Solid-phase adsorption of antigens for efficient production of antibodies reactive with native and fixed tissue antigens.

A study has been made of the efficacy of different immunization protocols using low antigen levels for the generation of monoclonal antibodies capable of detecting antigens (ADCP) in processed tissues. Protocols using unprocessed native antigen immobilized on nitrocellulose were more efficient than soluble antigen in generating serum antibodies reactive with both native antigen and processed tissues. The derived monoclonal antibodies reacted with native but not processed antigen. The use of antigen immobilized on polyvinylidene (PVDF) and subsequently processed as for histochemistry was successful in generating monoclonal antibodies reactive with processed antigen.

Distribution of adenosine deaminase complexing protein (ADCP) in human tissues.

The normal distribution of adenosine deaminase complexing protein (ADCP) in the human body was investigated quantitatively by ADCP-specific radioimmunoassay (RIA) and qualitatively by immunohistochemistry. In these studies we used a specific rabbit anti-human ADCP antiserum. In all 19 investigated tissues, except erythrocytes, ADCP was found by RIA in the soluble and membrane fractions. From all tissues the membrane fractions contained more ADCP (expressed per mg protein) than the soluble fractions. High membrane ADCP concentrations were found in skin, renal cortex, gastrointestinal tract, and prostate. Immunoperoxidase staining confirmed the predominant membrane-associated localization of the protein. In serous sweat glands, convoluted tubules of renal cortex, bile canaliculi, gastrointestinal tract, lung, pancreas, prostate gland, salivary gland, gallbladder, mammary gland, and uterus, ADCP immunoreactivity was found confined to the luminal membranes of the epithelial cells. These data demonstrate that ADCP is present predominantly in exocrine glands and absorptive epithelia. The localization of ADCP at the secretory or absorptive apex of the cells suggests that the function of ADCP is related to the secretory and/or absorptive process.

Expression of integrins and E-cadherin in cells from menstrual effluent, endometrium, peritoneal fluid, peritoneum, and endometriosis.

OBJECTIVE: To detect the expression of integrins and E-cadherin in cells from peritoneal fluid (PF), endometrium, menstrual effluent, peritoneum, and endometriotic lesions during the early follicular phase of the menstrual cycle. DESIGN: An immunohistochemical study. SETTING: Tertiary care university medical center. PATIENTS: Sixteen patients undergoing a diagnostic laparoscopy as part of a subfertility work-up. All patients had regular and ovulatory cycles. INTERVENTIONS: A laparoscopy was performed in the early follicular phase (days 2 to 5). Simultaneously, samples were taken from endometrium, menstrual effluent, and PF, and a representative biopsy of an endometriotic lesion was obtained. If endometriosis was not noted, a peritoneal biopsy was obtained instead. MAIN OUTCOME MEASURES: The expression of cell adhesion molecules, including the integrin alpha 2 beta 1, alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, and alpha 6 beta 1 and E-cadherin, as determined by immunohistochemistry on frozen sections. RESULTS: All integrins tested could be detected in the endometrium samples and in endometriotic lesions. In menstrual effluent samples, positive staining for the integrins alpha 2 beta 1 and alpha 3 beta 1 was found in epithelial cells in 13 of 16 cases. Integrin alpha 5 beta 1 was detected in 11 of 16 samples, and integrins alpha 4 beta 1 and alpha 6 beta 1 were detected in 5 of 16 samples. In PF, integrin alpha 3 beta 1 was found in epithelial cells in 12 of 16 samples, integrin alpha 5 beta 1 in 5 of 16, and integrins alpha 4 beta 1 in 2 of 16. The antibody for E-cadherin showed positive staining of epithelial cells in 6 of 16 menstrual effluent samples. All endometrial tissue samples showed positive staining for E-cadherin. In PF, E-cadherin was detected in the epithelial cells of one sample. One peritoneum biopsy revealed positive staining for E-cadherin. CONCLUSION: Integrins alpha 2 beta 1, alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, and E-cadherin, important cell adhesion molecules, are expressed in endometriotic lesions and in cells and tissues that are potentially involved in the development of endometriosis. These cell adhesion molecules could be involved in the shedding of endometrial tissue during menstruation and the attachment of endometrial tissue fragments to the peritoneum.

