demecolcine and Colonic-Neoplasms

The molecular basis for the sensitivity of tumor cells to chemopreventive natural food compounds and commonly used chemotherapeutic agents is not well understood, not least because studies are frequently confounded by the diversity among cell lines or rely on experimental protein overexpression. Here we investigated the effects of n-butyrate, a cancer-preventive short-chain fatty acid produced by anaerobic bacteria in the gastrointestinal tract, on the human wild-type p53 and p21 expressing HCT116 colon carcinoma cell line and on HCT116 cells with either p53 or p21 alleles inactivated by homologous recombination. The effects of n-butyrate were then compared with those elicited by cytotoxic drugs and the natural chemopreventive phytoalexin of wine and grapes, resveratrol. We document that physiological concentrations of n-butyrate stimulate p21 expression and induce apoptosis independently of p53, and that the absence of p21 increases apoptosis drastically. The apoptosis is mediated through the mitochondria and is accompanied by mitochondrial proliferation and membrane potential changes. Adriamycin, etoposide, cisplatinum, colcemid and resveratrol induce distinct cellular responses; however, absence of p21 favors apoptosis-induction by adriamycin, etoposide and colcemid. Thus, control of p21 expression may support chemoprevention and certain tumor therapies.

Topics: Adenocarcinoma; Alleles; Amino Acid Chloromethyl Ketones; Anticarcinogenic Agents; Antineoplastic Agents; Apoptosis; Benzothiazoles; Butyrates; Cisplatin; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Proteinase Inhibitors; Demecolcine; Doxorubicin; Drug Resistance, Neoplasm; Etoposide; Fluorouracil; Gene Expression Regulation, Neoplastic; Genes, p53; Humans; Intracellular Membranes; Membrane Potentials; Mitochondria; Neoplasm Proteins; Recombination, Genetic; Resveratrol; Stilbenes; Thiazoles; Toluene; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Radiosensitization has previously been demonstrated in a human colon cancer cell line (HT-29) following a 2 h exposure to low, clinically relevant concentrations (0.05-0.5 microM) of fluorodeoxyuridine (FdUrd) (15). The sensitizer enhancement ratio value (measured at 10% survival) plateaued at approximately 1.7 between 16 and 32 h following removal of drug. Parallel studies investigating the effect of FdUrd on the distribution of cells throughout the cell cycle found that the percentage of cells in early S-phase increased to approximately 70% during the same period that maximal radiosensitization was noted. As a follow-up to these findings, experiments have been designed to investigate the contribution of this early S-phase delay to radiosensitization.. Synchronized populations of HT-29 cells have been obtained with three separate techniques. Two involve the induction of a reversible metaphase arrest (with high pressure N2O or colcemid) followed by a shakeoff of mitotic cells. The third uses a plant amino acid, mimosine, to induce a reversible block at the G1/S boundary. Flow cytometry was used to analyze the degree of synchrony based on bromodeoxyuridine (BrdUrd) uptake and propidium iodide (PI) staining. Radiation survival curves were obtained on these synchronized populations to investigate changes in radiosensitivity through the cell cycle. Additionally, levels of thymidylate synthase (TS), the primary target of FdUrd cytotoxicity, were measured in each phase of the cell cycle using the TS 106 monoclonal antibody against human TS.. Synchronization with mitotic shakeoff produced relatively pure populations of cells in G1; however, the degree of synchrony in early S-phase was limited both by cells remaining in G1 and by cells progressing into late S-phase. These techniques failed to reveal increased radiosensitivity in early S-phase at 10% survival. An 18 h exposure to mimosine resulted in populations that more closely resembled the early S-phase enrichment following FdUrd exposure and revealed increased radiosensitivity during early S-phase. TS levels were noted to be only 1.3 times higher in S phase than in G0/G1.. Radiation survival data from cells synchronized with mitotic shakeoff techniques suggest that early S-phase delay is unlikely to be the primary mechanism of FdUrd radiosensitization. In contrast, the increased sensitivity seen in early S-phase with mimosine synchronized cells is similar to that seen with FdUrd. Although confounding biochemical pertubations cannot be ruled out, these data continue to suggest an association between early S-phase enrichment and radiosensitization. The significance of TS inhibition as a mechanism of FdUrd radiosensitization remains unclear.

