Project description:We used Affymetrix microarrays to characterize gene expression profiles that were perturbed in common myeloid progenitor (CMP) cells due to enforced expression of full-length or truncated forms of MN1. Expression profiles of MN1-induced leukemias arising from whole bone marrow transduction were also compared with the profiles obtained from the CMP cells. Lineage negative mouse bone marrow cells were transduced with retroviral vectors expressing full-length or truncated MN1 proteins. After 2 days in culture, cells were FACS-sorted for GFP expression and cultured for a further 3 days in growth media. After 5 days total, RNA was isolated and processed for microarray analysis. RNA was also prepared for microarray analysis from leukemias arising in mice following whole bone marrow transduction with full-lenght MN1.
Project description:We used Affymetrix microarrays to characterize gene expression profiles that were perturbed in common myeloid progenitor (CMP) cells due to enforced expression of full-length or truncated forms of MN1. Expression profiles of MN1-induced leukemias arising from whole bone marrow transduction were also compared with the profiles obtained from the CMP cells.
Project description:The v-erbA oncogene belongs to a superfamily of transcription factors called nuclear receptors, which includes the retinoic acid receptors (RARs) responsible for mediating the effects of retinoic acid (RA). Nuclear receptors bind to specific DNA sequences in the promoter region of target genes and v-erbA is known to exert a dominant negative effect on the activity of the RARs. The repressor activity of v-erbA has been linked to the development of hepatocellular carcinoma (HCC) in a mouse model. We have used microarray analysis to identify genes differentially expressed in hepatocytes in culture (AML12 cells) stably transfected with v-erbA and exposed to RA. We have found that v-erbA can affect expression of RA-responsive genes. We have also identified a number of v-erbA-responsive genes that are known to be involved in carcinogenesis and which may play a role in the development of HCC. Experiment Overall Design: AML12 control cells and v-erbA-transfected AML12 cells were exposed to 1 µM RA for 3h or 24h. Using microarray analysis, we compared gene expression in the presence and absence of v-erbA and identified RA-regulated genes differentially expressed in the presence of v-erbA.
Project description:Analysis of gene expression in human macrophages infected with influenza A viruses expressing full length or truncated NS1 protein. The hypothesis tested was that C-terminal truncations of viral NS1 protein attenuate the capability of NS1 to limit activation of host antiviral and immune response genes. Cells were infected with influenza A/WSN/33 viruses expressing wild type NS1 protein (WSN-230), NS1 protein of 220 aa long (WSN-220) and NS1 protein of 202 aa long (WSN-202) on non-infected (Mock)
Project description:Analysis of gene expression in human macrophages infected with influenza A viruses expressing full length or truncated NS1 protein. The hypothesis tested was that C-terminal truncations of viral NS1 protein attenuate the capability of NS1 to limit activation of host antiviral and immune response genes. Cells were infected with influenza A/WSN/33 viruses expressing wild type NS1 protein (WSN-230), NS1 protein of 220 aa long (WSN-220) and NS1 protein of 202 aa long (WSN-202) on non-infected (Mock) Total RNA isolated from macrophages after 8 hours of infection with wild type or mutant influenza A virus (multiplicity of infection = 2)
Project description:The tumor suppressor gene TP53 is mutated in approximately half of all human tumors. Of those, around 10% are nonsense mutations that produce truncated and inactive p53 protein. Induction of translational readthrough is a promising approach for rescuing full-length p53 and thereby eliminate tumor cells with nonsense mutant TP53. To find novel nonsense mutant TP53 readthrough-inducing compounds with a tolerable toxicity profile, we performed an in silico screening of data at the National Cancer Institute database and identified 5-Fluorouracil (5-FU). We show here that 5-FU induces full-length p53 in human tumor cells carrying R213X nonsense mutant TP53 and that this activity is mediated by its metabolite 5-Fluorouridine (FUr). Ribo-seq analysis validated induction of translational readthrough by FUr. We also show that FUr is incorporated into RNA where it potentially allows base pairing of Arg tRNA at the R213X UGA premature termination codon. Full-length p53 rescued by FUr is transcriptionally active and triggers p53-dependent cell death. Moreover, treatment with 5-FU or FUr restores full-length p53 expression in TP53 R213X mutant human tumor xenografts in vivo. Our results suggest that induction of readthrough by 5-FU/FUr could contribute to therapeutic efficacy in patients with TP53 nonsense mutant tumors.
Project description:The tumor suppressor gene TP53 is mutated in approximately half of all human tumors. Of those, around 10% are nonsense mutations that produce truncated and inactive p53 protein. Induction of translational readthrough is a promising approach for rescuing full-length p53 and thereby eliminate tumor cells with nonsense mutant TP53. To find novel nonsense mutant TP53 readthrough-inducing compounds with a tolerable toxicity profile, we performed an in silico screening of data at the National Cancer Institute database and identified 5-Fluorouracil (5-FU). We show here that 5-FU induces full-length p53 in human tumor cells carrying R213X nonsense mutant TP53 and that this activity is mediated by its metabolite 5-Fluorouridine (FUr). Ribo-seq analysis validated induction of translational readthrough by FUr. We also show that FUr is incorporated into RNA where it potentially allows base pairing of Arg tRNA at the R213X UGA premature termination codon. Full-length p53 rescued by FUr is transcriptionally active and triggers p53-dependent cell death. Moreover, treatment with 5-FU or FUr restores full-length p53 expression in TP53 R213X mutant human tumor xenografts in vivo. Our results suggest that induction of readthrough by 5-FU/FUr could contribute to therapeutic efficacy in patients with TP53 nonsense mutant tumors.