Photophatase and tensin homologue deleted on chromosome ten (PTEN) gene mutations in haematological malignancies.

WELBOURN, John T. (2007). Photophatase and tensin homologue deleted on chromosome ten (PTEN) gene mutations in haematological malignancies. Doctoral, Sheffield Hallam University (United Kingdom)..

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PTEN is a tumour suppressor protein named after its homology with phosphatase and tensin and its frequent deletion on chromosome 10. PTEN was discovered to be mutated in several solid tumours such as breast brain and ovarian cancers. This study sought to establish the role of PTEN mutation in haematological malignancies. Methods were developed to extract DNA from archival material consisting of formalin-fixed, paraffin-embedded tissue sections and methanol-fixed, May-Grunwald and Giemsa-stained bone marrow smears. To optimise amplification of extracted DNA, PCR primers were developed to produce a range of product sizes from human p-globin, prothrombin and PTEN genes. Techniques were developed for RTPCR amplification of cDNA from cultured haematological cell lines. Electrophoresis methods were developed to demonstrate heteroduplex formation using non-denaturing concentrations of denaturing agents and different gel media and running temperatures. Primers were designed to flank exon sequences and a representative group of myeloid and lymphoid malignancies were screened for PTEN mutations. Exons 5, 7 and 8 were initially amplified for heteroduplex analysis following reports of frequent mutation. No heteroduplex bands were observed in samples assayed. The possible insensitivity of heteroduplex analysis to mutations in sub populations of mutant tumour cells led to the development of SSCP techniques. These techniques were optimised for analysis of DNA that was extracted from frozen archived bone marrow. PCR reactions were developed to amplify each PTEN exon. No suitable primers were found for exon 2 analysis due to extensive non-specific amplification. A total of 912 SSCP assays were performed, screening a range of lymphoid and myeloid malignancies for mutations in all PTEN exons except exon two. Only two possible aberrant conformations were indicated, both in exon 5. The exon 5 PCR product from both positive samples were DNA sequenced using forward and reverse primers. Exon 5 of seven other leukaemia samples were sequenced as controls to detect possible lack of sensitivity of SSCP analysis to PTEN mutations. All samples showed exon 5 DNA sequences in agreement with published sequences. These results suggest that small deletions and point mutations in the PTEN gene are very rare in haematological malignancy. The expression of PTEN mRNA in cultured haematological malignancy cells was established by the earlier RTPCR amplification using cultured cells. Western blotting was performed to establish PTEN protein expression in primary malignancies. All samples assayed showed protein expression. Concerns regarding the contribution from residual normal cells to the PTEN expression picture in tumour cell samples led to the assay of protein by immunofluorescence. PTEN protein was demonstrated in all successful reactions with one CGL and one AML M3 showing higher levels of expression that control cells. These results suggest that in haematological malignancy, the PTEN gene is not mutated and PTEN protein is expressed in leukaemic cells.Research by other workers has demonstrated the importance of PTEN in normal haemopoesis. In mouse models, normal PTEN expression is essential for normal compartmentalisation and to prevent the acquisition of a leukaemic picture. This study suggests that if PTEN dysfunction is involved in the development of haematological malignancy, then it is by a subtle mechanism such as a change in expressed protein levels. It may be that, with the small number of oncogenic mutations necessary to promote a leukaemic phenotype, mutated PTEN does not provide a selective advantage to promote further the survival of the malignant cell population.

Item Type: Thesis (Doctoral)
Thesis advisor - Blair, Maria
Thesis advisor - Woodroofe, Nicola
Additional Information: Thesis (Ph.D.)--Sheffield Hallam University (United Kingdom), 2007.
Research Institute, Centre or Group - Does NOT include content added after October 2018: Sheffield Hallam Doctoral Theses
Depositing User: EPrints Services
Date Deposited: 10 Apr 2018 17:22
Last Modified: 26 Apr 2021 12:36

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