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S Chakrabarty

Publications and source records attributed to S Chakrabarty.

At least 73 records · Page 4Linked to original sources
PubMed1989

Diverse cellular responses elicited from human colon carcinoma cells by transforming growth factor-beta.

We have recently characterized the growth-inhibitory and cellular responses [carcinoembryonic antigen (CEA) secretion, protein secretion, protein expression, fibronectin and laminin synthesis] of the human colon carcinoma MOSER cell line to transforming growth factor-beta (TGF-beta) (Cancer Res., 47: 2950, 1987; 48: 4059, 1988). We have also recently isolated a subline (MOSER R2) from the parental MOSER cells which, unlike the parental line, is relatively resistant to the growth-inhibitory effect of TGF-beta (Biochem. Biophys. Res. Commun., 150: 711, 1988). We now report on the characterization of the cellular responses of this resistant MOSER R2 subline to TGF-beta and compare its responses to that of the highly growth-inhibition-sensitive MOSER cell line. In view of the reported relationship between CEA expression and differentiation in colon cancer and the ability of colon-derived substrata material to modulate the phenotypic properties of colon cancer cells, additional characterization and direct comparison of the effects of TGF-beta on the two cell lines were also performed with respect to (a) cellular expression of CEA and CEA cross-reactive glycoproteins; and (b) colon-derived substrata material. Unlike the growth-inhibition-sensitive MOSER cells, TGF-beta had no effects on fibronectin/laminin synthesis nor on the cellular morphology of the resistant MOSER R2 cells. TGF-beta was also unable to modulate protein secretion and deposition of substrata material by these cells. However, several other responses of the resistant cells to TGF-beta were found to be similar to that of the sensitive MOSER cells. These responses include: (a) a prolonged and stable secretion of CEA; (b) a prolonged and stable induction of elevated cellular expression of CEA and CEA cross-reactive glycoproteins; and (c) enhancement of the expression of three cellular proteins with molecular weights corresponding to 52,000, 48,000, and 42,000. We further report that the differences observed in the responses to TGF-beta in the two cell lines were not due to differences in TGF-beta binding or other receptor parameters such as the expression of distinct TGF-beta receptor subspecies.

Carcinoembryonic Antigen↗
PubMed1989

Examination of urokinase protein/transcript levels and their relationship with laminin degradation in cultured colon carcinoma.

Conditioned medium derived from the colon cancer cell lines was ineffective in solubilizing immobilized radiolabeled laminin. However, substantial degradation was observed in the presence of plasminogen and could be largely blocked by preincubation with polyclonal anti-urokinase antibody. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized products generated either by the conditioned medium or by authentic urokinase supplemented with plasminogen yielded identical results. Analysis of the spent medium for urokinase by an enzyme-linked immunosorbent assay method revealed a similar profile to that achieved with the laminin degradation assays for the six cell lines tested. However, Northern analysis of urokinase-specific mRNA indicated that protein levels could not be entirely predicted by steady-state levels of the transcript. In a previous study, undifferentiated colon cancer cell lines expressed larger amounts of the plasminogen activator into the conditioned medium compared with their well-differentiated counterparts. However, these earlier studies were performed using cells grown in defined medium which lacked epidermal growth factor (EGF). EGF has been reported to affect plasminogen activator levels. Consequently, to investigate the role of EGF in the modulation of urokinase protein/activity, cell types representative of well- and poorly differentiated colon cancer were examined for their sensitivity of expression to this growth factor. In the absence of EGF, primitive cell types secreted, on average, 5 times more urokinase than their well-differentiated counterparts. In response to EGF, however, well-differentiated cell lines exhibited 4- to 6-fold increases in these parameters while the primitive cell lines were refractory to the peptide. Consequently, the differences in urokinase protein expressed by the well- and poorly differentiated groups of cells were abolished by the presence of EGF. The expression of a well-differentiated phenotype by colon cancer cell types in vivo probably depends to some extent on laminin within a basement membrane. The data presented herein are consistent with the idea that depletion of this glycoprotein from a basement membrane by urokinase-dependent mechanisms may contribute to the undifferentiated phenotype seen with many of these malignancies.

Cell Line↗
PubMed1989

Antiproliferative activity of liposome-encapsulated transforming growth factor-beta against MDA-MB-435 human breast carcinoma cells.

We determined whether transforming growth factor-beta (TGF-beta) could be encapsulated in phospholipid liposomes and then would mediate antiproliferative activity against the sensitive, human breast cancer cell line, MDA-MB-435. TGF-beta was encapsulated in multilamellar liposomes consisting of phosphatidylcholine (PC) or PC and phosphatidylserine (PS) at a 7:3 molar ratio. It was captured in both the aqueous phase and the bilayer lipid (hydrophilic and lipophilic association) and was stable for at least 24 hr of incubation at 37 degrees C in medium that contained 5% fetal bovine serum. In calcium- and magnesium-free Hanks' balanced salt solution, TGF-beta in the internal aqueous compartment was stable for at least five days, even in the presence of trypsin and ethylenediamine tetraacetic acid. TGF-beta (type 1 or 2) in liposomes was active as free-form TGF-beta in mediation of antiproliferative effects. The lipophilic nature of TGF-beta, which resulted in a high capture ratio in liposomes, coupled with exceptional stability, suggested that liposomes could be a carrier for the in vivo use of TGF-beta.

