Loss of heterozygosity on chromosome 9p21 is one of the most frequent genetic alterations identified in human cancer. The rate of point mutations of p16, a candidate suppressor gene of this area, is low in most primary tumours with allelic loss of 9p21. Monosomic cell lines with structurally unaltered p16 show methylation of the 5' CpG island of p16. This distinct methylation pattern was associated with a complete transcriptional block that was reversible upon treatment with 5-deoxyazacytidine. Moreover, de novo methylation of the 5' CpG island of p16 was also found in approximately 20% of different primary neoplasms, but not in normal tissues, potentially representing a common pathway of tumour suppressor gene inactivation in human cancers.
Many tumour types have been reported to have deletion of 9p21 (refs 1-6). A candidate target suppressor gene, p16 (p16INK4a/MTS-1/CDKN2), was recently identified within the commonly deleted region in tumour cell lines. An increasing and sometimes conflicting body of data has accumulated regarding the frequency of homozygous deletion and the importance of p16 in primary tumours. We tested 545 primary tumours by microsatellite analysis with existing and newly cloned markers around the p16 locus. We have now found that small homozygous deletions represent the predominant mechanism of inactivation at 9p21 in bladder tumours and are present in other tumour types, including breast and prostate cancer. Moreover, fine mapping of these deletions implicates a 170 kb minimal region that includes p16 and excludes p15.
In this study we established the simultaneous status of TP53, p16, p14ARF and PTEN tumor suppressor genes in 34 randomly chosen human glioma cell lines. Nine cell lines (26.4%) harbored mutations or deletions in all four tumor suppressor genes and 22 cell lines (64%) had alterations in at least three. Mutations/deletions were found at the following frequencies: TP53 (76.5%), p14ARF (64.7%), p16 (64.7%), PTEN (73.5%). Thus, there was a high incidence of alterations in the cellular pathways involving the p53 transcription factor (94.1%), the retinoblastoma protein (64.7%) and the PTEN phosphatase (73.5%) and 91% of cell lines carried mutations in two or more pathways. This provides the first clear genetic evidence that these tumor suppressors participate in biological pathways which are functioning separately/independently in glioma cells. The status of the gene alterations did not correlate with tumorigenicity in immunocompromized mice or any clinical parameters. Although the mutation rate was higher in glioma cell lines than that reported for glioma tissues, the alterations were molecularly representative of those found in adult de novo glioblastoma. This study highlights the importance of developing therapeutic approaches applicable to tumors with a broad range of genetic alterations and also provides an invaluable panel of glioma cell lines to make this possible.
Microsatellite DNA markers have been widely used as a tool for the detection of loss of heterozygosity and genomic instability in primary tumors. In a blinded study, urine samples from 25 patients with suspicious bladder lesions that had been identified cystoscopically were analyzed by this molecular method and by conventional cytology. Microsatellite changes matching those in the tumor were detected in the urine sediment of 19 of the 20 patients (95 percent) who were diagnosed with bladder cancer, whereas urine cytology detected cancer cells in 9 of 18 (50 percent) of the samples. These results suggest that microsatellite analysis, which in principle can be performed at about one-third the cost of cytology, may be a useful addition to current screening methods for detecting bladder cancer.
The tumor suppressor p53 is commonly inhibited under conditions in which the phosphatidylinositide 3-OH kinase/protein kinase B (PKB)Akt pathway is activated. Intracellular levels of p53 are controlled by the E3 ubiquitin ligase Mdm2. Here we show that PKB inhibits
Shunt-associated infections among adults often present with nonspecific clinical signs, and affected patients can have normal CSF leukocyte counts and lactate levels; therefore, a high index of suspicion and improved methods are required for diagnosing shunt-associated infection.
Advances in proteomic research allow the identification of several hundred protein components in complex biological specimens. Structural information is typically lost during proteomic investigations. For this reason, the rapid isolation of monoclonal antibodies specific to proteins of interest would allow the study of structurally intact biological specimens, thus providing complementary proteomic information. Here, we describe the design, construction, characterization, and use of a large synthetic human antibody phage display library (ETH-2-Gold) containing three billion individual antibody clones. A large repertoire of antibodies with similar biochemical properties was produced by appending short variable complementarity-determining region 3 (CDR3) onto three antibody germline segments (DP47, DPK22, and DPL16), which are frequently found in human antibodies. The ETH-2-Gold library exhibits efficient display of antibody fragments on filamentous phage, as assessed by immunoblot. Furthermore, the library is highly functional, since >90% of clones express soluble antibodies in bacteria and since good quality monoclonal antibodies have been isolated against 16 different antigens. The usefulness of the library as a tool for generating monoclonal antibodies for biomedical applications was tested using the C-domain of tenascin-C (a marker of angiogenesis) as antigen and showing that specific antibodies to this target were able to stain vascular structures in tumor sections.
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