Genome-scale functional genomics identify genes preferentially essential for multiple myeloma cells compared to other neoplasias.

Clinical progress in multiple myeloma (MM), an incurable plasma cell (PC) neoplasia, has been driven by therapies that have limited applications beyond MM/PC neoplasias and do not target specific oncogenic mutations in MM. Instead, these agents target pathways critical for PC biology yet largely dispensable for malignant or normal cells of most other lineages. Here we systematically characterized the lineage-preferential molecular dependencies of MM through genome-scale clustered regularly interspaced short palindromic repeats (CRISPR) studies in 19 MM versus hundreds of non-MM lines and identified 116 genes whose disruption more significantly affects MM cell fitness compared with other malignancies. These genes, some known, others not previously linked to MM, encode transcription factors, chromatin modifiers, endoplasmic reticulum components, metabolic regulators or signaling molecules. Most of these genes are not among the top amplified, overexpressed or mutated in MM. Functional genomics approaches thus define new therapeutic targets in MM not readily identifiable by standard genomic, transcriptional or epigenetic profiling analyses.

Reversible ON- and OFF-switch chimeric antigen receptors controlled by lenalidomide.

Cell-based therapies are emerging as effective agents against cancer and other diseases. As autonomous "living drugs," these therapies lack precise control. Chimeric antigen receptor (CAR) T cells effectively target hematologic malignancies but can proliferate rapidly and cause toxicity. We developed ON and OFF switches for CAR T cells using the clinically approved drug lenalidomide, which mediates the proteasomal degradation of several target proteins by inducing interactions between the CRL4CRBN E3 ubiquitin ligase and a C2H2 zinc finger degron motif. We performed a systematic screen to identify "super-degron" tags with enhanced sensitivity to lenalidomide-induced degradation and used these degradable tags to generate OFF-switch degradable CARs. To create an ON switch, we engineered a lenalidomide-inducible dimerization system and developed split CARs that required both lenalidomide and target antigen for activation. Subtherapeutic lenalidomide concentrations controlled the effector functions of ON- and OFF-switch CAR T cells. In vivo, ON-switch split CARs demonstrated lenalidomide-dependent antitumor activity, and OFF-switch degradable CARs were depleted by drug treatment to limit inflammatory cytokine production while retaining antitumor efficacy. Together, the data showed that these lenalidomide-gated switches are rapid, reversible, and clinically suitable systems to control transgene function in diverse gene- and cell-based therapies.

Functional Genomics Identify Distinct and Overlapping Genes Mediating Resistance to Different Classes of Heterobifunctional Degraders of Oncoproteins.

Heterobifunctional proteolysis-targeting chimeric compounds leverage the activity of E3 ligases to induce degradation of target oncoproteins and exhibit potent preclinical antitumor activity. To dissect the mechanisms regulating tumor cell sensitivity to different classes of pharmacological "degraders" of oncoproteins, we performed genome-scale CRISPR-Cas9-based gene editing studies. We observed that myeloma cell resistance to degraders of different targets (BET bromodomain proteins, CDK9) and operating through CRBN (degronimids) or VHL is primarily mediated by prevention of, rather than adaptation to, breakdown of the target oncoprotein; and this involves loss of function of the cognate E3 ligase or interactors/regulators of the respective cullin-RING ligase (CRL) complex. The substantial gene-level differences for resistance mechanisms to CRBN- versus VHL-based degraders explains mechanistically the lack of cross-resistance with sequential administration of these two degrader classes. Development of degraders leveraging more diverse E3 ligases/CRLs may facilitate sequential/alternating versus combined uses of these agents toward potentially delaying or preventing resistance.

The CDK inhibitor CR8 acts as a molecular glue degrader that depletes cyclin K.

Molecular glue compounds induce protein-protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation1. Unlike traditional enzyme inhibitors, these molecular glue degraders act substoichiometrically to catalyse the rapid depletion of previously inaccessible targets2. They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously. Here, through systematically mining databases for correlations between the cytotoxicity of 4,518 clinical and preclinical small molecules and the expression levels of E3 ligase components across hundreds of human cancer cell lines3-5, we identify CR8-a cyclin-dependent kinase (CDK) inhibitor6-as a compound that acts as a molecular glue degrader. The CDK-bound form of CR8 has a solvent-exposed pyridyl moiety that induces the formation of a complex between CDK12-cyclin K and the CUL4 adaptor protein DDB1, bypassing the requirement for a substrate receptor and presenting cyclin K for ubiquitination and degradation. Our studies demonstrate that chemical alteration of surface-exposed moieties can confer gain-of-function glue properties to an inhibitor, and we propose this as a broader strategy through which target-binding molecules could be converted into molecular glues.

Patterns of substrate affinity, competition, and degradation kinetics underlie biological activity of thalidomide analogs.

Pharmacologic agents that modulate ubiquitin ligase activity to induce protein degradation are a major new class of therapeutic agents, active in a number of hematologic malignancies. However, we currently have a limited understanding of the determinants of activity of these agents and how resistance develops. We developed and used a novel quantitative, targeted mass spectrometry (MS) assay to determine the relative activities, kinetics, and cell-type specificity of thalidomide and 4 analogs, all but 1 of which are in clinical use or clinical trials for hematologic malignancies. Thalidomide analogs bind the CRL4CRBN ubiquitin ligase and induce degradation of particular proteins, but each of the molecules studied has distinct patterns of substrate specificity that likely underlie the clinical activity and toxicities of each drug. Our results demonstrate that the activity of molecules that induce protein degradation depends on the strength of ligase-substrate interaction in the presence of drug, the levels of the ubiquitin ligase, and the expression level of competing substrates. These findings highlight a novel mechanism of resistance to this class of drugs mediated by competition between substrates for access to a limiting pool of the ubiquitin ligase. We demonstrate that increased expression of a nonessential substrate can lead to decreased degradation of other substrates that are critical for antineoplastic activity of the drug, resulting in drug resistance. These studies provide general rules that govern drug-dependent substrate degradation and key differences between thalidomide analog activity in vitro and in vivo.

Enhancing oxygen delivery to ovarian follicles by three different methods markedly improves growth in serum-containing culture medium.

Invitro ovarian follicle culture systems are routinely used to study folliculogenesis and may provide solutions for infertility. Mouse follicles are typically cultured in standard gas-impermeable culture plates under gas phase oxygen concentrations of 5% or 20% (v/v). There is evidence that these conditions may not provide adequate oxygenation for follicles cultured as non-attached intact units in medium supplemented with serum and high levels of FSH. Three different methods of enhancing follicle oxygenation were investigated in this study: increasing the gas phase oxygen concentration, inverting the culture plates and using gas-permeable culture plates. Follicles cultured under 40% O2 were significantly larger (P P P 2 . These effects were associated with reduced secretion of vascular endothelial growth factor (P P P invivo -matured follicles (~500µm in diameter). Such follicular development is not possible under hypoxic conditions.

Mechanism of luminal ATP activated chloride secretion in a polarized epithelium.

There are both secretory and absorptive pathways working in tandem to support ionic movement driving fluid secretion across epithelia. The mechanisms exerting control of fluid secretion in the oviduct is yet to be fully determined. This study explored the role of apical or luminal extracellular ATP (ATPe)-stimulated ion transport in an oviduct epithelium model, using the Ussing chamber short-circuit current (Isc) technique. Basal Isc in oviduct epithelium in response to apical ATPe comprises both chloride secretion and sodium absorption and has distinct temporal phases. A rapid transient peak followed by a sustained small increase above baseline. Both phases of the apical ATPe Isc response are sensitive to anion (HCO3-, Cl-) and cation (Na+) replacement. Additionally, the role of apical chloride channels, basolateral potassium channels and intracellular calcium in supporting the peak Isc current was confirmed. The role of ATP breakdown to adenosine resulting in the activation of P2 receptors was supported by examining the effects of non-hydrolyzable forms of ATP. A P2YR2 potency profile of ATP = UTP > ADP was generated for the apical membrane, suggesting the involvement of the P2YR2 subtype of purinoceptor. A P2X potency profile of ATP = 2MeSATP > alpha,beta-meATP > BzATP was also generated for the apical membrane. In conclusion, these results provide strong evidence that purinergic activation of apical P2YR2 promotes chloride secretion and is thus an important factor in fluid formation by the oviduct.

