frequency in the total population of leukemic cells in AML patients. After each cell division of LSCs, most of the daughter cells lose their capacity for self-renewal. Investigations into the role of Isocitrate dehydrogenase (IDH) mutations in AML provide some insight on the regulation of the proliferation of LSCs. The primary role of IDH is to convert isocitrate to alpha-keto-glutarate (α-KG). When IDH is mutated, it converts α-KG to 2-hydroxyglutarate (2-HG), an inhibitor of the TET pathway and Jumonji-C histone demethylases (JHDMs). The demethylating action of these enzymes removes the epigenetic gene-silencing markers, DNA methylation, H3K27me3 and H3K9me2 and can lead to the differentiation of LSCs. This enzymatic action is blocked by 2-HG in mutated IDH (mut-IDH) AML patients, who can be induced into remission with antagonists of 2-HG. These observations suggest that there exists in cells a natural enzymatic mechanism that uses demethylation to reverse epigenetic gene-silencing, leading to a loss of the self-renewal capacity of LSCs. This mechanism limits the proliferative potential of LSCs. Epigenetic agents that inhibit DNA and histone methylation exhibit a synergistic antineoplastic action on AML cells. It is possible that the therapeutic potential of this epigenetic therapy may be enhanced by demethylation enzymes, resulting in a very effective treatment for AML.

Epigenetic alterations play an important role in the development of acute myeloid leukemia (AML) by silencing of genes that suppress leukemogenesis and differentiation. One of the key epigenetic changes in AML is gene silencing by DNA methylation. The importance of this alteration is illustrated by the induction of remissions in AML by 5-aza-2'-deoxycytidine (5-AZA-CdR, decitabine), a potent inhibitor of DNA methylation. However, most patients induced into remission by 5-AZA-CdR will relapse, suggesting that a second agent should be sought to increase the efficacy of this epigenetic therapy. An interesting candidate for this purpose is 3-deazaneplanocin A (DZNep). This analog inhibits EZH2, a histone methyltransferase that trimethylates lysine 27 histone H3 (H3K27me3), a marker for gene silencing. This second epigenetic silencing mechanism also plays an important role in leukemogenesis as shown in preclinical studies where DZNep exhibits potent inhibition of colony formation by AML cells. We reported previously that 5-AZA-CdR in combination with DZNep exhibits a synergistic antineoplastic action against human HL-60 AML cells and the synergistic activation of several tumor suppressor genes. In this report, we showed that this combination also induced a synergistic activation of apoptosis in HL-60 cells. The synergistic antineoplastic action of 5-AZA-CdR plus DZNep was also observed on a second human myeloid leukemia cell line, AML-3. In addition, 5-AZA-CdR in combination with the specific inhibitors of EZH2, GSK-126, or GSK-343, also exhibited a synergistic antineoplastic action on both HL-60 and AML-3. The combined action of 5-AZA-CdR and DZNep on global gene expression in HL-60 cells was investigated in greater depth using RNA sequencing analysis. We observed that this combination of epigenetic agents exhibited a synergistic activation of hundreds of genes. The synergistic activation of so many genes that suppress malignancy by 5-AZA-CdR plus DZNep suggests that epigenetic gene silencing by DNA and histone methylation plays a major role in leukemogenesis. Targeting DNA and histone methylation is a promising approach that merits clinical investigation for the treatment of AML.

Introduction: Curative chemotherapy should target cancer stem cells (CSCs). The key characteristics of CSCs are a block in differentiation and an epigenetic signature similar to embryonic stem cells (ESCs). Differentiation by ESCs and CSCs is suppressed by gene silencing through the polycomb repressive complex 2 (PRC2) and/or DNA methylation. PRC2 contains the EZH2 subunit, which catalyzes the trimethylation of histone 3 lysine 27, a gene silencing marker. It is possible to reverse this 'double lock' mechanism using a combination of inhibitors of EZH2 and DNA methylation (5-aza-2'-deoxycytidine), which exhibits remarkable synergistic antineoplastic activity in preclinical studies., Areas Covered: The authors discuss several specific EZH2 inhibitors that have been synthesized with antineoplastic activity. One such inhibitor, EPZ-6438 (E7438), has been shown to be effective against lymphoma in a Phase I study. The indirect EZH2 inhibitor, 3-deazaneplanocin-A (DZNep), also exhibits remarkable anticancer activity due to its inhibition of methionine metabolism., Expert Opinion: Agents that target EZH2 warrant Phase I trials. Due to its positive pharmacodynamics, DZNep merits a high priority for clinical investigation. Agents that show positive results in Phase I studies should be advanced to clinical trials for use in combination with 5-aza-2'-deoxycytidine due to the interesting potential of this epigenetic therapy to target CSCs.

Background: The silencing of tumor suppressor genes (TSGs) by aberrant DNA methylation occurs frequently in acute myeloid leukemia (AML). This epigenetic alteration can be reversed by 5-aza-2'-deoxcytidine (decitabine, 5-AZA-CdR). Although 5-AZA-CdR can induce complete remissions in patients with AML, most patients relapse. The effectiveness of this therapy may be limited by the inability of 5-AZA-CdR to reactivate all TSGs due to their silencing by other epigenetic mechanisms such as histone methylation or chromatin compaction. EZH2, a subunit of the polycomb repressive complex 2, catalyzes the methylation of histone H3 lysine 27 (H3K27) to H3K27me3. 3-Deazaneplanocin-A (DZNep), an inhibitor of methionine metabolism, can reactivate genes silenced by H3K27me3 by its inhibition of EZH2. In a previous report, we observed that 5-AZA-CdR, in combination with DZNep, shows synergistic antineoplastic action against AML cells. Gene silencing due to chromatin compaction is attributable to the action of histone deacetylases (HDAC). This mechanism of epigenetic gene silencing can be reversed by HDAC inhibitors such as trichostatin-A (TSA). Silent TSGs that cannot be reactivated by 5-AZA-CdR or DZNep have the potential to be reactivated by TSA. This provides a rationale for the use of HDAC inhibitors in combination with 5-AZA-CdR and DZNep to treat AML., Results: The triple combination of 5-AZA-CdR, DZNep, and TSA induced a remarkable synergistic antineoplastic effect against human AML cells as demonstrated by an in vitro colony assay. This triple combination also showed a potent synergistic activation of several key TSGs as determined by real-time PCR. The triple combination was more effective than the combination of two agents or a single agent. Microarray analysis showed that the triple combination generated remarkable changes in global gene expression., Conclusions: Our data suggest that it may be possible to design a very effective therapy for AML using agents that target the reversal of the following three epigenetic "lock" mechanisms that silence gene expression: DNA methylation, histone methylation, and histone deacetylation. This approach merits serious consideration for clinical investigation in patients with advanced AML.

