Cholesterol sulfate accumulation in tumorigenic and nontumorigenic rat esophageal epithelial cells: evidence for defective differentiation control in tumorigenic cells

In this study the regulation of squamous cell differentiation in several rat esophageal epithelial cell lines is examined. Nontumorigenic RE-149 cells undergo a program of squamous cell differentiation at confluence. This program of differentiation is influenced by the concentration of calcium in the medium and by the presence of retinoic acid. High calcium concentration stimulates terminal cell division, as indicated by a reduction in colony-forming efficiency, and increases the expression of the differentiated phenotype as indicated by an increase in cholesterol sulfate accumulation and cross-linked envelope formation. Retinoic acid inhibits squamous cell differentiation as both cholesterol sulfate accumulation and cross-linked envelope formation are reduced. Two tumorigenic cell lines, RE-B2 and RE-2BT, do not undergo squamous cell differentiation in vitro. High calcium concentration in the medium did not significantly reduce colony-forming efficiency or induce cross-linked envelope formation. High calcium concentration or retinoic acid had only a limited effect on the accumulation of cholesterol sulfate. RE-B2T cells exhibit high levels of cholesterol sulfate and cholesterol sulfotransferase activity. These levels appear no longer controlled by calcium or retinoic acid, indicating that the synthesis of cholesterol sulfate occurs in a constitutive manner. The altered responses of RE-2B and B2T cells to calcium and retinoic acid suggest that these malignant cells have acquired one or more defects in the control of differentiation.     

Morphologic and neoplastic transformation of Syrian hamster embryo fibroblasts by diethylstilbestrol and its analogs

A mammalian cell culture system using normal, diploid Syrian hamster embryo fibroblasts was used as a model to study the ability of diethylstilbestrol (DES) and related compounds to induce neoplastic transformation. Like benzo(a)pyrene, a known chemical carcinogen, DES (0.01 to 10 micrograms/ml) induces morphological transformation of Syrian hamster embryo fibroblasts in vitro; the transformed colonies were indistinguishable from colonies of benzo(a)pyrene-transformed cells. The morphologically transformed colonies derived from either benzo(a)pyrene- or DES-treated cells were tumorigenic when injected into newborn hamsters. At doses of 0.01 to 1 microgram/ml for 48 hr, DES did not inhibit or stimulate cell proliferation. Structural analogs of DES were also tested in the Syrian hamster embryo transformation system. Like DES, tetrafluorodiethylstilbestrol, dimethylstilbestrol, and cis,cis-dienestrol morphologically transformed Syrian hamster embryo cells. The transformation frequency of dimethylstilbestrol was much less than that of DES, tetrafluorodiethylstilbestrol, or cis,cis-dienestrol. Hexestrol, dimethoxydiethylstilbestrol, and trans,trans-dienestrol did not transform these cells. No correlation could be demonstrated between reported estrogenic potency of the compounds and their cell-transforming capacity. Structure-activity relationships developed for these chemicals suggest that metabolism via specific pathways plays a role in DES-induced cell transformation.     

Induction of morphological transformation and unscheduled DNA synthesis in Syrian hamster embryo fibroblasts by hexachlorobutadiene and its putative metabolite pentachlorobutenoic acid

Hexachloro-1,3-butadiene (HCBD) is a well known environmental carcinogen. The genotoxic properties of HCBD and its monooxidation product pentachloro-3-butenoic acid (PCBA) were investigated by comparative induction of unscheduled DNA synthesis (UDS) and morphological transformation in the same cell system (Syrian hamster embryo fibroblasts). HCBD and PCBA induce unscheduled DNA synthesis both in the presence and absence of an exogenous metabolizing system. The lowest effective dose for UDS induction is smaller for PCBA (1 μg/ml) than for HCBD (2 μg/ml). The intensity of UDS induction is increased about 3-fold for both compounds after metabolic activation. HCBD and PCBA induce morphological transformation. The lowest effective dose for transformation differs considerably between PCBA (0.8 μg/ml) and HCBD (10 μg/ml). The results are indicative of a genotoxic mechanism for the carcinogenic actions of HCBD and PCBA.     

