Heterogeneity of colorectal cancer (CRC) in reference to KRAS proto-oncogene utilizing WAVE technology.

BACKGROUND: New drugs targeting specific genes required for unregulated growth and metastases have improved survival rates for patients with metastatic colorectal cancer. Resistance to monoclonal antibodies specific for the epidermal growth factor receptor (EGFR) has been attributed to the presence of activating point mutations in the proto-oncogene KRAS. The use of EGFR inhibitor monotherapy in patients that have KRAS wild type has produced response rates of only 10-20%. The molecular basis for clinical resistance remains poorly understood. We propose two possible explanations to explain these low response rates; 1) levels of resistant CRC cells carrying mutated KRAS are below the sensitivity of standard direct sequencing modalities (<5%) or 2) the standard practice of analyzing a single area within a heterogeneous tumor is a practice that can overlook areas with mutated KRAS. METHODS: In a collaborative effort with the surgical and molecular pathology departments, 3 formalin fixed paraffin embedded tissue blocks of human CRC were obtained from the human tissue bank maintained by the Lifespan Pathology Department and/or the human tissue bank maintained by the Molecular Pathology Core of the COBRE for Cancer Research Development. The three specimens previously demonstrated KRAS mutations detected by the Applied Biosystems Kit. The Wave system 4500 (high performance ion-pairing liquid chromatography (IP-HPLC)) was utilized to evaluate tissue for the presence of KRAS proto-oncogene mutations at codons 12 and 13. RESULTS: Initially, the sensitivity of WAVE technology was compared with direct sequencing by evaluating a dilutional series. WAVE detected mutant alleles at levels of 2.5% compared to 20% performed with standard direct sequencing. Samples from three patients were evaluated by WAVE technology. Eight samples from patient 1 were analyzed. In two of eight samples, no mutations were detected at concentrations as low as 5%. In one sample a mutation was noted by WAVE and not by direct sequencing. All four samples from patient 2 tested positive for Exon 12/13 mutations. Of the seven samples from patient 3, five were positive for Exon 12/13 mutations and two were negative for Exon 12/13 mutations. CONCLUSION: In these studies the analysis of three patients' colorectal cancer tissues were analyzed utilizing the WAVE technology. Results demonstrated a greater degree of sensitivity in mutation detection when compared to standard sequencing. These studies also demonstrated heterogeneity of expression of KRAS mutations between areas of the tissue samples at a genomic level. The low clinical response rates to EGFR inhibition might be explained by the variation in mutation presence, which was dependent upon the region examined. The heterogeneity demonstrated in these studies provides another phenotypic variant that will impact clinical care.

Detection of an altered form of cell-CAM 105 on rat transplantable and primary hepatocellular carcinomas.

Monoclonal (MAb 362.50) and polyclonal (anti-gp 105-2) antibodies have been used to examine the expression by transplantable (THC) and primary (PHC) hepatocellular carcinomas of a 105 kd rat hepatocyte cell adhesion molecule designated cell-CAM 105. Two-dimensional gel analysis of components immunoprecipitated with MAb 362.50 or anti-gp 105-2 antibodies from detergent extracts of cells surface labeled with 125I showed that cell-CAM 105 from three different THC exhibited a more basic pI than its counterpart from normal rat hepatocytes. Immunoprecipitation of detergent extracts from radioiodinated hepatocytes with anti-THC antisera raised in rabbits by four immunizations with THC cells showed that six THC lines which were negative when stained by indirect immunofluorescence with MAb 362.50 expressed sufficient levels of cell-CAM 105 to induce precipitating antibodies. In contrast, antisera collected after eight immunizations with THC 253.1 and THC 252.2, showed no detectable reactivity with cell-CAM 105 suggesting that these THC lines had completely lost the expression of this molecule. Immunofluorescence analysis of normal rat tissues indicated that cell-CAM 105 was also present in the brush border of the small intestine and a subset of tubules in the kidney, raising the possibility that THC cells were expressing an isoform normally found in nonhepatic tissues. However, cell-CAM 105 isolated from kidney showed a mobility on two-dimensional gels that was distinct from both the THC and hepatocyte forms of this molecule. Indirect immunofluorescence analysis of PHC induced by ethionine in a choline-deficient diet or by the Solt/Farber protocol showed that 52% and 65% of the persistent hepatic nodules induced by ethionine in a choline-deficient diet and by the Solt/Farber protocol, respectively, were unreactive with MAb 362.50. Immunoprecipitation analysis of PHC induced by ethionine or diethylnitrosamine and choline-deficient diet showed that one of four PHC was expressing an altered form of cell-CAM 105 with the more basic pI characteristic of the THC form of this molecule. Taken together, these results suggest that quantitative and qualitative changes in the expression of cell-CAM 105 may constitute an important step in the acquisition of the malignant phenotype.

