transforming-growth-factor-alpha and Colonic-Neoplasms

There is now clear-cut evidence that polypeptide growth factors control the proliferation of the normal gastrointestinal mucosa. Epidermal growth factor (EGF) stimulates normal growth throughout the gastrointestinal tract, and accelerates the healing of ulcerated epithelium. While the effects of gastrin were at first thought to be similarly widespread, the gastrin target now appears to be restricted to the enterochromaffin-like cells in the stomach. Isolated reports suggest that several other hormones, including fibroblast growth factor and the insulin-like growth factors, have similar proliferative effects. In contrast, indirect evidence suggests that somatostatin and transforming growth factor-beta inhibit the growth of the gastrointestinal mucosa. The same growth factors profoundly affect the growth of some gastrointestinal carcinomas. Prolonged hypergastrinaemia increases the risk of development of gastric endocrine tumours, but has no effect on the incidence of gastric adenocarcinoma. Gastrin also stimulates the in vivo growth of 50% of gastric and colorectal carcinoma xenografts, but has no consistent effect on the growth of carcinoma cell lines in vitro. EGF, on the other hand, significantly stimulates proliferation of many gastrointestinal cell lines in culture. Interest has recently focused on autocrine stimulation of gastrointestinal carcinoma growth. Elevated levels of EGF receptor, and of EGF or related mRNAs, have been demonstrated in gastric carcinomas, and the growth of some gastrointestinal cell lines is inhibited by antibodies against EGF, and by antisense oligonucleotides based on EGF mRNA. Similarly gastrin/cholecystokinin antagonists inhibit the growth of several colon carcinoma cell lines, although the spectrum of antagonist potencies suggests that classical gastrin and cholecystokinin receptors are not necessarily involved. Continued research on antagonists may therefore lead to novel therapies for gastrointestinal cancers.

Topics: Adult; Animals; Colonic Neoplasms; Digestive System Physiological Phenomena; Epidermal Growth Factor; ErbB Receptors; Gastrins; Gastrointestinal Hormones; Gastrointestinal Neoplasms; Glucagon-Like Peptides; Humans; Peptide YY; Peptides; Somatostatin; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor alpha and beta 1 (TGF alpha and TGF beta 1) are representative members of two distinct and expanding families of polypeptide growth factors. TGF alpha is an epithelial cell mitogen, whereas TGF beta 1 inhibits epithelial cell growth; the role of these factors in contributing to the transformed phenotype is uncertain. Steady state mRNA expression for these growth factors and their receptors in a panel of human colon cancers and adjacent normal mucosa is presented. Based in part on results from transgenic mice in which TGF alpha is selectively overproduced in the mammary gland, a possible role for TGF alpha as a tumor promoter in the process of transformation is discussed.

Topics: Animals; Cell Transformation, Neoplastic; Colon; Colonic Neoplasms; Female; Gastrointestinal Neoplasms; Humans; Male; Mice; Mice, Transgenic; Multigene Family; Proto-Oncogenes; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Transforming Growth Factor alpha; Transforming Growth Factor beta

Colorectal cancer is the second-most frequent cause of cancer mortality in the United States. Human epidemiology and laboratory studies indicate that aspirin may be an effective colorectal cancer chemopreventive agent. This study was designed to determine whether treatment with 81 mg of aspirin per day for 3 months would alter two putative surrogate end point biomarkers of chemoprevention of colorectal cancer [i.e., mucosal prostaglandin E2 (PGE2) formation and transforming growth factor alpha (TGF-alpha) expression] in normal-appearing rectal mucosa from individuals with a history of adenomatous polyps. Rectal biopsies were obtained by flexible sigmoidoscopy at three sequential time points: (a) after a 1-month placebo run-in period (baseline), (b) after 3 months of ingesting 81 mg of aspirin (as a single tablet) once per day, and (c) after 3 months of ingesting a placebo tablet once per day (washout period). Daily aspirin significantly suppressed PGE2 formation, but this significant suppression was completely reversed when aspirin was withdrawn. The extent of TGF-alpha staining in rectal crypts was also reduced significantly (P = 0.039) by daily aspirin. After a 3-month placebo-washout period, however, the mean extent of TGF-alpha staining was not significantly different from either baseline or the aspirin time point. Thus, 81 mg of aspirin daily significantly reduced rectal mucosal PGE2 formation and TGF-alpha expression in patients with a history of adenomatous polyps. These putative surrogate end point biomarkers may be useful intermediate end points in future colorectal cancer chemoprevention trials.

Topics: Adenomatous Polyps; Aspirin; Biomarkers; Biopsy, Needle; Colonic Neoplasms; Dinoprostone; Double-Blind Method; Drug Administration Schedule; Female; Humans; Immunohistochemistry; Intestinal Mucosa; Male; Middle Aged; Prospective Studies; Reference Values; Statistics, Nonparametric; Transforming Growth Factor alpha

TGFαL3-SEB chimeric protein is a synthetic protein, which is produced by combining the third loop (L3) of transforming growth factor-α (TGF-α) with staphylococcal enterotoxin type B. To the best of our knowledge, anti-cancer activity of this chimeric protein against colon cancer that overexpresses epidermal growth factor receptor (EGFR) has not yet been studied. Thus, in the present study, the anti-tumor effects of TGFαL3-SEB chimeric protein on HT-29 colon cancer cells were evaluated.. The TGFαL3-SEB chimeric protein was previously designed and cloned in Escherichia coli (E. coli) [1,2]. The level of expression and the purity of this novel protein were examined for further analysis. For this purpose, the cells were treated with different concentrations (25, 50 and 75 μg/ml) of TGFαL3-SEB and then the proliferation was detected using the MTT assay. The apoptosis-inducing potential of TGFαL3-SEB in HT-29 and HEK-293 cells was evaluated by flow cytometry using Annexin V/PI double staining method; in addition, bax/bcl2 mRNA ratio, caspase-3 and caspase-9 activity were also assessed.. In the present study, TGFαL3-SEB chimeric protein was produced in E. coli. After effective purification, its growth inhibitory effect was evaluated. Our results indicated that the incubation of HT-29 colon cancer cell with 25, 50 and 75 μg/ml of TGFαL3-SEB for 24 h leads to significant reduction of proliferation in a dose-dependent manner (P < 0.05). Further analysis indicated that exposure of EGFR expressing HT-29 cells to TGFαL3-SEB leads to significant increase of the caspase-3 and caspase-9 activity in a concentration-dependent manner (P < 0.05). Bax/bcl-2 ratio also confirmed that TGFαL3-SEB has the pro-apoptotic effect. Flow cytometry analysis of TGFαL3-SEB treated cells showed that in addition to apoptotic cells, necrotic cells were also increased significantly at the concentration of 25, 50 and 75 μg/ml (P < 0.05).. In conclusion, our results demonstrated that TGFαL3-SEB chimeric protein induced cell death through both mechanisms of apoptosis and necrosis in HT-29 colon cancer cells. This paper has highlighted that TGFαL3-SEB has the potential to target EGFR expressing cancer cell.

Topics: Apoptosis; Cell Survival; Colonic Neoplasms; Dose-Response Relationship, Drug; Enterotoxins; Escherichia coli; Growth Inhibitors; HEK293 Cells; HT29 Cells; Humans; Recombinant Fusion Proteins; Transforming Growth Factor alpha

How cells in primary tumors initially become pro-metastatic is not understood. A previous genome-wide RNAi screen uncovered colon cancer metastatic suppressor and WNT promoting functions of TMED3, a member of the p24 ER-to-Golgi protein secretion family. Repression of canonical WNT signaling upon knockdown (kd) of TMED3 might thus be sufficient to drive metastases. However, searching for transcriptional influences on other family members here we find that TMED3 kd leads to enhanced TMED9, that TMED9 acts downstream of TMED3 and that TMED9 kd compromises metastasis. Importantly, TMED9 pro-metastatic function is linked to but distinct from the repression of TMED3-WNT-TCF signaling. Functional rescue of the migratory deficiency of TMED9 kd cells identifies TGFα as a mediator of TMED9 pro-metastatic activity. Moreover, TMED9 kd compromises the biogenesis, and thus function, of TGFα. Analyses in three colon cancer cell types highlight a TMED9-dependent gene set that includes CNIH4, a member of the CORNICHON family of TGFα exporters. Our data indicate that TGFA and CNIH4, which display predictive value for disease-free survival, promote colon cancer cell metastatic behavior, and suggest that TMED9 pro-metastatic function involves the modulation of the secretion of TGFα ligand. Finally, TMED9/TMED3 antagonism impacts WNT-TCF and GLI signaling, where TMED9 primacy over TMED3 leads to the establishment of a positive feedback loop together with CNIH4, TGFα, and GLI1 that enhances metastases. We propose that primary colon cancer cells can transition between two states characterized by secretion-transcription regulatory loops gated by TMED3 and TMED9 that modulate their metastatic proclivities.

Topics: Colonic Neoplasms; Epistasis, Genetic; Gene Expression Regulation; Humans; Neoplasm Metastasis; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Signal Transduction; Transforming Growth Factor alpha; Vesicular Transport Proteins; Wnt Signaling Pathway; Zinc Finger Protein GLI1

Colon cancer is one of the most common cancers in the developed countries. The association between transforming growth factor TGFα and human cancer incidence has been suggested, yet, the regulatory roles of TGFα and the molecular mechanisms remain unknown, especially in colon cancer. We aim to investigate the functional regulations of TGFα in colon cancer progression. Two colon cancer cell lines were applied, and plasmid overexpression and siRNA-mediated depletion techniques were used to verify the role of TGFα in colon cancer. Cell proliferation was analyzed by MTS assay and colony formation assay, and western blot assay was used to examine protein expression. Migration, invasion, and reporter assays were also carried out to study the regulations of TGFα in colon cancer. Our results evidenced that expression of TGFα facilitates short-term and long-term proliferations of colon cancer cells. Moreover, TGFα was suggested as a migration-and-invasion promoting factor of colon cancer. Finally, our data indicated that TGFα modulates epithelial-mesenchymal transition (EMT) markers and NFκB signaling pathway in colon cancer cells. We provide the first time evidence of the promoting role TGFα plays in colon cancer tumorigenesis with proposed regulatory mechanisms involving EMT alteration and NFκB signaling pathway.

Topics: Carcinogenesis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colonic Neoplasms; Disease Progression; Epithelial-Mesenchymal Transition; Humans; Neoplasm Invasiveness; NF-kappa B; Transforming Growth Factor alpha

Epidermal growth factor receptors (EGFR) are required for tumor promotion by Western diet. The metalloprotease, ADAM17 activates EGFR by releasing pro-EGFR ligands. ADAM17 is regulated by G-protein-coupled receptors, including CXCR4. Here we investigated CXCR4-ADAM17 crosstalk and examined the role of ADAM17 in tumorigenesis.. We used CXCR4 inhibitor, AMD3100 and ADAM17 inhibitor, BMS566394 to assess CXCR4-ADAM17 crosstalk in colon cancer cells. We compared the expression of CXCR4 ligand, CXCL2, and ADAM17 in mice fed Western diet versus standard diet. Separately, mice were treated with marimastat, a broad-spectrum ADAM17 inhibitor, or AMD3100 to assess EGFR activation by ADAM17 and CXCR4. Using Apc-mutant Min mice, we investigated the effects of ADAM17/10 inhibitor INCB3619 on tumorigenesis. To assess the effects of colonocyte ADAM17, mice with ADAM17 conditional deletion were treated with azoxymethane (AOM). ADAM17 expression was also compared in colonocytes from primary human colon cancers and adjacent mucosa.. CXCL12 treatment activated colon cancer cell EGFR signals, and CXCR4 or ADAM17 blockade reduced this activation. In vivo, Western diet increased CXCL12 in stromal cells and TGFα in colonocytes. Marimastat or AMD3100 caused >50% reduction in EGFR signals (P < 0.05). In Min mice, INCB3619 reduced EGFR signals in adenomas and inhibited intestinal tumor multiplicity (P < 0.05). In the AOM model, colonocyte ADAM17 deletion reduced EGFR signals and colonic tumor development (P < 0.05). Finally, ADAM17 was upregulated >2.5-fold in human malignant colonocytes.. ADAM17 is a Western diet-inducible enzyme activated by CXCL12-CXCR4 signaling, suggesting the pathway: Western diet→CXCL12→CXCR4→ADAM17→TGFα→EGFR. ADAM17 might serve as a druggable target in chemoprevention strategies. Clin Cancer Res; 23(2); 549-61. ©2016 AACR.

Topics: ADAM17 Protein; Animals; Carcinogenesis; Cell Line, Tumor; Chemokine CXCL2; Colonic Neoplasms; Diet, Western; ErbB Receptors; Humans; Ligands; Mice; Piperidines; Receptors, CXCR4; Signal Transduction; Spiro Compounds; Transforming Growth Factor alpha

Although cetuximab, an anti-EGF receptor (EGFR) monoclonal antibody, is an effective treatment for patients with KRAS wild-type metastatic colorectal cancer (mCRC), its clinical use is limited by onset of resistance.. We characterized two colorectal cancer models to study the mechanisms of acquired resistance to cetuximab.. Following chronic treatment of nude mice bearing cetuximab-sensitive human GEO colon xenografts, cetuximab-resistant GEO (GEO-CR) cells were obtained. In GEO-CR cells, proliferation and survival signals were constitutively active despite EGFR inhibition by cetuximab treatment. Whole gene expression profiling identified a series of genes involved in the hepatocyte growth factor (HGF)-MET-dependent pathways, which were upregulated in GEO-CR cells. Furthermore, activated, phosphorylated MET was detected in GEO-CR cells. A second colorectal cancer cell line with acquired resistance to cetuximab was obtained (SW48-CR). Inhibition of MET expression by siRNA restored cetuximab sensitivity in GEO-CR and SW48-CR cells, whereas exogenous activation of MET by HGF stimulation in cetuximab-sensitive GEO and SW48 cells induced resistance to cetuximab. Treatment of GEO-CR and SW48-CR cells with PHA665752, a selective MET inhibitor, inhibited cell growth, proliferation, and survival signals and impaired cancer cell migration. Overexpression of TGF-α, a specific EGFR ligand, was involved in the acquisition of cetuximab resistance in GEO-CR and SW48-CR cells. In fact, TGF-α overexpression induced the EGFR-MET interaction, with subsequent MET phosphorylation and activation of MET downstream effectors in GEO-CR and SW48-CR cells.. These results suggest that overexpression of TGF-α through induction of EGFR-MET interaction contributes to cetuximab resistance in colorectal cancer cells. The combined inhibition of EGFR and MET receptor could represent a strategy for preventing and/or overcoming cetuximab resistance in patients with colorectal cancer.

Topics: Animals; Antibodies, Monoclonal, Humanized; Cell Line, Tumor; Cell Proliferation; Cetuximab; Colonic Neoplasms; Drug Resistance, Neoplasm; ErbB Receptors; Gene Expression Regulation, Neoplastic; Hepatocyte Growth Factor; Humans; Mice; Proto-Oncogene Proteins c-met; Signal Transduction; Transforming Growth Factor alpha

The transforming growth factor-β-signaling pathway has been identified as being involved in colorectal cancer.. The aim of this study was to determine how diet and lifestyle factors in combination with genetic variation in the transforming growth factor-β-signaling pathway alters colorectal cancer risk.. We used data from 2 population-based case-control studies.. Participants included patients with colon cancer (n = 1574) and controls (n = 1970) and patients with rectal cancer ( n = 791) and controls (n = 999).. The primary outcomes measured were newly diagnosed cases of colon or rectal cancer.. Colon and rectal cancer risk increased with the number of at-risk genotypes within the transforming growth factor-β-signaling pathway (OR 3.68, 95% CI 2.74,4.94 for colon cancer; OR 3.89, 95% CI 2.66,5.69 for rectal cancer). A high at-risk lifestyle score also resulted in significant increased risk with number of at-risk lifestyle factors (OR 2.99, 95% CI 2.32,3.85 for colon cancer; OR 3.37, 95% CI 2.24,5.07 for rectal cancer). The combination of high-risk genotype and high-risk lifestyle results in the greatest increase in risk (OR 7.89, 95% CI 4.45,13.96 for colon cancer; OR 8.75, 95% CI 3.66,20.89 for rectal cancer).. The study results need validation in other large studies of colon and rectal cancer.. In summary, our data suggest that there is increased colon and rectal cancer risk with increasing number of at-risk genotypes and at-risk lifestyle factors. Although the integrity of the pathway can be diminished by a number of high-risk genotypes, this risk can be offset, in part, by maintaining a healthy lifestyle.

Topics: Adult; Aged; Colonic Neoplasms; DNA, Neoplasm; Female; Genetic Variation; Genotype; Humans; Incidence; Life Style; Male; Middle Aged; Polymerase Chain Reaction; Rectal Neoplasms; Retrospective Studies; Signal Transduction; Transforming Growth Factor alpha; United States

We have previously demonstrated that expression of the novel gene schlafen-3 (Slfn-3) correlates with intestinal epithelial cell differentiation (Patel VB, Yu Y, Das JK, Patel BB, Majumdar AP. Biochem Biophys Res Commun 388: 752-756, 2009). The present investigation was undertaken to examine whether Slfn-3 plays a role in regulating differentiation of FOLFOX-resistant (5-fluorouracil + oxaliplatin) colon cancer cells that are highly enriched in cancer stem cells (CSCs). Transfection of Slfn-3 in FOLFOX-resistant colon cancer HCT-116 cells resulted in increase of alkaline phosphatase activity, a marker of intestinal differentiation. Additionally, Slfn-3 transfection resulted in reduction of mRNA and protein levels of the CSC markers CD44, CD133, CD166, and aldehyde dehydrogenase 1 in both FOLFOX-resistant HCT-116 and HT-29 cells. This was accompanied by decreased formation of tumorosphere/colonosphere (an in vitro model of tumor growth) in stem cell medium and inhibition of expression of the chemotherapeutic drug transporter protein ABCG2. Additionally, Slfn-3 transfection of FOLFOX-resistant HCT-116 and HT-29 cells reduced Hoechst 33342 dye exclusion. Finally, Slfn-3 transfection inhibited the expression of transforming growth factor-α in both FOLFOX-resistant colon cancer cells, but stimulated apoptosis in response to additional FOLFOX treatment. In summary, our data demonstrate that Slfn-3 expression inhibits multiple characteristics of CSC-enriched, FOLFOX-resistant colon cancer cells, including induction of differentiation and reduction in tumorosphere/colonosphere formation, drug transporter activity, and autocrine stimulation of proliferation. Thus Slfn-3 expression may render colon CSCs more susceptible to cancer chemotherapeutics.

Topics: AC133 Antigen; Aldehyde Dehydrogenase 1 Family; Antigens, CD; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Autocrine Communication; Cell Adhesion Molecules, Neuronal; Cell Cycle Proteins; Cell Differentiation; Colonic Neoplasms; Drug Resistance, Neoplasm; ErbB Receptors; Fetal Proteins; Fluorouracil; Glycoproteins; HCT116 Cells; HT29 Cells; Humans; Hyaluronan Receptors; Isoenzymes; Leucovorin; Neoplasm Proteins; Neoplastic Stem Cells; Organoplatinum Compounds; Peptides; Proteins; Retinal Dehydrogenase; RNA, Messenger; Signal Transduction; Transfection; Transforming Growth Factor alpha

Colon cancer is a major cause of cancer deaths. Dietary factors contribute substantially to the risk of this malignancy. Western-style diets promote development of azoxymethane-induced colon cancer. Although we showed that epidermal growth factor receptors (EGFR) controlled azoxymethane tumorigenesis in standard fat conditions, the role of EGFR in tumor promotion by high dietary fat has not been examined.. A/J x C57BL6/J mice with wild-type Egfr (Egfr(wt)) or loss-of-function waved-2 Egfr (Egfr(wa2)) received azoxymethane followed by standard (5% fat) or western-style (20% fat) diet. As F(1) mice were resistant to azoxymethane, we treated mice with azoxymethane followed by one cycle of inflammation-inducing dextran sulfate sodium to induce tumorigenesis. Mice were sacrificed 12 weeks after dextran sulfate sodium. Tumors were graded for histology and assessed for EGFR ligands and proto-oncogenes by immunostaining, Western blotting, and real-time PCR.. Egfr(wt) mice gained significantly more weight and had exaggerated insulin resistance compared with Egfr(wa2) mice on high-fat diet. Dietary fat promoted tumor incidence (71.2% versus 36.7%; P < 0.05) and cancer incidence (43.9% versus 16.7%; P < 0.05) only in Egfr(wt) mice. The lipid-rich diet also significantly increased tumor and cancer multiplicity only in Egfr(wt) mice. In tumors, dietary fat and Egfr(wt) upregulated transforming growth factor-alpha, amphiregulin, CTNNB1, MYC, and CCND1, whereas PTGS2 was only increased in Egfr(wt) mice and further upregulated by dietary fat. Notably, dietary fat increased transforming growth factor-alpha in normal colon.. EGFR is required for dietary fat-induced weight gain and tumor promotion. EGFR-dependent increases in receptor ligands and PTGS2 likely drive diet-related tumor promotion.

