GD3, a prominent ganglioside of human melanoma. Detection and characterisation by mouse monoclonal antibody.

Mouse monoclonal antibody AbR24 has a high degree of specificity for human melanoma cells when tested on viable cultured cells using the protein A mixed hemagglutinin serological assay. The antigen detected by this antibody has been isolated from melanoma cells and shown to be GD3 ganglioside by compositional and partial structural analysis and by comparison with authentic GD3 in thin layer chromatography (TLC). AbR24 reacts with authentic GD3, but not with any other ganglioside tested. Using TLC and reactivity with AbR24, a wide range of cells and tissues was examined for the presence of GD3. A new serological assay, termed glycolipid-mediated immune adherence, was devised for assaying the reactivity of AbR24 with gangliosides. Melanomas (cultured cells or tumor tissue) were shown to have GD3 and GM3 as major gangliosides. Other cells and tissues examined also contained GD3, but usually only in low amounts. Melanomas (and MOLT-4, a T cell line) were characterized by a simplified ganglioside profile with GD3 and GM3 as major components. The apparent discrepancy between the ubiquitous presence of GD3 and the serological specificity of AbR24 for melanoma cells can be explained in terms of localization and concentration of GD3 in different cells.

Modes of binding and internalization of monoclonal antibodies to human melanoma cell lines.

The process of monoclonal antibody (MAb) binding to tumor cells is greatly influenced by the biology of the respective antigen. This was concluded from an analysis of binding and release of MAbs and MAb fragments to melanoma cells at different concentration levels and different temperatures. With an antigen known to be stably expressed at the cell surface (i.e., Mr 97,000 protein) rapid binding of MAbs was observed at both 0 degrees C and 37 degrees C, and this was reversed by treatment with isoosmolar acid buffer. With another group of antigens, MAb binding increased continuously up to considerable levels at 37 degrees C, but not at 0 degrees C. Concomitantly, the portion of radioactive MAb not desorbable by acid buffer treatment increased, pointing to temperature-dependent internalization. With still another group of (glycolipid) antigens, the highest MAb binding was obtained with fixed cells at 0 degrees C. In this situation MAb release was particularly rapid, thus pointing to a shedding process.

Inhibition of human melanoma cell growth in vitro by monoclonal anti-GD3-ganglioside antibody.

Malignant melanoma cell growth in vitro was blocked by the monoclonal antibody R-24, which detects the disialoganglioside GD3 on melanoma cells. GD3 expression varies greatly in cultured human malignant melanoma cells. The level of ganglioside GD3 was measured by quantitative absorption tests. In our observations, six melanoma cell lines expressing high levels of GD3 on the cell surface showed growth inhibition and rounding up in the presence of R-24 antibody. Four melanoma cell lines with low levels of GD3 and seven nonmelanoma cell lines with no detectable GD3 remained unchanged in morphology and continued to grow. The data presented indicate that complement is not involved in the growth inhibition observed here. Because ganglioside GD3 was detected in all 16 tissue specimens of primary and metastatic human malignant melanoma examined by immunofluorescence tests, the possible relevance of this finding in vivo is discussed.

Immunohistochemical localization of ganglioside GD3 in human malignant melanoma, epithelial tumors, and normal tissues.

The specific tissue distribution of melanoma-associated ganglioside II3-alpha-N-acetylneuraminosyl-alpha 2----8-N-acetylneuraminosyllactosylceramide (GD3) was studied on 175 cryopreserved, unfixed human tissue sections with R-24 mouse monoclonal antibody by indirect immunoperoxidase staining. A striking specificity of monoclonal antibody R-24 for malignant melanoma tissues was established. Ganglioside GD3 was detected in all 21 tissue sections of 21 patients with primary melanoma and in all 37 probes of 24 patients with metastatic malignant melanoma. The majority of tumor cells in the samples of primary malignant melanoma expressed GD3; however, GD3 expression was more heterogeneous in samples of metastatic lesions even in different metastases of the same patient. Of 11 nevi, 9 reacted with monoclonal antibody R-24, while melanocytes in the basal layer of normal skin stained only weakly and irregularly. None of the 32 normal and 12 fetal human tissue types were R-24 positive, but a strong cytoplasmic staining was observed with single cells in the dermis and in the interstitial tissue of the gastrointestinal tract, in the interlobular septa of the thymus, and in other distinct locations. Only two malignant carcinoid tumors of 38 nonmelanomatous tumors tested reacted with monoclonal antibody R-24.