Monoclonal antibodies recognizing oval cells induced in the liver of rats by N-2-fluorenylacetamide or ethionine in a choline-deficient diet.

In this paper, we describe the production of a panel of monoclonal antibodies which define antigens which distinguish between hepatocytes and oval cells. These antibodies were obtained from hybridomas constructed from the spleens of mice immunized by a novel protocol designed to suppress response to unwanted or immunodominant epitopes. Of the antibodies obtained, four, 258.7, 270.11, 258.34, and 270.38, were directed to antigens of morphologically defined oval cells, while two, 258.26 and 270.26, defined cytoplasmic antigens of hepatocytes. Examination of frozen sections of normal, regenerating adult and fetal liver and livers from rats fed 2-acetylaminofluorene or ethionine in a choline-deficient diet indicates that morphologically defined oval cells may in fact comprise a phenotypically complex set of cells composed of at least three antigenically distinct subpopulations. The patterns of expression of the antigens defined by these antibodies suggest two possible pathways of liver cell differentiation.

Alterations in the expression of a hepatocyte cell adhesion molecule by transplantable rat hepatocellular carcinomas.

Alterations in the expression of normal cell surface components on 13 transplantable hepatocellular carcinomas were examined using a heteroantiserum [anti-Mr 105,000 glycoprotein (gp 105)] reactive with a family of nine wheat germ agglutinin binding components from normal rat hepatocytes with an average molecular weight of 105,000. Analysis by two-dimensional polyacrylamide gel electrophoresis of components immunoprecipitated by anti-gp105 antiserum from detergent extracts of transplantable hepatocellular carcinoma cells surface labeled with 125I revealed qualitative and quantitative changes in the expression of anti-gp105-reactive components with the most consistent change being the apparent loss of a pair of acidic (pl 4.1 to 4.3) glycoproteins by all 13 transplantable hepatocellular carcinoma lines. One-dimensional peptide maps of fragments produced following digestion with V8 protease indicated that these acidic components were closely related in structure but differed significantly from other anti-gp105-reactive components. Immunodepletion analysis with monoclonal antibodies and heteroantisera reactive with individual components recognized by anti-gp105 antiserum showed that the two acidic glycoproteins were antigenically and structurally identical to cell-CAM 105, a Mr 105,000 glycoprotein involved in cell-cell adhesion of rat hepatocytes. Antibodies raised against purified cell-CAM 105 were specific in immunoprecipitation assays for the acidic components, strongly inhibited reaggregation of hepatocytes, and displayed no reactivity by indirect immunofluorescence or immunoprecipitation analysis with transplantable hepatocellular carcinoma cells. These results suggest that major alterations in the expression of cell-CAM 105 may be a consistent feature of the malignant phenotype.

Comparison of the structural characteristics of cell-CAM 105 from hepatocytes with those of an altered form expressed by rat transplantable hepatocellular carcinomas.

