Chris Bartholomew

The Evi-1 proto-oncogene encodes a transcriptional repressor activity associated with transformation.

The myeloid transforming gene Evi-1 encodes a protein with two zinc finger domains, designated ZF1 and ZF2, with distinct DNA binding specificities. For the first time we demonstrate that Evi-1 has transcriptional repressor activity which is directly proportional to the amount of Evi-1 protein in cells. Repression has been observed with two distinct promoters: the minimal HSV-1 tk promoter and a VP16 inducible adenovirus E1b minimal promoter. Optimal repression is DNA binding dependent and is mediated by either ZF1 or a heterologous GAL4 DNA binding domain (GAL4DBD) but is significantly less efficient through the ZF2 binding site. Both GAL4DBD/Evi-1 fusion and non-fusion proteins have been used to map the repressor activity to a proline-rich region located within amino acids 514 – 724 between the ZF1 and ZF2 domains. Constitutive expression of mutant proteins lacking the repressor domain are defective for transformation of Rat1 fibroblasts demonstrating that this region is required for the oncogenic activity of the Evi-1 protein. These studies show that the Evi-1 gene encodes a transcriptional repressor and has important implications for the mechanism of action of the Evi-1 protein both in development and in the progression of some myeloid leukaemias.

Evi-1 ZF1 DNA binding activity and a second distinct transcriptional repressor region are both required for optimal transformation of Rat1 fibroblasts

The Evi-1 gene encodes a zinc finger transcriptional repressor protein that normally plays a role in development and is frequently activated in myeloid leukaemias. Evi-1 has two distinct DNA binding domains, ZF1 and ZF2, and a defined repressor domain but the function of the remainder of the molecule is unknown. The ZF2 and repressor domains have been shown to be required for transformation and we show here that ZF1 is also required. An alternative splice variant of Evi-1, designated Δ324, encodes a protein which lacks a portion of the ZF1 DNA binding domain and the intervening amino acids 239–514 (designated IR) located between ZF1 and the repressor domain. We show that Δ324 can neither bind ZF1, repress transcription through this site nor transform Rat1 fibroblasts. Reconstitution studies demonstrate that the defect in Δ324 is partially complemented by recreating the ZF1 DNA binding activity. However, full function also requires the IR region which has transcriptional repressor activity. This study shows therefore, that ZF1, ZF2 and repressor domains and the IR region all contribute to the transformation efficiency of the Evi-1 protein.

Detection of functional PTEN lipid phosphatase protein and enzyme activity in squamous cell carcinomas of the head and neck, despite loss of heterozygosity at this locus.

The human tumour suppressor gene PTEN located at 10q23 is mutated in a variety of tumour types particularly metastatic cases and in the germline of some individuals with Cowdens cancer predisposition syndrome. We have assessed the status of PTEN and associated pathways in cell lines derived from 19 squamous cell carcinomas of the head and neck. Loss of heterozygosity is evident at, or close to the PTEN gene in 5 cases, however there were no mutations in the remaining alleles. Furthermore by Western analysis PTEN protein levels are normal in all of these SCC-HN tumours and cell lines. To assess the possibility that PTEN may be inactivated by another mechanism, we characterized lipid phosphatase levels and from a specific PIP3 biochemical assay it is clear that PTEN is functionally active in all 19 human SCCs. Our data strongly suggest the possibility that a tumour suppressor gene associated with development of SCC-HN, other than PTEN, is located in this chromosomal region. This gene does not appear to be MXI-1, which has been implicated in some other human tumour types. PTEN is an important negative regulator of PI3Kinase, of which subunit alpha is frequently amplified in SCC-HN. To examine the possibility that PI3K is upregulated by amplification in this tumour set we assessed the phosphorylation status of Akt, a downstream target of PI3K. In all cases there is no detectable increase in Akt phosphorylation. Therefore there is no detectable defect in the PI3K pathway in SCC-HN suggesting that the reason for 3q26.3 over-representation may be due to genes other than PI3K110α.

Differential regulation of the STARD1 subfamily of START lipid trafficking proteins in human macrophages

The STARD1 subfamily of ‘START’ lipid trafficking proteins can reduce macrophage lipid content and inflammatory status (STARD1; StAR), and traffic cholesterol from endosomes (STARD3/MLN64). During macrophage differentiation, STARD1 mRNA and protein increase with sterol content, while the reverse is true for STARD3. Sterol depletion (methyl beta‐cyclodextrin) enhances STARD3, and represses STARD1 expression. Agonists of Liver X receptors, peroxisome proliferator activated receptor‐gamma and retinoic acid X receptors increase STARD1 expression, while hypocholesterolaemic agent, LY295427, reveals both STARD1 and STARD3 as putative SREBP‐target genes. Pathophysiological ‘foam cell’ formation, induced by acetylated or oxidized LDL, significantly reduced both STARD1 and STARD3 gene expression. Differential regulation of STARD1 and D3 reflects their distinct roles in macrophage cholesterol metabolism, and may inform anti‐atherogenic strategies.

Differential expression of vitellogenin and oestrogen receptor genes in the liver of zebrafish, Danio Rerio.

