Clofarabine (2-chloro-2'-fluoro-2'-deoxyarabinosyladenine, ClF) is a 
second-generation 2'-deoxyadenosine analogue that is structurally related to 
cladribine (2-chloro-2'-deoxyadenosine, 2CdA) and fludarabine 
(9-beta-d-arabinosyl-2-fluoroadenine, F-ara-A). It demonstrates potent 
antitumour activity at much lower doses than parent compounds with high 
therapeutic efficacy in paediatric blood cancers. Our previous studies in breast 
cancer cells indicate that 2CdA and F-ara-A are involved in epigenetic 
regulation of gene transcription. We therefore investigated whether ClF 
influences methylation and expression of selected tumour suppressor genes, such 
as adenomatous polyposis coli (APC), phosphatase and tensin homologue (PTEN), 
and retinoic acid receptor beta 2 (RARbeta2), as well as expression of p53, p21 
and DNA methyltransferase 1 (DNMT1) in MCF-7 and MDA-MB-231 breast cancer cell 
lines with different invasive potential. Promoter methylation and gene 
expression were estimated using methylation-sensitive restriction analysis 
(MSRA) and real-time PCR, respectively. ClF demonstrated potent growth 
inhibitory activity in MCF-7 and MDA-MB-231 cells after 96h treatment with IC50 
determined as equal to 640nM and 50nM, respectively. In both breast cancer cell 
lines, ClF led to hypomethylation and up-regulation of APC, PTEN and RARbeta2 as 
well as increase in p21 expression. Only in non-invasive MCF-7 cells, these 
changes were associated with down-regulation of DNMT1. Our results provide first 
evidence of ClF implications in epigenetic regulation of transcriptional 
activity of selected tumour suppressor genes in breast cancer. It seems to be a 
new important element of ClF anticancer activity and may indicate its potential 
efficacy in epigenetic therapy of solid tumours, especially at early stages of 
carcinogenesis.