Topics: Cell Cycle; Cell Survival; Colonic Neoplasms; Demecolcine; Floxuridine; Humans; Mimosine; Nitrous Oxide; Nucleotides; Radiation Tolerance; Radiation-Sensitizing Agents; S Phase; Thymidylate Synthase; Tumor Cells, Cultured

In this communication we suggest two simple ways to represent the information on the cell cycle obtained by flow cytometry, offering some advantages over the traditional plots. We show that the results of a single experiment, when reduced to the percentages of cells in G1, S, and G2M phases, can be completely expressed with a single point in a G1-G2 plan where iso-S lines are also drawn. By the use of this plot, the time course of the complete kinetics of a cell population may be shown in a single drawing. The second plot, we suggest, integrates information on cell cycle, represented by the distribution mean, with cell number.

Topics: Cell Cycle; Colonic Neoplasms; Demecolcine; DNA; Flow Cytometry; Humans; Mathematics

Giant axonal neuropathy skin fibroblasts, which are characterized by a selective and partial disorganization of vimentin filaments [1] exhibited, when compared with normal skin fibroblasts, less fibrin clot retractile (FCR) activity and spreading within the fibrin clot both during active growth and resting stage. Skin fibroblasts derived from patients affected with adenomatosis of the colon and rectum, which display a disorganized actin network [2], exhibited reduced FCR activity and spreading within the fibrin clot only during resting stage. FCR inhibition was also obtained by treating the cells with colcemid, cytochalasin B (CB) and dihydrocytochalasin B. The data suggest that FCR activity is under the control of different cytoskeletal structures. For the first time, a direct involvement of intermediate-sized filaments could be demonstrated in the interaction between fibroblasts and an organic substratum.

Topics: Actins; Adenoma; Cell Movement; Clot Retraction; Colonic Neoplasms; Cytochalasins; Cytoskeleton; Demecolcine; Humans; Nervous System Diseases; Rectal Neoplasms; Skin; Vimentin

To provide a rapid method for examining cell cycle dynamics, we utilized continuous exposure of Chinese hamster ovary cells and human colon cancer cells to colcemid to block cycling cells in metaphase, suppressing re-entry into G1. Changes in cell cycle compartment distribution were monitored by DNA flow cytometry. Analysis of the rate of G2 + M compartment accumulation after addition of colcemid permitted calculation of all cycle transit parameters. These compared favorably with data in the same cell lines determined by the fraction of labeled mitoses technique. Serial assessment of DNA flow cytometry after addition of colcemid permits rapid quantitation of cycle traverse rates.

Topics: Animals; Cell Cycle; Cells, Cultured; Colonic Neoplasms; Cricetinae; Demecolcine; DNA; Female; Humans; Kinetics; Mitosis; Ovary

The lethal and cytokinetic effects of mitomycin C (MC) as a function of drug concentration and exposure time were assessed in cultured human colon adenocarcinoma (LoVo) cells using colony formation to determine cell survival and DNA flow cytometry to examine cell cycle perturbation. MC evoked threshold-exponential type 1-hr dose-dependent survival curves in both exponential and stationary growth phases (Dq = 0.4 microgram/ml; Do = 1.0 microgram/ml). In exponentially growing cultures, a given exposure dose of MC induced equitoxic effects regardless of the specific drug concentration and exposure time used with uninterrupted treatment. However, dose fractionation experiments revealed the ability of LoVo cells to partially repair sublethal damage from MC exposure. Cell cycle progression was reversibly delayed or blocked in G2, S, and G1 phases in this order of sensitivity, with a frozen cycle distribution after greater than or equal to 24 hr treatment with 5 microgram of MC per ml. The reversible delay in S-phase traverse without a significant subsequent G2 block may be exploitable for administration of S-phase-specific drugs to maximize cell kill.

Topics: Adenocarcinoma; Cell Cycle; Cell Line; Cell Survival; Colonic Neoplasms; Demecolcine; Dose-Response Relationship, Drug; Drug Administration Schedule; Humans; Interphase; Mitomycins; Neoplasms, Experimental

The cytokinetic response of a human colon carcinoma cell line to cis-dichlorodiammineplatinum(II) was investigated using flow cytometry of DNA content, autoradiography after pulse and continuous tritiated thymidine exposure, and mitotic accumulation after continuous Colcemid treatment. With increasing concentration and exposure time, cis-dichlorodiammineplatinum(II) delayed and then blocked cycle traverse in S and G2 phases. After prolonged treatment with high concentrations of cis-dichlorodiammineplatinum(II), an additional block in G1 or at the G1-S boundary was established. Irreversibility of cell cycle distribution changes after prolonged observation periods suggests cell death in G2, S, and G1 compartments.

Topics: Cell Cycle; Cell Line; Cell Survival; Cisplatin; Colonic Neoplasms; Demecolcine; DNA, Neoplasm; Humans; Kinetics; Mitosis