Subhas Chakrabarty

Heterogeneity of human colon carcinoma.

SummaryIn order to better understand colon cancer, a model system reflecting the heterogenous nature of this disease was developed and used in the development of new cytotoxic and non-cytotoxic therapeutic approaches. A large bank of colon carcinoma cell lines was established from primary human colon carcinomas and grouped based on their tumorigenicity in athymic mice, their growth rates in soft agarose and in tissue culture, and their secreted levels of carcinoembryonic antigen. These cell lines were later characterized based on cell surface proteins and antigens detected with antisera raised against a differentiated colon carcinoma cell line. Although these biochemical markers correlated with the biological classification of these cell lines, there was still extensive heterogeneity within each group in all properties examined. This colon carcinoma cell system was used to study natural vs. selected resistance to the anticancer drug mitomycin C (MMC). The differing IC50 values in vitro were reflected in the inhibition by MMC of xenograft growth in athymic mice. A new, more readily bioactivatable analogue of MMC was tried and shown to be more active in vitro and in vivo, suggesting that rapid efflux of the drug before activation may be important in examining causes of resistance to MMC. Another approach to the treatment of colon cancer is the use of non-cytotoxic agents such as growth factors and differentiation agents to restore normal growth to the malignant cells. We have isolated and characterized two types of polypeptides from colon carcinoma cells and conditioned medium from these cells. The first, transforming growth factors (TGFs) confer a transformed phenotype on non-transformed fibroblasts while the second, tumor inhibitory factors (TIFs), inhibits the anchorage independent growth of transformed cells. The fact that extracts of colon carcinoma cells contain both activities suggests that the heterogeneity of the cell lines could be due to different levels of TGFs and TIFs produced. The effectiveness of differentiation agents to restore normal growth control using a transformed mouse embryo cell line was examined. Treatment of these cells with differentiation agents restored normal growth control to these cells. An increased synthesis of TGFs resulted from these treatments. Therefore, differentiation agents may be useful in non-cytotoxic treatment. The use of this model system for human colon carcinoma will hopefully lead to more effective drugs for the treatment of colon cancer in man.

Comparison of the antiproliferative effects of transforming growth factor-β,N,N-dimethylformamide and retinoic acid on a human colon carcinoma cell line

In this study we have compared the anti-proliferative effects of transforming growth factor-beta (TGF-beta), N,N-dimethylformamide (DMF) and retinoic acid (RA) on a moderately-differentiated colon carcinoma cell line (JVC). TGF-beta, DMF and RA inhibited the anchorage-independent growth and induced morphological changes in JVC cells. EC50 values of 40 pM TGF-beta, 0.5% DMF and 5 nM RA were obtained for growth inhibition. In addition all three agents enhanced cellular fibronectin levels in a time- and dose-dependent manner. Inhibition of cell proliferation as well as fibronectin induction by all three agents were reversible. Combinations of any two agents at suboptimal doses, added simultaneously to JVC cells gave additive inhibitory response on growth. These data indicate that the effects of TGF-beta in this colon carcinoma cell line are similar to those of the two differentiation promoting agents DMF and RA.

Assessment of tumor cell sensitivity to mitomycin C by “B23 translocation” assay

Mouse leukemia cells (p388D1) were grown in medium containing various amounts of mitomycin C for 4 h. Cellular localization of protein B23 was detected using an immunofluorescence technique. Translocation of protein B23 from nucleoli to the nucleoplasm was observed with increasing dose of mitomycin C. To study the correlation of B23 translocation and drug resistance, three human colon carcinoma cell lines, HCT116, HCT116b (a line that is natively or intrinsically resistant to mitomycin C), and HCT116-44 (a line with an acquired resistance to mitomycin C), were employed. These cells were incubated with 1--150 micrograms/ml of mitomycin C. The drug concentration that caused 50% of the cells to have complete translocation (IC50) was determined for each cell line. The IC50 values of HCT116, HCT116b and HCT116-44 were 6, 10 and 50 micrograms/ml, respectively. These IC50 values correlate well with the mitomycin C resistant phenotype of these tumor cells as determined by other in vivo and in vitro assays (Willson, et al. (1985) Cancer Res., 45, 5281-5286). These results identify an inverse relationship between the ease of protein B23 translocation and the degree of mitomycin C resistance in human colon carcinoma cells. This relationship applies to cells that have either acquired mitomycin C resistance or intrinsic resistance to the drug.