Secretory epithelial cell marker on gastrointestinal tumors and in human secretions defined by a monoclonal antibody.

A new marker for human secretory epithelial cell types (Exo-1) has been defined by a mouse monoclonal antibody (Pa-G14). The antibody was raised against a human exocrine pancreatic tumor cell line (Capan-1) and tested against 46 cultured human cell types and 228 freshly frozen human tissue sections. It reacted specifically with 28 normal and 55 secretory neoplastic epithelial tissues tested. Eleven different secretory epithelial cell types expressed this antigen, as well as human fetal tissues of the gut and bronchi. One hundred and twenty samples of normal tissues, cells, and tumors of nonexocrine origin were Exo-1 negative. In normal secretory tissues staining was most pronounced at the apical poles and as shown by immunoelectron microscopy in the case of the duodenum, at the microvilli. In cultured Exo-1 positive tumor cells the antigen was not demonstrable on the cell surface but in the cytoplasm after acetone/methanol fixation only. The antigen was identified biochemically as a polar neutral glycolipid and detected in human salivary, bronchial, pancreatic, and intestinal secretions by an enzyme-linked immunosorbent assay, but was not found in sera of healthy controls or patients with gastrointestinal and other tumors. Antigen Exo-1 represents a novel common antigen for normal and tumorous glandular epithelial cells.

Melanoma-associated gangliosides in the fish genus Xiphophorus.

Gangliosides from benign and malignant melanomas and from normal skin of the fish genus Xiphophorus were isolated and analyzed by thin-layer chromatography. Individual ganglioside components were characterized by mapping according to their sialic acid content and by cleavage with neuraminidases. In all three tissues examined, sulfatide and the gangliosides NeuAc-GalCer (GM4), II3NeuAc-LacCer (GM3), II3NeuAc-GgOse3Cer (GM2), and II3(NeuAc)2-LacCer (GD3) were found. Ganglioside GD3 yielded a positive reaction, following immunoadsorption with mouse monoclonal antibody R24 on thin-layer plates. Two alkali-labile disialoganglioside species were specifically recognized by mouse monoclonal antibody D1.1, thus indicating the presence of O-acetyl-neuraminic acid residues. One of them, a major ganglioside component of the malignant melanoma, was identified as O-acetyl-GD3, since it could be converted to the R24-positive GD3 ganglioside after alkaline saponification. The other one appears to be restricted to the malignant tumor and represents a novel melanoma-associated ganglioside derivative. It was characterized as O-acetyl(NeuAc)2-nLc4Cer by exoglycosidase cleavage, by proving its neutral carbohydrate backbone as type II-chain lacto-series oligosaccharide using mouse monoclonal antibody 1B2, and by its cross-reaction with antibody R24 following alkaline treatment. Using antibody R24 and cryopreserved tissue sections of both benign and malignant amelanotic melanomas from albino fishes, it was demonstrated that one of the main melanoma-associated gangliosides, GD3, was exposed predominantly in the malignant tumor. Thus, the chemical nature and even the immunohistochemical localization of the gangliosides in fish melanomas proved to be very similar to those of the known gangliosides in the phylogenetically distant human melanomas.

A common epithelial cell surface antigen (EPM-1) on gastrointestinal tumors and in human sera.