Breast Neoplasms↗
PubMed1989

Clonal stimulation or inhibition of human colon carcinomas and human renal carcinomas mediated by transforming growth factor-beta 1.

We examined various human carcinomas and cells populating a single human neoplasm to determine whether they exhibit a heterogeneous response to the effects of transforming growth factor-beta 1 (TGF-beta). Using recently established human colon carcinoma and renal cell carcinoma under defined in vitro conditions, we observed intertumoral and intratumoral heterogeneity and polarity of responses to TGF-beta (growth inhibition or stimulation) that did not correlate with the metastatic phenotype of the cancer cells as assessed in athymic nude mice. TGF-beta mediated both cytostatic and cytolytic effects against sensitive tumor cells, and these responses were not related to the effects of TGF-beta on the cell-cycle traverse. The human colon carcinoma and renal cell carcinoma, however, exhibited differences in the expression of TGF-beta receptors.

Animals↗
PubMed1988

Induction of carcinoembryonic antigen secretion and modulation of protein secretion/expression and fibronectin/laminin expression in human colon carcinoma cells by transforming growth factor-beta.

We have recently reported that TGF-beta induces a response similar to that of planar polar differentiation promoters in human colon carcinoma MOSER cells. N,N-Dimethylformamide and TGF-beta had similar effects on MOSER cells with respect to reversible inhibition of growth (both in monolayer culture and semisolid medium), induction of fibronectin expression and the induction of morphological alterations (Cancer Res., 47:2950-2954, 1987). Since the expression of carcinoembryonic antigen (CEA) has been reported to be modulated by planar polar compounds that promote differentiation in colon carcinomas, we addressed the issue of whether the differentiation-like effects of TGF-beta on these cells would also encompass modulation of CEA expression in the MOSER cells. The biological modulating effects of TGF-beta on extracellular matrix glycoprotein expression and the expression and secretion of cellular proteins were also studied in view of the reported modulating effects of this growth factor on untransformed, noncolonic cells. In this communication we report that TGF-beta induced the synthesis of fibronectin and laminin but not collagen IV. TGF-beta also induced CEA secretion in a dose-dependent manner. Elevated CEA secretion was detected following 48 h of TGF-beta treatment and a 16-fold increase in CEA secretion was observed following 7 days of treatment. The cells were committed to secrete CEA following one dose of TGF-beta treatment. The enhanced expression of four cellular proteins (Mr 42,000, Mr 48,000, Mr 52,000, and Mr 55,000) and the enhanced secretion of three proteins (Mr 66,000, Mr 200,000, and Mr 400,000) were also induced. Some of these protein alterations were detected as early as 6-24 h following TGF-beta treatment. It is concluded that TGF-beta modulated the production and secretion of CEA, the synthesis of fibronectin and laminin, and the expression and secretion of several cellular proteins in the colon carcinoma MOSER cells. To our knowledge, this is the first report on the modulation of CEA and laminin by TGF-beta in tissue-cultured cells, and is the first report on the modulation of cellular proteins by this growth factor in human colon carcinoma cells.

Carcinoembryonic Antigen↗
PubMed1988

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.

Animals↗
PubMed1988

Comparison of the antiproliferative effects of transforming growth factor-beta, 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.

Cell Division↗
PubMed1988

Alterations of the biological characteristics of a colon carcinoma cell line by colon-derived substrata material.

This study documents the ability of substrata material derived from well but not poorly differentiated colon carcinoma cells to alter the biological characteristics of a separate colon carcinoma cell line (MOSERSF). To assess changes induced by the presence of these substrata, MOSERSF cells were screened for (a) morphological features, (b) secretion of carcinoembryonic antigen (CEA), (c) alteration of urokinase levels, and (d) sensitivity to the growth-inhibitory peptide transforming growth factor beta. Morphologically, MOSERSF cells grown on plastic displayed a rounded shape and could be detached by agitation. Subculturing of these cells onto substrata laid down by well differentiated (mature) colon carcinoma cells resulted in cell attachment and spreading. These changes did not manifest themselves when cells were plated on material derived from poorly differentiated (primitive) colon cells. Conditioned medium from MOSERSF cells grown on plastic or on colon-derived material from the well and poorly differentiated colon cells were compared for CEA levels. Substrata derived from undifferentiated cells were without effect on assayable CEA (substrata absent, 1.4 ng/ml/10(6) cells/72 h; substrata present, 1.4-1.7 ng/ml/10(6) cells/72 h). However, growth of MOSERSF cells on material deposited by well differentiated colon cells resulted in a 3-fold increase in the level of CEA. Spent medium was also analyzed for urokinase. A high level of the protease (20.3 ng/ml/10(6) cells/72 h) was expressed by MOSERSF cells. The concentration of the enzyme was reduced by over 50% when MOSERSF cells were propagated on substrata laid down by well differentiated cells. An enhanced sensitivity to the growth-retarding effects of transforming growth factor beta was seen with certain substrata. On plastic, transforming growth factor beta inhibited proliferation of MOSERSF cells with a median effective concentration of 0.65 ng/ml. However, on substrata from mature but not primitive cells, MOSERSF cells exhibited an increased sensitivity to the peptide (median effective concentration, 0.16 ng/ml). Colon-derived material obtained from both well differentiated and poorly differentiated colon carcinoma cells was compared after [35S]-methionine metabolic labeling. More [35S]methionine was incorporated into the material from the "mature" colon cells. The substrata could also be distinguished by quantitative differences in a number of high molecular weight proteins. Immunofluorescence of colon-deposited material revealed the presence of laminin and fibronectin.

