Screening method

#The present invention relates to a method for identifying genes, which are involved in the modulation of apoptosis in cells. In particular, the invention relates to a novel method for identifying and characterising those genes, and the molecular mechanisms involved in the GM-CSF mediated inhibition of cell death.

#Programmed cell death or apoptosis is a genetically programmed process by which cells die under both physiological and a variety of pathological conditions (Kerr et al, Br. J. Cancer, 26, 239-257, 1972). It serves as the counter-balancing force to mitosis during adult life and is a major contributor to the sculpting of physiological structures during the many processes of development (Wyllie et al, Int. Rev. Cytol, 68, 251-305, 1980). It is characterised by a number of well-defined biochemical hallmarks. These include DNA fragmentation, caused by the activation of an endogenous endonuclease enzyme (Wyllie, Nature, 284, 555-556,1980; Enari et al., Nature, 391, 43-50, 1998). The result is a DNA ladder pattern that can be readily visualised in agarose cells. Coupled with DNA fragmentation is cell shrinkage (Wesselbory et al., Cell Immunol. 148, 234-41, 1993) where water is actively extruded from the cell. The apoptotic cell then undergoes fragmentation into apoptotic bodies that are engulfed by neighbouring cells or cells of the reticuloedothelial system.

#The number of factors which are know to induce survival in particular cell types is ever increasing (e.g. IL2, IL3, IL4, IL5, IL8, GM-CSF, insulin like growth factor 1, NGF, VEGF, PDGF, SCF, LIF, EGF etc.). Many of these survival factors appear to share a commonality in the survival pathway (Datta SR et al. Genes and Development 13: 2905-2927, 1999). For an extracellular stimuli, to confer survival on a cell, it must inhibit the endogenous apoptotic machinery. The model predicts that there is a series of temporal events that occur upon survival factor/receptor interaction. The first of these is tyrosine phosphorylation at the plasma membrane due either to intrinsic receptor tyrosine kinase activity (e.g. the insulin growth factor 1 receptor), or indirectly coupled to tyrosine kinases or alternatively directly coupled to several transmembrane G protein-coupled receptors.

#FIG. 1 shows the correlation between the induction of apoptosis, inhibition of apoptosis by GM-CSF and the inhibition of GM-CSF-mediated survival by an inhibitor, for example gliotoxin. The central shaded region indicates changes associated with all three treatments.

#FIG. 2 shows the caspase activity present in the supernatant fraction of human neutrophils which have undergone spontaneous apoptosis. The activity is measured using absorbance at 405 nm and normalised per mg of protein. FIG. 2 shows the caspase activity during the incubation period. There is a dramatic increase between 8 and 20 h of culture.

#FIG. 3 shows a morphological determination of apoptosis. Cells are removed from culture and centrifuged at 300 g for 10 minutes at 4° C. The pellet is kept on ice, washed in ice cold hanks buffer and then re-suspended in cell lysis buffer. Details are given in example 1. Apoptosis as measured by morphology, increased from approximately 6 hours following isolation of the cells. There is a large increase in the number of cells undergoing spontaneous apoptosis between 6-8 hours resulting close to 50% of the cells showing apoptotic morphology at 8 hours.

#Table 1 shows a morphological determination of apoptosis. The table shows that although there is a small amount of apoptosis occuring two hours post isolation, there is a large increase in the number of cells undergoing spontaneous apoptosis between 6-8 hours, with close to 50% of the cells showing apoptotic morphology at 8 hours.

#Table 2 shows that an increase in superoxide production, as detected by flow cytometry occurs during the spontaneous apoptosis of neutrophils.

#Table 3 summarizes those genes regulated early in GM-CSF mediated inhibition of neutrophil apoptosis, as identified by Suppression Subtractive hybridisation (SSH). A sample of clones differentially expressed in these libraries are sequenced as described. A number of clones correspond to known genes, whilst many others are unique and do not have any identity with any expressed sequence in the public databases. The sequence identities are presented in table 3.