Tumorigenic behaviour of c-Ha-ras oncogene transfected CaCo 2 cells is associated with increased proteolytic potency.

BACKGROUND: Point mutations within the family of the ras genes are detected in approximately 50% of human colorectal adenomas and carcinomas. Therefore, it is generally accepted that the occurrence of ras-point mutations constitute an important step in colorectal carcinogenesis. In addition, many studies have demonstrated that the tumorigenicity of the human colorectal carcinoma cell line, CaCo 2, strongly increases after transfection with the c-Ha-ras oncogene. This cell line is suitable for gaining more insight into the mechanism of c-Ha-ras induced tumorigenesis. MATERIAL AND METHODS: Proliferation, differentiation, and proteolytic capacity of c-Ha-ras oncogene transfected CaCo 2 cells were studied in vitro. RESULTS: It was found that gelatinolytic capacity and production of urokinasetype plasminogen activator increased, whereas the production of tissue-type plasminogen activator was similar. Proliferative activity, as measured by the potential doubling time, did not alter. The expression of the differentiation markers sucraseiso-maltase, mucin, and chromogranin A was not different from that of the parental CaCo 2 cell line, which indicates that an increased tumorigenic capacity of c Ha-ras oncogene transfected CaCo 2 cells is not accompanied by loss of differentiation. CONCLUSION: These data demonstrate that the highly increased tumorigenic capacity of c Ha-ras oncogene-transfected CaCo 2 cells is associated with an enchanced proteolytic capacity.

Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion.

A new procedure is described to generate single-stranded DNA by exonuclease III (Exo III) digestion for bromodeoxyuridine (BrdU) immunocytochemistry on tissue sections. We compared this procedure with the most widely used procedure of DNA denaturation with 2 N HCl. In vivo and in vitro pulse and continuous labelling of tissues and cells were used. The specimens were fixed in formalin, ethanol, glutaraldehyde, Carnoy's, Bouin's or Zamboni's fixative and embedded in paraffin or used unfixed as cryostat sections or cytospin preparations. After Exo III digestion, BrdU substituted DNA was detected irrespective of the fixation procedure applied. The optimal protocol for nuclease digestion appeared to be simultaneous incubation, of 10 Units Exo III per ml EcoRI buffer and anti-BrdU monoclonal antibody at 37 degrees C. The advantages of Exo III digestion for BrdU immunocytochemistry compared to acid denaturation were: less non-specific nuclear background reactivity, no DNA renaturation, less DNA loss, optimal nuclear morphology, increase in antibody efficiency and the possibility for simultaneous detection of acid-sensitive tissue constituents. Disadvantages of the Exo III digestion are decreased sensitivity and the need for more rigorous pepsin pretreatment. We conclude that Exo III digestion of DNA is an appropriate alternative for acid denaturation for BrdU immunocytochemistry on sections of pulse-labelled specimens.

L-CAM expression in normal, premalignant, and malignant colon mucosa.

L-CAM is a cell adhesion molecule which is expressed at the intercellular borders of most epithelial cells. L-CAM has been demonstrated to act as an invasion suppressor in carcinoma cell lines. In order to determine whether or not L-CAM expression might distinguish between invasive and non-invasive or metastatic and non-metastatic colon neoplasms, we studied L-CAM expression in normal colon mucosa, colon adenomas with various degrees of dysplasia, and colon carcinomas by immunohistochemistry, using the 6F9 monoclonal anti-L-CAM antibody. Normal mucosa showed evenly distributed distinct L-CAM immunoreactivity along intercellular borders. In adenomas and carcinomas, a similar though weaker expression was observed. This pattern showed a tendency to decrease in parallel with decreasing differentiation. However, no correlation was found with Dukes stage or area within the tumour. In some carcinomas, L-CAM was expressed at the luminal surface of the cells. In others, L-CAM expression was not found. These results suggest that L-CAM expression is disregulated or lost as an early event in the development of colon neoplasia and indicate that L-CAM expression does not correlate with invasive or metastatic potential.

