Human Colon Tumor Cell Research Articles

Inhibition of a Specific DNA Repair System and Nitrosourea Cytotoxicity in Resistant Human Cancer Cells

In this report we present evidence which suggests that pretreatment of a highly nitrosourea-resistant human colon tumor cell line with non-cytotoxic doses of streptozotocin (STZ) prior to, or simultaneously with, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) exposure produces synergistic increases in cytotoxicity of several logs over the cytotoxicity produced by exposure to BCNU alone. STZ pretreatment or simultaneous treatment with BCNU allowed BCNU-induced DNA interstrand crosslinks to form in this cell line, in which BCNU alone did not induce DNA interstrand-crosslinks. Reversal of the schedule (i.e., STZ following BCNU) was less effective in producing synergistic cell kill or increased DNA interstrand crosslinking. Replacement of BCNU with fresh BCNU was not effective in producing increased cell kill and produced no observable interstrand crosslinking. Direct assays for guanine O6-DNA alkyltransferase activity confirmed that more than 75% inhibition of this important DNA repair system occurred following exposure to 2.5 mM STZ and that the inhibition was virtually complete when STZ pretreatment was combined with BCNU exposure.

Inhibition of a Protease Associated with Tumour Cells and the Probable Mechanism of Regulation of this Interactionin Vivo

The regulation of activity of a protease on the surface of human colonic tumour cells implanted in nude mice has been studied. A synthetic fluorescent probe was used to locate cells possessing active guanidinobenzoatase in frozen sections of tumour bearing tissues. These studies demonstrated the presence of intracellular protein inhibitors of guanidinobenzoatase which could be extracted in isotonic saline and transferred to the outer surface of the tumour cells. This study showed that the inhibition of guanidinobenzoatase was pH dependent and that the tumour cell may regulate the activity of its surface guanidinobenzoatase by exporting lactic acid.

Crypt cell antigens (CCA): new carbohydrate markers for human colon cancer cells.

A panel of monoclonal antibodies produced against surface membrane components of the human colon tumor cell line Caco-2 was found to define oncofetal crypt cell antigens (CCA) expressed by fetal intestinal cells, adult small intestinal crypt cells, and human and rat colonic adenocarcinomas. The epitopes recognized by these antibodies have been identified as O-linked oligosaccharide chains associated with specific glycoproteins in cultured intestinal cells and in colon tumors in vivo. Analysis of a large group of normal and diseased human intestinal specimens has demonstrated a marked heterogeneity in CCA expression which correlated with the degree of organization of the tumor cells in the tissue, suggesting that the CCA represent useful histological and clinical markers for colon cancer.

Interferon-induced modulation of cellular growth and antigen expression by human colon tumour cells

Interferon-induced modulation of cellular growth and antigen expression by human colon tumour cells

Different epidermal growth factor growth responses and receptor levels in human colon carcinoma cell lines

The growth response to epidermal growth factor (EGF) and the numbers and types of EGF receptors were studied in three human colon tumor cell lines from each of two groups of cell lines that differ markedly in their growth properties and extent of differentiation. Aggressively growing and poorly differentiated colon cells (group I) did not respond to EGF alone, while less aggressively growing and more differentiated cells (group III) responded with increased growth when EGF was added to their chemically defined, serum-free medium. The average number of EGF receptors (EGF-R) measured at the surface of group III cell lines by radioligand binding assays, was eight-fold higher than that measured for group I cell lines. These observations provide evidence for possible autocrine mechanisms that maintain available EGF-R levels in more differentiated group III colon tumor cells and down-regulate EGF-R levels in group I colon tumor cells.

Potentiation of x ray sensitivity by combinations of sodium butyrate and buthionine sulfoximine

The ability of various concentrations of the differentiation-inducing agent sodium butyrate (NAB, 0–2 mM) to produce radiosensitization in human colon tumor cells when combined with varying concentrations of the irreversible inhibitor of gamma-glutamyl cysteine synthetase, buthionine sulfoximine (BSO, 0–0.75 mM) was studied. We have previously shown that high concentrations of each agent in combination (2 mM NAB + 0.5 mM BSO) produced a supra-additive effect in terms of radiosensitization as indicated by a decrease in the quasi-threshold value (D q) of the single dose survival curve; we wished to define responses at other concentrations. Cells were adapted in vitro to growth in medium containing NAB for 3 passages prior to x-irradiation and BSO was given acutely 24 hrs before the x-irradiations. The most effective combination was 0.3 mM NAB + 0.75 mM BSO. These data suggest that adaptation of tumor cells to chronic low levels of a differentiation-inducing agent such as NAB followed by administration of BSO just prior to irradiation might be an effective combination in producing increased response of solid tumors.

Cell adhesion and migration of different human colon cell lines and primary tumors.

