Effects of mitomycin on human colon carcinoma cells.

Subpopulations of malignant cells from primary cultures of human colon carcinoma were characterized with respect to their response to mitomycin (MMC). Growth inhibition assays indicated values of 2.06, 0.93, and 0.33 microM for the concentration of drug giving 50% inhibition of growth for sublines HCT 116b, HCT 116, and HCT 116a, respectively. Alkaline elution of filter-bound DNA from cells exposed to MMC in vitro showed a positive correlation between the amount of DNA cross-linking and growth inhibition as a function of drug concentration. Comparable DNA cross-linking was obtained at MMC concentrations of 10 microM for HCT 116b and 5 microM and HCT 116. The cross-linking of DNA from HCT 116a cells at 5 microM MMC was approximately equal to that from HCT 116 cells at doses between 10 and 20 microM MMC. Cross-link removal as a function of time after drug removal of MMC-treated cells was also measured. There was little difference in the rates of alkaline DNA elution after drug removal between HCT 116b and HCT 116a, suggesting that the ability to repair cross-links was not responsible for the differential sensitivities of the cells to MMC. The relative sensitivities of the subpopulations to MMC were reflected in vivo by MMC treatment of nude BALB/c mice bearing xenografts of the cultured sublines.

Enhancement of growth of human colon tumor cell lines by feeder layers of murine fibroblasts.

The effects of feeder layers of C3H/10T 1/2 cells on the growth of human and mouse cell lines were tested. Feeder layers increased colony formation by cultured cancerous cells in semisolid medium over controls grown in semisolid medium without feeder layers. Maximal colony formation was also attained at a faster rate when feeder layers were used. Plating efficiency by cancerous cells obtained directly from xenografts was increased threefold to fivefold in tissue culture when feeder cells were present as confluent monolayers.

Characterization of the inhibitory effects of transforming growth factor-beta on a human colon carcinoma cell line.

The effects of transforming growth factor-beta (TGF beta) on a human colon carcinoma cell line (MOSER) were investigated. TGF beta, at low concentrations (between 0.1 and 1.0 ng/ml), inhibited the proliferation of MOSER cells both in monolayer culture and soft agarose, in a dose-dependent manner. MOSER cells adapted to growth in chemically defined serum-free medium were more sensitive to the inhibitory effects of TGF beta than cells maintained in serum-supplemented medium. Morphological changes in MOSER cells, observed with TGF beta, were similar to those seen with the chemical differentiation agent N,N-dimethylformamide. Also in similarity to the effects of N,N-dimethylformamide, TGF beta induced a time- and concentration-dependent increase in soluble extracellular fibronectin. Binding studies with [125I]TGF beta revealed a relatively low number of binding sites on MOSER cells (13%) compared with mouse embryo fibroblastic (AKR-2B) cells. Thus far, other colon carcinoma cell lines, some displaying TGF beta receptors, have been reported to be unresponsive to TGF beta. This study is therefore the first to demonstrate a TGF beta-responsive colon carcinoma cell line.

Comparison of growth requirements of two human intratumoral colon carcinoma cell lines in monolayer and soft agarose.

The human colon, intratumoral subpopulations HCT 116 and HCT 116a were established in chemically defined medium supplemented with transferrin, insulin, epidermal growth factor (EGF), triiodothyronine, hydrocortisone, and sodium selenite. The responsiveness of the adapted cell lines to these growth factors was compared in anchorage-dependent and -independent assays. HCT 116 cells maintained in serum-free conditions were further adapted to growth factor deprivation, and the effects of these polypeptides were determined in anchorage-independent assays. In monolayer, HCT 116 cells adapted to grow in serum-free medium responded to transferrin but not to EGF or insulin. Similarly adapted HCT 116a cells were, however, insensitive to transferrin addition but manifested a 300 and 500% increase in growth rates with EGF and insulin, respectively. Optimal growth of HCT 116 cells was seen in the presence of insulin and transferrin, while maximum proliferation of HCT 116a cells depended on combined insulin, transferrin, and EGF. In soft agarose, both HCT 116 and HCT 116a subpopulations showed a stringent requirement for transferrin. No combination of growth factors without transferrin supported colony formation. These data suggest that (a) these colon tumor subpopulations may be subject to separate growth controls, and (b) there may be an important role for transferrin in anchorage-independent growth and possibly in the maintenance of malignant characteristics.

Heterogeneity of malignant cells from a human colonic carcinoma.

Three subpopulations of malignant cells were isolated from a primary cell culture of a single human colonic carcinoma. The variant cells were established as cell lines designated HCT 116, HCT 116a, and HCT 116b, respectively. In vitro characterizations of the variant lines included growth in 0.5% agarose and growth on confluent layers of mouse fibroblasts. HCT 116a showed the highest colony formation in agarose and on confluent fibroblasts, while colony formation by HCT 116 was higher than that of HCT 116b in both of these systems. All of the variant lines were tumorigenic in athymic nude mice given injections of 10 x 10(6) cells, but the time between inoculation and tumor development (latency period) was approximately 10 times longer for HCT 116b as for HCT 116a and 8 times longer than for HCT 116. HCT 116b was not tumorigenic at an inoculum of 5 x 10(6) cells, while both HCT 116 and 116a were tumorgenic at this level. However, HCT 116a was clearly more tumorigenic than was HCT 116 on the basis of the number of animals developing tumors at inoculate of both 10 x 10(6) and 5 x 10(6) cells and on the basis of their differences in latency periods. While all the cell lines had near diploid numbers of chromosomes, each line showed a distinct histological pattern when grown as xenografts in athymic nude mice.

