Abstract
The ecotropic viral integration site 1 (EVI\) gene is highly conserved through evolution and codes for various isoforms including a 145 kDa transcription repressor protein characterised by two DNA binding zinc finger domains. During murine embryogenesis, the EVI\ gene is expressed in several organs and its inactivation leads to severe malformation, defects in multiple organs and embryonic death at mid-gestation. EVI1 is inappropriately activated in murine and human myeloid leukaemia and contributes to cell transformation and tumourigenesis.In this study the biological activity of EVI\ has been examined in fibroblast cells. Fibroblasts were selected for further investigation because previous studies show: 1) EVI 1 transforms Rati fibroblast cells and differential gene expression has been determined by microarray analysis and 2) EVI 1 is expressed in all fibroblasts examined. Here the role of a potential EVI\ target gene, RasGRP2, in mediating transforming activity has been examined and the biological activity of EVI\ in fibroblasts has been investigated by gene knockdown studies.
Microarray studies have shown that RasGRP2 is induced in transformed Rati cells mediated by constitutive Evil expression. RasGRP2 is a Rapl guanine nucleotide exchange factor which has previously been shown to be activated by frequent retroviral insertions in murine leukaemia. Quantitative real-time polymerase chain reaction (QRT-PCR) analysis verified a 4X increase in RasGRP2 expression in Evil transformed Rati cells, confirming differential expression. Enforced expression of RasGRP2, using a recombinant retroviral vector in Rati cells, produced low levels of a 72 kDa protein. However, Rati cells expressing the RasGRP2 transgene were
indistinguishable from parent and vector control cells by a variety of parameters including rate of proliferation and transformation, determined by the production of macroscopic colonies in soft agar. Furthermore, no differences in RasGRP2 enzyme activity, determined by measuring cellular levels of Rapl-GTP, were observed in either Rati cells expressing the transgene or Evil transformed Rati cells relative to parental and vector control cells. Interleukin-3-dependent leukaemia cell lines, derived from retrovirus induced murine myeloid leukaemia, were also examined for both Evil and RasGRP2 gene expressions. Eight of ten cell lines expressed elevated levels of Evil relative to murine bone marrow cells (selected as standard) whereas only two of ten cell lines show increased levels of RasGRPl. Together these results suggest that RasGRP2 does not mediate the transforming activity of Evil in Rati fibroblasts. Furthermore they show that although RasGRP2 is regulated directly or indirectly by Evil in Rati fibroblast cells, no association is observed in leukaemic cell lines. Therefore, it is unlikely that RasGRP2 mediates Evil biological activity in leukaemia progression either.
In the second part of this study Evil short hairpin RNA’s were used to knockdown (KD) Evil expression in NIH3T3 fibroblasts and primary mouse embryonic fibroblasts (MEF). The biological activity of the KD vector was verified by its ability to reduce protein levels and revert the transformed phenotype in Evil transformed Rati fibroblasts. QRT-PCR revealed stable expression of the KD vector in both NIH3T3 and MEF cells reduced Evil mRNA levels by 70%. Evil KD in NIH3T3 cells has no impact on cell proliferation, migration or cell cycle but increases apoptosis in low serum, as determined by measuring caspase 3 catalytic activity. Evil KD in MEF cells inhibits cell proliferation and causes a moderate reduction in cell viability. Surprisingly, PCR array analysis of cell cycle regulators in
MEF cells did not identify any changes in gene expression between Evil KD and control cells. These results show Evil has a role in stimulating cell proliferation and protection from apoptosis in cells that normally express this gene.
| Date of Award | 2010 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Christopher Bartholomew (Supervisor) |