Arsenic causes distinct gene expression changes in macrophages polarized in vitro with either interferon-γ or interleukin-4.

Arsenic exposure is correlated with atherosclerosis in epidemiological studies and in animal models. We have previously shown that arsenic exposure enhanced the atherosclerotic plaque size, increased the plaque lipid content, and decreased the plaque smooth muscle cell and collagen contents in the apolipoprotein E knockout (apoE-/-) mice. However, the percentage of plaque-resident macrophages, the primary drivers of atherosclerosis remained unchanged. Therefore, we hypothesized that although arsenic does not change the quantity of macrophages, it alters the macrophage transcriptome towards a proatherogenic state. To test this hypothesis, we used bone marrow-derived macrophages, polarized them to either interferon-γ (IFN-É£) stimulated, proinflammatory or interleukin-4 (IL-4) stimulated, alternatively activated macrophages in the presence or absence of 0.67 µM (50 ppb) arsenic and performed RNA sequencing. Arsenic exposure altered the gene expression of the macrophages in a subtype-specific manner. Most differentially expressed genes (88%) were altered specifically in either IFN-É£- or IL-4-stimulated macrophages, whereas in the remaining 12% of genes that changed in both cell types, did so in opposite directions. In IL-4-stimulated macrophages, arsenic significantly downregulated the genes involved in cholesterol biosynthesis and the chemokines CCL17/CCL22, whereas in IFN-É£-stimulated macrophages, the genes associated with the liver X receptor (LXR) pathway were downregulated by arsenic. Using a bone marrow transplant experiment, we validated that the deletion of LXRα from the hematopoietic compartment rescued arsenic-enhanced atherosclerosis in the apoE-/- mouse model. Together, these data suggest that arsenic modulates subtype-specific transcriptomic changes in macrophages and further emphasize the need to define macrophage heterogeneity in atherosclerotic plaques in order to evaluate the proatherogenic role of arsenic.

Sex-Specific Effects of Prenatal and Early Life Inorganic and Methylated Arsenic Exposure on Atherosclerotic Plaque Development and Composition in Adult ApoE-/- Mice.

BACKGROUND: Epidemiologic studies indicate that early life arsenic exposures are linked to an increased risk of cardiovascular diseases. Different oxidation and methylation states of arsenic exist in the environment and are formed in vivo via the action of arsenic (+3 oxidation state) methyltransferase (As3MT). Methylated arsenicals are pro-atherogenic postnatally, but pre- and perinatal effects are unclear. This is particularly important because methylated arsenicals are known to cross the placenta. OBJECTIVES: We tested the effects of early life exposure to inorganic and methylated arsenicals on atherosclerotic plaque formation and its composition in apolipoprotein E knock-out (apoE-/-) mice and evaluated whether apoE-/- mice lacking As3MT expression were susceptible to this effect. METHODS: We exposed apoE-/- or apoE-/-/As3MT-/- mice to 200 ppb inorganic or methylated arsenic in the drinking water from conception to weaning and assessed atherosclerotic plaques in the offspring at 18 wk of age. Mixed regression models were used to estimate the mean difference in each outcome relative to controls, adjusting for sex and including a random effects term to account for within-litter clustering. RESULTS: Early life exposure to inorganic arsenic, and more profoundly methylated arsenicals, resulted in significantly larger plaques in the aortic arch and sinus in both sexes. Lipid levels in these plaques were higher without a substantial difference in macrophage numbers. Smooth muscle cell content was not altered, but collagen content was lower. Importantly, there were sex-specific differences in these observations, where males had higher lipids and lower collagen in the plaque, but females did not. In mice lacking As3MT, arsenic did not alter the plaque size, although the size was highly variable. In addition, control apoE-/-/As3MT-/- mice had significantly larger plaque size compared with control apoE-/-. CONCLUSION: This study shows that early life exposure to inorganic and methylated arsenicals is pro-atherogenic with sex-specific differences in plaque composition and a potential role for As3MT in mice. https://doi.org/10.1289/EHP8171.

