Biomedical subjects

M You

Publications and source records attributed to M You.

98 records · Page 6Linked to original sources

PubMedDate not supplied

Inactivation of both Rb and p53 pathways in mouse lung epithelial cell lines.

Aberrant expression of key cell cycle regulatory genes is essential for the immortalization and transformation of cells in vitro. We examined 20 mouse lung epithelial cell lines (2 nontumorigenic, 5 nonmetastatic, and 13 metastatic) for mutations or alterations in the expression of key components of the Rb pathway (pRb and p16INK4a) and the p53 pathway (p53 and p19ARF). Seven cell lines had a mutation in exons 5 to 8 of p53. p19ARF was inactivated in the remaining 13 cell lines, primarily by homozygous deletion. Rb expression was present and unaltered in all cell lines, with both phosphorylated and unphosphorylated protein forms detectable. p16INK4a transcripts were undetectable in all cell lines tested except LM1. Loss of p16INK4a expression was a result of homozygous deletion in 11 out of 20 lung cell lines and promoter-exon 1 hypermethylation in 6 out of the remaining 8 cell lines. Other related components that were examined in this study included p21WAF1 and cyclin D1. Compared to normal lung tissue, p21WAF1 expression levels were reduced or undetectable in all cell lines, which did not correlate with loss of p53 function, but did correlate with inactivation of either p53 or p19ARF. Although cyclin D1 expression was variable between cell lines, transcript levels were decreased by at least 50% in the nontumorigenic lines C10 and E10 compared to the tumorigenic cell lines. These results demonstrate mutually exclusive relationships between p53 and p19ARF and between Rb and p16INK4a, but perhaps not between cyclin D1 and p16INK4a, and further describe the nature of involvement of both pathways in mouse lung tumorigenesis.

Animals↗

PubMedDate not supplied

MR findings in isolated oculomotor nerve palsy associated with infectious mononucleosis caused by Epstein-Barr virus infection.

A 20-year-old man presented with fever, bilateral swelling of the cervical and inguinal lymph nodes, and splenomegaly. Later he noted diplopia. CSF was unremarkable. High serum antibodies to Epstein-Barr virus (EBV) were discovered. MRI revealed an abnormal lesion in the right third cranial nerve, which was considered to be the pathological lesion associated with his clinically apparent oculomotor nerve dysfunction.

Adult↗

PubMedDate not supplied

Brief communication, P53 accumulation in N-methyl-N-nitrosourea-induced rat mammary tumors.

P53 aberrant protein expression and mutation is a component of many human tumors but less information is available regarding involvement in relevant animal models. We have examined invasive mammary epithelial neoplasms for p53 aberrant protein expression from Sprague-Dawley rats induced by two intrajugular injections of N-methyl-N-nitrosourea (NMU, 50 mg/kg), 7 days apart, beginning at 44-49 days of age. Positive nuclear staining was observed in 8/10 mammary tumor frozen sections using PAb 421, a mouse monoclonal antibody, compared to a mixed mouse IgG negative control antibody. Paraffin sections from two additional sets of similarly induced rat mammary tumors also showed positive nuclear staining (22/37 tumors) with CM-5, a rabbit polyclonal antibody. Our results indicate that elevated cellular content of p53 is a common event in invasive palpable mammary tumors induced by NMU in this dual-injection model system.

Adenocarcinoma↗

PubMedDate not supplied

Activation of protooncogenes in mouse lung tumors.

The detection of activated protooncogenes in mouse lung tumors has led to a major advance in our understanding of carcinogenesis of the lung at the molecular level. A high frequency of activated K-ras protooncogenes has been detected in tetranitromethane (TNM)- and 1,3-butadiene-induced lung tumors in B6C3F1 mice. In the past several years, we have pursued protooncogene activation in spontaneous and chemically induced tumors of strain A mice. The strain A mouse has a high incidence of spontaneous lung tumors and is susceptible to tumor induction by chemical carcinogens. We have detected and characterized the activated protooncogenes in the DNA of both spontaneously occurring and chemically induced lung tumors of strain A mice. Activated K-ras genes were detected using the NIH/3T3 transfection assay, and the activating mutations were identified by utilizing the polymerase chain reaction (PCR) and direct sequence analysis. A strong selectivity of mutations in the K-ras genes were observed in chemically induced lung tumors, as compared to spontaneous tumors, indicating that the carcinogens directly induced point mutations in the K-ras protooncogene. These findings suggest that the strain A mouse lung tumor model appears to be a very sensitive system to identify the mechanism by which chemical carcinogens activate the K-ras gene in lung tissue in vivo.

Animals↗

PubMedDate not supplied

Additional evidence that the K-ras protooncogene is a candidate for the major mouse pulmonary adenoma susceptibility (Pas-1) gene.

A locus for mouse pulmonary adenoma susceptibility, Pas-1, has been mapped on distal chromosome 6, where the K-ras gene is located. Allele-specific activation and expression of the K-ras allele from the susceptible parent has been observed in lung tumors from F1 hybrid mice. We report here genetic mapping of lung tumor susceptibility genes in urethane-treated A x B and B x A recombinant inbred (RI) mice using microsatellite markers to seek further evidence for the K-ras gene as candidate for Pas-1. The K-ras genotype differs between the A/J and C57BL/6J progenitors of the RI strains, and distal chromosome 6 contained a major lung tumor susceptibility determinant in the RI mice. Additional evidence that Pas-1 is K-ras involved linkage analysis of (A/JOLaHsd x BALB/ cOLaHsd) F2 intercross mice whose parents shared the same K-ras genotype. In contrast to the results with the A x B and B x A RI strains, no distal chromosome 6 site was significantly associated with tumor development in these F2 mice. In addition to this major locus, linkage analysis of the RI mice revealed additional quantitative trait loci for susceptibility on chromosomes 10, 17, and 19. These loci may serve as modifiers of Pas-1. The relationship between the K-ras genotype and the frequency of K-ras mutations in urethane-induced lung tumors from the RI mice was also explored. All 18 tumor DNAs from RI mice with high susceptibility contained an AT-->TA transversion at the second base of K-ras codon 61. This was also true for DNAs from 27 of 27 (100%) tumors in mice with high intermediate susceptibility. In RI strains with a low intermediate susceptibility, the DNA from 39 of 47 (83%) tumors contained an AT-->TA transversion at codon 61, and only 13 of 21 (62%) tumors had this mutation in the most resistant group. This reflects a positive correlation between the frequency of K-ras mutations in lung tumors of A x B or B x A RI strains and their susceptibility to lung carcinogenesis. Since K-ras appears to be Pas-1, these results suggest that some RI mice that have the resistant K-ras or Pas-1 allele undergo tumor development by a K-ras-independent route.

