Gemcitabine/Cisplatin Treatment Induces Concomitant SERTAD1, CDKN2B and GADD45A Modulation and Cellular Changes in Bladder Cancer Cells Regardless of the Site of TP53 Mutation.

Simultaneous use of cisplatin (CIS) and gemcitabine (GEN) for treating bladder cancer has increased because of their complementary effects. However, the molecular mechanisms underlying the activities of these two antineoplastic drugs are not fully known. Here, molecular biology techniques and microscopy were used to investigate transcriptomic and morphological changes in low and high-grade urinary bladder transitional carcinoma cell lines [RT4 - wild type TP53; 5637 - two TP53 mutations, one in codon 72 (Arg-Pro) and other in codon 280 (Arg-Thr) and T24 - in-frame deletion of tyrosine 126 in the TP53 allele] simultaneously treated with CIS/GEN. Gene expression profile was evaluated by PCR arrays; cell morphology by scanning and transmission electron microscopy, and apoptosis was analyzed using fluorescent dye. Results showed concomitantly upregulation of CDKN2B (G1/S transition), GADD45A (DNA repair and apoptosis) and SERTAD1 (regulation of transcription) gene, increased number of nuclear chamfers and apoptotic cells, and reduced number of microfilaments, organelles and in the size of the nucleus in 5637 and T24 cells after simultaneous treatment with CIS/GEN. In conclusion, independently of the TP53 mutation status and tumor grade, CIS/GEN induced gene modulation accompanied by changes in cell morphologies, which confirm the antiproliferative activity of the treatment protocol. These findings help to understand the pathways modulated by these antineoplastic agents and may provide insights for anti-cancer chemotherapy.

Increased mitochondrial DNA copy number in occupations associated with low-dose benzene exposure.

BACKGROUND: Benzene is an established leukemogen at high exposure levels. Although low-level benzene exposure is widespread and may induce oxidative damage, no mechanistic biomarkers are available to detect biological dysfunction at low doses. OBJECTIVES: Our goals were to determine in a large multicenter cross-sectional study whether low-level benzene is associated with increased blood mitochondrial DNA copy number (mtDNAcn, a biological oxidative response to mitochondrial DNA damage and dysfunction) and to explore potential links between mtDNAcn and leukemia-related epigenetic markers. METHODS: We measured blood relative mtDNAcn by real-time polymerase chain reaction in 341 individuals selected from various occupational groups with low-level benzene exposures (> 100 times lower than the Occupational Safety and Health Administration/European Union standards) and 178 referents from three Italian cities (Genoa, Milan, Cagliari). RESULTS: In each city, benzene-exposed participants showed higher mtDNAcn than referents: mtDNAcn was 0.90 relative units in Genoa bus drivers and 0.75 in referents (p = 0.019); 0.90 in Milan gas station attendants, 1.10 in police officers, and 0.75 in referents (p-trend = 0.008); 1.63 in Cagliari petrochemical plant workers, 1.25 in referents close to the plant, and 0.90 in referents farther from the plant (p-trend = 0.046). Using covariate-adjusted regression models, we estimated that an interquartile range increase in personal airborne benzene was associated with percent increases in mtDNAcn equal to 10.5% in Genoa (p = 0.014), 8.2% (p = 0.008) in Milan, 7.5% in Cagliari (p = 0.22), and 10.3% in all cities combined (p < 0.001). Using methylation data available for the Milan participants, we found that mtDNAcn was associated with LINE-1 hypomethylation (-2.41%; p = 0.007) and p15 hypermethylation (+15.95%, p = 0.008). CONCLUSIONS: Blood MtDNAcn was increased in persons exposed to low benzene levels, potentially reflecting mitochondrial DNA damage and dysfunction.

A novel synthetic oleanolic acid derivative with amino acid conjugate suppresses tumour growth by inducing cell cycle arrest.

