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.
It is now known that the analysis of chromatin texture can be used in oncology as a sensitive detection method, either to define diagnostic classifications or to locate a lesion along a defined trend curve. However, the functional significance of these variations in textural features remains sometimes unclear. Several drugs have been shown to be able to modulate chromatin structure. Among them, the phosphatase inhibitor okadaic acid at low concentration can increase accessibility to DNA in chromatin of carcinoma cells. This paper demonstrates that short exposures (0-3 h) to a 10-nM dose of okadaic acid induced an increased sensitivity to DNase I digestion in human CEM leukaemic cell nuclei and that this sensitization was associated to variations of nuclear texture characteristics, as evaluated by image cytometry. CEM cells treated with okadaic acid for 0-3h displayed changes in chromatin supraorganization with a more homogeneous and fine chromatin texture, as compared to control cells. This suggests that the appearance of an open configuration of chromatin structure as evaluated by biochemical methods corresponds to a more decondensed texture of nuclei measured by image cytometry. Longer exposures (6-24h) of CEM cells to 10 nM okadaic acid lead to apoptosis. As reported previously for camptothecin-treated HL60 cells, okadaic acid-treated CEM cells display biphasic nuclear chromatin texture changes, i.e. a decondensation phase followed by the appearance of typical apoptotic cells with a smaller nuclear area and a highly condensed chromatin. Finally, using the multidrug-resistant CEM-VLB cell line, it was confirmed that these multidrug-resistant cells also display cross-resistance to okadaic acid, as this compound was unable to induce either increased DNase I sensitivity, apoptosis, or altered nuclear texture in this particular cell line.
There is a constant need of features able to characterize potentially metastatic cells among the heterogeneous cell subpopulations which constitute a tumor. Image cytometry of metastatic tumor cells give rise to variable results, partly because of a heterogeneous origin of cells, or potential drug effects. The aim of this work was to characterize nuclear changes observed in metastatic cell clones issued in vitro from the same parental cell population The nuclear phenotypes of 6 cell sublines isolated from a rat rhabdomyosarcoma cell line and differing in their metastatic ability were evaluated by image cytometry on Feulgen‐stained preparations. Densitometric [5], geometric [3] and textural [9] features were computed from each nuclear image. For each cell subline, a metastatic score, ranging from 0 to 10, was calculated on the basis of in vitro invasivity data, by measuring the number of pulmonary metastases observed after s.c. graft of tumor cells in rats. Data obtained were compared to karyotype, growth characteristics, and oncogene expressions of cell lines. The nuclear DNA content, the chromosome numbers, the cell sublines doubling times, and the distribution of cells within the cell cycle appear unrelated with this score. On the contrary, increase in metastatic ability is accompanied by changes in chromatin pattern as assessed by textural features. Progressive increase in chromatin condensation can be observed in cell sublines with increasing metastatic score. These results were confirmed by an unsupervised multivariate partitioning of rhabdomyosarcoma cells which identified two separate subsets whose distributions within the analyzed cell lines correlate with their metastatic ability. These data suggest that, in rat rhabdomyosarcoma cell sublines, metastatic ability could be associated with nuclear morphological changes at the level of chromatin texture.
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