Lab Invest

Guanylin mRNA expression in human intestine and colorectal adenocarcinoma.

Cohen MB. Hawkins JA. Witte DP.
Division of Pediatric Gastroenterology and Nutrition, Children's Hospital Research Foundation, and University of Cincinnati, Ohio 45229, USA.
Guanylin is a mammalian peptide ligand that binds to the enterocyte receptor guanylyl cyclase C and mediates Cl- and HCO3- efflux via the cystic fibrosis transmembrane conductance regulator. To identify the regional localization of guanylin mRNA in the human intestine, we performed in situ hybridization using a guanylin-specific riboprobe. The pattern of guanylin mRNA distribution is complex and includes all epithelial lineages at various points along the duodenal-to-colonic axis. Guanylin mRNA expression is most prominent in the distal small intestine and colon. In the normal colon, guanylin mRNA is robustly expressed in superficial epithelial cells; in colorectal adenocarcinoma, however, guanylin mRNA expression is absent. Guanylin mRNA is detectable in several intestinal tumor cell lines, although at much lower levels than those seen in the human intestine. The pattern of guanylin expression is consistent with the possibility of region-specific functions for guanylin within the human intestine. Furthermore, the diminished expression of guanylin mRNA in adenocarcinoma of the colon and in colon cancer cell lines, along with the chromosomal localization of guanylin to the tumor modifier region 1p34-35, raises the possibility that loss of guanylin activity leads to or is a result of adenocarcinoma formation.

Lysyl oxidase-like protein localizes to sites of de novo fibrinogenesis in fibrosis and in the early stromal reaction of ductal breast carcinomas.

Decitre M. Gleyzal C. Raccurt M. Peyrol S. Aubert-Foucher E. Csiszar K. Sommer P.
Institut de Biologie et Chimie des Proteines, Centre National de la Recherche Scientifique, and Universite Claude Bernard Lyon I, France.
Lysyl oxidase (LO) initiates the first step in the crosslinking of collagens and elastin and has also been shown to function as a tumor suppressor. The purpose of the present work was to determine whether the products of a newly described LO-like gene (LOXL) that encodes a close homolog of LO, the LO-like (LOL) protein, is associated with extracellular matrix remodeling during fibrotic disorders. Specific antibody against LOL identified proteins of approximately 30, 42, 52 and 68 kd in various cells and in bovine aorta. These proteins were immunochemically distinct from the recombinant LO expressed by fibroblasts and from the bovine aorta LO. The LO gene (LOX) and LOXL were transiently up-regulated at early stages of liver granuloma development in Schistosoma mansoni-infected mice, although the peak of LOL mRNA synthesis preceded that of LO. LOL protein and LO were colocalized at sites of fibrogenesis in human lung fibrosis and in the stromal reaction of bronchiolo-alveolar carcinomas and of in situ ductal breast tumors. In conclusion, the LOL protein was identified as a secreted protein and localized in the extracellular matrix in active fibrotic diseases and in the early stromal reaction of breast cancer.

Biologic determinants of uveal melanoma metastatic phenotype: role of intermediate filaments as predictive markers.

Hendrix MJ. Seftor EA. Seftor RE. Gardner LM. Boldt HC. Meyer M. Pe'er J. Folberg R.
Department of Anatomy, Iowa Cancer Center, The University of Iowa College of Medicine, Iowa City 52242-1109, USA.
The long-range goal of our research is to develop intervention strategies based on newly discovered biologic mechanisms responsible for the invasive dissemination of metastatic uveal melanoma. To accomplish this goal, we have focused on the biologic relevance of novel marker proteins contributing to the uveal melanoma metastatic phenotype. The expression of vimentin intermediate filaments (IFs), a mesenchymal marker, is typical of melanomas, whereas carcinomas typically express keratin IFs, which are markers for epithelia. Thus, cells that coexpress both IFs are regarded as "interconverted" in that they display both mesenchymal and epithelial phenotypes. Although the biologic functions of IFs have remained enigmatic, there is substantial support to suggest that the significance of vimentin/keratin coexpression is linked with poor patient outcome in cutaneous melanoma. Our data demonstrate that human uveal melanoma cell lines (isolated from primary choroidal or ciliary body melanomas and from foci of metastatic uveal melanoma to the liver), which contain predominant populations of cells that coexpress vimentin/keratins 8 and 18 (keratins 8,18) IFs, were 6-fold more invasive through collagenous extracellular matrices in vitro, compared with uveal melanoma cells expressing vimentin only, and were 8- to 13-fold more invasive than normal uveal melanocytes. Colocalization of vimentin/keratins 8,18 in cell cultures was corroborated by immunohistochemistry in histologic sections of tumors from which the cell lines were derived. Minor populations of these cells also coexpressed keratins 13 and 17. Experimental down-regulation of the predominant keratins 8,18 in the interconverted cells, using 16-mer antisense oligonucleotides, resulted in a significant decrease in the migratory ability of the cells-similar to levels achieved by cells positive only for vimentin. These findings provide justification for additional studies of the association between coexpression of IFs vimentin/keratins 8,18 and uveal melanoma metastasis.