Defining the human C2H2 zinc finger degrome targeted by thalidomide analogs through CRBN.

The small molecules thalidomide, lenalidomide, and pomalidomide induce the ubiquitination and proteasomal degradation of the transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) by recruiting a Cys2-His2 (C2H2) zinc finger domain to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase. We screened the human C2H2 zinc finger proteome for degradation in the presence of thalidomide analogs, identifying 11 zinc finger degrons. Structural and functional characterization of the C2H2 zinc finger degrons demonstrates how diverse zinc finger domains bind the permissive drug-CRBN interface. Computational zinc finger docking and biochemical analysis predict that more than 150 zinc fingers bind the drug-CRBN complex in vitro, and we show that selective zinc finger degradation can be achieved through compound modifications. Our results provide a rationale for therapeutically targeting transcription factors that were previously considered undruggable.

Genome-wide screen identifies cullin-RING ligase machinery required for lenalidomide-dependent CRL4CRBN activity.

Lenalidomide mediates the ubiquitination and degradation of Ikaros family zinc finger protein 1 (IKZF1), IKZF3, and casein kinase 1α (CK1α) by facilitating their interaction with cereblon (CRBN), the substrate receptor for the CRL4CRBN E3 ubiquitin ligase. Through this mechanism, lenalidomide is a clinically effective treatment of multiple myeloma and myelodysplastic syndrome (MDS) with deletion of chromosome 5q [del(5q) MDS]. To identify the cellular machinery required for lenalidomide-induced CRL4CRBN activity, we performed a positive selection, genome-scale clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) screen in a lenalidomide-sensitive myeloma cell line. CRBN was the top-ranking gene, with all CRBN-targeting guide RNAs (gRNAs) ranking as the 6 highest-scoring gRNAs. A counterscreen using an IKZF3 degron reporter to assay lenalidomide-induced protein degradation highlighted regulators of cullin-RING ligase neddylation and 2 E2 ubiquitin-conjugating enzymes as necessary for efficient lenalidomide-induced protein degradation. We demonstrated that loss of UBE2M or members of the constitutive photomorphogenesis 9 (COP9) signalosome results in altered neddylation of cullin 4A and impairs lenalidomide-dependent CRL4CRBN activity. Additionally, we established that UBE2D3 and UBE2G1 play distinct roles in substrate ubiquitination by CRL4CRBN, with UBE2D3 acting to prime targets via monoubiquitination and UBE2G1 functioning to extend polyubiquitin chains with lysine 48 linkages. The validation of UBE2D3 and UBE2G1 highlights the functional capacity of CRISPR-Cas9 screening to identify E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase complex pairings. More broadly, these findings establish key proteins required for lenalidomide-dependent CRL4CRBN function in myeloma and inform potential mechanisms of drug resistance.

Corrigendum to: Hypoxia limits mouse follicle growth in vitro.

Ovarian follicle culture is useful for elucidation of factors involved in the regulation of follicular function. We examined the effects of gas phase oxygen concentration, an oil overlay, serum type and medium supplementation with FSH, insulin-transferrin-selenium (ITS) and I-ascorbic acid on cultured preantral mouse follicle growth in a spherical, non-attached follicle culture system. Follicle growth in 5% oxygen was significantly (P<0.01) inferior to growth in 20% oxygen in terms of follicle diameter. This was likely due to hypoxia, as evidenced by significantly (P<0.05) increased follicle secretion of vascular endothelial growth factor (VEGF), a marker of cell hypoxia. Follicular growth was not (P>0.05) affected by an oil overlay, ITS supplementation or serum type. Culture in medium with 5% mouse serum, 1 IU mL-1 FSH, 25 µgmL-1 l-ascorbic acid and 20% oxygen without an oil overlay supported the growth of follicles to a maximum diameter of 380 µm in 6 days. Compared with mature preovulatory mouse follicles in vivo that often have diameters >500 µm within the same time frame, in vitro-grown follicles clearly exhibit limited growth. Thus, adequate oxygenation is an essential factor in the process of optimising follicle growth.

Multiplex CRISPR/Cas9-Based Genome Editing in Human Hematopoietic Stem Cells Models Clonal Hematopoiesis and Myeloid Neoplasia.

