Method of screening cell death modulators

Cell death is required for many normal physiological functions for both development and homeostasis. The ability of tumor cells to elude programmed cell death, also known as apoptosis, is a hallmark of most types of cancer. A wide variety of cellular proteins, including cell surface receptors, adaptors, proteases and mitochondrial components, regulate a fine balance between cell survival and death by apoptosis. Mutations of these sensor and/or effector proteins may tip the balance, resulting in the uncontrolled survival and proliferation of cancer cells. Uncovering apoptosis mechanisms will result in new strategies to exploit apoptosis for therapeutic benefit. Email:marketing@medicilon.com.cn web:www.medicilon.com

The invention provides methods and compositions for identifying agents which modulate cell death assay, indicated e.g. by the expression of caspase-2 and/or caspase-7, in GDNF family growth factor deprived neuronal or nonneuronal cells. The methods for identifying such agents find particular application in drug development.

Programmed cell death is a process by which unwanted cells are intentionally removed, due to either physiological or pathological reasons. Morphological appearance of the dying cells and the death program (molecular and cellular death pathways) can differ remarkably between cell types and death stimuli. Currently, two death pathways have been described in detail: the death receptor (extrinsic) and mitochondrial (intrinsic) pathway. The extrinsic pathway is activated by tumour necrosis factor receptor superfamily death receptor ligation (Vincenz, 2001). The death-inducing signaling complex, assembled directly at the death receptors, activates the initiator caspase-8 that in turn activates caspases-3, -6 and -7. Activation of the intrinsic death pathway leads to release of cytochrome c (but also other apoptotic molecules) from the mitochondrial intermembrane space to the cytosol. Cytosolic cytochrome c triggers formation of the apoptosome that activates the initiator caspase-9 followed by activation of caspase-3, -6 and -7. It was shown recently that caspase-2 is activated upstream of mitochondria and may participate in the activation of mitochondria-related death events.

Mitochondrial death pathway is triggered by different modes of cellular stress and in some cells by removal (deprivation) of survival (trophic) factors. The well-characterized example of such cells is the neonatal mouse or rat sympathetic neurons that critically depend on nerve growth factor (NGF) for survival. Withdrawal of NGF from the cultured sympathetic neurons leads to the following events. The protein levels and phosphorylation of transcription factor c-Jun are increased, pro-apoptotic protein Bax is translocated from the cytosol to the mitochondria Deckwerth et al., 1996; Putcha et al., 1999), cytochrome c is released from the mitochondria to the cytosol together with Smac/DIABLO, a protein that releases caspases from the Inhibitor of Apoptosis Proteins (Deshmukh et al., 2002). As a result, caspase-9, caspase-3 but also caspase-2 are activated. All these events are critically required for the NGF deprivation-induced death. The neurons then exhibit classical features of apoptosis, including condensation of chromatin, cleavage of DNA but also increased autophagy and die finally in the culture by secondary necrosis.

In addition to these two, several other death pathways exist but these remain largely unknown. Cells in which the intrinsic apoptotic pathway is blocked can still be induced to die, both in vitro and in vivo, often with nonapoptotic ultrastructure . Recently, novel death pathways have been proposed for the dependence receptors that trigger death by a novel mechanism, when not occupied with their cognate ligands, whereas ligation of the receptors blocks death. In the case of the Deleted in Colorectal Cancer receptor, this mechanism includes direct interaction of caspases with the receptor and does not require the death receptors or mitochondrial pathways. Certainly further death pathways exist.

NGF is currently the best-characterized neurotrophic factor. Although many more neurotrophic factors are known that promote survival of different types of neurons, the death pathways activated by their withdrawal are virtually unstudied. Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor that promotes survival of several neuronal populations, including neonatal rat sympathetic neurons. NGF and GDNF signal via different receptor systems: TrkA/p75 for NGF and Ret/GFRα1 complex for GDNF. We compared the death programs triggered in the same cell type (sympathetic neurons) by removal of two different neurotrophic factors (NGF or GDNF). Surprisingly we found that the death pathways activated in these two cases differ considerably.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for identifying agents which modulate cell death assay, indicated e.g. by the expression of caspase-2 and/or caspase-7, in GDNF family growth factor deprived neuronal or nonneuronal cells. The methods for identifying such agents find particular application in commercial drug screens.

In particular, the invention provides a method of screening cell death modulators of neuronal or nonneuronal cells in a GDNF family growth factor dependent cell culture system comprising the steps of removing the GDNF family growth factor from the culture system, introducing a candidate modulator agent into the culture system, and determining the activity of caspase-2 and/or caspase-7.