Cytarabine (cytosine arabinoside) is one of the most effective drugs for the treatment of acute myeloid leukemia. The standard dose of cytarabine used to treat this leukemia is 100 mg per square meter. In an attempt to improve the effectiveness of cytarabine against acute myeloid leukemia, a high-dose treatment (3,000 mg per square meter) was introduced into therapy. The side effects of high-dose cytarabine was a major concern, especially its neurological toxicity. A review of recent clinical trials indicates that this high-dose cytarabine can be replaced by the intermediate-dose of 1,000 mg per square meter without loss of efficacy and with less toxicity. This is an important step to improve the efficacy of cytarabine for the treatment of acute myeloid leukemia. Despite the improvements in the therapy for this leukemia, the current overall survival rate for adult patients is less than 30%. To optimize the cytarabine therapy, it is important to determine how some leukemic stem cells survive treatment. Preclinical data suggest that survival of the leukemic stem cells could be due to the long 12 hour interval between infusions of cytarabine, which permits some leukemic cells to escape its S phase specific action. Among the other factors that can lead to leukemic cell survival are the high levels in the liver and spleen of cytidine deaminase, the enzyme that inactivates cytarabine and drug resistance due to deficiency in deoxycytidine kinase, the enzyme that activates the prodrug, cytarabine. Several approaches are proposed in this commentary to overcome these impediments with the goal of increasing the effectiveness of cytarabine for the treatment of acute myeloid leukemia.

Epigenetic analysis shows that many genes that suppress malignancy are silenced by aberrant DNA methylation in lung cancer. Many of these genes are interesting targets for reactivation by the inhibitor of DNA methylation, decitabine (5-aza-2'-deoxycytidine, DAC). A pilot study on intense dose DAC showed promising results in patients with metastatic non-small cell lung cancer (NSCLC). However, subsequent clinical studies using low dose DAC were not very effective against NSCLC and interest in this therapy diminished. Recently, interesting responses were observed in a patient with NSCLC following treatment with a combination of the related inhibitor of DNA methylation, 5-azacytidine, and an inhibitor of histone deacetylation. This finding has generated a renewed interest in the epigenetic therapy of lung cancer. Preclinical studies indicate that DAC has remarkable chemotherapeutic potential for tumor therapy. This epigenetic agent has a delayed and prolonged epigenetic action on tumor cells. This delayed action should be taken into consideration in the design and evaluation of clinical studies on DAC. Future research should be directed at finding the optimal dose-schedule of de DAC for the treatment of NSCLC.

5-Aza-2'-deoxycytidine (5-AZA-CdR, decitabine), an epigenetic drug that inhibits DNA methylation, is currently used to treat myelodysplastic syndrome (MDS), and is under investigation for treating acute myeloid leukemia (AML) and other malignancies. 5-AZA-CdR can reactivate tumor suppressor genes silenced by aberrant DNA methylation, a frequent event in all types of cancer. Because this epigenetic change is reversible, it is a good target for 5-AZA-CdR therapy. We have reviewed the preclinical data of 5-AZA-CdR to analyze the concentrations and exposure times required to eradicate cancer stem cells. We analyzed the dose-schedules used in animal models that show potent antineoplastic activity of 5-AZA-CdR. We attempted to correlate the preclinical data with the responses obtained in clinical trials of 5-AZA-CdR in patients with cancer. The pharmacokinetics and drug distribution of 5-AZA-CdR are key parameters because adequate therapeutic drug levels are required to eliminate cancer stem cells in all anatomic compartments. The plasma half-life of 5-AZA-CdR in humans is approximately 20 minutes due to the high levels in the liver of cytidine deaminase, the enzyme that inactivates this analogue. This provides a rationale to use an inhibitor of cytidine deaminase in combination with 5-AZA-CdR. Low-dose 5-AZA-CdR is effective for MDS and AML and can induce complete remissions (CR). However, maintenance of CR with low-dose 5-AZA-CdR is difficult. Based on analyses of preclinical and clinical data, low dose 5-AZA-CdR has the potential to be an effective form of therapy in some patients with cancer. For patients who do not respond to low dose therapy we recommend dose-intensive treatment with 5-AZA-CdR. Patients who are candidates for intensive dose 5-AZA-CdR should have a good bone marrow status so as to permit adequate recovery from myelosuppression, the major toxicity of 5-AZA-CdR. Solid tumors are also interesting targets for therapy with 5-AZA-CdR. Both low dose and intensive therapy with 5-AZA-CdR can reduce the proliferative potential of tumor stem cells in animal models. We propose novel dose schedules of 5-AZA-CdR for investigation in patients with cancer. The full chemotherapeutic potential of 5-AZA-CdR to treat cancer merits further clinical investigation and can only be realized when its optimal dose-schedule is determined.

5-Aza-2'-deoxycytidine (5-AZA-CdR, decitabine, Dacogen®) and 5-azacytidine (5-AC, Vidaza®) are epigenetic agents that have been approved for the clinical treatment of the hematological malignancy myelodysplastic syndrome (MDS) and are currently under clinical evaluation for the treatment of acute myeloid leukemia (AML). Most investigators currently classify 5-AZA-CdR and 5-AC as inhibitors of DNA methylation, which can reactivate tumor suppressor genes silenced by this epigenetic event. Examination of the pharmacology of these analogues reveals important differences with respect to their molecular mechanism of action. The action of 5-AZA-CdR is due to its incorporation into DNA. 5-AC is a riboside analogue that is incorporated primarily into RNA. A small fraction of 5-AC is converted to its deoxyribose form by ribonucleotide reductase and subsequently incorporated into DNA. The incorporation of 5-AC into RNA can interfere with the biological function of RNA and result in an inhibition protein synthesis. Microarray analysis revealed that both these analogues target the expression of different cohorts of genes. Preclinical studies show that 5-AZA-CdR is a more effective antileukemic agent than 5-AC. One explanation for this observation is that 5-AC blocks the progression of some leukemic cells from G1 into S phase, and this protects these cells from the chemotherapeutic action of this riboside analogue related to its incorporation into DNA. However, differences in chemotherapeutic efficacy of these related analogues have not been clearly demonstrated in clinical trials in patients with hematological malignancies. These observations should be taken into consideration in the design of new clinical trials using 5-AZA-CdR or 5-AC in patients with MDS and AML.

