Krystyna Fabianowska-Majewska

Common resistance mechanisms to deoxynucleoside analogues in variants of the human erythroleukaemic line K562

Resistant variants of the human leukaemic line K562 were developed using selection with the deoxynucleoside analogues cytosine arabinoside, 2‐chlorodeoxyadenosine, fludarabine and gemcitabine. The resistant lines displayed a high degree of cross resistance to all deoxynucleoside analogues, with little or no cross resistance to other agents. There was a profound accumulation defect of all nucleoside analogues in the resistant variants but no significant defect in nucleoside transport in any of the variants. 5′ nucleotidase activity was strongly increased and deoxycytidine kinase activity was moderately reduced in all of the resistant variants, resulting in reduced accumulation of triphosphate analogues. In addition a deletion in one of the alleles of the deoxycytidine kinase was detected in the fludarabine‐resistant line. Ribonucleotide reductase activity was found to be strongly increased in the gemcitabine‐selected line and purine nucleoside phosphorylase was increased in the 2‐chlorodeoxyadenosine‐selected line. Free nucleotide pools were increased in the 2‐chlorodeoxyadenosine‐selected line. There was no expression of the mdr1 gene by the resistant lines. Karyotypic analysis and FISH experiments using a 6q21 specific probe showed alterations in the 6(q16‐q22) region which contains the 5′‐nucleotidase gene. Early events in the activation and degradation of deoxynucleoside analogues appear to constitute common mechanisms of resistance to these compounds.

Epigenetic mechanisms in anti-cancer actions of bioactive food components – the implications in cancer prevention

The hallmarks of carcinogenesis are aberrations in gene expression and protein function caused by both genetic and epigenetic modifications. Epigenetics refers to the changes in gene expression programming that alter the phenotype in the absence of a change in DNA sequence. Epigenetic modifications, which include amongst others DNA methylation, covalent modifications of histone tails and regulation by non‐coding RNAs, play a significant role in normal development and genome stability. The changes are dynamic and serve as an adaptation mechanism to a wide variety of environmental and social factors including diet. A number of studies have provided evidence that some natural bioactive compounds found in food and herbs can modulate gene expression by targeting different elements of the epigenetic machinery. Nutrients that are components of one‐carbon metabolism, such as folate, riboflavin, pyridoxine, cobalamin, choline, betaine and methionine, affect DNA methylation by regulating the levels of S‐adenosyl‐L‐methionine, a methyl group donor, and S‐adenosyl‐L‐homocysteine, which is an inhibitor of enzymes catalyzing the DNA methylation reaction. Other natural compounds target histone modifications and levels of non‐coding RNAs such as vitamin D, which recruits histone acetylases, or resveratrol, which activates the deacetylase sirtuin and regulates oncogenic and tumour suppressor micro‐RNAs. As epigenetic abnormalities have been shown to be both causative and contributing factors in different health conditions including cancer, natural compounds that are direct or indirect regulators of the epigenome constitute an excellent approach in cancer prevention and potentially in anti‐cancer therapy.

Folic acid enforces DNA methylation-mediated transcriptional silencing of PTEN, APC and RARbeta2 tumour suppressor genes in breast cancer.

Folate, one of the most studied dietary compounds, has recently become the main topic of debates on food fortification. Although low folate levels may be associated with increased risk of cancer development, simultaneously several reports indicate a detrimental effects mediated by high folate concentrations. Using the methylation sensitive restriction analysis (MSRA) and real-time RT-PCR we tested the effect of folic acid on DNA promoter methylation and expression of PTEN, APC and RARbeta2 tumour suppressor genes in MCF-7 and MDA-MB-231 breast cancer cell lines with different invasive capacity. The tested genes encode proteins involved in regulation of oncogenic intracellular signaling pathways. The results show that the increasing concentrations of folic acid lead to a dose-dependent down-regulation of tumour suppressor genes which may be linked to the increased DNA methylation detected within their promoter regions. The effects were more remarkable in non-invasive MCF-7 cells where we also observed 30% up-regulation of DNMT1 expression at the highest folate concentration used. Our findings show that caution need to be used when introducing folic acid supplementation since it may lead to cancer progression.