The structural features of the adult rat hepatocyte (ARH) forms of cell-CAM 105, a Mr 105,000 cell adhesion molecule, were compared using a variety of immunochemical and biochemical techniques with altered forms of more basic pI present on two transplantable hepatocellular carcinomas (THC 1682c and THC AS-30D). Immunoprecipitation analysis with polyclonal (anti-gp 105-2) and monoclonal (MAb) antibodies specific for cell-CAM 105 (MAb 362.50) demonstrated that ARH and THC cell-CAM 105 were indistinguishable in several respects including: (a) binding to wheat germ agglutinin; (b) labeling with NaIO4/NaB3H4; (c) susceptibility to digestion with endoglycosidases (endoglycosidase H and F and peptide N-glycosidase F N-glycanase); (d) rate of turnover on the cell surface; and (e) differential resistance of upper and lower forms to trypsin digestion in the presence or absence of calcium. Digestion with Clostridium perfringens or Vibrio cholerae neuraminidase did not equalize pI but instead decreased the size and increased the pI of both ARH and THC cell-CAM 105. Comparison of two-dimensional tryptic peptide maps, however, revealed five unique peptides in the THC AS-30D map and one peptide in the THC 1682c map, peptides which were only apparent in maps of deglycosylated ARH cell-CAM 105. Based on these results, it was concluded that there were significant differences in the glycosylation of ARH and THC cell-CAM 105. Biosynthetic labeling with 32PO4 and 35SO4 showed that both ARH and THC molecules were phosphorylated but not sulfated. Comparison of 32P-labeled peptides produced by digestion with V-8 protease revealed significant differences in the phosphorylation of the upper and lower forms from ARH and showed that the pattern of phosphorylation on THC cell-CAM 105 most closely resembled ARH upper form. Pulse-chase analysis of ARH cell-CAM 105 further indicated that only a subpopulation of the molecules labeled with [35S]methionine were phosphorylated.

Antigenic relationship between oval cells and a subpopulation of hepatic foci, nodules, and carcinomas induced by the "resistant hepatocyte" model system.

Although proliferation of small ductular-like cells, designated oval cells, is often observed during the early stages of chemically induced hepatocarcinogenesis, their role during the carcinogenic process remains controversial. To investigate the possibility that oval cells may give rise to preneoplastic lesions that ultimately progress to hepatocellular carcinomas, we have carried out phenotypic analysis with a panel of monoclonal antibodies to determine if there is an antigenic relationship between oval cells and hepatic foci, nodules, and tumors induced by the resistant hepatocyte model system. In this model, rats are given a single dose (200 mg/kg) of diethylnitrosamine, followed by a brief exposure to 2-acetylaminofluorene and a partial hepatectomy. We found that approximately 10% of the early focal lesions observed 28 days after diethylnitrosamine expressed either one or both of the oval cell antigens designated OC.2 and OV-6. By 28 weeks after diethylnitrosamine, 16 of 16 hepatic nodules heterogeneously expressed OV-6 whereas 5-10% of the persistent nodules contained scattered small hepatocyte-like cells that expressed OC.2. Examination of resistant hepatocyte-induced primary hepatocellular carcinomas with an expanded panel of monoclonal antibodies demonstrated that most cells comprising 29 of 29 tumors expressed OV-6 and that 15-20% of the OV-6-positive tumors contained subpopulations of cells also expressing 3 additional oval cell antigens, OC.2, OC.3, and OV-1. All of the tumors examined expressed normal levels of the hepatocyte antigens, H.1 and HBD.1, and had dramatically reduced levels of H.2, H.4, and cell CAM 105 but showed elevated levels of the transferrin receptor, gamma-glutamyltranspeptidase, and the normal hepatocyte antigen, H.5. In conclusion, our findings demonstrate an antigenic relationship between oval cells and a subpopulation of hepatic foci, nodules, and tumors in the resistant hepatocyte model, suggesting that at least some primary tumors may be derived from oval cells in this model system.

Improved detection of the early stages of colon cancer by determining both free circulating and immune complex-bound antigens reactive with monoclonal antibody.

The measurement of both immune complex-bound and free unbound tumor-associated antigen was evaluated independently on a panel of sera from colon cancer patients by radioimmunoassay (RIA). A monoclonal antibody (mAb 46.3) raised against secreted antigens from human colon cancer cells in vitro was utilized in the RIA. When circulating immune complexes alone were analyzed, the data demonstrated that 5 of 5 (100%) Dukes' A patients and 11 of 16 (69%) Dukes' B patients had elevated levels of immune complexes reactive with mAb 46.3. Analysis of free circulating antigens demonstrated elevated levels of mAb 46.3-reactive antigen present in 5 of 5 (100%) Dukes' A patients and 15 of 16 (95%) Dukes' B patients. However, by analyzing total reactivity, defined by combining results from RIA with free and immune complex-bound antigen, the sensitivity of detection for Dukes' B increased to 16 of 16 (100%). Total antigen levels in sera from patients with benign diseases (ulcerative colitis, Crohn's disease, adenoma) were not significantly different from normal controls. Analysis of both free and bound antigen in RIA is, therefore, a more sensitive indicator than RIA with immune complex alone. For the advanced stages of disease, only 1 of 5 (20%) Dukes' C and 0 of 5 (0%) Dukes' D sera were positive for reactive immune complexes. When the combined RIA was evaluated, 3 of 5 (60%) and 1 of 5 (20%) Dukes' C and D sera, respectively, were positive with mAb 46.3. Taken together, these results show that RIA with mAb 46.3 is a sensitive indicator for the early stages of colon cancer.

