EVI1 is a nuclear zinc finger protein essential to normal development, which participates in acute myeloid leukaemia progression and transforms Rat1 fibroblasts. In this study we show that enforced expression of Evi1 in Rat1 fibroblasts protects from paclitaxel‐induced apoptosis, consistent with previously published studies. Surprisingly, however, these cells show increased sensitivity to hydrogen peroxide (H2O2)‐induced apoptosis, demonstrated by elevated caspase 3 catalytic activity. This effect is caused by a reduction in carbonic anhydrase III (caIII) production. caIII transcripts are repressed by 92–97% by Evi1 expression, accompanied by a similar reduction in caIII protein. Reporter assays with the rat caIII gene promoter show repressed activity, demonstrating that Evi1 either directly or indirectly modulates transcription of this gene in Rat1 cells. Targeted knockdown of caIII alone, with Dicer‐substrate short inhibitory RNAs, also increases the sensitivity of Rat1 fibroblasts to H2O2, which occurs in the absence of any other changes mediated by Evi1 expression. Enforced expression of caIII in Evi1‐expressing Rat1 cells reverts the phenotype, restoring H2O2 resistance. Together these data show that Evi1 represses transcription of caIII gene expression, leading to increased sensitivity to H2O2‐induced apoptosis in Rat1 cells and might suggest the basis for the development of a novel therapeutic strategy for the treatment of leukaemias and solid tumours where EVI1 is overexpressed.
Obesity-associated conditions represent major global health and financial burdens and understanding processes regulating adipogenesis could lead to novel intervention strategies. This study shows that adhesion G-protein coupled receptor 56 (GPR56) gene transcripts are reduced in abdominal visceral white adipose tissue derived from obese Zucker rats versus lean controls. Immunostaining in 3T3-L1 preadipocytes reveals both mitotic cell restricted surface and low level general expression patterns of Gpr56. Gpr56 transcripts are differentially expressed in 3T3-L1 cells during adipogenesis. Transient knockdown (KD) of Gpr56 in 3T3-L1 cells dramatically inhibits differentiation through reducing the accumulation of both neutral cellular lipids (56%) and production of established adipogenesis Pparγ 2 (60-80%), C/ebpα (40-78%) mediator, and Ap2 (56-80%) marker proteins. Furthermore, genome editing of Gpr56 in 3T3-L1 cells created CW2.2.4 and RM4.2.5.5 clones (Gpr56 −/−
SummaryMECOM oncogene expression correlates with chronic myeloid leukaemia (CML) progression. Here we show that the knockdown of MECOM (E) and MECOM (ME) isoforms reduces cell division at low cell density, inhibits colony-forming cells by 34% and moderately reduces BCR-ABL1 mRNA and protein expression but not tyrosine kinase catalytic activity in K562 cells. We also show that both E and ME are expressed in CD34 + selected cells of both CML chronic phase (CML-CP), and non-CML (normal) origin. Furthermore, MECOM mRNA and protein expression were repressed by imatinib mesylate treatment of CML-CP CD34 + cells, K562 and KY01 cell lines whereas imatinib had no effect in non-CML BCR-ABL1 Àve CD34 + cells. Together these results suggest that BCR-ABL1 tyrosine kinase catalytic activity regulates MECOM gene expression in CML-CP progenitor cells and that the BCR-ABL1 oncoprotein partially mediates its biological activity through MECOM. MECOM gene expression in CML-CP progenitor cells would provide an in vivo selective advantage, contributing to CML pathogenesis.
Marek’s disease (MD) is an immunosuppressive and highly contagious lymphoproliferative disease caused by Marek’s disease virus (MDV) in poultry. Lymphoblastoid cell lines (LCLs) generated ex vivo from MD lymphomas are considered excellent models to study virus-host molecular interactions. LCLs mostly have latently infected MDV genome, but many of them also have varying populations of lytically-infected cells, thus making them very suitable to examine the molecular events associated with the switch from latent to lytic infection. MDV-encoded phosphoprotein 38 (pp38) is readily detectable in lytically-infected LCLs and hence considered as a biomarker for lytic infection. Whilst previous studies have suggested that pp38 is essential for the early cytolytic infection of B-cells, its role in the switch from latent to lytic infection of LCLs is still unclear. pp24, another phosphorylated protein in the same protein complex, shares the same promoter and N-terminal 65 amino acids as pp38. In this study we employed CRISPR activation (CRISPRa) technology for targeted activation of pp38/pp24 in LCLs to investigate their role in inducing lytic infection. Our results show that enforced expression of pp38/pp24 through CRISPRa induces orchestrated upregulation of other MDV genes including ICP4, gB, Meq and pp14 as well as differential expression of host genes thereby facilitating lytic infection. Our results also show that pp38/pp24 expression induces the lytic switch through inhibiting apoptosis.
Carbonic anhydrase isozyme CA3 protects cells against oxidative stress. Ectopic expression of murine Ca3, but not Ca2, protects proto-oncogene Evi1 expressing Rat1 fibroblast cells (ca3low) against hydrogen peroxide (H 2 O 2 ) induced stress. Ca3 is S-glutathionylated via glutathione adducts with cysteines 181 and 186. Substitution of both Ca3 cysteines with serine fails to protect cells from oxidative stress. Insertion of cysteine at 181 and 186 in Ca2 is insufficient for conferring efficient anti-oxidant activity. This shows for the first time that S-glutathionylation of cys181 and cys186 residues is required for Ca3 anti-oxidant activity but that additional factors are also required.
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