Citations

Background— Excitation-contraction coupling in striated muscle requires proper communication of plasmalemmal voltage-activated Ca 2+ channels and Ca 2+ release channels on sarcoplasmic reticulum within junctional membrane complexes. Although previous studies revealed a loss of junctional membrane complexes and embryonic lethality in germ-line junctophilin-2 (JPH2) knockout mice, it has remained unclear whether JPH2 plays an essential role in junctional membrane complex formation and the Ca 2+ -induced Ca 2+ release process in the heart. Our recent work demonstrated loss-of-function mutations in JPH2 in patients with hypertrophic cardiomyopathy. Methods and Results— To elucidate the role of JPH2 in the heart, we developed a novel approach to conditionally reduce JPH2 protein levels using RNA interference. Cardiac-specific JPH2 knockdown resulted in impaired cardiac contractility, which caused heart failure and increased mortality. JPH2 deficiency resulted in loss of excitation-contraction coupling gain, precipitated by a reduction in the number of junctional membrane complexes and increased variability in the plasmalemma–sarcoplasmic reticulum distance. Conclusions— Loss of JPH2 had profound effects on Ca 2+ release channel inactivation, suggesting a novel functional role for JPH2 in regulating intracellular Ca 2+ release channels in cardiac myocytes. Thus, our novel approach of cardiac-specific short hairpin RNA–mediated knockdown of junctophilin-2 has uncovered a critical role for junctophilin in intracellular Ca 2+ release in the heart.

AbstractA 55-year-old man presented with fever, night sweats, and weight loss of about 20 lbs. in the prior 6 months. Physical examination revealed multiple cervical, axillary, and inguinal lymphadenopathy. The spleen was enlarged. A complete blood count revealed leukocytosis with absolute lymphocytosis: 30,000/μL. Peripheral blood-flow cytometric analysis showed a clonal lymphocyte population with immunophenotypes positive for CD5, CD20(dim), and monotypic kappa light chain. Fluorescence in situ hybridization (FISH) analysis revealed del(11q22.3), but negative for t(11:14). What should be used to treat his chronic lymphocytic leukemia (CLL) disease?

PurposeLenalidomide is an oral immunomodulatory drug with multiple effects on the immune system and tumor cell microenvironment leading to inhibition of malignant cell growth. Based on encouraging reports of lenalidomide in relapsed and refractory chronic lymphocytic leukemia (CLL), we investigated the first-line use of single-agent lenalidomide in CLL.Patients and MethodsUsing a starting dose of lenalidomide 10 mg/d for 21 days of a 28-day cycle and weekly 5-mg dose escalations to a target of 25 mg, we encountered severe toxicities (tumor lysis, fatal sepsis) in the first two patients enrolled. The study was halted and the protocol amended to a more conservative regimen: starting dose of lenalidomide 2.5 mg with monthly escalations to a target dose of 10 mg, and extended tumor lysis prophylaxis and monitoring. Gene expression profiles from patient samples before and after 7 days of lenalidomide were performed.ResultsTwenty-five patients were enrolled on the amended protocol. No further tumor lysis events were reported. Tumor flare was common (88%) but mild. Grade 3 to 4 neutropenia occurred in 72% of patients, with only five episodes of febrile neutropenia. The overall response rate was 56% (no complete responses). Although rapid peripheral lymphocyte reductions were observed, rebound lymphocytoses during the week off-therapy were common. Lenalidomide-induced molecular changes enriched for cytoskeletal and immune-related genes were identified.ConclusionLenalidomide is clinically active as first-line CLL therapy and is well-tolerated if a conservative approach with slow dose escalation is used. A lenalidomide-induced molecular signature provides insights into its immunomodulatory mechanisms of action in CLL.