Hematologic malignancies are driven by combinations of genetic lesions that have been difficult to model in human cells. We used CRISPR/Cas9 genome engineering of primary adult and umbilical cord blood CD34+ human hematopoietic stem and progenitor cells (HSPCs), the cells of origin for myeloid pre-malignant and malignant diseases, followed by transplantation into immunodeficient mice to generate genetic models of clonal hematopoiesis and neoplasia. Human hematopoietic cells bearing mutations in combinations of genes, including cohesin complex genes, observed in myeloid malignancies generated immunophenotypically defined neoplastic clones capable of long-term, multi-lineage reconstitution and serial transplantation. Employing these models to investigate therapeutic efficacy, we found that TET2 and cohesin-mutated hematopoietic cells were sensitive to azacitidine treatment. These findings demonstrate the potential for generating genetically defined models of human myeloid diseases, and they are suitable for examining the biological consequences of somatic mutations and the testing of therapeutic agents.

Reporting of sex and gender in randomized controlled trials in Canada: a cross-sectional methods study.

BACKGROUND: Accurate reporting on sex and gender in health research is integral to ensuring that health interventions are safe and effective. In Canada and internationally, governments, research organizations, journal editors, and health agencies have called for more inclusive research, provision of sex-disaggregated data, and the integration of sex and gender analysis throughout the research process. Sex and gender analysis is generally defined as an approach for considering how and why different subpopulations (e.g., of diverse genders, ages, and social locations) may experience health conditions and interventions in different or similar ways.The objective of this study was to assess the extent and nature of reporting about sex and/or gender, including whether sex and gender analysis (SGA) was carried out in a sample of Canadian randomized controlled trials (RCTs) with human participants. METHODS: We searched MEDLINE from 01 January 2013 to 23 July 2014 using a validated filter for identification of RCTs, combined with terms related to Canada. Two reviewers screened the search results to identify the first 100 RCTs that were either identified in the trial publication as funded by a Canadian organization or which had a first or last author based in Canada. Data were independently extracted by two people from 10% of the RCTs during an initial training period; once agreement was reached on this sample, the remainder of the data extraction was completed by one person and verified by a second. RESULTS: The search yielded 1433 records. We screened 256 records to identify 100 RCTs which met our eligibility criteria. The median sample size of the RCTs was 107 participants (range 12-6085). While 98% of studies described the demographic composition of their participants by sex, only 6% conducted a subgroup analysis across sex and 4% reported sex-disaggregated data. No article defined "sex" and/or "gender." No publication carried out a comprehensive sex and gender analysis. CONCLUSIONS: Findings highlight poor uptake of sex and gender considerations in the Canadian RCT context and underscore the need for better articulated guidance on sex and gender analysis to improve reporting of evidence, inform policy development, and guide future research.

Hypoxia limits mouse follicle growth in vitro.

Ovarian follicle culture is useful for elucidation of factors involved in the regulation of follicular function. We examined the effects of gas phase oxygen concentration, an oil overlay, serum type and medium supplementation with FSH, insulin-transferrin-selenium (ITS) and l-ascorbic acid on cultured preantral mouse follicle growth in a spherical, non-attached follicle culture system. Follicle growth in 5% oxygen was significantly (P < 0.01) inferior to growth in 20% oxygen in terms of follicle diameter. This was likely due to hypoxia, as evidenced by significantly (P < 0.05) increased follicle secretion of vascular endothelial growth factor (VEGF), a marker of cell hypoxia. Follicular growth was not (P > 0.05) affected by an oil overlay, ITS supplementation or serum type. Culture in medium with 5% mouse serum, 1 IU mL-1 FSH, 25 µg mL-1 l-ascorbic acid and 20% oxygen without an oil overlay supported the growth of follicles to a maximum diameter of 380 µm in 6 days. Compared with mature preovulatory mouse follicles in vivo that often have diameters >500 µm within the same time frame, in vitro-grown follicles clearly exhibit limited growth. Thus, adequate oxygenation is an essential factor in the process of optimising follicle growth.

The past, present and future in scaffold-based tendon treatments.

