Abstract. Image cytometric study of pathological specimens or cell lines has suggested that epigenetic mechanisms are likely to play a major role in determining chromatin patterns evaluable through nuclear texture analysis. We previously reported that nuclear textural changes observed in the OV1-VCR etoposide-resistant ovarian carcinoma cell line were associated with an increased acetylated histone H4 level. In this study we analyzed the effects of treatments with the HDAC inhibitor trichostatin A (TSA) or with nickel subsulfide on histone H4 acetylation, nuclear texture, and MDR1 gene expression in drug-sensitive IGROV1 and drug-resistant OV1-VCR cell lines. In IGROV1 cells, TSA induced an increase in acetylated H4 level associated with a chromatin textural decondensation and an increase in MDR1 gene expression. In OV1-VCR cells, a similar increase in H4 acetylation was observed, but nuclear texture or MDR1 gene expression remained unchanged. ChIP analysis revealed that MDR1 gene expression remained stable in TSA-treated OV1-VCR cells despite a localized increase in H4 acetylation at the promoter level. Analysis of the methylation status of MDR1 promoter showed an increase in DNA methylation at 3 specific sites in OV1-VCR cells, that could participate to TSA low responsiveness in these cells. Treatment with nickel subsulfide induced a decrease in H4 acetylation without any effect on nuclear texture characteristics in both cell lines. In OV1-VCR cells, nickel subsulfide induced a significant down-regulation of the MDR1 gene expression. These results indicate that modulation of histone H4 acetylation level can be associated with up-or down-regulation of the MDR1 gene in OV1 cells. However, this modulation does not always result in chromatin pattern alterations and these data emphasize the complexity of chromatin texture regulation in tumor cells.

Background: Texture analysis of chromatin patterns by image cytometry can be used in the development and refinement of diagnosis and prognosis of cancers and in the follow-up of therapies. However, little is known about the biological mechanisms underlying these patterns. Epigenetic mechanisms as histone posttranslational modifications and particularly histone acetylation could play a major role in the determination of these chromatin patterns and then influence nuclear texture measurements. Methods: This study examined the consequences of treatment by the histone deacetylase inhibitor trichostatin A (TSA) on the nuclear texture in human cell lines sensitive and resistant to chemotherapy. Small cell lung carcinoma H69 cells and their variant H69-VP, which is resistant to etoposide, were incubated with 100 ng/ml of TSA for 0 to 24 h. Nuclear texture was evaluated by image cytometry and compared with the histone H4 acetylation level measured by western blotting and expression of c-jun gene evaluated by reverse transcription and real-time polymerase chain reaction. Results: TSA treatment induced an increase in histone H4 acetylation level in both cell lines. However, at the level of

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This original study shows that laser tweezer Raman spectroscopy is a label-free rapid tool to identify living cells that underwent epigenetic changes.

Melanoma is the most severe skin cancer characterized by a bad prognosis at metastatic stages due to resistance to most classical chemotherapies. Invasion of melanoma cells into the surrounding microenvironment locally and at distance of the primary tumour, is facilitated by expression of proteases that degrade the extracellular matrix. Matrix metalloproteinases (MMP) have been long thought as potential therapeutic targets as they are involved in several steps of tumour progression. However, based on this general concept, broad spectrum MMP inhibitors showed weak anticancer potential. Furthermore, MMPs are also expressed by stroma and infiltrating cells. Although, inflammatory conditions lead to uncontrolled expression of MMPs leading to massive matrix destruction, these enzymes are also essential for immune cells to migrate towards the tumour site, and hence mount an anti-tumoral response. During stromal reaction, MMPs also act as non-matrix deteriorating enzymes, and thus modulating the inflammatory response through limited proteolysis of cytokines and chemokines. MMPs contribution to these processes depends on their activity and their expression. Besides the classic control level of transcription by a variety of growth factors and cytokines, the contribution of epigenetic mechanisms on MMPs expression was demonstrated of great importance to extend our knowledge about the role of these enzymes in a specific context such as melanoma progression. Understanding MMPs regulation by epigenetic drugs in melanoma and infiltrated cells will provide a new platform to develop efficient therapies. The therapeutic implication of epigenetic mechanisms to switch a pro-tumoral inflammatory towards an immune anti-tumoral response will be an exciting challenge in which MMPs expression could play a major role.