Adenoma↗

PubMedDate not supplied

Expression of cell cycle proteins in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced mouse lung tumors.

Cyclin D1 dysregulation and differential inactivation of p16INK4a and Rb have been observed in human lung cancer. In chemically induced mouse lung tumors, the p16INK4a gene is a target of inactivation, and Rb is reduced at the mRNA level (Northern blot) although similar at the protein level (Western blot) when compared to normal lung tissues. The expression of cyclin D1, cdk4, p16INK4a, and Rb protein was examined by immunohistochemistry in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mouse lung tumors. Immunohistochemical staining revealed exclusive nuclear staining of both cyclin D1 and cdk4 that was light to moderate in normal mouse lung tissues, but intense in lung adenomas and adenocarcinomas. Western blot analysis confirmed the increased expression of cyclin D1 and cdk4 in lung tumors compared to normal lung. Immunohistochemical analyses of lung tumors showed focal areas which lacked p16INK4a staining. Expression of p16INK4a, as determined by RT-PCR, was variable in lung tumors. Mutations in p16INK4a were not found by SSCP analysis. Immunohistochemical analyses of normal lung tissues showed intense staining for Rb protein in alveolar epithelial cells and in other lung cell types; however, in the lung tumors the staining intensity was reduced and the distribution was altered. Expression of Rb was detected in normal lung tissues but was barely detectable by Northern blot hybridization in lung tumors. Western blot analysis indicated the presence of both hypophosphorylated and hyperphosphorylated Rb protein in lung tumors and in normal lung tissues. These results suggest that alterations in the cell cycle proteins, cyclin D1, cdk4, p16INK4a, and Rb, may play a role in the acquisition of autonomous growth by adenomas. Furthermore, they demonstrate the importance of immunohistochemical studies to examine expression in tissues that contain multiple cell types, such as the lung, and in tumors that by nature are heterogeneous.

Adenocarcinoma↗

PubMedDate not supplied

Detection of differentially expressed genes in mouse lung adenocarcinomas.

Increasing evidence suggests that altered gene expression is associated with the induction and maintenance of malignancy in various organs including mouse lung adenocarcinomas. A competitive cDNA library screening (CCLS) was used to examine gene expression in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung adenocarcinomas from (C3H/HeJ x A/J])F1 mice. Comparisons of RNA expression in lung adenocarcinomas to those of normal surrounding lung tissue revealed altered expression in 220 clones from more than 50,000 clones screened. Fifty clones were selected for quantitative reverse transcriptase-polymerase chain reaction (PCR) analysis to verify altered expression. PCR primers were designed based on partial sequence analysis of the clones. Twenty-two clones were found to be differentially expressed in lung adenocarcinomas compared with normal lungs. GenBank database analysis showed that 14 of the 22 clones were homologous with known genes, whereas 8 clones contained novel sequences. Thirteen clones were down regulated in tumors compared to normal lung tissues, and 9 were overexpressed. The clones underexpressed or absent include adipocyte p27, carbonic anhydrase III, carbonyl reductase, cytochrome CYP2E1, skelemin, myosin, major urinary protein, and contrapsin. Overexpressed clones include Bruton's tyrosine kinase, cyclin D3, poly(A)-binding protein, alpha-fetoprotein, transferrin, and mouse B2 family repetitive sequence. Further examination of biologic implications of the differentially expressed genes in lung adenocarcinomas is necessary to understand their role(s) in mouse lung carcinogenesis.

Adenocarcinoma↗

PubMedDate not supplied

Detection of oncogene mRNA sequences in cultured cells by in situ hybridization.

The present study was undertaken to determine the feasibility of using in situ hybridization techniques to identify oncogene transcription in cultured cells. Following in situ hybridization with 32P-labeled v-src and v-Ha-ras DNA probes, src and Ha-ras related transcripts were identified in cell lines transfected with v-src and Ha-ras, respectively. In both the v-src and c-Ha-ras transfected cell lines, the number of silver grains over individual cells were significantly higher (p less than 0.001, t-test) than in a non-transfected, non-tumorigenic, rat esophageal epithelial cell line. There was a highly variable number of silver grains above individual cells. Significantly fewer silver grains were counted over cells that had been preincubated with either non-labeled v-src or v-Ha-ras DNA or that were pretreated with RNase A. Both oncogene transfected cell lines contained approximately 10 times more oncogene related mRNAs than non-transfected cells as judged by the numbers of silver grains over individual cells. Filter-hybridization analysis of the transfected and non-transfected cell lines confirmed that the expression of src and Ha-ras transcripts was higher in the transfected cell lines than in the non-transfected cell line. Therefore, the in situ hybridization technique would appear useful for the identification of oncogene transcripts in single cells and could potentially be applied to cytological preparations of human cells and to human tumor cells in culture.

Animals↗