Clustering of discrete cell properties essential for tumorigenicity and metastasis. II. Studies of Syrian hamster embryo fibroblasts transformed by Rous sarcoma virus

Tumorigenic and metastasizing activities (TGA; MA) and susceptibility, or resistance to H2O2 and PGE-releasing activity (H2O2R + PGEs+ phenotype) have been examined in 6 Syrian hamster embryo cell strains transformed in vitro with Rous sarcoma viruses (Schmidt-Ruppin and Prague strains). Early observations of extremely high level of TGA and even MA of RSV-SR-transformants never selected in vivo have been confirmed. The correspondence of these properties with a high level of expression of H2O2R + PGEs+ phenotype and its clustering character were demonstrated in 4 RSV-SR transformants, while significantly lower expression of all these characteristics, including TGA, was observed in 2 RSV-Prague transformants. High level of spontaneous MA was noticed in some RSV-SR transformants. A tumor cell line induced in vivo by RSV-SR did not differ from the cell strain transformed in vitro by RSV-SR. Inhibition of H2O2R + PGEs+ phenotype in one of RSV-SR transformants was obtained with non-toxic doses of BCNU and indomethacin, leading to a marked decrease of TGA.     

Induction of morphological transformation and micronuclei in Syrian hamster embryo fibroblasts by 1,2-dioxetanes. Correlation with single-strand breaks in HL-60 cells

The photochemical genotoxic and cell-transforming potentialof 4-hydroxymethyl-3,3,4-trimethyl-1,2dioxetane (HTMD) and 3-(N-[4-pyridino]carbamoyl)methyl-3,4,4-trimethyl-1,2-dioxetane, (APD), in mammalian cell was studied. Both dioxetanes,which are efficient sources of triplet-excited ketones on thermaldecompsition, induced morphological transformation in Syrianhamster embryo (SHE) fibroblasts. Unscheduled DNA synthesisin SHE and in HeLa cells could not be detected with these dioxetanes,but the number of micronuclei scored after the first mitosiswas dose-dependently increased. Single-strand breaks but notMicrococcus luteus u.v.-endonuclease sensitive sites were observedby alkaline elution in HL-60 cells when treated with sub-lethaldoses of HTMD and APD. A possible mechanism for the transformationmediated by DNA and chromosomal damage as well as the intermediacyof triplet carbonyls in these events are discussed.     

Suppression of tumorigenicity in hybrids of tumorigenic Chinese hamster cells and diploid mouse fibroblasts: dependence on the presence of at least three different mouse chromosomes and independence of hamster genome dosage

Somatic cell hybrids were generated between Chinese hamster cell lines (Cl-4 and TK 17-O) with a near-diploid number of partially abnormal chromosomes and embryonic mouse fibroblasts (BALB/c). Hybrids harboring a near-diploid, near-triploid, and near-tetraploid set of hamster chromosomes plus 22 to 30 mouse chromosomes were analyzed for the expression of the transformed or tumorigenic phenotype, respectively, indicated by their capacity to form colonies in soft agar and by tumor formation after s.c. injection into nude mice. The hybrids showed (partial) suppression of tumorigenicity and of anchorage independence. The minimum number of hybrid cells required to initiate tumor growth in nude mice was 100- to 50,000-fold higher, and the latency period was 3- to 6-fold longer in comparison with the highly tumorigenic parental hamster cells. Suppression of tumorigenicity was also found in intraspecific Chinese hamster hybrids involving tumorigenic cells (E 36-O and TK 17-O) and embryonic hamster fibroblasts. To identify those mouse chromosomes associated with suppression of tumorigenicity, we investigated the expression of mouse isozyme genes and the presence of mouse chromosomes in interspecific suppressed hybrids and their tumorigenic hybrids described previously. No single mouse chromosome, even if present in two copies, and no combination of two different mouse chromosomes was sufficient to suppress tumorigenicity in these hybrids. This conclusion is based on either the presence of these chromosomes in hybrids isolated from tumors or their absence in suppressed hybrids.     

