calyculin-a and Lung-Neoplasms

The analysis of chromosomal aberrations by premature chromosome condensation (PCC) induced by Calyculin A (Cal) is feasible in tumor biopsies from patients and has the potential to predict sensitivity to radiotherapy. As hyperthermia (HT) improves radiotherapy outcome in certain tumor sites, it was investigated whether PCC induction is still possible after temperatures reached in the clinic. Human cervical carcinoma (CaSki) and lung carcinoma (SW-1573) cells were incubated with Cal to induce PCC immediately after 1 h treatment at temperatures ranging from 41 degrees C to 43 degrees C and after recovery for up to 24 h after treatment with 43 degrees C. Levels of phosphorylated Cdc2 (at the Tyr15 residue), histone H3 (at the Ser10 residue) and Cyclin B1 were investigated by immunoblotting. The amount of cells positive for phosphorylated histone H3 was determined by flow cytometry. Temperatures > or =42.5 degrees C inhibited the induction of PCC by Cal, while recovery of PCC-induction was observed at >20 h after treatment in both cell lines. The phosphorylation status of Cdc2 as well as of histone H3 in cells treated with Cal directly after HT at 43 degrees C was similar to that of cells treated with Cal alone or treated with Cal 24 h after HT at 43 degrees C. HT alone did not affect the levels of phosphorylated Cdc2, while phosphorylation levels of histone H3 were increased as compared with control status of these two proteins. Phosphorylated and total Cyclin B1 levels were not influenced by any of the treatments. Flow cytometric analysis confirmed that HT at 43 degrees C did not interfere with phosphorylation of histone H3. Our data indicate that HT transiently inhibits PCC induction by Cal in a temperature-dependent manner. Therefore, an interval of at least 24 h after HT should be applied before taking tumor biopsies for karyogram analysis of patients treated with temperatures above 42.5 degrees C.

Topics: Carcinoma, Squamous Cell; CDC2 Protein Kinase; Cell Line, Tumor; Chromatin Assembly and Disassembly; Chromosome Aberrations; Cyclin B; Cyclin B1; Cyclin-Dependent Kinases; Female; Fever; Histones; Humans; Hyperthermia, Induced; Lung Neoplasms; Marine Toxins; Oxazoles; Phosphorylation; Uterine Cervical Neoplasms

Exon 2 of the Bcl-x gene undergoes alternative splicing in which the Bcl-xS splice variant promotes apoptosis in contrast to the anti-apoptotic splice variant Bcl-xL. In this study, the regulation of the alternative splicing of pre-mRNA of Bcl-x was examined in response to emetine. Treatment of different types of cancer cells with emetine dihydrochloride downregulated the level of Bcl-xL mRNA with a concomitant increase in the mRNA level of Bcl-xS in a dose- and time-dependent manner. Pretreatment with calyculin A, an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), blocked emetine-induced alternative splicing in contrast to okadaic acid, a specific inhibitor of PP2A in cells, demonstrating a PP1-mediated mechanism. Our finding on the regulation of RNA splicing of members of the Bcl-2 family in response to emetine presents a potential target for cancer treatment.

Topics: Alternative Splicing; bcl-X Protein; Breast Neoplasms; Cell Line, Tumor; Cycloheximide; Down-Regulation; Emetine; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Marine Toxins; Okadaic Acid; Oxazoles; Prostatic Neoplasms; Protein Phosphatase 1; Protein Phosphatase 2; Up-Regulation; Uterine Cervical Neoplasms

