Divergent phenotypes induced by expression of bcl-XS: Cytokinetic effects and death without caspases.

bcl-XS, a member of the bcl-2 family, has been shown to induce and/or sensitize some cells to undergo programmed cell death and to negate the anti-apoptotic activity of bcl-XL and bcl-2 by mechanisms which are still uncertain. Using a tetracycline-regulated expression system, we have shown that expression of bcl-XS is able to elicit divergent phenotypic responses in different cell lines. In K12 rat colon carcinoma cells, we observe two phenotypic responses. A small fraction of cells undergo spontaneous programmed cell death while the majority of cells undergo a form of cytostasis. This stasis is the result of a redistribution of cells out of the S-phase, and into the G1-phase of the cell cycle. Expression of bcl-XS also decreased the viability of K12 cells, as demonstrated by a log decline in clonogenic survival. This decrease in viability is not prevented by caspase inhibition. Expression of bcl-XS was determined to be sufficient to induce acute cell death in 3T3 cells and the manner in which these cells die is both morphologically and biochemically different from Fas/CD95 induced apoptosis. Bcl-XS expression causes loss of the inner mitochondrial membrane potential (DeltaPsim) but does not induce caspase activation. In addition to the loss of DeltaPsim, electron microscopy showed gross structural perturbations in the mitochondria upon expression of bcl-XS. These data suggested that cytochrome c was being released from the mitochondria and should, therefore, trigger the activation of pro-caspase-9 and apoptosis. However, the lack of caspase activation. appeared to be due to a unique phenomenon also caused by expression of bcl-XS, depletion of cellular cytochrome c. In vitro pro-caspase activation assays proved that addition of exogenous cytochrome c to cytosolic extracts prepared from bcl-XS expresssing 3T3 cells is sufficient to trigger 35S-pro-caspase-9 processing. Furthermore, transient transfection experiments showed that cell death induced by bcl-XS is not inhibited by dominant negative caspase 9 but, rather, that cell death induced by bax or bak expression is inhibited. Consequently, bcl-XS must kill 3T3 cells through a pathway that does not require liberation and/or action of bax/bak nor through a caspase-dependent pathway.Ph.D.Biological SciencesCellular biologyHealth and Environmental SciencesOncologyPharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/132350/2/9963783.pd

An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo

The application of RNA interference (RNAi) to mammalian systems has the potential to revolutionize genetics and produce novel therapies. Here we investigate whether RNAi applied to a well-characterized gene can stably suppress gene expression in hematopoietic stem cells and produce detectable phenotypes in mice. Deletion of the Trp53 tumor suppressor gene greatly accelerates Myc-induced lymphomagenesis, resulting in highly disseminated disease(1,2). To determine whether RNAi suppression of Trp53 could produce a similar phenotype, we introduced several Trp53 short hairpin RNAs (shRNAs) into hematopoietic stem cells derived from Emu-Myc transgenic mice, and monitored tumor onset and overall pathology in lethally irradiated recipients. Different Trp53 shRNAs produced distinct phenotypes in vivo, ranging from benign lymphoid hyperplasias to highly disseminated lymphomas that paralleled Trp53(-/-) lymphomagenesis in the Emu-Myc mouse. In all cases, the severity and type of disease correlated with the extent to which specific shRNAs inhibited p53 activity. Therefore, RNAi can stably suppress gene expression in stem cells and reconstituted organs derived from those cells. In addition, intrinsic differences between individual shRNA expression vectors targeting the same gene can be used to create an 'epi-allelic series' for dissecting gene function in vivo

Survival signalling by Akt and eIF4E in oncogenesis and cancer therapy

Evading apoptosis is considered to be a hallmark of cancer, because mutations in apoptotic regulators invariably accompany tumorigenesis. Many chemotherapeutic agents induce apoptosis, and so disruption of apoptosis during tumour evolution can promote drug resistance. For example, Akt is an apoptotic regulator that is activated in many cancers and may promote drug resistance in vitro. Nevertheless, how Akt disables apoptosis and its contribution to clinical drug resistance are unclear. Using a murine lymphoma model, we show that Akt promotes tumorigenesis and drug resistance by disrupting apoptosis, and that disruption of Akt signalling using the mTOR inhibitor rapamycin reverses chemoresistance in lymphomas expressing Akt, but not in those with other apoptotic defects. eIF4E, a translational regulator that acts downstream of Akt and mTOR, recapitulates Akt's action in tumorigenesis and drug resistance, but is unable to confer sensitivity to rapamycin and chemotherapy. These results establish Akt signalling through mTOR and eIF4E as an important mechanism of oncogenesis and drug resistance in vivo, and reveal how targeting apoptotic programmes can restore drug sensitivity in a genotype-dependent manner