Topics: Animals; Azoxymethane; Blood Glucose; Colonic Neoplasms; Cyclooxygenase 2; Dextran Sulfate; ErbB Receptors; Gene Expression Regulation, Neoplastic; Insulin; Insulin Resistance; Ligands; Mice; Mice, Inbred C57BL; Point Mutation; Transforming Growth Factor alpha

The transforming growth factor alpha (TGFalpha)/epidermal growth factor receptor (EGFR) signaling pathway appears to play a critical role in colon cancer progression, but the cellular and molecular mechanisms that contribute to metastasis remain unknown. KM12C colon cancer cell clones expressing high (C9) or negligible (C10) levels of TGFalpha were implanted into the cecal walls of nude mice. C9 tumors formed autocrine and paracrine EGFR networks, whereas C10 tumors were unable to signal through EGFR. The tumor microenvironment of C9, but not C10, contained cells enriched in vascular endothelial growth factor (VEGF) A, interleukin-8, and matrix metalloproteinases-2 and -9 and had a high vascular surface area. C9 tumors recruited a macrophage population that co-expressed F4/80 and lymphatic vessel endothelial hyaluronic acid receptor and produced VEGFC. The mean lymphatic density of C9 tumors was threefold higher than that of C10 tumors. C9, but not C10, tumor cells metastasized to regional lymph nodes in all mice and to the liver in 5 of 10 mice. Forced expression of TGFalpha in C10 tumor cells led to the generation of autocrine and paracrine EGFR signaling, macrophage recruitment, enhanced blood and lymphatic vascular surface areas, and increased lymphatic metastasis. Collectively, these data show that activation of TGFalpha-EGFR signaling in colon cancer cells creates a microenvironment that is conducive for metastasis, providing a rationale for efforts to inhibit EGFR signaling in TGFalpha-positive colon cancers.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Colonic Neoplasms; Enzyme-Linked Immunosorbent Assay; ErbB Receptors; Fluorescent Antibody Technique; Humans; Immunohistochemistry; Male; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neovascularization, Pathologic; Signal Transduction; Transfection; Transforming Growth Factor alpha

The cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway may have a pathogenic role in colorectal cancer (CRC). Recent studies suggest that 5-aminosalicylic acid (5-ASA) reduces the risk of inflammatory bowel disease-related CRC, but the mechanism by which 5-ASA interferes with CRC cell growth remains unknown. In this study, we have examined whether the negative effect of 5-ASA on CRC cells is dependent on COX-2/PGE2 axis inhibition. We show that 5-ASA down-regulates both constitutive and TNF-alpha or IL-1beta-induced COX-2 in HT-115 and HT-29 cells. Inhibition of COX-2 by 5-ASA occurs at the RNA and protein level, and is associated with a significant decrease in PGE2 synthesis, arrest of growth and enhanced death of CRC cells. However, exogenous PGE2 does not revert the 5-ASA-mediated CRC cell proliferation block. 5-ASA also inhibits the growth of DLD-1, a COX-deficient CRC cell line, thus suggesting that the anti-proliferative effect of 5-ASA on CRC cells is not strictly dependent on the inhibition of COX-2/PGE2. Taken together our data indicate that 5-ASA causes both a COX-2-dependent and -independent inhibition of CRC cell growth.

Topics: Apoptosis; Cell Line, Tumor; Colonic Neoplasms; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dinoprostone; Dose-Response Relationship, Drug; Humans; Interleukin-1beta; Mesalamine; Transforming Growth Factor alpha

Using a mouse predisposed to neoplasia by a germ line mutation in Apc (Apc(Min)), we tested whether induced hyperplasia is sufficient to increase intestinal tumor multiplicity or size in the intestine. We found that hyperplasia in the jejunum correlated with a significant increase in tumor multiplicity. However, tumor multiplicity was unchanged in the hyperplastic colon. This result indicates that even an intestine predisposed to neoplasia can, in certain regions including the colon, accommodate net increased cell growth without developing more neoplasms. Where hyperplasia correlated with increased tumor multiplicity, it did not increase the size or net growth of established tumors. This result suggests that the event linking hyperplasia and neoplasia in the jejunum is tumor establishment. Two novel observations arose in our study: the multiple intestinal neoplasia (Min) mutation partially suppressed both mitosis and transforming growth factor alpha-induced hyperplasia throughout the intestine; and zinc treatment alone increased tumor multiplicity in the duodenum of Min mice.

Topics: Animals; Apoptosis; Colonic Neoplasms; Duodenal Neoplasms; Ethylnitrosourea; Female; Genes, APC; Hyperplasia; Ileal Neoplasms; Jejunal Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Mitosis; Mutation; Transforming Growth Factor alpha; Transgenes; Zinc

We determined whether phosphorylated epidermal growth factor receptor (EGFR) expressed on tumor-associated endothelial cells is a primary target for therapy with EGFR tyrosine kinase inhibitors (TKIs). Human colon cancer cells SW620CE2 (parental) that do not express EGFR or human epidermal growth factor receptor 2 (HER2) but express transforming growth factor alpha (TGF-alpha) were transduced with a lentivirus carrying nontargeting small hairpin RNA (shRNA) or TGF-alpha shRNA. The cell lines were implanted into the cecum of nude mice. Two weeks later, treatment began with saline, 4-[R]-phenethylamino-6-[hydroxyl] phenyl-7H-pyrrolo [2,3-D]-pyrimidine (PKI166), or irinotecan. Endothelial cells in parental and nontargeting shRNA tumors expressed phosphorylated EGFR. Therapy with PKI166 alone or with irinotecan produced apoptosis of these endothelial cells and necrosis of the EGFR-negative tumors. Endothelial cells in tumors that did not express TGF-alpha did not express EGFR, and these tumors were resistant to treatment with PKI166. The response of neoplasms to EGFR antagonists has been correlated with EGFR mutations, HER2 expression, Akt activation, and EGFR gene copy number. Our present data using colon cancer cells that do not express EGFR or HER2 suggest that the expression of TGF-alpha by tumor cells leading to the activation of EGFR in tumor-associated endothelial cells is a major determinant for the susceptibility of neoplasms to therapy by specific EGFR-TKI.

Topics: Animals; Apoptosis; Blotting, Western; Camptothecin; Cecum; Cell Proliferation; Colonic Neoplasms; Drug Therapy, Combination; Endothelial Cells; Enzyme Inhibitors; ErbB Receptors; Humans; Immunoenzyme Techniques; In Situ Nick-End Labeling; Irinotecan; Lymphatic Metastasis; Male; Mice; Mice, Nude; Necrosis; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrimidines; Pyrroles; Receptor, ErbB-2; RNA, Small Interfering; Topoisomerase I Inhibitors; Transforming Growth Factor alpha

Epidermal growth factor receptor (EGFR) is widely expressed in a number of solid tumors including colorectal cancers. Overexpression of this receptor is one means by which a cell can achieve positive signals for survival and proliferation; another effective means is by constitutive activation of EGFR. We have elucidated the role of constitutive EGFR signaling in malignant progression by stably transfecting colon cancer cells with a human transforming growth factor-alpha cDNA (a ligand for EGFR) under repressible control by tetracycline. We show that constitutive expression of transforming growth factor-alpha and its subsequent constitutive activation of EGFR allows for cancer cell survival in response to environmental stress in vitro and in vivo as well. The reversal of constitutive EGFR activation results in the loss of downstream mitogen-activated protein kinase and Akt activation, and a reduction in xenograft size that is associated with decreased proliferation and increased apoptosis. We used CI-1033, a small molecule antagonist of EGFR, to dissect an activation pathway that shows the ability of ERBb2 to activate Akt, but not Erk in the face of EGFR antagonism. This novel escape mechanism is a possible explanation of why anti-EGFR therapies have shown disappointing results in clinical trials.

Topics: Animals; Apoptosis; Cell Growth Processes; Cell Line, Tumor; Colonic Neoplasms; Drug Resistance, Neoplasm; Enzyme Activation; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Humans; Immunohistochemistry; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Morpholines; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Signal Transduction; Transfection; Transforming Growth Factor alpha; Xenograft Model Antitumor Assays

A role of the epidermal growth factor receptor (EGFR) family has been suggested in colon cancer etiology, progression, and/or severity. Our recently identified pan-erbB inhibitor EGFR-related protein (ERRP) targets EGFRs by attenuating their activation and subsequent signaling leading to cellular growth inhibition. In the present study, we evaluated the therapeutic effectiveness of ERRP on early and intermediate stages of colon cancer by examining regression of chemically induced aberrant crypt foci (ACF) in the colon of CF1 mice and intestinal adenomas in APC(Min+/-) (Min) mice. After formation of ACF or adenomas, the mice were injected (i.p.) with ERRP (50 microg/mouse) for 10 consecutive days. This treatment significantly reduced the number of ACF from 25.0 +/- 3.0 (controls) to 14.9 +/- 1.6 (ERRP-treated; P = 0.011) and also reduced their size (P < 0.01). In Min mice, ERRP caused the regression of adenomas throughout the small intestine (P < 0.05) and reduced their size (P < 0.001). This could partly be attributed to inhibition of proliferation and stimulation of apoptosis in the intestinal mucosa and was associated with decreased activation of several EGFR family members, suppression of downstream effector nuclear factor kappaB and down-regulation of cyclooxygenase-2. ERRP-induced attenuation of EGFR activation could be due to increased sequestration of the ligand(s) by ERRP, rendering them unavailable for binding to and activation of the receptor. In conclusion, our data show that ERRP is effective in regressing both early and intermediate intestinal lesions and could be an effective therapeutic agent for colon cancer.

Topics: 1,2-Dimethylhydrazine; Adenoma; Animals; Apoptosis; Carcinogens; Cell Growth Processes; Colonic Neoplasms; Drosophila Proteins; ErbB Receptors; Female; Intestinal Mucosa; Male; Mice; Mice, Inbred C57BL; Neoplasm Staging; NF-kappa B; Precancerous Conditions; Receptors, Estrogen; Recombinant Fusion Proteins; Transforming Growth Factor alpha

Currently, pathologic stage is the main prognostic indicator of colorectal carcinoma, but the current staging system is insufficient to predict the risk of recurrence or the need for adjuvant treatment for the patients with Dukes'A and B disease. Biologic prognostic markers may supplement the staging system and provide a basis for the decision of therapeutic strategies according to individual tumor biology. This study was to investigate the correlations of c-erbB-2, epithelial growth factor receptor (EGFR), and transforming growth factor-alpha (TGF-alpha) expression to recurrence of Dukes'A and B colorectal carcinoma.. The expression of c-erbB-2, EGFR and TGF-alpha in 26 specimens of Dukes'A and 62 specimens of Dukes'B colorectal adenocarcinoma was detected by SP immunohistochemistry. Of the 88 patients underwent curative resection between 1989 and 1999, 44 had recurrence, and 44 had not. The patients were followed up for at least 5 years or till recurrence. The tumor location, Dukes staging, age, sex, and depth of bowel wall invasion matched as closely as possible between the 2 groups.. The overexpression rate of c-erbB-2 was higher in recurrence group than in non-recurrence group (43.2% vs. 25.0%, P=0.072); the overexpression rates of EGFR and TGF-alpha were significantly higher in recurrence group than in non-recurrence group (63.6% vs. 27.3%, P=0.001; 65.9% vs. 43.2%, P=0.032). The co-overexpression rate of EGFR and TGF was significantly higher in recurrence group than in non-recurrence group (36.4% vs. 11.4%, P=0.006). Multivariate analysis showed that the overexpression of EGFR was associated with postoperative recurrence of colorectal carcinoma.. The expression of EGFR may be associated with postoperative recurrence of Dukes'A and B colorectal carcinoma.

Topics: Adenocarcinoma; Adult; Aged; Colonic Neoplasms; ErbB Receptors; Female; Humans; Liver Neoplasms; Lung Neoplasms; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Proportional Hazards Models; Receptor, ErbB-2; Rectal Neoplasms; Transforming Growth Factor alpha

The effect of conjugated linoleic acid (CLA) on peritoneal metastasis was examined by in vitro treatment of cancer cells and mouse peritoneal metastasis models. First, cell growth of MKN28 human gastric cancer cells and Colo320 human colon cancer cells was suppressed by CLA in a dose-dependent manner with an increment in apoptosis. CLA significantly inhibited invasion into type IV collagen-coated membrane of MKN28 and Colo320 cells (p < 0.05). CLA-induced growth inhibition was recovered by the exposure to antisense S-oligodeoxynucleotide for peroxisome proliferator-activated receptor (PPAR)-gamma in both cell lines. BALB/c nu-nu mice were inoculated with MKN28 and Colo320 cells into their peritoneal cavity, and administrated with CLA intraperitoneally (weekly, 4 times). CLA treatment did not affect food intake or weight gain of mice. CLA treatment significantly decreased metastatic foci of both cells in the peritoneal cavity (p < 0.005). Survival rate in mice inoculated with MKN28 or Colo320 cells was significantly recovered by CLA treatment (p = 0.0025 and 0.0052, respectively). Protein production in MKN28 and Colo320 cells treated with CLA showed a decrease in epidermal growth factor receptor and transforming growth factor-alpha and an increase in Bax. These findings suggest that CLA inhibits metastasis of human gastric and colon cancer cells.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Colonic Neoplasms; Dose-Response Relationship, Drug; ErbB Receptors; Humans; Injections, Intraperitoneal; Linoleic Acids, Conjugated; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Oligodeoxyribonucleotides, Antisense; Peritoneal Neoplasms; PPAR gamma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stomach Neoplasms; Survival Rate; Transforming Growth Factor alpha

We examined the effects of IL-15 and TGF-alpha on the inhibition of macrophage infiltration into colon cancer tissue, and secretion of amphoterin from colon cancer cells. Production of IL-15 and/or TGF-alpha was associated with depletion of tumor-associated macrophages (TAMs) in both Dukes' B and C tumors (P = 0.0324 and 0.0051, respectively). Production of IL-15 and/or TGF-alpha was also associated with amphoterin mRNA expression in colon cancer tissues with TAM depletion in both Dukes' B and C tumors (P = 0.0167 and P = 0.0062, respectively). WiDr human colon cancer cells treated with IL-15 and/or TGF-alpha induced reduction of nucleus-localized amphoterin and an increase in cytosolic and membranous amphoterin. Moreover, IL-15 and/or TGF-alpha treatment increased amphoterin secretion by WiDr cells. Most notably, IL-15 and TGF-alpha treatment induced the increase of cytosol/membrane localization and secretion of amphoterin and the most pronounced effect among the treatments carried out. These results suggested that IL-15/TGF-alpha promotes depletion of TAMs and secretion of amphoterin in colon cancer.

Topics: Antigens, CD; Antigens, Differentiation, Myelomonocytic; Cell Count; Colonic Neoplasms; HMGB1 Protein; Humans; Immunohistochemistry; Interleukin-15; Macrophages; Transforming Growth Factor alpha

Cyclooxygenase (COX)-generated prostaglandin E(2) (PGE(2)) plays critical roles in colorectal carcinogenesis. Recently, we have shown that PGE(2) and transforming growth factor-alpha synergistically induces the expression of amphiregulin (AR) in colon cancer cells (Shao, J., Evers, B. M., and Sheng, H. (2003) Cancer Res. 63, 5218-5223). In this study, we demonstrated synergistic actions of PGE(2) and the receptor tyrosine kinase signaling system in AR expression and in tumorigenic potential of colon cancer cells. Activation of the Ras/Raf/MAPK pathway induced AR transcription in colon cancer LS-174 cells that was enhanced by PGE(2) in a synergistic fashion. The cAMP-responsive element within the AR promoter was required for the synergistic activation of AR transcription. An Sp1 element was responsible for the basal transcription of AR and significantly enhanced the synergy between PGE(2) and the epidermal growth factor receptor (EGFR) signaling system. Furthermore, activation of both PGE(2) and EGFR signaling pathways synergistically promoted the growth and migration of colon cancer cells. Our results suggest that COX-2/PGE(2) may exert pro-oncogenic effects through synergistic induction of receptor tyrosine kinase-dependent signaling pathway, thus, provide a novel mechanism for the combinatorial treatment of colonic neoplasms targeting both COX-2/PGE(2) and the EGFR system that has demonstrated remarkable advantages.

Topics: Antineoplastic Agents; Cell Division; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Cyclic AMP Response Element-Binding Protein; Cyclooxygenase 2; Dinoprostone; Drug Synergism; ErbB Receptors; Humans; Isoenzymes; MAP Kinase Signaling System; Membrane Proteins; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases; Proto-Oncogene Proteins c-raf; ras Proteins; Response Elements; Sp1 Transcription Factor; Transforming Growth Factor alpha

Tumor-stromal interactions, which are regulated by stromal-derived HGF and tumor-derived HGF inducers, are essential for tumor cell acquisition of such malignant properties as invasion and metastasis. NK4, a proteolytic cleavage product of HGF, has antitumor activities as both an HGF antagonist and an angiogenesis inhibitor. In this study, we examined the in vitro and in vivo behaviors of mouse colon adenocarcinoma C T26 cells modified by gene transfer to secrete NK4, and investigated the influence of NK4 on expression of HGF and HGF inducers associated with tumor-stromal interactions. In vitro cell proliferation rates of NK4 transfectant (C T26-NK4) and mock transfectant (C T26-NEO) were essentially the same, and scattering and invasion were stimulated by HGF in C T26-NEO, but not in C T26-NK4. In syngeneic BALB/c female mice, subcutaneous tumor growth of C T26-NK4 was potently suppressed, and the survival was prolonged significantly. Immunohistochemistry showed significantly decreased microvessels and increased apoptotic cells in C T26-NK4 tumor compared with control. Interestingly, HGF, strongly expressed in C T26-NEO tumor stroma, was reduced in C T26-NK4. In vitro, conditioned medium of C T26-NK4 inhibited fibroblast-derived HGF production, which was increased by that of C T26-NEO. Moreover, although similar constitutive expression levels of PDGF and TGF-alpha (both HGF inducers) were detected in C T26-NK4 and C T26-NEO in semiquantitative RT-PCR analyses, the expression was up-regulated by HGF in C T26-NEO, but not C T26-NK4. These results suggest that NK4 may exert antitumor activities not only by antagonizing HGF, but also by inhibiting HGF amplification via tumor-stromal interactions. Continuous, abundant NK4 production induced at a tumor site by gene transfer should show multiple antitumor activities with potential therapeutic benefit.

Topics: Adenocarcinoma; Animals; Cell Division; Cell Line, Tumor; Colonic Neoplasms; Female; Fibroblasts; Gene Expression Regulation, Neoplastic; Hepatocyte Growth Factor; Humans; Mice; Mice, Inbred BALB C; Mitogens; Neoplasm Transplantation; Phenotype; Platelet-Derived Growth Factor; Recombinant Proteins; Stromal Cells; Transfection; Transforming Growth Factor alpha

Recently, we showed that autocrine transforming growth factor alpha (TGFalpha) controls the epidermal growth factor receptor (EGFR)-mediated basal expression of integrin alpha2, cell adhesion and motility in highly progressed HCT116 colon cancer cells. We also reported that the expression of basal integrin alpha2 and its biological effects are critically controlled by the constitutive activation of the ERK/MAPK pathway (Sawhney, R. S., Sharma, B., Humphrey, L. E., and Brattain, M. G. (2003) J. Biol. Chem. 278, 19861-19869). In the present report, we further examine the downstream signaling mechanisms underlying EGFR/ERK signaling and integrin alpha2 function in HCT116 cells. Selective MEK inhibitors attenuated TGFalpha-mediated basal activation of p70S6K (S6K) specifically at Thr-389, indicating that this S6K site is downstream of ERK/MAPK signaling. Cells were treated with the selective protein kinase C (PKC) inhibitor bisindolylmaleimide to determine the role of PKC in S6K activation. The Thr-421 and Ser-424 phosphorylation sites of S6K were specifically inhibited by bisindolylmaleimide, which also blocked integrin alpha2 expression, cell adhesion, and motility. These data establish a novel cell motility function of S6K via PKC activation in a cancer cell. In addition, we examined whether mammalian target of rapamycin signaling controls S6K activation. Rapamycin inhibited constitutive S6K phosphorylation specifically at Thr-389, Thr-421, and Ser-424 sites. The assignment of these phosphorylation sites on S6K to biological functions was unequivocally confirmed by transfection of cells with specific single phosphorylation site dominant negative mutants. These experiments show for the first time that autocrine TGFalpha regulates cell adhesion function by multiple signaling pathways via specific phosphorylation sites of S6K in cancer cells.