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.

Characterization of transforming growth factor-beta-resistant subclones isolated from a transforming growth factor-beta-sensitive human colon carcinoma cell line.

Previous work indicated that transforming growth factor-beta (TGF-beta) elicits proliferation-inhibitory effects in the human colon carcinoma cell line MOSER. This paper describes the isolation and characterization of spontaneously arising subclones from this TGF-beta-sensitive parental line which were relatively refractory to the inhibitory effects of TGF-beta. While the parental cell line responded to TGF-beta with an inhibition of cellular proliferation in monolayer culture and in soft agarose, an increase in extracellular fibronectin, and a down-regulation of c-myc protooncogene expression, these responses were absent or attenuated in the sublines. However, the resistant clones retained the ability to specifically bind TGF-beta. N,N-Dimethylformamide and retinoic acid, two other agents associated with induction of a partial differentiation-like response in the MOSER parental cells (similar to that elicited by TGF-beta), inhibited the monolayer proliferation of both the parental cells and the TGF-beta-resistant sublines. Thus, the refractoriness observed in the isolated clones was relatively specific for TGF-beta.

Mitomycin C resistance in a human colon carcinoma cell line associated with cell surface protein alterations.

A human colon carcinoma cell line resistant to mitomycin C (MMC) was obtained by repeated exposure of a previously described sensitive parental line, HCT 116, to MMC in vitro. Xenografts grown from the MMC-resistant phenotype were not inhibited in MMC-treated animals, while MMC treatment produced growth inhibition in parental cell xenografts. The MMC-resistant phenotype exhibited a greater amount of a Mr 148,000 cell surface protein than did the parental line. The increase in this Mr 148,000 cell surface protein correlated positively with the degree of MMC resistance. Alkaline elution of filter-bound DNA from resistant cells exposed to MMC in vitro showed a decrease in DNA cross-link formation such that a 10-fold higher MMC concentration was required to produce similar cross-link formation in the resistant cell as compared to the parental cell. The development of MMC resistance was not associated with in vitro cross-resistance to other natural product cytotoxic drugs. This model for resistance to MMC will be useful in future studies to define the mechanisms for MMC action and resistance in human colon carcinoma cells.

Identification of endogenous inhibitory growth factors from a human colon carcinoma cell line.

A line of human colon carcinoma cells, designated MOSER, was established which synthesized tumor-inhibitory factor (TIF) and transforming growth factor (TGF) activity. Both activities were found in serum-free conditioned medium and in cell extracts. The activities coelute on Bio-Gel P-10 in acetic acid, but can be completely separated by reverse-phase high-pressure liquid chromatography. The TIF and TGF activities were acid and heat stable and were sensitive to trypsin and dithiothreitol. MOSER cell TIF prevented the anchorage-independent growth of the more differentiated colon carcinoma cell lines tested but did not affect the less differentiated lines. Using anchorage-dependent growth conditions, the effect of TIF appeared to be noncytotoxic and partially reversible. Purified TGF stimulated the growth of normal rat kidney fibroblasts and the slow-growing CBS colon carcinoma cell line but did not stimulate MOSER cell growth. MOSER cells contain both positive (TGF) and negative (TIF) factors with relative concentrations that may be important parameters in the regulation of cell growth.

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.

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.

Promotion of differentiation in human colon carcinoma cells by vasoactive intestinal polypeptide.

The effects of vasoactive intestinal polypeptide (VIP) and dibutyryl cyclic adenosine 3':5'monophosphate (dbcAMP) on two human colon carcinoma cell lines, HCT 116 and GEO, were investigated. VIP and dbcAMP inhibited the growth of both cell lines in monolayer culture in a dose-dependent manner. Within 6 h of treatment with 1 mM dbcAMP or 0.3 microM VIP, numerous mucin-like droplets were secreted by GEO cells. VIP and dbcAMP also increased carcinoembryonic antigen (CEA) secretion. In both cell lines, a 9-fold increase in conditioned medium CEA levels was observed at 1 mM dbcAMP and a 2.6-fold increase at 1.5 microM VIP. Time- and concentration-dependent evaluation in cAMP levels were elicited by VIP in the two cell lines. Immunocytochemical studies for cell-surface glycoprotein detection in GEO cells showed that VIP induced a morphological and functional organization of mucin-secreting cells. These results indicate that VIP and dbcAMP have antiproliferative and strong differentiation-promoting effects in colon cancer cells. This is the first report of VIP-induced mucin secretion in colon tumor cells.

