The p53 status of cultured human premalignant oral keratinocytes.

Around 60% of oral squamous cell carcinomas (SCCs) have been shown to harbour p53 mutations, and other studies have demonstrated mutant p53 genes in normal and dysplastic squamous epithelium adjacent to these SCCs. In line with these earlier studies we show here that DOK, a keratinocyte cell line derived from a dysplasia, displays elevated levels of p53 protein and harbours a 12 bp in-frame deletion of the p53 gene spanning codons 188-191. In contrast, the coding region of the p53 gene was normal in a series of six benign recurrent laryngeal papillomas and a series of four premalignant oral erythroplakia biopsies and their cell cultures. All but one of these lesions were free of malignancy at the time of biopsy, in contrast to the premalignant lesions studied by previous investigators, but keratinocytes cultured from these lesions all displayed a partially transformed phenotype that was less pronounced than that of DOK. Since three out of four of the erythroplakia patients developed SCC within 1 year of biopsy, these lesions were by definition premalignant. The availability of strains of partially transformed keratinocytes from premalignant erythroplakias which possess normal p53 genes should enable us to test the role of mutant p53 in the progression of erythroplakia to SCC. The premalignant tissues and cultures were also tested for the presence of human papillomavirus (HPV), which is known to inactivate p53 function in some cases. Only the benign papillomas were shown to contain high levels of either HPV 6 or HPV 11 E6 DNA, but not both, and none of the samples contained detectable levels of HPV 16, HPV 18 or HPV 33 E6 DNA or L1 DNA of several other HPV types. There was therefore no evidence to suggest that p53 was being inactivated by a highly oncogenic HPV in these samples.     

Immunohistochemical detection of antigen in human primary and metastatic melanomas by the monoclonal antibody 140.240 and its possible prognostic significance

An indirect immunofluorescence technique was used to study the tissue distribution of the epitope recognized by the monoclonal antibody 140.240 which identifies a p97-like melanoma-associated oncofetal antigen. Cryostat sections of various normal and neoplastic human tissues were examined. The presence of antigenic activity was demonstrated in 20 of 39 (51%) primary skin melanomas, in 21 of 52 (40%) metastatic melanomas, and in 20 of 44 (45%) nevi. The reactive nevi were restricted to intradermal, junctional, compound and spindle cell types. Of the 110 samples of 12 major tumor types other than melanoma tested, only 1 of 6 epidermoid tumors, 1 of 4 benign breast tumors and 1 of 5 prostatic tumors gave weak staining. This antibody also reacted with sweat glands and fetal small intestine tissue, but not with other adult or fetal normal tissues. Intrapatient as well as interpatient heterogeneity in the epitope expression was present in primary as well as metastatic tumor lesions surgically removed from patients with melanoma. Evaluation of the immunohistologic data and the clinical outcome of patients with melanoma reveals that the expression of the epitope recognized by this antibody is associated with a more favorable prognosis.     

Alteration of glycine N-methyltransferase activity in fetal, adult, and tumor tissues.

Glycine N-methyltransferase activity has been examined in a number of fetal and adult organs, as well as in several rodent hepatomas, using both enzymatic and immunological techniques. In fetal rabbit liver, the activity first appears at a low level at about 20 days postfertilization and rises to high levels after birth, reaching maximum in the adult liver. In fast-growing hepatomas, the activity could not be detected by either enzymatic or immunological assay. It could be detected in the slower-growing hepatomas, but in considerably diminished levels compared with that of normal adult rat liver. Immunoassays gave no evidence for inactive forms of the enzyme in the tissues that had no enzymatic activity. Transfer RNA methyltransferase assays carried out simultaneously showed an inverse relationship to the glycine N-methyltransferase activity. The levels of transfer RNA methyltransferase activity were high in fetal and tumor tissues and lower in normal adult tissues.     