DNA methylation and histone methylation are both involved in epigenetic regulation of gene expression and their dysregulation can play an important role in leukemogenesis. Aberrant DNA methylation has been reported to silence the expression of tumor suppressor genes in leukemia. Overexpression of the histone methyltransferase, EZH2, a subunit of the polycomb group repressive complex 2 (PRC2), was observed to promote oncogenesis. This is due to aberrant gene silencing by the trimethylation of histone H3 lysine 27 (H3K27me3) by EZH2. Since both these epigenetic silencing events are reversible, they are interesting targets for chemotherapeutic intervention by using an inhibitor of DNA methylation, such as 5-aza-2'-deoxcytidine (5-AZA-CdR), and 3-deazaneplanocin-A (DZNep), an inhibitor of the EZH2. Human HL-60 and murine L1210 leukemic cells exposed in vitro to 5-AZA-CdR and DZNep in combination showed a synergistic loss of clonogenicity in a colony assay as compared to each agent alone. This positive chemotherapeutic interaction was also observed in mice with L1210 leukemia. Quantitative PCR showed that the combination also produced a remarkable synergistic activation of the tumor suppressor genes, CDKN1A and FBXO32. Microarray analysis showed that 5-AZA-CdR plus DZNep produced a synergistic activation of >150 genes. Our results indicate that 5-AZA-CdR plus DZNep can reactivate target genes that are silenced by two distinct epigenetic mechanisms leading to a loss of the proliferative potential of leukemic cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

New approaches should be sought to treat high-risk acute lymphoblastic leukemia (ALL). Since aberrant DNA methylation plays an important role in leukemogenesis of ALL, it can be targeted by 5-aza-2'-deoxycytidine (5-AZA-CdR), a potent inhibitor of DNA methylation. 5-AZA-CdR is a prodrug that is activated by deoxycytidine kinase (DCK). Leukemic cells lacking DCK are drug-resistant. In a previous phase I study, we reported that 5-AZA-CdR could induce remissions in ALL. However, some patients developed drug-resistance due to deficiency in DCK. These observations aroused our interest in 3-deazauridine (3-DU), a CTP synthetase inhibitor that is effective against leukemic cells deficient in DCK. In this report, we observed that 3-DU enhanced the in vitro antineoplastic action of 5-AZA-CdR on human leukemic cells by increasing its incorporation into DNA. Using an optimized dose-schedule we showed that this combination could cure some mice bearing L1210 leukemia, even in the presence of a subpopulation of drug-resistant (L1210/ARA-C) leukemic cells lacking DCK. 3-DU alone also cured some mice with L1210/ARA-C leukemia. In a pilot study on 3 relapsed patients with advanced ALL, the combination of 5-AZA-CdR and 3-DU produced a marked reduction in leukemic blasts, confirming our preclinical observations. Furthermore, after several treatments with these agents all three patients developed drug-resistance to 5-AZA-CdR as determined by an in vitro drug sensitivity test. In two patients we showed by enzymatic analysis that the drug-resistance was due to deficiency in DCK. Our preclinical and clinical results provide a strong rationale to further investigate the combination of 5-AZA-CdR and 3-DU for the treatment of advanced ALL., (Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.)

Background: Most patients with advanced Ewing's sarcoma (EWS) respond poorly to conventional chemotherapy, indicating the need for new treatment approaches. Epigenetic events, such as promoter hypermethylation and chromatin histone deacetylation, silence the expression of tumor suppressor genes (TSGs) and play an important role in tumorigenesis. These epigenetic changes can be reversed by using 5-aza-2'-deoxycytidine (5AZA-CdR), a potent inhibitor of DNA methylation, in combination with an inhibitor of histone deacetylase (HDAC)., Results: Here, we used a clonogenic assay to evaluate the in vitro antineoplastic activity of 5AZA-CdR in combination with different HDAC inhibitors on EWS cells. We observed that the HDAC inhibitors, MS-275, trichostatin-A, phenylbutyrate, LAQ824 and depsipeptide, enhanced the antineoplastic action of 5AZA-CdR on EWS cells. The combination of 5AZA-CdR and MS-275 showed marked synergy, and was correlated with significant reactivation of the expression of two TSGs, E-cadherin and tumor suppressor lung cancer-1 (TSLC1), in a EWS cell line., Conclusion: These results suggest the value of future clinical studies investigating the combination of 5AZA-CdR and MS-275 in patients with advanced EWS.

Soy has been used in traditional medicine for the treatment of various diseases, including cancer. The isoflavones present in soy have been shown in animal models to have cancer-preventing activity. However, the therapeutic effects of isoflavones against cancer are still unclear. We have evaluated the in vitro and in vivo antileukemic activity of genistein (1), a major isoflavone present in soy. We observed that it produced a dose- and time-dependent antineoplastic activity against myeloid and lymphoid leukemic cell lines. In addition, genistein treatment of the leukemic cells reactivated tumor suppressor genes that were silenced by aberrant DNA methylation. A genistein-enriched diet produced a moderate, but significant, antileukemic effect in mice. The limited extent of this in vivo response may have been due to the rapid metabolic inactivation of genistein in mice. Due to the longer half-life of genistein in humans, a soy-enriched diet has the potential to produce plasma levels of this isoflavone in the range of the concentrations used in vitro that produced an antileukemic activity.

5-Aza-2'-deoxycytidine (5-AZA-CdR), a potent inhibitor of DNA methylation, is an effective agent for the treatment of leukemia. The aim of this study was to investigate the antileukemic activity of this epigenetic agent in combination with genistein, a nontoxic isoflavone with chemopreventive activity. The combined treatment produced a synergistic loss of clonogenicity in human myeloid (HL-60) and lymphoid (MOLT-3) leukemic cell lines. Genistein alone showed a significant antileukemic activity against murine 5-AZA-CdR-resistant cells, and this effect was enhanced when used in combination with 5-AZA-CdR. The combined treatment also produced a synergistic increase in life span of mice with L1210 leukemia. These results suggest that genistein may have the potential to increase the clinical efficacy of 5-AZA-CdR for the treatment of leukemia.