Patterns of alpha- and beta-catenin and E-cadherin expression in colorectal adenomas and carcinomas.

Previous in vitro and in vivo model studies have shown that when E-cadherin expression in carcinoma cells is reduced, invasive behaviour ensues. The situation in human cancer in vivo, however, appears to be more complex, as immunohistochemically determined E-cadherin expression in various carcinomas, including colorectal cancer, does not always correlate with invasive growth. Loss of cell adhesion during invasion in spite of E-cadherin expression might be associated with a defective cadherin-catenin complex. The expression of alpha- and beta-catenin in comparison with E-cadherin was therefore examined in colorectal adenomas and carcinomas and in lymph node and liver metastases. In normal colonic mucosa, alpha- and beta-catenin immunoreactivity occurred along the lateral plasma membranes of the epithelial cells, in a pattern identical to E-cadherin staining. A similar pattern was found in colorectal adenomas and in most malignancies. In general, in neoplastic epithelia, the majority of the cancer cells displayed a normal (matching) pattern of E-cadherin and catenin expression. It is concluded that the patterns of expression of E-cadherin and alpha- and beta-catenin are very similar in colorectal neoplasms. This observation indicates that invasion in colorectal cancer is not paralleled by consistent loss of expression of the components of the cadherin-catenin complex.

L-CAM expression in lymph node and liver metastases of colorectal carcinomas.

L-CAM, also known as E-cadherin, is a cell adhesion molecule expressed on the plasma membranes of epithelial cells at the intercellular interface. From in vitro gene transfection experiments the idea has been conceived that loss of L-CAM expression might be related to the invasive capacity as well as metastatic potential of tumour cells. In several tumours a relation between the grade of differentiation and L-CAM expression has been noticed: loss of differentiation appears to be associated with loss of L-CAM immunoreactivity. Also, in lymph node metastases of poorly differentiated carcinomas loss of L-CAM expression was demonstrated. In this study we describe L-CAM expression in lymphogenous and haematogenous metastases of large bowel adenocarcinomas, using an indirect immunoperoxidase method with the monoclonal anti-L-CAM antibody 6F9. All the metastases studied--lymphogenous as well as haematogenous--demonstrated L-CAM immunoreactivity in a pattern comparable to that of primary tumours. Intratumour heterogeneity in expression was noted, with normal intercellular, apical (non-functional), and focally negative areas in the same tumour. The data indicate that primary tumours and their metastases do not differ strikingly in their pattern of L-CAM expression. This would be consistent with transient rather than constitutive down-regulation of L-CAM in invasive and metastatic cancer cells.

Epithelial cells in peritoneal fluid--of endometrial origin?

OBJECTIVE: Our purpose was to examine the immunohistochemical properties of epithelial cells in peritoneal fluid and to compare the staining characteristics with cells of endometrium, menstrual effluent, peritoneum, and endometriotic lesions. STUDY DESIGN: Samples of menstrual effluent, endometrium, and peritoneal fluid and biopsy specimens of endometriotic lesions and peritoneum from 16 patients were examined. Monoclonal antibodies against vimentin, cytokeratin 18 and 19, and the monoclonal antibody BW495/36, staining an epithelial marker present in endometrium and absent in peritoneal epithelium, were used. RESULTS: All but one sample of menstrual effluent and peritoneal fluid cells stained positively with antibodies against vimentin and cytokeratin 18 and 19. BW495/36 stained 14 of 16 menstrual effluent samples and nine of 16 peritoneal fluid cell samples. Endometriotic specimens showed staining with all markers. No major differences in staining properties were observed in menstrual effluent, endometrium, and peritoneal fluid cells between patients with or without endometriosis. CONCLUSION: These results support the contention of transport of menstrual detritus to the peritoneal cavity in women with patent fallopian tubes.

Extracellular matrix components induce endocrine differentiation in vitro in NCI-H716 cells.

