Analysis of Multistage Mouse Skin Carcinogenesis by Retroviral Mediated Gene Transfer

Boeger-Brown, Uta (1993) Analysis of Multistage Mouse Skin Carcinogenesis by Retroviral Mediated Gene Transfer. PhD thesis, University of Glasgow.

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Abstract

In the present study I have explored the feasibility of using retroviral mediated gene transfer to study the role of given genes during chemically induced carcinogenesis of the mouse skin. Retroviral vectors expressing parts of the murine c-Ha-ras gene as antisense RNA were compared to their sense counterparts for their ability to revert the transformed phenotype of cells containing a mutant Ha-ras gene. I have shown that one of the antisense retroviral constructs (pZN(X)RAS-l, containing exons 1 and 2 and 1.3 kb of the 5' untranslated region of murine c-Ha-ras) was effective in altering the transformed phenotype of NIH3T3 cells transformed by a Ha-ras gene containing a mutation at codon 61. Expression of this antisense H2-ras retroviral construct altered the ratio of morphologically transformed to untransformed colonies compared to vector alone controls. Furthermore the ability to form colonies in soft agar of the Ha-ras transformed NIH3T3 cells was reduced by 68% after infection with pZN(X)RAS-l compared to vector alone infectants. Infection of cells with the sense counterpart of pZN(X)RAS-1 did not reduce the ability of these cells to grow in soft agar. No significant reduction in soft agar cloning efficiency was observed with the other antisense or sense retroviral constructs. Metalloproteinases have been implied to play a role in progression of tumours to an invasive and metastatic phenotype. The rat transin cDNA was introduced into murine epithelial cells. However no evidence was observed of transin expression influencing progression towards invasion or metastasis as assayed by a spontaneous metastasis assay following subcutaneous injection of cells in athymic nude mice. The feasibility of histochemically tagging mouse keratinocytes through constitutive expression of a bacterial lacZ gene expressed within a retroviral vector was examined. Results suggested successful infection and expression of the lacZ gene in keratinocytes in vitro and in vivo. However a retroviral vector containing both the v-Ha-ras and lacZ genes failed to induce tumours in mouse skin. This appeared to be due to lack of efficient expression of the v-Ha-ras gene in murine epithelial cells. The results presented in this study show the potential and limitations of retroviral vectors to study an in vivo tumour model system such as mouse skin carcinogenesis. The successful reversion of the transformed phenotype of cells in vitro using antisense Ha-ras expression may suggest this approach could be used to reverse or inhibit transformation in such tumour model systems.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Allan Balmain
Keywords: Oncology
Date of Award: 1993
Depositing User: Enlighten Team
Unique ID: glathesis:1993-74992
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Sep 2019 14:43
Last Modified: 27 Sep 2019 14:43
URI: http://theses.gla.ac.uk/id/eprint/74992

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