Several cell lines derived from colon tumors (HT-29, WiDr, PT4, PT5) and from human liver metastases (LM3) and primary human colon tumor cells (PTR) were compared with regard to their ability to migrate and to attach to different substrates (collagen G, laminin, fibronectin). Cells from the WiDr cell lines migrated actively, whereas the other cell lines, and LM3 and the PTs showed almost no migratory activity. The attachment efficiency was best in all cell lines assayed when tested on collagen followed by laminin and fibronectin as substrates. The HT-29 cells showed the strongest adhesion to all substrates, while the adhesiveness of PT4, PT5, and LM3 was reduced. The WiDr cells which migrated best showed the lowest adhesion to the substrates.

The cytotoxic principles of Prunella vulgaris, Psychotria serpens, and Hyptis capitata: ursolic acid and related derivatives.

Bioassay-directed fractionation of the cytotoxic antileukemic extracts of Prunella vulgaris, Psychotria serpens, and Hyptis capitata has led to the isolation of ursolic acid as one of the active principles. Ursolic acid showed significant cytotoxicity in the lymphocytic leukemia cells P-388 and L-1210 as well as the human lung carcinoma cell A-549. It also demonstrated marginal cytotoxicity in the KB and the human colon (HCT-8) and mammary (MCF-7) tumor cells. Esterification of the hydroxyl group at C-3 and the carboxyl group at C-17 led to compounds with decreased cytotoxicity in the human tumor cell lines, but with equivalent or slightly increased activity against the growth of L-1210 and P-388 leukemic cells.

Changes in X-Ray Sensitivity and Glutathione Content of Human Colon Tumor Cells after Exposure to the Differentiation-Inducing Agent Sodium Butyrate

Clone A human colon cancer cells were exposed to concentrations of sodium butyrate (NAB, 0-2 mM) for three passages in vitro, and responses to either graded single doses or split doses of 250 kVp X rays were determined. The survival data were fit to the single-hit, multitarget model of inactivation. For the graded single dose experiments, we found that NAB produced a decrease in the magnitude of the quasi-threshold (Dq) parameter after a concentration of about 0.9 mM was exceeded. Similarly, in split dose experiments, the amount of sublethal damage recovery (SLDR) was reduced in a concentration-dependent manner as shown by a decrease in the Dq parameter. However, the inhibition of SLDR occurred with no apparent threshold NAB concentration. NAB did not affect potentially lethal damage recovery. Paradoxically, increasing concentrations of NAB produced an exponential increase in the intracellular glutathione content, which could be blocked by exposure of the cells to buthionine sulfoximine (BSO). BSO treatment of NAB-adapted cells led to additional cell killing, again most noted by changes in the Dq parameter. We postulate that these responses are associated with NAB-induced changes in chromatin structure, particularly the association between DNA and nucleosomal histones H3 and H4.

X ray responses of a human colon tumor cell line after exposure to the differentiation-inducing agent N-methylformamide: Concentration dependence and reversibility characteristics

The combination of differentiation-inducing agents with conventional cytotoxic agents has been suggested as a potential cancer therapeutic strategy. In this regard, we have chronically exposed (3 passages) a human colon tumor line (clone A) to varying concentrations (0–170 mM) of N-methylformamide and examined the change in sensitivity to ionizing radiation in vitro. The linear-quadratic formalism of survival was used to characterize the single graded dose survival curves. This equation yields two constants (α and β) relating to cellular inactivation produced by either single events (α) or by the combination of two events (β). As the N-methylformamide concentration increased, the a parameter increased while the β parameter concomitantly decreased, yielding a concentration dependent radiosensitization which was most marked in the low dose region of the survival curve. Upon removal of NMF, the original radiation resistance was regained within 2–3 cell culture doubling times.

Modification of the hypoxic fraction of a xenografted human colon tumor by differentiation-inducing agents.

Xenografted tumors were produced in nude mice by injection of HCT-15 human colon tumor cells. The hypoxic fractions of control tumors as determined from x-ray survival curves were approximately 18%. Other tumors were treated (every day X 9) with daily injections of N-methylformamide (150 mg/kg) or sodium butyrate (2,000 mg/kg). For both agents, it was found that the hypoxic fractions were less than 0.05% and less than 1.7%, respectively. These data indicate that selected differentiation-inducing agents could be of value for treatment of human solid tumors that contain hypoxic cells.

