Nuclear chromatin patterns in 3 glucocorticoid-resistant RPMI 8226 human myeloma cell sub-lines: correlations with cell growth and immunological phenotype.

Nuclear morphological alterations associated with glucocorticoid resistance in human myeloma were evaluated by image cytometry in three human myeloma RPMI 8226 cell sub-lines. Resistance was induced by drug selection using prednisone (8226p), methylprednisolone (8226m) and dexamethasone (8226d), respectively. All these three cell sub lines displayed significant glucocorticoid-resistance without cross-resistance to doxorubicin. Nuclear geometry and texture were analyzed on G0/G1-selected cell nuclei and data compared with cell growth characteristics and membrane expression of CD23, CD38, CD44 and CD58 antigens. When compared to the parental RPMI 8226 cell line, glucocorticoid-resistant cells display a progressive chromatin condensation with heterogeneously distributed large chromatin clumps, a phenomenon not observed in the multidrug-resistant CEM-VLB cells. These alterations were correlated to the resistance index against glucocorticoids and to the expressions of CD38, and of CD44 variant forms CD44v5 and CD44v7-8 antigens. These data suggest that glucocorticoid resistance in RPMI 8226 cells could be associated with sub-visual specific higher-order chromatin organization changes. Furthermore, these alterations are correlated to the expression of membrane markers associated with tumors aggressiveness.

Nicotine induces chromatin changes and c-Jun up-regulation in HL-60 leukemia cells.

Although nicotine has been implicated as a potential factor in the pathogenesis of human cancer, its mechanisms of action regarding cancer development remain largely unknown. HL-60 cells were used to investigate the effects of a short-term treatment with nicotine at concentrations found in the blood of smokers. The findings show that nicotine induces chromatin decondensation, histone H3 acetylation and up-regulation of the c-Jun transcription factor mRNA. This increase is inhibited by mecamylamine, a nicotinic receptor antagonist, suggesting that nicotine alters cellular function directly via nicotinic acetylcholine receptors and may then play a role in cell physiology and tumor promotion.

Induction of apoptosis by bleomycin in p53-null HL-60 leukemia cells.

The role of p53 in apoptosis and the contrasting p53 status in tumors prompted us to investigate the bleomycin-induced apoptosis in p53-null human leukemia HL-60 cells (bleomycin at 160 microM for 7.5 h). Cells with apoptotic phenotype increased from 0.87% in controls to 9.40% in bleomycin-treated cells. Both the enzymes, caspase-3 and -8, were activated. Furthermore, the apoptotic phenotypes totally disappeared with zVAD-fmk, a caspase inhibitor. Besides, cytochrome c release from mitochondria happened simultaneously to apoptotic phenotypes, shrinkage of mitochondria but being independent of the mitochondrial permeability transition, since cyclosporine A and bongkrekic acid were inefficient on induced apoptosis. On the other hand, incubations with bleomycin (BLM) did not result in detectable changes in the expression of Bcl-2- and Bax-mRNA neither Bcl-2- or Bax-proteins. In conclusion, we suggest that BLM can produce apoptosis independently of p53 through three mechanisms: i) at the nuclear level by its endonuclease activities; ii) at the cell membrane, by activating caspases; and iii) at the mitochondria by releasing cytochrome c. These results indicate that BLM-induced apoptosis in HL-60 cells results from the activation of a mitochondria-dependent caspase cascade which includes also the activation of the initiator caspase-8.

Nuclear texture and chromatin structure in OV1/VCR human multidrug-resistant cell line.

Previous studies have demonstrated that multidrug-resistant leukemic cells displayed nuclear texture changes. In this work, the human ovarian carcinoma cell line IGROV1 and its multidrug-resistant variant OV1/VCR were studied. Cell smears of these cell populations were analysed by image cytometry. As compared to sensitive cells, OV1/VCR display a chromatin global decondensation as assessed by textural features analysis. In order to correlate this decondensation with alterations in chromatin structure, DNase I was used. OV1/VCR DNA displayed an increased DNase I sensitivity, suggesting an increased chromatin accessibility. Furthermore, OV1/VCR cells displayed an increased level in acetylated histone H4, a mechanism known to be associated with transcriptionally active chromatin and relaxed chromatin conformation.

