Macroautophagy is an evolutionary conserved lysosomal pathway involved in the turnover of cellular macromolecules and organelles. In spite of its essential role in tissue homeostasis, the molecular mechanisms regulating mammalian macroautophagy are poorly understood. Here, we demonstrate that a rise in the free cytosolic calcium ([Ca(2+)](c)) is a potent inducer of macroautophagy. Various Ca(2+) mobilizing agents (vitamin D(3) compounds, ionomycin, ATP, and thapsigargin) inhibit the activity of mammalian target of rapamycin, a negative regulator of macroautophagy, and induce massive accumulation of autophagosomes in a Beclin 1- and Atg7-dependent manner. This process is mediated by Ca(2+)/calmodulin-dependent kinase kinase-beta and AMP-activated protein kinase and inhibited by ectopic Bcl-2 located in the endoplasmatic reticulum (ER), where it lowers the [Ca(2+)](ER) and attenuates agonist-induced Ca(2+) fluxes. Thus, an increase in the [Ca(2+)](c) serves as a potent inducer of macroautophagy and as a target for the antiautophagy action of ER-located Bcl-2.

A chemotherapeutic vitamin D analogue, EB1089, kills tumor cells via a caspase-independent pathway that results in chromatin condensation and DNA fragmentation. Employing transmission-and immunoelectronmicroscopy as well as detection of autophagosome-associated LC3-b protein in the vacuolar structures, we show here that EB1089 also induces massive autophagy in MCF-7 cells. Interestingly, inhibition of autophagy effectively hindered apoptosis-like nuclear changes and cell death in response to EB1089. Furthermore, restoration of normal levels of beclin 1, an autophagyinducing tumor suppressor gene that is monoallelically deleted in MCF-7 cells, greatly enhanced the EB1089-induced nuclear changes and cell death. Thus, EB1089 triggers nuclear apoptosis via a pathway involving Beclin 1-dependent autophagy. Surprisingly, tumor cells depleted for Beclin 1 failed to proliferate suggesting that even though the monoallelic depletion of beclin 1 in human cancer cells suppresses EB1089-induced autophagic death, one intact beclin 1 allele is essential for tumor cell proliferation.

Very little is known about the signaling pathways mediating vitamin D-induced cell death. A single family of proteases, the caspases, has until recently been considered the pivotal executioner of all programmed cell death (8). Therefore it is interesting to note that breast cancer cells treated with vitamin D compounds die in the complete absence of effector caspase activation (4). Despite the lack of the caspase activation, dying cells present several characteristics of apoptosis, i.e. rounding, shrinkage, and detachment of cells as well as DNA strand breaks and DNA fragmentation (4, 9, 10). Furthermore, antiapoptotic proteins Bcl-2 and Bcl-X L can rescue breast cancer cells from death induced by the active form of vitamin D or its analogs (4). This apoptosis-like death program appears also independent of cysteine cathepsins and p53 tumor suppressor protein (4, 11). Instead, the elevation in the intracellular free calcium ([Ca 2ϩ ] i ) brought about by vitamin D compounds correlates with the induction of apoptosis in breast cancer cells (9,12,13).Data from studies employing various pharmaceutical modulators of calcium homeostasis have suggested that the elevation in [Ca 2ϩ ] i is a sufficient signal to induce apoptosis in several model systems, even though it may also have the opposite effect in other systems (14). Further supporting the idea that the elevation in [Ca 2ϩ ] i may mediate apoptosis, studies based on modulated expression of calcium-binding proteins, calbindin-D 28k or glucose-regulated proteins GRP78 and GRP94, have shown that Ca 2ϩ buffering can confer protection against various apoptotic stimuli (15-19). The calcium-dependent neutral cysteine proteases, calpains, are frequently activated in apoptosis models involving elevated [Ca 2ϩ ] i (20 -22). Two forms of calpains, -calpain and m-calpain or type I and type II calpain, respectively, are ubiquitously expressed in human cells (23)(24)(25). The active forms of the enzymes consist of a variable large subunit (80 kDa) and a common small subunit (30 kDa). To become active, calpains require an elevation in [Ca 2ϩ ] i , and the autoproteolytic cleavage of the enzymes further enhances their activity. Whereas m-calpain requires Ca 2ϩ at a millimolar range, micromolar concentrations are enough for the activation of -calpain (in vitro; lower in cells). So far no difference in the substrate specificity of the two isozymes has been found. Growing evidence suggests that calpains may play a central role in the execution of apoptosis either upstream or * This work was supported by the Danish Medical Research Council, the Danish Cancer Society (to I. S. M. and M. J.), and by Grant CA67317 from the NCI, National Institutes of Health (to I. N. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Present address: Molecular Biology, Novo Nordisk A/S, Bagsvaerd DK 2880, Denmar...

Insulin receptor (IR) and insulin-like growth factor I receptor (IGF-IR) are both from the same subgroup of receptor tyrosine kinases that exist as covalently bound receptor dimers at the cell surface. For both IR and IGF-IR, the most described forms are homodimer receptors. However, hybrid receptors consisting of one-half IR and one-half IGF-IR are also present at the cell surface. Two splice variants of IR are expressed that enable formation of two isoforms of the IGF-IR/IR hybrid receptor. In this study, these two splice variants of hybrid receptors were studied with respect to binding affinities of insulin, insulin-like growth factor I (IGF-I), and insulin-like growth factor II (IGF-II). Unlike previously published data, in which semipurified receptors have been studied, we found that the two hybrid receptor splice variants had similar binding characteristics with respect to insulin, IGF-I, and IGF-II binding. We studied both semipurified and purified hybrid receptors. In all cases we found that IGF-I had at least 50-fold higher affinity than insulin, irrespective of the splice variant. The binding characteristics of insulin and IGF-I to both splice variants of the hybrid receptors were similar to classical homodimer IGF-IR.