Identification and characterization of a rat hepatic oncofetal membrane glycoprotein.

A major interest of our laboratory is to delineate the pathways leading to experimentally induced liver cancer in the rat. Although the cellular progenitors of primary hepatocellular carcinoma remain controversial, current findings suggest that proliferation of chemically initiated liver epithelial cells gives rise to hepatic nodules, a rare population of which eventually progress to carcinoma. Presently, the availability of cell surface markers that are closely associated with malignant progression is needed for the identification, isolation, and further characterization of these rare malignant cells. In this paper, we describe two new monoclonal antibodies (MAbs), MAb 324.5 and MAb 324.9, that recognize a novel oncofetal membrane glycoprotein, designated TuAg1. MAbs 324.5 and 324.9 were produced using three different transplantable hepatocellular carcinoma cell lines during immunization and screening. MAb 324.5 and MAb 324.9 were shown to be reactive with different epitopes on TuAg1 by competitive immunoprecipitation assays combined with results from immunodepletion analysis and one-dimensional V-8 peptide maps. TuAg1 showed variations in molecular weight from 78,000 to 92,000, and a marked heterogeneity in pI, with charge variants ranging between 4.3 and 6.0. The 324.5-epitope was not expressed at detectable levels in any adult normal tissues or during liver regeneration but was transiently expressed during fetal liver development as shown by indirect immunofluorescence analysis of frozen tissue sections. In contrast, the 324.9-epitope was observed on nerve fibers and ganglia and on sperm tails in the adult rat and also appeared independently of the 324.5-epitope during fetal development. Although normal hepatocytes did not express TuAg1, isolated hepatocytes became positive during the first 24 h of primary culture. Attempts to modulate the in vitro expression of TuAg1 were unsuccessful; however, TuAg1 was lost within 7 days following ectopic transplantation of cultured hepatocytes into the pancreas. During the carcinogenic process, TuAg1 was expressed by a rare population of hepatic nodules, by many primary liver tumors, and by all lung metastases and transplantable hepatocellular carcinomas examined to date. Taken together, these observations suggest that the in vivo constitutive expression of this novel oncofetal membrane antigen is closely associated with acquisition of the malignant phenotype during hepatocarcinogenesis.

Expression of hepatocyte and oval cell antigens in hepatocellular carcinomas produced by oncogene-transfected liver epithelial cells.

We have established an in vivo/in vitro system in which epithelial cells ("oval cells") isolated from livers of rats fed a carcinogenic diet for a very brief period are placed in culture and transfected with an oncogene. Injection s.c. into nude mice of oval cells transfected with the activated c-Ha-ras (EJ oncogene) produces tumors with morphological features of differentiated hepatocellular carcinomas. Using monoclonal antibodies that can recognize hepatocyte, oval cell, and tumor antigens, we investigated the expression of these antigens in oval cells in culture, transfected with either the EJ oncogene or the normal c-Ha-ras allele and in tumors derived from the oncogene-transfected cells. We show that EJ-transfected cells and most particularly the tumors they produce expressed hepatocyte and oval cell antigens not detectable in untransfected cells or cells transfected with the normal c-Ha-ras gene. Furthermore, we found that in cloned tumor cells, the expression of hepatocyte antigens could be induced by changes in culture conditions and was accompanied by a decrease in the expression of oval cell markers. Trabecular hepatocellular carcinomas had higher reactivity toward monoclonal antibodies recognizing hepatocyte antigens while tumors with glandular architecture reacted predominantly with monoclonal antibodies against oval cells. We conclude that, in addition to its tumorigenic effect, the EJ oncogene induced the differentiation of tumor cells toward the hepatocyte lineage. In addition, the data provide further confirmation that oval cells can serve as progenitors of differentiated hepatocellular carcinomas.

TuAg.1 is the liver isoform of the rat colon tumor-associated antigen pE4 and a member of the immunoglobulin-like supergene family.

