Cancer Letters

Cancer Letters

Volume 127, Issues 1–2, 15 May 1998, Pages 113-121
Cancer Letters

Intracellular calcium distribution in apoptosis of HL-60 cells induced by harringtonine: intranuclear accumulation and regionalization

https://doi.org/10.1016/S0304-3835(98)00023-8Get rights and content

Abstract

Harringtonine (HT), an anticancer drug with high chemotherapeutic efficiency to human chronic granulocytic/myelomonocytic leukemia, has been reported to rapidly induce apoptosis in HL-60 cells in a wide scope/range of dosage by investigators from our lab and others. In the present studies, by using video enhancement contrast (VEC) microscopy, we dynamically analyzed changes in intracellular calcium distribution in a single HL-60 cell over the period from the initiation of apoptosis to the obvious appearance of chromatin condensation. The results from this paper demonstrated the striking distinction of intracellular calcium distribution at different time points after treatment with HT. Before treatment in normal HL-60 cells the highest [Ca2+]i accumulation was observed in the peri-nuclear area and the lowest was observed in the nucleus; after treatment with 1 μg/ml HT for 30 min intracellular calcium diffused all over the cell compartments, while intranuclear calcium increased comparatively and significantly. The phenomenon of intranuclear calcium accumulation was further confirmed by using laser scanning confocal microscopy (LSCM). In addition, co-localization of the highest calcium region with condensed chromatin in apoptotic HL-60 cells was also observed by LSCM. Our results suggest that two sequential alterations of intracellular calcium distribution occurred in apoptotic HL-60 cells induced by HT, i.e. (a) accumulation of calcium in the nucleus and (b) regionalization in a specific nuclear region.

Introduction

Apoptosis or programmed cell death has been recognized as a mode of cell deletion, which is characterized by typical morphological and biochemical alterations, including chromatin condensation, blebbing of the plasma membrane and nuclear envelope, cell shrinkage and fragmentation of DNA to oligonucleosomal-sized fragments [1]. It occurs during development in the embryo [2], thymic maturation [3]and in immature thymocytes treated with glucocorticoid hormones [4]or exposure to a certain environmental toxin [5]. In addition, a wide variety of chemotherapeutic drugs, which are generally considered to have distinct targets in cells, have been shown to induce cell death by a common mechanism of apoptosis [6]. Therefore, it is important to elucidate the mechanism of apoptosis induced by different anticancer drugs in order to improve their clinical effects.

As the most important intracellular signal molecular calcium is thought to play a multiple role in the complex pathways leading to apoptosis in different in vitro and in vivo experimental systems. However, the definitive role of calcium in mediating apoptotic cell death is still controversial. Many studies have demonstrated that sustained increases in intracellular calcium precede apoptosis 7, 8. For example, apoptosis of thymocytes induced by glucocorticoid could be mimicked by a calcium ionophore [9]and an inhibitor of endoplasmic reticulum Ca2+-ATPase, thapsigargin [10], suggesting that glucocorticoids exert their effects in part by a calcium-dependent mechanism. Further investigation by McConkey et al. [11]in the same experimental system revealed that glucocorticoids induced an early, gradual and sustained increase in cytosolic Ca2+. Intracellular Ca2+ levels were enhanced approximately eight-fold (from 85 to 750 nm) over a 90-min period and remained constant for at least an additional 30 min [11]. The increase in [Ca2+]i resulted in DNA fragmentation while chelation of extracellular Ca2+ with EGTA inhibited DNA degradation. These data indicated that the increase in cytosolic Ca2+ should be due to an influx of Ca2+ from the extracellular medium. In addition, an increase in intracellular Ca2+ due to an influx of extracellular calcium was also found to be involved in radiation-induced apoptosis [7].

In contrast to thymocyte apoptosis, cell death in HL-60 cells induced by anticancer drugs [12]or cytotoxic T cell-mediated target cell death through Fas–Fas L interaction [13]did not accompany an obvious calcium influx and could be completed even in the presence of extracellular calcium chelator EGTA. Under these conditions, the fact that buffering of intracellular Ca2+ protected stimulated cells from undergoing apoptosis also indicated that pre-existing intracellular Ca2+ plays an essential role in the induction of apoptosis. It is well known that the typical morphological and biochemical characteristics of apoptosis, i.e. chromatin condensation and cleavage of host chromatin into oligonucleosome-length fragments, take place in the nucleus. This suggests the possibility that apoptosis in HL-60 cells may be accompanied by an elevation in nuclear Ca2+ due to an influx from the cytosol fraction.

Although Lam et al. (unpublished data) demonstrated that the depletion of the intracellular calcium store is necessary for apoptosis in some cell types, due to a lack of powerful methods, there is no definitive evidence to support the distribution changes of intracellular calcium in apoptosis, with which no alteration of total intracellular Ca2+ is accompanied.

To study the spatial distribution of intracellular Ca2+ in a single HL-60 cell during the process of apoptosis for up to 2 h, fluorescent images of an individual Fluo-3 AM-loaded HL-60 cell were processed by VEC microscopy at a low light level. The results revealed that HT-triggered apoptosis in HL-60 cells was characterized by the accumulation of calcium in the nucleus and subsequent regionalization in a specific nuclear region of condensed chromatin; however, a dramatic increase in the total intracellular-free Ca2+ in the apoptotic HL-60 cells was not found.

Section snippets

Cell culture

Human HL-60 myelogenous leukemia cells were grown at 37°C in RPMI 1640 (GIBCO, USA) medium containing 10% heat-inactivated fetal bovine serum (Sigma, USA) in an atmosphere containing 5% CO2. Exponentially-growing cells were used in all experiments.

Simultaneous staining of HL-60 cells with Hoechst 33342 and Fluo-3 AM

For simultaneously recording the changes in intracellular calcium and apoptotic characteristics in an identical cell, HL-60 cells were loaded with Fluo-3 AM (Molecular Probes, USA) and Hoechst 33342 (Molecular Probes, USA) as described by Xue et al.

BAPTA-AM and not EGTA inhibits HT-induced apoptosis in HL-60 cells

Some researchers have reported that exposure of HL-60 cells to HT (0.02–5 μg/ml) for 2 h leads to apoptosis in a concentration-dependent manner 15, 16. In the present study, in order to follow the dynamic changes in intracellular calcium in a single apoptotic HL-60 cell, we made some modifications to the methods for induction of apoptosis by HT. As described in Section 2, apoptotic HL-60 cells pre-loaded with Fluo-3 AM and Hoechst 33342 and continuously excited by very weak UV light were

Discussion

Early studies from many laboratories over the past 5 years have demonstrated that the most important signal element, Ca2+, might be involved in triggering and regulating apoptosis. Working with immature thymocytes, McConkey shows that glucocorticoid-stimulated apoptosis is associated with enhanced Ca2+ influx [11]. Apoptosis in other systems also appears to involve alterations in intracellular Ca2+. For example, a rapid and sustained Ca2+ increase preceded the cytolysis of the target of

Acknowledgements

This work was supported by grants from the National Education Committee Doctor's Foundation of China and the National Natural Science Foundation of China (Nos. 397301608 and 39670204).

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