Abstract
Evil (ecotropic viral integration site 1) was first discovered as a common retroviral integration site in myeloid tumours in AKXD mice. It is a 145kDa nuclear protein with two zinc finger domains containing ten motifs. Evil encodes a transcription factor and is located on chromosome 3, band q26.2. The gene spans 16 exons with 14 coding exons which cover 47 kb of genomic DNA. Human and mice sequences of the Evil gene show a great degree of homology with 91% nucleotide sequence identity and 94% homology at the amino acid level. There are different naturally occurring splice variants of Evil present in both humans and mice.Evil has been shown to be associated with both murine and human myeloid leukaemias. The inappropriate expression of Evil appears to occur mostly by chromosomal rearrangements such as translocations and inversions involving chromosome 3 and this contributes to the development or progression of myeloid leukaemia. The exact mechanism of disease development and progression is not known but may involve the abnormal expression of its different isoforms, proliferative defects and also may involve the abnormal repression of genes necessary for cellular maturation.
I have generated different splice variant specific primers and probes to investigate the expression levels in normal haematopoietic progenitor cells, CML-chronic phase (CML-CP), CML-blast crisis (CML-BC) samples and also in CML-BC cell line K562. The results show that all the isoforms are expressed in each case but the expression pattern varies.
To study the biological significance of EVI1 in CML cells we have knocked down (KD) its gene expression in K562 cells. I used two recombinant lentivirus containing EVI1 shRNA specifically targeting different regions of the mRNA. 80-90% KD in mRNA and protein level was achieved and the effect of EVI1 depletion in K562 cells was investigated by various cellular assays.
Here I show that the KD of both EVI1 and MDS1-EVI1 (ME) isoforms reduces cell division at low cell density, inhibits colony forming cells (CFC) by 34% and moderately reduces BCR-ABL oncogene mRNA and protein but not tyrosine kinase catalytic activity in K562 cells. Furthermore, EVI1 mRNA and protein expression are repressed by Imatinib mesylate (IM) treatment in K562 and KYOl cells lines. Also observed EVI1 mRNA repression by IM treatment in CD34+ CML-CP cells. EVI1 KD K562 cells are partially resistant to IM treatment, although the mechanism of resistance is unknown. BCR-ABL might regulate EVI1 expression and EVI1 promotes cell proliferation and survival, so its sustained expression by BCR-ABL suggests its involvement in CML pathology, which suggests that EVI1 expression might be regulated by BCR-ABL tyrosine kinase.
| Date of Award | 2011 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Christopher Bartholomew (Supervisor) |