Resistance to diverse apoptotic triggers in multidrug resistant HL60 cells and its possible relationship to the expression of P-glycoprotein, Fas and of the novel anti-apoptosis factors IAP (inhibitory of apoptosis proteins)
Introduction
It is now recognized that the inability of the cells to undergo apoptosis contributes in several ways to the genesis and progression of cancer and may also represent a critical cause of tumor drug resistance. Induction of apoptosis seems in fact a major modality through which anti-cancer agents may eliminate their cell targets [1], [2], [3]. However, different factors (e.g. p53, Bcl-2 family proteins, caspases and many others) involved in apoptosis can be altered in cancer cells, thereby rendering them less prone to drug-induced cell death [1], [2], [3]. It has also been suggested that, besides exporting a wide range of xenobiotics from cells, the multidrug transporter P-glycoprotein (P-gp) may also confer resistance to apoptosis from different stimuli, such as anti-neoplastic agents, Fas ligation, serum starvation and UV or γ irradiation, through specific mechanisms affecting the activation of the cell death effector enzymes caspases [4], [5], [6]. Conversely, this inhibitory effect might be reversed using specific P-gp antagonists, such as anti-P-gp antibodies or the pharmacological inhibitor verapamil [4], [5]. Nevertheless, the interrelationships between the factors of apoptosis and those of drug resistance are far from being completely understood.
In the present paper, we compared the acute myeloid leukemia cell line HL60 with its multidrug resistant (MDR), P-gp overexpressing, variant HL60R. We examined the relationship between the sensitivity of these cell lines to different apoptotic stimuli (doxorubicin (Dox), cisplatin, Fas ligation, tumor necrosis factor (TNF)-α, TRAIL (TNF-related apoptosis-inducing ligand), interferon-γ or serum deprivation) and the function of P-gp. We focused also on the possible importance of the expression of the genes of the inhibitory of apoptosis proteins (IAP) family in this respect. First described as baculoviral genes, various cellular homologues of IAPs have been identified in eukaryotes. Human IAPs include neuronal apoptosis inhibitory protein (NAIP), X-linked IAP (XIAP), c-IAP-1, c-IAP-2, survivin and other members described more recently [7], [8], [9], [10], [11], [12]. IAPs are endowed with different activities (e.g. on cytokinesis and mitotic spindle function during cell division), but have also shown a remarkable ability to block apoptosis induced by a wide spectrum of non-related triggers, including different anti-tumor agents [7], [8], [9], [10], [11], [12]. On the other hand, most IAPs seem to be able to directly bind and potently inhibit the terminal effector cell death proteases, caspase 3 and 7, as well as to suppress the activation of caspase 9, the initiator caspase in the cytochrome c/mitochondrial pathway for apoptosis [13], [14]. Thus, given these premises, IAPs might potentially play a key role in resistance to anti-cancer drugs.
Section snippets
Tumor cells and cell culture
The characteristics of the acute myeloid leukemia cell line HL60 and of its Dox resistant and MDR variant HL60R have been described previously [15]. The cells were routinely maintained in RPMI 1640 (HyClone Europe Ltd, Cramlington, UK) supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM l-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin (all from HyClone Europe). The cells were grown in a humidified atmosphere at 37°C in 5% CO2.
Agents
Dox hydrochloride was obtained from
Responsiveness to inducers of apoptosis
The concentrations of Dox or cisplatin which produced 50% (IC50) or 70% (IC70) inhibition of cell growth in HL60 or HL60R are shown in Table 1. They indicate that HL60R is resistant both to Dox and, at a lesser but significant extent, also to cisplatin. Verapamil sensitized HL60R cells to Dox, but not to cisplatin, which is not a substrate of P-gp. On the other hand, also in presence of the P-gp inhibitor, the IC50 or IC70 of Dox were still about ten-fold higher in HL60R than in HL60 (Table 1).
Discussion
In contrast to their parental cells, the MDR HL60R cells lacked sensitivity to cell death induction from diverse stimuli, including Dox, cisplatin, agonistic anti-Fas antibody administration and serum withdrawal. Clearly, the cells relied on more than one mechanism to resist apoptosis. For example, there was a loss of surface Fas expression in HL60R. This result did not seem to be instrumental for the drug resistance of HL60R, since there was no evidence that the activation of the Fas/FasL
Acknowledgements
This paper was partially supported by COFIN MURST 1998.
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