Genetic Analysis of the Immortal Phenotype in Human Squamous Cell Carcinoma

Edington, Kirsten Gillian (1994) Genetic Analysis of the Immortal Phenotype in Human Squamous Cell Carcinoma. PhD thesis, University of Glasgow.

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There is accumulating evidence that the escape from the phenomenon of in vitro senescence, or immortality, is an important step in tumour progression. Senescence is tightly linked to terminal differentiation and is genetically programmed; furthermore, the genes regulating senescence are candidate tumour suppressor genes. The work described in this thesis investigates immortality in a human head and neck squamous cell carcinoma (SCC) system. Prior to this work there were no cell lines available where loss of heterozygosity could be studied in SCC, and few in other systems. In addition, most SCC cell lines had been derived from recurrent tumours and/or without feeder layers of irradiated mouse Swiss 3T3 fibroblasts. Therefore it was not possible to distinguish the genetic events causing the tumour and those resulting from therapy and adaptation to tissue culture. Twenty-two primarily untreated tumours were collected along with blood samples and seven immortal cell lines (the BICR cell lines) were isolated. We have also obtained several premalignant erythroplakia cultures, so it is now possible to grow all the stages of SCC progression from normal keratinocytes through premalignant erythroplakias to carcinomas and lymph node metastases. DNA fingerprinting confirms that blood, fibroblast and cell line DNA came from the same patient and each cell line is unique. Similarly keratin & involucrin staining and electron micrographs show they are SCCs. Immortality appears to be a late-emerging phenotype during carcinogenesis as the erythroplakia cells eventually senesce and it is difficult to establish cell lines from early (TNM clinical stage T2) tumours relative to late (T4) tumours. Erythroplakias and T2 cell lines are non-tumorigenic in nude mice, whereas late-stage lines are tumorigenic. Human papilloma viruses (HPV) 16 & 18 can immortalise keratinocytes. Their transforming genes E6 & E7 bind p53 and pRb respectively and thus p53 and pRb may be involved in senescence. None of the cell lines contain HPV 16 or 18, however loss of heterozygosity (LOH) analysis confirms data showing high frequencies of p53 mutation obtained by Bums et al (1993). Interestingly, although it had been previously shown that see cell lines have normal RB mRNA and no abnormalities in pRB size, location or phosphorylation status, 3/6 informative cases exhibit LOH at RB. Chromosomes 1, 4, 6 & 9 have been shown by several other groups to induce senescence in various target cells when introduced by microcell-mediated chromosome transfer. These chromosomes were investigated in the BICR cultures, again by LOH analysis. Regions Iq, 4p and 6q do not show a significant degree of LOH at any stage, but there are high levels of loss at both 9p and separately at 9q in later stage immortal cultures. The information allows tentative assignment of a tumour suppressor gene involved in SCC progression and possibly in escape from senescence to 9p22-23.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Robert McFarlene
Keywords: Oncology
Date of Award: 1994
Depositing User: Enlighten Team
Unique ID: glathesis:1994-75318
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 21:13
Last Modified: 19 Nov 2019 21:13

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