Colon Cancer Lines Research Articles

Increased levels of phorbin, c‐myc, and ornithine decarboxylase RNAs in human colon cancer

Our previous work on protein kinase C (PKC) and colon cancer has shown altered levels of PKC activity in human colon tumors, as well as activation of PKC by colon tumor promoters such as bile acids. To understand further the role of PKC in colon carcinogenesis, we analyzed the expression of phorbin, a gene induced by PKC activation, in a series of different stages of human colon tumors. As shown by northern blot analyses of poly (A)+ RNA, higher levels of phorbin RNA were seen in 26 colon tumor samples than in their adjacent normal colonic mucosa. There also appeared to be a correlation between the abundance of phorbin RNA in the tumors and the extent of invasion (tumor-to-normal tissue phorbin RNA ratio = 4.2, 8.0, and 11.9 for Dukes' A, B, and C, respectively). Phorbin RNA was also abundant in a human colon cancer line (HT29). We also examined the expression of other mitogen-responsive genes (c-myc, ODC, and beta-actin) in a set of 19 colon tumor samples. All tumors displayed significant (mean 3.8-fold) increases in the level of c-myc RNA compared with their adjacent normal colonic mucosa. About 47% and 16% of these tumor samples also showed increased levels of ODC (mean 3.1-fold) and beta-actin (mean 1.6-fold) RNA, respectively. The increased levels of c-myc, ODC, and beta-actin RNA did not correlate with the extent of tumor invasion. Taken together, these results demonstrate that human colon tumors usually display increased levels of both phorbin and c-myc RNAs. The marked increases in phorbin RNA suggest that this could serve as a useful biomarker in studies on human colon cancer.

Schedule-dependency of in vivo modulation of 5-fluorouracil by leucovorin and uridine in murine colon carcinoma.

The effect of leucovorin (LV) given in various doses and schedules on the in vivo antitumor activity and toxicity of 5-fluorouracil (5FU) was studied in two murine colon cancer lines, i.e., Colon 26 (relatively resistant to 5FU) and Colon 38 (5FU sensitive), maintained in Balb-c and C57B1/6 mice, respectively. Mice were treated weekly with 5FU at the maximum tolerated dose, alone and in combination with LV. In Colon 26, neither simultaneous administration of 5FU and LV nor 5FU combined with delayed administration of LV potentiated the antitumor activity of 5FU. LV given twice - 1 hr before (50 mg/kg) and then together (50 mg/kg) with 5FU (100 mg/kg) - gave significantly better delay of tumor growth of both tumor lines than 5FU did alone (100 mg/kg). No differences were found after a total LV dose of 100 or 200 mg/kg. Delayed administration of uridine (3500 mg/kg) allowed the use of higher 5FU doses, which improved the antitumor effect on Colon 26. Systemic toxicity led to moderate weight loss in treated mice, but was comparable for mice treated with 5FU alone or combined with LV. Hematological toxicity consisted of moderate leukopenia (nadir 40%), which was observed with the most active schedule and was less severe than with 5FU alone. This schedule did not cause thrombocytopenia, but after discontinuation the thrombocyte count showed an overshoot. Addition of uridine to this schedule reduced hematological toxicity only slightly. It is concluded that LV potentiated the antitumor activity of 5FU against two solid tumor lines, i.e., a relatively resistant and a sensitive murine colon carcinoma, and that toxicity was moderate.

ELISA assay for colon cancer

AbstractThe high mortality rate for patients with colon cancer results largely from the inability to diagnose the condition in its early stages. The assay described in this report may be useful in early detection of colon cancer. In the study we use the cotton‐top tamarin, Saguinus oedipus, as an animal model for studying human colon cancer. This New World monkey develops a high incidence of colitis and colon cancer. Antibodies that recognize adenocarcinoma cells and antigens from these cells can be detected in the sera of tamarins with colon cancer. The antibodies are not found in sera of healthy animals. In one procedure, tamarin adenocarcinoma cells are used to coat the wells of a microtiter plate. In an alternate procedure, a glycoprotein adenocarcinoma antigen is used. The antigen is extracted from a cultured tamarin colon cancer line by detergent solubilization followed by affinity chromatography. Test sera are examined by adding biotinylated immunoglobulin serum fractions to the coated wells. The wells are washed and an avidin‐enzyme conjugate is added. Positive sera are identified by the addition of the substrate. The presence of the autoantibodies in the sera may be taken as evidence that the animal has colon cancer.

