Genome wide in silico SNP-tumor association analysis

BACKGROUND: Carcinogenesis occurs, at least in part, due to the accumulation of mutations in critical genes that control the mechanisms of cell proliferation, differentiation and death. Publicly accessible databases contain millions of expressed sequence tag (EST) and single nucleotide polymorphism (SNP) records, which have the potential to assist in the identification of SNPs overrepresented in tumor tissue. METHODS: An in silico SNP-tumor association study was performed utilizing tissue library and SNP information available in NCBI's dbEST (release 092002) and dbSNP (build 106). RESULTS: A total of 4865 SNPs were identified which were present at higher allele frequencies in tumor compared to normal tissues. A subset of 327 (6.7%) SNPs induce amino acid changes to the protein coding sequences. This approach identified several SNPs which have been previously associated with carcinogenesis, as well as a number of SNPs that now warrant further investigation CONCLUSIONS: This novel in silico approach can assist in prioritization of genes and SNPs in the effort to elucidate the genetic mechanisms underlying the development of cancer

Dexamethasone inhibits the HSV-tk/ ganciclovir bystander effect in malignant glioma cells

BACKGROUND: HSV-tk/ ganciclovir (GCV) gene therapy has been extensively studied in the setting of brain tumors and largely relies on the bystander effect. Large studies have however failed to demonstrate any significant benefit of this strategy in the treatment of human brain tumors. Since dexamethasone is a frequently used symptomatic treatment for malignant gliomas, its interaction with the bystander effect and the overall efficacy of HSV-TK gene therapy ought to be assessed. METHODS: Stable clones of TK-expressing U87, C6 and LN18 cells were generated and their bystander effect on wild type cells was assessed. The effects of dexamethasone on cell proliferation and sensitivity to ganciclovir were assessed with a thymidine incorporation assay and a MTT test. Gap junction mediated intercellular communication was assessed with microinjections and FACS analysis of calcein transfer. The effect of dexamethasone treatment on the sensitivity of TK-expressing to FAS-dependent apoptosis in the presence or absence of ganciclovir was assessed with an MTT test. Western blot was used to evidence the effect of dexamethasone on the expression of Cx43, CD95, CIAP2 and Bcl(XL). RESULTS: Dexamethasone significantly reduced the bystander effect in TK-expressing C6, LN18 and U87 cells. This inhibition results from a reduction of the gap junction mediated intercellular communication of these cells (GJIC), from an inhibition of their growth and thymidine incorporation and from a modulation of the apoptotic cascade. CONCLUSION: The overall efficacy of HSV-TK gene therapy is adversely affected by dexamethasone co-treatment in vitro. Future HSV-tk/ GCV gene therapy clinical protocols for gliomas should address this interference of corticosteroid treatment

Recombinant AAV-mediated HSVtk gene transfer with direct intratumoral injections and Tet-On regulation for implanted human breast cancer

BACKGROUND: HSVtk/ganciclovir (GCV) gene therapy has been extensively studied in tumors and relies largely on the gene expression of HSVtk. Most studies, however, have failed to demonstrate any significant benefit of a controlled gene expression strategy in cancer treatment. The Tet-On system is commonly used to regulate gene expression following Dox induction. We have evaluated the antitumor effect of HSVtk/ganciclovir gene therapy under Tet-On regulation by means of adeno-associated virus-2 (AAV-2)-mediated HSVtk gene transfer with direct intratumoral injections in mice bearing breast cancer tumors. METHODS: Recombinant adeno-associated virus-2 (rAAV) was constructed and transduced into MCF-7 cell line. GCV treatment to the rAAV infected MCF-7 cells was performed by MTT assay under the doxycycline (Dox) induction or without Dox induction at a vp (viral particle) number of ≥10(4 )/cell. The virus was administered intratumorally to nude mice that had also received GCV intraperitoneally. The antitumor effects were evaluated by measuring tumor regression and histological analysis. RESULTS: We have demonstrated that GCV treatment to the infected MCF-7 cells under the Dox induction was of more inhibited effects than those without Dox induction at ≥10(4 )vp/cell. In ex vivo experiments, tumor growth of BALB/C nude mice breast cancer was retarded after rAAV-2/HSVtk/Tet-On was injected into the tumors under the Dox induction. Infiltrating cells were also observed in tumors after Dox induction followed by GCV treatment and cells were profoundly damaged. The expression of HSVtk gene in MCF-7 cells and BALB/C nude mice tumors was up-regulated by Tet-On under Dox induction with reverse transcription-PCR (RT-PCR) analysis. CONCLUSION: The antitumor effect of rAAV-mediated HSVtk/GCV gene therapy under the Dox induction with direct intratumoral injections may be a useful treatment for breast cancer and other solid tumors