Environmental oestrogens are responsible for adverse effects in fish that affect reproduction. Availability of model fish to study the differential effects of endogenous and exogenous oestrogens and to test for oestrogenic activity of chemicals would be advantageous. Zebrafish could provide such a model, but the organisation and expression of vitellogenins (VTGs) and oestrogen receptors (ERs) are not completely understood. VTGs are synthesised in the liver and provide a sensitive biomarker of oestrogenic activity since they are thought to be under the regulation of the ER. There are multiple genes for VTGs and an in silico analysis of their distribution in the Zebrafish genome has identified six genes: VTG-1, VTG-2, VTG-4, VTG-5, VTG-7 located on chromosome 22 and VTG-3 on chromosome 11. VTG-specific, quantitative, real-time, reverse-transcriptase polymerase chain reaction assays were developed and used to measure differential expression in the livers of mature male and female zebrafish. Following normalisation in female fish, relative expression of VTG-5 mRNA is highest and is 1.3×, 1.6× and 2× higher than VTG-4, VTG-2 and VTG-1, respectively, while expression of VTG-3 and VTG-7 is very low. Expression of VTGs in male fish was either undetectable or very low (VTG-4 and VTG-5). ERα and ERβ2 were expressed at higher levels than ERβ1 in females, but only ERβ2 was expressed in appreciable quantity in males. Expression of ERα in males was significant but only at the limit of detection (<0.1% of female fish), while ERβ1 could not be detected. The very low level of expression of ERα in males raises questions about the accepted mechanism of oestrogenic induction of VTG in male fish.

The Evi1 proto-oncoprotein blocks endomitosis in megakaryocytes by inhibiting sustained cyclin-dependent kinase 2 catalytic activity

The 3q21q26 syndrome leukaemias are characterised by dystrophic megakaryocytes, elevated platelet counts, ectopic EVI1 protein production and poor prognosis. To investigate the molecular basis of this disease, we developed a model system to examine the biological activity of EVI1 in a megakaryocyte progenitor cell line. For this purpose, Evi1 was conditionally expressed in human erythroleukaemia cells (HEL) that progress along the megakaryocyte lineage in the presence of 12‐O‐tetradecanoylphorbol 13‐acetate (TPA). TPA‐stimulated HEL cells normally undergo: (1) growth arrest; (2) altered morphology; (3) endomitosis and (4) characteristic changes in gene expression, including reduction of the erythroid‐specific glycophoryn A and elevation of the specific glycoproteins GPIIIa and GPVI. Enforced Evi1 expression alone had no effect upon HEL cell proliferation or differentiation but a phenotype was manifest upon stimulation to differentiate. Evi1‐expressing, TPA‐treated HEL cells still showed growth arrest, had reduced and enhanced glycophoryn A and GPIIIa mRNAs, respectively, but failed to significantly elevate GPVI mRNA. This was accompanied by inhibition of endomitosis and altered cell morphology. Sustained CDK2 catalytic activity, typically associated with megakaryocyte endomitosis, was dramatically decreased in TPA‐stimulated Evi1‐expressing HEL cells because of significantly reduced levels of cyclin A. Therefore, enforced Evi1 expression could inhibit megakaryocyte differentiation although retention of some characteristic molecular changes, in combination with a block in endomitosis and altered morphology, suggest a defect in lineage progression. These results suggest that ectopic Evi1 expression contributes to a defective megakaryocyte differentiation programme and is likely to contribute to the phenotype observed in 3q21q26 syndrome leukaemias.

UXT interacts with the transcriptional repressor protein EVI1 and suppresses cell transformation.

The EVI1 transcriptional repressor is critical to the normal development of a variety of tissues and participates in the progression of acute myeloid leukaemias. The repressor domain (Rp) was used to screen an adult human kidney yeast two‐hybrid library and a novel binding partner designated ubiquitously expressed transcript (UXT) was isolated. Enforced expression of UXT in Evi1‐expressing Rat1 fibroblasts suppresses cell transformation and UXT may therefore be a negative regulator of Evi1 biological activity. The Rp‐binding site for UXT was determined and non‐UXT‐binding Evi1 mutants (Evi1Δ706–707) were developed which retain the ability to bind the corepressor mCtBP2. Evi1Δ706–707 transforms Rat1 fibroblasts, showing that the interaction is not essential for Evi1‐mediated cell transformation. However, Evi1Δ706–707 produces an increased proportion of large colonies relative to wild‐type, showing that endogenous UXT has an inhibitory effect on Evi1 biological activity. Exogenous UXT still suppresses Evi1Δ706–707‐mediated cell transformation, indicating that it inhibits cell proliferation and/or survival by both Evi1‐dependent and Evi1‐independent mechanisms. These observations are consistent with the growth‐suppressive function attributed to UXT in human prostate cancer. Our results show that UXT suppresses cell transformation and might mediate this function by interaction and inhibition of the biological activity of cell proliferation and survival stimulatory factors like Evi1.

Enhanced sensitivity to hydrogen peroxide‐induced apoptosis in Evi1 transformed Rat1 fibroblasts due to repression of carbonic anhydrase III

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.

BCR-ABL1 tyrosine kinase sustained MECOM expression in chronic myeloid leukaemia

MECOM 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 −ve 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.