Simultaneous production of IGF-I and EGF competing growth factors by HT-29 human colon cancer line.

The conditioned medium from the HT-29 human colonic adenocarcinoma cell line contains a potent mitogenic activity that can markedly stimulate the proliferation of both rat and human fibroblasts in the absence of serum. Fractionation of conditioned medium on Bio-Gel P-100 shows that HT-29 cells simultaneously produce 2 different types of endogenous growth factors. The first one (molecular mass of 35, 8 and 5.5 kDa) exhibits an IGF-I competing activity which is positively correlated to mitogenic activity. This mitogen is recognized by anti-IGF-I antibodies but is resistant to reducing agents. It is distinct from IGF-II, insulin and PDGF. The second one (molecular mass of 40- and 20-kDa) is able to displace EGF binding to its receptor. This factor is immunologically recognized by anti-EGF antibodies but with a lower affinity as compared to EGF. This suggests that this endogenous HT-29-growth factor is related to but distinct from native EGF. Although more active in radioreceptor assay than in radioimmunoassay, the EGF-competing factor is distinct from TGF alpha or beta since it is unable to induce anchorage-independent growth of NRK or FR3T3 target cells in the presence or absence of exogenous EGF. Moreover, free functional EGF receptors are available at the HT-29 cell surface.

Enterocytic differentiation and glucose utilization in the human colon tumor cell line Caco-2: modulation by forskolin.

The human colon cancer line Caco-2 exhibits after confluency a concomitant increase of glycogen accumulation and an enterocytic differentiation. The purpose of this work was to investigate whether forskolin (FK), an activator of adenylate cyclase, would induce a permanent glycogenolysis and, if so, whether it would result in modifications of the differentiation pattern of the cells. FK activates adenylate cyclase in Caco-2 cells with an ED50 of 7 X 10(-6)M. Three different treatment protocols with FK (10(-5)M) were applied: 1) the cells were treated during all the time in culture (20 days); 2) the treatment was started after confluency; 3) the treatment was interrupted after confluency. The presence of FK results in a permanent stimulation of cAMP accumulation (10 to 20 fold the basal values) and in a permanently reduced glycogen content (30 or 50% of the control values). The rates of glucose consumption are increased three and five fold in protocols 1 and 3 respectively. These metabolic changes are associated with morphological changes (tightening of the intercellular spaces and shortening of the brush border microvilli) and with a dual inhibition of the activities of brush border hydrolases: a) an inhibition of the post-confluent increase of activity of sucrase, aminopeptidase N and alkaline phosphatase in the brush border enriched fraction; b) an inhibition of the post-confluent increase of activity of sucrase in the cell homogenate. A comparison of the results obtained in each protocol shows that the morphological modifications and the decrease of the enzyme activities in the brush border fraction are regularly associated with an increased cAMP accumulation, whereas the inhibition of the differentiation of sucrase is a direct consequence of the increase in glucose consumption and decrease in glycogen stores.

Carcinoembryonic antigen release from colorectal cancer cells in tissue culture

Limited data are available concerning the physicochemical and immunological characteristics of carcinoembryonic antigen spontaneously released from cancer cells in tissue culture. The Hoffman—LaRoche Z-gel assay for the detection of circulating carcinoembryonic antigen (CEA) in serum was modified for the detection of CEA in tissue culture supernatants. A number of mycoplasma free epithelial lines established from colorectal cancer specimens were evaluated for cell-bound and spontaneously released CEA. Further studies were performed using tissue culture supernatants from colon cancer line SW480, which spontaneously released microgram quantities of CEA into the cell culture supernatant. By competitive inhibition analysis this CEA appeared identical with the Roche CEA standard. Gel filtration studies on Sephadex G-200 and affinity chromatography with concanavalin A sepharose-4B revealed the supernatant CEA and the Roche standard to be similar. These data suggest that this system offers potential for further characterization of this oncofetal protein and for study of its biosynthesis and “spontaneous” release from cancer cell surface. The molecular basis of such activity on the genetic level would have significant importance in basic biological studies of malignant transformation.