Purpose: Epigenetic silencing of tumor suppressor genes (TSGs) by aberrant DNA methylation and chromatin deacetylation provides interesting targets for chemotherapeutic intervention by inhibitors of these events. 5-Aza-2'-deoxycytidine (decitabine, 5AZA-CdR) is a potent demethylating agent, which can reactivate TSGs silenced by aberrant DNA methylation. LAQ824 (LAQ) is a novel inhibitor of histone deacetylase (HDAC) that shows antineoplastic activity and can activate genes that produce cell cycle arrest. Both 5AZA-CdR and LAQ as single agents are currently under clinical investigation in patients with cancer. Previous reports indicate that the "cross-talk" between inhibitors of DNA methylation and HDAC can result in a synergistic activation of silent TSGs. These observations suggest that combination of these inhibitors may be an effective form of epigenetic therapy for breast cancer. The objective of our study was to determine if the combination of 5AZA-CdR and LAQ would show additive or synergistic antineoplastic activity on human MDA-MB-231 and MCF-7 breast carcinoma cells. The antineoplastic activity of these agents was evaluated by clonogenic assay and inhibition of DNA synthesis., Results: The combination produced greater antineoplastic activity for the MDA-MB-231 tumor cells than either agent alone. For the MCF-7 tumor cells, there were signs of antagonism between 5AZA-CdR and LAQ when administered simultaneously. When a sequential schedule (first 5AZA-CdR followed by LAQ) was used, there were no signs of antagonism of the antineoplastic action for the MCF-7 tumor cells. The mechanism of this interaction is probably due to the reduction of progression of MCF-7 tumor cells into S phase by LAQ. This would interfere with the antineoplastic action of 5AZA-CdR, since it is an S phase specific agent., Conclusions: These studies demonstrated the importance of the schedule of administration of 5AZA-CdR and LAQ and may have application for future clinical trials on the treatment of breast cancer with these agents.

Hepatitis C virus (HCV) infection represents an important global health problem. Current antiviral therapeutics for HCV have proven inadequate in stemming the disease process. A novel therapeutic strategy involves the use of deoxyribozymes, also known as DNA enzymes or DNAzymes. These catalytic DNA molecules, designed to target and cleave specific RNA sequences, have shown promise in in vitro experimental models for various diseases and may serve as an alternative or adjunct to current HCV drug therapy. We designed and tested several deoxyribozymes that can bind and cleave highly conserved RNA sequences encoding the HCV core protein in in vitro systems. One of these deoxyribozymes reduced the level of our HCV RNA target by 32% and 48% after 24 h of cell exposure when tested in human hepatoma and epithelial cell lines, respectively. As this deoxyribozyme showed significant cleavage activity against HCV core protein target RNA in human cells, it may have potential as a therapeutic candidate for clinical trial in HCV infected patients.

Epigenetic events, such as aberrant DNA methylation, have been demonstrated to silence the expression of many genes that suppress malignancy. Since the event is reversible, it is an interesting target for intervention with specific inhibitors of DNA methylation, such as 5-aza-2'-deoxycytidine (5-AZA-CdR, decitabine). 5-AZA-CdR is a prodrug that requires activation via phosphorylation by deoxcytidine kinase. The nucleotide analog is incorporated into DNA, where it produces an irreversible inactivation of DNA methyltransferase. 5-AZA-CdR is an S-phase-specific agent. The demethylation of DNA by this analog in neoplastic cells can lead to the reactivation of silent tumor-suppressor genes, induction of differentiation or senescence, growth inhibition, and loss of clonogenicity. 5-AZA-CdR was demonstrated to be a potent antineoplastic agent against leukemia and tumors in animal models. Preliminary clinical trials of 5-AZA-CdR using different dose-schedules have shown interesting antineoplastic activity in patients with leukemia, myelodysplastic syndrome (MDS), and non-small cell lung cancer (NSCLC). Pharmacokinetic studies have shown that 5-AZA-CdR has a short in vivo half-life of 15 to 25 minutes. The major toxicity produced by this analog is granulocytopenia. To exploit the full chemotherapeutic potential of 5-AZA-CdR for the treatment of cancer, its optimal dose-schedule has to be found. This will require a good understanding of the pharmacology of this analog and its action on both normal and neoplastic cells.

The preclinical pharmacology of 5-aza-2'-deoxycytidine (decitabine, 5AZA-CdR) is reviewed. 5AZA-CdR, an analogue of deoxycytidine, is a prodrug that requires metabolic activation by deoxycytidine kinase. The active inhibitor in the cell is its triphosphate form (5AZA-dCTP), which incorporates very readily into DNA to produce an inhibition of DNA methyltransferase. The mechanism responsible for the antileukemic action of 5AZA-CdR is related to its reversal of epigenetic silencing by aberrant DNA methylation of genes that suppress leukemiogenesis. 5AZA-CdR is an S-phase-specific agent. At concentrations in the range of micromolars this analogue can induce terminal differentiation and loss of clonogenicity of human leukemic cells. Drug resistance to 5AZA-CdR occurs primarily by reduction in deoxycytidine kinase activity or increase in the activity of cytidine deaminase, the enzyme that inactivates this analogue. 5AZA-CdR is a very potent antileukemic agent in animal models, more effective than the related antileukemic drug, cytosine arabinoside. In humans, 5AZA-CdR has a short half-life of 15 to 25 minutes due to rapid inactivation by liver cytidine deaminase. The major toxicity produced by 5AZA-CdR is myelosuppression. Preliminary clinical studies in patients with hematologic malignancies indicate that 5AZA-CdR is an active chemotherapeutic agent. The optimal dose-schedule for this interesting epigenetic agent with a novel mechanism of action remains to be determined. Translation of the pharmacology of 5AZA-CdR into therapeutic regimens based on scientific rationale can be used to obtain this objective.