Using the Apolipoprotein E Knock-Out Mouse Model to Define Atherosclerotic Plaque Changes Induced by Low Dose Arsenic.

Arsenic exposure increases the risk of atherosclerosis, the gradual occlusion of the large arteries with fibro-fatty plaque. While epidemiologic data provide convincing evidence this is true at higher exposures, it is unclear whether this may occur at low arsenic exposures, near the maximum contaminant level of 10 ppb. We have previously shown that 200 ppb arsenite in the drinking water increased the atherosclerosis in apolipoprotein E knock-out (apoE-/-) mice after 13 weeks, but the effects of lower concentrations were unknown. Therefore, here, we analyzed the effects of oral exposure to arsenite from 10 to 200 ppb after 13 weeks. Importantly, we found that even at the lowest concentration of arsenite, there was a significant increase in atherosclerotic plaque size. In our previous studies, we found that arsenite exposure resulted in decreased smooth muscle cells (SMCs) and collagen within the plaque. This change is indicative of a less stable phenotype that could increase the risk of rupture and subsequently, myocardial infarct or stroke in humans. In addition, we observed that lipid increased within the plaque without concomitant increase in macrophage content, suggesting that the macrophages were retaining more lipid intracellularly. We also assessed these plaque components in apoE-/- mice exposed to 10-200 ppb arsenite. Interestingly, we observed that macrophage lipid accumulation occurred at lower concentrations than the decreased SMC/collagen content. Together these data suggest that in the apoE-/- model, low arsenite concentrations are pro-atherogenic and that macrophage lipid homeostasis is more sensitive to arsenite-induced perturbation than the SMCs.

The genomic landscape of two Burkitt lymphoma cases and derived cell lines: comparison between primary and relapse samples.

Relapse occurs in 10-40% of Burkitt lymphoma (BL) patients that have completed intensive chemotherapy regimens and is typically fatal. While treatment-naive BL has been characterized, the genomic landscape of BL at the time of relapse (rBL) has never been reported. Here, we present a genomic characterization of two rBL patients. The diagnostic samples had mutations common in BL, including MYC and CCND3. Additional mutations were detected at relapse, affecting important pathways such as NFκB (IKBKB) and MEK/ERK (NRAS) signaling, glutamine metabolism (SIRT4), and RNA processing (ZFP36L2). Genes implicated in drug resistance were also mutated at relapse (TP53, BAX, ALDH3A1, APAF1, FANCI). This concurrent genomic profiling of samples obtained at diagnosis and relapse has revealed mutations not previously reported in this disease. The patient-derived cell lines will be made available and, along with their detailed genetics, will be a valuable resource to examine the role of specific mutations in therapeutic resistance.

Effects of Inorganic Arsenic, Methylated Arsenicals, and Arsenobetaine on Atherosclerosis in the Mouse Model and the Role of As3mt-Mediated Methylation.

BACKGROUND: Arsenic is metabolized through a series of oxidative methylation reactions by arsenic (3) methyltransferase (As3MT) to yield methylated intermediates. Although arsenic exposure is known to increase the risk of atherosclerosis, the contribution of arsenic methylation and As3MT remains undefined. OBJECTIVES: Our objective was to define whether methylated arsenic intermediates were proatherogenic and whether arsenic biotransformation by As3MT was required for arsenic-enhanced atherosclerosis. METHODS: We utilized the apoE−/− mouse model to compare atherosclerotic plaque size and composition after inorganic arsenic, methylated arsenical, or arsenobetaine exposure in drinking water. We also generated apoE−/−/As3mt−/− double knockout mice to test whether As3MT-mediated biotransformation was required for the proatherogenic effects of inorganic arsenite. Furthermore, As3MT expression and function were assessed in in vitro cultures of plaque-resident cells. Finally, bone marrow transplantation studies were performed to define the contribution of As3MT-mediated methylation in different cell types to the development of atherosclerosis after inorganic arsenic exposure. RESULTS: We found that methylated arsenicals, but not arsenobetaine, are proatherogenic and that As3MT is required for arsenic to induce reactive oxygen species and promote atherosclerosis. Importantly, As3MT was expressed and functional in multiple plaque-resident cell types, and transplant studies indicated that As3MT is required in extrahepatic tissues to promote atherosclerosis. CONCLUSION: Taken together, our findings indicate that As3MT acts to promote cardiovascular toxicity of arsenic and suggest that human AS3MT SNPs that correlate with enzyme function could predict those most at risk to develop atherosclerosis among the millions that are exposed to arsenic. https://doi.org/10.1289/EHP806.