Retinoic acid restores normal growth control to a transformed mouse embryo fibroblast cell line

The effects of retinoic acid on a transformed mouse embryo fibroblast cell line (AKR-MCA) were examined. Treatment with retinoic acid restored a non-transformed phenotype to this transformed cell line in a dose dependent manner. Retinoic acid (RA) treated AKR-MCA cells showed a non-transformed morphology, a slower growth rate, and did not grow with anchorage independence. A 38,000 Da protein was phosphorylated to a high degree in the AKR-MCA transformed cell line compared to the non-transformed AKR-2B cell line. RA treatment greatly reduced the level of phosphorylation of this protein in AKR-MCA cells. Growth arrested AKR-MCA cells showed a mitogenic response to nutrient replenishment, but not to epidermal growth factor (EGF). Treatment of AKR-MCA cells with RA restored their ability to respond to EGF while the response to nutrient replenishment was lost. This pattern of growth control was similar to that of the non-transformed AKR-2B cells.

Fibrin solubilizing properties of certain anionic and cationic detergents

The fibrinolytic (fibrin dissolving) properties of several anionic, cationic, nonionic and zwitterionic detergents were assessed in an in vitro fibrin agarose assay. Of the 4 anionic detergents tested, only sodium dodecyl sulfate (SDS) was found to be fibrinolytic. SDS was fibrinolytic either in the absence or presence of factor XIII. Four other cationic detergents were found to possess similar fibrinolytic properties. These cationic detergents were cetyltrimethylammonium bromide (CTAB), mix alkyltrimethyl ammonium bromide (MTAB), hexadecyltrimethylammonium bromide (HTAB) and cetylpyridium chloride (CPC). The nonionic (digitonin, triton X-100/tween 20) and zeitterionic (CHAPS, zeittergent 3-08) detergents were not fibrinolytic. Detergents mediated fibrinolysis, unlike that of tissue type plasminogen activator and urokinase, was independent of the presence of plasminogen. Non-detergents such as polyethylene glycol and highly charged compounds such as poly-1-lysine and poly-1-glutamic acid were not fibrinolytic. Fibrinolytic activity was observed for SDS and the cationic detergents at concentrations ranging from 0.1-10 percent. The effects of these fibrinolytic detergents (SDS, CTAB, MTAB, HTAB and CPC) on clot formation and on pre-formed clots were then assessed, using freshly drawn human venous blood. Incorporation of these detergents into blood inhibited the formation of clots in a concentration dependent manner. The detergents were also able to dissolve pre-formed clots in a similar fashion. SDS was found to be most potent in these properties.

Selective nuclear protein phosphorylation/dephosphorylation in subpopulations of human colonic carcinoma cells

The nuclear protein and phosphoprotein profiles from 3 subpopulations of human colonic carcinoma cells which expressed different levels of neoplastic properties were characterized by two-dimensional electrophoresis. The silver stained nuclear protein profiles were found to be remarkably similar among the subpopulations. However, 2 types of nuclear proteins were found to be selectively modified by phosphorylation/dephosphorylation reactions. The dephosphorylation of type I and the phosphorylation of type II nuclear proteins were found to be associated with the HCT 116a subpopulation which expressed a high level of neoplastic properties. Conversely, the phosphorylation of type I and the dephosphorylation of type II nuclear proteins were found to be associated with the HCT 116b subpopulation, which expressed a low level of neoplastic properties. The HCT 116 subpopulation, which expressed an intermediate level of neoplastic properties, was found to possess an intermediate phosphoprotein profile relative to that of the other two subpopulations. Selective modification of cellular proteins by phosphorylation/dephosphorylation reactions may be involved in the generation of tumor cell diversity and heterogeneity.

