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In human cells the length of telomeres depends on telomerase activity. This activity and the expression of the catalytic subunit of human telomerase reverse transcriptase (hTERT) is strongly up-regulated in most human cancers. hTERT expression is regulated by different transcription factors, such as c-Myc, Mad1 and Sp1. In this study, we demonstrated that 15d-PG J2 and rosiglitazone (an endogenous and synthetic peroxisome proliferators activated receptor γ (PPARγ) ligand, respectively) inhibited hTERT expression and telomerase activity in CaCo-2 colon cancer cells. Moreover, both ligands inhibited c-Myc protein expression and its E-box DNA binding activity. Additionally, Mad1 protein expression and its E-box DNA binding activity were strongly increased by 15d-PG J2 and, to a lesser extent, by rosiglitazone. Sp1 transcription factor expression and its GC-box DNA binding activity were not affected by both PPARγ ligands. Results obtained by transient transfection of CaCo-2 cells with pmaxFP-Green-PRL plasmid constructs containing the functional hTERT core promoter (including one E-box and five GC-boxes) and its E-box deleted sequences, cloned upstream of the green fluorescent protein reporter gene, demonstrated that 15d-PG J2, and with minor effectiveness, rosiglitazone, strongly reduced hTERT core promoter activity. E-boxes for Myc/Mad/Max binding showed a higher activity than GC-boxes for Sp1. By using GW9662, an antagonist of PPARγ, we demonstrated that the effects of 15d-PG J2 are completely PPARγ independent, whereas the effects of rosiglitazone on hTERT expression seem to be partially PPARγ independent. The regulation of hTERT expression by 15d-PG J2 and rosiglitazone, through the modulation of the Myc/Max/Mad1 network, may represent a new mechanism of action of these substances in inhibiting cell proliferation. [ABSTRACT FROM AUTHOR]

Abstract: 4-Hydroxynonenal (HNE), produced during oxidative stress, has an antiproliferative/differentiative effect in several tumor cells. Recently, it has been observed that oxidative stress accelerates telomere loss. The length of telomeres depends on the telomerase activity, and the catalytic subunit of telomerase (hTERT) is strongly up-regulated in most human cancers and inhibited by differentiating agents. In this paper the inhibitory effect of HNE on telomerase activity and hTERT expression in three human leukemic cell lines (HL-60, U937, ML-1) is reported. To investigate the molecular mechanism involved in hTERT down-regulation by HNE, the expression of several transcription factors was also studied: in all these cell lines, c-Myc was inhibited, Mad-1 was up-regulated, and Sp-1 was not affected. Moreover, in p53 wild-type ML-1 cells, HNE up-regulated p53 expression. In HL-60 cells, DNA binding activity of c-Myc and Mad-1 to the E-box sequence of the hTERT promoter was inhibited and up-regulated, respectively. In summary, HNE inhibits telomerase activity via decreased hTERT promoter activity, by modulating c-Myc/Mad-1 transcription factor expression. [Copyright &y& Elsevier]

Abstract: In this study, we have shown the intracellular distribution of choline and phosphatidylcholine fluorescent derivatives in human breast carcinoma cells using confocal microscopy. The fluorescent choline derivatives ethanamimium 2-hydroxy-N,N-dimethyl-N-[2-N-(2,1,3-benzoxadiazol-4-amine,-N-methyl,-7- nitro)-ethyl] bromide (NBD-choline) and C6–NBD–phosphatidylcholine (C6–NBD–PC) were used in this work. NBD-choline was easily internalised into drug sensitive MCF-7 and in multidrug resistant MCF-7/DX cells. The probe was found to localise in the endoplasmic reticulum of sensitive cells and in the Golgi of multidrug resistant cells. In contrast, very low accumulation was found in normal MCF10A cells. For C6–NBD–PC, a similar pattern of localisation was found in tumour cells, but a significant uptake was also observed in normal cells. Unlike NBD-choline, C6–NBD–PC appears not to discriminate between normal and tumour cells. These results are consistent with previously published results showing higher levels of 11C-choline uptake in malignant lesions seen with positron emission tomography (PET) in vivo imaging. Our results suggest that using NBD-choline and laser scanning confocal fluorescence microscopy (LSCFM) could be a useful tool to study choline metabolism in cancer cells and to consolidate PET imaging findings. [Copyright &y& Elsevier]