Reduced Expression of Brain-Enriched microRNAs in Glioblastomas Permits Targeted Regulation of a Cell Death Gene

Glioblastoma is a highly aggressive malignant tumor involving glial cells in the human brain. We used high-throughput sequencing to comprehensively profile the small RNAs expressed in glioblastoma and non-tumor brain tissues. MicroRNAs (miRNAs) made up the large majority of small RNAs, and we identified over 400 different cellular pre-miRNAs. No known viral miRNAs were detected in any of the samples analyzed. Cluster analysis revealed several miRNAs that were significantly down-regulated in glioblastomas, including miR-128, miR-124, miR-7, miR-139, miR-95, and miR-873. Post-transcriptional editing was observed for several miRNAs, including the miR-376 family, miR-411, miR-381, and miR-379. Using the deep sequencing information, we designed a lentiviral vector expressing a cell suicide gene, the herpes simplex virus thymidine kinase (HSV-TK) gene, under the regulation of a miRNA, miR-128, that was found to be enriched in non-tumor brain tissue yet down-regulated in glioblastomas, Glioblastoma cells transduced with this vector were selectively killed when cultured in the presence of ganciclovir. Using an in vitro model to recapitulate expression of brain-enriched miRNAs, we demonstrated that neuronally differentiated SH-SY5Y cells transduced with the miRNA-regulated HSV-TK vector are protected from killing by expression of endogenous miR-128. Together, these results provide an in-depth analysis of miRNA dysregulation in glioblastoma and demonstrate the potential utility of these data in the design of miRNA-regulated therapies for the treatment of brain cancers

TNFRSF1B +676 T>G polymorphism predicts survival of non-Small cell lung cancer patients treated with chemoradiotherapy

<p>Abstract</p> <p>Background</p> <p>The dysregulation of gene expression in the TNF-TNFR superfamily has been involved in various human cancers including non-small cell lung cancer (NSCLC). Furthermore, functional polymorphisms in <it>TNF-α </it>and <it>TNFRSF1B </it>genes that alter gene expression are likely to be associated with risk and clinical outcomes of cancers. However, few reported studies have investigated the association between potentially functional SNPs in both <it>TNF-α </it>and <it>TNFRSF1B </it>and prognosis of NSCLC patients treated with chemoradiotherapy.</p> <p>Methods</p> <p>We genotyped five potentially functional polymorphisms of <it>TNF-α </it>and <it>TNFRSF1B </it>genes [<it>TNF-α </it>-308 G>A (rs1800629) and -1031 T>C (rs1799964); <it>TNFRSF1B </it>+676 T>G (rs1061622), -1709A>T(rs652625) and +1663A>G (rs1061624)] in 225 NSCLC patients treated with chemoradiotherapy or radiotherapy alone. Kaplan-Meier survival analysis, log-rank tests and Cox proportional hazard models were used to evaluate associations between these variants and NSCLC overall survival (OS).</p> <p>Results</p> <p>We found that the <it>TNFRSF1B </it>+676 GG genotype was associated with a significantly better OS of NSCLC (GG <it>vs. </it>TT: adjusted HR = 0.38, 95% CI = 0.15-0.94; GG <it>vs. </it>GT/TT: adjusted HR = 0.35, 95% CI = 0.14-0.88). Further stepwise multivariate Cox regression analysis showed that the <it>TNFRSF1B </it>+676 GG was an independent prognosis predictor in this NSCLC cohort (GG <it>vs. </it>GT/TT: HR = 0.35, 95% CI = 0.14-0.85), in the presence of node status (N_2-3<it>vs. </it>N_0-1: HR = 1.60, 95% CI = 1.09-2.35) and tumor stage (T_3-4<it>vs. </it>T_0-2: HR = 1.48, 95% CI = 1.08-2.03).</p> <p>Conclusions</p> <p>Although the exact biological function for this SNP remains to be explored, our findings suggest a possible role of <it>TNFRSF1B </it>+676 T>G (rs1061622) in the prognosis of NSCLC. Further large and functional studies are needed to confirm our findings.</p