Clustering of discrete cell properties essential for tumorigenicity and metastasis. I. Studies of Syrian hamster embryo fibroblasts spontaneously transformed in vitro

The expression of two discrete cell properties related to the host natural effector mechanisms, i.e., resistance to damage by H2O2, a cytotoxic product of activated macrophages, and the ability to secrete PGE, which inhibits NK-cell cytotoxicity, has been examined in parental Syrian hamster embryo cells spontaneously transformed in vitro (STHE strain) and in 18 in vivo selected sublines. In all cell variants, resistance to H2O2 and PGE-releasing activity were either both expressed, or not expressed at all. Parental STHE cells and 5 variants selected in vivo, which were equally highly susceptible to H2O2-induced damage, did not release any detectable amount of PGE upon contact with NK cells. In contrast, 13 other STHE variants selected in vivo and characterized by their resistance to H2O2, all released PGE upon contact with NK cells. Thus, these two biochemically unrelated cell phenotypic characteristics are likely to be either simultaneously selected in vivo, or united in cluster which pre-exist or appear in rare cell variants of the parental cell population in the conditions of in vivo natural selection pressure.     

Comparison of nude mice with the host species for evaluation of the tumorigenicity of guinea pig and hamster cells transformed in vitro by chemical carcinogens.

Nude mice (nu/nu) were compared with the species of origin for determination of the tumorigenicity of cells from chemical carcinogen-transformed and noncarcinogenic chemically treated, nontransformed guinea pig and Syrian hamster cultures. The incidence and time of appearance of progressively growing tumors were similar in the host species and in nude mice after injection of 10(7) transformed cells. Inoculation of 10(8) nontransformed cells routinely was nontumorigenic in the host species and in nude mice. The nude mouse has potential as a sensitive and reliable alternative host to the species of origin to elaluate the tumorigenicity of xenogeneic mammalian cells from cell culture model systems of carcinogenesis.     

Pathogenesis of tumor desmoplasia. II. Collagens synthesized by line 1 and line 10 guinea pig carcinoma cells and by syngeneic fibroblasts in vitro

For the investigation of the pathogenesis of desmoplasia, the capacities to synthesize collagen in vitro of 2 bile duct carcinomas (lines 1 and 10) of Sewall-Wright inbred strain 2 guinea pigs and of syngeneic dermal fibroblasts were studied. Line 10 cells synthesized collagen type IV as judged by sensitivity to bacterial collagenase, by immunoprecipitation, by migration of pro alpha (IV) chains and pepsin-resistant fragments on sodium dodecyl sulfate-polyacrylamide gels, and by immunofluorescence. Line 1 cells also synthesized small amounts of collagenase-sensitive protein. Neither line 1 nor line 10 cells synthesized detectable collagen type I, III, or V. Only about 1% of [14C]proline incorporated by tumor cells was found in collagenase-sensitive protein. In contrast, dermal fibroblasts synthesized 4 and 128 times as much collagenase-sensitive protein as line 10 and line 1 cells, respectively, amounting to 20% of total protein synthesized. Fibroblasts produced mostly collagen types I and III, in a ratio of 7:1, and smaller amounts of collagen type V. Thus lines 1 and 10 carcinoma cells produce primarily basement membrane collagen, whereas interstitial collagens, abundant in desmoplastic tumor stroma, are fibroblast products.     

Interleukin-4 enhances proliferation of human pancreatic cancer cells: evidence for autocrine and paracrine actions

Interleukin-4 (IL-4) is an immunomodulatory cytokine, which can inhibit the growth of tumour cells. Pancreatic cancer cells and tissues express high levels of IL-4 receptors. The aim of this study was to characterise the effects of IL-4 on the growth and signalling pathways of pancreatic cancer cells. Cell growth was determined by cell counting and MTT assays in association with fluorescence-activated cell sorter analysis, IL-4 expression using ELISA and real-time PCR techniques, and signal transduction using immunoprecipitation or immunoblot analysis. We now report for the first time that IL-4 significantly enhanced the growth of five out of six cultured pancreatic cancer cell lines in a dose-dependent manner in association with an increased fraction of cells in S-phase. Surprisingly, all six cell lines expressed endogenous IL-4, and IL-4 was detectable in the supernatant. Incubating cells with neutralising IL-4 antibodies resulted in a significant inhibition of basal growth in three cell lines, including IL-4-unresponsive MIA PaCa-2 cells, which however expressed the highest endogenous IL-4 levels. Interleukin-4 enhanced activity of MAPK, Akt-1, and Stat3 in IL-4-responsive, but not in IL-4-unresponsive MIA PaCa-2 cells; however, IL-4 enhanced tyrosine phosphorylation of insulin receptor substrate-1 and -2 in all cell lines. Our results demonstrate for the first time that pancreatic cancer cells produce IL-4 and that IL-4 can act as a growth factor in pancreatic cancer cells. Together with the observation that neutralising IL-4 antibodies can inhibit the growth of these cells, our results suggest that IL-4 may act as an autocrine growth factor in pancreatic cancer cells and also give rise to the possibility that cancer-derived IL-4 may suppress cancer-directed immunosurveillance in vivo in addition to its growth-promoting effects, thereby facilitating pancreatic tumour growth and metastasis.     