Oleanolic acid (3beta-hydroxy-olean-12-en-28-oic acid; OA) has a wide variety of bioactivities and is used for medicinal purposes in many Asian countries. Various derivatives of OA have been synthesized in attempts to improve the potency. Here we describe the anti-tumour activity of a novel OA derivative, N-[(3beta)-3-(acetyloxy)-28-oxoolean-12-en-28-yl]-glycine methyl ester (AOA-GMe). AOAGMe was a more potent inhibitor of the growth of B16 melanoma cells than its parent compound OA, both in-vitro and in-vivo. AOA-GMe also exhibited dose-dependent inhibition of human K562 leukaemia cells, but had almost no toxicity in normal human peripheral blood mononuclear cells. AOA-GMe induced cell cycle arrest in G0/G1 and blocked G1-S transition, which correlated well with marked decreases in levels of cyclin D, cyclin-dependent kinase CDK4 and phosphorylated retinoblastoma protein, and increases in the cyclin-dependent kinase inhibitor p15. OA did not show such activities. These results suggest that AOA-GMe may induce growth arrest in tumour cells through regulation of proteins involved in the cell cycle.

Zebularine inhibits human acute myeloid leukemia cell growth in vitro in association with p15INK4B demethylation and reexpression.

OBJECTIVE: The p15INK4B tumor suppressor is frequently silenced by promoter hypermethylation in myelodysplastic syndrome and acute myeloid leukemia (AML). Clinically approved DNA methylation inhibitors, such as 5-aza-2'-deoxycytidine, can reverse p15INK4B promoter methylation, but widespread clinical use of these inhibitors is limited by their toxicity and instability in aqueous solution. The cytidine analog zebularine is a stable DNA methylation inhibitor that has minimal toxicity in vitro and in vivo. We evaluated zebularine effects on p15INK4B reactivation and cell growth in vitro to investigate a potential role for zebularine in treating myeloid malignancies. METHODS: We examined the specific effects of zebularine on reexpression of transcriptionally silenced p15INK4B and its global effects on cell cycle and apoptosis in AML cell lines and primary patient samples. RESULTS: Zebularine treatment of AML193, which has a densely methylated p15INK4B promoter, results in a dose-dependent increase in p15INK4B expression that correlates with CpG island promoter demethylation and enrichment of local histone acetylation. We observed enhanced p15INK4B induction following co-treatment with zebularine and the histone deacetylase inhibitor Trichostatin A. Zebularine inhibits cell proliferation, arrests cells at G(2)/M, and induces apoptosis at dosages that effectively demethylate the p15INK4B promoter. Zebularine treatment of KG-1 cells and AML patient blasts with hypermethylated p15INK4B promoters also reactivates p15INK4B reexpression and induces apoptosis. CONCLUSION: Zebularine is an effective inhibitor of p15INK4B methylation and cell growth in human AML in vitro. Our results extend the spectrum of zebularine effects to nonepithelial malignancies and provide a strong rationale for evaluating its clinical utility in the treatment of myeloid malignancies.

Cigarette smoke condensate and dioxin suppress culture shock induced senescence in normal human oral keratinocytes.

The aryl hydrocarbon receptor is a ligand activated transcription factor which regulates biological responses to a variety of environmental pollutants, such as dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) and cigarette smoke. The purpose of this study was to determine whether cigarette smoke condensate (CSC) is capable of activating the AHR in normal human oral keratinocytes (NHOK) and inhibiting their ability to senesce. Towards this end, NHOK were isolated from human subjects and were cultured in the presence or absence of either TCDD or CSC. While neither TCDD nor CSC treatments altered the lifespan of NHOK in culture, both were capable of suppressing a culture induced premature senescence as indicated by their ability to decrease the mRNA and protein levels of the senescence markers p16(INK4a), p53 and p15(INK4b). A role of the AHR in mediating these events is indicated by the observations that the TCDD and CSC-induced decreases in p15(INK4b), p16(INK4a) and p53 expression was accompanied by a corresponding increase in the expression levels of the AHR target gene, CYP1A1. In addition, cotreatment with the AHR antagonist, 3'-methoxy-4'-nitroflavone (MNF) blocked the effects of TCDD and CSC on p53 and CYP1A1 expression. The findings of this study indicate that in NHOK, CSC is capable of altering a key cell fate decision, i.e., commitment to premature senescence, that is in part, dependent on the AHR. These results support the idea that progression of CSC-induced tumorigenesis may include an AHR-mediated inhibition of senescence that contributes to immortalization and agents that block the actions of the AHR may be effective components of novel cancer therapeutics.

Upregulation of chicken p15INK4b at senescence and in the developing brain.