TuAg.1 is a tumor-associated membrane glycoprotein first identified in rat hepatocellular carcinoma by monoclonal antibodies (mAbs) 324.5 and 324.9. This oncofetal antigen is also expressed by hepatocytes in cell culture but not normal adult hepatocytes in vivo. Affinity chromatography and preparative continuous elution slab-gel electrophoresis were used to separate TuAg.1 from co-purified actin and immunoglobulin. TuAg.1 was recovered as a series of bands Mr 82,000-90,000, which were pooled and subjected to CNBr digestion for primary amino acid sequence analysis. Computer database analysis of TuAg.1 peptide sequence revealed homology to the rat colon carcinoma-associated antigen pE4, a member of the immunoglobulin gene superfamily. Oligonucleotide primers derived from sequences shared by TuAg.1 and pE4 were used in reverse transcription-PCR to amplify tumor-specific products corresponding to TuAg.1 cDNA. Northern blot analysis with one of these products confirmed the oncofetal expression of transcripts related to TuAg.1/pE4 and indicated an RNA species of different size expressed only in normal liver. Identity between TuAg.1 and pE4 was further confirmed by immunochemical analysis with mAb 324.5 and mAb E4. Both antibodies were reactive with the same protein on transplantable hepatocellular carcinoma AS30D but recognized different epitopes. The reactivity of human tumor cells with mAb 324.5 and 324.9 indicates the presence of a related TuAg.1 molecule expressed in human neoplasia as well.

Effect of neuraminidase and papain treatment on lectin-induced agglutination of Novikoff tumor cells and assay of lectin receptor activity of the glycopeptides released from the cell surface by papain.

Lectins, plant proteins that bind specific saccharide determinants, have been utilized to examine the effect of neuraminidase digestion on the structure and/or expression of oligosaccharide moieties present at the periphery of Novikoff ascites hepatoma cells. Five lectins were utilized: concanavalin A (Con A), specific for alpha-D-manno- or alpha-D-glucopyranosyl residues; wheat germ agglutinin, specific for 2-acetamido-2-deoxy-D-glucopyranosyl residues; Ricinus communis agglutinin I (RCAI), specific for D-glucopyranosyl residues; R. communis agglutinin II (RCAII), specific for D-galacto- or 2-acetamido-2-deoxy-D-galactopyranosyl residues; and soybean agglutinin, specific for 2-acetamido-2-deoxy-D-galactopyranosyl residues. Neuraminidase treatment of Novikoff cells did not alter their agglutination by Con A or wheat germ agglutinin. Similar treatment produced only a 2-fold increase in their agglutination by RCAI but a 12-fold increase in their agglutination by RCAII, indicating that 2-acetamido-2-deoxy-D-galactopyranosyl residues become expressed upon neuraminidase treatment. This conclusion was confirmed by the observation that neuraminidase-treated Novikoff cells acquired agglutinability by soybean agglutinin. Binding studies using ferritin-conjugated RCAII indicated that neuraminidase treatment exposed cryptic cell surface receptors for RCAII. To ascertain the role of cell surface glycoproteins in lectin-induced agglutination of Novikoff cells, glycopeptides cleaved from the cell surface by papain were assayed for lectin receptor activity. The cell surface glycopeptides exhibited receptor activity for Con A, wheat germ agglutinin and RCAI but not for RCAII and soybean agglutinin. A cell surface macrosialoglycopeptide fraction, resolved by gel filtration and ion-exchange chromatography, possessed a major portion of the Con A and RCAI receptor activity.

Characterization of a family of glycoproteins associated with the bile canalicular membrane of normal hepatocytes but not expressed by two transplantable rat hepatocellular carcinomas.

