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S Chakrabarty

Publications and source records attributed to S Chakrabarty.

At least 91 records · Page 5Linked to original sources
PubMed1985

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.

Autoradiography↗
PubMed1985

Modulation of cellular phosphoprotein profiles in transformation and redifferentiation of murine AKR embryonic fibroblastic cells.

Cellular phosphoprotein profiles from normal mouse embryonic fibroblast AKR-2B cells were compared to those of their permanently, chemically transformed malignant counterparts AKR-MCA cells, and AKR-2B cells reversibly transformed by transforming growth factor (AKR-TGF). Similar 32P-phosphorylation profiles were observed for both the AKR-TGF and AKR-MCA cells which were distinct from that of the normal AKR-2B cells. Dimethylformamide (DMF)-induced differentiation of the AKR-MCA cells resulted in a restoration of the normal AKR-2B phosphorylation profile to the malignant AKR-MCA cells.

Animals↗
PubMed1985

Selective phosphorylation of cytosol proteins associated with transformation and restoration of normal phenotype in AKR mouse embryo fibroblasts.

Phosphoproteins from cytosol preparations of methylcholanthrene-transformed AKR mouse (AKR-MCA) cells were compared to those of their untransformed counterparts, AKR-2B cells, by two-dimensional electrophoresis following an in vitro 32P phosphorylation procedure using endogenous kinases and substrates. Five proteins were phosphorylated in the AKR-MCA cells which were not observed in the AKR-2B cells, while six proteins were phosphorylated in the untransformed cells which were not observed in the malignant cells. Treatment of AKR-MCA cells with 1% N,N-dimethylformamide induced the reversion of the malignant cells to a phenotype similar to that of untransformed AKR-2B cells (S. Chakrabarty et al., Cancer Res., 44: 2181, 1984). Treatment of AKR-MCA cells with dimethyl formamide resulted in the restoration of five of the AKR-2B-associated phosphorylations and abolished 2 of the AKR-MCA-associated phosphorylations. AKR-2B cells have been shown to respond to transforming growth factors with reversible phenotypic transformation (R. F. Tucker et al., Cancer Res., 43: 1581, 1983). Transforming growth factor treatment of AKR-2B cells induced all five of the AKR-MCA-associated phosphoproteins and the loss of all six of the AKR-2B phosphoproteins. Epidermal growth factor treatment of AKR-2B cells resulted in the phosphorylation of several proteins which were not observed in either AKR-MCA or untreated AKR-2B cells. Some, but not all, of the AKR-2B-associated phosphorylations were also observed in epidermal growth factor-treated cells. The results of these studies demonstrated qualitative and/or quantitative changes in cytosolic protein kinase-phosphatase activities between transformed and normal AKR-2B cells. Treatment of AKR-MCA cells with dimethylformamide resulted in the restoration of some of the normal AKR-2B cell-associated protein kinase-phosphatase activities.

Animals↗
PubMed1985

Effects of BMY 25282, a mitomycin C analogue, in mitomycin C-resistant human colon cancer cells.

BMY 25282, a newly designed analogue of mitomycin C (MMC), was assessed for its non-cross-resistant cytotoxic and biochemical action against MMC-resistant human colon carcinoma cells. The analogue has an amidine substituted at position 7 of MMC and has a more efficient intracellular activation to its active species than MMC. In this study we demonstrated that BMY 25282 can overcome MMC resistance in a series of previously described human colon carcinoma cells resistant to MMC (Cancer Res., 44: 5880, 1984). The non-cross-resistance of the analogue in the model was confirmed in vivo by treating tumor xenograft-bearing athymic mice with equitoxic doses of MMC or BMY 25282. We further investigated the formation of interstrand DNA cross-link (IDC) formation by BMY 25282 and MMC. MMC-sensitive cells contained 3 to 8 times as many IDCs as resistant colon carcinoma cells, while no significant differences in IDCs were found between the MMC-sensitive or -resistant cells incubated with BMY 25282. When MMC-sensitive or -resistant cells were exposed to the 70% inhibition concentration of either MMC or BMY 25282, no differences were seen with respect to IDC formation. These studies demonstrate that BMY 25282 is able to overcome MMC resistance in a series of human colon carcinoma cells and that IDC formation in the MMC-sensitive or -resistant cells parallels cytotoxicity for both MMC and the analogue.