Sulforaphane Alone and in Combination with Clofarabine Epigenetically Regulates the Expression of DNA Methylation-Silenced Tumour Suppressor Genes in Human Breast Cancer Cells.

Background/Aim: Sporadic breast cancer is frequently associated with aberrant DNA methylation patterns that are reversible and responsive to environmental factors, including diet. In the present study, we investigated the effects of sulforaphane (SFN), a phytochemical from cruciferous vegetables, on the methylation and expression of PTEN and RARbeta2 tumour suppressor genes as well as on the expression of regulators of DNA methylation reaction, DNMT1, p53, and p21, in MCF-7 and MDA-MB-231 human breast cancer cells with different invasive potential. We also evaluate the role of SFN epigenetic effects in support of therapy with clofarabine (ClF) that was recently shown to modulate the epigenome as well. Methods: Promoter methylation and gene expression were estimated using methylation-sensitive restriction analysis and real-time PCR, respectively. Results: In both MCF-7 and MDA-MB-231 cells, SFN at IC50 (22 and 46 µM, respectively) and a physiologically relevant 10 µM concentration lead to hypomethylation of PTEN and RARbeta2 promoters with concomitant gene up-regulation. The combination of SFN and ClF enhances these effects, resulting in an increase in cell growth arrest and apoptosis at a non-invasive breast cancer stage. Conclusions: Our findings provide evidence that SFN activates DNA methylation-silenced tumour suppressor genes in breast cancer cells and may contribute to SFN-mediated support of therapy with an anti-cancer drug, ClF, increasing its applications in solid tumours.

The Role of Sulforaphane in Epigenetic Mechanisms, Including Interdependence between Histone Modification and DNA Methylation

Carcinogenesis as well as cancer progression result from genetic and epigenetic changes of the genome that leads to dysregulation of transcriptional activity of genes. Epigenetic mechanisms in cancer cells comprise (i) post-translation histone modification (i.e., deacetylation and methylation); (ii) DNA global hypomethylation; (iii) promoter hypermethylation of tumour suppressor genes and genes important for cell cycle regulation, cell differentiation and apoptosis; and (iv) posttranscriptional regulation of gene expression by noncoding microRNA. These epigenetic aberrations can be readily reversible and responsive to both synthetic agents and natural components of diet. A source of one of such diet components are cruciferous vegetables, which contain high levels of a number of glucosinolates and deliver, after enzymatic hydrolysis, sulforaphane and other bioactive isothiocyanates, that are involved in effective up-regulation of transcriptional activity of certain genes and also in restoration of active chromatin structure. Thus a consumption of cruciferous vegetables, treated as a source of isothiocyanates, seems to be potentially useful as an effective cancer preventive factor or as a source of nutrients improving efficacy of standard chemotherapies. In this review an attempt is made to elucidate the role of sulforaphane in regulation of gene promoter activity through a direct down-regulation of histone deacetylase activity and alteration of gene promoter methylation in indirect ways, but the sulforaphane influence on non-coding micro-RNA will not be a subject of this review.

The Effects of Nucleoside Analogues on Promoter Methylation of Selected Tumor Suppressor Genes in MCF-7 and MDA-MB-231 Breast Cancer Cell Lines

The effects of 2-chloro-2′-deoxyadenosine, 9-β-D-arabinofuranosyl-2-fluoroadenine, and 5-aza-2′-deoxycytidine on promoter methylation of the selected tumor suppressor genes (i.e., ERα, BRCA1, RARβ2, E-cadherin, PTEN, and APC) were estimated using methylation-sensitive restriction analysis. The studies were carried out in hormone-responsive, low-invasive cell line MCF-7 and hormone-insensitive, highly invasive cell line MDA-MB-231. The results demonstrate an implication of the tested adenosine analogues and 5-aza-dCyd in regulation of DNA methylation process. Moreover, the effects of nucleoside analogues on PTEN promoter methylation suggest distinct mechanism of regulation of the epigenetic DNA modification in low-invasive compared to highly invasive breast cancer cells.

The levels of 7,8-dihydrodeoxyguanosine (8-oxoG) and 8-oxoguanine DNA glycosylase 1 (OGG1) - A potential diagnostic biomarkers of Alzheimer's disease.

