Aims: To determine the contribution of Kirsten-ras and other abnormalities in the signalling pathway from growth factor to RAS in colorectal cancer and to develop a means of targeting a ras mutation of probable prognostic importance. Methods: Sections of 105 consecutive early colorectal cancers were obtained. DNA was extracted and codons 110-134 of the TGF- β RII receptor gene were amplified using PCR and screened for microsatellite instability. Codons 12 and 13 of the Kirsten-ras gene were identified by direct sequencing. Immunohistochemical staining was used to identify expression of pan RAS proteins, the growth factors EGF and TGF- β, and their common receptor, EGF-R. A ribozyme transcription unit was constructed to target the mRNA produced from Kirsten ras encoding a valine mutation at codon 12. The ribozyme was transcribed in colorectal cell lines using a novel expression/reporter plasmid. Two control ribozyme constructs were also used: one lacked ribozyme activity, the other was not transcribed in colorectal cells. Ribozyme expression and Kirsten-ras mRNA levels in transfected cells were identified by an RNase protection assay. Cell viability after transfection was determined by labelled thymidine uptake. Results: A ras pathway abnormality was identified in almost 90% of the tumours while microsatellite instability at the TGF- β RII gene was identified in only 3 (2.9%). Kirsten-Ras mutations were present in 30 (28.6%). Cleavage of target mRNA by the ribozyme in vitro and transcription in transfected cells were confirmed. However, there was no demonstrable reduction in Kirsten-ras mRNA in treated cells and no specific effect on cell viability.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:541294 |
| Date | January 2009 |
| Creators | Tilsed, J. V. T. |
| Publisher | Institute of Cancer Research (University Of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://publications.icr.ac.uk/10067/ |
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