Topics: Autocrine Communication; Binding Sites; Biotinylation; Blotting, Western; Cell Adhesion; Cell Culture Techniques; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation; Genes, Dominant; Humans; Immunoblotting; Indoles; Integrin alpha2; Maleimides; Mitogen-Activated Protein Kinase Kinases; Mutation; Phosphorylation; Plasmids; Protein Kinase C; Ribosomal Protein S6 Kinases, 70-kDa; Serine; Signal Transduction; Sirolimus; Threonine; Time Factors; Transfection; Transforming Growth Factor alpha

Serine proteases are now considered as crucial contributors to the development of human colon cancer. We have shown recently that thrombin is a potent growth factor for colon cancer cells through activation of the aberrantly expressed protease-activated receptor 1 (PAR1). Here, we analyzed the signaling pathways downstream of PAR1 activation, which lead to colon cancer cell proliferation in HT-29 cells. Our data are consistent with the following cascade of events on activation of PAR1 by thrombin or specific activating peptide: (a) a matrix metalloproteinase-dependent release of transforming growth factor-alpha (TGF-alpha) as shown with TGF-alpha blocking antibodies and measurement of TGF-alpha in culture medium; (b) TGF-alpha-mediated activation of epidermal growth factor receptor (EGFR) and subsequent EGFR phosphorylation; and (c) activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and subsequent cell proliferation. The links between these events are shown by the fact that stimulation of cell proliferation and ERK1/2 on activation of PAR1 is reversed by the MMP inhibitor batimastat, TGF-alpha neutralizing antibodies, EGFR ligand binding domain blocking antibodies, and the EGFR tyrosine kinase inhibitors AG1478 and PD168393. Therefore, transactivation of EGFR seems to be a major mechanism whereby activation of PAR1 results in colon cancer cell growth. Finally, PAR1 activation induces Src phosphorylation, which is reversed by using the Src tyrosine kinase inhibitor PP2, suggesting that Src activation plays a permissive role for PAR1-mediated ERK1/2 activation and cell proliferation probably acting downstream of the EGFR. These data explain how thrombin exerts robust trophic action on colon cancer cells and underline the critical role of EGFR transactivation.

Topics: Antibodies, Monoclonal; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Enzyme Activation; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Phosphorylation; Receptor, PAR-1; src-Family Kinases; Transcriptional Activation; Transforming Growth Factor alpha

Recently, we have shown that autocrine transforming growth factor-alpha (TGF-alpha) controls the expression of integrin alpha2, cell adhesion to collagen IV and motility in highly progressed HCT116 colon cancer cells (Sawhney, R. S., Zhou, G-H. K., Humphrey, L. E., Ghosh, P., Kreisberg, J. I., and Brattain, M. G. (2002) J. Biol. Chem. 277, 75-86). We now report that expression of basal integrin alpha2 and its biological effects are controlled by constitutive activation of the extracellular signal-regulated/mitogen-activated protein kinase (ERK/MAPK) pathway. Treatment of cells with selective mitogen-activated protein kinase kinase (MEK) inhibitors PD098059 and U0126 showed that integrin alpha2 expression, cell adhesion, and activation of ERK are inhibited in a parallel concentration-dependent fashion. Moreover, autocrine TGF-alpha-mediated epidermal growth factor receptor activation was shown to control the constitutive activation of the ERK/MAPK pathway, since neutralizing antibody to the epidermal growth factor receptor was able to block basal ERK activity. TGF-alpha antisense-transfected cells also showed attenuated activation of ERK. Using a real time electric cell impedance sensing technique, it was shown that ERK-dependent integrin alpha2-mediated cell micromotion signaling is controlled by autocrine TGF-alpha. Thus, this study implicates ERK/MAPK signaling activated by endogenous TGF-alpha as one of the mechanistic features controlling metastatic spread.

Topics: Butadienes; Chromones; Colonic Neoplasms; Enzyme Inhibitors; Flavonoids; Humans; Integrin alpha2; Mitogen-Activated Protein Kinases; Morpholines; Nitriles; Transforming Growth Factor alpha; Tumor Cells, Cultured

Trefoil peptides (TFFs) are now considered as scatter factors, proinvasive and angiogenic agents acting through cyclooxygenase-2 (COX-2)- and thromboxane A2 receptor (TXA2-R)-dependent signaling pathways. As expression and activation levels of the epidermal growth factor receptor (EGFR) predict the metastatic potential of human colorectal cancers, the purpose of this study was to establish whether the EGF receptor tyrosine kinase (EGFR-TK) contributes to cellular invasion induced by TFFs in kidney and colonic cancer cells. Both the dominant negative form of the EGFR (HER-CD533) and the EGFR-TK inhibitor ZD1839 (Iressa) abrogated cellular invasion induced by pS2, spasmolytic polypeptide (SP) and the src oncogene, but not by ITF and the TXA2-R. Similarly, EGFR-TK inhibition by ZD1839 reversed the invasive phenotype promoted by the constitutively activated form of the EGFR (EGFRvIII) and the EGFR agonists transforming growth factor alpha (TGFalpha), amphiregulin and EGF. We also provide evidence that TFFs, EGFRvIII, and TGFalpha trigger common proinvasive pathways using the PI3'-kinase and Rho/Rho- kinase cascades. These findings identify the EGFR-TK as a key signaling element for pS2- and SP-mediated cellular invasion. It is concluded that although pS2, SP and ITF belong to the same family of inflammation- and cancer-associated regulatory peptides, they do not control identical signaling networks.

Topics: Amphiregulin; Animals; Cells, Cultured; Colonic Neoplasms; Dogs; EGF Family of Proteins; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Genes, src; Glycoproteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Kidney; Mucins; Muscle Proteins; Mutation; Neoplasm Invasiveness; Neuropeptides; Peptides; Phosphatidylinositol 3-Kinases; Proteins; Quinazolines; Receptors, Thromboxane; Signal Transduction; Transforming Growth Factor alpha; Trefoil Factor-1; Trefoil Factor-2; Trefoil Factor-3; Tumor Suppressor Proteins

Growth factor independence is a hallmark of malignancy that is attributed to the development of autocrine growth factor loops in cancer cells. However, growth factor-dependent normal cells also exhibit autocrine activity, thus raising the issue of how endogenously produced activity in cancer cells differs in a manner that leads to growth factor independence. We have examined this issue by comparing growth factor-independent HCT116 human colon carcinoma cells with a growth factor-dependent subcompartment of malignant cells designated HCT116b that was isolated from the same patient tumor. Therefore, the development of the growth factor-independent phenotype represents clonal progression within the tumor in vivo. The growth factor independence of HCT116 cells was shown to be dependent on autocrine transforming growth factor (TGF)-alpha activity, yet the isoparental HCT116b subcompartment showed similar levels of TGF-alpha expression as HCT116 when cells were in exponential growth. When both cell lines were growth arrested by nutrient deprivation, HCT116b cells required nutrient replenishment and growth factors for reinitiation of DNA synthesis, whereas HCT116 cells required only nutrient replenishment. In contrast to growth factor-dependent HCT116b cells, the HCT116 cells showed up-regulation of TGF-alpha expression during growth arrest as a result of enhanced transcription. This increased TGF-alpha expression in quiescent HCT116 cells was associated with constitutive epidermal growth factor receptor (EGFR) activation in the growth-arrested state, whereas growth-arrested HCT116b cells did not show EGFR activation. TGF-alpha antisense transfection of HCT116 cells showed that EGFR activation was due to increased TGF-alpha expression. Pretreatment of growth-arrested HCT116 cells with AG1478, a selective inhibitor of EGFR tyrosine kinase activity, blocked the reinitiation of DNA synthesis, demonstrating that growth factor independence was due to the increased TGF-alpha expression and EGFR activation of these cells in growth arrest relative to growth factor-dependent HCT116b cells. Importantly, the level of EGFR activation in growth-arrested HCT116 cells was only slightly higher than that of exponential cells, indicating that it was inappropriate EGFR activation in growth arrest rather than the amplitude of activation that generated growth factor independence.

Topics: Animals; Cell Division; Colonic Neoplasms; Disease Progression; DNA, Neoplasm; ErbB Receptors; Growth Substances; Humans; Mice; Mice, Nude; Nuclear Proteins; Promoter Regions, Genetic; Protein Binding; RNA, Messenger; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured; Up-Regulation

Prostaglandin E(2) (PGE(2)), a major product of cyclooxygenase enzymes, is implicated in colorectal carcinogenesis and has been shown to stimulate the growth of human colorectal carcinoma cells. Here, we show that PGE(2) activated the cyclic AMP/protein kinase A pathway, which induced the expression of amphiregulin (AR), an epidermal growth factor family member, through activation of a cyclic AMP-responsive element in the AR promoter. AR exerted a mitogenic effect on LS-174 cells and partially mediated the PGE(2)-induced growth stimulation. In addition, PGE(2), in collaboration with transforming growth factor-alpha or K-Ras oncogene, synergistically induced AR expression and activated receptor tyrosine kinase-dependent signaling pathways. Our results provide novel mechanisms for cyclooxygenase-2 pro-oncogenic activity and suggest that PGE(2) may act with major oncogenic pathways in a synergistic fashion to activate the epidermal growth factor receptor signaling system through a ligand-dependent autocrine pathway.

Topics: Amphiregulin; Cell Division; Colonic Neoplasms; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclooxygenase 2; Dinoprostone; Drug Synergism; EGF Family of Proteins; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Isoenzymes; Membrane Proteins; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases; Signal Transduction; Transforming Growth Factor alpha; Tumor Cells, Cultured

The purpose of this study was to identify a mediator produced by human colon cancer cells that is responsible for the induction of hyperplasia in the adjacent mucosa.. Seventy human colon cancer surgical specimens were immunostained to determine the presence of cytokines that can induce hyperplasia in the adjacent mucosal. Human colon cancer cells with low and high metastatic potential were implanted into the cecal wall of nude mice. The resulting lesions were studied by immunohistochemistry to detect possible mediators of mucosal hyperplasia.. Immunostaining of 70 colon cancer specimens from 70 patients suggested that mucosal hyperplasia and distant metastasis were associated with the expression of interleukin (IL)-15 and, to a lesser extent, transforming growth factor alpha. The production of IL-15 by colon cancer cells was not associated with the infiltration of natural killer cells into the tumors. Cecal tumors produced in nude mice by human colon cancer cells with low and high metastatic potential (KM12C and KM12SM cells, respectively) expressed similar levels of transforming growth factor alpha, and expression of IL-15 was detected only in the metastatic KM12SM cells and was associated with hyperplasia of the surrounding mucosa. The expression of the IL-15 receptor in rat intestinal epithelial cells (IEC6 cells) was confirmed by immunoblotting with antibodies against IL-15 receptor alpha and IL-2 receptor beta and gamma subunits and by a binding assay using (125)I-labeled IL-15 (K(d) = 0.011 nM). IL-15 stimulated proliferation of the IEC6 cells, even under serum starvation. Treatment of IEC6 cells with IL-15 decreased doxorubicin-mediated cytotoxicity. In IEC6 cells treated with either IL-15- or KM12SM-conditioned medium, immunoblotting revealed a decrease in the production of p21Waf1, Bax, and Bak and an increase in the production of cyclin E, proliferating cell nuclear antigen, the phosphorylated active form of AKT, basic fibroblast growth factor, and vascular endothelial growth factor, changes associated with cell growth, survival, and induction of angiogenesis.. These data indicate that IL-15 produced by metastatic colon carcinoma cells can induce hyperplasia in the mucosa adjacent to colon cancer, thus contributing to angiogenesis and progression of the disease.

Topics: Animals; Cell Division; Cell Line; Cell Line, Tumor; Cells, Cultured; Colonic Neoplasms; Culture Media, Conditioned; Disease Progression; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Humans; Hyperplasia; Immunoblotting; Immunohistochemistry; Interleukin-15; Kinetics; Mice; Mice, Inbred BALB C; Mice, Nude; Mucous Membrane; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; Proliferating Cell Nuclear Antigen; Rats; Receptors, Interleukin-2; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Transforming Growth Factor alpha

Transforming growth factor alpha (TGF-alpha) has been localized in neuroendocrine L-cells of the colon and rectum in previous studies. We examined whether neuroendocrine tumours with L-cell differentiation express TGF-alpha.. Immunohistochemistry was performed for proglucagon- and pro-pancreatic polypeptide derivatives, as well as for TGF-alpha, and epidermal growth factor receptor (EGFR) using paraffin sections from 16 neuroendocrine tumours of the colon and rectum. Also, in situ hybridization for TGF-alpha and proglucagon was carried out.. A strong expression of TGF-alpha at the protein level can be shown for neuroendocrine tumours of the hindgut. In one third of our cases we found a strong hybridization signal and in two thirds a moderate signal for TGF-alpha. The immunohistological phenotype concerning gut hormones is highly heterogeneous. Glucagon-like peptide 2 (GLP2) in our series was the most sensitive immunohistological hormone marker.. The immunophenotype of colorectal neuroendocrine tumours regarding hormone markers is heterogeneous. The expression of TGF-alpha corresponds to the immunohistological profile of normal L-cells. TGF-alpha, especially in the neuroendocrine L-cells, most probably acts as a multifunctional trophic factor responsible for cellular integrity and survival, and not as an oncogenic growth factor.

Topics: Adult; Aged; Aged, 80 and over; Colonic Neoplasms; Enteroendocrine Cells; ErbB Receptors; Female; Gene Expression; Glucagon; Humans; Immunohistochemistry; In Situ Hybridization; Male; Middle Aged; Neuroendocrine Tumors; Pancreatic Polypeptide; Proglucagon; Protein Precursors; Rectal Neoplasms; Transforming Growth Factor alpha

LT97, a permanent cell line consisting of epithelial cells with an early premalignant genotype was established from small colorectal polyps. LT97 cells have lost both alleles of the APC tumour suppressor gene. In addition, they carry a mutated Ki-ras oncogene, while TP53 is normal. LT97 growth characteristics are thus representative of early adenomas. They had to be passaged as multicellular aggregates indicating a dependency of survival on cell-cell contact and in accordance with their premalignant genotype were not capable of growth in soft agar. LT97 cells did express both the EGF-receptor and small amounts of TGF(alpha) establishing an autocrine growth or survival pathway. However, in spite of autocrine TGF(alpha) production, growth was strongly dependent on exogenous growth factors--mainly EGF, insulin and HGF. Inhibition of the EGF-receptor kinase induced apoptosis at an IC(50) concentration of 4 micromolar indicating that TGF(alpha) activated survival pathways in the early adenoma cell.

Topics: Adenoma; Apoptosis; Cell Division; Colonic Neoplasms; Flow Cytometry; Genes, ras; Humans; Mutation; Precancerous Conditions; Transforming Growth Factor alpha; Tumor Cells, Cultured

The aim of this study was to determine whether constitutive ErbB2 activation controls growth and apoptosis in colon cancer cells. Growth arrested GEO cells showed constitutive activation of ErbB2 in the absence of exogenous growth factors or serum supplementation. Higher levels of heregulin and ErbB2 activation were observed in the growth-arrested state and cell cycle re-entry was independent of exogenous growth factors. Blockade of ErbB2 activation by heregulin neutralizing antibodies and by AG879 resulted in prevention of cell cycle re-entry. This indicated that autocrine heregulin activity was responsible for growth factor independence and for cell cycle re-entry. Activation of ErbB2 was the result of heregulin mediated interaction with ErbB3 and generated downstream activation of the ERK and the PI3K/AKT pathways. Heregulin neutralizing antibody treatment of growth arrested GEO cells also generated apoptosis as reflected by PARP cleavage and DNA fragmentation indicating a cell survival signal was also induced by the constitutively activated ErbB2. The activation of AKT but not the MAPK pathway was responsible for cell survival in these cells.

Topics: Adenocarcinoma; Apoptosis; Autocrine Communication; Cell Cycle; Chromones; Colonic Neoplasms; Culture Media; Culture Media, Serum-Free; Dimerization; DNA Fragmentation; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Gene Expression Regulation, Neoplastic; Genes, erbB-2; Growth Substances; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Morpholines; Neoplasm Proteins; Neuregulin-1; Neutralization Tests; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Poly(ADP-ribose) Polymerases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptor, ErbB-3; Signal Transduction; Sirolimus; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

The inappropriate expression of TGFalpha in growth arrest contributes to malignant progression in human colon carcinoma cells. Early stage, non-progressed colon tumor cells show a down-regulation of TGFalpha in growth arrest and require both nutrients and growth factors for re-entry into the cell cycle. In contrast, highly progressed cells up-regulate TGFalpha during growth arrest and require only nutrients for re-entry. Given the importance of TGFalpha in malignant progression, this work addressed the regulation of TGFalpha expression in the early stage colon carcinoma cell line, FET. Growth-arrested FET cells down-regulated the expression of TGFalpha, EGFr and, in turn, EGFr activation. These quiescent cells continued to express high levels of IGF-IR protein, but IGF-IR activation was undetectable. Cell cycle re-entry required exogenous growth factor activation of the IGF-IR by insulin or IGF-I. This IGF-IR activation resulted in S phase re-entry and was accompanied by an approximate threefold induction of TGFalpha expression along with EGFr activation at 1 h following release from growth arrest. Activation of IGF-IR occurred within 5 min of cell-cycle re-entry. Previously identified DNA binding proteins which bind to a unique TGFalpha/EGF response element within the TGFalpha promoter were similarly induced following IGF-IR activation. The addition of EGFr neutralizing antibodies abolished the activated IGF-IR stimulated S phase re-entry. Moreover, disruption of the growth arrest associated down-regulation of TGFalpha in FET cells by constitutive TGFalpha expression abrogated the requirement for IGF-IR activation for cell cycle re-entry. Consequently, this study indicates, for the first time, that IGF-IR activation up-regulates components of the TGFalpha autocrine loop resulting in TGFalpha-mediated EGFr activation which was critical for IGF-IR mediated re-entry into the cell cycle from the growth-arrested state.

Topics: Cell Division; Colonic Neoplasms; DNA; ErbB Receptors; Gene Expression; Humans; Insulin; Insulin-Like Growth Factor I; Receptor, IGF Type 1; S Phase; Signal Transduction; Transforming Growth Factor alpha; Tumor Cells, Cultured

Among the proteins of the epidermal growth factor family, transforming growth factor-alpha (TGF-alpha) may be an especially reliable indicator of metastasis or prognosis in human colorectal carcinomas. Moreover, anomalous forms of TGF-alpha have been detected in several tissues of cancer origin, suggesting a role of these forms in the development of the disease. This study was designed to identify the presence of TGF-alpha precursors in different colon cancer cell lines by mean of immunocytochemistry and western blotting techniques. Pro-TGF-alpha was detected in all cell lines tested. Staining for pro-TGF-alpha was observed in cytoplasm. Monoclonal antibody to TGF-alpha detected two bands of 20 and 21 kDa. Polyclonal antibody to pro-TGF-alpha revealed five bands ranging from 15 to 24 kDa. All these proteins were also detected in nonmalignant cells expressing a transfected rat pro-TGF-alpha gene. In conclusions, transformation in these human colon carcinoma cells is not due to the presence of anomalous forms of TGF-alpha precursors.