A common epithelial cell surface marker (EPM-1) was defined by two monoclonal antibodies (Pa-25 and Pa-42), raised against a pancreatic tumor cell line (Capan-1). Both monoclonal antibodies were tested on 49 cultured human cell lines and 244 tissue samples and reacted with all 76 tissue samples of 20 different normal epithelial cell types and with 57 of 63 epithelial tumor tissue samples of nonendocrine origin. Included were eight exocrine pancreatic carcinomas. There the percentage of EPM-1 positive tumor cells correlated with tumor grading. EPM-1 was detectable on the cell surface of cultured human cell lines of the pancreas, liver, colon, and mamma. Some epithelial tumor cell lines did not express EPM-1 on the cell surface, but in the cytoplasm. 100 nonepithelial and epithelial endocrine tissue samples as well as 25 nonepithelial cultured tumor and normal cells were unreactive with monoclonal antibodies Pa-25 and Pa-42. These included cells of neuronal, endocrine, and mesenchymal origin. EPM-1 activity was purified from pancreas tumor cells Capan-1 and from human sera by high-performance liquid chromatography and its molecular weight amounts to about Mr 400,000. EPM-1 was detectable in bronchial, intestinal, and pancreatic secretions and saliva and serum by an enzyme-linked immunosorbent assay test. EPM-1 values were high in the sera of normal individuals, but low or not detectable in most sera (21 of 31) of patients with gastrointestinal tumors. EPM-1 represents a novel differentiation marker for epithelial cell types, which should have a central role in the biology of normal and tumorous epithelial cells.

Expression of epithelial antigens Exo-1 and EPM-1 in human epidermal keratinocyte maturation and benign and malignant neoplasia.

Exo-1, a polar neutral glycolipid, and EPM-1, a high molecular weight glycoprotein, are developmental antigens of human epithelial cells, initially described as components both on the cell surface and in secretions of gastrointestinal epithelial and respective tumors. In order to assess the biological significance of both antigens for epithelial cell differentiation and neoplastic transformation, their expression during human skin development and benign and malignant neoplasia was analyzed in fresh frozen tissue specimens of skin biopsies and of human epidermal keratinocytes growing in experimental model systems. Antigen expression was assessed immunohistochemically with specific monoclonal antibodies. During fetal development Exo-1 was temporarily expressed in intermediate cells but was absent in normal adult human skin. Exo-1 expression reemerged in neoplasias, both benign and malignant, but was restricted to spinous-like differentiated cells. Similarly, Exo-1 was not expressed in transplants of normal keratinocytes mimicking the normal epidermis but was clearly visible in differentiated areas of transplants of malignantly transformed keratinocytes. EPM-1 appeared first in basal epidermal cells in the second half of gestation and remained detectable in the stratum basale of adult skin. While squamous cell carcinomas continued to express EPM-1, it was not detectable in basal cell epitheliomas and in normal epidermis after invasion by neuroectodermal tumor cells. In experimental models, EPM-1 was present in the basal layers of normal human keratinocytes and of transformed keratinocytes with benign growth characteristics whenever a well stratified and keratinized epidermis-like epithelium had formed in transplants. In transformed keratinocytes with malignant growth behavior, EPM-1 was expressed irregularly, as in squamous cell carcinomas in situ. Thus, expression of Exo-1 is a marker for an early embryonic differentiation pathway of human keratinocytes and in adult tissue reveals abnormal differentiation associated with certain stages of hyperproliferation. EPM-1 expression is part of developmental programs and is influenced by microenvironmental interactions and alterations of tissue homeostasis.

CEA-immunoscintigraphy with 99m-technetium correlates with tumour cell differentiation in colorectal cancer.

The clinical usefulness of immunoscintigraphy with the monoclonal anti-CEA (carcinoembryonic antigen) antibody BW431/26, directly labelled with 99m-Technetium in targeting colorectal carcinomas was investigated in 43 patients. In addition, tumour cell grading and CEA-expression were examined immunohistochemically. Best imaging results were obtained in pelvic tumour lesions (sensitivity 80%). Tumour grading correlated with radioimmunoimaging, well differentiated tumours being detectable at a higher rate (P = 0.09). Immunoscintigraphy preceded the findings of conventional diagnostic methods in 3 patients. In 4 cases immunoscintigraphy was decisive for patients management. Therefore, immunoscintigraphy with 99m-Technetium is valuable in directing patients management if conventional diagnostic methods remain undecisive.