Carcinoembryonic Antigen↗
PubMed1987

Modulation of fibronectin, laminin, and cellular adhesion in the transformation and differentiation of murine AKR fibroblasts.

The functional relationship between membrane/cell surface expression of fibronectin and laminin and transformation/differentiation was examined in an AKR mouse fibroblastic cell model. This model consisted of the untransformed AKR-2B cells, their chemically transformed counterpart (AKR-MCA cells) and the chemically differentiated form of the AKR-MCA cells. The transformed AKR-MCA cells were found to express more surface laminin and less fibronectin than the untransformed AKR-2B cells. The transformed AKR-MCA cells were slower to attach and spread on both plastic and type IV collagen-coated dishes in comparison to the AKR-2B cells. However, a higher percentage of the AKR-MCA cells ultimately attached and spread on the type IV collagen-coated dishes. The induction of differentiation in the AKR-MCA cells by N,N-dimethylformamide (DMF) restored fibronectin to the surface of the AKR-MCA cells but reduced laminin expression only slightly. The DMF-treated AKR-MCA cells resembled the AKR-2B cells in that they rapidly attached and spread on plastic dishes and dishes coated with type IV collagen. They also resembled the AKR-MCA cells in that a high proportion ultimately attached and spread on the collagen-coated dishes.

Animals↗
PubMed1987

Molecular comparisons of acquired and native mitomycin C resistance in human colon carcinoma cells.

Acquired mitomycin C (MMC) resistant phenotype in the HCT 116 human colon carcinoma cell line is associated with an inability of the resistant cells to activate MMC (Cancer Res., 46 (1986) 3456). This report shows that the natively resistant human colon carcinoma cell line (HCT 116b), which had never been exposed to the drug, also exhibited a deficiency in MMC activation. The deficient activation of MMC in the natively resistant cells (like their acquired resistant counterpart) was circumvented by the MMC analog BMY28282 which was designed with a low quinone reduction potential and thus a greater ease of reductive activation. Interestingly, the development of acquired MMC resistance in the HCT 116 to a level of resistance similar to that of the natively resistant cells also resulted in several other molecular alterations that paralleled the molecular phenotype of the natively MMC resistant HCT 116b cells. These alterations included a significant increase in membrane Ricinus communis I binding carbohydrate moieties (Mr 80,000-92,000) and decrease in the expression of specific membrane antigens (Mr 55,000-57,000). The decrease in phosphorylation of specific nuclear phosphoproteins was also observed. This included a major nuclear phosphoprotein with approximate Mr- and pI-values corresponding to 63,000/5.0, and three groups of lower molecular weight phosphoproteins with approximate Mr- and pI-values corresponding to 22,000/6.4, 22,000/6.8, and 22,000/7.4. The results of these studies suggested that similar resistant mechanism(s) might exist in these acquired and natively MMC resistant cell lines which possessed similar level of resistance to MMC.

Biotransformation↗
PubMed1987

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.

Carbohydrates↗
PubMed1986

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.

Animals↗
PubMed1986

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.

Animals↗
PubMed1986

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.

Antigens, Neoplasm↗
PubMed1986

Circumvention of deficient activation in mitomycin C-resistant human colonic carcinoma cells by the mitomycin C analogue BMY25282.

BMY25282, a newly designed analogue of mitomycin C (MMC) with the substitution of an amidine group at position 7 of MMC, can circumvent MMC resistance in a series of human colonic carcinoma cells that were selected for resistance to MMC (J.K.V. Willson et al., Cancer Res., 45:5281-5286, 1985). In this study MMC resistance was found to be associated with an inability of the resistant cells to activate MMC. However, both the MMC-sensitive and -resistant cells were observed to metabolize BMY25282 extensively in vitro to a reactive species capable of alkylating 4-(p-nitrobenzyl)pyridine (a trapping agent for activated drug). The results of these studies suggested that the deficient cellular reductive activating mechanism was associated with MMC resistance and that analogue BMY25282 was able to overcome this deficiency in MMC-resistant cells by virtue of its enhanced activation.

Animals↗