Xenoantisera were used to investigate the expression of normal cell-surface components on transplantable rat hepatocellular carcinoma (253 and 311c) cells. Cell-surface components, immunoprecipitated from non-ionic detergent extracts of 125I-labeled hepatocytes by xenoantisera raised against rat hepatocytes, 253 cells, or 311c cells, were analyzed by 2-dimensional polyacrylamide gel electrophoresis. Comparison of the gel patterns revealed that anti-hepatocyte antiserum was reactive with a component having an apparent molecular weight of 105,000 (gp105) that was not recognized by xenoantisera against 253 or 311c cells. This component was isolated by affinity adsorption to Sepharose-conjugated wheat germ agglutinin, reconstituted in liposomes, and used to immunize a rabbit. The resulting antiserum, anti-gp105, was reactive with a family of glycoproteins that had an apparent molecular weight of 105,000 but differed in isoelectric points. Immunohistochemical techniques revealed that gp105 was localized in the bile canalicular domain of the hepatocyte plasma membrane. Immunodepletion analysis, blocking of immunoprecipitation by extracts of tumor cells, and labeling of cells by immunohistochemical techniques indicated that gp105 was not expressed at the surface of 253 or 311c cells. These studies suggest that the altered expression of gp105 on 253 and 311c hepatocellular carcinoma cells may be associated with the abnormal tissue architecture and growth patterns exhibited by these transplantable tumors.

Association of an 80 kDa protein with C-CAM1 cytoplasmic domain correlates with C-CAM1-mediated growth inhibition.

Decreased expression of C-CAM, a member of the CEA family of immunoglobulin like cell adhesion molecules, occurs in carcinomas of the colon, liver and prostate. Down regulation of C-CAM during the early stages of carcinogenesis in rat liver and human prostate has also been reported. We have recently shown that restoration of the expression of the isoform with long cytoplasmic domain, C-CAM1, leads to suppression of the tumorigenicity of prostatic carcinoma cells in vivo and growth suppression in vitro. These observations suggest that C-CAM1 may play an important role in regulating cell growth in normal tissues. Previous studies have demonstrated that the function of many members of the Ig-supergene family is dependent on interactions with cytoplasmic proteins. In the present study, we have used a bifunctional cross-linker to identify cellular proteins that interact directly with C-CAM1. Immunoblot analysis of WGA bound membrane proteins crosslinked with DSS identified a 180 kDa complex composed of C-CAM and an 80 kDa protein designated CAP-80 (C-CAM Associated Protein). Immunoprecipitation with anti-C-CAM antibodies showed that CAP-80 was co-precipitated with C-CAM from detergent solubilized, WGA-purified proteins. To assess the specificity of CAP-80 binding, the ability of CAP-80 to form stable complexes with C-CAM1 mutants expressed in insect cells was tested. Deletion of the cytoplasmic domain of C-CAM1 abolished complex formation whereas deletion of the extracellular Ig domains had no effect. These results suggest that a CAP-80 homologue (ICAP-80) is present in insect cells and ICAP-80 interacts with the cytoplasmic domain of C-CAM1. Replacement of Tyr488, a residue in the cytoplasmic domain known to be phosphorylated in vivo, with Phe did not diminish the association between C-CAM1 and ICAP-80, suggesting that Tyr488 phosphorylation is not required for association. The ability of various C-CAM1 mutants to associate with ICAP-80 correlated with their growth inhibitory activities, suggesting that ICAP-80/CAP-80 may play an important role in C-CAM1-mediated growth inhibition.

Expression and androgen regulation of C-CAM cell adhesion molecule isoforms in rat dorsal and ventral prostate.

C-CAM is an epithelial cell adhesion molecule with two major splice variants that differ in the length of the cytoplasmic domain. C-CAM1 (long (L)-form) strongly suppresses the tumorigenicity of human prostate carcinoma cells. In contrast, C-CAM2 (short (S)-form) does not exhibit tumor-suppressive activity. In the present study we have investigated the functional significance of L-form and S-form C-CAM in rat prostate by examining their expression and distribution in different prostate lobes and their response to androgen deprivation. RNase protection assays with a probe for both C-CAM isoforms detected high levels of C-CAM messages in the rat dorso-lateral prostate (DLP). L- and S-form proteins, localized by indirect immunofluorescence using isoform-specific antipeptide antibodies, were co-expressed on the apical surface of prostate epithelial cells in normal DLP. Androgen depletion did not significantly change the steady state levels of C-CAM message and protein expression in the DLP, although there was a change in the pattern of protein expression in these lobes. In contrast, C-CAM isoform messages and proteins were undetectable in normal ventral prostate (VP) but increased markedly in this lobe in response to castration, producing isoform ratios similar to those in DLP. These results demonstrate that coordinate expression of C-CAM isoforms is maintained in the VP following androgen depletion and suggest that androgen suppresses C-CAM expression in VP but not in DLP. These results suggest that balanced expression of L- and S-form C-CAM is important for normal prostate growth and differentiation.