Paracrine signaling between cholangiocytes and stromal cells regulates biliary remodeling. Cholangiocytes have neuroepithelial characteristics and serotonin receptor agonists inhibit their growth, but whether they are capable of serotonin biosynthesis is unknown. We hypothesized that cholangiocytes synthesize serotonin and that cross talk between liver myofibroblasts (MF) and cholangiocytes regulates this process to influence biliary remodeling. Transwell cultures of cholangiocytes ± MF, and tryptophan hydroxylase-2 knockin (TPH2KI) mice with an inactivating mutation of the neuronal tryptophan hydroxylase (TPH) isoform, TPH2, were evaluated. Results in the cell culture models confirm that cholangiocytes have serotonin receptors and demonstrate for the first time that these cells express TPH2 and produce serotonin, which autoinhibits their growth but stimulates MF production of TGF-β1. Increased TGF-β1, in turn, counteracts autocrine inhibition of cholangiocyte growth by repressing cholangiocyte TPH2 expression. Studies of TPH2KI mice confirm that TPH2-mediated production of serotonin plays an important role in remodeling damaged bile ducts because mice with decreased TPH2 function have reduced biliary serotonin levels and exhibit excessive cholangiocyte proliferation, accumulation of aberrant ductules and liver progenitors, and increased liver fibrosis after bile duct ligation. This new evidence that cholangiocytes express the so-called neuronal isoform of TPH, synthesize serotonin de novo, and deploy serotonin as an autocrine/paracrine signal to regulate regeneration of the biliary tree complements earlier work that revealed that passive release of serotonin from platelets stimulates hepatocyte proliferation. Given the prevalent use of serotonin-modulating drugs, these findings have potentially important implications for recovery from various types of liver damage.

The spindle assembly checkpoint (SAC) is essential for proper sister chromatid segregation. Defects in this checkpoint can lead to chromosome missegregation and aneuploidy. An increasing body of evidence suggests that aneuploidy can play a causal role in tumorigenesis. However, mutant mice that are prone to aneuploidy have only mild tumor phenotypes, suggesting that there are limiting factors in the aneuploidy-induced tumorigenesis. Here we provide evidence that p53 is such a limiting factor. We show that aneuploidy activates p53 and that loss of p53 drastically accelerates tumor development in two independent aneuploidy models. The p53 activation depends on the ataxia-telangiectasia mutated (ATM) gene product and increased levels of reactive oxygen species. Thus, the ATM-p53 pathway safeguards not only DNA damage but also aneuploidy.

Abstract Malignant cells are capable of influencing the microenvironment in a manner that facilitates tumor cell survival. Bidirectional crosstalk between chronic lymphocytic leukemic (CLL) cells and marrow-derived mesenchymal stromal cells (MSCs) activates both cell types. In this study, we observed that the conditioned medium (CM) obtained from CLL cells was able to induce Akt activation in MSC. Subsequent studies investigated the mechanism of MSC activation mediated by CLL-CM. Platelet-derived growth factor receptors (PDGFRs) were selectively activated in MSCs by CLL-CM and found to be critical receptors for CLL-CM–driven MSC proliferation and MSC Akt activation. The known ligands of PDGFR, platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF), were detected in CLL-CM, but PDGF was the predominant ligand involved in the CM-mediated PDGFR activation. Both PDGF and VEGF were found to be elevated in the plasma of CLL patients with a positive association for high-risk factors and more advanced stage. Finally, we demonstrated that PDGF induced MSC VEGF production through a phosphatidylinositol 3-kinase (PI3K)–dependent mechanism. These results show that PDGF-PDGFR signaling influences at least the MSC in the microenvironment of CLL and may play a role in the induction of an angiogenic switch known to be permissive for disease progression.

Abstract This study represents a multiplex cytokine analysis of serum from a 10-month randomized, controlled trial of 238 subjects that investigated the effects of selenomethionine and/or celecoxib in subjects with mild or moderate esophageal squamous dysplasia. The original chemoprevention study found that, among those with mild dysplasia, selenomethionine treatment favorably altered dysplasia grade. The current analysis found that selenomethionine downregulated interleukin (IL)-2 by 9% (P = 0.04), whereas celecoxib downregulated IL-7 by 11% (P = 0.006) and upregulated IL-13 by 17% (P = 0.008). In addition, an increase in IL-7 tertile from baseline to t10 was significantly associated with an increase in dysplasia grade, both overall [odds ratio (OR), 1.47; P = 0.03] and among those with mild dysplasia at t0 (OR, 2.53; P = 0.001). An increase in IL-2 tertile from baseline to t10 was also nonsignificantly associated with worsening dysplasia for all participants (OR, 1.32; P = 0.098) and significantly associated with worsening dysplasia among those with mild dysplasia at baseline (OR, 2.0; P = 0.01). The association of increased IL-2 with worsening dysplasia remained significant in those on selenomethionine treatment who began the trial with mild dysplasia (OR, 2.52; P = 0.03). The current study shows that selenomethionine supplementation decreased serum IL-2 levels, whereas celecoxib treatment decreased IL-7 levels and increased IL-13 levels during a 10-month randomized chemoprevention trial. An increase in IL-2 or IL-7 was associated with increased severity of dysplasia over the course of the trial, especially in those who began the trial with mild dysplasia. The favorable effect of selenomethionine on esophageal dysplasia in the original trial may have been mediated in part by its effect in reducing the levels of IL-2. Cancer Prev Res; 3(7); 810–7. ©2010 AACR.