Animals↗
PubMed1985

Selective protein phosphorylation in heterogeneous subpopulations of human colon carcinoma cells.

Endogenous membrane and cytosolic and nuclear protein phosphorylations were compared among three well-characterized subpopulations of human colonic carcinoma cells that were originally isolated from a single human primary colon tumor. These intratumoral subpopulations of cells were found to differ significantly in their biological properties. Analysis of phosphoproteins by two-dimensional electrophoresis following 32P phosphorylation of subcellular fractions in a cell-free system or labeling intact cells in vivo revealed significant differences in the selective phosphorylation of membrane, cytosol, and nuclear proteins. The two-dimensional membrane, cytosol, and nuclear phosphoprotein profiles distinguished the three subpopulations of colonic carcinoma cells from each other. Silver-staining proteins from the three subpopulations were also compared. The two-dimensional, silver-stained electrophoretic profiles of nuclear proteins were essentially the same for all three subpopulations. The silver-stained electrophoretic profile of membrane and cytosolic proteins revealed only minor differences in the expression of polypeptides. Nevertheless, these changes could also distinguish the three subpopulations. The results of this study suggest that minor differences in the expression of cytosolic and membrane proteins exist in intratumoral subpopulations of colonic cells. However, a significantly greater degree of heterogeneity was found to be associated with post-translational modification of proteins by phosphorylation and/or dephosphorylation. These modifications could play an important role in determining the expression of different biological properties among subpopulations of malignant cells.

Cell Line↗
PubMed1984

Heterogeneity of human colon carcinoma.

In 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 (TGF's) confer a transformed phenotype on non-transformed fibroblasts while the second, tumor inhibitory factors (TIF's), 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 TGF's and TIF's 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 TGF's 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.

Animals↗
PubMed1984

Restoration of normal growth control and membrane antigen composition in malignant cells by N,N-dimethylformamide.

The effects of the differentiation agent, N,N-dimethylformamide (DMF), on malignant AKR-MCA cells were studied. The properties of DMF-treated AKR-MCA cells were compared to those of the normal parental AKR-2B mouse embryo fibroblasts. AKR-MCA cells grown in 1% DMF were found to be more similar to their normal counterparts than to untreated AKR-MCA cells by several criteria. These criteria included the loss of the transformed morphology, a 2-fold reduction of doubling time, a 10-fold reduction of saturation density, and the complete loss of the ability to grow with anchorage independence. The expression of high-molecular-weight membrane antigens (Mr 110,000 to 450,000), which was found to be greatly reduced in AKR-MCA cells in comparison to normal AKR-2B cells, was restored by treatment of AKR-MCA cells with DMF. The expression of a low-molecular-weight AKR-MCA cell-associated membrane antigen, on the other hand was found to be suppressed. Studies on the mitogenic response of these cells indicated that AKR-MCA and AKR-2B cells may be regulated by different types of growth control. Growth-arrested AKR-MCA cells did not respond to epidermal growth factor, but responded to nutrient replenishment. AKR-2B cells, on the other hand, responded to epidermal growth factor, but did not respond to nutrient replenishment. Treatment of AKR-MCA cells with DMF restored their ability to respond to epidermal growth factor, while their ability to respond to nutrient replenishment was lost. The results of this study indicated that DMF treatment induced the normalization of malignant AKR-MCA cells with regard to membrane antigen composition and growth control properties.

Animals↗
PubMed1983

Identification of cytosolic antigens from GW-39 adenocarcinoma cells by crossed immunoelectrophoresis and immunofluorescence.