Consequences of altered TGF-beta expression and responsiveness in breast cancer: evidence for autocrine and paracrine effects.

To characterize the impact of increased production of TGF-beta in a xenograft model of human breast cancer, TGF-beta-responsive MDA-231 cells were genetically modified by stable transfection so as to increase their production of active TGF-beta1. Compared with control cells, cells that produced increased amounts of TGF-beta proliferated in vitro more slowly. In vivo, however, tumors derived from these cells exhibited increased proliferation and grew at an accelerated pace. To evaluate the role of autocrine TGF-beta signaling, cells were also transfected with a dominant-negative truncated type II TGF-beta receptor (TbetaRII). Disruption of autocrine TGF-beta signaling in the TGF-beta-overexpressing cells reduced their in vivo growth rate. Co-inoculation of Matrigel with the TGF-beta-overexpressing cells expressing the truncated TbetaRII compensated for their diminished in vivo growth capacity, compared with the TGF-beta-overexpressing cells with an intact autocrine loop. Tissue invasion by the tumor was a distinctive feature of the TGF-beta-overexpressing cells, whether or not the autocrine loop was intact. Furthermore, tumors derived from TGF-beta-overexpressing cells, irrespective of the status of the autocrine TGF-beta-signaling pathway, had a higher incidence of lung metastasis. Consistent with the suggestion that TGF-beta's enhancement of invasion and metastasis is paracrine-based, we observed no significant differences among the cell clones in an in vitro invasion assay. Thus, in this experimental model system in vitro assays of cell proliferation and invasion do not accurately reflect in vivo observations, perhaps due to autocrine and paracrine effects of TGF-beta that influence the important in vivo-based phenomena of tumor growth, invasion, and metastasis.     

Dependence on exogenous metabolic activation for induction of unscheduled DNA synthesis in Syrian hamster embryo cells by diethylstilbestrol and related compounds

Diethylstilbestrol (DES) induces morphological and neoplastic transformation of Syrian hamster embryo cells in vitro in the absence of any measurable induction of gene mutations, which is consistent with the lack of genotoxicity of DES in a number of other assays. However, a few reports of a genotoxic activity of DES in certain systems have been published. In order to understand these differences, we have investigated whether DES induces unscheduled DNA synthesis (UDS) in Syrian hamster embryo cells under the conditions which result in cell transformation and have examined the role of an exogenous metabolic activation system on DES-induced UDS. DES, over a concentration range of 1 to 10 micrograms/ml, failed to induce any detectable UDS in the cells, while other known transforming agents, including UV irradiation (6 to 24 J/sq m), benzo(a)pyrene (0.1 to 1.0 micrograms/ml), and aflatoxin B1 (10 to 100 micrograms/ml), induced significant levels of UDS. In contrast, UDS was induced in a dose-dependent manner by DES (1 to 10 micrograms/ml) after addition of an Aroclor-induced rat liver postmitochondrial supernatant fraction and other cofactors for exogenous metabolic activation. In order to probe the basis for this alteration in UDS induction, the ability of structural analogues and metabolites of DES to induce UDS was examined. In the absence of exogenous activation, the only oxidative metabolite of DES detected in the presence of the cells was cis,cis-dienestrol, which did not induce UDS by itself. In the presence of exogenous activation, cis,cis-dienestrol and its trans,trans-isomer induced UDS but not to a greater extent than DES. With the addition of the exogenous metabolizing system, increased metabolism of DES to cis,cis-dienestrol and additional polar derivatives of DES or dienestrol, possibly hydroxylated derivatives, were observed. With exogenous metabolic activation, tetrafluoro-DES and hexestrol, which differ in their ability to be peroxidatively metabolized to quinone and phenoxyradical intermediates, both induced UDS, although tetrafluorodiethylstilbestrol at 10 micrograms/ml stimulated a higher level of UDS. None of the DES-related compounds examined was active in the UDS assay without exogenous metabolic activation, but all of the compounds can potentially form phenoxyradical intermediates by a peroxidase-mediated reaction. The compounds which can be further oxidized to a quinone were most active in inducing UDS. These results are consistent with the hypothesis that this peroxidase-mediated pathway is important in the induction of UDS, although secondary metabolites may also be involved.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quantitative structure-activity relationships: analysis of interactions of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2-substituted analogues with rat, mouse, guinea pig, and hamster cytosolic receptor