Previous studies have demonstrated that several splice variants are derived from both the caspase 9 and Bcl-x genes in which the Bcl-x splice variant, Bcl-x(L) and the caspase 9 splice variant, caspase 9b, inhibit apoptosis in contrast to the pro-apoptotic splice variants, Bcl-x(s) and caspase 9. In a recent study, we showed that ceramide induces the dephosphorylation of SR proteins, a family of protein factors that regulate alternative splicing. In this study, the regulation of the alternative processing of pre-mRNA of both caspase 9 and Bcl-x(L) was examined in response to ceramide. Treatment of A549 lung adenocarcinoma cells with cell-permeable ceramide, D-e-C(6) ceramide, down-regulated the levels of Bcl-x(L) and caspase 9b mRNA and immunoreactive protein with a concomitant increase in the mRNA and immunoreactive protein levels of Bcl-x(s) and caspase 9 in a dose- and time-dependent manner. Pretreatment with calyculin A (5 nm), an inhibitor of protein phosphatase-1 (PP1) and protein phosphatase 2A (PP2A) blocked ceramide-induced alternative splicing in contrast to okadaic acid (10 nm), a specific inhibitor of PP2A at this concentrations in cells, demonstrating a PP1-mediated mechanism. A role for endogenous ceramide in regulating the alternative splicing of caspase 9 and Bcl-x was demonstrated using the chemotherapeutic agent, gemcitabine. Treatment of A549 cells with gemcitabine (1 microm) increased ceramide levels 3-fold via the de novo sphingolipid pathway as determined by pulse labeling experiments and inhibition studies with myriocin (50 nm), a specific inhibitor of serine palmitoyltransferase (the first step in de novo synthesis of ceramide). Treatment of A549 cells with gemcitabine down-regulated the levels of Bcl-x(L) and caspase 9b mRNA with a concomitant increase in the mRNA levels of Bcl-x(s) and caspase 9. Again, inhibitors of ceramide synthesis blocked this effect. We also demonstrate that the change in the alternative splicing of caspase 9 and Bcl-x occurred prior to apoptosis following treatment with gemcitabine. Furthermore, doses of D-e-C(6) ceramide that induce the alternative splicing of both caspase 9 and Bcl-x-sensitized A549 cells to daunorubicin. These data demonstrate a role for protein phosphatases 1 (PP1) and endogenous ceramide generated via the de novo pathway in regulating this mechanism. This is the first report on the dynamic regulation of RNA splicing of members of the Bcl-2 and caspase families in response to regulator

Topics: Adenocarcinoma; Alternative Splicing; Base Sequence; bcl-X Protein; Carboxylic Acids; Caspase 9; Caspases; Ceramides; DNA Primers; Enzyme Inhibitors; Fumonisins; Humans; Lung Neoplasms; Marine Toxins; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Protein Phosphatase 1; Protein Phosphatase 2; Proto-Oncogene Proteins c-bcl-2; Sphingolipids; Tumor Cells, Cultured

A reduced cation reabsorption across the alveolar epithelium decreases water reabsorption from the alveoli and could diminish clearing accumulated fluid. To test whether hypoxia restricts cation transport in alveolar epithelial cells, cation uptake was measured in rat lung alveolar type II pneumocytes (AII cells) in primary culture and in A549 cells exposed to normoxia and hypoxia. In AII and A549 cells, hypoxia caused a PO2-dependent inhibition of the Na-K pump, of Na-K-2Cl cotransport, and of total and amiloride-sensitive 22Na uptake. Nifedipine failed to prevent hypoxia-induced transport inhibition in both cell types. In A549 cells, the inhibition of the Na-K pump and Na-K-2Cl cotransport occurred within approximately 30 min of hypoxia, was stable >20 h, and was reversed by 2 h of reoxygenation. There was also a reduction in cell membrane-associated Na-K-ATPase and a decrease in Na-K-2Cl cotransport flux after full activation with calyculin A, indicating a decreased transport capacity. [14C]serine incorporation into cell proteins was reduced in hypoxic A549 cells, but inhibition of protein synthesis with cycloheximide did not reduce ion transport. In AII and A549 cells, ATP levels decreased slightly, and ADP and the ATP-to-ADP ratio were unchanged after 4 h of hypoxia. In A549 cells, lactate, intracellular Na, and intracellular K were unchanged. These results indicate that hypoxia inhibits apical Na entry pathways and the basolateral Na-K pump in A549 cells and rat AII pneumocytes in culture, indicating a hypoxia-induced reduction of transepithelial Na transport and water reabsorption by alveolar epithelium. If similar changes occur in vivo, the impaired cation transport across alveolar epithelial cells might contribute to the formation of hypoxic pulmonary edema.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Amiloride; Animals; Carrier Proteins; Cations, Monovalent; Cell Hypoxia; Cell Membrane; Cells, Cultured; Enzyme Inhibitors; Epithelial Cells; Humans; Kinetics; Lung Neoplasms; Marine Toxins; Oxazoles; Potassium; Pulmonary Alveoli; Rats; Sodium; Sodium-Potassium-Chloride Symporters; Sodium-Potassium-Exchanging ATPase; Tumor Cells, Cultured