The adeno-associated virus 2 (AAV), a single-stranded DNA-containing, nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. However, the single-stranded nature of the viral genome significantly impacts upon the transduction efficiency, because the second-strand viral DNA synthesis is the rate-limiting step. We hypothesized that a host-cell protein interacts with the single-stranded D sequence within the inverted terminal repeat structure of the AAV genome and prevents the viral second-strand DNA synthesis. Indeed, a cellular protein has been identified that interacts specifically and preferentially with the D sequence at the 3 end of the AAV genome. This protein, designated the single-stranded Dsequence-binding protein (ssD-BP), is phosphorylated at tyrosine residues and blocks AAV-mediated transgene expression in infected cells by inhibiting the leading strand viral DNA synthesis. Inhibition of cellular protein tyrosine kinases by genistein results in dephosphorylation of the ssD-BP, leading not only to significant augmentation of transgene expression from recombinant AAV but also to autonomous replication of the wild-type AAV genome. Dephosphorylation of the ssD-BP also correlates with adenovirus infection, or expression of the adenovirus E4orf6 protein, which is known to induce AAV DNA replication and gene expression. Thus, phosphorylation state of the ssD-BP appears to play a crucial role in the life cycle of AAV and may prove to be an important determinant in the successful use of AAV-based vectors in human gene therapy.The adeno-associated virus 2 (AAV), a single-stranded DNAcontaining parvovirus, so far has not been shown to be associated with any pathology in humans (1, 2). The wild-type (wt) AAV genome has been shown to integrate in a sitespecific manner into human chromosome 19q13.3-qter (3-5). Thus, recombinant AAV vectors have emerged as useful alternatives to the more commonly used retroviral and adenoviral vectors for human gene therapy (6-10). However, recent studies from two independent laboratories have suggested that after infection, the leading strand viral DNA synthesis is a rate-limiting step in the efficient transduction by AAV vectors (11,12). AAV inverted terminal repeats (ITRs) contain 145 nt each, the terminal 125 nt of which are palindromic and form T-shaped hairpin (HP) structures. The 3Ј-HP structure serves as a primer for AAV DNA replication. AAV ITRs also contain an additional domain, designated the D sequence, which is a stretch of 20 nt that is not involved in HP formation (1, 2, 13). Our previous studies have indicated that the D sequence plays a crucial role in the efficient rescue, selective replication, and encapsidation of the AAV genome (14, 15). We hypothesized that a cellular protein(s) might interact with the D sequence and prevent the second-strand viral DNA synthesis. Indeed, using electrophoretic mobility-shift assays (EMSAs), we have recently provided evidence for the existence of a hitherto unknown host-c...
Purpose: The objective of this study was to use gene expression profiling to identify novel biomarkers that are predictive of aggressive behavior in clear cell renal cell carcinoma (CCRCC). Experimental Design: Candidate genes were discovered using Human Genome U133 Plus 2 Arrays and validated on independent samples by quantitative reverse transcription-PCR (RT-PCR). Both the discovery and the validation cohorts included nonaggressive primary CCRCC, aggressive primary CCRCC, metastatic CCRCC, and nonneoplastic kidney adjacent to tumor. Results: Aggressive primary and metastatic CCRCC displayed no significant differences in gene expression. In contrast, we identified significant differences in gene expression between nonaggressive andaggressive CCRCC (including metastatic CCRCC).Thirty-fourof the 35 transcriptsthat displayed the most significant differential expression by microarray analysis also displayed significant differential expression inindependent validation studies using quantitative RT-PCR (P < 0.001 for 31candidates and P < 0.005 for the remaining three candidates). Hierarchical clustering of the quantitative RT-PCR data using our candidate markers accurately grouped 88% (23 of 26) of aggressive and metastatic CCRCC samples, 100% (14 of 14) of nonaggressive CCRCC samples, and100% (15 of15) of nonneoplastic samples into separate clusters. Finally, we evaluated theability of protein expression levels of one of our candidate markers (survivin) to predict survival among a cohort of 183 CCRCC patients treated surgically at Mayo Clinic from 1990 to 1992. In multivariate analysis, expression of survivin (BIRC5) was inversely associated with cancer-specific survival (P = 0.017). Conclusion: We used a combination of genomic profiling and validation by quantitative PCR to identify a panel of candidate biomarkers for determining CCRCC aggressiveness. Our data also indicate that the gene expression alterations that result in aggressive behavior and metastatic potential can be identified in the primary tumor.
Background: To discover prostate cancer biomarkers, we profiled gene expression in benign and malignant cells laser capture microdissected (LCM) from prostate tissues and metastatic prostatic adenocarcinomas. Here we present methods developed, optimized, and validated to obtain high quality gene expression data.
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