Tungsten Promotes Sex-Specific Adipogenesis in the Bone by Altering Differentiation of Bone Marrow-Resident Mesenchymal Stromal Cells.

Tungsten is a naturally occurring metal that increasingly is being incorporated into industrial goods and medical devices, and is recognized as an emerging contaminant. Tungsten preferentially and rapidly accumulates in murine bone in a concentration-dependent manner; however the effect of tungsten deposition on bone biology is unknown. Other metals alter bone homeostasis by targeting bone marrow-derived mesenchymal stromal cell (MSC) differentiation, thus, we investigated the effects of tungsten on MSCsin vitroandin vivoIn vitro, tungsten shifted the balance of MSC differentiation by enhancing rosiglitazone-induced adipogenesis, which correlated with an increase in adipocyte content in the bone of tungsten-exposed, young, male mice. Conversely, tungsten inhibited osteogenesis of MSCsin vitro; however, we found no evidence that tungsten inhibited osteogenesisin vivo Interestingly, two factors known to influence adipogenesis are sex and age of mice. Both female and older mice have enhanced adipogenesis. We extended our study and exposed young female and adult (9-month) male and female mice to tungsten for 4 weeks. Although tungsten accumulated to a similar extent in young female mice, it did not promote adipogenesis. Interestingly, tungsten did not accumulate in the bone of older mice; it was undetectable in adult male mice, and just above the limit of detect in adult female mice. Surprisingly, tungsten enhanced adipogenesis in adult female mice. In summary, we found that tungsten alters bone homeostasis by altering differentiation of MSCs, which could have significant implications for bone quality, but is highly dependent upon sex and age.

High-selenium lentil diet protects against arsenic-induced atherosclerosis in a mouse model.

BACKGROUND: Cardiovascular disease (CVD) is a major cause of death worldwide, and arsenic (As) intake, mainly through drinking water, is a well-known risk factor for CVD as well as other health problems. Selenium (Se) is a known antagonist to As toxicity. OBJECTIVE: We tested the potential of high-Se lentils from the Canadian prairies as a therapeutic food to alter the outcome of As-enhanced atherosclerosis. MATERIALS AND METHODS: Male ApoE(-/-) mice exposed to a moderate level of As (200ppb) in their drinking water, and control mice on tap water received one of three lentil diets: Se-deficient (0.009mg/kg), Se-adequate (0.16mg/kg) or Se-high (0.3mg/kg). After 13weeks, lesion formation in the aortic arch and sinus were assessed. Intralesional cellular composition, serum lipid levels and hepatic oxidative stress were assessed as well. RESULTS: Arsenic-exacerbated plaque formation was reduced in the sinus and completely abolished in the aortic arch of mice on the Se-fortified lentil diet, whereas lesions were increased in As-exposed mice on both the Se-deficient and Se-adequate diets. Notably, Se deficiency contributed to proatherogenic composition of serum lipids in As-exposed mice as indicated by high-density lipoprotein:low-density lipoprotein. At least adequate Se status was crucial for counteracting As-induced oxidative stress. CONCLUSION: This study is the first to show the potential of high-Se lentils to protect against As-triggered atherosclerosis, and this invites further investigations in human populations at risk from As contamination of their drinking water.

Arsenic Exposure Increases Monocyte Adhesion to the Vascular Endothelium, a Pro-Atherogenic Mechanism.

