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Title: Identification of Id family proteins as promoting factors in tumourigenesis of small cell lung cancer
Author: Kamalian, Laleh
ISNI:       0000 0004 2702 5515
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2010
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In an effort to find new genetic mechanisms involved in pathogenesis of Small Cell Lung Cancer (SCLC), a differential display technique was used in our molecular biology lab previously. This experiment showed Inhibitor of DNA/differentiation (Id) 1 gene differentially expressed in a malignant SCLC cell line, Lu-165, comparing to a normal bronchial epithelial cell line, Beas-2b. Id proteins are negative regulatory members of basis helix-loop-helix family of transcription factors that have important regulatory functions in cell cycle, differentiation in normal development and cancer. The main aim of this work was investigating the possible role of Id proteins in tumourigenesis of SCLC. The first part of this thesis established the expression status of Id proteins in SCLC using Western blot and immunohistochemistry techniques. Western blot showed over-expression of Id1, Id2 to Id3 in 8-9 out of 10 malignant SCLC cell lines comparing to Beas-2b cells. Immunoblot results for Id4 expression was not conclusive. Immunohistochemical staining of 26 pairs of SCLC/normal bronchial epithelium tissue sections from the same patient demonstrated significant over-expression of all four Id proteins in cytoplasm of malignant cells. But in the nuclei, Id1 was under-expressed, Id3 and Id4 were over-expressed and Id2 was equally expressed in malignant cells comparing to normal epithelium. Survival analysis of 100 immunohistochemically stained SCLC biopsy samples showed longer patients' survival correlated with higher levels of Id2 but no significant correlation with other Id proteins. The overall conclusion of this part of study suggested upregulation of Id proteins in SCLC and its association with malignant transformation. Second part of the study focused on Id3 and looked for the effects of decreasing its level in SCLC cell lines on malignant behaviour. Using si-RNA technique two cell lines were established that stably expressed 70% less Id3 than control cell line. These cell lines grew 1.7- and 6.7- fold lower numbers of colonies in soft agar assay and a significant 2.5 - 3 folds decrease in final cell count in proliferation assay for transfected cell lines comparing to empty vector cell line. Similar results were obtained from in-vivo experiment. Si-Id3 transfected cells produced significantly smaller subcutaneous tumours after injection into nude mice. Since levels of Id2 and Id3 hadn't changed in si-Id3 transfected cells, these results were concluded to be directly related to decreased levels of Id3. These findings collectively suggested the clear effect of Id3 knock down on proliferation and tumourigenesis in the SCLC malignant cell line. Flowcytometry didn't detect higher levels of apoptotic cells associated with lower levels of Id3. So, anti-proliferative effect of Id3 knock down is related more likely to cell cycle regulation. However, low-Id3 cells showed considerably more sensitivity to apoptotic induction by camptothecin, indicating a possible protective role of Id3 for malignant SCLC cells in cytotoxic conditions, which might be important when using apoptogenic chemotherapy to treat patients suffering from this cancer.
Supervisor: Ke, Y. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral