Immunohistochemical detection of the MUC1 gene product in human cancers grown in scid mice.
Schumacher U. Adam E.
Human Morphology, University of Southampton, Southampton, United Kingdom.
Alterations in mucin expression have been detected in many clinically relevant cancers and, in particular, the polymorphic epithelial mucin, encoded by the MUC1 gene, has attracted considerable attention. We investigated its expression in human breast, colon, ovarian, lung, and skin cancer cells and their metastases grown in severe combined immunodeficient (scid) mice using three different monoclonal antibodies (HMFG-1, HMFG-2, and SM3). Four of five breast cancer cell lines, three of five colon cancer cell lines, two of three small-cell carcinoma of the lung cell lines, and A 431 cells all expressed the MUC1 gene product. Neuraminidase predigestion often enhanced HMFG-1 immunoreactivity, which was more widespread and stronger than SM3 immunoreactivity. A considerable heterogeneity of MUC1 gene product expression was observed in the same tumors grown in different mice. The binding pattern between single-cell/small-cell clusters (up to 10 cells) and larger cell number aggregates varied. The results indicate that the MUC1 gene expression both in primary tumors and metastases is not tightly controlled within a particular tumor cell line. Because of this heterogeneous antigen expression in vivo, it appears impossible to target all metastatic deposits by a single monoclonal antibody directed against the MUC1 gene product. (J Histochem Cytochem 46:127-134, 1998)
p53 expression in human carcinomas: could flow cytometry be an alternative to immunohistochemistry?
Benini E. Costa A. Abolafio G. Silvestrini R.
Oncologia Sperimentale C, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy.
Several studies have shown that p53 expression has important clinical implications as an indicator of prognosis and response to chemotherapy or radiotherapy in different human tumor types. Determination of p53 expression by immunohistochemistry (IHC) has been incorporated into routine practice and its reliability has been consolidated. However, flow cytometric (FCM) analysis might represent an important objective and rapid approach. In the present study we determined p53 expression by IHC and FCM on a series of 118 human solid tumors. IHC determination was performed on histological sections and FCM analysis on cell suspensions. Low correlation coefficients (rs from 0.22 to 0.57) were observed between IHC and FCM data from individual tumors. By considering the IHC approach as the gold standard, high sensitivity and low specificity were found for FCM in detecting p53 expression. The FCM analysis of p53 expression and DNA content showed p53-positive cells in all cell cycle phases. Moreover, in most breast, lung, and colon aneuploid tumors (77%), p53-positive cells were detected only in the subpopulations with abnormal DNA content. In conclusion, FCM-p53 expression cannot be used alternatively to IHC determination, and its clinical relevance remains to be validated. Nevertheless, FCM may provide important information about p53 protein expression in the different subpopulations and cell cycle phases. (J Histochem Cytochem 46:41-47, 1998)
Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tissue sections.
Ikeda K. Monden T. Kanoh T. Tsujie M. Izawa H. Haba A. Ohnishi T. Sekimoto M. Tomita N. Shiozaki H. Monden M.
Department of Surgery II, Osaka University Medical School, Osaka, Japan.
We describe and discuss a method of protein extraction for Western blot analysis from formalin-fixed, paraffin-embedded tissue sections. From 5-mm2 50-micron-thick tissue sections, an abundance of proteins could be extracted by incubating the sections in lysis buffer containing 2% sodium dodecyl sulfate (SDS) at 100C for 20 min followed by incubation at 60C for 2 hr. Extracts yielded discernible protein bands ranging from 10 kD to 120 kD as identified by SDS-polyacrylamide gel electrophoresis (PAGE). Western blot analysis successfully detected membrane-bound protein such as E-cadherin, cytosolic protein such as beta-catenin, and nuclear proteins including proliferating cell nuclear antigen (PCNA), mutant-type p53, cyclin D1, cyclin E, and cyclin-dependent kinases (CDKs). With this technique, we could examine cyclin D1 and CDK2 expression in small adenomas compared with cancer tissues and normal mucosa. The simple method of protein extraction described here should make it possible to use large-scale archives of formalin-fixed, paraffin-embedded samples for Western blot analysis, and its application could lead to detailed analysis of protein expression. This new technique should yield valuable information for molecular biology.
Preferential activation of the epidermal growth factor receptor in human colon carcinoma liver metastases in nude mice.
Parker C. Roseman BJ. Bucana CD. Tsan R. Radinsky R.
Department of Cell Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 44106, USA.