Epidemiological studies have shown that arsenic exposure increases atherosclerosis, but the mechanisms underlying this relationship are unknown. Monocytes, macrophages and platelets play an important role in the initiation of atherosclerosis. Circulating monocytes and macrophages bind to the activated vascular endothelium and migrate into the sub-endothelium, where they become lipid-laden foam cells. This process can be facilitated by platelets, which favour monocyte recruitment to the lesion. Thus, we assessed the effects of low-to-moderate arsenic exposure on monocyte adhesion to endothelial cells, platelet activation and platelet-monocyte interactions. We observed that arsenic induces human monocyte adhesion to endothelial cells in vitro. These findings were confirmed ex vivo using a murine organ culture system at concentrations as low as 10 ppb. We found that both cell types need to be exposed to arsenic to maximize monocyte adhesion to the endothelium. This adhesion process is specific to monocyte/endothelium interactions. Hence, no effect of arsenic on platelet activation or platelet/leukocyte interaction was observed. We found that arsenic increases adhesion of mononuclear cells via increased CD29 binding to VCAM-1, an adhesion molecule found on activated endothelial cells. Similar results were observed in vivo, where arsenic-exposed mice exhibit increased VCAM-1 expression on endothelial cells and increased CD29 on circulating monocytes. Interestingly, expression of adhesion molecules and increased binding can be inhibited by antioxidants in vitro and in vivo. Together, these data suggest that arsenic might enhance atherosclerosis by increasing monocyte adhesion to endothelial cells, a process that is inhibited by antioxidants.

Tungsten targets the tumor microenvironment to enhance breast cancer metastasis.

The number of individuals exposed to high levels of tungsten is increasing, yet there is limited knowledge of the potential human health risks. Recently, a cohort of breast cancer patients was left with tungsten in their breasts following testing of a tungsten-based shield during intraoperative radiotherapy. While monitoring tungsten levels in the blood and urine of these patients, we utilized the 66Cl4 cell model, in vitro and in mice to study the effects of tungsten exposure on mammary tumor growth and metastasis. We still detect tungsten in the urine of patients' years after surgery (mean urinary tungsten concentration at least 20 months post-surgery = 1.76 ng/ml), even in those who have opted for mastectomy, indicating that tungsten does not remain in the breast. In addition, standard chelation therapy was ineffective at mobilizing tungsten. In the mouse model, tungsten slightly delayed primary tumor growth, but significantly enhanced lung metastasis. In vitro, tungsten did not enhance 66Cl4 proliferation or invasion, suggesting that tungsten was not directly acting on 66Cl4 primary tumor cells to enhance invasion. In contrast, tungsten changed the tumor microenvironment, enhancing parameters known to be important for cell invasion and metastasis including activated fibroblasts, matrix metalloproteinases, and myeloid-derived suppressor cells. We show, for the first time, that tungsten enhances metastasis in an animal model of breast cancer by targeting the microenvironment. Importantly, all these tumor microenvironmental changes are associated with a poor prognosis in humans.

Genetic deletion of LXRα prevents arsenic-enhanced atherosclerosis, but not arsenic-altered plaque composition.

Arsenic exposure has been linked to an increased incidence of atherosclerosis. Previously, we have shown in vitro and in vivo that arsenic inhibits transcriptional activation of the liver X receptors (LXRs), key regulators of lipid homeostasis. Therefore, we evaluated the role of LXRα in arsenic-induced atherosclerosis using the apoE(-/-) mouse model. Indeed, deletion of LXRα protected apoE(-/-) mice against the proatherogenic effects of arsenic. We have previously shown that arsenic changes the plaque composition in apoE(-/-) mice. Arsenic decreased collagen content in the apoE(-/-) model, and we have observed the same diminution in LXRα(-/-)apoE(-/-) mice. However, the collagen-producing smooth muscle cells (SMCs) were decreased in apoE(-/-), but increased in LXRα(-/-)apoE(-/-). Although transcriptional activation of collagen remained the same in SMC from both genotypes, arsenic-exposed LXRα(-/-)apoE(-/-) plaques had increased matrix metalloproteinase activity compared with both control LXRα(-/-)apoE(-/-) and apoE(-/-), which could be responsible for both the decrease in plaque collagen and the SMC invasion. In addition, arsenic increased plaque lipid accumulation in both genotypes. However, macrophages, the cells known to retain lipid within the plaque, were unchanged in arsenic-exposed apoE(-/-) mice, but decreased in LXRα(-/-)apoE(-/-). We confirmed in vitro that these cells retained more lipid following arsenic exposure and are more sensitive to apoptosis than apoE(-/-). Mice lacking LXRα are resistant to arsenic-enhanced atherosclerosis, but arsenic-exposed LXRα(-/-)apoE(-/-) mice still present a different plaque composition pattern than the arsenic-exposed apoE(-/-) mice.