C-CAM1 expression: differential effects on morphology, differentiation state and suppression of human PC-3 prostate carcinoma cells.

Studies in rat prostate and liver have suggested that C-CAM1 is involved in the formation and maintenance of histotypic associations in tissues and possibly tumors. Most recently, C-CAM1 has been shown to suppress tumorigenicity of prostate and colon carcinoma cells. However, the mechanisms whereby C-CAM1 suppresses growth and the relationship of this activity to its proposed role in histotypic interactions remain largely unknown. In the present study, we have analysed the growth, phenotypic, morphological and ultrastructural characteristics of four human PC-3 prostate carcinoma cell lines transduced with C-CAM1 retrovirus. We report that three of four lines regained their tumorigenic phenotype in vivo while maintaining high levels of C-CAM1 expression and a growth retarded phenotype in vitro. These findings suggested that high levels of C-CAM1 expression were negatively influencing recovery during reconstitution after freezing or during the latency period after subcutaneous injection and that loss of suppression resulted from changes in expression of other molecules required for full disclosure of C-CAM1 mediated growth inhibition. Results from Northern blot and immunofluorescence analyses of tumor nodules demonstrated that C-CAM1 decreased rather than enhanced phenotypic differentiation and induced ultrastructural and morphological changes that occurred independently of tumor suppression.

Karyotypic analysis of adult pluripotent stem cells.

Three categories of precursor cells have been identified in postnatal mammals: tissue-committed progenitor cells, germ layer lineage-committed stem cells and lineage-uncommitted pluripotent stem cells. Progenitor cells are the immediate precursors of differentiated tissues. Germ layer lineage stem cells can be induced to form multiple cell types belonging to their respective ectodermal, mesodermal, and endodermal embryological lineages. Pluripotent stem cells will form somatic cell types from all three primary germ layer lineages. Progenitor cells demonstrate a finite life span before replicative senescence and cell death occur. Both germ layer lineage stem cells and pluripotent stem cells are telomerase positive and display extensive capabilities for self-renewal. Stem cells which undergo such extensive replication have the potential for undergoing mutations that may subsequently alter cellular functions. Gross mutations in the genome may be visualized as chromosomal aneuploidy and/or chromosomes that appear aberrant. This study was designed to determine whether any gross genomic mutations occurred within the adult pluripotent stem cells. Karyotypic analysis was performed using pluripotent stem cells purified from adult male rats using established procedures. Giemsa Banding was used in conjunction with light microscopy to visualize metaphase chromosome spreads. To date over 800 metaphase spreads have been analyzed. We found that the metaphase spreads averaged 42 chromosomes and concluded that these pluripotent stem cells isolated from adult rats have a normal karyotype.

Selective proliferation of chemically altered rat liver epithelial cells following hepatic transplantation.

Although proliferation of oval cells is often observed during the early stages of chemical hepatocarcinogenesis, the role of these putative hepatic stem cells during the neoplastic process is unknown. In earlier studies our laboratory showed that feeding a choline-deficient (CD) diet containing 0.05% 2-acetylaminofluorene (CD-AAF) to rats produced three subpopulations of oval cells that antigenically resemble biliary duct cells, fetal liver cells, and transitional cells. In the present investigation we have employed a semiallogeneic transplantation protocol in order to study the fate of these nonparenchymal epithelial cells (NPEC) beyond the 4-week endpoint imposed by the lethality of CD-AAF diet. An enriched NPEC suspension containing gamma-glutamyl-transpeptidase (GGT)-positive oval cells (greater than 75%) was isolated from ACI rats maintained on CD-AAF diet for 3 weeks. The donor cells were transplanted via the portal vein into livers of male F1 progeny (LExACI) that had been fed a CD diet for 7 days prior to receiving a partial hepatectomy and the cell suspension. Host rats were then fed either a CD or choline-supplemented (CS) diet for 12 weeks and killed. Colonies of donor-derived cells identified in frozen sections by their lack of reactivity with ACI anti-LE alloantiserum in indirect immunofluorescence (IF) assays were only observed in rats continuously fed the CD diet. Histochemical analysis indicated that the donor-derived colonies expressed GGT, a preneoplastic marker for liver cancer. IF assays using MAbs previously shown to be capable of distinguishing between oval cells and mature hepatocytes indicated that the donor-derived colonies consisted of a mixture of cells with phenotypes resembling those of mature and immature hepatocytes rather than those of oval or ductal cells. Although the cellular origin of the GGT+ donor-derived colonies has not been unequivocally resolved, our results demonstrate that the livers of rats fed a CD-AAF diet contain a chemically altered call population that can be induced to proliferate by a CD diet. In contrast, a CD diet did not promote colonization when normal hepatocytes were employed as the donor cell population, suggesting that the GGT+ oval cells and not the few contaminating GGT- hepatocytes (1%) in the CD-AAF donor cell suspension were the preneoplastic precursors that gave rise to donor-derived colonies. This transplantation protocol will be useful to define the biological potential of chemically altered liver cells during carcinogenesis.

