Tumour drug-resistant ABCB1 gene expression is regulated at the chromatin level through epigenetic mechanisms. We examined the effects of the histone deacetylase inhibitor trichostatin A (TSA) on ABCB1 gene expression in small cell lung carcinoma (SCLC) drugsensitive (H69WT) or etoposide-resistant (H69VP) cells. We found that TSA induced an increase in ABCB1 expression in drugsensitive cells, but strongly decreased it in drug-resistant cells. These up-and downregulations occurred at the transcriptional level. Protein synthesis inhibition reduced these modulations, but did not completely suppress them. Differential temporal patterns of histone acetylation were observed at the ABCB1 promoter: increase in H4 acetylation in both cell lines, but different H3 acetylation with a progressive increase in H69WT cells but a transient one in H69VP cells. ABCB1 regulations were not related with the methylation status of the promoter À50GC, À110GC, and Inr sites, and did not result in further changes to these methylation profiles. Trichostatin A treatment did not modify MBD1 binding to the ABCB1 promoter and similarly increased PCAF binding in both H69 cell lines. Our results suggest that in H69 drug-resistant SCLC cell line TSA induces downregulation of ABCB1 expression through a transcriptional mechanism, independently of promoter methylation, and MBD1 or PCAF recruitment.

Nuclear morphological alterations associated with multidrug resistance (MDR) were evaluated by image cytometry in various human leukemic cell sub-lines: 3 cell lines with P-gp-mediated resistance (CEM-VLB, HL60/Vinc, K562-Dox), the non-Pgp-mediated MDR HL60/AR leukemic cell line with over-expression of MRP, and the at-MDR CEM-VMI leukemic cell line with alteration of topoisomerase II. All these MDR cell sub-lines were obtained by drug selection and were compared with their sensitive counterparts and with the hamster LR73-R cell line obtained by transfection of mouse mdrl cDNA. All MDR cell sub-lines obtained by drug selection displayed decreased DNA Feulgen stainability as compared with their respective sensitive parental cell line, a phenomenon not observed in the transfected LR73-R cells. Nuclear texture analysis on G0/G1-selected cell nuclei revealed 2 types of textural phenotype. The first phenotype was characterized by chromatin decondensation with small but compact chromatin clumps, and was observed in drug-selected P-gp-mediated MDR cells (CEM-VLB, HL60-Vinc, K562-Dox) and in the non-P-gp-mediated MDR HL60/AR cell line. The second phenotype was characterized by a condensed and homogeneous chromatin pattern, and was observed in the at-MDR CEM-VMI cell line. LR73-R cells transfected with mdrl cDNA did not display any significant changes in textural phenotype as compared with sensitive LR73 cells, suggesting that P-gp over-expression alone cannot account for the cytological modifications observed in MDR cells. These data suggest that multidrug resistance could be associated with specific nuclear morphological changes which appeared to be a consequence of alterations occurring during selection by cytotoxic drugs rather than of P-gp over-expression.

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.

We previously demonstrated that the alpha 1(I) polypeptide chain of collagen can bind and activate polymorphonuclear neutrophils (PMN). In the present experiments, performed in culture grade 96-well plastic plates coated with collagen, fibronectin, or other proteins, adhesion was assessed by staining the adhering cells after 30 min with crystal violet and measuring absorbance at 560 nm, and activation of PMNs was assessed by measuring the amount of O2-formed. Adhesion occurred at 17 and 37 degrees C but activation at 37 degrees C only. Monoclonal antibody anti-CD 18 inhibited adhesion, showing that the receptor of collagen I on PMNs is a beta 2 integrin. On the other hand, adhesion of PMNs to fibronectin was inhibited by monoclonal antibodies to CD18 and to CD11b.

Human polymorphonuclear neutrophils (PMNs), purified on Ficoll-Hypaque cushions, were incubated for 5 min with calf skin acid-soluble collagen and the released superoxide anions (O2-) measured spectrophotometrically by reduction of ferricytochrome c or by chemiluminescence analysis. This collagen stimulated the release of O2- unless it had been treated with pepsin. The stimulatory activity remained in denatured collagen, was contained only in the alpha 1(I) chain and was present in the alpha 1(I)-CB 6 (CNBr-cleaved) peptide, which is C-terminal. The activity was linearly dependent on the collagen concentration up to about 200 micrograms/ml. In addition, this collagen induced a release of beta-glucuronidase and N-acetyl-beta-glucosaminidase from PMNs.

Contact between type I collagen purified from several species and human polymorphonuclear neutrophils (PMNs) triggers the production of O2.- by these cells. The activity of collagen is located in the alpha 1(I)-CB6 cyanogen bromide-cleaved (CB)-peptide, which is the C-terminal CB-peptide of the alpha 1(I) chain. Experiments based on the competitive inhibition of O2.- production by simultaneous incubation of PMNs with type I collagen and synthetic peptides identical to the conserved sequences of this collagen demonstrated that the binding of collagen to PMNs and the subsequent activation of these cells depend on the simultaneous presence of two sequences: Arg-Gly-Asp [residues 915, 916 and 917 of the complete alpha 1(I) chain, located in the helical part] Asp-Gly-Gly-Arg-Tyr-Tyr (residues 1034-1039, located in the C-terminal non-helical telopeptide).

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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