Expression pattern of the neu (NGL) gene-encoded growth factor receptor protein (p185neu) in normal and transformed epithelial tissues of the digestive tract

The neu gene (also called NGL, erbB-2, and HER-2) encodes a 185-190 kDa transmembrane glycoprotein, p185neu, which has tyrosine-specific kinase activity and is homologous to but distinct from the epidermal growth factor receptor. The normal expression of neu mRNA and protein has been demonstrated in epithelial tissues of adult animals. Also, activation of the neu oncogene has been implicated in a variety of human adenocarcinomas. In the present study, we examined the expression of the p185neu protein in normal and transformed digestive tract tissues and in a panel of digestive tract-derived cell lines. By immunohistochemistry, strong reactivity was observed in the mucosal epithelium of the stomach, small intestine, and colon of both rodents and humans. In the small intestine, there was prominent p185neu expression by mucosal epithelium of the villus, with little or no staining in the crypts. Prominent expression was observed in the liver parenchyma, the endocrine and exocrine portions of the pancreas, and in the salivary gland. Immunoreactive p185neu was also demonstrated in fetal human intestinal epithelium. Tissue sections of selected benign and malignant colonic neoplasms were also examined. Immunoreactivity was consistently greater in adenomatous polyps than in adjacent normal colonic epithelium or areas showing malignant degeneration. By radioimmunoprecipitation, there was decreased expression in cell lines derived from more anaplastic colonic tumors. The p185neu protein is expressed widely in normal and transformed epithelial tissues of the digestive tract of the adult rat and human. This finding suggests that p185neu, a putative growth factor receptor, may play a role in the regulation of normal growth and function or in the malignant transformation of these cells.     

Expression of the epitope recognized by the monoclonal antibody 44-3A6 during human fetal development.

In this report we describe the expression of the adenocarcinoma associated antigen recognized by the monoclonal antibody 44-3A6, in various tissues during normal human fetal development. Conventional, formalin-fixed and paraffin-embedded sections of normal organs were examined from fetuses ranging from 9 to 42 weeks of gestation. Immunohistochemical localization of antigen-antibody complexes was accomplished using the avidin-biotin complex (ABC) method using horseradish peroxidase. The monoclonal antibody (MAb) 44-3A6 detects a cell surface 40 kD protein which is frequently expressed by adenocarcinomas and by select normal glandular tissues. Detectable expression of this protein was seen at different time periods during fetal development depending on the tissue. This expression was confined to a relatively small range of cell types and tissues; immunostaining was noted in select epithelial cells of the aerodigestive tract, exocrine pancreas, neural tissues, renal tubules, and transitional urothelium, as well as in other tissues. This immunostaining generally, but not invariably, corresponded with patterns previously reported in benign and/or malignant neoplasms of adult tissues. In most instances, once expression occurred within a tissue, it continued through gestation. These data show that this tumor associated gene product is differentially expressed in a broad range of normal developing human fetal tissues.     

Ha-ras p21-GTP levels remain constant during primary keratinocyte differentiation

Several lines of evidence that indicate that mutation of the Ha-ras oncogene is the initiating event in mouse skin carcinogenesis. Keratinocytes known to possess a mutated Ha-ras have been shown to be resistant to differentiation. Thus, overstimulation of the Ha-ras signaling pathway appears to block normal keratinocyte differentiation, and we hypothesized that for normal keratinocytes to terminally differentiate, the Ha-ras signaling cascade must be turned off. In the present studies, we measured the level and activity state of Ha-ras p21 protein in cultured keratinocytes undergoing calcium-induced differentiation. We have employed Western blot analysis to demonstrate that Ha-ras p21 protein levels remain constant during primary newborn and adult keratinocyte differentiation. The overall level of Ha-ras p21 was higher in immortalized, benign, and malignant mouse keratinocyte cell lines than in normal keratinocytes but did not change within each cell type when subjected to differentiating conditions. The percentage of Ha-ras p21 protein in its active, GTP-bound form also remained unchanged during primary adult keratinocyte differentiation and in immortalized, benign, and malignant keratinocytes subjected to differentiating conditions. Our results indicate that terminal differentiation of primary adult mouse keratinocytes occurred in the presence of constant levels of Ha-ras p21-GTP, suggesting that the Ha-ras signaling pathway may be blocked at a point distal to a step involving the Ha-ras p21 protein itself. © 1995 Wiley-Liss Inc.     