Endocrine cells occur in +/- 30% of colorectal adenocarcinomas. The significance of this phenomenon in terms of tumor behavior is still controversial. Endocrine differentiation in colorectal cancer cell lines is almost confined to tumor xenografts in vivo, suggesting that endocrine differentiation might be regulated by epithelial-stromal interactions. This hypothesis was studied in the cecal adenocarcinoma-derived cell line NCI-H716 by comparing the expression of chromogranin A protein and messenger RNA in vivo and in vitro and by attempts to induce differentiation in vitro. We found that chromogranin A expression, which was strongest in vivo, could be significantly enhanced in vitro by culturing tumor cells in the presence of native extracellular matrix, on fibroblast feeder layers, and in a defined medium with basic fibroblast growth factor. The results suggest that the extracellular matrix induces endocrine differentiation through factors (e.g., basic fibroblast-growth factor) that may be produced by stromal cells and after secretion bind to the extracellular matrix.

Distribution of adenosine deaminase-complexing protein in murine tissues.

It has been suggested that mouse and rat lack adenosine deaminase-complexing protein because in these species exclusively the small molecular weight form of adenosine deaminase (ADA-S) is found. This suggestion is based on the assumption that the adenosine deaminase binding capacity is an inherent functional characteristic of adenosine deaminase-complexing protein. We report on the presence of adenosine deaminase-complexing protein immunoreactivity in mouse and rat determined with a species cross-reactive polyclonal anti-adenosine deaminase-complexing protein serum. In the mouse the tissue and subcellular distribution and the electrophoretic mobility in starch and polyacrylamide gels of the protein correspond with those of adenosine deaminase-complexing protein, but it does not bind the small molecular weight form of adenosine deaminase. Furthermore, in human, mouse, and rat kidney cortex adenosine deaminase and adenosine deaminase-complexing protein did not colocalize by immunohistochemistry. It is suggested that the function of adenosine deaminase-complexing protein is not adenosine deaminase-related.

In vivo and in vitro invasion in relation to phenotypic characteristics of human colorectal carcinoma cells.

In this study we investigated the tumorigenicity, growth pattern and spontaneous metastatic ability of a series of nine human colorectal carcinoma cell lines after subcutaneous and intracaecal xenografting in nude mice. CaCo2 cells were found to be poorly tumorigenic to non-tumorigenic in either site; the other cell lines were tumorigenic in both sites. SW1116, SW480 and SW620 did not show local invasive in the NCI-H716 and LS174T cells were both invasive in the caecum, but only NCI-H716 was invasive in the subcutis. HT29 and 5583 (S and E) cells were invasive in the caecum and from that site metastatic to the lungs and/or the liver. HT29 and 5583S cells were both invasive in the subcutis, but 5583E cells were not. Of each category of in vivo behaviour in the caecum, one cell line was further investigated with regard to invasion in vitro (into embryonic chick heart fragments), E-cadherin expression in vivo and in vitro and in vitro production of u-PA and t-PA. These parameters were chosen in view of their purported role in extracellular matrix degradation and intercellular adhesion, which are all involved in the invasive and metastatic cascade. Invasion in vitro was not predictive for invasion or metastasis in vivo. In the cell line which showed invasion in embryonic chick heart tissue, heterogeneous E-cadherin expression was observed in vitro together with a relatively high production of u-PA. The non-invasive cell lines showed in vitro homogeneous expression of E-cadherin with a relatively low production of u-PA. In vivo expression of E-cadherin was either absent or heterogeneous. We conclude that: (1) colorectal carcinoma xenografts show site-specific modification of in vivo invasive and metastatic behaviour; (2) invasion in vitro does not correlate with invasion and metastasis in vivo; (3) in vitro non-invasion might be associated with homogeneous E-cadherin expression and low production of u-PA; (4) E-cadherin expression in vitro differs from E-cadherin expression in vivo. The results support the notion that the microenvironment in which cancer cells grow is one of the factors involved in the regulation of invasive and metastatic behaviour.

Human Caco-2 cells transfected with c-Ha-Ras as a model for endocrine differentiation in the large intestine.