Evidence indicates that oxidative stress contributes to neuronal cell death in Alzheimers disease (AD). Increased oxidative DNA damage l, as measured with 8-oxoguanine (8-oxoG), and reduced capacity of proteins responsible for removing of DNA damage, including 8-oxoguanine DNA glycosylase 1 (OGG1), were detected in brains of AD patients. In the present study we assessed peripheral blood biomarkers of oxidative DNA damage, i.e. 8- oxoG and OGG1, in AD diagnosis, by comparing their levels between the patients and the controls. Our study was performed on DNA and serum isolated from peripheral blood taken from 100 AD patients and 110 controls. For 8-oxoG ELISA was employed. The OGG1 level was determined using ELISA and Western blot technique. Levels of 8-oxoG were significantly higher in DNA of AD patients. Both ELISA and Western blot showed decreased levels of OGG1 in serum of AD patients. Our results show that oxidative DNA damage biomarkers detected in peripheral tissue could reflect the changes occurring in the brain of patients with AD. These results also suggest that peripheral blood samples may be useful to measure oxidative stress biomarkers in AD.

Alteration of DNA Methylation Status in K562 and MCF-7 Cancer Cell Lines by Nucleoside Analogues

The effects of 2-chloro-2′-deoxyadenosine, β-D-arabinofuranosyl-2-fluoroadenine, and 5-aza-2′-deoxycytidine on promoter methylation of the selected tumor suppressor genes (i.e., ERα, BRCA1, E-cadherin, PTEN, and APC) were estimated using methylation-sensitive restriction analysis (MSRA) in K562 cells (human erythroleukemic cell line) and MCF-7 cells (human breast cancer cell line). In both cell lines all tested drugs completely reduced methylation of PTEN and APC promoters. The results indicate that the tested nucleoside analogues, which are known inhibitors of DNA synthesis, also are implicated in indirect (or direct in the case of 5-aza-dCyd) regulation of post-replicative DNA modifications (i.e., DNA methylation).

Studies on the Methylation Status of CpG Sequences Located in Promoters of Selected Tumour Suppressor Genes in Breast Cancer Cells

In the tested samples of sporadic breast cancer (100 cases), hypermethylation of CpG sequences located in ERα promoter was observed in 62 cases. It correlated with: (i) deficiency of ERα protein in 45%, (ii) hypermethylation of BRCA1 promoter in 95%, and (iii) nonmethylated E-cadherin promoter in 90%. Fifty-eight percent of the patients with nonmethylated E-cadherin promoter (56 cases) did not show metastasis to lymphatic nodes. The analysis of the methylation level of the promoter of ERα, BRCA1, and E-cadherin, frequently connected with their activity, shows that it can be an important parameter in the diagnosis and therapeutic strategies in sporadic breast cancer.

Inhibition of DNA Methylation by 5-Aza-2′-Deoxycytidine Correlates with Induction of K562 Cells Differentiation

DNA methylation is a postreplication modification which, in higher organisms, is one of the mechanisms that regulates gene expression and controls cell differentiation 1 . Recently, the role of DNA methylation in the differentiation process (i.e. in the programmed expression of tissue-specific genes) and the influence of 5-aza-2’-deoxycytidine (5-aza-dCyt) on the process has been under active investigation2 , 3 . Changes in methylation patterns have been shown to be relevant to Friend erythroleukemia cells treated with potent inhibitors of C-5 DNA methylransferase: 5-azacytidine and 5-aza-dCyt 4 . We have previously reported that 5-aza-dCyt, in human lymphocytes 5 and murine leukemic cells L1210 , led to a decrease of endogenous C-5 DNA methyltransferase activity, which was expressed by lower level of incorporated methyl groups into synthetic double-stranded poly dI-dC. Most of other authors’ results suggest that hypomethylation of DNA is involved in molecular mechanisms of gene expression, apoptosis and differentiation of cells. The present studies were designed to assay the effect of 5-aza-dCyt on the level of cytosine methylation in genomic DNA of erythroleukemic cell line, K562, and to check if this effect correlates with induction of the cells differentiat ion. For this reason in the present assays, we used methylation-dependent restriction analysis of genomic DNA and