Comparison of the effects of transforming growth factor beta, N,N-dimethylformamide, and retinoic acid on transformed and nontransformed fibroblasts.

In order to compare the effects of transforming growth factor (TGF beta) with those of the differentiation promoters N,N-dimethylformamide (DMF) and retinoic acid (RA), the antiproliferative and fibronectin-inducing activities of the three agents were examined. AKR-2B mouse embryo fibroblasts and their chemically transformed counterpart AKR-MCA cells were used as the model system. Growth in monolayer culture of both cell lines was inhibited by TGF beta (EC50 approximately 1 ng/ml), DMF (EC50 approximately 0.5%), and RA (EC50 approximately 1 microM) in a concentration-dependent manner. Time-dependent elevation in fibronectin expression was also observed with all three agents. The EC50 for growth inhibition of both cell lines by TGF beta agreed well with that obtained for stimulation of fibronectin synthesis. A 3-h exposure to TGF beta is sufficient to obtain the maximal fibronectin level observed at 48 h in AKR-2 B cells but not in AKR-MCA cells. Our results indicate that in this system the effects of TGF beta are similar to those of the chemical differentiation inducers DMF and RA. Furthermore, our data also suggest that the TGF beta signal may be processed differently by nontransformed and transformed fibroblasts.

Characterization of human colon carcinoma cell lines isolated from a single primary tumour.

The initiation of a cultured human colon carcinoma line on a feeder layer of confluent fibroblasts is described. Attempts to initiate cultures without fibroblast feeder layers were not successful. Two sub-lines (designated HCT C and HCT C Col) were isolated and weaned from cells growing on the surface of the feeder layer. The sub-lines had different morphologies, secreted different levels of carcinoembryonic antigen (CEA) into the medium of confluent cultures and had slightly different karyotypes. Both sub-lines grew in semi-solid medium and formed xenografts when injected s.c. into athymic nude mice. Analysis of radioiodinated cell membrane components indicated small, but significant differences between the sub-lines.

Modulation of c-myc by transforming growth factor-beta in human colon carcinoma cells.

Previous work indicated that transforming growth factor-beta elicits proliferation-inhibitory and differentiation-like effects in the human colon carcinoma cell line MOSER. We report for the first time that the proto-oncogene c-myc is repressed in response to transforming growth factor-beta in a human colon carcinoma cell line. We also describe a subline of these cells which are relatively resistant to the transforming growth factor-beta-induced effects on proliferation in monolayer and in soft agarose, but which retain the ability to specifically bind transforming growth factor-beta. Analysis of molecular and cellular alterations in this subline may aid in elucidating the mechanism of action of transforming growth factor-beta.

Differential sensitivity of subclasses of human colon carcinoma cell lines to the growth inhibitory effects of transforming growth factor-beta 1.

In this study we have employed a model system comprising three groups of colon carcinoma cell lines to examine the growth-inhibitory effects of two molecular forms of transforming growth factor-beta (TGF-beta), TGF-beta 1 and TGF-beta 2. Aggressive, poorly differentiated colon carcinoma cells of group I did not respond to growth inhibitory effects of TGF-beta 1 or TGF-beta 2, while less aggressive, well-differentiated cells of group III displayed marked sensitivity to both TGF-beta 1 and TGF-beta 2 in monolayer culture as well as in soft agarose. One moderately well-differentiated cell line from group II which has intermediate growth characteristics failed to respond to TGF-beta 1 or TGF-beta 2, but the growth of two other cell lines in this group was inhibited. TGF-beta 1 and TGF-beta 2 were equally potent, 50% growth inhibition for responsive cell lines being observed at a concentration of 1 ng/ml (40 pM). Antiproliferative effects of TGF-beta 1 and TGF-beta 2 in responsive cell lines of groups II and III were associated with morphological alterations and enhanced, concentration-dependent secretion of carcinoembryonic antigen. Radiolabeled TGF-beta 1 bound to all three groups of colon carcinoma cells with high affinity (Kd between 42 and 64 pM). These data indicate for the first time a strong correlation between the degree of differentiation of colon carcinoma cell lines and sensitivity to the antiproliferative and differentiation-promoting effects of TGF-beta 1 and TGF-beta 2.

Initiation and characterization of cultures of human colonic carcinoma with different biological characteristics utilizing feeder layers of confluent fibroblasts.

The tissue culture of human colonic carcinoma with and without feeder layers of confluent C3H 10T1/2 mouse fibroblast was compared. In a series of 27 different tumor specimens, 21 long-term cultures (6 mos. or more) were obtained by utilizing feeder layers. Only 3 long-term cultures were obtained from the same set of specimens when feeder layers were not employed. Several of the long-term cultures were established as cell lines weaned from fibroblast feeder layers. These lines could be classified into 3 groups based upon their expression of several biological properties including tumorigenicity in nude mice, degree of differentiation of the tumors growing in nude mice, growth in semisolid medium, morphology in vitro and production of carcinoembryonic antigen in vitro.