Modulation of Drug Resistance in Small Cell Lung Cancer

Milroy, Robert (1990) Modulation of Drug Resistance in Small Cell Lung Cancer. MD thesis, University of Glasgow.

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The aim of these studies was to modulate drug resistance in small cell lung cancer (SCLC) both in the laboratory and in the clinic with verapamil. Nine small cell lung cancer cell lines have been established from both pretreatment and relapse biopsies and include a pair of cell lines from the same patient. Problems in establishing cell lines related to lack of viable tumour material and overgrowth by fibroblasts. All the cell lines have been characterised and demonstrate features characteristic of SCLC in vitro. All cell lines expressed the small cell marker enzymes dopa-decarboxylase and the BB isoenzyme of creatine kinase though the levels varied between cell lines. Pathological, immunocytochemical and ultrastructural studies were used to identify both epithelial and neuroendocrine features. Thus the cell lines could be classified into classic or variant phenotypes. Detailed cytogenetic analysis of the cell lines confirmed their human origin and all were shown to contain the 3p deletion characteristic of lung tumours. The modal chromosome number ranged from 39 to 53. The cell lines all grow as non-adherent aggregates except for a monlayer variant of LS112. Measurement of growth rates and chemosensitivity therefore proved difficult since the aggregates could not be dispersed into a viable single cell suspension. Initially measurements were attempted by computerised spheroid image analysis. This technique was slow but provided limited information for three of the cell lines (LS106, LS111 and LS112FL). However, the other cell lines tested did not grow well in this system. A tetrazolium dye-based microtitration assay was therefore modified for use with both adherent and non-adherent cell lines. The assay uses as an end point the ability of live, but not dead cells, to reduce a yellow water-soluble tetrazolium dye (MTT) to a purple water-insoluble formazan product (MTT-formazan). Initial work with this assay indicated a number of deficiencies and, in particular, at high cell density or low pH the relationship between MTT-formazan production and cell number was not linear. It was shown that by determining the optimal concentration of MTT for each cell line and by addition of a buffer at a high pH (10.5) to control the pH of the formazan product a linear relationship could be obtained. When intact aggregates were incubated with MTT, crystals of MTT-formazan were produced by the entire viable cell population of the aggregate. Furthermore, doxorubicin was shown to penetrate throughout the viable cell population of the aggregate. Thus the assay could be applied to the intact SCLC aggregates thereby avoiding the need to disrupt the clusters with the consequent loss of cell interaction and viability. The cell lines were slow growing with doubling times of between 4 and 6 days. There was a 60-fold range in sensitivity to doxorubicin (ID50, 17.5 - 1050nM). Intrinsic sensitivity to doxorubicin did not relate either to any specific characteristic of the cell lines or to the history of the patient from whose biopsy the cell line was established. However, for the pair of cell lines established from the same patient the line derived from the relapse biopsy (LS310) was five-fold more resistant to doxorubicin than the line derived from the original chemosensitive pretreatment biopsy (LS274). For the multidrug resistant SCLC cell line, H69LX10, resistance modification by verapamil was shown to be dose dependent and was maximal (10-fold) at about 6-7 muM. At clinically achievable levels of verapamil (1uM) the effect was only 2-fold. These results indicate that verapamil may have limited activity in the clinic. However, the D-stereoisomer of verapamil was shown to be an equally effective resistance modulator as the racemic mixture. Since D-verapamil is thought to be less cardiotoxic than L-verapamil it is possible that use of D-verapamil in the clinic will allow increased plasma levels of the modulator without undue cardiotoxicity. Quinidine and bepridil were also identified as modulators that have significant activity in H69LX10 cells in the laboratory at levels achievable in the clinic. An increase in sensitivity to doxorubicin (2-3 fold) in the presence of verapamil (6.6muM) was demonstrated in 5 of the 9 newly established SCLC cell lines. The sensitising effect of verapamil did not relate to patient history or to the intrinsic sensitivity of the cell line to doxorubicin. Thus, the most sensitive line (LS277) established from an untreated patient showed a 2.3-fold increase in sensitivity whereas the most resistant line (LS310) from a relapse biopsy showed no change in sensitivity when co-incubated with verapamil.

Item Type: Thesis (MD)
Qualification Level: Doctoral
Keywords: Medicine, Pharmacology, Oncology
Date of Award: 1990
Depositing User: Enlighten Team
Unique ID: glathesis:1990-78175
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 30 Jan 2020 15:38
Last Modified: 30 Jan 2020 15:38

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