The identification of metastasis-related gene products in a rodent mammary tumour model
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| Title: | The identification of metastasis-related gene products in a rodent mammary tumour model |
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| Authors: | Oates, Adam John |
| Summary: | The rat mammary epithelial cell line, Rama 37, yields benign, non-metastasising, adenomatous tumours in syngeneic Furth-Wistar rats. Following transfection of this stably diploid cell line with genomic DNA fragments from a human, metastasising breast cancer cell line, a number of animals, when injected subcutaneously with the newly-transfected cells develop secondary tumours. From one such secondary lung tumour a cell line was established, designated Ca2-5-LT1. This cell line when introduced into the host, also showed the ability to metastasise. To determine key changes in gene expression that occur during the progression from Rama 37, the benign tumour-inducing cell line, to the metastatic derivative Ca2-5-LT1, a general method of subtractive hybridisation has been employed. This technique, in conjunction with Northern blotting and nucleic acid-sequencing procedures have been used to identify mRNAs expressed differentially between the benign, Rama 37 and metastatic, Ca2-5-LT1 cell lines. From the differentially expressed cDNA clones identified, one clone has a 9- fold higher level of specific mRNA in the metastatic line, than in the nonmetastatic parental Rama 37 cell line. This CDNA, when sequenced, was found to correspond to the mRNA for rat osteopontin, a secreted phosphoprotein, previously implicated in metastasis in other systems. The levels of osteopontin mRNA in a number of other malignant, metastatic mammary cell lines produced spontaneously, and by transfection of DNA from metastatic human mammary cell lines were also increased dramatically, whilst control transfectant cells and benign cells failed to show an increase in osteopontin expression. A comprehensive analysis of osteopontin expression in normal rat tissues was performed, with osteopontin predominantly located on the surfaces of specific populations of epithelial cells possibly suggesting that osteopontin may have a normal physiological role mediating the interactions between these epithelial surfaces and the external environment and/or surrounding tissue components. To determine whether elevated expression of osteopontin is responsible alone for initiating the metastatic phenotype in the Ca2-5-LT1 cell line, Rama 37 cells were transfected with an expression construct in which an osteopontin mRNA represented by the respective cDNA was driven by the Cytomegalovirus intermediate early promoter. Transfected cells expressing elevated levels of osteopontin were then assayed in the syngeneic Furth-Wistar rats, and in 55% of the rats which produced primary tumours, lung metastases developed. These experiments show that increasing the expression of osteopontin in a previously benign cell line is sufficient to produce a metastatic phenotype in the rat mammary model employed in this project. |
| URL: | https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.283448 |
| Database: | OpenDissertations |
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