We have found that insulin-like growth factor I (IGF-I) can protect fibroblasts from apoptosis induced by UV-B light. Antiapoptotic signalling by the IGF-I receptor depended on receptor kinase activity, as cells overexpressing kinase-defective receptor mutants could not be protected by IGF-I. Overexpression of a kinasedefective receptor which contained a mutation in the ATP binding loop functioned as a dominant negative and sensitized cells to apoptosis. The antiapoptotic capacity of the IGF-I receptor was not shared by other growth factors tested, including epidermal growth factor (EGF) and thrombin, although the cells expressed functional receptors for all the agonists. However, EGF was antiapoptotic for cells overexpressing the EGF receptor, and expression of activated pp60 v-src also was protective. There was no correlation between protection from apoptosis and activation of mitogen-activated protein kinase, p38/HOG1, or p70 S6 kinase. On the other hand, protection by any of the tyrosine kinases against UV-induced apoptosis was blocked by wortmannin, implying a role for phosphatidylinositol 3-kinase (PI3 kinase). To test this, we transiently expressed constitutively active or kinase-dead PI3 kinase and found that overexpression of activated phosphatidylinositol 3-kinase (PI3 kinase) was sufficient to provide protection against apoptosis. Because Akt/PKB is believed to be a downstream effector for PI3 kinase, we also examined the role of this serine/threonine protein kinase in antiapoptotic signalling. We found that membrane-targeted Akt was sufficient to protect against apoptosis but that kinasedead Akt was not. We conclude that the endogenous IGF-I receptor has a specific antiapoptotic signalling capacity, that overexpression of other tyrosine kinases can allow them also to be antiapoptotic, and that activation of PI3 kinase and Akt is sufficient for antiapoptotic signalling.

The caspase-3 (CPP32, apopain, YAMA) family of cysteinyl proteases has been implicated as key mediators of apoptosis in mammalian cells. Gelsolin was identified as a substrate for caspase-3 by screening the translation products of small complementary DNA pools for sensitivity to cleavage by caspase-3. Gelsolin was cleaved in vivo in a caspase-dependent manner in cells stimulated by Fas. Caspase-cleaved gelsolin severed actin filaments in vitro in a Ca2+-independent manner. Expression of the gelsolin cleavage product in multiple cell types caused the cells to round up, detach from the plate, and undergo nuclear fragmentation. Neutrophils isolated from mice lacking gelsolin had delayed onset of both blebbing and DNA fragmentation, following apoptosis induction, compared with wild-type neutrophils. Thus, cleaved gelsolin may be one physiological effector of morphologic change during apoptosis.

Double-stranded short interfering RNAs (siRNA) induce post-transcriptional silencing in a variety of biological systems. In the present study we have investigated the structural requirements of chemically synthesised siRNAs to mediate efficient gene silencing in mammalian cells. In contrast to studies with Drosophila extracts, we found that synthetic, double-stranded siRNAs without specific nucleotide overhangs are highly efficient in gene silencing. Blocking of the 5'-hydroxyl terminus of the antisense strand leads to a dramatic loss of RNA interference activity, whereas blocking of the 3' terminus or blocking of the termini of the sense strand had no negative effect. We further demonstrate that synthetic siRNA molecules with internal 2'-O-methyl modification, but not molecules with terminal modifications, are protected against serum-derived nucleases. Finally, we analysed different sets of siRNA molecules with various 2'-O-methyl modifications for stability and activity. We demonstrate that 2'-O-methyl modifications at specific positions in the molecule improve stability of siRNAs in serum and are tolerated without significant loss of RNA interference activity. These second generation siRNAs will be better suited for potential therapeutic application of synthetic siRNAs in vivo.

Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule recruited to the membrane by activated growth factor receptors. The p85 subunit of PI 3-kinase links the catalytic p110 subunit to activated growth factor receptors and is required for enzymatic activity of p110. In this report, we describe the effects of expressing novel forms of p110 that are targeted to the membrane by either N-terminal myristoylation or C-terminal farnesylation. The expression of membrane-localized p110 is sufficient to trigger downstream responses characteristic of growth factor action, including the stimulation of pp70 S6 kinase, Akt/Rac, and Jun N-terminal kinase (JNK). These responses can also be triggered by expression of a form of p110 (p110*) that is cytosolic but exhibits a high specific activity. Finally, targeting of p110* to the membrane results in maximal activation of downstream responses. Our data demonstrate that either membrane-targeted forms of p110 or a form of p110 with high specific activity can act as constitutively active PI 3-kinases and induce PI 3-kinasedependent responses in the absence of growth factor stimulation. The results also show that PI 3-kinase activation is sufficient to stimulate several kinases that appear to function in different signaling pathways.Phosphatidylinositol (PI) 3-kinase activity has been implicated in the regulation of a number of different cellular responses, including the regulation of cell growth. Oncogenic transformation or the stimulation of cells with growth factors results in an increased level in the phospholipid products of PI 3-kinase (for reviews, see references 4, 23, and 50). Mutants of platelet-derived growth factor (PDGF) receptor or certain oncogenes which fail to activate PI 3-kinase are deficient in triggering either mitogenic responses or oncogenic transformation, respectively. These data suggest that PI 3-kinase is an important mediator of signaling events that regulate cell growth and cellular transformation.PI 3-kinase is a heterodimeric complex consisting of 85-and 110-kDa subunits (p85 and p110) (23). The p85 subunit consists of multiple domains including a Src homology 3 (SH3) domain, a breakpoint cluster region domain, and two SH2 domains. The two SH2 domains bind tyrosine-phosphorylated receptors and in this manner recruit the p85-p110 complex to activated receptors. The two SH2 domains are separated by the inter-SH2 (iSH2) region. The iSH2 domain mediates the interaction of p85 with p110, and this interaction is required for the enzymatic activity of p110 (28) (Fig. 1). It is possible that the role of p85 is to target p110 to the membrane, where its lipid substrates reside (23).PI 3-kinase has been implicated in the regulation of many other cellular processes, including the reorganization of the actin cytoskeleton (24,30,40,59), receptor internalization (21), histamine secretion (63), neutrophil activation (56), platelet activation (64), cell migration (31), glucose transport (41), and vesicular sorting (48). PI 3-kinase was implicated in the...