An epitope localized in c-Src negative regulatory domain is a potential marker in early stage of colonic neoplasms.

Sakai T. Kawakatsu H. Fujita M. Yano J. Owada MK.
Division of Hemopoiesis, Institute of Hematology, Jichi Medical School, Tochigi, Japan.
In previous work, we established a new monoclonal antibody that specifically recognizes the active form of c-Src tyrosine kinase (Kawakatsu et al, 1996). To determine whether c-Src is active in colorectal tumorigenesis, we examined the expression of an active form of c-Src in human normal mucosa, hyperplastic polyps, adenomas, and adenocarcinomas. The tissue distribution of the active form of c-Src was studied by immunohistochemistry using this antibody, termed Clone 28. Among 66 cases of adenoma tested, 61 (92%) showed positive staining (adenoma with mild atypia, 3 of 3; adenoma with moderate atypia, 38 of 42; adenoma with severe atypia, 20 of 21). In contrast to the frequent and intense staining in adenomas, adenocarcinoma showed weak staining with less frequency in 4 of 16 (25%) cases. The number of specimens with positive staining in well- and moderately differentiated adenocarcinomas was limited to an early stage. The active form of c-Src mainly localized to the nuclear membrane and the perinuclear region. These results provide the first direct evidence that the activation of c-Src appears to be an early event in colonic carcinogenesis in situ. The findings of the present study thus allow us to propose a molecular mechanism involving c-Src activation in the process of malignant transformation of the human colonic neoplastic cells.

A particular characteristic of IgH complementarity-determining region 3 suggests autoreactive B-cell origin of primary gastric B-cell lymphomas.

Year 1998
Yumoto N. Furukawa M. Kurosu K. Mikata A.
First Department of Pathology, School of Medicine, Chiba University, Japan.
To clarify the origin of tumor cells and the possible role of antigen in the pathogenesis of mucosa-associated lymphoid tissue (MALT) lymphoma, we analyzed the third complementarity determining region of IgH gene in nine primary gastric B-cell lymphomas by PCR. The presence of vacA gene of Helicobacter pylori was also determined. These cases were diagnosed histopathologically as three low (extranodal marginal zone B-cell lymphoma)- and three high-grade MALT lymphomas and three diffuse large-cell lymphomas without evidence of MALT lymphoma. All cases showed a monoclonally rearranged JH band. A single dominant clone was detected by sequencing the IgH CDR3 regions in eight cases. In the remaining cases. In the remaining case, two major sequences were identified. Complementarity-determining region 3 (CDR3) sequences in lymphoma cell clones of seven cases showed 61% to 81% homology with autoantibody-associated lymphocyte clones including rheumatoid factor. The N-D-N region in the low-grade MALT lymphoma group was significantly longer than in the high-grade lymphoma groups. Although vacA gene of H. pylori was detected in five of nine cases, all lymphoma cell clones showed no association with foreign antigen. These results indicate that autoantigen may be responsible for the clonal selection of lymphoma cells, and thus autoimmunity may play a role in the pathogenesis of primary gastric B-cell lymphomas.

Immunoglobulin VH genes of high-grade mucosa-associated lymphoid tissue lymphomas show a high load of somatic mutations and evidence of antigen-dependent affinity maturation.