Selective Modifications of Cellular Proteins in Intratumoral Subpopulations of Human Colonic Carcinoma Cells

Molecular alterations of cellular proteins were assessed in three previously described subpopulations of human colon carcinoma cells that were originally isolated from a single human primary colon carcinoma. The subpopulations designated HCT 116b, HCT 116, and HCT 116a exhibited significant differences in their expression of several biological properties. Immunoautoradiographic analysis of membrane components, fractionated by SDS-PAGE and electrophoretically transferred onto nitrocellulose sheets, distinguished the most aggressive tumor cell subpopulation from the intermediate and least aggressive subpopulations. An elevated expression of antigens of molecular weight in the range of 116-120 kd, 92 kd, 55-66 kd, and 31 kd was found to be associated with the most aggressive subpopulation of HCT 116a cells. The binding pattern of RCA I and WGA lectins to membrane components, fractionated by SDS-PAGE and fixed on nitrocellulose, also distinguished the three subpopulations of cells. Elevated bindings of RCA I to 80-92 kd components of HCT 116b cells and elevated bindings of WGA to 120 kd components of HCT 116a cells distinguished the three subpopulations from one another. Analysis of cellular phosphoprotein profiles, following labeling of the cells with 32Pi in the presence of phosphate-free medium, further distinguished the most aggressive subpopulation from the intermediate and least aggressive subpopulations. High level of phosphorylation of 120 kd and 250 kd nuclear components, 30 and 90 kd cytosolic components, and low level of phosphorylation of lower molecular weight membrane components were found to be associated with the most aggressive subpopulation. It is concluded that differences in the expression of membrane antigens, differences in the glycosylation of membrane components, and the selective phosphorylation and/or dephosphorylation of cellular proteins exist in subpopulations of intratumoral colonic carcinoma cells with different biological properties. These biochemical alterations of cellular proteins may play an important role in the generation of phenotypic diversity and heterogeneity of malignant cells.

Comparison of immunoelectrophoretic techniques for the analysis of cytosol antigens

Crossed immunoelectrophoresis was employed in the analysis of cytosol antigens of a human colon adenocarcinoma cell line. Other immunoelectrophoretic techniques - crossed immunoelectrophoresis in the presence of Ampholine (incorporation of Ampholine in the first dimension electrophoresis gel), crossed immunotachophoresis and crossed immunoelectrofocusing - were also investigated ad compared with crossed immunoelectrphoresis in an attempt to select the optimal immunoelectrophoretic system for the analysis of cytosol antigens. The results of these comparisons showed that each technique offered its own advantages. The maximum number of immunoprecipitin peaks were detected by crossed immunoelectrophoresis. Both crossed immunoelectrophoresis in the presence of Ampholine and crossed immunotachophoresis provided the greatest resolution of electrophoretically similar antigens. Crossed immunoelectrofocusing provided IP values for these antigens. It was concluded that these techniques, when employed in combination, provided a more complete analysis of the complex cytosol antigenic mixture and may be useful when employed in combination to other antigen-antibody systems.

The use of 125I-lectin probes in defining plasma membrane carbohydrate moieties in 3 subpopulations of human colonic carcinoma cells.

The molecular mechanism(s) responsible for the generation of phenotypic diversity within tumors is not understood. Since the cell surface/plasma membrane components are involved in a variety of important biological function such as growth and differentiation regulation which may be mediated through intercellular and/or extracellular matrix interaction, the plasma membranes from 3 human colonic carcinoma cell lines (originally isolated from a single primary tumor) were purified and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Membrane carbohydrate moieties were also characterized by a panel of 5 125I-labeled lectin probes following their electrophoretic fractionation and transfer onto nitrocellulose. Though these cell lines possessed diverse biological properties, their Coomassie blue stained electrophoretic protein profiles were found to be very conserved. The altered quantitative expression of only 1 membrane protein (Mr = 46 KD) was found to be associated with the more neoplastic HCT 116a cells which distinguished this cell line from the less neoplastic HCT 116b and HCT 116 cells. All 5 lectin binding profiles, on the other hand, clearly and easily distinguished the HCT 116a cells from the HCT 116b and HCT 116 cells. Thus, heterogeneity in terms of differences in membrane carbohydrate moieties was more obvious.

Transforming growth factors from human colonic carcinoma cells induced molecular alterations in untransformed mouse AKR embryo fibroblasts

Over 700 polypeptide spots could be detected by two-dimensional electrophoretic analyses of membranes prepared from the murine AKR fibroblastic cells. Out of this abundance of polypeptides, only 9 polypeptide spots were found to be differentially expressed between the untransformed AKR-2B cells and their methylcholanthrene-transformed counterparts, the AKR-MCA cells. Treatment of the untransformed AKR-2B cells with transforming growth factors, prepared from the serum-free conditioned medium of HCT 116 MOSER human colonic carcinoma cells, induced the altered expression of 6 of these polypeptides which paralleled the electrophoretic profile of their permanently transformed counterparts, the AKR-MCA cells.