Topics: Adenocarcinoma; Colonic Neoplasms; Growth Substances; Humans; Immunoblotting; Immunohistochemistry; Lymphatic Metastasis; Protein Precursors; Transforming Growth Factor alpha; Tumor Cells, Cultured

Transforming growth factor alpha (TGFalpha) is widely expressed in malignant as well as normal cells and is involved in regulating cell growth and differentiation. Although processing of TGFalpha has been extensively studied in normal cells, there is little information regarding TGFalpha cleavage in malignant cells. Therefore, we compared the processing of TGFalpha in two human colon carcinoma cell lines. We found that there was a defective cleavage pattern for the TGFalpha precursor resulting in retention of partially processed TGFalpha on the cell surface of both the HCT116a2alphaS3 and CBS4alphaS2 cell lines. This raised the possibility that signaling from the resulting defective cleavage species could differ from that of soluble TGFalpha. The membrane-associated TGFalpha induced higher phosphorylation of EGFR on the cell surface of adjacent cells than equivalent levels of mature TGFalpha. The interaction of membrane bound TGFalpha precursor with the EGFR caused a slower internalization of activated EGFR relative to the internalization of the soluble TGFalpha/EGFR complexes. In addition, the tethered TGFalpha was resistant to the ability of protein-tyrosine phosphatases (PTPs) to reduce EGFR tyrosine phosphorylation, also contributing to higher activation of EGFR. The enhanced activation of EGFR by the tethered form of TGFalpha was reflected by higher activation of Grb2, SHC and Erk downstream mediators of EGF receptor signaling. The higher activation of EGFR by membrane tethered TGFalpha indicates that defective TGFalpha processing provides a mechanism whereby malignant cells can obtain a growth advantage over normal cells.

Topics: Cell Membrane; Colonic Neoplasms; ErbB Receptors; Humans; Mitogen-Activated Protein Kinase Kinases; Neutralization Tests; Phosphorylation; Protein Precursors; Protein Processing, Post-Translational; Protein Tyrosine Phosphatases; Signal Transduction; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

Angiogenesis plays a key role in tumor growth and metastasis. The transforming growth factor alpha (TGF-alpha)-epidermal growth factor receptor (EGFR) autocrine pathway controls in part the production of angiogenic factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in cancer cells. In this study, we have evaluated the antiangiogenic and antitumor activity of monoclonal antibody (MAb) C225, an anti-EGFR chimeric human-mouse MAb, alone and in combination with a human VEGF antisense (AS) 21-mer phosphorothioate oligonucleotide (VEGF-AS) in human GEO colon cancer cells. MAb C225 treatment determined a dose-dependent inhibition of VEGF, bFGF, and TGF-alpha production by GEO cells in vitro. Treatment with VEGF-AS caused a selective inhibition in VEGF expression by GEO cells in vitro. Treatment of immunodeficient mice bearing established, palpable GEO xenografts for 3 weeks with VEGF-AS or with MAb C225 determined a cytostatic reversible inhibition of tumor growth. In contrast, a prolonged inhibition of tumor growth was observed in all mice treated with the two agents, in combination with a significant improvement in mice survival compared with controls (P < .001), to MAb C225 (P < .001), or to VEGF-AS (P < .001) treated mice. All mice died within 4, 6, and 8 weeks after tumor cell injection in the control, VEGF-AS and MAb C225 groups, respectively. In contrast, 50% of mice treated with the combination of VEGF-AS and MAb C225 were alive at 13 weeks. Ten % of mice treated with VEGF-AS plus MAb C225 were alive at 20 weeks and had no histological evidence of GEO tumors. Immunohistochemical analysis of GEO tumor xenografts demonstrated a significant reduction of VEGF expression after treatment with VEGF-AS with a parallel reduction in microvessel count. MAb C225 treatment determined a reduction in the expression of VEGF, bFGF, and TGF-alpha with a reduction in microvessel count. Finally, a significant potentiation in inhibition of VEGF expression and little or no microvessels were observed in GEO tumors after the combined treatment with the two agents.

Topics: Animals; Antibodies, Monoclonal; Blotting, Western; Colonic Neoplasms; Dose-Response Relationship, Drug; Endothelial Growth Factors; ErbB Receptors; Female; Fibroblast Growth Factor 2; Humans; Immunohistochemistry; Lymphokines; Mice; Mice, Inbred BALB C; Mice, Nude; Neovascularization, Pathologic; Oligonucleotides, Antisense; Thionucleotides; Transforming Growth Factor alpha; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Xenograft Model Antitumor Assays

A majority of human colon carcinomas coexpress the epidermal growth factor (EGF)-related peptides transforming growth factor alpha (TGFalpha), amphiregulin (AR) and CRIPTO-1 (CR). We have synthesized novel, antisense mixed backbone oligonucleotides (AS MBOs) directed against TGFalpha, AR and CR. We screened the EGF-related AS MBOs for their ability to inhibit the anchorage independent growth of GEO human colon carcinoma cells. The MBOs that showed a high in vitro efficacy were then used for in vivo experiments. TGFalpha, AR and CR AS MBOs were able to inhibit the growth of GEO tumor xenografts in nude mice in a dose-dependent manner. Furthermore, the AS MBOs were able to specifically inhibit the expression of the target mRNAs and proteins in the tumor xenografts. A more significant tumor growth inhibition was observed when mice were treated with a combination of the three AS MBOs as compared to treatment with a single AS MBO. Finally, tumors from mice treated with TGFalpha, AR and CR AS MBOs showed a significant reduction of microvessel count, as compared with tumors from untreated mice or from mice treated with a single AS MBO. These data suggest that combinations of AS oligonucleotides directed against different growth factors might represent a novel, experimental therapy approach of colon carcinomas.

Topics: Amphiregulin; Animals; Colonic Neoplasms; EGF Family of Proteins; Epidermal Growth Factor; Glycoproteins; GPI-Linked Proteins; Growth Inhibitors; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Mice; Mice, Nude; Neoplasm Proteins; Neovascularization, Pathologic; Oligonucleotides, Antisense; Thionucleotides; Transforming Growth Factor alpha; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

To determine whether colon crypt proliferative parameters were significantly altered by the stage of colon carcinogenesis or the type or location of colon tumors in rats, male Sprague-Dawley rats received an injection of the carcinogen 1,2-dimethylhydrazine (12 mg DMH base/kg body weight) or DMH vehicle once a week for 8 weeks, then were killed 24 weeks later. Three hours before sacrifice, rats were injected with 1 mg/kg body weight colchicine to arrest mitotic cells at metaphase. Transverse sections of the colon mucosa were taken 6 cm from the anus and at least 3 cm from any tumor, fixed in formalin, then stained with hematoxylin & eosin (H&E) for analyses of proliferative parameters. Only complete, mid-axial crypts were scored for mitotic count (MC), crypt proliferative zone (PZ) height and crypt height (CH). Serial tumor sections were stained with H&E for histological evaluation or used in immunohistochemical detection of transforming growth factor alpha (TGF alpha). DMH treatment significantly increased MC, PZ and CH regardless of tumor status. The PZ and CH of rats with a carcinoma located in the distal colon were significantly increased compared with DMH-treated rats without an adenocarcinoma (AC) or with rats which had a tumor located in the proximal colon. Distal colon ACs were found to be well differentiated and to have greater TGF alpha immunoreactivity than the generally less differentiated proximal colon carcinomas. Distal colon AC production and systemic circulation of a soluble colon crypt stimulating factor such as TGF alpha may explain the significant increase in PZ and CH in histologically normal colonic mucosa located away from the tumor.

Topics: 1,2-Dimethylhydrazine; Adenocarcinoma; Animals; Carcinogens; Cell Division; Cell Size; Colon; Colonic Neoplasms; Intestinal Mucosa; Male; Mitotic Index; Rats; Rats, Sprague-Dawley; Transforming Growth Factor alpha

Transforming growth factor-alpha (TGF-alpha) is synthesized as a membrane-bound precursor protein, pro-TGF-alpha, that is converted to a soluble form by 2 endoproteolytic cleavages. Several factors have been implicated in the regulation of the second rate-limiting step, including protein kinase C (PKC). Earlier results indicated a potential role for the conventional class of PKC isozymes in the observed increase in TGF-alpha in the conditioned media of 2 human colon carcinoma cell lines. The present study addresses the potential role of specific PKC isozymes in this process using sense and anti-sense expression vectors for PKC isozymes. Two human colon carcinoma cell lines, HCT 116 and GEO, were transfected with plasmids, leading to the over-expression of PKC-alpha, -betaI or -betaII; and the secretion of TGF-alpha into the conditioned medium was determined. Over-expression of either PKC-betaI or PKC-betaII in these cell lines enhanced the levels of TGF-alpha in the media 2- to 5-fold. Over-expression of PKC-alpha did not alter the amount of TGF-alpha in the media to a significant extent. Transfection of HCT 116 cells with the anti-sense PKC-betaI cDNA resulted in a reduction in PKC-betaI protein expression. This was accompanied by a decrease in the amount of TGF-alpha in the conditioned media. Our results indicate that modulation of PKC-beta protein levels alters the amount of TGF-alpha found in the conditioned media from these colon carcinoma cells.

Topics: Colonic Neoplasms; Culture Media, Conditioned; Gene Expression Regulation, Neoplastic; Humans; Isoenzymes; Protein Kinase C; Protein Kinase C beta; Protein Kinase C-alpha; Recombinant Proteins; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

Colonic enterocytes, like many epithelial cells in vivo, are polarized with functionally distinct apical and basolateral membrane domains. The aims of this study were to characterize the endogenous epidermal growth factor (EGF)-like ligands expressed in two polarizing colon cancer cell lines, HCA-7 Colony 29 (HCA-7) and Caco-2, and to examine the effects of cell polarity on EGF receptor-mediated mitogenesis. HCA-7 and Caco-2 cells were grown on plastic, or as a polarized monolayer on Transwell filters. Cell proliferation was measured by 3H-thymidine incorporation and EGF receptor (EGFR) binding was assessed by Scatchard analysis. EGFR ligand expression was determined by Northern blot analysis, reverse transcription polymerase chain reaction, metabolic labelling and confocal microscopy. We found that amphiregulin (AR) was the most abundant EGFR ligand expressed in HCA-7 and Caco-2 cells. AR was localized to the basolateral surface and detected in basolateral-conditioned medium. Basolateral administration of neutralizing AR antibodies significantly reduced basal DNA replication. A single class of high-affinity EGFRs was detected in the basolateral compartment, whereas the apical compartment of polarized cells, and cells cultured on plastic, displayed two classes of receptor affinity. Basolateral administration of transforming growth factor alpha (TGF-alpha) or an EGFR neutralizing antibody also resulted in a dose-dependent stimulation or attenuation, respectively, of DNA replication. However, no mitogenic response was observed when these agents were added to the apical compartment or to confluent cells cultured on plastic. We conclude that amphiregulin acts as an autocrine growth factor in HCA-7 and Caco-2 cells, and EGFR ligand-induced proliferation is influenced by cellular polarity.

Topics: Amphiregulin; Antibodies, Monoclonal; Antineoplastic Agents; Autocrine Communication; Caco-2 Cells; Cell Division; Cell Polarity; Colonic Neoplasms; DNA; DNA Replication; EGF Family of Proteins; ErbB Receptors; Glycoproteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

Autocrine transforming growth factor alpha (TGFalpha) activity and control mechanisms for its expression were examined in a representative clonal isolate (CBS4) of a well-differentiated human colon carcinoma cell line designated CBS. CBS4 cells expressed TGFalpha and its receptor, epidermal growth factor receptor (EGFr). Blockade of EGFr and TGFalpha by neutralizing antibodies inhibited clonal growth and the initiation of DNA synthesis from quiescence in CBS4 cells. Therefore, TGFalpha is an autocrine growth factor for CBS4 cells. Several studies have indicated that activation of the EGFr by exogenous EGF stimulates TGFalpha expression. However, in CBS4 cells EGF did not induce TGFalpha mRNA expression, indicating that EGF does not affect TGFalpha transcription in these cells. Exogenous treatment of exponentially growing cells with either EGF or EGFr blocking antibody enhanced release of TGFalpha protein into the conditioned medium. This indicated that the release of TGFalpha into the conditioned medium by exogenous EGF was at least partially due to the displacement of TGFalpha from the TGFalpha/EGFr complexes. Similarly to exponentially growing cells, the EGFr blocking antibody and EGF also enhanced TGFalpha release into the medium of CBS4 cells after release from quiescence. These results indicated that exogenous EGF had little if any effect on TGFalpha expression in these cells and suggested that TGFalpha expression might be under endogenous TGFalpha control. Blockade of the autocrine TGFalpha loop by TGFalpha neutralizing antibody suppressed TGFalpha mRNA both in exponentially growing and quiescent cells, demonstrating that autocrine TGFalpha is autoregulatory in this system.

Topics: Antibodies; Cell Division; Clone Cells; Colonic Neoplasms; DNA Replication; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

Autocrine transforming growth factor alpha (TGFalpha) is an important positive growth effector in malignant cells and plays a significant role in generating the growth factor-independent phenotype associated with malignant progression. However, the molecular mechanisms by which TGFalpha confers a growth advantage in progression is poorly understood. The highly tumorigenic cell line HCT116 up-regulates TGFalpha mRNA expression during growth arrest, whereas the poorly tumorigenic growth factor-dependent FET cell line down-regulates TGFalpha mRNA expression as it becomes quiescent. We have identified a 25-bp sequence at -201 to -225 within the TGFalpha promoter which mediates the differential regulation of TGFalpha expression during quiescence establishment in these two cell lines. This same sequence confers TGFalpha promoter responsiveness to exogenous growth factor or autocrine TGFalpha. The abberant upregulation of TGFalpha mRNA in quiescent HCT116 cells may allow them to return to the dividing state under more stringent conditions (nutrient replenishment alone) then quiescent FET cells (requires nutrients and growth factors). Antisense TGFalpha approaches showed that the dysregulated TGFalpha expression in quiescent HCT116 cells is a function of the strong TGFalpha autocrine loop (not inhibited by blocking antibodies) in these cells.

Topics: Base Sequence; Cell Cycle; Cell Division; Cell Line; Chloramphenicol O-Acetyltransferase; Clone Cells; Cloning, Molecular; Colonic Neoplasms; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Genomic Library; Humans; Insulin; Kinetics; Leukocytes; Molecular Sequence Data; Phenotype; Promoter Regions, Genetic; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; RNA, Messenger; Transcription, Genetic; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

To evaluate the association among known angiogenic growth factors or factors related to the plasminogen activation system and clinicopathological factors in patients with colorectal cancer, we examined the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor-alpha (TGF-alpha), urokinase-type plasminogen activator (u-PA), u-PA receptor (u-PA-R) and plasminogen activator inhibitor-1 (PAI-1) in clinical specimens of colorectal cancers by Northern blot analysis. In comparison with the expression of these angiogenesis-related genes in 7 paired samples of colorectal cancers and the adjacent normal mucosa, VEGF mRNA level was significantly higher in the cancer tissues than in the adjacent normal mucosa (p < 0.05). We analyzed expression of these genes in 44 cases of primary colorectal cancers. Among the 3 angiogenic growth factors we examined, VEGF mRNA expression was significantly higher in the cancer tissues with blood vessel invasion or with lymphatic vessel invasion than in those without, respectively (p < 0.05). On the other hand, u-PA-R mRNA expression was significantly higher in the cancers with blood vessel invasion than in those without (p < 0.05). In addition, there was a correlation between the expression levels of VEGF and u-PA-R mRNA in the cancer tissues we have examined. Using immunohistochemistry, strong staining of VEGF or u-PA-R was observed in the cancer cells invading the microvessels. Our findings suggest that malignant transformation might accompany the upregulation of VEGF expression in colorectal cancers and that VEGF and u-PA-R might contribute cooperatively to increase angiogenesis around the tumor as well as the metastasis via microvessels.

Topics: Adult; Aged; Aged, 80 and over; Cell Line; Colon; Colonic Neoplasms; Colorectal Neoplasms; DNA Probes; Endothelial Growth Factors; Female; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Intestinal Mucosa; Lymphokines; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor alpha; Urokinase-Type Plasminogen Activator; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

To determine whether the expression of transforming growth factor alpha (TGF-alpha), its receptor (epidermal growth factor receptor [EGFr]), p53 nuclear protein, and proliferation influences prognosis of patients with liver metastases, a study was performed in 45 liver metastases and 33 corresponding primary colorectal carcinomas in patients referred for liver surgery. The expression of TGF-alpha, EGFr, p53 nuclear protein, and proliferation rate was correlated with clinicopathological characteristics and survival after partial liver resection. In liver metastases, TGF-alpha expression was low in 42%, intermediate in 35%, and high in 23%. TGF-alpha expression was higher in liver metastases derived from lymph node-positive primary carcinomas, in synchronous and in irresectable liver metastases compared with those derived from lymph node-negative primary carcinomas, metachronous, and resectable liver metastases. Nuclear p53 expression was found in 83% of primary tumors and 71% of liver metastases. p53 expression did not correlate with the various clinicopathological characteristics. Ki67 expression was not associated with clinicopathological characteristics in primary and metastatic tumors. In the 38 patients in whom a partial liver resection was performed, median survival was 25 months in patients with a higher TGF-alpha expression in the metastasis than in the primary tumor and 60 months in patients with comparable or lower TGF-alpha expression in the metastasis than in the primary tumor (P = .036). Median survival after liver resection was 21 months in patients with p53-negative liver metastases and 58 months in patients with p53-positive metastases (P = .043). By multivariate analysis, p53 and EGFr expression on liver metastases were the best predictors of disease-free survival after partial liver resection, with relative risks of 2.38 and 3.33, respectively. In patients with colorectal liver metastases, referred for liver surgery, a higher TGF-alpha expression is associated with unfavorable tumor characteristics, whereas p53 and absence of EGFr expression is associated with a better survival after partial liver resection.

Topics: Adenocarcinoma; Adult; Aged; Cell Division; Cell Nucleus; Colonic Neoplasms; Colorectal Neoplasms; Disease-Free Survival; ErbB Receptors; Female; Follow-Up Studies; Humans; Ki-67 Antigen; Liver Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Prognosis; Survival Analysis; Time Factors; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

Matrix metalloproteinases (MMPs) and growth factors such as hepatocyte growth factor (HGF) are implicated in tumoral progression of several digestive cancers. The rat DHD/K12 colonic cancer cell line is very invasive in vivo. We showed by RT-PCR and western immunoblotting the presence of HGF receptor, c-Met, in DHD/K12 cells. Then, we detected by zymography and western blots the secretion of MMP-2 and MMP-9 in the conditioned medium of these cells. After 24 or 48 h of culture in medium supplemented with HGF, transforming growth factor-alpha (TGF-alpha) or sodium butyrate, MMP production by DHD/K12 cells was stimulated by HGF and TGF-alpha and inhibited by sodium butyrate. Knowing the capacity of MMPs to degrade the extracellular matrix and thus to favour tumoral invasion, results suggest that HGF is implicated in the aggressive behaviour of DHD/K12 cells since it increased MMPs secretion by these cells.

Topics: Animals; Butyrates; Colonic Neoplasms; Extracellular Matrix; Hepatocyte Growth Factor; Metalloendopeptidases; Neoplasm Invasiveness; Proto-Oncogene Proteins c-met; Rats; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor alpha; Tumor Cells, Cultured

There is strong evidence that selenium protects against certain human cancers, but the underlying mechanism is unknown. Glutathione peroxidase (GPX1) and thioredoxin reductase (TR), the most abundant antioxidant selenium-containing proteins in mammals, have been implicated in this protection. We analyzed the expression of TR and GPX1 in the following model cancer systems: (1) liver tumors in TGFalpha/c-myc transgenic mice; (2) human prostate cell lines from normal and cancer tissues; and (3) p53-induced apoptosis in a human colon cancer cell line. TR was induced while GPX1 was repressed in malignancies relative to controls in transgenic mice and prostate cell lines. In the colon cell line, p53 expression resulted in elevated GPX1, but repressed TR. The data indicate that TR and GPX1 are regulated in a contrasting manner in the cancer systems tested and reveal the p53-dependent regulation of selenoprotein expression. The data suggest that additional studies on selenoprotein regulation in different cancers are required to evaluate future implementation of selenium as a dietary supplement in individuals at risk for developing certain cancers.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Line; Colonic Neoplasms; Enzyme Induction; Epithelial Cells; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Genes, myc; Glutathione Peroxidase; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred CBA; Mice, Transgenic; Prostate; Prostatic Neoplasms; Proto-Oncogene Proteins c-myc; Thioredoxin-Disulfide Reductase; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Previously, we reported that unaggressive, growth factor-dependent FET human colon carcinoma cells downregulated their transforming growth factor alpha (TGFalpha) expression in a quiescent state (G0/G1) induced by growth factor and nutrient deprivation (Mulder, 1991, Cancer Res., 51:2256-2262). In contrast, highly aggressive, growth factor-independent HCT116 human colon carcinoma cells aberrantly upregulated this autocrine activity in the quiescent state (Mulder, 1991, Cancer Res., 51:2256-2262; Howell et al., 1998, Mol. Cell. Biol., 18:303-313). In this report, the role of autocrine TGFalpha and the mechanism of its regulation of expression during reentry into the cell cycle from a noncycling growth state were determined in FET cells. Optimal induction of DNA synthesis from a quiescent state in FET cells is dependent upon autocrine TGFalpha as well as exogenous transferrin and insulin. Reentry into the cell cycle resulting from treatment with exogenous transferrin and insulin resulted in approximately 3-fold induction of TGFalpha expression within 1 hr. TGFalpha induction was controlled at the transcription level, and the cis-controlling element was localized to the region between bp -370 - -201 relative to the translation start codon within the TGFalpha promoter. Thus neutralization of autocrine TGFalpha protein revealed that the induced TGFalpha autocrine activity was necessary for DNA synthesis and acted only in the early G1 phase of the cell cycle. Blockade of autocrine TGFalpha expression early in the cell cycle resulted in the reduction of DNA synthesis, whereas treatment with neutralization antibody at later times had no effect. This suggested that autocrine TGFalpha functions to initiate cell growth from noncycling states. This was further confirmed by the dependence of FET cells upon autocrine TGFalpha for colony formation in experiments where the plating density was sufficiently low to generate a lag phase in tissue culture. In contrast, TGFalpha autocrine activity was not required for exponential phase cells, as evidenced by the failure of TGFalpha neutralizing antibody to inhibit proliferation in this growth state. Taken together, these results suggest that autocrine TGFalpha acts primarily in the process of growth initiation by moving cells from a noncycling state back into the cell cycle, rather than supporting cell growth already initiated.