The competitive receptor binding affinities of thirteen 2-substituted 3,7,8-trichlorodibenzo-p-dioxins to hepatic cytosol from rat, mouse, guinea pig, and hamster were determined with [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin as the radioligand. Multiple parameter linear regression analysis of the binding data from the four species gave the following equations: pEC50 (rat) = 7.196 + 0.600 pi - 0.255 delta Es - 1.683 HB pEC50 (mouse) = 6.365 + 1.641 pi + 1.206 sigma 0 pEC50 (hamster) = 7.416 + 1.026 pi + 0.509 delta Es + 0.748 sigma 0 pEC50 (guinea pig) = 6.892 + 1.035 pi where pi, delta Es, HB, sigma 0, and Vw are physicochemical parameters for substituent lipophilicity, steric effect, hydrogen bonding capacity, electronegativity, and van der Waals volume (relative to H), respectively. These equations demonstrate that there are important species differences in the receptor protein binding site interactions with the substituted analogues. These data, coupled with the known species differences in the molecular properties of the receptor proteins, are evidence for a heterologous nature of the receptor between mammalian species. Multiple parameter linear regression analysis of the relative aryl hydrocarbon hydroxylase (AHH) induction potencies of these analogues in rat hepatoma H-4-II E-cells in culture gave the following equation. The correlation pEC50 (AHH induction) = 3.208 + 0.950 pEC50 (rat binding) - 0.955 delta B5 between receptor binding and AHH induction was dependent on a steric parameter (delta B5, STERIMOL) and the results suggest that an additional substituent-dependent process (e.g., an activation step) may be required after initial ligand-receptor binding for the ultimate expression of the receptor-mediated response (i.e., AHH induction).     

Acquisition of glutamine synthetase expression in human hepatocarcinogenesis: relation to disease recurrence and possible regulation by ubiquitin-dependent proteolysis

BACKGROUND: The authors previously reported increased ubiquitin (Ub) immunoreactivity in hepatocellular carcinomas (HCCs) and suggested a possible correlation between changes in ubiquitinated protein levels and multistep hepatocarcinogenesis. The current study was performed to identify one of these ubiquitinated proteins (42 kDa) and to analyze the clinical significance of its accumulation. METHODS: The protein was purified using two-dimensional gel electrophoresis and identified by amino acid sequence analysis. The authors studied the expression of this protein in 101 HCCs and 23 precancerous lesions by immunohistochemical methods and in 26 HCCs by immunoblot analysis. A survival analysis was performed on patients with advanced HCC using the Kaplan-Meier method with approximate chi-square statistics for the log rank test. RESULTS: The target protein for ubiquitination was identified as glutamine synthetase (GS). Accumulation of GS was found in 19 of 49 advanced HCCs (38.8%) by immunohistochemical methods and in 9 of 16 (56.3%) by immunoblot analysis, whereas the frequency was much lower in early HCCs (12.9% and 33.3%, respectively) and precancerous lesions (4.3% by immunostaining). In the Ub immunoblot analysis of strongly GS positive specimens, an intense 42-kDa ubiquitinated band was observed. Nine of 21 (42.9%) nodule-in-nodule type HCCs showed a GS positive, high-grade component within a GS negative, low-grade component, indicating the acquisition of GS expression during progression. Among 23 patients with a single advanced HCC nodule, the relapse free survival time was significantly shorter in the GS positive group than in the GS negative group. CONCLUSIONS: The results of this study demonstrate the acquisition of GS expression during hepatocarcinogenesis and the possible regulation of GS enzyme activity by a Ub-dependent proteolytic system. Moreover, GS might play a significant role in promoting the metastatic potential of HCC.     