Year 1998
Hallas C. Greiner A. Peters K. Muller-Hermelink HK.
Institute of Pathology, University of Wurzburg, Germany.
High-grade mucosa-associated lymphoid tissue (MALT) B-cell lymphoma of the stomach shares several features with its low-grade counterpart. The latter is nearly invariably associated with Helicobacter pylori, and the tumor cells of all MALT lymphomas normally express surface antigen receptors; thus, it is possible that the high-grade type, like the low-grade type, is still influenced by interaction with antigen. In the present study, we analyzed the immunoglobulin heavy chain variable (V)-region genes from eight cases of high-grade MALT lymphoma and one case of Burkitt's lymphoma of the stomach. The V-region genes revealed somatic mutations in all cases, leading to the conclusion that high-grade MALT lymphomas derive from antigen-experienced (post-) germinal center B-cells. Nonrandom distribution of replacement and silent mutations within the gene segments in seven of the eight MALT lymphomas indicated that these V-region genes were selected by antigen, at least for some period of time. Five of the cases showed an unusual mutation pattern that was suggestive of selection by autoantigen or superantigen rather than heterogeneous antigen. Analysis for intraclonal variations revealed evidence of ongoing mutations in two cases. In these cases, the tumor clones probably derived from cells affected by a germinal center B-cell reaction, as the microenvironment of the germinal center is required for maintenance of an active hypermutation mechanism. On the other hand, in another two cases, no evidence of intraclonal variations was found. Thus, either these tumor clones were derived from postgerminal center B-cells, or the hypermutation mechanism in the germinal center ceased after some period of time. Given the mutation pattern, it is possible that high-grade MALT lymphomas emerge from further transformation of low-grade MALT lymphomas with accumulation of additional mutations in the complementarity-determining regions.

Analysis of differential gene expression in human colorectal tumor tissues by RNA arbitrarily primed-PCR: a technical assessment.

Year 1998
Tortola S. Capella G. Marcuello E. Gunther K. Aiza G. Masramon L. Reymond MA. Peinado MA.
Department of Cancer and Metastasis, Institut de Recerca Oncologica, Hospital Duran i Reynals, Barcelona, Spain.
RNA arbitrarily primed (RAP)-PCR is a powerful tool for studying differential gene expression in cancer cells. Systematic analysis of human tumor samples may provide a list of markers with potential application to the diagnosis, prognostic assessment, and treatment of the disease. Nevertheless, because of characteristics inherent to the samples and technique, artifactual results are likely. We have assessed the effects of several factors on RAP-PCR performance to determine the sensitivity and reproducibility of the technique, as well as the accuracy of its results, under different conditions in human cell lines and in a series of 129 paired human normal colonic mucosa-colorectal carcinoma samples. Our results show that RAP-PCR provides reliable fingerprints in a relatively wide spectrum of circumstances, including variations in RNA concentration and contamination by DNA. Densitometric analysis indicated that relative band-intensity variations more than 20% were reproducible in 95% of the cases. Serial analysis of paired normal-tumor cases yielded a number of bands that were recurrently either underexpressed or overexpressed in tumor relative to normal mucosa. These differentially expressed bands are prime targets of research because they represent candidate tumor-specific up- or down-regulated genes with a relevant role in carcinogenesis.

Amplification of c-erbB-2 in gastric cancer: detection in formalin-fixed, paraffin-embedded tissue by fluorescence in situ hybridization.