Tumor suppressor genes that have been silenced by aberrant DNA methylation are potential targets for reactivation by novel chemotherapeutic agents. The potent inhibitor of DNA methylation and antileukemic agent, 5-aza-2'-deoxycytidine (5-AZA-CdR, Decitabine), can reactivate silent tumor suppressor genes. One hindrance to the curative potential of 5-AZA-CdR is its rapid in vivo inactivation by cytidine deaminase (CD). An approach to overcome this obstacle is to use 5-AZA-CdR in combination with zebularine (Zeb), a potent inhibitor of CD. Zeb also possesses independent antineoplastic activity due to its inhibition of DNA methylation. We tested the capacity of 5-AZA-CdR and Zeb alone and in combination to inhibit growth and colony formation of different leukemic cell lines. 5-AZA-CdR and Zeb in combination produced a greater inhibition of growth against murine L1210 lymphoid leukemic cells, and a greater reduction in colony formation by L1210 and human HL-60 myeloid leukemic cells, than either agent alone. The ability of these agents to reactivate the tumor suppressor gene, p57KIP2, was also tested using RT-PCR. The combination produced a synergistic reactivation of p57KIP2 in HL-60 leukemic cells. A methylation-specific PCR assay showed that this combination also induced a significantly greater demethylation level of the p57KIP2 promoter than either drug alone. The in vivo antineoplastic activity of the agents was evaluated in mice with L1210 leukemia. A greater increase in survival time of mice with L1210 leukemia was observed with the combination than with either agent alone using three different dose schedules. The enhanced activity observed with 5-AZA-CdR plus Zeb in both murine and human leukemic cells lines provides a rationale for the clinical investigation of these drugs in patients with advanced leukemia. The probable mechanism of this drug interaction involves inhibition of CD by Zeb and the complementary inhibition of DNA methylation by both agents.

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.

The antineoplastic activity of 5-aza-2'-deoxycytidine (5-AZA-CdR) and docetaxel (Taxotere, Taxo) alone or in combination against human MDA-MB-231 breast, Calu-6 lung and DU-145 prostate carcinoma cell lines was evaluated by clonogenic assay. We also investigated by RT-PCR the capacity of these agents to re-activate the expression of E-cadherin and maspin, two tumor suppressor genes that were silenced by DNA methylation. 5-AZA-CdR and Taxo in combination produced a greater loss of clonogenicity than either agent alone. In MDA-MB-231 breast carcinoma cells, Taxo did not interfere with the re-activation of E-cadherin and maspin genes by 5-AZA-CdR. These results provide a rationale for clinical trials on the combination of 5-AZA-CdR and Taxo in patients with advanced cancer.

Background: Inactivation of genes that suppress neoplasia by aberrant DNA methylation is a key event that occurs during the development of leukemia. The inhibitor of DNA methylation, 5-aza-2'-deoxycytidine (5AZA), which can re-activate these genes, is under clinical investigation for therapy of leukemia. The objective of this study was to determine the concentrations of 5AZA that will re-activate target silent genes in human leukemic cell lines., Materials and Methods: RT-PCR was used to evaluate the effect of concentrations of 1 to 100 ng/ml of 5AZA on the re-activation of p15 and p73 in KG1a myeloid leukemic cells and E-cadherin in HL-60 myeloid leukemic cells. The effect of 5AZA on inhibition of growth, DNA synthesis and colony formation in these cell lines was also investigated., Results: The extent of activation of the target genes was dependent on the concentration of 5AZA. For p15, pronounced activation was observed at 10 ng/ml or greater. For p73 and E-cadherin significant activation was observed at 100 ng/ml of 5AZA. Maximal inhibition of growth, DNA synthesis and colony formation occurred at 100 ng/ml., Conclusion: The in vitro antineoplastic and gene re-activation activity of 5AZA is dependent on the concentration of this analog. These data may be helpful in the design of the optimal dose-schedule of 5AZA for the clinical therapy of leukemia.

Epigenetic changes, such as aberrant DNA methylation that silences tumor suppressor genes (TSGs), can play an important role in the development of leukemia. The DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5-AZA-CdR), can reactivate these silent TSGs and is an interesting agent to investigate for therapy of leukemia. It has been reported that the effectiveness of 5-AZA-CdR to reactivate TSG can be enhanced by inhibitors of histone deacetylase (HDIs). HDIs can convert a compact chromatin structure to an open configuration that facilitates gene expression. An interesting HDI is phenylbutyrate (PB), which has shown some clinical activity for the therapy of leukemia. In this report we have investigated the antineoplastic activity of 5-AZA-CdR and PB alone and in combination on murine L1210 lymphoid leukemic cells. The in vitro treatment of 5-AZA-CdR and PB in combination produced a greater inhibition of growth, DNA synthesis, and also a greater reduction on colony formation on both L1210 and human HL-60 leukemic cells as compared to either drug alone. The combination also produced a synergistic activation of the TSG, p15CDN2B, in the L1210 cells. In mice with L1210 leukemia the combination showed enhanced antineoplastic activity. We also observed an enhancement of the antineoplastic activity of this combination in mice with L1210 leukemia. These data provide a rationale to investigate 5-AZA-CdR and PB in patients with advanced leukemia.

Troxacitabine (beta-L-Dioxolane-cytidine; Troxatyl) is a beta-L-nucleoside analog, which has shown preclinical antitumor activity in human xenograft tumor models and antileukemic response in patients with relapsed myeloid leukemia. Troxacitabine is activated by cellular kinases and incorporated into DNA, inhibiting its replication. In contrast to other cytosine nucleoside analogs, troxacitabine is resistant to inactivation by cytidine deaminase (CD). In this study we have investigated the effects of increased intracellular levels of CD on the antineoplastic action of troxacitabine and the related antileukemic drug, cytosine arabinoside (ARA-C). Retroviral transduction of the human CD gene in A549 lung carcinoma cells (A549-CD cells) markedly increased the expression of this gene. The A549-CD cells were more resistant to the cytotoxic action of ARA-C than the wild type A549 cells as determined by clonogenic assays. In contrast, the CD-transduced cells were as or more sensitive to the cytotoxic action of troxacitabine than the wild type cells. These results suggest that troxacitabine may be an effective antineoplastic agent against tumors with high levels of CD that show drug resistance to cytosine nucleoside analogs.