Topics: Autocrine Communication; Carcinoma; Cell Cycle; Cell Division; Colonic Neoplasms; DNA; G1 Phase; Humans; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured

CBS human colon carcinoma cells are poorly tumorigenic in athymic nude mice, whereas FET colon carcinoma cells are non-tumorigenic. Both cell lines have well differentiated properties in tissue culture. Transforming growth factor alpha (TGF-alpha) was ectopically expressed by stable transfection of a TGF-alpha cDNA under repressible tetracycline control. The TGF-alpha-transfected cells showed enhanced clonal initiation and shortened lag phase growth in tissue culture without an alteration in doubling time in exponential phase relative to untransfected cells. Furthermore, the TGF-alpha transfectants showed increased independence from exogenous growth factors in clonal growth assays and induction of DNA synthesis after release from quiescence. Growth factor independence was associated with sustained epidermal growth factor receptor activation in quiescent TGF-alpha-transfected cells and the requirement of exogenous insulin for stimulation of quiescent cells to re-enter the cell cycle. Higher cloning, reduced lag time in tissue, and the acquisition of growth factor independence for DNA synthesis without a change in doubling time of TGF-alpha-transfected cells indicate that autocrine TGF-alpha functions by facilitating re-entry into the cell cycle from sub-optimal growth states rather than promoting or controlling the proliferation of actively cycling cells. The modulation of growth regulation by autocrine TGF-alpha was associated with increased malignant properties as TGF-alpha transfectants showed increased tumorigenicity in athymic nude mice. The administration of tetracycline reversed the effects of TGF-alpha expression in these cells both in vivo and in vitro, indicating that the alterations of the biological properties were due to the expression of TGF-alpha. Since these cells are continuously grown in a completely chemically defined medium without serum supplementation, it was possible to assign the mechanism underlying the generation of growth factor independence to the replacement of a requirement for exogenous insulin in parental cells by autocrine TGF-alpha.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Animals; Autocrine Communication; Carcinoma; Cell Adhesion; Cell Cycle; Cell Transformation, Neoplastic; Colonic Neoplasms; ErbB Receptors; Humans; Mice; Mice, Nude; Mitogens; Neoplasms, Experimental; Proteins; Recombinant Proteins; Shc Signaling Adaptor Proteins; Src Homology 2 Domain-Containing, Transforming Protein 1; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

Many studies have been conducted to assess the potential preneoplastic nature of colonic aberrant crypt foci (ACF), but still the biological significance of these foci and their relationship to colon neoplasia remains to be elucidated. In the present paper a battery of variables suggested to be indicative for colon cancer development has been studied in relation to ACF in rats. These include: (i) the degree of dysplasia; (ii) the type of mucus production; (iii) the cellular immunohistochemical expression and distribution of transforming growth factors alpha and beta and their respective receptors, epidermal growth factor receptor and transforming growth factor beta receptors I and II and phosphorylated cellular tyrosine. The parameters have been investigated in ACF selected from a previous study where the foci were induced under different circumstances, leading to disparities in the number as well as the crypt multiplicity obtained. The present study showed that for all parameters investigated, apart from sialomucin production, the different experimental conditions had no effect on the individual ACF, irrespective of the number and distribution of the different categories of ACF among the various diets. However, it was shown that the degree of dysplasia correlated strongly with crypt multiplicity and that all the investigated ACF lacked expression of transforming growth factor alpha and expressed a reduced amount of transforming growth factor beta compared with normal crypts. These observations may indicate that ACF are preneoplastic lesions and supports the suggestion that they may, at least in the rat, have the potential to gradually progress to tumors, but no single ACF showed particular characteristics indicating specific proneness to tumor development. The study could not confirm the presence of sialomucin-producing ACF as a valid marker for tumor development.

Topics: Animals; Cell Transformation, Neoplastic; Colon; Colonic Neoplasms; Dietary Fats; Dietary Fiber; Dietary Sucrose; ErbB Receptors; Gene Expression Regulation; Growth Substances; Intestinal Mucosa; Male; Mucins; Precancerous Conditions; Rats; Receptors, Growth Factor; Receptors, Transforming Growth Factor beta; Sialomucins; Transforming Growth Factor alpha; Transforming Growth Factor beta

We have demonstrated that anti-sense phosphorothioate oligodeoxynucleotides (AS S-oligos) directed against the EGF-like growth factors CRIPTO (CR), amphiregulin (AR) or transforming-growth-factor-alpha(TGFalpha) mRNA, are equipotent in their ability to inhibit the growth of human colon-carcinoma GEO cells. In this study, we evaluated the effect of combinations of these AS S-oligos and conventional anti tumor drugs, such as 5-fluorouracil (5-FU), adriamycin (ADR), mitomycin C (MIT) and cis-platinum (CDDP), on GEO cell growth. Dose-dependent growth inhibition was observed by treatment either with AS S-oligos or with anti-tumor drugs, using a clonogenic assay. Furthermore, an additive growth inhibitory effect occurred when GEO cells were exposed to the AS S-oligos after treatment with different concentrations of either 5-FU, MIT, ADR or CDDP. For example, treatment of GEO cells with a combination of low concentrations of 5-FU and any of the 3 AS S-oligos resulted in up to 70% growth inhibition. However, treatment of GEO cells with AS S-oligos before exposure to 5-FU or CDDP resulted in reduced efficacy of both drugs. Flow-cytometric analysis of DNA content demonstrated that treatment with the AS S-oligos caused a slight reduction of the percentage of cells in the S-phase of the cell cycle. These data suggest that combinations of AS S-oligos directed against EGF-related growth factors and of conventional anti-tumor drugs may result in efficient inhibition of colon-carcinoma cell growth.

Topics: Amphiregulin; Antineoplastic Agents; Cell Division; Cisplatin; Colonic Neoplasms; DNA, Neoplasm; Doxorubicin; Drug Combinations; Drug Synergism; EGF Family of Proteins; Epidermal Growth Factor; Flow Cytometry; Fluorouracil; Glycoproteins; GPI-Linked Proteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Mitomycin; Neoplasm Proteins; Oligonucleotides, Antisense; RNA, Messenger; Thionucleotides; Transforming Growth Factor alpha; Tumor Cells, Cultured

Growth factor receptor-signaling pathways are potentially important targets for anticancer therapy. The interaction of anticancer agents with specific molecular targets can be identified by correlating target expression patterns with cytotoxicity patterns. We sought to identify new agents that target and inhibit the activity of the epidermal growth factor (EGF) receptor and of c-erbB2 (also called HER2 or neu), by correlating EGF receptor, transforming growth factor (TGF)-alpha (a ligand for EGF receptor), and c-erbB2 messenger RNA (mRNA) expression levels with the results of cytotoxicity assays of the 49000 compounds in the National Cancer Institute (NCI) drug screen database.. The levels of mRNAs were measured and used to generate a molecular target database for the 60 cell lines of the NCI anticancer drug screen. The computer analysis program, COMPARE, was used to search for cytotoxicity patterns in the NCI drug screen database that were highly correlated with EGF receptor, TGF-alpha, or c-erbB2 mRNA expression patterns. The putative EGF receptor-inhibiting compounds were tested for effects on basal tyrosine phosphorylation, in vitro EGF receptor tyrosine kinase activity, and EGF-dependent growth. Putative ErbB2-inhibiting compounds were tested for effects on antibody-induced ErbB2 tyrosine kinase activity.. EGF receptor mRNA and TGF-alpha mRNA levels were highest in cell lines derived from renal cancers, and c-erbB2 mRNA levels were highest in cells derived from breast, ovarian, and colon cancers. Twenty-five compounds with high correlation coefficients (for cytotoxicity and levels of the measured mRNAs) were tested as inhibitors of the EGF receptor or c-erbB2 signaling pathways; 14 compounds were identified as inhibitors of these pathways. The most potent compound, B4, inhibited autophosphorylation (which occurs following activation) of ErbB2 by 50% in whole cells at 7.7 microM.. Novel EGF receptor or c-erbB2 pathway inhibitors can be identified in the NCI drug screen by correlation of cytotoxicity patterns with EGF receptor or c-erbB2 mRNA expression levels.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Division; Cell Line; Cluster Analysis; Colonic Neoplasms; Drug Screening Assays, Antitumor; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Ovarian Neoplasms; Receptor, ErbB-2; RNA, Messenger; Structure-Activity Relationship; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured

8-Chloro-cyclic AMP (8-Cl-cAMP) is a cAMP analogue that specifically down-regulates type I protein kinase A, a signaling protein directly involved in cell proliferation and neoplastic transformation, and that causes growth inhibition in a variety of human cancer cell types. In this report, we have investigated the effects of 8-Cl-cAMP on the expression of several growth factors in human colon (GEO and LS174T) and breast (MDA-MB468) cancer cell lines. 8-Cl-cAMP treatment caused in the three cancer cell lines a significant dose- and time-dependent inhibition in the expression of various endogenous autocrine growth factors, such as transforming growth factor alpha, amphiregulin, and CRIPTO, and of two angiogenic factors, such as vascular endothelial growth factor and basic fibroblast growth factor, at both the mRNA and protein levels. Furthermore, 8-Cl-cAMP treatment markedly inhibited the ability of all three cell lines to invade a basement membrane matrix in a chemoinvasion assay. Finally, 8-Cl-cAMP-induced inhibition of GEO tumor growth in nude mice was accompanied by a significant suppression of transforming growth factor alpha, amphiregulin, CRIPTO, basic fibroblast growth factor, and vascular endothelial growth factor production by the tumor cells, and of neoangiogenesis, as detected by factor VIII staining of host blood cells. These results demonstrate that 8-Cl-cAMP is a novel anticancer drug that inhibits the production of various autocrine and paracrine tumor growth factors that are important in sustaining autonomous local growth and facilitate invasion and metastasis.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Amphiregulin; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Division; Colonic Neoplasms; Colorectal Neoplasms; EGF Family of Proteins; Endothelial Growth Factors; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Glycoproteins; GPI-Linked Proteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Lymphokines; Membrane Glycoproteins; Mice; Mice, Nude; Neoplasm Proteins; Neovascularization, Pathologic; Transforming Growth Factor alpha; Transplantation, Heterologous; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

GEO is a well-differentiated colon cancer cell line that coexpresses the epidermal growth factor-like growth factors CRIPTO (CR), amphiregulin (AR), and transforming growth factor alpha (TGF-alpha). Antisense 20-mer phosphorothioate oligodeoxynucleotides (AS S-oligos) directed against CR, AR, and TGF-alpha mRNAs were equipotent in their ability to inhibit both the anchorage-dependent growth and the anchorage-independent growth (AIG) of GEO cells, with a 50% inhibitory concentration of about 5 micrometer in the AIG assay. A supraadditive effect was observed when a combination of S-oligos was used. For example, a combination of two different AS S-oligos (either AR + CR, or TGF-alpha + CR, or TGF-alpha + AR) at a concentration of 1 micrometer each (total concentration, 2 micrometer) resulted in 50% inhibition of GEO cells AIG, whereas the use of each AS S-Oligo at a 1 or 2 micrometer concentration resulted respectively in about 10 and 20% growth inhibition. A combination of the three AS S-oligos was even more effective, resulting in about 60% inhibition of GEO cells AIG at a concentration of 1 micrometer each (3 micrometer total concentration). The AS S-oligos were also able to inhibit specifically the expression of either AR, CR, or TGF-alpha proteins in GEO cells, as assessed using immunocytochemistry or Western blot analysis. Finally, a supraadditive growth inhibitory effect of the AS S-oligos and an epidermal growth factor receptor-blocking antibody (monoclonal antibody 528) was observed. These data suggest that the use of a combination of AS S-oligos directed against different growth factors and antibodies directed against their receptors might result in an efficient inhibition of colon carcinoma cell growth.

Topics: Amphiregulin; Antibodies, Monoclonal; Colonic Neoplasms; EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; Glycoproteins; GPI-Linked Proteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Neoplasm Proteins; Oligonucleotides, Antisense; Thionucleotides; Transforming Growth Factor alpha; Tumor Cells, Cultured

Xenografted tumours were produced in nude mice by injection of HCT-8 human colon tumour cells. At average volumes of about 750 mm3, animals were injected with fast green vital dye, and 20 min later, tumours were excised and dissected into viable (stained) and necrotic portions (unstained). Viable and necrotic regions were then examined for cell yields, colony forming efficiencies, and levels of basic fibroblast growth factor (FGF-2), transforming growth factors-beta 1 and -alpha (TGF-beta 1, TGF-alpha), platelet derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) using enzyme-linked immunoassay (ELISA) procedures. Levels in the viable and necrotic regions were compared to levels in unseparated tumours. The average extent of necrosis in HCT-8 tumours of this size was 64%. The data for cell yields, colony forming efficiencies FGF-2, VEGF, TGF-beta 1 and TGF-alpha indicated that values determined in the unseparated tumours could be understood on the basis of the weighted average between viable and necrotic tissue, with the higher values occurring in the viable tissue. Low levels of FGF-2 and VEGF were found in the necrotic portions of the tumour while no measurable levels of TGF-beta 1 and TGF-alpha could be determined. PDGF levels were, however, equivalent in both the viable and necrotic regions indicating that necrotic tissue could be an important reservoir for this growth factor.

Topics: Colonic Neoplasms; Endothelial Growth Factors; Enzyme-Linked Immunosorbent Assay; Fibroblast Growth Factor 2; Growth Substances; Humans; Lymphokines; Necrosis; Neoplasms, Experimental; Platelet-Derived Growth Factor; Tissue Distribution; Transforming Growth Factor alpha; Transforming Growth Factor beta; Transplantation, Heterologous; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Two morphologically distinct cell lines, GP2d and GP5d, derived from the same adenocarcinoma of the colon, have been established and characterised. Both clones have the same genetic changes, consistent with the usual pattern of tumour progression in colon cancer. The cells also have an inverted duplication of bands 10q11 to 10q21, but Southern blot analysis failed to identify any translocations involving the ret protooncogene, which maps to this region. GP2d grew by spreading from the edges of microcolonies to form a confluent layer of cells. GP5d grew in discrete islands of cells forming multi-layered colonies. These differing patterns of growth correlated with variation in expression or cellular distribution of alpha 2-integrin, desmoplakin and e-cadherin. Only GP2d responded to exogenously added epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha) or insulin with an increase in cell numbers, even though both cell lines possessed similar numbers of EGF receptors. Analysis of EGF receptor ligand expression showed that GP5d cells expressed relatively more TGF alpha mRNA than did GP2d; in contrast, amphiregulin mRNA, which was abundant in GP2d, was virtually undetectable in GP5d. Even though GP5d failed to exhibit a growth response to EGF, it underwent a marked epithelial-mesenchymal transition when treated with EGF, indicating separation of growth and morphological responses to EGF.

Topics: Adenocarcinoma; Base Sequence; Cell Adhesion Molecules; Cell Division; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Humans; Ligands; Molecular Sequence Data; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

As colon epithelial cells migrate up the cylindrical colonic crypt, they terminally differentiate and lose their ability to divide. Elevated levels of the epithelial cell mitogen TGF alpha have been found at the top of the crypt by other investigators, causing us to speculate that colon epithelial cells lose mitogenic response to TGF alpha as they differentiate. We tested this hypothesis by using the HT29 colon carcinoma sublines U4 and U4H as models of one colonocyte lineage, fluid-transporting enterocytes. TGF alpha was mitogenic for the U4 cells, but inhibited the growth of the more differentiated U4H cells. However, p44 MAP kinase was activated by TGF alpha in both U4 and U4H cells, as well as in two control undifferentiated HT29 sublines which showed no change in proliferation in response to TGF alpha. In addition, TGF alpha activated the EGF receptor in each line by increasing its tyrosine phosphorylation. No relationship was found in these four lines between response to TGF alpha and level of expression of either the EGF receptor or two EGF receptor ligands, TGF alpha and amphiregulin. Activated EGF receptors initiate both growth-inhibitory and mitogenic signals in these cells since blocking some of the EGF receptors on TGF alpha-growth-inhibited U4H cells and TGF alpha-unresponsive U9 cells overrode the inhibitory signals and made both U9 and U4H cells sensitive to mitogenesis by added TGF alpha. These data imply that upon reaching stages of greater maturation, colon enterocytes lose proliferative response to TGF alpha because of changes in signaling by their EGF receptors.

Topics: Amphiregulin; Blotting, Northern; Calcium-Calmodulin-Dependent Protein Kinases; Cell Differentiation; Cell Division; Cell Line; Colon; Colonic Neoplasms; EGF Family of Proteins; Enzyme Activation; Epithelial Cells; Epithelium; ErbB Receptors; Gene Expression; Glycoproteins; Growth Substances; Humans; Immunoglobulin G; Intercellular Signaling Peptides and Proteins; Intestinal Absorption; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

The human colon cancer cell lines HCT 116 (poorly differentiated) and GEO (well differentiated) express the mitogenic peptide transforming growth factor alpha (TGF-alpha). The secretion of TGF-alpha was enhanced by phorbol 12-myristate 13-acetate (PMA), indicating the possible role of protein kinase C (PKC) in the formation of mature TGF-alpha. Cells were metabolically labeled with 35S-cysteine and the formation of the mature 6 kDa TGF-alpha polypeptide from the 17 kDa pro-TGF-alpha precursor was determined. The conversion of pro-TGF-alpha was complete in 2-4 hr with the HCT 116 cells showing faster kinetics of TGF-alpha formation than GEO cells. HCT 116 cells secreted more TGF-alpha than GEO cells and the rate and extent of formation of TGF-alpha was enhanced by PMA in both cell lines. The expression of several PKC isozymes by HCT 116 and GEO cells was examined by immunoblotting. The expression of all isozymes examined was higher in HCT 116 cells compared with GEO cells. Calphostin C, an inhibitor of PKC, reduced the enzyme activity and significantly inhibited the PMA-induced secretion of TGF-alpha by both cell lines. Two agonists of PKC that act on specific PKC isozymes, thymeleatoxin and 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), stimulated the release of TGF-alpha into the medium to the same extent as PMA. Since dPPA has been reported to stimulate PKC-beta 1 specifically, our results suggest a potential role for PKC-beta in the processing of pro-TGF-alpha by these 2 human colon carcinoma cell lines.