Year 1998
Ooi A. Kobayashi M. Mai M. Nakanishi I.
Department of Pathology, Faculty of Medicine, Kanazawa University, Japan.
A total of 396 adenocarcinomas of the stomach were examined immunohistochemically using antibodies specific for the internal domain of human c-erbB-2 protein. Forty of these (consisting of 35 well-differentiated and 5 undifferentiated types) overexpressed c-erbB-2, as evidenced by cytoplasmic membrane staining; these tumors were further examined by fluorescence in situ hybridization using a cosmid probe for 17q11.2-12 (c-erbB-2 locus) on formalin-fixed, paraffin-embedded tissues. This technique enabled us not only to detect c-erbB-2 amplification on a cell-by-cell basis, but also to compare the gene amplification with the protein expression, observe topographical distribution, and establish quantitative pictures of the amplification in vivo condition. In all 40 tumors, we observed gene amplification and found that the cancer cell with the amplification corresponded to the cells overexpressing c-erbB-2. In eight mucosal carcinomas, signals were coalesced to one or two clusters, indicating that the amplified gene resided in a homogeneous staining region (HSR). Among 32 carcinomas invading beyond muscularis mucosae, approximately half were mostly composed of cells with the amplification; the others had populations of tumor cells with and without amplification, which were sometimes in separate zones or areas and sometimes mixed together. In 22 cancers, the amplified genes were exclusively in HSR; however, cancer cells with multiple scattered signals-suggesting that the amplified genes were translocated to other chromosomes-were found exclusively in 6 tumors and concurrently with HSR in 4 tumors. These findings suggest that: (a) the amplification produces c-erbB-2 in the HSR form generally early in the carcinogenesis of gastric adenocarcinomas; and (b) in the process of cancer development, the amplified gene is lost in some cancer cells by uneven segregation of HSR in mitosis, whereas in others, it is kept in HSR or translocated to other chromosomal positions, thereby preserving overexpression of c-erbB-2 protein.

Elevated c-Src protein expression is an early event in colonic neoplasia.

Year 1998
Iravani S. Mao W. Fu L. Karl R. Yeatman T. Jove R. Coppola D.
Department of Pathology, Moffitt Cancer Center, Tampa, Florida, USA.
The molecular events regulating the development and progression of colonic neoplasia are currently being delineated. Recent studies have implicated c-Src protein kinase activation as an early event in the malignant transformation of colonic epithelial cells. However, increased c-Src activity has also been reported in colon carcinomas as well as in metastatic hepatic and extrahepatic colon carcinomas. To further investigate the potential role of c-Src in the progression of colonic neoplasia, we analyzed c-Src levels by immunohistochemistry in 27 colorectal resection specimens. Mouse monoclonal antibody to c-Src protein was applied to 3-micron sections from formalin-fixed, paraffin-embedded tissues using the avidin-biotin-peroxidase method. The combination of adenomatous (AD) and adjacent carcinomatous mucosa (CA) specimens were present in 20 of 27 patients. In 15 cases, synchronous metastatic (MT) lesions were available for evaluation. Strong c-Src expression was evident in 95% of AD (n = 20), in contradistinction to 32% of MT (n = 19) and 14% of CA (n = 22). Weak-to-moderate c-Src expression was seen in adjacent normal colonic mucosa (NM) in 96% of cases. Signed rank test univariate analysis revealed a statistically significant difference in c-Src expression between NM/AD (p = 0.0001), NM/CA (p = 0.0001), NM/MT (p = 0.0006), AD/CA (p = 0.0001), and AD/MT (p = 0.0002). No significant correlation between levels of c-Src expression and patient survival, tumor size, histologic grade, or tumor configuration was observed using the Cox's Regression Model. Similar results were obtained by analysis of c-Src protein levels and c-Src kinase activity as measured by Western blot and in vitro kinase assays of representative cases. Our results indicate that: (a) elevated c-Src expression is an important early event during colorectal carcinogenesis; (b) its activation may be involved in tumor progression in a subset of colonic carcinomas; and (c) additional molecular events are necessary for invasion to occur.

Protein kinase Calpha controls the adhesion but not the antiproliferative response of human colon carcinoma cells to transforming growth factor beta1: identification of two distinct branches of post-protein kinase Calpha adhesion signal pathway.

Year 1998
Chakrabarty S. Rajagopal S. Moskal TL.
Department of Laboratory Medicine, University of Texas, M. D. Anderson Cancer Center, Houston 77030, USA.
Transforming growth factor beta1 (TGFbeta1) inhibits cellular proliferation and induces the expression of the matrix adhesion molecules fibronectin (FN) and laminin (LM) in a concurrent manner, followed by the induction of the intercellular adhesion molecule carcinoembryonic antigen (CEA) (collectively designated as adhesion responses) in TGFbeta1-responsive human colon carcinoma cells. Exactly how TGFbeta1 controls cellular adhesion and proliferation is poorly understood. In the present report, we showed that down-regulating protein kinase Calpha (PKCalpha) expression blocked the induction of these adhesion responses by TGFbeta1, showing that PKCalpha is a postreceptor focal point controlling the induction of these molecules. Down-regulating PKCalpha expression, however, had minimal effect on the antiproliferative response to TGFbeta1 or the induction of p21/WAF1, a marker associated with the antiproliferative effect of TGFbeta1, demonstrating that the adhesion signal pathway is distinct from that of antiproliferation. Blockade of FN induction blocked the induction of CEA but not the induction of LM. Blockade of LM induction, on the other hand, had no effect on the induction of FN and CEA. These results established the existence of two distinct and parallel postPKCalpha adhesion signal pathways, one leading to the induction of LM and the other leading to the induction of FN and CEA.

