calyculin-a and Carcinoma--Squamous-Cell

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

Using patch clamp and ion-selective fluorescence dye techniques, we investigated the influence of actin cytoskeleton rearrangements on the activity of calcium entry channels in plasma membrane of human carcinoma A431 cells. It is shown that disruption of actin microfilaments by cytohalasin D has no significant effect on calcium release from the stores and its entry from the extracellular space. It also does not interfere with the activation of inositol 1,4,5-trisphosphate (IP3)-dependent high-selective low-conductance calcium channels Imin. The treatment of cells with calyculin A induces the formation of actin filament layer beneath plasma membrane and also inhibits Imin activation and calcium entry through the plasma membrane, though calcium efflux from the stores was nearly unchanged. Thus, it is concluded that calcium signalling in A431 cells can be modulated by actin cytoskeleton rearrangements, and may be well described in terms of "conformational coupling" model.

Topics: Actins; Calcium; Calcium Channel Agonists; Calcium Channels; Calcium Signaling; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Cytochalasin D; Cytoskeleton; Electric Conductivity; Extracellular Space; Humans; Inositol 1,4,5-Trisphosphate; Ion Channel Gating; Marine Toxins; Oxazoles; Patch-Clamp Techniques; Thapsigargin; Tumor Cells, Cultured

Intrinsic radiosensitivity of tumor cells from biopsies, assayed by colony formation after in vitro irradiation, has shown significant correlations with outcome after radiotherapy. Alternatives to the colony assay have been sought due to its long and cumbersome nature. We have previously shown good correlations between colony formation and radiation-induced chromosome aberrations in human tumor cell lines. In addition, we and others have shown on cell lines that premature chromosome condensation (PCC) induced with phosphatase inhibitors can be used to aid rapid assessment of aberrations in interphase cells, reducing the selection problem with metaphases. The purpose of this study was to translate the in vitro results to human cancer, with the aim of developing a rapid assay for intrinsic radiosensitivity.. The problem of admixtures of normal and malignant cells in biopsies was addressed using magnetic bead separation (MACS) employing antibodies to human fibroblasts. This proved to be a reliable and efficient method, enriching mean tumor cell fractions from 20 to almost 80%. PCC could be induced in human normal and tumor cell lines, and in sorted or unsorted suspensions from biopsies, with the phosphatase inhibitor calyculin A. Maximum PCCs were achieved after 1-week culture of biopsy-derived cells. Mean fractions of aneuploid tumor cell PCCs were, however, less than 1%. PCCs were predominantly from S and G2 phase, of which only G2 were scorable for aberrations. Almost no G1 PCCs were found. More scorable PCCs were found after 1h of calyculin A than metaphases after 5h of colcemid, but these were calculated to be too few to yield reliable estimates of chromosome damage after radiation.CONLCUSIONS: Tumor cells can be satisfactorily separated from fibroblasts in fresh suspensions from cancer biopsies, but poor growth of tumor cells in short term culture and low yields of PCCs combine to prevent the routine use of such cytogenetic assays for pre-treatment prediction of radiotherapy outcome.

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Cell Separation; Chromosome Aberrations; Fibroblasts; Flow Cytometry; Head and Neck Neoplasms; Humans; Marine Toxins; Oxazoles; Radiation Tolerance; Tumor Cells, Cultured

To determine whether protein phosphorylation and dephosphorylation can affect apoptosis in oral epithelial cells we examined the effects of protein phosphatase inhibitors, okadaic acid (OA) and calyculin A (CA), on cultured human oral squamous carcinoma (SCC) cell line, SCC-25 cells. After reaching subconfluence these cells were exposed to varying concentrations of the protein phosphatase inhibitors, OA and CA. Both OA and CA induced cell death in SCC-25 cells in a dose-dependent fashion as determined by phase-contrast microscopy and WST-1 cell viability assay. By using the Hoechst 33342 staining, marked nuclear condensation and fragmentation of chromatin was observed. DNA ladder formation also was detected in SCC-25 cells by treatment with OA and CA. The induced nuclear fragmentation and DNA ladder formation were dose-dependent with maximal effect at concentrations of 20 nM OA and 2 nM CA, respectively. OA also induced DNA ladder formation in other human oral SCC cell lines, SCCKN and SCCTF. To further determine if new gene transcription and protein synthesis are required for OA-induced apoptosis in SCC-25 cells, the cells were treated for 48 h with varying concentrations of cycloheximide in the presence of 20 nM OA. Cycloheximide did not protect the cells against OA-induced cytotoxicity and DNA ladder formation. Based on the known selectivity of OA and CA, the present results indicate that the pathway of the apoptosis in the cultured oral SCC cells is in part regulated by protein phosphatase type 1 and type 2A. Our results also indicate that new protein synthesis is not involved in OA-induced apoptosis in SCC-25 cells.

Topics: Apoptosis; Carcinoma, Squamous Cell; Enzyme Inhibitors; Humans; Marine Toxins; Mouth Neoplasms; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Tumor Cells, Cultured

Of nine biological factors (ATP, bradykinin, vasopressin, substance P, angiotensin II, norepinephrine, epinephrine, 12-tetradecanoylphorbol 13-acetate (TPA), and A23187 calcium ionophore) examined, bradykinin, as well as ATP, TPA, and A23187, significantly increased the phosphorylation of epidermal growth factor (EGF) receptors and reduced the binding of EGF to their high-affinity site. The reduction in EGF binding by bradykinin, ATP, and TPA was similarly reversed by concomitant incubation with staurosporine, a protein kinase C inhibitor, implying that the phosphorylation of EGF receptors was catalyzed probably by a protein kinase C of the same or similar type in each case. This possibility was confirmed by the fact that the major phosphorylation site of EGF receptors by the stimulation with either bradykinin, ATP, or TPA was the same (Thr-654). Different from the stimulations with ATP and TPA, the effect of bradykinin of decreasing the high-affinity EGF binding was transient (a minimum binding at 2.5 min); the reduced EGF binding was, however, sustained for up to 30 min in the presence of calyculin A, a phosphoprotein phosphatase inhibitor. Moreover, the homogenate prepared from bradykinin-stimulated A-431 cells had stronger dephosphorylation activity for phosphorylated EGF receptors than that from control cells. These results suggest that bradykinin stimulates both the protein kinase C system and a phosphoprotein phosphatase(s) activity in A-431 cells. Such biphasic effects of bradykinin to phosphorylate and dephosphorylate EGF receptors via protein kinase C and a phosphoprotein phosphatase, respectively, imply a homeostatic control of receptor function in regulating phosphorylation level by the same bioactive factor.

Topics: Alkaloids; Binding, Competitive; Bradykinin; Carcinoma, Squamous Cell; ErbB Receptors; Humans; Marine Toxins; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase C; Staurosporine; Tumor Cells, Cultured