Characterization and reactivity of a monoclonal antibody that recognizes a 76 kilodalton human colon tumor antigen

A monoclonal antibody (MAb 76) was produced by immunizing mice with a soluble cytoplasmic protein fraction from a human adenocarcinoma of the colon. MAb 76 showed specific immunoreactivity against a 76 kDa protein in immunoblot studies using total colon tumor cytosol proteins. Immunoprecipitation of phosphorylated cytosolic protein products with MAb 76 and subsequent analysis on SDS containing polyacrylamide gels revealed a single 38 kDa band, indicating that the 76 kDa antigen is associated with a 38 kDa phosphoprotein species. Indirect immunofluorescence analysis of primary tumor specimens and human colon tumor cell lines showed positive immunoreactivity with 6 7 human colon adenocarcinoma tissues and 15 18 human colon tumor cell lines. MAb 76 was unreactive with normal colon, liver and lung specimens from human, mouse and hamster. The epitope-bearing monomer detected by MAb 76 is immunologically conserved in a high percentage of colon tumor cells and tissues and may represent a cellular product that is characteristic of the transformed colon cell phenotype.

Tumor-promoter-enhanced destruction of noninvasive human benign colon tumor cells by cocultivated carcinoma cells.

Colon carcinoma cells are first found as microscopic foci within benign tumors or adenomas. The carcinoma must invade the adenoma which protrudes into the colon lumen before it can infiltrate the bowel wall. A quantitative model for this process has been developed in tissue culture in which human colon carcinoma cells destroy cocultivated adenoma colonies. 43 adenoma colonies were assayed by cocultivation with carcinoma cells. Constitutive secretion of the urokinase form of plasminogen activator by carcinoma cells apparently plays some role in adenoma destruction as inhibition of this protease by the competitive inhibitor benzamidine reversibly inhibited adenoma destruction (p less than 0.01). Elevation of plasminogen activator secretion by addition of the tumor promoter 12-tetradecanoylphorbol-13-acetate significantly enhanced the destruction of colonies cultured from tubular adenomas with only mild dysplasia (p less than 0.025) and from villous, villotubular and tubular adenomas with moderate to severe dysplasia (p less than 0.0005).

Biosynthesis of glycosaminoglycans in the human colonic tumor cell line Caco‐2: structural changes occurring with the morphological differentiation of the cells

The human colon cancer cell line Caco-2 cultured in vitro displayed morphological differentiation which was shown to be a growth-related event. We have investigated this phenomenon further in relation to the cell surface glycosaminoglycans produced by growing (5-day, i.e., prior to differentiation) and confluent (9-day, i.e., after morphological and functional differentiation) cultures. Neosynthesized [35S]glycosaminoglycans were purified on DEAE-cellulose; at confluency, they were bound more strongly to the column than the corresponding fractions from the growing cells. Analysis of Kav values of heparan sulfate and chondroitin sulfates from growing and confluent cells indicated an increase in chain length of both glycosaminoglycans in morphologically differentiated cells. Heparan sulfate was the main 35S-labeled glycosaminoglycan of the cell surface of both 5-day and 9-day cultures. Paper chromatography of the unsaturated disaccharides obtained by chondroitinase digestion showed that chondroitin sulfate chains were primarily 6-sulfated in the 2 studied extracts. Heparan sulfate chains were isolated as chondroitinase-resistant material and treated with nitrous acid. Analysis of N- and O-sulfate group-related radioactivity showed an increase in the amount of 35S-label in the form of N-sulfate groups and an increase in the O-35S-sulfation pattern in heparan sulfate from morphologically differentiated cells. Thus, the structural features of both chondroitin sulfates and heparan sulfate were significantly different when the growing cells became morphologically differentiated.

Effects of the Differentiating Agents Sodium Butyrate and N-Methylformamide on the Oxygen Enhancement Ratio of Human Colon Tumor Cells

We have previously shown that chronic adaptation of human tumor cells to the differentiation-inducing agents N-methylformamide (NMF) and sodium butyrate (NAB) increases the sensitivity of oxic cells to graded single doses of X rays. These studies were carried out to define the sensitivity of hypoxic cells after adaptation. Clone A colon tumor cells were grown for three passages in medium containing 170 mM NMF or 2 mM NAB and irradiated in suspension culture, after gassing with either oxygen (60 min) or ultrapure nitrogen (90 min), and complete survival curves were generated. Using the linear-quadratic equation to describe the data, it was found that NMF and NAB produced increased X-ray killing of hypoxic cells. At the 10% level of survival, the dose-modifying factors were about 1.20 and 1.25 for NMF- and NAB-adapted hypoxic cells, respectively, as compared to hypoxic control cells. However, since both oxic and hypoxic cells exhibited increased sensitivity after NMF and NAB adaptation, there was no major change in the oxygen enhancement ratio.

Biosynthesis of glycosaminoglycans in the human colonic tumor cell line Caco-2: structural changes occurring with the morphological differentiation of the cells

Biosynthesis of glycosaminoglycans in the human colonic tumor cell line Caco-2: structural changes occurring with the morphological differentiation of the cells

Effects of glutathione depletion on the cytotoxicity of agents toward a human colonic tumour cell line.