Expression of epithelial antigens EPM-1 and EXO-1 in normal, transitional, inflammatory and neoplastic colorectal mucosa.

EPM-1 (a high molecular weight glycoprotein) and EXO-1 (a carbohydrate epitope expressed on polar neutral glycolipids and mucins) are two developmental antigens of normal and neoplastic human epithelia and were characterised by monoclonal antibodies. Their distribution was investigated in normal and pathological human colorectal mucosa. In normal mucosa, EPM-1 and EXO-1 showed characteristic expression patterns. EPM-1 was differentially expressed along the crypt villus axis with maximum at the crypt basis. EXO-1 was present throughout the whole mucosa. The characteristic gradient of EPM-1 expression along the crypt axis in normal mucosa was no longer detectable in benign polyps. Intact gradient of EPM-1 staining discriminated between neoplastic changes of the benign adenomatous polyp and mucosal inflammation. Neoplastic mucosa in benign polyps and especially atypical glands in highly differentiated tumours showed essentially identical expression patterns. In colorectal carcinomas the overall reactivities for EPM-1 and EXO-1 were independently associated with the histopathological grade of tumour differentiation.

Expression and regulation by interferon-gamma of the membrane-bound complement regulators CD46 (MCP), CD55 (DAF) and CD59 in gastrointestinal tumours.

The membrane-bound complement inhibitors CD46 (membrane cofactor protein), CD55 (decay-accelerating factor) and CD59 (protectin) protect tumour cells against lysis by activated complement. In this study, a total of 14 (3 gastric, 3 colonic and 8 pancreatic) gastrointestinal tumour cell lines were examined for the expression of CD46, CD55 and CD59 with respect to the regulatory efficacy of interferon-gamma (IFN-gamma). The effects of IFN-gamma on mRNA and protein expression levels of CD46, CD55 and CD59 were evaluated by Northern blot hybridisation, RT-PCR, flow cytometry and immunostaining. In unstimulated cell lines, CD46 and CD59 transcripts were expressed at comparable levels, whereas the basal expression of CD55 mRNA was heterogeneous. The complement inhibitor proteins were detected in all cell lines using specific antibodies. Additional immunohistochemical stainings of gastrointestinal tissue specimens supported these findings. IFN-gamma evoked a weak induction of certain transcripts in a subset of the cell lines. Upregulation of protein expression was only observed in HT29 cells for CD55 and CD59 and was accompanied by a marked increase of the corresponding transcripts. We conclude that membrane-bound complement inhibitors are broadly expressed in gastrointestinal tumour cells and vary in their susceptibility to IFN-gamma. Thus, they may be involved in tumour escape mechanisms in gastric, pancreatic and colorectal cancer.

Ah receptor mediating induction of cytochrome P450IA1 in a novel continuous human liver cell line (Mz-Hep-1). Detection by binding with [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin and relationship to the activity of aryl hydrocarbon hydroxylase.