Tendon injuries represent a significant clinical burden on healthcare systems worldwide. As the human population ages and the life expectancy increases, tendon injuries will become more prevalent, especially among young individuals with long life ahead of them. Advancements in engineering, chemistry and biology have made available an array of three-dimensional scaffold-based intervention strategies, natural or synthetic in origin. Further, functionalisation strategies, based on biophysical, biochemical and biological cues, offer control over cellular functions; localisation and sustained release of therapeutics/biologics; and the ability to positively interact with the host to promote repair and regeneration. Herein, we critically discuss current therapies and emerging technologies that aim to transform tendon treatments in the years to come.

Somatic mutation as a mechanism of Wnt/ß-catenin pathway activation in CLL.

One major goal of cancer genome sequencing is to identify key genes and pathways that drive tumor pathogenesis. Although many studies have identified candidate driver genes based on recurrence of mutations in individual genes, subsets of genes with nonrecurrent mutations may also be defined as putative drivers if they affect a single biological pathway. In this fashion, we previously identified Wnt signaling as significantly mutated through large-scale massively parallel DNA sequencing of chronic lymphocytic leukemia (CLL). Here, we use a novel method of biomolecule delivery, vertical silicon nanowires, to efficiently introduce small interfering RNAs into CLL cells, and interrogate the effects of 8 of 15 mutated Wnt pathway members identified across 91 CLLs. In HEK293T cells, mutations in 2 genes did not generate functional changes, 3 led to dysregulated pathway activation, and 3 led to further activation or loss of repression of pathway activation. Silencing 4 of 8 mutated genes in CLL samples harboring the mutated alleles resulted in reduced viability compared with leukemia samples with wild-type alleles. We demonstrate that somatic mutations in CLL can generate dependence on this pathway for survival. These findings support the notion that nonrecurrent mutations at different nodes of the Wnt pathway can contribute to leukemogenesis.

Autologous CLL cell vaccination early after transplant induces leukemia-specific T cells.

BACKGROUND: Patients with advanced hematologic malignancies remain at risk for relapse following reduced-intensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (allo-HSCT). We conducted a prospective clinical trial to test whether vaccination with whole leukemia cells early after transplantation facilitates the expansion of leukemia-reactive T cells and thereby enhances antitumor immunity. METHODS: We enrolled 22 patients with advanced chronic lymphocytic leukemia (CLL), 18 of whom received up to 6 vaccines initiated between days 30 and 45 after transplantation. Each vaccine consisted of irradiated autologous tumor cells admixed with GM-CSF-secreting bystander cells. Serial patient PBMC samples following transplantation were collected, and the impact of vaccination on T cell activity was evaluated. RESULTS: At a median follow-up of 2.9 (range, 1-4) years, the estimated 2-year progression-free and overall survival rates of vaccinated subjects were 82% (95% CI, 54%-94%) and 88% (95% CI, 59%-97%), respectively. Although vaccination only had a modest impact on recovering T cell numbers, CD8+ T cells from vaccinated patients consistently reacted against autologous tumor, but not alloantigen-bearing recipient cells with increased secretion of the effector cytokine IFN-γ, unlike T cells from nonvaccinated CLL patients undergoing allo-HSCT. Further analysis confirmed that 17% (range, 13%-33%) of CD8+ T cell clones isolated from 4 vaccinated patients by limiting dilution of bulk tumor-reactive T cells solely reacted against CLL-associated antigens. CONCLUSION: Our studies suggest that autologous tumor cell vaccination is an effective strategy to advance long-term leukemia control following allo-HSCT. TRIAL REGISTRATION: Clinicaltrials.gov NCT00442130. FUNDING: NCI (5R21CA115043-2), NHLBI (5R01HL103532-03), and Leukemia and Lymphoma Society Translational Research Program.

Small conductance potassium channels drive ATP-activated chloride secretion in the oviduct.