Intraflagellar transport (IFT) machinery mediates the bidirectional movement of cargos that are required for the assembly and maintenance of cilia. However, little is known about how IFT is regulated in vivo. In this study, we show that the small guanosine triphosphatase (GTPase) adenosine diphosphate ribosylation factor–like protein 13 (ARL-13) encoded by the Caenorhabditis elegans homologue of the human Joubert syndrome causal gene ARL13B, localizes exclusively to the doublet segment of the cilium. arl-13 mutants have shortened cilia with various ultrastructural deformities and a disrupted association between IFT subcomplexes A and B. Intriguingly, depletion of ARL-3, another ciliary small GTPase, partially suppresses ciliogenesis defects in arl-13 mutants by indirectly restoring binding between IFT subcomplexes A and B. Rescue of arl-13 mutants by ARL-3 depletion is mediated by an HDAC6 deacetylase-dependent pathway. Thus, we propose that two conserved small GTPases, ARL-13 and ARL-3, coordinate to regulate IFT and that perturbing this balance results in cilia deformation.

PurposeBiliary cancers overexpress epidermal growth factor receptor (EGFR), and angiogenesis has been correlated with poor outcome. Erlotinib, an EGFR tyrosine kinase inhibitor, and bevacizumab, a vascular endothelial growth factor (VEGF) inhibitor have each been shown to have activity in biliary cancer. The primary objective of this study was to evaluate the response rate by Response Evaluation Criteria in Solid Tumors (RECIST). Secondary end points included overall survival (OS), time to progression (TTP), VEGF levels, and molecular studies of EGFR and k-ras.Patients and MethodsEligible patients had advanced cholangiocarcinoma or gallbladder cancer. Patients were treated with bevacizumab 5 mg/kg intravenously on days 1 and 15 and erlotinib 150 mg by mouth daily on days 1 through 28. Responses were evaluated by RECIST. VEGF levels were collected, and samples were analyzed for EGFR mutation by polymerase chain reaction.ResultsFifty-three eligible patients were enrolled at eight sites. Of 49 evaluable patients, six (12%; 95% CI, 6% to 27%) had a confirmed partial response. Stable disease was documented in another 25 patients (51%). Rash was the most common grade 3 toxicity. Four patients had grade 4 toxicities. Median OS was 9.9 months, and TTP was 4.4 months. Low repeats (< 16) in EGFR intron 1 polymorphism and G>G k-ras Q38 genotype (wild type) were associated with improved outcomes.ConclusionCombination chemotherapy with bevacizumab and erlotinib showed clinical activity with infrequent grade 3 and 4 adverse effects in patients with advanced biliary cancers. On the basis of preliminary molecular analysis, presence of a k-ras mutation may alter erlotinib efficacy. The combination of bevacizumab and erlotinib may be a therapeutic alternative in patients with advanced biliary cancer.

Abstract The low efficiency of reprogramming and genomic integration of virus vectors obscure the potential application of induced pluripotent stem (iPS) cells; therefore, identification of chemicals and cooperative factors that may improve the generation of iPS cells will be of great value. Moreover, the cellular mechanisms that limit the reprogramming efficiency need to be investigated. Through screening a chemical library, we found that two chemicals reported to upregulate E-cadherin considerably increase the reprogramming efficiency. Further study of the process indicated that E-cadherin is upregulated during reprogramming and the established iPS cells possess E-cadherin-mediated cell–cell contact, morphologically indistinguishable from embryonic stem (ES) cells. Our experiments also demonstrate that overexpression of E-cadherin significantly enhances reprogramming efficiency, whereas knockdown of endogenous E-cadherin reduces the efficiency. Consistently, abrogation of cell–cell contact by the inhibitory peptide or the neutralizing antibody against the extracellular domain of E-cadherin compromises iPS cell generation. Further mechanistic study reveals that adhesive binding activity of E-cadherin is required. Our results highlight the critical role of E-cadherin-mediated cell–cell contact in reprogramming and suggest new routes for more efficient iPS cell generation.