The Ah receptor regulates induction of cytochrome P450IA1 and mediates certain toxicities of polyhalogenated aromatics such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). It has been characterized previously in continuous cell lines, notably the mouse hepatoma line Hepa 1, the human squamous cell carcinoma line A431, and the human liver cell line Hep G2. The present work extends our knowledge of the Ah receptor in continuous human liver cell lines. Ah receptor can be detected in Mz-Hep-1, a hepatitis B virus-negative cell line derived from a Thorotrast-induced hepatocellular carcinoma. The mean concentration of Ah receptor in Mz-Hep-1 cells was 341 +/- 22 fmol/mg cytosol protein (mean +/- SEM, nine separate determinations). This is equivalent to approximately 30,000 sites per cell. The concentration of Ah receptor in Mz-Hep-1 cells is similar to that in Hepa 1 cells and approximately three times higher than that in Hep G2 cells. The Mz-Hep-1 Ah receptor sedimented in continuous sucrose gradients at approximately 9 S. Specificity of binding by [3H]TCDD was demonstrated by competitive binding of non-radiolabeled 2,3,7,8-tetrachlorodibenzofuran, 3-methylcholanthrene (MC), and dibenz[a,h]anthracene in 50-fold molar excess. Phenobarbital, which is not a substrate for P450IA1, did not compete with [3H]TCDD for binding to Mz-Hep-1 Ah receptor. Dexamethasone and estradiol also did not compete with [3H]TCDD for binding, suggesting non-identity of Ah receptor with glucocorticoid or estrogen receptor. In separate experiments, glucocorticoid receptor was identified in Mz-Hep-1 cells. By Scatchard plot analysis, the apparent equilibrium dissociation constant (Kd) for binding of [3H]TCDD to Mz-Hep-1 Ah receptor was estimated to be 4.4 nM, compared to 0.8 nM in Hepa 1 cells. By Woolf plot analysis the Kd was 5.4 nM, compared to 1.2 nM in Hepa 1 cells. The [3H]TCDD.Ah receptor complex extracted from nuclei of Mz-Hep-1 cells incubated with [3H]TCDD in culture at 37 degrees sedimented at approximately 6 S under conditions of high ionic strength. Aryl hydrocarbon hydroxylase (AHH) activity was detectable in Mz-Hep-1 cells after pretreatment with inducing chemicals. Mz-Hep-1 cells have the highest concentrations of Ah receptor in any continuous human liver cell line thus far investigated. The Mz-Hep-1 Ah receptor is similar physicochemically to that described in murine systems. AHH activity is inducible in Mz-Hep-1 cells.

New cell lines of gastric and pancreatic cancer: distinct morphology, growth characteristics, expression of epithelial and immunoregulatory antigens.

Two new cell lines from stomach cancers and one from a pancreatic carcinoma are presented. MZ-GC-1 was established from a hepatic metastasis of a well differentiated gastric adenocarcinoma. MZ-GC-2 was derived from ascites induced by a poorly differentiated gastric adenocarcinoma. MZ-PC-1 originated from the pleural effusion of a moderately well differentiated pancreatic ductal adenocarcinoma. MZ-GC-1 cells were adherent and partially polarized, connected tightly via desmosomes. In contrast MZ-GC-2 cells consisted of slightly adherent or floating subpopulations and displayed no desmosomes. MZ-PC-1 cells were adherent and showed polarized growth, connected by apical junctional complexes. Cell doubling times were 7 days for MZ-GC-1 and 45 h for MZ-GC-2 and MZ-PC-1 cells. MZ-GC-2 and MZ-PC-1 gave rise to nude mouse tumours, resembling the original lesions. Chromosome analysis of the cell lines revealed a high range of numerical abnormalities. Each cell line had cytokeratin patterns fitting well to typical in vivo patterns. Furthermore the cell lines expressed a panel of antigens typical for gastrointestinal epithelia. Unique for MZ-PC-1 were high amounts of secreted Ca19-9. gamma-Interferon enhanced HLA-class I antigens up to twofold and induced ICAM-1 expression on each cell line. HLA-class II antigens were differentially enhanced by gamma-interferon. Due to their distinct characteristics the three tumour cell lines may be useful models in the investigation of the cell biology and immunogenicity of gastrointestinal tumours.

[Primary infection with Epstein-Barr virus in a 77-year-old patient with B symptoms and hepatitis -- a case report]. / 77-jähriger Patient mit Epstein-Barr-Virus-Erstinfektion mit B-Symptomatik und Hepatitis -- ein Fallbericht.