Rabbits were immunized with a cytosolic fraction prepared from human adenocarcinoma cells of the colon (GW-39). The antibodies obtained were analyzed using crossed immunoelectrophoresis and were found to precipitate 23 distinct antigens in the cytosolic fraction from colon tumor cells. After preabsorption with human serum and plasma as well as acetone powders prepared from 2 normal human tissues and 4 normal hamster tissues, 1 major immunodominant cytosol antigen (CA-3) and two less intense immunoprecipitin peaks (CA-1 and CA-5) remained detectable by the crossed immunoelectrophoretic method. The preabsorptions with normal tissues were sufficiently complete to remove anti-carcinoembryonic antigen antibodies and showed that antigens CA-1, CA-3 and CA-5 are immunologically distinct from carcinoembryonic antigen. Indirect immunofluorescence localization studies with preabsorbed anti-cytosol antibodies and FITC-conjugated second antibody showed that these antigens were expressed in the cytoplasm and at the cell surface in several human colon tumor cell lines, their subclones and in primary colon tumor specimens.

Adenocarcinoma↗
PubMed1983

Isolation and partial characterization of a 700 kilodalton human colon carcinoma associated antigen.

An antigen of high molecular weight (CA-3) was isolated from the cytosol fraction of GW-39 human colon tumor cells by antibody affinity chromatography. CA-3 was characterized by an acidic pI value of 4.5-4.9, a molecular weight of 700 kilodaltons and a sedimentation coefficient of 13S. It contained all of the commonly occurring amino acids and had an acidic to basic amino acid ratio of 1.4. CA-3 was resistant to dissociation by reducing agents as well as by sodium dodecylsulfate. Quantitation of CA-3 by a radioimmunoassay employing rabbit anti-CA-3 antiserum revealed a marked elevation of CA-3 in the cytosol extracts of human primary colon carcinoma in comparison to normal colon. The molecular properties of CA-3 are compared to those of carcinoembryonic antigen, high molecular weight colon specific antigen CSAp and two other high molecular weight proteins, fibronectin and conglutinin. Colon antigen CA-3 appears to be different from these other molecules in terms of its molecular weight, sedimentation value, isoelectric point and amino acid composition.

Animals↗
PubMed1981

Biological profile of the steroid 5 alpha-stigmastane-3 beta, 5, 6 beta-triol-3-monobenzoate.

The steroid 5 alpha-stigmastane-3 beta, 5, 6 beta-triol-3-monobenzoate at a dose of 40 mg/kg/day showed significant antifertility activity when administered orally in a single or in consecutive doses during pre-implantation stage of pregnancy in mice. In the rabbit interceptive activity was obtained when the test steroid was administered on Day 8 of pregnancy in a single dose level. The compound failed to show any distinct estrogenic or androgenic property following bioassay method. But in McPhial test, the compound showed some progestagenic activity.

Animals↗
PubMed1981

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.

Adenocarcinoma↗
PubMed1981

Isolation and partial characterization of allergen C from Kentucky blue grass pollen.

An allergenic component, designated as Allergen C, was isolated from the retentate (R) of the aqueous extract of defatted Kentucky blue grass (KBG) pollen by a combination of preparative isoelectrofocussing and gel filtration. Allergen C was found to be a glycoprotein with molecular size of the order of 11,000 daltons and had a pI value of approximately 9.7. It possessed all the allergenic determinants collectively present in R. The allergenic activity was found to be associated with the protein moiety of the molecule. Allergen C was one of the 15 antigenic components detectable in R by crossed immunoelectrophoresis with a hyperimmune sheep antiserum to R. Observations based on enzymatic degradation of the molecule suggested that the determinants recognized by murine IgE antibodies were different from those which combined with rabbit precipitating antibodies.

Allergens↗
PubMed1981

Allergenic relationships of two different antigens of Kentucky bluegrass pollen.

