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. 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 E(mu)-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 E(mu)-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.     

Effects of ACTH and diabetes on phospholipid metabolism in adrenal mitochondria.

Incorporation of 32P into adrenal mitochondrial phospholipids (PL) incrased in ACTH-treated rats, but it decreased in diabetics, inspite of the fact that these animals showed adrenal overacity. Since diabetics did not show increased 11 beta-hydroxylation. as opposed to ACTH-treated rats, it is suggested that the stimulation of this enzyme activity by exogenous ACTH is related to an increased turnover of PL at the mitochondrial membrane. The process is impaired in diabetics and prevents the stimulation of 11 beta-hydroxylation.     

RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels

Therapeutics that discriminate between the genetic makeup of normal cells and tumour cells are valuable for treating and understanding cancer. Small molecules with oncogene-selective lethality may reveal novel functions of oncoproteins and enable the creation of more selective drugs. Here we describe the mechanism of action of the selective anti-tumour agent erastin, involving the RAS-RAF-MEK signalling pathway functioning in cell proliferation, differentiation and survival. Erastin exhibits greater lethality in human tumour cells harbouring mutations in the oncogenes HRAS, KRAS or BRAF. Using affinity purification and mass spectrometry, we discovered that erastin acts through mitochondrial voltage-dependent anion channels (VDACs)--a novel target for anti-cancer drugs. We show that erastin treatment of cells harbouring oncogenic RAS causes the appearance of oxidative species and subsequent death through an oxidative, non-apoptotic mechanism. RNA-interference-mediated knockdown of VDAC2 or VDAC3 caused resistance to erastin, implicating these two VDAC isoforms in the mechanism of action of erastin. Moreover, using purified mitochondria expressing a single VDAC isoform, we found that erastin alters the permeability of the outer mitochondrial membrane. Finally, using a radiolabelled analogue and a filter-binding assay, we show that erastin binds directly to VDAC2. These results demonstrate that ligands to VDAC proteins can induce non-apoptotic cell death selectively in some tumour cells harbouring activating mutations in the RAS-RAF-MEK pathway.     

Mid-anaphase arrest in S. cerevisiae cells eliminated for the function of Cin8 and dynein

S. cerevisiae anaphase spindle elongation is accomplished by the overlapping function of dynein and the kinesin-5 motor proteins, Cin8 and Kip1. Cin8 and dynein are synthetically lethal, yet the arrest phenotypes of cells eliminated for their function had not been identified. We found that at a non-permissive temperature, dyn1Δ cells that carry a temperature-sensitive cin8 – 3 mutation arrest at mid-anaphase with a unique phenotype, which we named TAN (two microtubule asters in one nucleus). These cells enter anaphase, but fail to proceed through the slow phase of anaphase B. At a permissive temperature, dyn1Δ, cin8 – 3 or dyn1Δcin8 – 3 cells exhibit perturbed spindle midzone morphologies, with dyn1Δcin8 – 3 anaphase spindles also being profoundly bent and nonrigid. Sorbitol, which has been suggested to stabilize microtubules, corrects these defects and suppresses the TAN phenotype. We conclude that dynein and Cin8 cooperate in anaphase midzone organization and influence microtubule dynamics, thus enabling progression through the slow phase of anaphase B. Received 10 August 2008; received after revision 22 October 2008; accepted 27 October 2008     

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.     

Effects of ACTH and diabetes on phospholipid metabolism in adrenal mitochondria

Summary Incorporation of 32P into adrenal mitochondrial phospholipids (PL) increased in ACTH-treated rats, but it decreased in diabetics, inspite of the fact that these animals showed adrenal overactivity. Since diabetics did not show increased 11 -hydroxylation, as opposed to ACTH-treated rats, it is suggested that the stimulation of this enzyme activity by exogenous ACTH is related to an increased turnover of PL at the mitochondrial membrane. This process is impaired in diabetics and prevents the stimulation of 11 -hydroxylation.     

Tyrosine-containing motif that transduces cell activation signals also determines internalization and antigen presentation via type III receptors for IgG.

Type III receptors for IgG (Fc gamma RII; ref. 1), high-affinity IgE receptors (Fc epsilon RI; ref. 2), as well as the T- and B-cell antigen receptors, consist of multiple components with specialized ligand-binding and signal transduction functions. Fc gamma RII alpha (ligand-binding) and gamma (signal-transducing) subunits are expressed in macrophages, a cell type involved in the uptake of antigen, its processing and the presentation of the resulting peptides to major histocompatibility complex class II-restricted T lymphocytes. Here we show that murine Fc gamma RIII, transfected into Fc gamma R-negative antigen-presenting B-lymphoma cells, mediate rapid ligand internalization and strongly increase the efficiency of antigen presentation when antigen is complexed to IgG. Efficient internalization and antigen presentation via Fc gamma RIII did not require the cytoplasmic domain of the ligand-binding alpha-chain, but did require the gamma-subunit. Using chimaeric molecules, we show that gamma-chain contains a signal for receptor internalization and that the mutation of either of the two tyrosine residues present in its cytoplasmic domain prevents efficient internalization and antigen presentation of immune complexes. Thus, associated chains and their tyrosine-containing motif are not exclusively involved in cell activation, but also determine multimeric receptor internalization.