In vivo tungsten exposure alters B-cell development and increases DNA damage in murine bone marrow.

High environmental tungsten levels were identified near the site of a childhood pre-B acute lymphoblastic leukemia cluster; however, a causal link between tungsten and leukemogenesis has not been established. The major site of tungsten deposition is bone, the site of B-cell development. In addition, our in vitro data suggest that developing B lymphocytes are susceptible to tungsten-induced DNA damage and growth inhibition. To extend these results, we assessed whether tungsten exposure altered B-cell development and induced DNA damage in vivo. Wild-type mice were exposed to tungsten in their drinking water for up to 16 weeks. Tungsten concentration in bone was analyzed by inductively coupled plasma mass spectrometry and correlated with B-cell development and DNA damage within the bone marrow. Tungsten exposure resulted in a rapid deposition within the bone following 1 week, and tungsten continued to accumulate thereafter albeit at a decreased rate. Flow cytometric analyses revealed a transient increase in mature IgD(+) B cells in the first 8 weeks of treatment, in animals of the highest and intermediate exposure groups. Following 16 weeks of exposure, all tungsten groups had a significantly greater percentage of cells in the late pro-/large pre-B developmental stages. DNA damage was increased in both whole marrow and isolated B cells, most notably at the lowest tungsten concentration tested. These findings confirm an immunological effect of tungsten exposure and suggest that tungsten could act as a tumor promoter, providing leukemic "hits" in multiple forms to developing B lymphocytes within the bone marrow.

Exposure to moderate arsenic concentrations increases atherosclerosis in ApoE-/- mouse model.

Arsenic is a widespread environmental contaminant to which millions of people are exposed worldwide. Exposure to arsenic is epidemiologically linked to increased cardiovascular disease, such as atherosclerosis. However, the effects of moderate concentrations of arsenic on atherosclerosis formation are unknown. Therefore, we utilized an in vivo ApoE(-/-) mouse model to assess the effects of chronic moderate exposure to arsenic on plaque formation and composition in order to facilitate mechanistic investigations. Mice exposed to 200 ppb arsenic developed atherosclerotic lesions, a lower exposure than previously reported. In addition, arsenic modified the plaque content, rendering them potentially less stable and consequently, potentially more dangerous. Moreover, we observed that the lower exposure concentration was more atherogenic than the higher concentration. Arsenic-enhanced lesions correlated with several proatherogenic molecular changes, including decreased liver X receptor (LXR) target gene expression and increased proinflammatory cytokines. Significantly, our observations suggest that chronic moderate arsenic exposure may be a greater cardiovascular health risk than previously anticipated.

Inhibition of cytidine deaminase by zebularine enhances the antineoplastic action of 5-aza-2'-deoxycytidine.

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.

Importance of dose-schedule of 5-aza-2'-deoxycytidine for epigenetic therapy of cancer.

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.

Enhancement of antineoplastic action of 5-aza-2'-deoxycytidine by zebularine on L1210 leukemia.

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.

Enhancement of antineoplastic action of 5-aza-2'-deoxycytidine by phenylbutyrate on L1210 leukemic cells.

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.

Activation of expression of p15, p73 and E-cadherin in leukemic cells by different concentrations of 5-aza-2'-deoxycytidine (Decitabine).

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.