Endocrine cells occur in approximately 30% of all colorectal adenocarcinomas, and this feature appears to correlate with a relatively poor prognosis. To study the factors regulating endocrine differentiation in colorectal cancer, which may bear resemblance to the regulation of endocrine differentiation in normal intestinal mucosa, models in which differentiation can be manipulated are essential. However, endocrine features in colorectal cancer cell lines are scarce and are almost exclusively observed in xenografts, presumably as a result of differentiation induction by stromal components. We attempted to demonstrate endocrine differentiation in the colonic adenocarcinoma cell line Caco-2, which is frequently used as a model for enterocytic differentiation. In vitro endocrine tumor cells were not encountered. In vivo studies were cumbersome, because of the low take rate of Caco-2 cells. We did manage to establish nude mouse xenografts of Caco-2 cells by inoculating cells in collagen gel and by suppressing natural killer cell activity. In an attempt to induce a better take rate and to investigate the effect of Ras oncoprotein overexpression on endocrine differentiation, Caco-2 cells were transfected with a point-mutated c-Ha-Ras gene. The cell line Caco-2 EJ6, generated from these experiments, could be xenografted in nude mice with a high take rate, yielding a moderately well differentiated adenocarcinoma, morphologically identical to the tumors derived from untransfected Caco-2 cells. The xenografts displayed goblet cell, enterocytic, Paneth cell and endocrine differentiation. In vitro endocrine differentiation was observed neither under standard conditions nor with extracellular matrix components as differentiation inducers. We conclude that the Caco-2 cell line and its c-Ha-Ras transfected subline Caco-2 EJ6 in vivo display endocrine differentiation. Ras overexpression does not enhance endocrine differentiation. Due to its favorable growth properties in vivo, Caco-2 EJ6 is a suitable model for studies on endocrine differentiation in colorectal cancer.

Adenosine deaminase complexing protein (ADCP) expression and metastatic potential in prostatic adenocarcinomas.

The expression of the adenosine deaminase complexing protein (ADCP) was investigated by immunohistochemistry in the normal and hyperplastic human prostate, in 30 prostatic adenocarcinomas, and in seven human prostatic adenocarcinoma cell lines grown as xenografts in athymic nude mice. In the normal and hyperplastic prostate, ADCP was localized exclusively in the apical membrane and the apical cytoplasm of the glandular epithelial cells. In prostatic adenocarcinomas, four distinct ADCP expression patterns were observed: diffuse cytoplasmic, membranous, both cytoplasmic and membranous, and no ADCP expression. The expression patterns were compared with the presence of metastases. We found an inverse correlation between membranous ADCP immunoreactivity and metastatic propensity. Exclusively membranous ADCP immunoreactivity occurred only in non-metastatic tumours. In contrast, the metastatic tumours showed no or diffuse cytoplasmic ADCP immunoreactivity. This suggests that immunohistochemical detection of ADCP might predict the biological behaviour of prostatic cancer. However, the occurrence of membranous ADCP immunoreactivity in the xenograft of a cell line (PC-EW), derived from a prostatic carcinoma metastasis, indicates that not only the tendency to metastasize modulates ADCP expression.

Expression of a marker for colonic crypt base cells is correlated with poor prognosis in human colorectal cancer.

BACKGROUND: There is a need for markers in colorectal cancer which will allow subclassification of stage groups into subgroups with high versus low risk of recurrent disease. AIMS: To develop monoclonal antibodies that recognise antigens or immature crypt base cells, on the assumption that in a neoplasm undifferentiated but not the terminally differentiated cells will be responsible for tumour progression. METHODS: Colon crypt cells which were isolated from human colonic mucosa by EDTA/EGTA incubation were studied. By stepwise harvesting, crypt base cell enriched fractions were obtained, and after incubation with antibodies against dominant antigens, used as immunogens. RESULTS: Of one crypt base cell specific antibody (5E9), the reactive epitope appeared to be a non-terminal carbohydrate in the mucin O-glycans of the colon. The epitope did not seem to be colon specific, but was expressed in a variety of other tissues. In colorectal carcinomas, 5E9 immunoreactivity identified a subgroup of patients with a tendency for worse prognosis. CONCLUSION: A mucin associated maturation epitope was identified in colonic crypt base cells, the expression of which in Dukes' stage B3 colorectal carcinoma may be associated with poor prognosis.