Epstein-Barr virus (EBV) infection has a prevalence of 90 % and is - depending on the immune status of the host - associated with a broad spectrum of clinical manifestations. By presenting a case report we would like to demonstrate an unusual clinical course of a primary infection with EBV in an elderly patient. A 77-year-old patient was admitted to hospital in reduced health condition because of a persisting bronchopulmonary infection with B symptoms. The patient had already been treated with antibiotics. Because of elevated liver enzymes, a liver biopsy was performed. Histopathology revealed moderate acute hepatitis with cholangitis und endothelialitis, pointing to an EBV-induced hepatitis. Serological examinations confirmed the diagnosis, revealing a primary infection with positive EBV VCA IgM and IgG. EBV PCR of the liver tissue was positive, viral genome could be demonstrated within lymphocytes. A short period later the patient was discharged to reconvalescence. This case report demonstrates an unusual primary infection with EBV at the age of 77 with atypical clinical symptoms and hepatitis. The relevance of EBV in the differential diagnosis of atypical infectious diseases with hepatitis of unknown aetiology is strengthened on taking data reports from the literature into consideration.

Analysis of the biosynthesis of HLA-DR glycoproteins in human malignant melanoma cell lines.

Human malignant melanoma cell line SK-MEL-37 expresses HLA-DR antigens having a characteristic 2-chain structure, with heavy chains (alpha) of approximately 32,000 daltons and light chains (beta) of approximately 28,000 daltons. Nonequilibrium pH-gradient electrophoresis (NEPHGE) on HLA-DR immunoprecipitated by rabbit anti-HLA-DR sera from [35S]-methionine-labeled, pulse-chased cells showed 2 closely spaced heavy-chain components (1, 2) and 4 light-chain spots (3-6). From nonchased samples, numerous more-basic components running in the heavy chain region were also precipitated. In particular, a very basic, 30,000 dalton component (pl approximately 8.5) was prominent (component 7); this spot probably corresponds to the invariant (Ii) peptide previously demonstrated in lymphoid cell HLA-DR precipitates (9) and the M-1 peptide of Shackelford and Strominger (10). None of these components (alpha, beta, or component 7) was precipitated from extracts of HLA-DR-negative melanoma cells. Pulse-chase experiments and the use of different labeled sugar precursors showed that component 7 is a partially glycosylated intracellular precursor, possibly of the HLA-DR alpha-chain. None of the immunoprecipitates, even from cells pulse-labeled for only 15 min, contained a peptide migrating in the 55,000 to 60,000 m.w. region. It was concluded that melanoma HLA-DR is not synthesized via a polyprotein precursor. In contrast to these results, obtained with rabbit anti-HLA-DR sera, a mouse monoclonal anti-HLA-DR was found to precipitate only biosynthetically completed alpha (1, 2) and beta (3-6) chains.

Immunoreactivity of monoclonal anti-melanoma antibodies in relation to the amount of radioactive iodine substituted to the antibody molecule.

The damage to monoclonal anti-melanoma antibodies caused by iodination was investigated by comparing the results obtained using the chloramine-T method and the 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenyl-glycoluril (IODOGEN) method at different levels of iodine substitution to the molecule. The level of substitution at which losses in immunoreactivity occurred was evaluated in each monoclonal antibody (MAb) studied. This phenomenon was not dependent on the method of substitution, provided that mild conditions of reaction were used. Lineweaver-Burk plots and--in cases of alterations in binding affinity--Scatchard plots were found to provide an adequate description of the binding behaviour of individual MAbs after labelling. Immunoreactivity was shown to be determined not only by the proportion of bona fide reactive MAb molecules, but also by a substitution-dependent decrease in affinity constants. The practical consequences of altered binding parameters were demonstrated by quantitating specific antibody accumulation in melanoma transplants in vivo.

[The treatment of solid tumors with monoclonal antibodies]. / Behandlung solider Tumoren mit monoklonalen Antikörpern.

Patients with B-cell lymphomas, gastrointestinal tumors and melanomas were treated in pilot and phase-I clinical studies with monoclonal antibodies directed to tumor-associated antigens in recent years. Side effects of this new treatment approach were minor. Tumor regressions were observed in all three groups of tumors.

[Malignant melanoma of the mucous membranes of the upper aerodigestive tract. Clinical, histological and immunohistochemical characteristics]. / Maligne Melanome der Schleimhäute des oberen Aerodigestivtraktes. Klinische, histologische und immunhistochemische Charakteristika.