Two purified allergens, allergen C and KBG-1, isolated from Kentucky bluegrass (KBG) pollen, were found to be antigenically distinct with respect to rabbit precipitating antisera produced separately to each allergen. On the other hand, allergen C and KBG-1 appeared to be allergenically identical in terms of the specificities evaluated with murine reaginic antisera produced to each of these allergens; whereas they appeared to be only partially identical when evaluated with respect to the IgE antibodies present in a pool of human sera from individuals allergic to KBG pollen. Therefore, not all of the allergenic determinants recognized by the human IgE antibodies were detected by the murine reaginic antisera. This study serves to emphasize that for a meaningful evaluation of allergenic specificities any antisera prepared in animals and intended for the standardization of allergens must recognize allergenic specificities which are similar or identical to those to which the human IgE antibodies are directed.

Allergens↗
PubMed1981

Detection of cross-reactive allergens in Kentucky bluegrass pollen and six other grasses by crossed radioimmunoelectrophoresis.

Using crossed immunoelectrophoretic analysis of an aqueous extract of Kentucky bluegrass (KBG)pollen employing hyperimmune rabbit and sheep precipitating antibodies to KBG and various grasses we detected at least 33 antigenic components. The majority of these antigens were extractable from the pollen within a period of 14 min and possessed anodal electrophoretic mobility. The extensive recognition of antigens in the KBG extract by antibodies raised to false oat, and to the combined extracts of timothy, orchard, meadow, velvet and rye grasses indicate that these grasses contain many cross-reactive antigens. One of the KBG antigens with cathodal mobility--the major component of the previously isolated allergen C--was identified as immunologically identical to timothy Ag 30. Crossed radioimmunoelectrophoretic analysis revealed differences in the extent to which IgE antibodies present in sera of individuals allergic to KBG bound to various antigens of KBG pollen. Those antigens that were recognized as allergens by all of the allergic sera examined were regarded as candidates for future isolation and characterization.

Allergens↗
PubMed1980

Isolation of a purified allergen from Kentucky blue grass pollen.

An allergenic fraction, designated as C-I-2-6, was isolated from among the allergenic constituents present in the dialysate of the aqueous extract of defatted Kentucky blue grass (KBG) pollen by a combination of gel filtration, ion-exchange chromatography and preparative isoelectrofocusing. This fraction was shown in consist of glycoprotein with a molecular size corresponding to 10,000 daltons and of two compounds with closely related pI values (4.5--4.9); its protein moiety contained all the amino acids except cysteine. As demonstrated by crossed immunoelectrophoresis with a sheep anti-R serum, fraction C-I-2-6 shared the antigenic determinants of 2 of the 15 antigenic components of the nondialysable components, i.e. retentate (R) of the aqueous extract of KBG pollen. Remarkably, in spite of the markedly lower molecular weight than R, fraction C-I-2-6 possessed all the allergenic determinants of R responsible for inducing murine IgE antibodies.

Allergens↗
PubMed1980

Allergens of Kentucky Blue Grass pollen. II. Isolation of hapten-like components from Kentucky Blue Grass pollen by preparative isoelectrofocussing.

Components with hapten-like properties were isolated from the nondialyzable fraction, i.e. the retentate (R) and the dialyzable fractions of the aqueous extract of Kentucky Blue Grass pollen (KBC aq.ext.), by preparative isoelectrofocussing on Sephadex G-100 gel. These haptenic components could not elicit the passive cutaneous anaphylaxis (PCA) reactions in rats passively sensitized with a murine reaginic antiserum to R, but could inhibit completely and specifically the PCA reaction which is normally elicitable with R. It was concluded that the specificity of the murine IgE antibodies was directed to a determinant(s) which was common to either allergenic or haptenic fractions. Moreover, by employing a pool of human sera from individuals allergic to KBG pollen in the radioallergosorbent test procedure, it was apparent that most of the haptenic fractions lacked some of the specificities present on allergenic components of R that were recognized by the human IgE antibodies. Evidence was obtained to suggest that the electrophoretic heterogeneity of allergenic components present in various fractions of KBG aq.ext. may be due primarily to differences in their net charge, rather than to differences in their allergenic specificity.

Absorption↗