Levels of glutathione (GSH) in tumour tissue may be important in determining the clinical response to certain anticancer agents. Recent reports have suggested that D,L-buthionine-S,R-sulphoximine (BSO), a specific inhibitor of GSH synthesis, may be used to deplete tumour cell GSH and thus increase the therapeutic ratio of these agents. We have previously shown that 1-naphthol is a potential antitumour agent, and that its possible metabolite 1,4-naphthoquinone is thiol reactive and capable of redox cycling. It was therefore of interest to investigate the effect of pretreatment with BSO, on the toxicity of these agents, to tumour cells. For comparison we included three other cytotoxic agents, melphalan, helenalin and menadione, the toxicities of which are reported to be modulated by intracellular GSH. Depletion of GSH using BSO did not effect the toxicity of 1-naphthol, or 1,4-NQ but did produce slight potentiation of the cytotoxicities of menadione, helanalin and melphalan. The lack of effect of BSO on 1-naphthol and 1,4-NQ is not easily explained but if one also considers the modest potentiation of cytotoxicity+ achieved with the other agents studied, the potential use of BSO in combined chemotherapy is at best rather modest.

Effects of nucleoside analogs and sodium butyrate on recovery from potentially lethal x ray damage in human colon tumor cells

The effects of 5-azacytidine (5-aza-CR) and 5-aza-2'-deoxycytidine (5-aza-CdR) both alone and in combination with sodium butyrate (NaB) on intrinsic radiation sensitivity and ability to recover from potentially lethal damage (PLDR) were studied in two subpopulations of cells (clones A and D) from a heterogeneous human colon adenocarcinoma (DLD-1). Growth for three passages in medium containing 1 mM NaB alone enhanced radiation cell killing in the low dose ("shoulder") region of the survival curve for both cell lines. Neither 1.0 microM 5-aza-CR nor 0.25 microM 5-aza-CdR alone enhanced cell killing. However, treatment of these cells with a combination of either 5-aza-CR or 5-aza-CdR and NaB enhanced radiation cell killing at a clinically relevant dose level of 2.0 Gy by approximately 25% for both clone A and clone D cells. Also, while exposure to these differentiation-inducing agents separately enhanced the expression of PLDR in both tumor subpopulations, treatment with either of the combinations reversed this increase in PLDR. These results indicate that the gene-activating agents 5-aza-CR, 5-aza-CdR, and NaB may interact to modify the radiation sensitivity of two human tumor cell lines. Such combinations may prove useful clinically, if enhanced X ray cell killing of tumor cells can be achieved without a concomitant enhancement of recovery from potentially lethal X ray damage.

Calcium and vitamin D modulate mouse colon epithelial proliferation and growth characteristics of a human colon tumor cell line.

The role of calcium and vitamin D in the prevention of colorectal cancer is only now being investigated at the organismal, cellular, and molecular biologic levels. Recent epidemiologic studies have supported the hypothesis that dietary calcium and vitamin D may be related to a reduced risk for colon cancer. The evidence from laboratory investigations in animals and in cell culture also indicate a possible preventative effect. Addition of calcium and vitamin D to the roster of developmental cancer chemopreventative agents for further research is warranted.

Effects of sodium butyrate on human colonic adenocarcinoma cells. Induction of placental-like alkaline phosphatase.

We have examined the effects of the "differentiating agent," sodium butyrate, on the induction of alkaline phosphatase in human colonic tumor cell line LS174T. Culture of these cells in the presence of 2 mM butyrate caused this activity to increase from less than 0.0001 unit/mg of protein to greater than 0.7 unit/mg of protein over an 8-day period. This induction proceeded in a nonlinear fashion with a lag time of 2-3 days occurring before enzymatic activity began to rise. These increases in activity were accompanied by elevations in the content of a placental-like isozyme of alkaline phosphatase as demonstrated by "Western" immunoblots. Dome formation, indicative of differentiation in cultured cells, also required 3 days treatment with butyrate before becoming evident. The rate of biosynthesis of the enzyme, examined using metabolic labeling with L-[35S]methionine and immunoprecipitation, was found to increase continuously between days 2 and 6 of butyrate treatment. "Northern" blot analysis indicated that treatment of these cells with butyrate caused greater than 20-fold induction of a 2700-base mRNA that hybridized to a cDNA probe for placental alkaline phosphatase. The mRNA for alkaline phosphatase produced by these cells upon butyrate treatment was approximately 300-400 bases smaller than the mRNA for alkaline phosphatase found in placenta. Human small intestine also contained two mRNAs that hybridized relatively weakly with the placental alkaline phosphatase probe. These results indicate that a placental alkaline phosphatase-like protein and mRNA are induced by butyrate in LS174T cells with a time course consistent with cellular differentiation preceding induction.