Topics: Colonic Neoplasms; Humans; Isoenzymes; Naphthalenes; Phorbol Esters; Protein Kinase C; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha; Tumor Cells, Cultured

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) are potent mitogens that contribute to abnormal growth regulation in colon cancer. Growth factors have been shown to regulate transmembrane nutrient uptake as an adaptive response to support cellular proliferation.. The transport of L-arginine by the SW480 primary human colon adenocarcinoma cell line was characterized by assaying the uptake of [3H]L-arginine in the presence and absence of sodium. Kinetic studies were performed over a range of L-arginine concentrations to determine transport affinity (Km) and maximal transport velocity (Vmax). To further characterize the specific transporters, [3H]L-arginine uptake was measured in the presence of selected amino acids, hormones, and under conditions of varying external pH. To investigate the effects of EGF and TGF alpha, cells were incubated with increasing doses of growth factors (1, 10, 50 ng/ml) and L-arginine transport was measured at various time intervals (8, 12, 24 h). Proliferation was assessed by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 3 days after growth factor stimulation.. The majority of carrier-mediated L-arginine transport was via a sodium-independent process (65-70%), whereas the remainder was sodium-dependent (28-30%). Diffusion contributed a small amount to total L-arginine uptake (2%). Kinetic studies of arginine transport revealed a single high-affinity Na(+)-independent transporter with a Km = 55.8 +/- 5.8 microM and a Vmax = 710.6 +/- 87.3 pM/mg protein/30 s. Na(+)-independent arginine uptake was pH-insensitive and markedly inhibited by system y+ substrates L-homoarginine, L-lysine, and L-ornithine. A single Na(+)-dependent transporter with a Km = 19.8 +/- 2.3 microM and a Vmax = 159.1 +/- 8.9 pM/mg protein/30 s was identified. Na(+)-dependent arginine uptake was inhibited by system BO,+ substrates L-lysine, L-ornithine, L-leucine, L-cysteine, and L-glutamine, but not by 2-methylaminoisobutyric acid. In addition, Na(+)-dependent arginine uptake was pH- and hormone-insensitive. Incubation with EGF or TGF alpha had no effect on Na(+)-independent L-arginine uptake; however, Na(+)-dependent uptake was enhanced 60% by EGF (10 ng/ml, p < 0.05) and 100% by TGF alpha (10 ng/ml, p < 0.05), whereas cellular proliferation was increased 27% by EGF (10 ng/ml, p < 0.05) and 37% by TGF alpha (10 ng/ml, p < 0.01).. L-arginine transport in the SW480 colon cancer cell line is principally mediated by the Na(+)-independent system y+ and to a lesser extent by the Na(+)-dependent system BO,+. Furthermore, EGF and TGF alpha preferentially stimulate L-arginine uptake via the Na(+)-dependent transporter, ostensibly to accommodate for the mitogenic stimulus.

Topics: Adenocarcinoma; Amino Acids; Arginine; Carrier Proteins; Cell Division; Cell Membrane Permeability; Colonic Neoplasms; Epidermal Growth Factor; Humans; Hydrogen-Ion Concentration; Ion Channel Gating; Nitric Oxide; Radioisotopes; Time Factors; Transforming Growth Factor alpha; Tumor Cells, Cultured

Transforming growth factor alpha (TGF alpha), a polypeptide growth-stimulating factor, has been implicated to play a role in the progression of gastrointestinal (GI) cancer. It has been suggested that TGF alpha expression in tumors or TGF alpha in the biological fluids of cancer patients may have tumor marker value. The serum levels of TGF alpha in GI cancer patients have not been reported. In this study, the serum TGF alpha levels of 100 GI cancer patients, as well as 74 healthy individuals, were determined by a TGF alpha-specific RIA kit. All of the cancer patient sera and 67% of the normal sera had detectable levels of TGF alpha. The TGF alpha concentrations in GI cancer patients ranged from 119 to 760 pg/ml, with a mean value of 269 +/- 102 pg/ml. Fifty normal individuals had detectable levels of TGF alpha, and their levels ranged from 120 to 207 pg/ml, with a mean value of 147 +/- 18 pg/ml. Differences in serum TGF alpha concentration between cancer patients and healthy individuals were found to be statistically significant, as evaluated by Mann-Whitney and Student's t tests. Serum TGF alpha levels were found to be significantly elevated in all disease stages of gastric, pancreas, colon, and rectal cancers, and only in the late stages of esophageal cancer. Serum carcinoembryonic antigen levels were significantly elevated only in the late stages of these diseases. The potential of serum TGF alpha as a tumor marker for GI malignancy, therefore, warrants further investigation.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoembryonic Antigen; Case-Control Studies; Colonic Neoplasms; Esophageal Neoplasms; Female; Gastrointestinal Neoplasms; Humans; Male; Middle Aged; Neoplasm Staging; Pancreatic Neoplasms; Rectal Neoplasms; Sensitivity and Specificity; Stomach Neoplasms; Transforming Growth Factor alpha

Two cell line models for colon goblet cells expressed 6- to 14-fold elevated levels of the EGF receptor, 3- to 5-fold levels of TGF alpha and 11- to 15-fold levels of amphiregulin compared with 2 cell lines which model colon enterocytic differentiation, suggesting a role for the EGF receptor and its ligands in goblet cell growth control. Two HT29 colon carcinoma sublines were used to model normal goblet cells at different stages of maturation. TGF alpha induced a 2-fold increase in growth of the HD8 subline but inhibited the growth of the more differentiated HD6 subline by 40%. EGF receptors were activated in each line by ligand, but signal transduction varied sharply. Both MAP kinase isoforms, p44 and p42, were markedly activated in HD8 cells for at least 20 min, while only a marginal activation was seen in HD6 cells. In contrast, the more differentiated HD6 cells showed an increase in 105 kDa MBP kinase activity with EGF treatment, while HD8 cells displayed constitutively elevated levels of this kinase. Thus, activated EGF receptors initiated different signalling pathways in model cell lines for colon goblet cells at different stages of maturation. TGF alpha protein levels have been shown by other investigators to be restricted to the top of the cylinder-like colonic crypt, where cells terminally differentiate and cease division, an unexpected location for an epithelial cell mitogen. Our data with model cell lines imply that normal colon goblet cells lose proliferative response to TGF alpha as they differentiate and the elevated levels of TGF alpha at the top of the colonic crypt in vivo serve to inhibit goblet cell growth.

Topics: Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma; Cell Differentiation; Cell Division; Colon; Colonic Neoplasms; ErbB Receptors; Humans; Signal Transduction; Transforming Growth Factor alpha; Tumor Cells, Cultured

The authors determined the molecular mechanisms for the failure of transforming growth factor-beta (TGF-beta) to inhibit the growth of SW1116 and SW48 colon cancer cell lines.. Transforming growth factor-beta is a bifunctional regulator of cell growth that typically stimulates proliferation of mesenchymal cells, but inhibits proliferation of normal epithelial cells. In the colon, TGF-beta appears to arrest proliferation of enterocytes as they leave the intestinal crypt and move to the villus tip. Transforming growth factor-beta actions are mediated by binding to heteromeric complexes of type I and type II TGF-beta receptors. Loss of TGF-beta responsiveness may contribute to uncontrolled cell growth and cancer.. The effects of TGF-beta 1 on DNA synthesis were measured by incorporation of tritiated thymidine into DNA of cultures of moderately differentiated adenocarcinoma (SW48) and poorly differentiated adenocarcinoma (SW1116) colon cell lines and a mink lung epithelial cell line (CCL-64). The effects of TGF-beta on the expression of c-myc, TGF-alpha, and TGF-beta in SW48 cells, SW1116 cells, and CCL-64 cells (c-myc only) were measured by Northern blot analysis. Expression of TGF-beta receptors in the cell lines was measured using competitive binding assays, receptor affinity labelling techniques, and reverse transcriptase-polymerase chain reaction.. Incubation with TGF-beta 1 (50 ng/mL) did not decrease serum-stimulated uptake of [3H]-thymidine into actively growing cultures of SW48 or SW1116 cells, but suppressed DNA synthesis of actively growing CCL-64 cells by 90%. Similarly, incubation with TGF-beta 1 (12 ng/mL) for 4 hours did not substantially alter the mRNA levels of c-myc, TGF-alpha, and TGF-beta 1 in either colon tumor cell line, although levels of c-myc mRNA in CCL-64 cells were reduced by TGF-beta 1 treatment. Competitive displacement of [125I]-TGF-beta 1 binding detected high levels (16,500 TGF-beta receptors per cell) of specific, high-affinity (200 pmol/L half-displacement) TGF-beta receptors on CCL-64 cells. In marked contrast, very low levels of TGF-beta 1 binding to SW1116 cells (250 receptors per cell) and SW48 cells (260 receptors per cell) were detected. Autoradiograms of CCL-64 cells affinity labelled with [125I]TGF-beta 1 revealed the presence of type I, type II, and type III TGF-beta receptors. No TGF-beta receptors were identified on SW1116 cells, and only very low levels of the nonsignaling type III TGF-beta receptors were detected on SW48 cells. Reverse transcriptase-polymerase chain reaction amplification detected mRNAs for type I, type II, and type III TGF-beta receptors in CCL-64 cells. SW48 cells, and SW1116 cells.. These results suggest that the lack of growth inhibition by TGF-beta in SW48 and SW1116 colon cancer cells may be caused by a lack of expression of functional TGF-beta receptors.

Topics: Activin Receptors, Type I; Base Sequence; Cell Division; Colonic Neoplasms; DNA, Neoplasm; Epithelium; Genes, myc; Humans; Molecular Sequence Data; Polymerase Chain Reaction; Protein Serine-Threonine Kinases; Proteoglycans; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Gastrin is transcriptionally responsive to EGF stimulation (Merchant et al., 1991, Mol. Cell. Biol., 11:2686-2696). Consequently, we hypothesized that previously recognized gastrin autocrine loops (Hoosein et al., 1990, Exp. Cell. Res., 186:15-21), might be controlled by autocrine TGF alpha in human colon carcinoma cells. Therefore, we examined the interaction between these two autocrine growth factors in two colon carcinoma cell lines which utilize TGF alpha. The FET cell line requires exogenous TGF alpha/EGF for optimal growth and has a classical TGF alpha autocrine loop which is disrupted by TGF alpha or epidermal growth factor receptor (EGFr) antibodies. The HCT 116 cell line is not dependent on exogenous TGF alpha/EGF and exhibits a nonclassical TGF alpha autocrine loop which is not disrupted by neutralizing antibodies to either TGF alpha itself or the EGFr. Basal gastrin mRNA production is significantly higher in HCT 116 than FET as measured by RNase protection assay. In the FET cells, exogenous EGF stimulates gastrin mRNA production but not in HCT 116. When the TGF alpha autocrine loop in HCT 116 is disrupted by constitutive expression of antisense TGF alpha mRNA, the gastrin mRNA level is significantly repressed. In xenografts derived from these antisense clones, TGF alpha reverted to high expression, and the gastrin mRNA level was again increased. This interaction between the strong TGF alpha loop in HCT 116 and the gastrin autocrine loop may confer a growth advantage to these colon cells. Such interactions between growth factors may promote enhanced tumorigenicity to transformed cells with these strong, nonclassical autocrine loops.

Topics: Animals; Colonic Neoplasms; DNA, Antisense; Epidermal Growth Factor; Gastrins; Gene Expression Regulation; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Polymerase Chain Reaction; RNA, Messenger; Transfection; Transforming Growth Factor alpha; Transplantation, Heterologous; Tumor Cells, Cultured

The purpose of this study was to determine whether production of liver metastasis by human colon carcinoma (HCC) cells depends on the response of tumor cells to organ-derived growth factors. HCC cells were isolated from several surgical specimens whose malignant potential differed (Dukes' stage B or D tumors), adapted to grow in culture, and assessed for expression of the epidermal growth factor receptor (EGF-R). Northern blot analyses revealed that highly metastatic HCC cells expressed >5-fold the number of EGF-R mRNA transcripts as low metastatic cells. The level of mRNA correlated with the amount of EGF-R protein as detected by Western blotting, immunohistochemistry, and Scatchard analyses. HCC growth response in vitro to picograms of transforming growth factor alpha was associated with functional cell surface EGF-Rs as determined by receptor tyrosine kinase activity assays. The EGF-R gene was not amplified or rearranged in highly metastatic cells. However, fluorescence in situ hybridization analysis showed that the copy number of chromosome 7 was higher in the highly metastatic cells. HCC cells were selected in vitro for low or high expression of EGF-R. Subsequent to injection into nude mice, only cells with high expression of EGF-R produced a high incidence of liver metastasis. These data demonstrate that expression of EGF-R by HCC cells directly correlates with their ability to produce hepatic metastasis.

Topics: Animals; Carcinoma, Squamous Cell; Cell Division; Chromosome Mapping; Chromosomes, Human, Pair 7; Colonic Neoplasms; ErbB Receptors; Humans; Liver Neoplasms; Male; Mice; Mice, Nude; Neoplasm Metastasis; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor alpha; Transplantation, Heterologous; Tumor Cells, Cultured

Chemotherapy has little efficacy in the treatment of advanced colorectal carcinoma. Biological investigation has made evident several autocrine stimulation loops; the best documented one involves epidermal growth factor (EGF): this growth factor stimulates cell proliferation and cell secretion of proteolytic enzymes. Suramin and somatostatin are able to disrupt these loops of stimulation. Clinical studies performed with octreotide, a somatostatin analogue, and suramin have been unsuccessful until now.

Topics: Antineoplastic Agents; Cell Division; Colonic Neoplasms; Epidermal Growth Factor; Humans; Neoplasm Invasiveness; Neoplasm Proteins; Somatostatin; Suramin; Transforming Growth Factor alpha; Tumor Cells, Cultured

Gliomas, squamous carcinomas and different adenocarcinomas from breast, colon and prostate might have an increased number of epidermal growth factor (EGF) receptors. The receptors are, in these cases, candidates for binding of receptor specific toxic conjugates that might inactivate cellular proliferation. The purpose of this study was to evaluate whether it is reasonable to try ligand-dextran based conjugates for therapy.. EGF or TGF alpha were conjugated to dextran and binding, internalization, retention and degradation of eight types of such conjugates were analyzed in EGF-receptor amplified glioma cells. The conjugates were labelled with radioactive nuclides to allow detection and two of the conjugates were carrying boron in the form of carboranyl amino acids or aminoalkyl-carboranes. Comparative binding tests, applying 125I-EGF, were made with cultured breast, colon and prostate adenocarcinoma, glioma and squamous carcinoma cells. Some introductory tests to label with 76Br for positron emission tomography and with 131I for radionuclide therapy were also made.. The dextran part of the conjugates did not prevent receptor specific binding. The amount of receptor specific binding varied between the different types of conjugates and between the tested cell types. The dextran part improved intracellular retention and radioactive nuclides were retained for at least 20-24 h. The therapeutical effect improved when 131I was attached to EGF-dextran instead of native EGF.. The improved cellular retention of the ligand-dextran conjugates is an important property since it gives extended exposure time when radionuclides are applied and flexibility in the choice of time for application of neutrons in boron neutron capture therapy (BNCT). It is possible that ligand-dextran mediated BNCT might allow, if the applied neutron fields covers rather wide areas around the primary tumor, locally spread cells that otherwise would escape treatment to be inactivated.

Topics: Adenocarcinoma; Animals; Boron Compounds; Boron Neutron Capture Therapy; Carcinoma; Colonic Neoplasms; Dextrans; Drug Carriers; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Iodine Radioisotopes; Male; Mammary Neoplasms, Experimental; Models, Biological; Neoplasms; Neoplasms, Experimental; Prostatic Neoplasms; Rats; Rats, Sprague-Dawley; Transforming Growth Factor alpha; Tumor Cells, Cultured

Gene expression of epidermal growth factor receptor (EGFR), epidermal growth factor (EGF) and transforming growth factor-alpha (TGF alpha) in human colonic carcinoma cell lines (HT10, LST174 and Lovo) was studied by using Northern blot technique. Total RNAs were isolated from these cell lines, separated by 1% agarose gel electrophoresis, transferred onto a membrane and hybridized with EGFR, EGF and TGF alpha probes. EGF and EGFR mRNAs were found in all three cell lines, and TGF alpha mRNA was seen in LST174 and HT10 cell lines but not in Lovo. The results indicate that autocrine stimulation by growth factor exists in human colonic carcinoma cell lines and it may be one of the important causes for the uncontrolled growth of carcinoma cell.

Topics: Blotting, Northern; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Humans; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

Male, Sprague-Dawley rats were injected subcutaneously with the colon carcinogen 1,2-dimethylhydrazine (DMH) at a dosage of 9.5 mg DMH base/per kg rat body weight once weekly for 8 weeks; control rats received an equivalent volume of the vehicle. Analyses of variance showed that in carcinogen-treated as well as in non-carcinogen-treated rats, the proliferative zone height and the crypt height in colonic crypts located over the aggregates of lymphoid nodules (ALN) were significantly higher than in colonic crypts located away from the ALN. Immunohistochemical localization of transforming growth factor alpha (TGF alpha) showed that this mitogenic factor was found in cells in the proliferative zone of colonic crypts located over the ALN, but TGF alpha was not detectable in cells in the proliferative zone of colonic crypts located away from the ALN. Examination of histological sections of the colon taken through the ALN of DMH-treated rats revealed that eight out of 25 DMH-treated rats had microscopic adenocarcinomas (AC) within the ALN, but in the same rats no microscopic AC were seen in histological sections taken away from the ALN. Furthermore, there was no evidence of an adenomatous precursor to these microscopic, endophytic AC, suggesting that the endophytic AC arose de novo. Therefore, because of (i) the significantly higher proliferative activity in colonic crypts located over the ALN, (ii) the localization of TGF alpha in the proliferative zone of the colonic crypts associated with ALN and (iii) the high incidence of endophytic AC associated with ALN, it seems likely that factors emanating from the ALN are promotional to carcinogenesis in the colonic epithelium that is located in close proximity to the ALN.

Topics: 1,2-Dimethylhydrazine; Adenocarcinoma; Animals; Carcinogens; Cell Cycle; Cell Division; Cocarcinogenesis; Colon; Colonic Neoplasms; Dimethylhydrazines; Epithelium; Hyperplasia; Immunohistochemistry; Lymphoid Tissue; Male; Rats; Rats, Sprague-Dawley; Transforming Growth Factor alpha

Human colon carcinoma cell lines secrete transforming growth factor-alpha (TGF-alpha). Previous work indicated that the apparent m.w. of the TGF-alpha secreted by these cells ranged between 5 and 25 kDa. The more differentiated GEO cell line secreted a higher percentage of high m.w. TGF-alpha than did the poorly differentiated HCT 116 cell line. In addition, the HCT 116 cells secreted 5-fold more TGF-alpha. Treatment of HCT 116 and GEO cells with a phorbol ester (TPA) resulted in a 4-fold increase in TGF-alpha in the conditioned media of both cell types. The TPA-induced release of TGF-alpha was blocked by an inhibitor of elastase-like enzymes. This suggested a role for protein kinase C (PKC) in TGF-alpha processing in colon carcinoma cells. Direct measurement of PKC activity indicated that the HCT 116 cells (which secrete more fully processed TGF-alpha) had 10-fold more PKC activity than GEO cells. The presence of an elastase-like activity in detergent extracts and the ability of an elastase inhibitor to block the TPA-induced secretion of TGF-alpha suggests that PKC and an elastase-like enzyme are involved in the processing and secretion of TGF-alpha by human colon carcinoma cell lines.

Topics: Colonic Neoplasms; Detergents; Enzyme Activation; Humans; Pancreatic Elastase; Protease Inhibitors; Protein Kinase C; Sensitivity and Specificity; Stimulation, Chemical; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha; Tumor Cells, Cultured

Transforming growth factor alpha (TGF-alpha) has multifunctional biological effects on a variety of mesenchymal and epithelial cells. It is a potent mitogen for a number of normal and transformed cell types, regulates extracellular matrix (ECM) production and promotes breast, kidney and lung morphogenesis. To clarify the role of ECM proteins in the morphogenetic and mitogenic effects of TGF-alpha, we have used a human colon carcinoma cell line (SW1222) which expresses EGF receptor. Here we show that TGF-alpha at 1 ng/ml increases the proliferation of SW1222 cells, but only when they are cultured on plastic rather than collagen-coated plates. Higher concentrations of TGF-alpha (10 ng/ml) did not increase cell proliferation but significantly enhanced the crypt-like glandular differentiation when cells were grown in 3-dimensional collagen gel (p = 0.027). These effects were accompanied by increased expression of alpha 2 beta 1 and alpha 3 beta 1 integrin molecules, which are receptors for extracellular matrix proteins, and by a statistically significant increase in binding of SW1222 cells to type-1 collagen. The effects of TGF-alpha both on binding to type-1 collagen and on morphological differentiation in 3-dimensional collagen gel were inhibited by monoclonal antibodies recognizing the alpha 2 beta 1 integrin. These data indicate that the morphogenetic or mitogenic activities of TGF-alpha are critically dependent on cellular interactions with extracellular matrix proteins and are primarily mediated by the alpha 2 beta 1 integrin receptor. Inappropriate expression of this growth factor, seen in tumours whose cell-matrix interactions are greatly impaired, could have deleterious effects on the maintenance of normal tissue architecture and growth control.

