Retroviral transfer of the p16INK4a cDNA inhibits C6 glioma formation in Wistar rats

BACKGROUND: The p16INK4A gene product halts cell proliferation by preventing phosphorylation of the Rb protein. The p16INK4a gene is often deleted in human glioblastoma multiforme, contributing to unchecked Rb phosphorylation and rapid cell division. We show here that transduction of the human p16INK4a cDNA using the pCL retroviral system is an efficient means of stopping the proliferation of the rat-derrived glioma cell line, C6, both in tissue culture and in an animal model. C6 cells were transduced with pCL retrovirus encoding the p16INK4a, p53, or Rb genes. These cells were analyzed by a colony formation assay. Expression of p16INK4a was confirmed by immunohistochemistry and Western blot analysis. The altered morphology of the p16-expressing cells was further characterized by the senescence-associated beta-galactosidase assay. C6 cells infected ex vivo were implanted by stereotaxic injection in order to assess tumor formation. RESULTS: The p16INK4a gene arrested C6 cells more efficiently than either p53 or Rb. Continued studies with the p16INK4a gene revealed that a large portion of infected cells expressed the p16INK4a protein and the morphology of these cells was altered. The enlarged, flat, and bi-polar shape indicated a senescence-like state, confirmed by the senescence-associated beta-galactosidase assay. The animal model revealed that cells infected with the pCLp16 virus did not form tumors. CONCLUSION: Our results show that retrovirus mediated transfer of p16INK4a halts glioma formation in a rat model. These results corroborate the idea that retrovirus-mediated transfer of the p16INK4a gene may be an effective means to arrest human glioma and glioblastoma.     

Unraveling the Phylogenetic Relationships of the Eccoptochilinae,an Enigmatic Array of Ordovician Cheirurid Trilobites

The Cheiruridae are a diverse group of trilobites and several subfamilies within the clade have been the focus of recent phylogenetic studies. This paper focuses on the relationships of one of those subfamilies, the Ordovician Eccoptochilinae. We analyze sixteen species from six genera within the traditionally defined group, using the pilekiid Anacheirurus frederici as an outgroup. To assess the monophyly of the Eccoptochilinae seven sphaerexochine species, Kawina arnoldi, Sphaerexochus arenosus, S. atacius, S. latifrons, S. mirus, S. parvus, and S. scabridus were included in the analysis as well. The results of this analysis show that the genus Eccoptochile represents a paraphyletic grade and species traditionally assigned to Parasphaerexochus and Skelipyx plot within Pseudosphaerexochus. Also, representative species of Sphaerexochinae plot within the traditionally defined Eccoptochilinae, suggesting Eccoptochilinae itself is paraphyletic. To resolve this, we propose all species of Pseudosphaerexochus be placed within Sphaerexochinae and Eccoptochilinae be restricted to a monotypic Eccoptochile clavigera.     

Cathepsin-mediated Necrosis Controls the Adaptive Immune Response by Th2 (T helper type 2)-associated Adjuvants

Immunologic adjuvants are critical components of vaccines, but it remains unclear how prototypical adjuvants enhance the adaptive immune response. Recent studies have shown that necrotic cells could trigger an immune response. Although most adjuvants have been shown to be cytotoxic, this activity has traditionally been considered a side effect. We set out to test the role of adjuvant-mediated cell death in immunity and found that alum, the most commonly used adjuvant worldwide, triggers a novel form of cell death in myeloid leukocytes characterized by cathepsin-dependent lysosome-disruption. We demonstrated that direct lysosome-permeabilization with a soluble peptide, Leu-Leu-OMe, mimics the alum-like form of necrotic cell death in terms of cathepsin dependence and cell-type specificity. Using a combination of a haploid genetic screen and cathepsin-deficient cells, we identified specific cathepsins that control lysosome-mediated necrosis. We identified cathepsin C as critical for Leu-Leu-OMe-induced cell death, whereas cathepsins B and S were required for alum-mediated necrosis. Consistent with a role of necrotic cell death in adjuvant effects, Leu-Leu-OMe replicated an alum-like immune response in vivo, characterized by dendritic cell activation, granulocyte recruitment, and production of Th2-associated antibodies. Strikingly, cathepsin C deficiency not only blocked Leu-Leu-OMe-mediated necrosis but also impaired Leu-Leu-OMe-enhanced immunity. Together our findings suggest that necrotic cell death is a powerful mediator of a Th2-associated immune response.     

