21% of adenocarcinomas and squamous cell carcinomas were positive according to our criteria (grade 2 or higher), of which 2% of adenocarcinoma and 4% of squamous cell carcinomas were highly positive (grade 3) (Table?1). TA tumor samples, albeit inefficiently. Conclusions While a causative role has not been established, these data suggest that a JSRV-like virus might infect humans. With next generation sequencing approaches, a JSRV-like virus in human lung cancers may be identified which could have profound implications for prevention, diagnosis and therapy. and endogenous JSRV sequences in human tissues [26-28]; however, there is no Thiarabine clear consensus on the association of JSRV with human lung cancer as other studies report no correlation between JSRV and human lung cancer [29-31]. Interestingly, a number of epidemiological studies have found that workers in abattoirs and meat processing plants have an increased risk of developing lung cancer that is postulated to be due to exposure to oncogenic viruses of food animals such as JSRV and bovine papilloma virus [32-34]. Given that JSRV Env is capable of inducing tumors in both sheep and mice, and due to the controversy surrounding the role of JSRV in human lung cancer, we decided to examine multiple types of human lung tumor samples for the presence of JSRV Env by immunohistochemical staining of lung cancer tissue arrays with an Env-specific monoclonal antibody and by PCR amplification with and sequences can reproducibly be amplified from genomic DNA extracted from human lung cancer tissue arrays, albeit inefficiently. Methods Tissue samples Human lung cancer tissue arrays (LC2085a) containing lung tumors from 188 patients and 20 samples of normal tissue were purchased from US Biomax (Rockville, MD). Of the 208 core tissues, there were 72 adenocarcinomas, 72 squamous cell carcinomas, 22 small cell Thiarabine carcinomas, 2 Rabbit polyclonal to ACADL large cell carcinomas, 10 normal lung tissues and 10 normal adjacent tissues. A human nasopharyngeal cancer tissue array (NH1001, US Biomax) containing 15 squamous cell carcinomas, 3 basal cell carcinomas, 2 adenocarcinomas, 15 papillomas, 6 polyps, 3 each of hyperplasia and inflammation and 1 adjacent normal tissue was used as a control. Note that all specimens on the lung and nasopharyngeal cancer tissue arrays were of Chinese origin. In addition, 10 examples of adenocarcinoma in-situ and 10 non-neoplastic lung tissue specimens (unstained slides and tissue cores) were obtained from formalin fixed paraffin embedded tissue blocks at the Roswell Park Cancer Institute (Buffalo, NY). Approval for the use of human tissue samples was obtained from the Roswell Park Cancer Institute. Tissue arrays were subjected to immunohistochemical analysis as described below. Cell lines Human A549 cells (ATCC CRL-1573) and the ovine pulmonary adenocarcinoma cell line, JS7, (kindly provided by Dr. Mark Ackerman, Iowa State University, USA) were propagated in Dulbeccos modified Thiarabine Eagle medium supplemented with 10% fetal bovine serum, 2?mM?L-glutamine and 1% penicillin/streptomycin. HBE135-E6E7 cells (ATCC CRL-2741) were grown in keratinocyte-serum free medium (Invitrogen) with 5?ng/ml human recombinant EGF and 0.05?mg/ml bovine pituitary extract. Cells were maintained at 37C in 5% CO2. Immunohistochemistry Paraffin embedded tissue was dewaxed and rehydrated using xylene followed by decreasing concentrations of ethanol. Citrate buffer was used for antigen retrieval and tissues were blocked using 5% bovine serum albumin (BSA). Tissue sections were incubated at 4C overnight with either a 1:50 dilution Thiarabine of a highly specific anti-JSRV Env monoclonal antibody , an isotype control antibody or supernatant from an unrelated antibody-producing hybridoma as described previously . Primary antibodies were Thiarabine detected using a 1:50 dilution of an anti-mouse secondary antibody conjugated to biotin (Santa Cruz Technologies). Sigma Fast 3,3-diaminobenzidine tablets (Sigma, St. Louis, MO) were used to visualize protein localization in the tissue. Hemotoxylin was used as a counterstain. Tissues were analyzed by three independent observers and were graded as 0 for.