In mammalian cells, products of the INK4a-ARF locus play major roles in senescence and tumour suppression in different contexts, whereas the adjacent INK4b gene is more generally associated with transforming growth factor beta (TGF-beta)-mediated growth arrest. As the chicken genome does not encode an equivalent of INK4a, we asked whether INK4b and/or ARF contribute to replicative senescence in chicken cells. In chicken embryo fibroblasts (CEFs), INK4b levels increase substantially at senescence and the gene is transcriptionally silenced in two spontaneously immortalised chicken cell lines. By contrast, ARF levels are unaffected by prolonged culture or immortalisation. These expression patterns resemble the behaviour of INK4a and ARF in human fibroblasts. However, short-hairpin RNA (shRNA)-mediated knockdown of chicken INK4b or ARF provides only modest lifespan extension, suggesting that other factors contribute to senescence in CEFs. As well as underscoring the importance of the INK4b-ARF-INK4a locus in senescence, these findings imply that the encoded products have assumed different roles in different evolutionary niches. Although ARF RNA is not detectable in early chicken embryos, the INK4b transcript is expressed in the roof-plate of the developing hind-brain, consistent with a role in limiting cell proliferation.

Effects of 5-aza-2'-deoxycytidine on proliferation, differentiation and p15/INK4b regulation of human hematopoietic progenitor cells.

The demethylating agents 5-azacytidine and 5-aza-2'-deoxycytidine (DAC) have been shown to induce differentiation and inhibit growth of leukemic myeloid cells at low concentrations. However, the effect of DAC in changing the differentiation and proliferation behavior of normal human myeloid progenitors has rarely been investigated. Therefore, we established an in vitro model of normal hematopoietic differentiation, using CD34+ cells from mobilized peripheral blood, to study proliferation and colony formation, expression of several myeloid maturation markers and of the inhibitor of cyclin-dependent kinases p15/INK4b. Upon DAC treatment, cell growth was significantly decreased in a dose-dependent manner, without an increase in cytotoxicity. DAC treatment also resulted in a substantial increase of lysozyme-positive cells, which could be enhanced by G-CSF, a modest increase of myeloperoxidase+ and CD15+ cells, as well as an increase of colony-forming cells (CFU-GM) compared to control cells. p15/INK4b protein expression was strongly upregulated upon myeloid maturation, and additional DAC treatment did not change p15 expression or the methylation status of the p15 promoter at the noncytotoxic concentrations used. Taken together, these data indicate a role of DAC in changing myeloid progenitor cell expansion and differentiation. This model appears suitable also for global analyses of multiple differentially methylated genes.

In vivo effects of decitabine in myelodysplasia and acute myeloid leukemia: review of cytogenetic and molecular studies.

Low-dose demethylating agents such as 5-aza-2'-deoxycytidine (decitabine, DAC) and 5-azacytidine (azacitidine, Vidaza) have been explored for the treatment of myelodysplasia, acute myeloid leukemia, and hemoglobinopathies since the early 1980s, aiming to revert a methylator phenotype. Originally, the treatment rationale in hemoglobinopathies was to achieve demethylation of the hypermethylated and hence silent gamma-globin gene locus, thus reactivating synthesis of hemoglobin F (HbF). In myelodysplastic syndrome (MDS), cytogenetic analyses are mandatory for risk stratification and for monitoring response to drug treatment. The current knowledge regarding cytogenetic subgroups as predictors of response to low-dose decitabine in MDS as well as cytogenetic responses caused by demethylating agents is summarized in this review. Decitabine treatment is associated with a response rate that is higher in patients with high-risk cytogenetics (i.e., complex karyotype and/or abnormalities of chromosome 7) than in patients with intermediate-risk cytogenetics (two abnormalities or single abnormalities excluding 5q-, 20q-, and -Y). Following decitabine treatment of patients with abnormal karyotype, approximately one-third achieve a major cytogenetic response that can be confirmed by FISH analyses, while in two-thirds of patients, the abnormal karyotype persists but hematologic improvement may be observed during continued treatment. The most frequently studied gene in myelodysplasia is the cell cycle regulator p15(INK4b). Hypermethylation of p15(INK4b) in MDS is reversed during treatment with decitabine, resulting in reactivation of this gene. In hemoglobinopathies, treatment with demethylating agents leads to reactivation of fetal HbF (the gamma-globin gene locus also possibly being another target for reactivation in MDS), and thus, HbF may potentially act as surrogate marker for activity of decitabine. Other, thus far unidentified hypermethylated genes may also be targets for demethylating agents.