Rapid low molecular weight polyethylene glycol embedding protocol for immunocytochemistry.

We describe an alternative polyethylene glycol (PEG) embedding procedure which utilizes PEG 200 for dehydration and PEG 600 for infiltration and embedding of perfusion-fixed rat liver. PEG 600 has a melting point of 22 degrees C, enabling infiltration of fixed tissue to be performed at room temperature. Sections (2 microM) cut in a cryostat at -20 degrees C and immobilized in agarose were readily labeled by immunoperoxidase protocols with monoclonal antibodies to hepatocyte membrane antigens. Subsequent examination by light microscopy or by electron microscopy after re-embedding in resin and ultra-thin sectioning showed excellent preservation of morphology, with minimal impairment of antigenicity.

Evaluation of periodate/lysine/paraformaldehyde fixation as a method for cross-linking plasma membrane glycoproteins.

Fixation by periodate/lysine/paraformaldehyde, a method purported to cross-link specifically plasma membrane glycoproteins, was evaluated using Novikoff rat ascites hepatocellular carcinoma cells. Cells were treated with periodate/lysine, periodate/glycine, and periodate/lysine/paraformaldehyde and subsequently reduced with NaB3H4. The glycoproteins labeled with 3H were resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and visualized by fluorography. The effects of reactant concentrations on 3H-labeling of cellular components, cell viability, and cross-linkage of 3H-labeled proteins were examined. The effect of increasing the localized density of plasma membrane glycoproteins on the extent of cross-linkage by periodate and lysine was investigated using cells in which patching of the plasma membrane glycoproteins had been induced by ferritin-conjugated concanavalin A/rabbit antiferritin antiserum. Also investigated was the periodate-independent to mixtures of periodate and lysine or glycine. Results of these studies did not support a mechanism of cross-linking involving reaction between the free base lysin and aldehyde groups on periodate oxidized carbohydrate residues but suggested a complex interaction between periodate oxidized plasma membrane glycoproteins and polymeric complexes of lysine and formaldehyde.U

Hepatocyte cell surface polarity as demonstrated by lectin binding.

We performed an investigation at the ultrastructural level of the differential distribution of lectin-binding sites among sinusoidal, lateral, and bile canalicular domains of adult rat hepatocytes. Lectin binding to hepatocyte glycocalices was studied in situ or after cellular dissociation by enzymatic (collagenase), chemical (EDTA), and mechanical methods, as well as during cell culture. Using thirteen biotinylated lectins and an avidin-biotin-peroxidase complex (ABC), we have identified lectin-binding sites that are predominantly localized in the bile canalicular [Ricinus communis agglutinin (RCA)] or sinusoidal [Phaseolus vulgaris (PHA)] domains in situ and in mechanically dissociated cells. Lens culinaris (LCA) staining was prominent on sinusoidal surfaces, slight along lateral surfaces, and completely absent in the bile canalicular domain. Concanavalin A (ConA) was unique in binding equally to all domains. Triticum vulgaris [wheat germ agglutinin (WGA)] was also bound to all domains, but most intensely to the bile canalicular region. Cells dissociated via collagenase or EDTA treatment exhibited a spherical morphology characterized by many surface microvilli and absence of morphological domains. Lectin binding to dissociated cells was uniformly distributed over the entire cell surface, suggesting a redistribution of lectin receptors that was independent of the separation procedure. Hepatocytes in culture exhibited a partial restoration of morphological domains, but lectin binding polarity was not re-established.