SIRNA-Directed In Vivo Silencing of Androgen Receptor Inhibits the Growth of Castration-Resistant Prostate Carcinomas

BACKGROUND: Prostate carcinomas are initially dependent on androgens, and castration or androgen antagonists inhibit their growth. After some time though, tumors become resistant and recur with a poor prognosis. The majority of resistant tumors still expresses a functional androgen receptor (AR), frequently amplified or mutated. METHODOLOGY/PRINCIPAL FINDINGS: To test the hypothesis that AR is not only expressed, but is still a key therapeutic target in advanced carcinomas, we injected siRNA targeting AR into mice bearing exponentially growing castration-resistant tumors. Quantification of siRNA into tumors and mouse tissues demonstrated their efficient uptake. This uptake silenced AR in the prostate, testes and tumors. AR silencing in tumors strongly inhibited their growth, and importantly, also markedly repressed the VEGF production and angiogenesis. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that carcinomas resistant to hormonal manipulations still depend on the expression of the androgen receptor for their development in vivo. The siRNA-directed silencing of AR, which allows targeting overexpressed as well as mutated isoforms, triggers a strong antitumoral and antiangiogenic effect. siRNA-directed silencing of this key gene in advanced and resistant prostate tumors opens promising new therapeutic perspectives and tools

Phosphorothioate antisense oligonucleotides induce the formation of nuclear bodies

Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20-30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150-300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides

Analysis of single nucleotide polymorphisms in the FAS and CTLA-4 genes of peripheral T-cell lymphomas

Angioimmunoblastic T-cell lymphoma (AILT) represents a subset of T-cell lymphomas but resembles an autoimmune disease in many of its clinical aspects. Despite the phenotype of effector T-cells and high expression of FAS and CTLA-4 receptor molecules, tumor cells fail to undergo apoptosis. We investigated single nucleotide polymorphisms (SNPs) of the FAS and CTLA-4 genes in 94 peripheral T-cell lymphomas. Although allelic frequencies of some FAS SNPs were enriched in AILT cases, none of these occurred at a different frequency compared to healthy individuals. Therefore, SNPs in these genes are not associated with the apoptotic defect and autoimmune phenomena in AILT

Comprehensive analysis of the 9p21 region in neuroblastoma suggests a role for genes mapping to 9p21–23 in the biology of favourable stage 4 tumours

Chromosome 9p21 is frequently deleted in many cancers. Previous reports have indicated that 9p21 LOH is an uncommon finding in neuroblastoma (NB), a tumour of childhood. We have performed an extensive analysis of 9p21 and genes located in this region (cyclin-dependent kinase inhibitor 2A – CDKN2A/p16INK4a, CDKN2A/p14ARF, CDKN2B/p15INK4b, MTAP, interferon α and β cluster). LOH was detected in 16.4% of 177 NB. The SRO was identified between markers D9S1751 and D9S254, at 9p21–23, a region telomeric to the CDKN2A and MTAP genes. A significantly better overall and progression-free survival was detected in stage 4 patients displaying 9p21–23 LOH. Hemizygous deletion of the region harbouring the CDKN2A and CDKN2B loci was identified in two tumours by means of fluorescent in situ hybridisation and MTAP was present by immunostaining in all but one tumour analysed. The transcriptional profile of tumours with 9p21–23 LOH was compared to that of NB displaying normal 9p21–23 status by means of oligonucleotide microarrays. Four of the 363 probe sets downregulated in tumours with 9p21–23 LOH were encoded by genes mapping to 9p22–24. The only well-characterised transcript among them was nuclear factor I-B3. Our results suggest a role for genes located telomeric of 9p21 in good risk NB