Topics: Antibodies, Monoclonal; Cadherins; Cell Differentiation; Cell Division; Collagen; Colonic Neoplasms; Extracellular Matrix Proteins; Humans; Integrin alpha3beta1; Integrins; Mitogens; Morphogenesis; Transforming Growth Factor alpha; Tumor Cells, Cultured

CRIPTO is an epidermal growth factor-related gene expressed in a majority of human colorectal tumors. To assess the role of CRIPTO in the growth control of human colon cancer, we have treated human colon carcinoma GEO and CBS cells, that possess high levels of CRIPTO, and WIDR colon cancer cells, that are negative for CRIPTO expression, with two antisense phosphorothioate oligodeoxynucleotides complementary to the 5' end of the human CRIPTO mRNA. Both antisense oligodeoxynucleotides significantly reduced endogenous CRIPTO protein levels and inhibited GEO and CBS cell growth in monolayer and in semisolid medium, whereas they did not affect WIDR cell growth. In addition, GEO, CBS and WIDR cells were infected with a recombinant retroviral vector containing the hygromycin-resistance gene and a 900 bp EcoRI-EcoRI coding fragment of the human CRIPTO cDNA oriented in the 3' to 5' direction. GEO and CBS CRIPTO antisense infectants exhibited a 60 to 70% reduction in CRIPTO protein expression, in monolayer growth and in soft agar cloning efficiency as compared to parental noninfected cells. In contrast, infection of WIDR cells with the CRIPTO antisense retrovirus did not alter their growth. Finally, GEO CRIPTO antisense infectants formed tumors in nude mice that were significantly smaller and had a larger latency period as compared to noninfected GEO cells.

Topics: Animals; Base Sequence; Cells, Cultured; Colonic Neoplasms; Epidermal Growth Factor; Female; Gene Expression; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Neoplasm Proteins; Oligonucleotides, Antisense; RNA, Antisense; Transforming Growth Factor alpha

We have previously shown that four human oesophageal squamous cell carcinoma (SCC) cell lines secrete significant quantities of transforming growth factor alpha (TGF-alpha) in vitro. Three of these lines are known to produce supernumerary low-affinity epidermal growth factor receptors (EGF-Rs). Using an 125I-EGF competitive binding assay and Scatchard analysis, we show that the fourth also overproduces low-affinity receptors. According to slot blot DNA analyses, the secretion of high levels of TGF-alpha is not associated with amplification of the TGF-alpha gene, and hyperproduction of the EGF-R is correlated with receptor gene amplification. Western blot analyses show that the c-Myc protein is overexpressed in two of the cell lines; and Southern and Northern blot analyses indicate that this overexpression occurs independently of c-myc gene amplification. Our results are consistent with an autocrine role for TGF-alpha and EGF-R in oesophageal carcinogenesis and support the possibility that c-myc overexpression may be required for the in vivo tumourigenicity of cells that produce high levels of TGF-alpha and the EGF-R.

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; Colonic Neoplasms; ErbB Receptors; Esophageal Neoplasms; Gene Amplification; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Neoplasm Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) are polypeptides which bind to the EGF receptor (EGFr) and may play a role in cell growth and carcinogenesis. Our study investigated the content of EGF, TGF-alpha, and EGFr in tumors of the stomach and the colon in comparison with the surrounding mucosa. EGF was detected in half of the stomach specimens with concentrations between 1 and 9 ng/g weight irrespective of histology. In the colon no EGF was found in the tumor or normal mucosa. In the stomach normal mucosa contained higher TGF-alpha concentrations (mean 22.4 ng/g) than the tumors (mean 11.8 ng/g), but the difference was not statistically significant because of a wide variation in mucosal values. By contrast, the colon mucosa displayed significantly higher TGF-alpha concentrations than the tumor tissues (33 ng/g versus 12 ng/g; P < 0.01). EGFr content in the gastric mucosa was lower compared to gastric carcinoma (48 fmol/g versus 75 fmol/g) yet not significantly different. In contrast, colorectal tumor specimens disclosed significantly higher concentrations than the mucosal tissues (mean of 155 fmol/g versus 80 fmol/g; P < 0.01). In conclusion, TGF-alpha should not be considered a tumorigenic but a physiological growth factor in the stomach and colon. An elevated EGFr content in colorectal tumors in comparison with the normal mucosa could lead to a growth advantage by an autostimulating mechanism.

Topics: Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Gastric Mucosa; Humans; Intestinal Mucosa; Reference Values; Stomach Neoplasms; Transforming Growth Factor alpha

Transforming growth factor alpha (TGF alpha) is a growth factor produced by colon cancer cells which may function as an autocrine growth regulator. Therefore, the proliferation and transformation of colon cancer cells might be attenuated by blocking the production of endogenous TGF alpha. GEO cells, from a human colon carcinoma cell line that expresses TGF alpha and functional epidermal growth factor (EGF) receptors, were infected with a replication-defective, recombinant amphotropic retroviral expression vector containing the neomycin-resistance gene and a 435-bp ApaI-EcoRI coding fragment of the human TGF alpha cDNA oriented in the 3' to 5' direction under the transcriptional control of the heavy-metal-inducible mouse metallothionein I promoter. Following antibiotic selection, G418-resistant colonies were pooled and expanded into a cell line (GEO TGF alpha AS cells). A 50 to 70% inhibition in the production of secreted and cell-associated TGF alpha protein was observed in GEO TGF alpha AS cells that had been maintained in CdCl2-supplemented medium. Moreover, a growth inhibition of 70% and 50% was observed in CdCl2-treated GEO TGF alpha AS cells under anchorage-dependent and anchorage-independent culture conditions, respectively. In contrast, CdCl2 treatment of parental GEO cells had no significant effect upon these parameters. Our results suggest that TGF alpha may be involved in modulating the in vitro cell growth and transformation of human colon cancer cells that express both this growth factor and its cognate receptor.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Colonic Neoplasms; Female; Flow Cytometry; Gene Expression; Genetic Vectors; Humans; Mice; Mice, Nude; Radioimmunoassay; Retroviridae; RNA, Antisense; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

FET cells are well differentiated human adenocarcinoma cells whose growth is partially inhibited (50-60%) by transforming growth factor-beta 1 (TGF-beta 1). In exponentially growing cultures, TGF-beta 1 induces the expression of transforming growth factor-alpha (TGF-alpha) by 3-fold. To determine whether this induction is the result of increased TGF-alpha promoter activity, FET cells were transiently transfected with a plasmid containing 2816 base pairs of the 5'-flanking region of the TGF-alpha gene linked to luciferase. Transfected FET cells treated with growth-inhibitory concentrations of TGF-beta 1 (10 ng/ml) showed up to a 10-fold increase in luciferase activity. The increase in luciferase activity was dose dependent through the normal physiological range of TGF-beta 1 (0.5-20 ng/ml), saturating at 10 ng/ml. This effect was also TGF-alpha promoter specific, inasmuch as the Rous sarcoma virus long terminal repeat used as a control remained relatively insensitive to the effects of TGF-beta 1. By using progressively smaller portions of the TGF-alpha promoter region, the TGF-beta 1-responsive element was mapped between base pairs -77 and -201 of the 5'-flanking region. TGF-beta 1 treatment also affected epidermal growth factor receptor levels. FET cells treated with TGF-beta 1 (10 ng/ml) for 48 h showed a 20% decrease in the number of epidermal growth factor receptors and a 2-fold increase in the number of high affinity epidermal growth factor receptors on their surface. These results indicate that TGF-beta 1 acts as a positive regulator of TGF-alpha transcription, and they suggest a possible mechanism by which these cells circumvent the growth-inhibitory effects of TGF-beta 1.

Topics: Adenocarcinoma; Base Sequence; Colonic Neoplasms; Culture Media, Serum-Free; Enzyme Induction; Humans; Luciferases; Molecular Sequence Data; Promoter Regions, Genetic; Transfection; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

This report examines the effects of inhibitors of cell proliferation on transforming growth factor alpha (TGF-alpha) expression in low-density cultures of poorly (PD) and well-differentiated (WD) human colon carcinoma cells, continuously maintained in serum-free medium. In contrast to results in certain untransformed cells, growth inhibitors such as transforming growth factor beta 1 (TGF-beta 1) and N,N-dimethylformamide up-regulated TGF-alpha mRNA and protein expression in these human colon carcinoma cells. Treatment of low-density WD cells with TGF-beta 1 (10 ng/ml) resulted in a 1.5-fold increase in TGF-alpha mRNA levels within 4 h of treatment. TGF-alpha mRNA levels increased to 2.7-fold above control values by 48 h after TGF-beta 1 addition. Additionally, over a TGF-beta 1 concentration range of 1-30 ng/ml, TGF-alpha protein levels were increased by 2-10-fold, despite the fact that the growth of the WD cells remained inhibited. Although TGF-beta 1 control of TGF-alpha expression was altered in these WD colon carcinoma cells, relative to that in untransformed cells previously examined, the cells retained the ability to up-regulate TGF-alpha expression in an epidermal growth factor-dependent manner. In similarity to the results with TGF-beta 1 in WD colon carcinoma cells, the differentiation agent N,N-dimethylformamide (0.7%) resulted in an increase of TGF-alpha mRNA of approximately 3.8-fold in PD colon carcinoma cells, as well as a 4.4-fold increase in TGF-alpha protein after 4 days of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cell Differentiation; Cell Division; Colonic Neoplasms; Culture Media, Serum-Free; Dimethylformamide; Epidermal Growth Factor; Humans; Phenotype; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

We previously immortalized human oral keratinocytes by transfection with recombinant human papillomavirus type 16 (HPV-16) DNA and established two cell lines. These transfected cells were morphologically different from the normal counterpart, contained intact HPV-16 DNA in an integrated form, and expressed numerous viral genes. These cells contained lower levels of wild-type p53 protein and higher levels of c-myc mRNAs compared to normal cells. However, they proliferated only in keratinocyte growth medium containing a low level of calcium and were not tumorigenic in nude mice. A HPV-16-immortalized cell line was exposed to either 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone or N-methyl-N'-nitro-N-nitrosoguanidine. Four chemically transformed cell colonies were isolated. These cells proliferated well in Dulbecco's minimum essential medium containing a physiological level of calcium. They contained, similar to the immortalized counterpart, integrated HPV-16 sequences and lower levels of both wild-type p53 protein and DCC messages compared to normal cells. Among the chemically transformed cells, two colonies obtained from 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone exposure demonstrated an enhanced proliferation capacity in nude mice and transcribed a substantially higher amount of HPV-16 E6/E7, epidermal growth factor receptors, and c-myc genes compared with the immortalized counterpart. These experiments indicate that malignant transformation of oral keratinocytes can be caused by a sequential combined effect of "high risk" HPV and tobacco-related carcinogens.

Topics: Base Sequence; Carcinogens; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cells, Cultured; Colonic Neoplasms; DNA Primers; ErbB Receptors; Exons; Genes, myc; Genes, p53; Genes, ras; Humans; Keratinocytes; Male; Methylnitronitrosoguanidine; Molecular Sequence Data; Mouth Neoplasms; Mutagenesis; Nicotiana; Nitrosamines; Oligonucleotides, Antisense; Papillomaviridae; Plants, Toxic; Polymerase Chain Reaction; Transforming Growth Factor alpha

The GEO colon carcinoma cell line is weakly tumorigenic in athymic mice and shows differentiated properties both in tissue culture and in xenografts. Proliferating monolayer cultures of GEO cells which normally require exogenous epidermal growth factor (EGF) for optimal growth displayed a marked inhibition in growth upon addition of antibodies that block binding to the EGF receptor or neutralize TGF-alpha. These results indicated that GEO cells utilize TGF-alpha in a weak autocrine loop. The availability of a weakly malignant model system in which TGF-alpha had demonstrable, but low level autocrine activity, permitted the investigation of the role of TGF-alpha in tumorigenesis by generating a stronger autocrine loop through the overexpression of the polypeptide. GEO cells were electroporated with an expression vector containing the human TGF-alpha cDNA, and stable clones were isolated that constitutively expressed the TGF-alpha cDNA in a strong autocrine loop. However, the growth rate of the parental cells in EGF-supplemented medium was the same as that of transfected cells with or without growth factor-supplemented medium. Thus, any biological changes generated by the overexpression of TGF-alpha were due to the autocrine nature of the growth mechanism rather than due to any decrease in doubling time leading to a faster growth rate. Transfected GEO cells showed an increase in anchorage-independent growth and formed tumors more readily in athymic nude mice indicating that TGF-alpha plays a role in progression of transformed properties.

Topics: Animals; Blotting, Northern; Blotting, Southern; Cell Division; Cell Transformation, Neoplastic; Clone Cells; Colonic Neoplasms; DNA; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Kinetics; Mice; Mice, Nude; RNA, Messenger; Transfection; Transforming Growth Factor alpha; Transplantation, Heterologous; Tumor Cells, Cultured

Transforming growth factor-alpha (TGF alpha) is able to elicit growth in many target cells expressing a functional epidermal growth factor (EGF) receptor. Other laboratories have reported that the TGF alpha precursor polypeptide (proTGF alpha) is inefficiently cleaved from many target cells, resulting in accumulation of proTGF alpha on the cell surface. Since it has been shown that noncleavable, mutated cell-associated TGF alpha can stimulate cell growth on receptor-bearing adjacent cells, we have tried to determine whether cell-associated TGF alpha populations might be involved in supporting autonomous cell growth regulatory mechanisms in a human colon carcinoma cell line, HCT116. To address this question, the levels of secreted and nonsecreted TGF alpha produced were determined. Cells grown to medium cell density (40-60% confluent) expressed the greatest percentage of cell-associated TGF alpha (50%). Incubation of HCT116 cells with 0.1 U/ml porcine pancreatic elastase resulted in the release of 67% of the cell-associated TGF alpha into their medium and caused the treated cells to acquire a newly established growth sensitivity to exogenous TGF alpha at a ligand concentration of 1.0 nM. Western blot analysis of EGF receptor phosphotyrosine levels showed a decrease in phosphotyrosine content after elastase treatment. Phosphotyrosine content was restored to basal levels if elastase treatment was followed by addition of exogenous TGF alpha or EGF. These results suggest that HCT116 cells use a "closed" autocrine loop between cell-associated TGF alpha species and their EGF receptor to stimulate their cell growth.

Topics: Blotting, Western; Cell Count; Cell Division; Colonic Neoplasms; ErbB Receptors; Humans; Pancreatic Elastase; Phosphorylation; Precipitin Tests; Recombinant Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured

The c-myc gene is amplified and the c-Ki-ras gene is mutated in the SW613-S human colon carcinoma cell line. Two cell types with different levels of c-myc amplification are present in the SW613-S cell population and representative cell clones can be isolated. The clones with a high level of amplification and expression of the c-myc gene are tumorigenic in nude mice whereas those with a low level are not. The tumorigenic clones secrete transforming growth factor alpha (TGF-alpha) in the culture medium whereas the non-tumorigenic clones do not produce any detectable amount. Accordingly the level of TGF-alpha mRNA is higher and the transcription rate of the gene is increased in the tumorigenic clones. The acquisition of the tumorigenic phenotype by cells of non-tumorigenic clones, following introduction of c-myc gene copies by transfection, is accompanied by an increase in the steady-state level of TGF-alpha mRNA. These findings suggest a role for an elevated level of TGF-alpha production in the tumorigenic phenotype of SW613-S cells. The possibility that this role is indirect is discussed.

Topics: Animals; Carcinoma; Cell Transformation, Neoplastic; Clone Cells; Colonic Neoplasms; Gene Amplification; Genes, myc; Humans; Mice; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured

A natural DNA oligomer (15-mer) was synthesized with a sequence complementary to the translation initiation codon region of the human TGF-alpha mRNA and mixed with Lipofectin to form unilamellar complexes. It was found that tumor cell growth was inhibited when HCT116 cells were treated with Lipofectin-DNA oligomer complexes or with Lipofectin alone. Uptake of 32P-labeled 15-mers into colon tumor cells was compared in the presence and absence of Lipofectin. The amount of labeled oligomer found in cells that received optimal ratios of Lipofectin to DNA was 4- to 10-fold higher than the amount found in cells that received 32P-labeled DNA alone. Although Lipofectin-antisense DNA oligomer treatment of HCT116 cells caused a dose-dependent inhibition of cell growth, there was a subsequent rise in target mRNA product. Because the mechanism of growth inhibition could not involve an inhibition of TGF-alpha expression, it was concluded that Lipofectin probably exerts a nonspecific, detergent-like effect upon the cell membrane, producing an enhancement of TGF-alpha processing and release.

Topics: Base Sequence; Biological Transport; Cell Division; Cell Line; Chloramphenicol O-Acetyltransferase; Colonic Neoplasms; DNA, Antisense; Epidermal Growth Factor; Humans; Insulin; Kinetics; Molecular Sequence Data; Oligonucleotides, Antisense; Phosphatidylethanolamines; Promoter Regions, Genetic; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

Many carcinoma cells secrete transforming growth factor alpha (TGF alpha). A 23 base anti-sense oligonucleotide that recognizes the TGF alpha mRNA inhibits both DNA synthesis and the proliferation of the colon carcinoma cell line LIM 1215. The effects of the anti-sense TGF alpha oligonucleotide are reversed by epidermal growth factor (EGF) at 20 ng/ml. When the LIM 1215 cells are grown under serum free conditions, the anti-sense TGF alpha oligonucleotides have their greatest effects at high cell density (2 x 10(5) cells/cm2), indicating that the secreted TGF alpha is acting as an exogenous growth stimulus. In addition, at higher cell densities, the kinase activity of the EGF receptor is activated and the receptor is down-modulated. The cell density dependent activation of the EGF receptor is inhibited by the application of the antisense TGF alpha oligonucleotides.

Topics: Amino Acid Sequence; Base Sequence; Blotting, Western; Cell Division; Colonic Neoplasms; DNA, Neoplasm; Down-Regulation; Epidermal Growth Factor; Molecular Sequence Data; Oligonucleotides, Antisense; Precipitin Tests; Radioligand Assay; Transforming Growth Factor alpha; Tumor Cells, Cultured

Human colon cancer cells produce and secrete a variety of polypeptide growth factors. The functional role of these growth factors, however, is poorly understood. Though the secretion of epidermal growth factor (EGF)-like activity and EGF-related molecules by human colon cancer cells in culture has been reported, it is not known whether colon cancer cells produce and secrete EGF, and the functional role of EGF in the growth control of these cells is also unknown. We have shown that EGF acts as a potent growth stimulator on the moderately differentiated Moser colon cancer cell line and as an inhibitor on the highly metastatic KM12SM cell line. In the present study, we show that EGF is produced by human colon cancer cells and characterize the levels of EGF mRNA expression and EGF protein secretion from 8 human colon cancer cell lines. The cell-surface EGF receptors on these cell lines were also characterized by radiolabeled ligand binding and Scatchard analyses. All the cell lines expressed EGF mRNA and secreted EGF. Both high- and low-affinity subtypes of EGF receptor were detected on 7 of the cell lines. These lines also secreted transforming growth factor (TGF)alpha. Some cell lines exhibited a proliferative response to treatment with either exogenous EGF or TGF alpha, while others did not respond to treatment with these growth factors. Antibody-blocking experiments, using anti-EGF or anti-EGF receptor antibody, suggested that these cell lines could be broadly classified into 2 groups in terms of their autocrine or paracrine growth regulation via the cell-surface EGF receptor: (1) cells that utilized EGF and/or TGF alpha; and (2) cells that did not utilize EGF or TGF alpha (via the cell-surface receptor), even though they secreted abundant amounts of these growth factors.

Topics: Blotting, Northern; Cell Division; Colonic Neoplasms; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Humans; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

Six human colon tumor cell lines were analyzed for their constitutive levels of the c-myc protein. The nuclear proto-oncogene, c-myc, was detected as an expressed product in all of the human colon tumor cell lines analyzed. The poorly differentiated cell lines HCT116, RKO and C showed c-myc levels that averaged 2-fold greater than their well-differentiated counterparts, i.e., GEO, CBS and FET. When c-myc levels and responses to serum induction were analyzed in the presence of inducers of differentiation, i.e., dimethylformamide, retinoic acid, sodium butyrate and TGF-beta, distinct patterns of sensitivity and resistance emerged. Nuclear c-myc levels were reduced in all the colon cell phenotypes treated with dimethylformamide or sodium butyrate. Only the well-differentiated human colon tumor cell lines were responsive to transforming growth factor-beta. Only one of the human colon tumor cell lines (GEO) responded to retinoic acid. Increased levels of c-myc protein were found to correlate well with greater growth rates and with poor differentiation class. Similarly, a parallel sensitivity to down-regulation of c-myc levels and attenuation of c-myc induction curves for inducers of differentiation were observed in growth sensitive human colon tumor cell lines.