Aberrant DNA methylation of the promoter region is a key mechanism for inactivation of genes that suppress tumorigenesis. Genes that are involved in every step of tumor formation can be silenced by this mechanism. Inhibitors of DNA methylation, such as 5-azadeoxycytidine (5AZA), can reverse this epigenetic event suggesting a potential use in cancer therapy. The structure of chromatin can also play an important role with respect to the regulation of gene expression. Chromatin containing hypoacetylated lysines in histones has a compact structure that is repressive for transcription. Inhibitors of histone deacetylase (HDAC) can convert chromatin to an open structure and activate certain genes that inhibit tumor growth. These HDAC inhibitors also have potential in cancer therapy. A 'cross-talk' between DNA methylation and histone deacetylation can occur and work in concert to silence gene expression. The molecular mechanism involves the attachment of a methylated CpG binding protein (MBP) to the methylated promoters and its recruitment of HDAC to form a complex that suppresses transcription. These two epigenetic modifications represent an interesting target for therapeutic intervention using 5AZA and HDAC inhibitors. These agents in combination have been shown to produce a synergistic reactivation of tumor suppressor genes and an enhanced antineoplastic effect against tumor cells, and should be investigated as a novel form of epigenetic therapy for cancer.

During the development of leukemia, genes that suppress growth and induce differentiation can be silenced by aberrant DNA methylation and by changes in chromatin structure that involve histone deacetylation. It has been reported that a positive interaction between DNA methylation and histone deacetylation takes place to inhibit transcription. Based on this observation, our working hypothesis was that a combination of inhibitors of these processes should produce an enhancement of their antineoplastic activity on leukemic cells. The cytosine nucleoside analog, 5-aza-2'-deoxycytidine (5AZA), is a potent inhibitor of DNA methylation, which can activate tumor suppressor genes in leukemic cells that have been silenced by aberrant methylation. In clinical trials, 5AZA was demonstrated to be an active antileukemic agent. Histone deacetylase inhibitors (HDI) can also activate gene expression in leukemic cell lines by producing changes in chromatin configuration, and show antineoplastic activity in preclinical studies. In this report, we investigated the in vitro antineoplastic activity of 5AZA, alone and in combination with the HDI, trichostatin A (TSA) and depsipeptide (FR901228, depsi), on the human myeloid leukemic cell lines, HL-60 and KG1a. The results showed that the combination of 5AZA with TSA or depsi produced a greater inhibition of growth and DNA synthesis and a greater loss of clonogenicity than either agent alone. These results suggest that 5AZA used in combination with HDI may be an interesting chemotherapeutic regimen to investigate in patients with acute myeloid leukemia that is resistant to conventional chemotherapy.

Genes that suppress tumorigenesis can be silenced by epigenetic events, such as aberrant DNA methylation and modification of chromatin structure. Inhibitors of DNA methylase and histone deacetylase (HDAC) can potentially reverse these events. The aim of this study was to determine the in vitro antineoplastic activity of 5-aza-2'-deoxycytidine (5-AZA-CdR), a potent inhibitor of DNA methylase, in combination with depsipeptide (depsi), an inhibitor of HDAC, on human breast carcinoma cells. We observed a synergistic antineoplastic interaction between 5-AZA-CdR and depsi in their capacity to inhibit colony formation of Hs578T and MCF-7 breast carcinoma cells. In order to understand the molecular mechanism of this interaction, we investigated the effect of these drugs on the activation of the 14-3-3sigma, E-cadherin and tissue inhibitor of metalloproteinase 3 (TIMP3) cancer-related genes, which were reported to be silenced by aberrant methylation in many breast tumor cell lines. 14-3-3sigma was reported to produce G cell cycle arrest following DNA damage. E-cadherin and TIMP3 function as suppressors of tumor metastasis. Semi-quantitative RT-PCR was used to determine the effect of the co-administration of 5-AZA-CdR and depsi on four breast carcinoma cell lines for the reactivation of these genes. We observed a synergistic activation of E-cadherin by the combination in Hs578T, MDA-MB-231 and MDA-MB-435 tumor cells. For 14-3-3sigma, we demonstrated an additive to synergistic activation by the combination for Hs578T and MDA-MB-435 tumor cells, respectively. In the MCF-7 tumor cells, the drug combination produced a synergistic activation of TIMP3. The association between the synergistic antineoplastic activity and the synergistic activation of the target genes in this study suggests that the mechanism of anticancer activity of 5-AZA-CdR, in combination with depsi, is probably related to their enhanced activation of different types of tumor suppressor genes that have been silenced by epigenetic events.(2), (Copyright 2003 Lippincott Williams & Wilkins)

During tumorigenesis, cancer-related genes can be silenced by aberrant DNA methylation and by changes in chromatin structure. It has been reported that 5-aza-2'-deoxycytidine, a potent inhibitor of DNA methylation, in combination with histone deacetylase inhibitors, can produce a synergistic reactivation of these genes. The aim of our study was to investigate the in vitro antineoplastic activity of 5-aza-2'-deoxycytidine in combination with depsipeptide, a potent histone deacetylase inhibitor, against MDA-MB-231 and MDA-MB-435 human breast carcinoma cell lines. We observed that the combination of 5-aza-2'-deoxycytidine and depsipeptide produced a synergistic antineoplastic effect against these tumor cells as compared to either agent administered alone. We also investigated the effect of this drug combination on the activation of maspin and gelsolin expression. These 2 genes whose function is to suppress tumor metastasis have been reported to be silenced by epigenetic events in breast cancer. Using semi-quantitative RT-PCR, we observed that 5-aza-2'-deoxycytidine in combination with depsipeptide produced a greater reactivation of both maspin and gelsolin as compared to each agent alone. The synergistic interaction between 5-aza-2'-deoxycytidine and depsipeptide on breast carcinoma cell lines provides a rationale to investigate this interesting drug combination in future clinical trials on patients with advanced breast cancer., (Copyright 2002 Wiley-Liss, Inc.)