Expression of ras proto-oncogene proteins in normal human tissues

The expression of ras proto-oncogenes in normal human tissues was studied by immunohistochemical staining and by immunoblotting using monoclonal antibodies. We detected p21ras protein in almost every fetal and adult tissue, but the level varied significantly among cell types. In some cell lineages, immature cells capable of proliferation contain more p21ras than do mature cells. By contrast, certain fully differentiated cells, such as neurons and the epithelial cells of endocrine glands, express abundant p21ras. Among mammalian tissues the highest level of ras protein was detected in brain. Crude synaptosomal membrane preparations from rat brain contain substantially more p21ras than do plasma membranes from rat liver. The observed distribution of p21ras suggests a role for these proteins both in cellular proliferation and in certain specialized cellular functions.     

Quantitation of Harvey ras p21 enhanced expression in human breast and colon carcinomas

Molecular studies have demonstrated increased expression of the Harvey (Ha) ras oncogene in human breast and colon carcinomas. With the use of a direct-binding liquid competition radioimmunoassay (RIA), capable of providing truly quantitative analysis of the 21,000-dalton (p21) ras oncogene and protooncogene products, absolute levels of Ha-ras p21 have been determined in human breast and colon carcinomas, benign lesions, and/or their respective normal tissues. Enhanced Ha-ras expression was documented in 66% of breast and 100% of colon carcinomas as compared with their normal counterparts, with levels in breast carcinomas ranging from 10.1 to 50.4 pg ras p21/micrograms protein and those in colon carcinomas ranging from 18.4 to 51.7 pg ras p21/micrograms protein. Some dysplastic lesions of the breast and colon also contained elevated Ha-ras p21. Relative levels of Ha-ras p21 expression, detected by competition RIA, correlated with percent Ha-ras p21-positive cells as determined by immunohistochemical assays. By use of liquid competition RIA and immunohistochemical assays, it has been shown that levels of ras p21 expression did not always correlate between primary and metastatic colon lesions of the same patient. The use of the quantitative RIA and semiquantitative immunohistochemical assays, in concert with cDNA probes for identification of specific ras point-mutated oncogenes or protooncogenes, may now provide the means for definitive quantitative analyses of ras p21 in human carcinomas and benign lesions.     

Monoclonal antibody defining fibroblasts appearing in fetal and neoplastic tissues

VIF3 is a hybridoma-derived mouse IgG monoclonal antibody (MoAb) generated in a fusion with the use of a malignant fibrous histiocytoma as the immunizing agent and shown to recognize a 70-kilodalton antigen expressed within connective tissues. Of 55 human tissue culture cell lines tested by indirect immunofluorescence, VIF3 was shown to bind to 20 of 35 (57%) sarcomas, 4 of 9 (44%) normal fibroblasts, and none of 11 carcinomas and other neoplasm-derived cell lines. A panel of over 259 human frozen tissue sections obtained from surgical pathology specimens, postmortem studies, and elective abortions was used to further determine the histopathologic specificity of VIF3. MoAb VIF3 was found to bind to 15 of 19 (79%) human sarcoma tissue sections tested. It was also shown to recognize an antigen expressed on a subset of fibroblasts dispersed within the stroma of carcinomas obtained from all 46 patients tested, as well as a subset of cells within 3 of 10 benign hyperplastic tissues (30%). VIF3-positive cells were detected in all 60 fetal tissues tested including amniotic membranes, placentas, and umbilical cords. In contrast, fibroblasts of normal adult tissues tested stained infrequently (22/97 or 23%) with this reagent. The results confirm that this MoAb is directed against a human connective tissue-specific marker. VIF3 detects a marker appearing on fetal fibroblasts that is typically not present in normal adult tissues, but reappears in association with neoplastic diseases. MoAb VIF3 therefore defines a fibroblast-oncofetal antigen and as such may serve as a probe for immunopathologic studies.     