Functional role of nicotinic acetylcholine receptors in apoptosis in HL-60 cell line.

The subunit composition of nicotinic acetylcholine receptors involved in apoptosis is an ongoing question. HL-60 cells were used in order to investigate the implication of nicotinic acetylcholine receptors in bleomycin-induced apoptosis. We found that bleomycin-induced apoptosis was significantly enhanced by nicotine and was blocked by nicotinic acetylcholine receptor antagonists, including alpha-bungarotoxin, a competitive antagonist of alpha 7 nicotinic receptor. Among the other agonists tested, 3-[2,4-dimethoxybenzylidene]anabaseine (GTS-21)-selective agonist for alpha 7-nicotinic acetylcholine receptor-, but not epibatidine or cytisine, enhanced bleomycin-induced apoptosis. In addition to these results, the detectable presence of alpha 7-mRNA supports a key role of alpha 7-nicotinic acetylcholine receptors in the modulation of the induced apoptosis by nicotine.

Prevalence of Chlamydia pneumoniae infection in squamous cell carcinoma of the head and neck.

On the basis of epidemiological data, an association between Chlamydia pneumoniae (Cp) infection and head and neck cancer might be suggested. The aim of the present study was to detect Cp-DNA within tumour tissue specimens by a two-step polymerase chain reaction. Investigation was planned on the Fleming's procedure for early termination when initial results were extreme. So, after ten consecutive patients, only one tumour contained Cp-DNA. Hence the prevalence could be regarded as inferior to 60% (2a=b=0.08), the threshold under which a direct role of Cp in head and neck cancer development does not seem to be likely.

Lymphocytes prime activation is required for nicotine-induced calcium waves.

Lymphocytes are reported to express nicotinic acetylcholine receptors (nAChR). However, no data are available on the expression of these nAChR on activated lymphocyte relatively to resting lymphocytes. In this study, we examined nAChR subunits expression in PHA-stimulated versus un-stimulated lymphocytes, and four leukemic cell lines. Cell stimulation with nicotine triggered calcium responses only in some experiments conducted with PHA-stimulated lymphocytes. Likewise, only the Jurkat and HL-60 cell lines displayed calcium waves upon nicotine stimulation, whereas the Raji and CCRF-CEM did not. All responding cells displayed an active form of the nicotinic a-7 nAChR. Indeed, use of 2 different sets of primers for the corresponding mRNA showed that expression of the full-length a-7 subunit mRNA was only present in PHA-stimulated lymphocytes for which calcium waves had been evidenced. Microscopy analysis of lymphocytes structure showed a direct relationship between their size, their a-7 nAChR expression, and calcium release upon nicotine stimulation. Then, this relationship suggested that lymphocytes need a prime activation to express the a-7 nAChR, and therefore to release calcium in response to nicotine.

Effects of the histone deacetylase inhibitor trichostatin A on nuclear texture and c-jun gene expression in drug-sensitive and drug-resistant human H69 lung carcinoma 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 chromatin texture, sensitive H69 cells displayed a progressive chromatin decondensation up to 24 h, whereas resistant H69-VP showed rapid (8 h) but transient changes. Similarly, expression of c-jun increased regularly in TSA-treated H69 cells. In H69-VP cells, an increase was also observed up to 12 h followed by a decrease after 24 h of treatment. CONCLUSIONS: Analysis of nuclear texture appeared to be a sensitive technique to detect chromatin pattern alterations induced by the histone deacetylase inhibitor TSA in the H69 cell line and enabled the observation of chromatin pattern discrepancies between chemotherapeutic drug-sensitive and drug-resistant cells during this treatment. When c-jun gene expression was analyzed as gene sensitive to epigenetic control, these textural differences seemed to be correlated to gene expression.