Increased epidermal growth factor receptor (EGF-R) gene expression and functional protein levels correlate with the metastatic potential of human colon carcinoma (HCC) cells in nude mice. The purpose of this study was to determine whether the production of liver metastases by HCC cells depends on the EGF-R activation status and whether different organ microenvironments influence this activation. Using two independent monoclonal antibodies specific for the activated (i.e., tyrosine-phosphorylated) EGF-R, increased immunoreactivity was observed in HCC cells growing as metastatic lesions in the livers of athymic nude mice. Staining was observed throughout these lesions, both peripherally and centrally. In contrast, little or no immunoreactivity for activated EGF-R was observed in primary tumors growing orthotopically in the cecum or ectopically in the subcutis of nude mice. Immunohistochemistry for total EGF-R levels (irrelevant of activation status) demonstrated similar levels of immunoreactivity in HCC tumors growing in the cecum, subcutis, or liver of nude mice, indicating that total EGF-R levels are not altered after growth in these different microenvironments. Controls included immunohistochemistry for total and activated EGF-R levels in HCC cells growing in vitro under serum-free or EGF-stimulated conditions and A431-epidermoid carcinoma growing in nude mice. Western blot analyses confirmed the specificity of the antibodies for the activated EGF-R. These results suggest that the production of liver metastasis by HCC cells depends in part on the response of tumor cells to organ-derived growth factors and hence the activation of specific cell surface tyrosine kinase receptors.
Detection and localization by in situ molecular biology techniques and immunohistochemistry of hepatitis C virus in livers of chronically infected patients.
Walker FM. Dazza MC. Dauge MC. Boucher O. Bedel C. Henin D. Lehy T.
Department of Pathology, Hopital Bichat-Claude Bernard, Paris, France.
Hepatitis C virus (HCV) detection in the livers of chronically infected patients remains a debatable issue. We used immunohistochemistry, in situ hybridization (ISH) alone or after microwave heating with FITC-labeled probes, RT-PCR with unlabeled primers followed by ISH (RT-PCR-ISH), and in situ RT-PCR with FITC-labeled primers (in situ RT-PCRd) to localize the virus in 38 liver biopsy specimens from 21 chronically infected HCV patients treated with interferon-alpha (IFN-alpha). Biopsies were taken at the beginning and end of IFN-alpha treatment and 1 year later. Results were compared with that of HCV-PCR in serum. RT-PCR-ISH and in situ RT-PCRd showed HCV signal in all liver biopsies even in responders with seronegative HCV PCR. This signal was intranuclear, diffuse, or peripheral, in hepatocytes, bile ductule cells, and lymphocytes. Cytoplasmic signals were occasionally observed. Whereas the percentage of labeled hepatocytes remained constant, the number of labeled lymphoid follicles decreased after INF-alpha therapy. Immunohistochemistry resulted in the same pattern of positivity but it was weaker and inconstant. This study indicates the persistency of HCV latency in IFN-alpha responders 1 year after IFN-alpha treatment cessation, a finding that certainly deserves confirmation.
Histochemical reactivity of normal, metaplastic, and neoplastic tissues to alpha-linked N-acetylglucosamine residue-specific monoclonal antibody HIK1083.
Nakamura N. Ota H. Katsuyama T. Akamatsu T. Ishihara K. Kurihara M. Hotta K.
Second Department of Internal Medicine, Shinshu University School of Medicine, Matsumoto, Japan.
Monoclonal antibody (MAb) HIK1083, which is obtained by immunizing mice with a preparation of rat gastric mucins, has been shown to bind specifically to alpha-linked N-acetylglucosamine (alpha-GlcNAc). We investigated the specificity of MAb HIK1083 by immunostaining normal human organs, mucinous metaplasia of human pancreas, adenocarcinomas of human stomach, pancreas, and colon, and normal rat organs. The specificity was investigated by making comparisons with (a) a stain that labels Class III concanavalin A (ConA)-reactive mucin (Class III mucin), i.e., paradoxical ConA (PCS), and (b) staining with horseradish peroxidase (HRP)-conjugated Griffonia simplicifolia agglutinin II (GSA-II). In normal human and rat organs and in mucinous metaplasia of human pancreas, immunostaining with MAb HIK1083 and PCS showed similar specificities for mucins in glandular mucous cells. In adenocarcinoma of stomach and pancreas, GSA-II showed the most widespread positivity, PCS showed the least, and MAb HIK1083 showed a reactivity between those two extremes. Colon adenocarcinomas were labeled only with GSA-II. These results demonstrate that MAb HIK1083 could be a useful screening tool for Class III mucin in normal, metaplastic, and carcinoma tissues, and that the alpha-GlcNAc residue is one of the specific sugar residues found in Class III mucin.