Topics: Butyrates; Butyric Acid; Cell Division; Cell Nucleus; Colonic Neoplasms; Dimethylformamide; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation, Neoplastic; Humans; Proto-Oncogene Mas; Proto-Oncogene Proteins c-myc; Transforming Growth Factor alpha; Tretinoin; Tumor Cells, Cultured

A synthetic DNA template has been constructed that is suitable for the quantitation of mRNAs encoding gastrin, transforming growth factor alpha (TGF alpha), cholecystokinin, and the 78-kDa gastrin-binding protein. The template was used to measure levels of gastrin and TGF alpha mRNA in 7 colonic and 2 gastric carcinoma cell lines by the polymerase chain reaction. All lines produced detectable gastrin and TGF alpha mRNA with amounts varying between 2.1 and 540 molecules of gastrin mRNA/10(3) cells and 1.1 and 28 molecules of TGF alpha mRNA/10(3) cells. These results are consistent with the hypothesis that both gastrin and TGF alpha act as autocrine growth factors in colon carcinoma cell lines.

Topics: 3T3 Cells; Animals; Base Sequence; CHO Cells; Colonic Neoplasms; Cricetinae; Gastrins; Humans; Mice; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Stomach Neoplasms; Transforming Growth Factor alpha; Tumor Cells, Cultured

Amphiregulin (AR) is a heparin-regulated, epidermal growth factor-like growth factor capable of stimulating the proliferation of non-tumorigenic cells while inhibiting cell proliferation in some human tumor cell lines in vitro. In the present study, we have investigated AR mRNA expression in normal, hyperproliferative, and neoplastic human epithelium. Our results demonstrate that, compared with the adjacent uninvolved epithelium, AR mRNA expression is markedly elevated in epidermal biopsies derived from three human psoriatic lesions as well as in biopsies derived from five human colon carcinomas and three human stomach carcinomas. Moreover, analysis of a colon carcinoma by in situ hybridization revealed that AR mRNA is localized to the epithelium.

Topics: Amphiregulin; Base Sequence; Blotting, Northern; Colonic Neoplasms; EGF Family of Proteins; Epithelial Cells; Epithelium; Glycoproteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Molecular Sequence Data; Oligodeoxyribonucleotides; Oligonucleotides, Antisense; Psoriasis; Reference Values; RNA, Messenger; Skin; Stomach Neoplasms; Transforming Growth Factor alpha

Four human colon adenocarcinoma cell lines, SNU-C1, SNU-C4, SNU-C5, and NCI-H716, that are capable of proliferating autonomously in serum-free medium containing no added peptide growth factors were identified. All four cell lines show epidermal growth factor (EGF) receptors (EGFRs), express transforming growth factor alpha (TGF-alpha) messenger RNA, and release anti-TGF-alpha-immunoreactive molecules. The blocking anti-EGFR monoclonal antibody (mAb) 225 blocks autonomous proliferation of SNU-C1 and SNU-C4 cells. In both of these cell lines, the inhibitory effect of mAb 225 is reversible by the addition of EGF, TGF-alpha, or conditioned medium from any of the four cell lines. In contrast, autonomous proliferation of SNU-C5 and NCI-H716 cells is not inhibited by mAb 225 and is not affected by exogenous EGF, TGF-alpha, or conditioned medium. Together, these data confirm the previous finding that anti-EGFR antibodies can inhibit the proliferation of some carcinoma cell lines that coexpress TGF-alpha and EGFR. However, here it is shown that the mechanisms of autonomous proliferation of colon carcinoma cell lines are heterogeneous and not always sensitive to antibody disruption of TGF-alpha/EGFR autocrine interactions.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Base Sequence; Cell Division; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Humans; Molecular Sequence Data; Radioimmunoassay; Radioligand Assay; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

A panel of rat colon adenocarcinoma cell lines (the Per series) were used to investigate the phenotype and karyotype changes induced by in vivo passage in the subcutis of athymic nude mice. One poorly and one well-differentiated tumor cell line were serially passaged through the athymic nude mouse and then back to the syngeneic rat host. Each of the primary and xenograft cell lines expressed fetal crypt cell ("CaCo") antigens. The well differentiated primary and xenograft lines (Per305, Per305N1, and Per305N2a) were different in each of their growth factor responsiveness in vitro [i.e. epidermal growth factor (EGF), bombesin, vasoactive intestinal peptide], their EGF receptor expression, their secretion of transforming growth factor-alpha, and their exhibition of anchorage independent (A-I) growth capabilities. The poorly differentiated primary and xenograft cell lines were also different but were all capable of A-I growth; their responsiveness to exogenous growth factor stimulation decreased with progressive in vivo passage, as did their basal unstimulated proliferation rate. Cytogenetic alterations detected were those associated with clinical specimens from various stages of malignancy, i. e. aneuploidy, structural aberrations, and marker chromosomes. Genetic and mitogenic individuality of each line demonstrated the diversity of the growth control mechanisms in neoplasms at different stages of progression.

Topics: Adenocarcinoma; Animals; Biomarkers, Tumor; Bombesin; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Karyotyping; Male; Mice; Mice, Nude; Neoplasm Transplantation; Phenotype; Receptors, Bombesin; Receptors, Neurotransmitter; Transforming Growth Factor alpha; Transplantation, Heterologous; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Amphiregulin (AR) and cripto are proteins that are structurally related to epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha). AR is also functionally related to this family of growth regulatory molecules and is able to bind and activate the 170-kDa EGF receptor (EGFR). Human EGFR-3 (HER3)/ERBB3 is a recently identified protein related to the EGFR that is widely expressed in breast carcinomas and is a candidate receptor for EGF-like growth factors. Differential expression of these putative ligands and receptors in transformed cells suggests that they may function in an autocrine manner to regulate tumor cell growth. Specific mRNA transcripts for TGF-alpha [4.8 kilobases (kb)], AR (1.4 kb), cripto (2.2 kb), and HER3 (6.2 kb) were expressed in a majority of human colon cancer cell lines. HER3 mRNA was detected in 55% of primary or metastatic human colorectal carcinomas but in only 22% of normal colon mucosa and 32% of normal liver samples. In contrast, cripto and AR mRNA were expressed in 60-70% of primary or metastatic human colorectal cancers but in only 2-7% of normal human colonic mucosa. Immunostaining also detected AR protein in primary and metastatic colorectal tumors but not in normal colon or uninvolved liver. These findings suggest that cripto and AR may be useful markers to discriminate between normal and malignant colonic epithelium and may provide a selective growth advantage for colorectal carcinomas.

Topics: Actins; Adenocarcinoma; Amphiregulin; Blotting, Northern; Cell Line; Colon; Colonic Neoplasms; EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Glycoproteins; GPI-Linked Proteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Liver; Membrane Glycoproteins; Neoplasm Proteins; Restriction Mapping; RNA; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha

Epidermal growth factor receptors and insulin like growth factor-I receptors were co-expressed on two gastric and three colorectal tumour cell lines. Previous studies have shown that gastrin receptors were also expressed at a low level or two of these cell lines. Both TGF alpha and IGF-I promoted cell growth in all of the cell lines tested. The cell doubling time of a colorectal cell line was reduced from 48 to 30-34 h. Furthermore the effects of the growth factors were additive. Each growth factor also increased the response of the cells to gastrin, but a combination of both growth factors and gastrin did not further increase growth.

Topics: Cell Division; Colonic Neoplasms; Drug Synergism; Drug Therapy, Combination; ErbB Receptors; Gastrins; Humans; Insulin-Like Growth Factor I; Mitogens; Receptors, Cell Surface; Receptors, Somatomedin; Stomach Neoplasms; Transforming Growth Factor alpha; Tumor Cells, Cultured

The functional role of epidermal growth factor (EGF) in epithelium-derived human colonic carcinoma cells was investigated by transfection with plasmid pUCDS3, which contained synthetic human EGF encoding sequences, into two human colonic carcinoma cell types with dissimilar phenotypic properties: the moderately differentiated and growth factor-responsive Moser and the highly metastatic KM12SM cells. The Moser cells exhibited a proliferative response to treatment with exogenous EGF, while the KM12SM cells did not. The constitutive expression of the human EGF gene in these colonic carcinoma cell types resulted in elevated expression of EGF mRNA, with concurrent production and secretion of a large amount of EGF, and downmodulation of transforming growth factor-alpha (TGF-alpha) secretion. Growth stimulation and down-modulation of both high and low affinity EGF receptors were observed in the EGF-transfected Moser clones. Results of experiments using anti-EGF and anti-EGF-receptor antibody to block the proliferation of EGF-transfected Moser clones suggested that autocrine stimulatory mechanisms involving both EGF and TGF-alpha were operative in these cells. By comparison, a growth-inhibitory effect, with no apparent EGF receptor modulation, was observed in the EGF-transfected KM12SM clones. Both the parental and EGF-transfected KM12SM clones possessed fewer EGF receptors than the Moser cells, and anti-EGF or anti-EGF-receptor antibody did not affect the cells' growth properties. These results suggested that the mechanisms of growth inhibition in the EGF-transfected KM12SM clones were non-autocrine or intracellular in nature. Thus, constitutive expression of the human EGF gene in two phenotypically different, epithelium-derived human colonic carcinoma cells resulted in divergent altered growth characteristics.

Topics: Cell Division; Colonic Neoplasms; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Humans; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

Antibodies to epidermal-growth-factor receptor (EGF) and transforming growth factor-alpha (TGF-alpha) were used to determine the role of endogenous TGF-alpha in the growth of 2 human colon-carcinoma cell lines. Both the GEO and HCT 116 colon-carcinoma cell lines secrete similar levels of TGF-alpha and have similar numbers of low-affinity binding sites for EGF. However, the HCT 116 cells lack the high-affinity EGF binding site present on the GEO cells. The anti-EGF receptor antibodies effectively blocked the binding of 125I-EGF to the GEO and HCT 116 cell lines. Growth of the GEO cell line was inhibited 50-80% by the anti-EGF receptor and anti-TGF-alpha antibodies. When the same antibodies, in sufficient amounts to block binding of TGF-alpha to the cells, were added to the HCT 116 cell line, no effect on growth was seen. These results suggest that while the GEO cell line utilizes TGF-alpha in an autocrine manner, the TGF-alpha secreted by the HCT 116 cells apparently does not play a role in the growth of these cells.

Topics: Animals; Antibodies; Cell Division; Cell Line; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Humans; Mice; Transforming Growth Factor alpha

A previous report from this laboratory indicated that a transformed fibroblast cell line up-regulated c-myc by as much as 14-fold as cultures approached saturating densities, whereas the untransformed counterparts displayed little alteration in c-myc expression (Cancer Res., 49: 2320, 1989). The results suggested a mechanism for the growth advantage of the transformed cells at postconfluent densities. Similarly, the present results indicate that regulation of c-myc expression during establishment of a quiescent state markedly differed in poorly differentiated versus well-differentiated human colon carcinoma cells. While c-myc expression increased 2- to 3-fold during this period in the poorly differentiated cells, expression levels for this protooncogene showed little variation in the well-differentiated cells. There was, however, no correlation between degree of differentiation and c-myc mRNA levels in growing cultures (i.e., cells in late log phase). Another proliferation-associated mRNA, transforming growth factor alpha (TGF-alpha), was also differentially regulated in the two groups of colon carcinoma cells as cultures approached quiescence. Further, addition of exogenous growth-stimulatory factors (epidermal growth factor plus insulin plus transferrin) to quiescent, well-differentiated cells resulted in an up-regulation of TGF-alpha mRNA levels by 9-fold over a 24-h period. In contrast, poorly differentiated cells displayed little alteration in TGF-alpha mRNA levels under similar conditions. The results suggest that inappropriate kinetic regulation of c-myc and TGF-alpha mRNAs at quiescence may be related to the growth factor independence of the poorly differentiated colon carcinoma cells. Furthermore, altered temporal regulation of c-myc and TGF-alpha expression appears to be more relevant to differentiation status in human colon carcinoma cells than are absolute expression levels.

Topics: Carcinoma; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; RNA, Messenger; Time Factors; Transforming Growth Factor alpha; Tumor Cells, Cultured; Up-Regulation

The role of autocrine growth factors in tumor cell growth has been difficult to prove. Our results indicate that more than one autocrine factor is required for the autonomous growth of the LIM 1215 colonic carcinoma cell line. Furthermore, the morphologic changes induced by epidermal growth factor (EGF) are also density dependent and appear to require a synergistic autocrine factor. The serum-free proliferation of the colonic carcinoma cell line LIM 1215 depends on cell density and the presence of EGF (A. Sizeland, S. Bol, and A.W. Burgess, Growth Factors 4:129-143, 1991). At cell densities below 10(4)/cm2, conditioned medium (from cells at a density of 10(5)/cm2) was required for the cells to elicit a mitogenic response to exogenous EGF. At higher cell densities (10(5)/cm2), the cells were independent of both exogenous EGF and conditioned medium. In addition, the EGF receptor was found to be phosphorylated on tyrosine in LIM 1215 cells proliferating at high density, suggesting that the autocrine production of transforming growth factor alpha (TGF alpha) and subsequent ligation to the EGF receptor was occurring. The proliferation of cells at high density was partly inhibited by TGF alpha antibodies but was almost completely inhibited by an antisense oligonucleotide to TGF alpha. The antisense inhibition could be overcome by the addition of EGF, indicating that the effect of the antisense TGF alpha oligonucleotide was on the production of autocrine TGF alpha. LIM 1215 cells were also observed to undergo morphologic changes (spreading and actin cable organization) in response to EGF. These changes were density dependent, but they occurred with a cell density dependence different from that of the proliferative response. These results suggest two possibilities: that the morphologic changes and proliferative responses have different sensitivities to the autocrine factors or that the actions of the autocrine factors are mediated through different signal transduction pathways.

Topics: Actins; Amino Acid Sequence; Antisense Elements (Genetics); Base Sequence; Blotting, Western; Cell Division; Cell Line; Colonic Neoplasms; Culture Media; DNA Replication; Epidermal Growth Factor; Humans; Kinetics; Molecular Sequence Data; Oligonucleotide Probes; Thymidine; Transforming Growth Factor alpha

The expression of TGF-alpha in human colon and lung carcinoma cell lines has been reported previously, but its expression in primary tumours has not been described in detail. We have used the radio-immunoassay method to measure the specific content of immunoreactive TGF-alpha in the acid ethanol extracts of normal and cancerous tissues of human colon and lung. The average TGF-alpha content of colon carcinomas is 4 times that of the normal mucosa, and for non-small cell lung carcinomas it is twice that of the normal parenchyma. Because of variability in the TGF-alpha expression among individuals and in different segments of colon and lobes of lung, the ratio of TGF-alpha content of paired tumour and normal tissue was also calculated. On average, the tumour/normal ratio for colon carcinoma is higher than that for lung carcinoma. Although 55% of colon tumours show a ratio 4 times, or greater, only 33% of lung carcinomas demonstrate this ratio. The level of TGF-alpha in both colon and lung carcinomas does not correlate with histological type stage, grade nor degree of desmoplasia of these tumours. Northern blot analysis of total cellular RNA confirms the expression of an approximately 4.8 kb TGF-alpha mRNA in normal colonic mucosa and lung parenchyma. However, in contrast to the results of radio-immunoassay, significant over-expression of TGF-alpha mRNA is uncommon in primary human colon carcinomas.

Topics: Blotting, Northern; Colonic Neoplasms; Gene Expression; Humans; Intestinal Mucosa; Lung; Lung Neoplasms; Nucleic Acid Hybridization; Radioimmunoassay; RNA; RNA, Messenger; Transforming Growth Factor alpha

Previously, we reported that exponentially proliferating cultures of well-differentiated human colon carcinoma cells responded to transforming growth factor beta 1 (TGF-beta) with growth inhibition, alterations in morphology, and increased secretion of the differentiation marker, carcinoembryonic antigen. Poorly differentiated cultures were unresponsive. Here we show that TGF-beta was ineffective in repressing nutrient-stimulated mitogenesis in quiescent, poorly differentiated cells. However, in quiescent, well-differentiated cells, TGF-beta repressed the mitogenic responses to both nutrients alone (by 90%) and to nutrients plus the exogenous stimulatory factors epidermal growth factor (E), insulin (I), and transferrin (T) (by 55-65%). Thymidine incorporation experiments indicated that TGF-beta reduced both the onset and peak mitogenic response to growth factors and/or nutrients in the well-differentiated cells. Additionally, TGF-beta repressed the growth factor (E + I + T)-stimulated upregulation of expression of both c-myc and of transforming growth factor alpha (TGF-alpha) mRNAs in quiescent, well-differentiated cells. TGF-beta also elicited a rapid (t1/2 approximately 1h) down-regulation of c-myc expression in the absence of prior growth factor (E + I + T) stimulation. In contrast, TGF-beta had no effect on c-myc or TGF-alpha mRNA expression in the poorly differentiated cells. The results suggest that TGF-beta exerts rapid inhibitory effects on proliferation-associated genes in quiescent and restimulated, well-differentiated cells. Expression of these genes (c-myc and TGF-alpha) may otherwise (in the absence of TGF-beta) play roles in the cellular signaling of mitogenic responses by growth stimulatory factors in well-differentiated colon carcinoma cells.

Topics: Blotting, Northern; Cell Line; Colonic Neoplasms; Down-Regulation; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Insulin; Mitosis; Proto-Oncogene Proteins c-myc; RNA; Transferrin; Transforming Growth Factor alpha; Transforming Growth Factor beta

The existence of an autocrine loop for self-stimulation of growth in malignant cells has been proposed for transforming growth factor-alpha (TGF alpha) and its receptor, the epidermal growth factor (EGF) receptor, in a variety of malignant cell types. Expression of both has been described in colon carcinoma. In order to investigate whether there is a correlation between TGF alpha and EGF receptor mRNA expression and differentiation, we studied the effects of differentiating agents on seven human colon carcinoma cell lines. All of the lines responded to the differentiating agents. In four of the seven lines there was increased EGF receptor mRNA two to five days after treatment with 2 mM sodium butyrate. In three of these lines TGF alpha mRNA and protein were also increased. In the one cell line treated with the differentiating agents DMF and DMSO, EGF receptor mRNA was also increased. [125I]-EGF binding to the cells was measured before and after treatment with butyrate. In two of three cell lines, increased EGF receptor mRNA was accompanied by a 2.4-fold increase in the number of binding sites per cell. In SW620 cells, no EGF receptor binding was detected before or after butyrate treatment. In the two cell lines in which butyrate increased EGF receptor binding, simultaneous treatment with EGF did not enhance growth. These data demonstrate increased expression of the TGF alpha/EGF receptor system after differentiation of colon carcinoma cell lines and suggest that their expression may be characteristic of a differentiated phenotype.

Topics: Alkaline Phosphatase; Blotting, Northern; Butyrates; Butyric Acid; Cell Differentiation; Cell Division; Cell Line; Colonic Neoplasms; Dimethyl Sulfoxide; Dimethylformamide; ErbB Receptors; Gene Expression Regulation, Neoplastic; Radioimmunoassay; RNA, Messenger; Transforming Growth Factor alpha

A cDNA encoding transforming growth factor type alpha (TGF alpha) was fused to the 5' end of a gene encoding a modified form of Pseudomonas exotoxin A (PE), which is devoid of the cell recognition domain (domain Ia). The chimeric molecule, termed TGF alpha-PE40, was expressed in Escherichia coli and isolated from the periplasm or inclusion bodies depending on the construction expressed. TGF alpha-PE40 was found to be extremely cytotoxic to cells displaying epidermal growth factor (EGF) receptors. Comparison with a similar molecule in which TGF alpha was placed at the carboxyl end of PE40 demonstrated the importance of the position of the cell recognition element; TGF alpha-PE40 was found to be about 30-fold more cytotoxic to cells bearing EGF receptors than PE40-TGF alpha. In addition, TGF alpha-PE40 was shown to be extremely cytotoxic to a variety of cancer cell lines including liver, ovarian, and colon cancer cell lines, indicating high levels of EGF receptor expression in these cells.

Topics: ADP Ribose Transferases; Bacterial Toxins; Base Sequence; Binding, Competitive; Carcinoma, Hepatocellular; Cell Survival; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Escherichia coli; Exotoxins; Female; Gene Expression; Liver Neoplasms; Molecular Sequence Data; Ovarian Neoplasms; Pseudomonas aeruginosa Exotoxin A; Recombinant Fusion Proteins; Structure-Activity Relationship; Transforming Growth Factor alpha; Transforming Growth Factors; Tumor Cells, Cultured; Virulence Factors