Malignant melanomas of the mucous membranes are rare tumors. They make up about 10% of all malignant melanomas of the head and neck; 15 of the authors' cases are reviewed in this article. Six of these had neck lymph node metastasis when first diagnosed. The tumors were surgically removed in all patients. Thirteen patients developed at least one tumor recurrence, ten patients distant metastasis. Eight patients died of the tumor condition; the mean survival time of all patients was 33.4 months. While the tumors could be classified histologically into four types, this had no bearing on the course of the disease. In many cases, primary tumor and metastasis or recurrent tumor differed histologically. Melanin pigment was found in 13 tumors. Mucosal melanomas can be regarded as a discrete tumor entity because their biological behavior differs from that of malignant melanomas of the skin. However there are no morphological differences between the two tumor entities. Ophthalmological and dermatological examinations must be performed in all patients with mucosal melanoma to exclude metastasis of the skin or choroid melanoma.

Stimulation of pancreas and gastric carcinoma cell growth by interleukin 3 and granulocyte-macrophage colony-stimulating factor.

Hematopoietic growth factors have recently been well characterized by complementary DNA cloning. For human epidermal growth factor, granulocyte-macrophage colony-stimulating factor recombinant proteins have been expressed in Escherichia coli. To reduce the toxic side effects of chemotherapy on the bone marrow, recombinant human granulocyte-macrophage colony-stimulating factor and recombinant human interleukin 3 were applied to patients suffering of gastrointestinal cancers. To determine the influence of recombinant human granulocyte-macrophage colony-stimulating factor and recombinant human interleukin 3 on human pancreas and gastric cancer cell cells in vitro, a sensitive microculture test system was established that allows precise quantification of proliferation. A more than twofold enhancement of proliferation was observed by interleukin 3 and granulocyte-macrophage colony-stimulating factor in two of two cell cultures derived from gastric carcinoma cells, while two of nine cultures from pancreas carcinoma cells have shown enhanced cell growth in the presence of recombinant human interleukin 3 or recombinant human granulocyte-macrophage colony-stimulating factor. In comparison, recombinant human epidermal growth factor increased cell growth in two of two gastric and in five of nine pancreas carcinoma cultures. In general, 1-10 ng/mL of the growth factors yielded the highest growth rate, but even 1-pg amounts produced increased cell growth. Expression of messenger RNA for granulocyte-macrophage colony-stimulating factor, interleukin 3, and the oncogene HER2/neu remained undetectable in all of the tested cell lines, while the various abundance of messenger RNA for the epidermal growth factor receptor was different in each cell line. The reported results imply that the hematopoietic growth factors interleukin 3 and granulocyte-macrophage colony-stimulating factor influence cellular growth of pancreas and gastric carcinoma cells by a paracrine mechanism and may possess a more general regulatory function than originally anticipated.

Distribution of Ha-ras alleles in patients with colorectal cancer and Crohn's disease.

The allele distribution of the Ha-ras gene on chromosome 11p was analysed by the restriction fragment length polymorphism of the enzymes Mspl/Hpall in 238 individuals. The investigation covered 116 patients with colorectal carcinoma and 122 patients with Crohn's disease, representing two patient populations with the same ethnic origin, one with a malignant and the other a benign disease of the same organ system. A total of 17 different alleles were detected belonging to the common, intermediate, and rare classes according to the original nomenclature of Ha-ras alleles. Patients with Crohn's disease showed no difference in the distribution of Ha-ras alleles when compared with expected frequencies. In patients with colorectal carcinoma, the frequency of rare alleles was significantly increased compared with the patients with Crohn's disease (chi 2 = 8.166; Fisher's exact test = 0.005) and with a reference population of 424 cancer free individuals (chi 2 = 49.312; Fisher's exact test = 0.000). Homozygosity was not detected for any rare allele. The occurrence of a rare Ha-ras allele was not linked to the location of the colorectal tumour. These results confirm the hypothesis that unique Ha-ras alleles represent an inherited factor which predisposes the development of colorectal cancer.