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Title: An investigation of the cellular responses to the unsymmetrically substituted polyamine analogues and identification of the pathway to cell death
Author: Fraser, Alison
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 2003
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HL-60 human promyelogenous leukaemic cells were used as a model to determine the cytotoxic potential of the unsymmetrically substituted polyamine analogues CHENSpm, CPENSpm and IPENSpm with a view to their use as chemotherapeutic agents. The cytotoxicity was compared with etoposide, an established cytotoxic drug. The analogues CHENSpm and IPENSpm induced cytotoxicity over 48 h, with decreases in cell number and protein content, with CPENSpm showing a growth inhibitory effect after 96 h. The cellular content of all 3 polyamines was decreased in all treatments, and this resulted from increases in the catabolic enzyme SS AT, and polyamine export, determined as active export by the presence of acetyl-polyamines and putrescine in the culture medium. All 3 polyamine analogues were detected intracellularly, and their internalisation was essential for their toxicity, as their co-incubation with transport inhibitors provided protection against analogue toxicity. Use of the transport inhibitors also confirmed the polyamine transporter as the route of analogue uptake. Analysis of the type of cell death induced by the unsymmetrically substituted polyamine analogues confirmed it as apoptosis, through morphological determination using DAPI staining, cell cycle analysis showing a pre G0/G1 peak, increased DNA fragmentation and induction of caspase-3-like enzymes. HL-60+ cells were used in conjunction with wild-type HL-60+ cells in an attempt to determine the pathway to apoptosis induced by these analogues. HL-60+ cells contained a stable vector that overexpressed bcl-2, an anti-apoptotic protein that prevents the mitochondrial release of apoptogenic factors including cytochrome c. These cells had shown virtually no toxicity to the analogues over all exposure periods, despite the intracellular accumulation of the analogues. Western blotting was used to probe for the presence of cytochrome c, an activator of apoptosis through the mitochondrial pathway, in the cytosol of both cell lines in response to both analogue and etoposide treatment. The detection of cytochrome c in the cytosol of wild-type HL-60 cells, but not the bcl-2 overexpressors, suggested that the mitochondrial route to apoptosis was being activated, and this was further confirmed by the increased activation of caspase-9, the initiator caspase in this route, with only basal levels of caspase-8, the initiator caspase in the death effector route, detected. These data strongly suggest that the exposure of HL-60 cells to unsymmetrically substituted analogues, CHENSpm and IPENSpm, results in the activation of apoptosis through the mitochondrial release of cytochrome c. Consideration of these analogues as future chemotherapeutic agents requires that their effects are decreased in normal cells. Two normal cell lines and primary human cell cultures were exposed to these analogues and results confirmed that the analogues were selectively cytotoxic towards malignant cells. Overall, these analogues were effective cytotoxic agents in the HL-60 human leukaemic cell model, and show promise as chemotherapeutic agents through a low dose and short exposure, which is directly comparable with etoposide. The identification of the mitochondrial pathway to apoptosis as the mechanism of action of these analogues means they would not be useful chemotherapeutic agents in tumours where anti-apoptotic proteins in the mitochondrial apoptotic pathway, such as bcl-2, are overexpressed. However, these analogues could be effective cytotoxic agents in other types of cancer, particularly those where the death effector pathway is suppressed due to a similar anti-apoptotic protein overexpression, and could be used either alone, or in conjunction with existing cytotoxic drugs.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Genetics