Current chemotherapy of advanced non-small cell lung cancer produces only a modest increase in survival time. New approaches are needed to improve its effectiveness. During tumorigenesis, silencing of tumor suppressor genes can occur by aberrant methylation. The DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5-AZA-CdR), can reactivate the expression of these genes. Nucleosomes containing unacetylated positively charged histones bind tightly to DNA producing a compact configuration, which inhibits transcription. Phenylbutyrate (PB), an inhibitor of histone deacetylase (HDAC), increases histone acetylation, neutralizing its positive charge and resulting in DNA with a more open structure, which favors transcription. It has been reported that 5-AZA-CdR in combination with HDAC inhibitor can increase the expression of silent tumor suppressor genes. The objective of our study was to determine if these agents, in combination, produce an enhancement of their antitumor activity. We evaluated the antineoplastic activity of 5-AZA-CdR and PB alone or in combination on human A549 and Calu-6 lung carcinoma cell lines by inhibition of DNA synthesis and clonogenic assays. 5-AZA-CdR and PB in combination produced a greater inhibition of DNA synthesis than either agent alone. Also, in a clonogenic assay the combination of these drugs showed a significant synergistic antitumor effect. These results provide a rationale to investigate the combination of 5-AZA-CdR and PB in patients with advanced lung cancer.

Deoxycytidine nucleoside analogs must be first phosphorylated to become active anticancer drugs. The rate-limiting enzyme in this pathway is deoxycytidine kinase (dCK). Cells deficient in this enzyme are resistant to these analogs. To evaluate the potential of dCK to be used as suicide gene for deoxycytidine nucleoside analogs, we transduced both human A-549 lung carcinoma and murine NIH3T3 fibroblast cell lines with this gene. The dCK-transduced cells showed an increase in cytotoxicity to the analogs, cytosine arabinoside (ARA-C), and 5-aza-2'-deoxycytidine (5-AZA-CdR). Unexpectedly, the related analog, 2',2'-difluorodeoxycytidine (dFdC), was less cytotoxic to the dCK-transduced cells than the wild-type cells. For the A-549-dCK cells, the phosphorylation of dFdC by dCK was much greater than control cells. In accord with the elevated enzyme activity, we observed a 6-fold increased dFdC incorporation into DNA and a more pronounced inhibition of DNA synthesis in the A-549-dCK cells. In an attempt to clarify the mechanism of dFdC, we investigated its action on A549 and 3T3 cells transduced with both cytidine deaminase (CD) and dCK. We reported previously that overexpression of CD confers drug resistance to deoxycytidine analogs. In this study, when the CD-transduced cells were also transduced with dCK they became relatively more sensitive to dFdC. In addition, we observed that dFdU, the deaminated form of dFdC, was cytotoxic to the A-549-dCK cells, but not the wild-type cells. Our working hypothesis to explain these results is that the mitochondrial thymidine kinase (TK2), an enzyme reported to phosphorylate dFdC, acts as an important modulator of dFdC-induced cell toxicity. These findings may further clarify the action of dFdC and the mechanism by which it induces cell death.

Although new agents and drug combinations have increased the response rate in advanced non-small cell lung cancer (NSCLC), long-term survivors are rare. There is an urgent need to develop new chemotherapeutic approaches for disease. In a previous pilot phase I-II study on 5-aza-2'-deoxycytidine (5-AZA-CdR) in patients with stage IV NSCLC, we observed several interesting responses, including one patient that was still alive (68 months) at the time of publication of our results. In the present report, we want to point out the long-term follow up of this patient, who survived 81 months, and discuss the interesting mechanism of action of 5-AZA-CdR that may have been responsible for this interesting response. 5-AZA-CdR is a potent inhibitor of DNA methylation. Recent progress in this field has shown that aberrant methylation of the promoter region of tumor suppressor genes inhibits their expression. This epigenetic event can contribute to tumorigenesis. Since 5-AZA-CdR can reactivate these genes by blocking DNA methylation, it has the potential to reverse tumorigenesis. This novel mode of action makes it an interesting agent to investigate for the chemotherapy of malignant disease, including lung cancer.

Hematopoietic toxicity produced by most anticancer drugs limits their potential for curative therapy. We have shown previously that the human cytidine deaminase (CD) gene can confer drug resistance in murine bone marrow cells (BMCs) to the nucleoside analog, cytosine arabinoside (ARA-C). In the present study, as the first objective we showed that the CD gene can also render drug resistance in BMCs to related analogs, 2',2'-difluorodeoxycytidine (dFdC) and 5-azadeoxycytidine (5-AZA-CdR). As a second objective, we investigated the potential of ex vivo selection with cytosine nucleoside analogs of CD-transduced BMC. The goal of this approach was to enrich the fraction of CD-transduced BMCs so as to increase the transgene expression and level of drug resistance before transplantation. This strategy may have the potential to circumvent the problem in clinical gene therapy of low level of gene transfer and adequate long-term gene expression. Using a bicistronic retroviral vector containing the CD and the green fluorescent protein (CDiGFP), we transduced murine L1210 leukemic cells. All three analogs, ARA-C, dFdC, and 5-AZA-CdR were demonstrated in vitro to enrich (>95%) the population of leukemic cells expressing the GFP transgene. However, with CD-transduced primary murine BMCs cultivated at high cell density we observed that in vitro selection with ARA-C was not possible due to release of CD into the culture medium at amounts that were sufficient to inactivate the analog. The CD-containing medium produced a chemoprotective effect on mock BMCs as shown by lack of significant growth inhibition in the presence of ARA-C. However, at low cell density in a cell mixture containing CD-transduced cells, the mock BMCs showed marked drug sensitivity to ARA-C as determined by clonogenic assay. Selection with ARA-C was shown to significantly increase the CD enzyme activity in transduced BMC. These results suggest that CD gene has the potential to be a good selectable marker and a possible tool for chemoprotection in cancer gene therapy.

