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Title: The development of tumour-specific assays for cellular response to anthracycline drugs using laser cytometry
Authors: Reeve, Louise J.
First Published: 1999
Award date: 1999
Abstract: Adjunctive chemotherapy for breast cancer is confounded by varying degrees of drug resistance in individual tumours. This could be overcome by patient-specific assays using layer cytometry to measure drug uptake in viable cells.;Preliminary studies confirmed the spectroscopic, fluorometric and DNA binding characteristics of commonly used anthracyclines and demonstrated that the emission and excitation max for doxorubican made it suitable for laser cytometric detection.;Flow cytometric (FCM) measurement of doxorubicin (0.1-100 mol.L-1) uptake into drug-sensitive and resistant human breast tumour cell lines (MCF-7/S and MCF-7/R) demonstrated dose- and time-dependent uptake in MCF-7/S but not MCF-7/R cells over 4-144 hours.;By 72h the drug had a cytostatic effect upon the MCF-7/S cells and a loss of population was seen, although viability and apoptosis (Annexin V-FITC binding) were no different from control (untreated) cells. Growth and viability of MCF-7/R cells were unaffected by doxorubicin.;Comparisons of viability probes found that the green fluorescent dye sytox was the most suitable for measuring cell viability simultaneously with doxorubicin uptake. Studies using laser scanning cytometry (LSC) showed light scatter computation was suited to single colour quantification, while fluorescence computation was ideal for two colour fluorescence detection. Comparisons between FCM and LSC of doxorubicin and calcein-AM uptake by MCF-7 cells, and BrdUrd uptake into human tumour nuclei, showed good correlation.;LSC was used to visualise uptake by MCF-7/S cells, of doxorubicin covalently coupled to albumin capsules (Cytocaps TM 0-2 mol.L-1). Maximum binding of CytocapsTM was seen at 24h and they entered the cells by 48h. CytocapsTM caused cells to become cytostatic by 72h and exhibited apoptosis by 96h.;Disruption of primary human breast tumours and FNAs using mechanical and enzymatic techniques yielded low numbers of viable cells.;In this study a model assay, using cell lines has been established to detect doxorubicin uptake in viable tumour cells, using LSC relocation to identify cells.
Type: Thesis
Rights: Copyright © the author. All rights reserved.
Appears in Collections:Theses, College of Medicine, Biological Sciences and Psychology
Leicester Theses

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