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Autocrine growth control in human carcinoma cells in vitro
Gregory, Bernard F.
This project examined methodologies used to establish primary cell cultures from non-small cell lung carcinoma (NSCLC) tumours. The objective was to enhance the in vitro proliferative potential of these clinically important tumours, particularly the squamous cell carcinoma (SCC) subtype. Existing techniques were successfully employed and refined here, permitting short-term maintenance in vitro and resulting in the establishment of a novel lung SCC cell line, DLRP, which was charaterised herein. The ability to generate long-term NSCLC cultures and cell lines on a routine basis however, remained elusive. The apparent defecit of relevant growth factors in currently available media for NSCLC was addressed. To this end, two likely sources of growth stimulators for malignant epithelial cells were explored. The first approach pursued literature reports of growth-promoting activités in the malignant effusions (MEs) and preliminary evidence in this project detected the presence in these fluids, of both stimulatory and inhibitory activity for NSCLC growth (including DLRP) in vitro. However, the active stimulatory species did not appear to be tumour-type specific and was conceivably a serum-derived systemic species. The second option evolved from the first, with evidence for the production of ‘self-stimulating’ autocrine growth factors by the human epidermoid carcinoma cell line, HEp-2. A bioassay was developed using HEp-2, which detected the presence of growth stimulators in MEs and HEp-2 conditioned medium (CM), based on their ability to stimulate the colony growth in monolayer of low density HEp-2 cells. Two separate fractions of HEp-2 CM, prepared by ultrafiltration (UF), i.e a 10-30 kDa and a >30 kDa fraction, both exhibited autocrine stimulatory activity (ASA). These fractions were distinguished by differential sensitivities to proteinase treatment. Production of several growth factors by HEp-2 is indicated by ASA elution profiles from gel-filtration, hydrophobic interaction, and heparin affinity chromatographic separations. Both heparin-binding and non-heparin-binding ASA are present in HEp-2 CM, but the heterologus elution profile of the heparin-binding ASA indicates that more than one high affinity heparin-binding mitogen is involved. Neutralising antibodies suggest that at least 60% of the ASA in the 10-30 MW fraction is attributable to a bFGF-like factor, and a similar species accounts for 40% of the > 30 kDa activity. Antisense studies designed to inhibit bFGF gene expression in HEp-2 cells did not support these findings. The possibility remains that one or more of the less well characterised heparin-binding growth factors are involved in HEp-2 autocrine growth control.
Keyword(s): Cell biology; Cell culture; Carcinogenesis; Carcinoma cells
Publication Date:
Type: Doctoral thesis
Peer-Reviewed: No
Language(s): English
Institution: Dublin City University
Citation(s): Gregory, Bernard F. (1994) Autocrine growth control in human carcinoma cells in vitro. PhD thesis, Dublin City University.
Publisher(s): Dublin City University. School of Biotechnology
File Format(s): application/pdf
Supervisor(s): Clynes, Martin
Related Link(s):
First Indexed: 2013-07-31 05:27:36 Last Updated: 2018-02-22 06:09:29