Detection of soluble Fas mRNA using in situ reverse transcription-polymerase chain reaction.

Year 1998
Lee SH. Kim SY. Lee JY. Shin MS. Dong SM. Na EY. Park WS. Kim KM. Kim CS. Kim SH. Yoo NJ.
Department of Pathology and Cancer Research Institute, Catholic University Medical College, Seoul, Korea.
Fas protein (Fas) is known to induce cell death by apoptosis in susceptible cells. Alternative splicing of the Fas gene produces soluble Fas protein (sFas), which is considered to block the function of Fas. The serum level of sFas is elevated in patients with various malignancies in a manner reflective of disease stage and tumor burden, but the precise cellular origin of sFas in vivo has not yet been clarified. To identify the cells that synthesize sFas mRNA on histologic specimens, we applied in situ reverse transcription-polymerase chain reaction (in situ RT-PCR) in 11 cases of gastric adenocarcinoma/metastatic lymph node. Furthermore, we studied the distribution of Fas using immunohistochemistry and Fas mRNA using in situ RT-PCR. In all primary tumors and 10 of 11 metastatic tumors, tumor cells expressed both Fas- and sFas mRNA. Lymphocytes infiltrated in the tumor tissues and the lymph nodes also revealed both mRNA signals. A clear correlation between the tissue distribution for Fas and its mRNA was also observed. These observations demonstrated that solid tumors in vivo can synthesize sFas mRNA and suggest that tumor cells are responsible in part for elevated sFas in human malignancies. However, the additional expression of sFas mRNA in tissue lymphocytes indicates the complex regulatory mechanisms of Fas-mediated apoptosis pathway in tumor pathogenesis and host defense. We also demonstrated that in situ RT-PCR can be a suitable method for in situ detection of alternatively spliced mRNA.

Somatic mutations of multiple endocrine neoplasia type 1 gene in the sporadic endocrine tumors.

Year 1998
Shan L. Nakamura Y. Nakamura M. Yokoi T. Tsujimoto M. Arima R. Kameya T. Kakudo K.
Department of Pathology, Wakayama Medical College, Wakayama City, Japan.
Endocrine tumors of the parathyroid and pancreas are encountered either as sporadic type or as part of multiple endocrine neoplasia type 1 (MEN 1). A high frequency of the loss of heterozygosity (LOH) has been observed in tumors of the sporadic type in the locus of the MEN 1 gene, which has recently been cloned and designated the menin gene. It would be of great interest to determine whether somatic mutations in the menin gene are responsible for the sporadic endocrine tumors. For this purpose, we have investigated the menin gene mutations in 21 sporadic parathyroid adenomas, 2 parathyroid carcinomas, 4 sporadic insulinomas, and 1 malignant VIP (vasoactive intestinal polypeptide)oma with WDHA (watery diarrhea, hypokalemia, and achlorhydria) syndrome, using PCR-single strand conformation polymorphism analysis and DNA sequencing. In none of these cases did the patient have a family history or other possible association with MEN 1. We have discovered somatic point mutations in two parathyroid adenomas (A340T and A541T), in one insulinoma (T429K), and in the malignant VIPoma (W198X). In addition, we have found two polymorphisms (D418D and V367V) in two parathyroid carcinomas and two parathyroid adenomas. Of these mutations and polymorphisms, three (A340T, T429K, and V367V) are first reported here, in the present article. Our results indicate that somatic mutations of the menin gene are responsible for a proportion of the sporadic parathyroid adenomas and pancreatic islet cell tumors.