Identification of a 170-kDa protein over-expressed in lung cancers

Lung cancer is the leading cause for cancer death in both male and female populations. Although many molecular markers for lung cancer have been developed and useful for early detection of lung cancer, their function remains unknown. In this paper, we report our findings that a 170-kDa protein (p170) is over-expressed in all types of human lung cancers compared with normal tissues and it is identified as a subunit of translation initiation factor eIF3 by cDNA cloning. Translation initiation factors are a family of proteins that promote the initiation step of protein synthesis and are regulators of cell growth at the translational level. Further studies showed that p170 mRNA is ubiquitously expressed with higher levels in adult proliferating tissues (e.g. bone marrow) and tissues during development (e.g. fetal tissues). This study suggests that p170 and eIF3 may be important factors for cell growth, development, and tumorigenesis.     

Detection of a human sarcoma-associated antigen with monoclonal antibodies

Hybridoma cells were derived from a mouse immunized with plasma membranes prepared from the fresh tumor tissues of a patient with malignant fibrous histiocytoma (MFH), a soft tissue sarcoma. Supernatants from the resultant hybridoma clones were screened for positive antibody binding to tumor membranes and negative binding to membrane preparations of normal tissues using a solid-phase radioimmunoassay. Two distinct monoclonal IgG1 (kappa) antibodies, 19-14 and 19-24, were identified that showed identical patterns of reactivity with a large panel of tissues. Both antibodies displayed high levels of binding to membranes prepared from a majority of MFH and osteogenic sarcoma tumors tested. Moderate levels of binding were obtained with melanoma, colorectal carcinoma, and first-trimester fetal membranes. Weak or no significant binding was observed with membranes from a variety of autologous and allogeneic normal adult tissues. Antibody reactivities could be specifically removed by absorption with MFH and osteosarcoma membranes but not with adult muscle membranes. An electrophoretic analysis of immunoprecipitated membrane antigens indicated that antibodies 19-14 and 19-24 reacted with the same protein, a monomer with an approximate molecular weight of 102,000. The antigen was detected in membrane preparations of MFH, osteosarcoma, and first trimester fetus, but was not present in normal adult spleen. However, a small amount of antigen of molecular weight 107,000 was precipitated from a normal adult liver preparation, which suggests that related antigens may be present in low levels in some normal tissues. Antibodies 19-14 and 19-24 also specifically bound to intact, cultured MFH cells, indicating that the relevant antigens were expressed on the outer cell surface.     

Receptors for hormones and growth factors and (onco)-gene amplification in human ovarian cancer.

Receptor status and gene amplification were studied in advanced human ovarian adenocarcinoma tissues, borderline and benign ovarian tumours and normal ovarian tissues. Sixty-five percent (53/82) of ovarian adenocarcinomas, 57% (8/14) of benign/borderline tumours and only 31% (5/16) of normal ovarian tissues studied showed specific 125I-EGF (epidermal growth factor) binding (median: 17; 10; and 0 fmol EGF-R/mg protein, respectively) and a significant increase in progesterone receptor (PgR) levels was observed in these groups (median: 5; 33; and 152 fmol/mg protein, respectively). No correlations were observed between the levels of EGF-R and the levels of either oestrogen receptors (ER) or PgR. All membrane samples of 25 adenocarcinomas studied by Scatchard analysis were positive for insulin-like growth-factor-I receptors (IGF-I-R) and contained higher IGF-I-R levels than membranes of 10 normal ovarian tissues, of which 9 were positive (median: 64 and 26 fmol IGF-I-R/mg membrane protein, respectively). Also, as measured by autoradiography, 37 adenocarcinoma tissues showed a higher expression of IGF-I-R (1.5+ to 4+) than sections derived from 10 normal ovarian tissues (1+). 125I-IGF-I binding was predominantly associated with epithelial tumour cells, the surrounding connective tissue was negative and in several samples high expression of IGF-I-R was found in areas of tumour necrosis. Southern blot analysis of DNAs isolated from 25 ovarian adenocarcinomas revealed no amplification of the IGF-I-R or the EGF-R gene. The HER2/neu gene was amplified only in 2 out of 3 histologically confirmed endometrioid adenocarcinomas studied but not in 22 other tumours. An amplification of the c-myc gene was observed in 28% (7/25) of the tumours. All c-myc-amplified tumours were PgR-negative. No rearrangement was observed for any of the genes studied. In conclusion, ovarian adenocarcinoma tissues show a decrease in PgR levels and an increased expression of IGF-I-R and EGF-R, in the absence of gene amplification, when compared to benign/borderline ovarian tumours and normal ovarian tissues. In addition, amplification of the c-myc and HER2/neu genes, without rearrangement of these genes, occurs in a minority of these tumours.     