Cytosolic thioredoxin reductase 1 is required for correct disulfide formation in the ER

Folding of proteins entering the secretory pathway in mammalian cells frequently requires the insertion of disulfide bonds. Disulfide insertion can result in covalent linkages found in the native structure as well as those that are not, so‐called non‐native disulfides. The pathways for disulfide formation are well characterized, but our understanding of how non‐native disulfides are reduced so that the correct or native disulfides can form is poor. Here, we use a novel assay to demonstrate that the reduction in non‐native disulfides requires NADPH as the ultimate electron donor, and a robust cytosolic thioredoxin system, driven by thioredoxin reductase 1 (TrxR1 or TXNRD1). Inhibition of this reductive pathway prevents the correct folding and secretion of proteins that are known to form non‐native disulfides during their folding. Hence, we have shown for the first time that mammalian cells have a pathway for transferring reducing equivalents from the cytosol to the ER, which is required to ensure correct disulfide formation in proteins entering the secretory pathway.     

A chemical-genetic screen reveals a mechanism of resistance to PI3K inhibitors in cancer

Linking the molecular aberrations of cancer to drug responses could guide treatment choice and identify new therapeutic applications. However, there has been no systematic approach for analyzing gene-drug interactions in human cells. Here we establish a multiplexed assay to study the cellular fitness of a panel of engineered isogenic cancer cells in response to a collection of drugs, enabling the systematic analysis of thousands of gene-drug interactions. Applying this approach to breast cancer revealed various synthetic-lethal interactions and drug-resistance mechanisms, some of which were known, thereby validating the method. NOTCH pathway activation, which occurs frequently in breast cancer, unexpectedly conferred resistance to phosphoinositide 3-kinase (PI3K) inhibitors, which are currently undergoing clinical trials in breast cancer patients. NOTCH1 and downstream induction of c-MYC over-rode the dependency of cells on the PI3K-mTOR pathway for proliferation. These data reveal a new mechanism of resistance to PI3K inhibitors with direct clinical implications.     

Epigenetic germline inheritance in mammals: looking to the past to understand the future

Life experiences can induce epigenetic changes in mammalian germ cells, which can influence the developmental trajectory of the offspring and impact health and disease across generations. While this concept of epigenetic germline inheritance has long been met with skepticism, evidence in support of this route of information transfer is now overwhelming, and some key mechanisms underlying germline transmission of acquired information are emerging. This review focuses specifically on sperm RNAs as causal vectors of inheritance. We examine how they might become altered in the germline, and how different classes of sperm RNAs might interact with other epimodifications in germ cells or in the zygote. We integrate the latest findings with earlier pioneering work in this field, point out major questions and challenges, and suggest how new experiments could address them.     

Sequence differences between alpha1C and alpha1S Ca2+ channel subunits reveal structural determinants of a guarded and modulated benzothiazepine receptor

The molecular basis of the Ca2+ channel block by (+)-cis-diltiazem was studied in class A/L-type chimeras and mutant alpha1C-a Ca2+ channels. Chimeras consisted of either rabbit heart (alpha1C-a) or carp skeletal muscle (alpha1S) sequence in transmembrane segments IIIS6, IVS6, and adjacent S5-S6 linkers. Only chimeras containing sequences from alpha1C-a were efficiently blocked by (+)-cis-diltiazem, whereas the phenylalkylamine (-)-gallopamil efficiently blocked both constructs. Carp skeletal muscle and rabbit heart Ca2+ channel alpha1 subunits differ with respect to two nonconserved amino acids in segments IVS6. Transfer of a single leucine (Leu1383, located at the extracellular mouth of the pore) from IVS6 alpha1C-a to IVS6 of alpha1S significantly increased the (+)-cis-diltiazem sensitivity of the corresponding mutant L1383I. An analysis of the role of the two heterologous amino acids in a L-type alpha1 subunit revealed that corresponding amino acids in position 1487 (outer channel mouth) determine recovery of resting Ca2+ channels from block by (+)-cis-diltiazem. The second heterologous amino acid in position 1504 of segment IVS6 (inner channel mouth) was identified as crucial inactivation determinant of L-type Ca2+ channels. This residue simultaneously modulates drug binding during membrane depolarization. Our study provides the first evidence for a guarded and modulated benzothiazepine receptor on L-type channels.