Comparison of lesions induced in the Syrian golden hamster by diethylnitrosamine, dimethylhydrazine, and dibutylnitrosamine: influence of subsequent butylated hydroxyanisole treatment

The target organ specificity of the carcinogens diethylnitrosamine [(DENA) CAS: 55-18-5], dimethylhydrazine [(DMH) CAS: 57-14-7], and dibutylnitrosamine [(DBN) CAS: 924-16-3] was examined in Syrian golden hamsters. Groups of male animals were given 8 weekly injections of one of these carcinogens and then were maintained on a basal diet or a diet supplemented with 1% butylated hydroxyanisole [(BHA) CAS: 25013-16-5], or they were given the respective carcinogens in the drinking water until they were sacrificed at week 34. While DENA specifically induced tracheal polyps and hepatocellular foci and nodules, DMH administration was associated with development of both hepatocellular and hemangiocellular liver lesions as well as forestomach papillomas and adenocarcinomas of the large intestine. DBN induced lesions in the urinary bladder, forestomach, and trachea, in addition to a few preneoplastic foci in the liver and lungs. In all organs studied, preneoplastic and neoplastic populations were essentially similar to those observed in other experimental animals, with colon and tracheal lesions demonstrating alteration in polysaccharide metabolism. While inhibiting the development of hepatocellular lesions, especially in the group initiated with DENA, and while itself inducing extensive papillomatous forestomach hyperplasia, BHA administration did not exert a significant modifying influence on tumorigenesis in other organs. The present results demonstrate the efficacy of Syrian golden hamster studies for investigation of comparative neoplasia. Of particular interest in this respect were differences in the degree of phenotypic instability demonstrated by glutathione S-transferase placental form-positive foci induced by the 3 carcinogens, which indicated a possible qualitative variation in "initiation."     

Forssman antigen content of guinea pig hepatomas induced by diethylnitrosamine: a quantitative approach to the search for tumor-specific antibodies

The Forssman antigen content of diethylnitrosamine-induced guinea pig hepatomas was found to be greater than that of either autogenous or allogeneic guinea pig liver. The autogenous normal liver was obtained from guinea pigs before tumor induction, and comparison of normal and neoplastic tissues was based on the capacity of these tissues to inhibit (absorb) the hemolytic activity of rabbit antitumor serum. A method is presented for distinguishing quantitative from qualitative antigenic differences in the search for tumor-specific antigens. The results of these experiments indicate that, in the absence of strict quantitation, quantitative differences may be mistaken for qualitative differences.     

Growth in serum-free medium of NIH3T3 cells transformed by the EJ-H-ras oncogene: evidence for multiple autocrine growth factors.

Rodent fibroblastic cells transformed by ras oncogenes can grow in serum-free (S-) medium. We have studied clonal lines of mouse NIH3T3 fibroblasts transfected with the EJ-H-ras oncogene, and observed that practically all become independent of exogenous growth and attachment factors shortly after transfection. Moreover, all the clones tested soon form anchorage-independent (AI) colonies in S- medium, and most give rise to spheroids able to grow in suspension. The cell-conditioned S- medium of the transformed (TR) cells stimulates autocrinally the AI and anchored growth of these cells, in the absence of serum, and it contains growth factors related to TGF-alpha (or EGF), PDGF and bFGF, and other uncharacterized factors. Some of these factors are not found, or are found only in very small amounts, in the S- medium of non-transformed NIH3T3 cells, which also stimulates the growth of the TR cells, in the absence of serum. In addition, the TR cells contain 4-6 times more cell-associated bFGF than the non-transformed cells and release more latent TGF-beta activatable by acid treatments. However, no active TGF-beta is secreted by either cell type. Activated TGF-beta and pure TGF-beta 1 stimulate the growth of the anchored TR and NIH3T3 cells, but inhibit the AI growth of the TR cells. Another inhibitor of this growth is also found in the concentrated medium of the NIH3T3 cells.