Detection of human papillomavirus DNA in biopsies of human oral tissue

We have employed molecular probes produced from DNA fragments of human papillomavirus, cloned into prokaryotic vectors, to detect virus nucleic acid sequences in extracts of human oral tissues. The study was conducted with duplicate coded snap-frozen tissue biopsies from which frozen sections had been taken to accurately assess the pathology of each particular sample. The results show that a large proportion of the oral biopsies contained DNA which hybridized to the viral DNA probes, even under conditions of high stringency. The presence of virus did not correlate with neoplasia in the tissues examined, but HPV like sequences were found in a high proportion (80%) of biopsies taken from areas of keratosis and lichen planus and also in 41 to 46% of normal and tumour tissues.     

Protein levels and gene expressions of the epidermal growth factor receptors, HER1, HER2, HER3 and HER4 in benign and malignant ovarian tumors

The epidermal growth factor receptors, HER1, HER2, HER3 and HER4 play a key role in the growth of malignant tumors. The receptors of the EGF receptor family are not cancer-specific proteins since these receptors are expressed to some extent in both normal and benign tissue, but this is not elucidated in detail in ovarian tissue. High tumor-to-normal-tissue concentration ratios would be favorable for molecular targeted anti-cancer treatment. The primary aim of the study was to analyze the potential differential protein content and gene expression of the four receptors in benign and malignant ovarian tumors. Tissue from 207 patients (101 malignant, 19 borderline, 64 benign ovarian tumors and 23 normal ovaries) were analyzed by quantitative ELISA for HER1-HER4 protein concentrations and by real-time PCR for HER1-HER4 gene expression. HER2 was also analyzed by immunohistochemistry. The HER2-4 receptor protein content and the median gene expression level was significantly higher in ovarian cancer patients compared to patients with benign ovarian tumors and normal ovaries (p<0.0000001). The protein content of the HER1 receptor was significantly lower in ovarian cancer compared to borderline tumors (p=0.012), benign ovarian tumors (p=0.049) and to normal ovaries (p=0.000069). A sound correlation between the protein levels and gene expressions was documented. In conclusion, decreased concentration of HER1 protein and increased HER2, HER3 and HER4 protein concentration were observed, as also elevated HER2-HER4 gene expression levels in ovarian cancer patients with barely any overlap of the HER3 and HER4 expression in malignant ovarian tumors compared to benign ovarian tissues.     

Expression of the major group-specific antigen (GS-A) of avian type-C viruses in normal chicken cells and tissues

The major group-specific antigen (gs-a) of avian type-C viruses was detected by radioimmunoassay in all tissue samples and cultured cells from leukosts-free chickens. Visceral tissues of individual embryos contained 7–36 ng antigen per mg protein. The mean values in adult tissues were 2–5 times lower than those of the embryos, except for spleen which gave exceedingly high values of about 50 ng per mg protein. The identity of the endogeneous gs-a with viral gs-a was established by several criteria. The gs-a levels of tissues and cells producing avian type-C viruses were 100–1,000 times higher. In such cells up to 1% of cellular protein can be gs-a. Differences in expression of gs-a and other malignancy-associated proteins in normal tissues may prove useful indicators of loosened gene regulation as an initial step towards malignancy.