Purpose: During tumorigenesis several cancer-related genes can be silenced by aberrant methylation. In many cases these silenced genes can be reactivated by exposure to the DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5-AZA-CdR). Histone acetylation also plays a role in the control of expression of some genes. The aim of this study was to determine the antineoplastic activities of 5-AZA-CdR and trichostatin A (TSA), either administered alone or in combination. in MDA-MB-231 breast carcinoma cells. The effects of these drugs (alone and in combination) on the expression of the tumor suppressor gene, retinoic acid receptor (RAR beta) and of the estrogen receptor alpha gene (ER alpha), whose expression is lost in the cell line used in the study, were also investigated., Methods: MDA-MB-231 cells were treated with 5-AZA-CdR and TSA and the antitumor activity of these drugs was determined by clonogenic assay. Total RNA was extracted from the treated cells and RT-PCR was used to determine the effect of the treatment on the expression of RAR beta and ER alpha. Methylation-sensitive PCR analysis was used to confirm that lack of expression of both genes was due to hypermethylation of their promoter regions. A single nucleotide primer extension assay was also used to quantify the reduction in DNA methylation following drug treatment., Results: Both 5-AZA-CdR and TSA alone showed significant antineoplastic activity. The combination of the two drugs was synergistic with respect to MDA-MB-231 cell kill. 5-AZA-CdR alone weakly activated the expression of both RAR beta and ER alpha. TSA alone only activated RAR beta, but not ER alpha. The combination of these agents appeared to produce a greater activation of both genes., Conclusions: The interesting interaction between 5-AZA-CdR and TSA in both cell kill and cancer-related gene reactivation provides a rationale for the use of inhibitors of DNA methylation and histone deacetylation in combination for the chemotherapy of breast cancer.

Hematopoietic toxicity is the dose-limiting side effect produced in cancer chemotherapy with deoxycytidine nucleoside analogs. Deletion of the deoxycytidine kinase (dCK), results in a drug resistance phenotype to these analogs. An interesting gene therapy strategy to confer drug resistance to cytosine nucleoside analogs would be to specifically inactivate the dCK in normal hematopoietic stem cell. In this study, we designed hammerhead ribozymes that can specifically cut and downregulate the murine dCK mRNA. Three different ribozymes were identified and shown to cleave in vitro the dCK RNA. After introduction of ribozyme cDNA into murine L1210 leukemic cells by retroviral transfer, two of the ribozymes showed some capacity in reducing dCK activity. However, analysis of transduced L1210 clones showed that the significant reduction in the dCK mRNA was not sufficient to confer drug resistance to cytosine arabinoside. Nevertheless, these results provide a new avenue of modulating the dCK enzyme activity and with improved modifications may have the potential for use in gene therapy to confer drug resistance to deoxycytidine analogs., (Copyright 2000 Academic Press.)

The expression of several cancer-related genes has been reported to be silenced by DNA methylation of their promoter region. 5-Aza-2'-deoxycytidine (5-AZA-CdR), a potent and specific inhibitor of DNA methylation, can reactivate the in vitro expression of these genes. In future clinical trials in tumor therapy with 5-AZA-CdR a method to quantitate its inhibition of methylation of specific tumor suppressor genes would provide important data for the analysis of the therapeutic efficacy of this analogue. We have modified the methylation-sensitive single-nucleotide primer extension assay reported by Gonzalgo and Jones (Nucleic Acids Res. 25, 2529-2531, 1997). Genomic DNA was treated with bisulfite and a fragment of the promoter region of the human retinoic acid receptor beta (RARbeta) gene, a tumor suppressor gene, was amplified using seminested PCR. Using two different primers we quantitated the inhibition of methylation produced by 5-AZA-CdR at two specific CpG sites in the RARbeta promoter in a human colon and a breast carcinoma cell line. The results obtained with the modified assay show a precise and reproducible quantitation of inhibition of DNA methylation produced by 5-AZA-CdR in tumor cells.

The transfer of drug resistance genes into hematopoietic cells is an experimental approach to protect patients from drug-induced myelosuppression. Because anti-cancer drugs are often administered in combination to increase their clinical efficacy, vectors that express two drug resistance genes are being developed to broaden the spectrum of chemoprotection. We have constructed a bicistronic vector, MFG/GST-IRES-CD (MFG/GIC) coexpressing rat glutathione S-transferase (GST) A3 isoform (rGST Yc1) and human cytidine deaminase (CD). Murine NIH 3T3 fibroblast cells transduced with this vector were evaluated for their resistance to nitrogen mustards and cytosine nucleoside analogs. GIC-transduced polyclonal cell populations (GIC cells) demonstrated marked increases in selenium-independent glutathione peroxidase (peroxidase) and CD activities, as well as increased resistance to melphalan (2.3-fold), chlorambucil (3.4-fold), and cytosine arabinoside (Ara-C) (8.1-fold). After selection with Ara-C, the peroxidase and CD activities of GIC cells were augmented 2.6- and 2.9-fold, respectively, in comparison with unselected cells, and the resistance to melphalan, chlorambucil, and Ara-C was further increased to 3.7-, 5.9-, and 53-fold, respectively. Melphalan selection of GIC cells likewise augmented their peroxidase (2.3-fold) and CD (1.9-fold) activities. GIC cells proliferated in the simultaneous presence of melphalan and Ara-C at drug concentrations that completely inhibited the growth of untransduced cells. The growth rate of unselected GIC cells exposed to the drug combination averaged 18% that of drug-free cultures. The growth rate of GIC cells exposed to the drug combination increased to 30% of controls after Ara-C selection and to 50% after melphalan selection. Our results suggest that retroviral transfer of MFG/GIC may be useful for chemoprotection against the toxicities of nitrogen mustards and cytosine nucleoside analogs.

The methylation of DNA is an epigenetic modification that can play an important role in the control of gene expression in mammalian cells. The enzyme involved in this process is DNA methyltransferase, which catalyzes the transfer of a methyl group from S-adenosyl-methionine to cytosine residues to form 5-methylcytosine, a modified base that is found mostly at CpG sites in the genome. The presence of methylated CpG islands in the promoter region of genes can suppress their expression. This process may be due to the presence of 5-methylcytosine that apparently interferes with the binding of transcription factors or other DNA-binding proteins to block transcription. In different types of tumors, aberrant or accidental methylation of CpG islands in the promoter region has been observed for many cancer-related genes resulting in the silencing of their expression. How this aberrant hypermethylation takes place is not known. The genes involved include tumor suppressor genes, genes that suppress metastasis and angiogenesis, and genes that repair DNA suggesting that epigenetics plays an important role in tumorigenesis. The potent and specific inhibitor of DNA methylation, 5-aza-2'-deoxycytidine (5-AZA-CdR) has been demonstrated to reactivate the expression most of these "malignancy" suppressor genes in human tumor cell lines. These genes may be interesting targets for chemotherapy with inhibitors of DNA methylation in patients with cancer and this may help clarify the importance of this epigenetic mechanism in tumorigenesis., (Copyright 2000 Wiley-Liss, Inc.)