The molecular mechanisms controlling fluid secretion within the oviduct have yet to be determined. As in other epithelia, both secretory and absorptive pathways are likely to work in tandem to drive appropriate ionic movement to support fluid movement across the oviduct epithelium. This study explored the role of potassium channels in basolateral extracellular ATP (ATP(e))-stimulated ion transport in bovine oviduct epithelium using the Ussing chamber short-circuit current (I(SC)) technique. Basal I(SC) in bovine oviduct epithelium comprises both chloride secretion and sodium absorption and was inhibited by treatment with basolateral K(+) channel inhibitors tetrapentlyammonium chloride (TPeA) or BaCl(2). Similarly, ATP-stimulated chloride secretion was significantly attenuated by pretreatment with BaCl(2,) tetraethylammonium (TEA), tolbutamide, and TPeA. Basolateral K(+) current, isolated using nystatin-perforation technique, was rapidly activated by ATP(e), and pretreatment of monolayers with thapsigargin or TPeA abolished this ATP-stimulated K(+) current. To further investigate the type of K(+) channel involved in the ATP response in the bovine oviduct, a number of specific Ca(2+)-activated K(+) channel inhibitors were tested on the ATP-induced ΔI(SC) in intact monolayers. Charbydotoxin, (high conductance and intermediate conductance inhibitor), or paxilline, (high conductance inhibitor) did not significantly alter the ATP(e) response. However, pretreatment with the small conductance inhibitor apamin resulted in a 60% reduction in the response to ATP(e). The presence of small conductance family member KCNN3 was confirmed by RT-PCR and immunohistochemistry. Measurements of intracellular calcium using Fura-2 spectrofluorescence imaging revealed the ability of ATP(e) to increase intracellular calcium in a phospholipase C-inositol 1,4,5-trisphosphate pathway-sensitive manner. In conclusion, these results provide strong evidence that purinergic activation of a calcium-dependent, apamin-sensitive potassium conductance is essential to promote chloride secretion and thus fluid formation in the oviduct.

SF3B1 and other novel cancer genes in chronic lymphocytic leukemia.

BACKGROUND: The somatic genetic basis of chronic lymphocytic leukemia, a common and clinically heterogeneous leukemia occurring in adults, remains poorly understood. METHODS: We obtained DNA samples from leukemia cells in 91 patients with chronic lymphocytic leukemia and performed massively parallel sequencing of 88 whole exomes and whole genomes, together with sequencing of matched germline DNA, to characterize the spectrum of somatic mutations in this disease. RESULTS: Nine genes that are mutated at significant frequencies were identified, including four with established roles in chronic lymphocytic leukemia (TP53 in 15% of patients, ATM in 9%, MYD88 in 10%, and NOTCH1 in 4%) and five with unestablished roles (SF3B1, ZMYM3, MAPK1, FBXW7, and DDX3X). SF3B1, which functions at the catalytic core of the spliceosome, was the second most frequently mutated gene (with mutations occurring in 15% of patients). SF3B1 mutations occurred primarily in tumors with deletions in chromosome 11q, which are associated with a poor prognosis in patients with chronic lymphocytic leukemia. We further discovered that tumor samples with mutations in SF3B1 had alterations in pre-messenger RNA (mRNA) splicing. CONCLUSIONS: Our study defines the landscape of somatic mutations in chronic lymphocytic leukemia and highlights pre-mRNA splicing as a critical cellular process contributing to chronic lymphocytic leukemia.

Effect of basolateral adenosine triphosphate on chloride secretion by bovine oviductal epithelium.

The composition of the fluid within the oviduct is largely determined by the secretory and absorptive activities of the oviduct epithelium. The present study explored the effects of basolateral nucleotide stimulation on ion transport in the bovine oviduct using the chamber short-circuit current technique. Basolateral application of ATP induced a rapid transient increase in ion secretion by oviduct epithelial monolayers in a concentration-dependent manner. The ATP-induced short-circuit current (I(SC)) response was preserved in the presence of amiloride, whereas it was reduced in the absence of extracellular chloride or in the presence of bumetanide. The channels underlying the chloride secretory response were identified as Ca(2+)-activated Cl(-) channels and CFTR. The ATP-induced Cl(-) secretory response was largely preserved in the absence of extracellular Ca(2+) but was significantly reduced in the presence of BAPTA-am (1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid-acetomethoxy ester), thapsigargin, or 2-APB (2-aminoethoxydiphenylborate), demonstrating an important role for intracellular Ca(2+) signaling in mediating these effects. A nucleotide potency profile of ATP = UTP (uridine triphosphate) > ADP, sensitivity to suramin, and cross-desensitization by basolateral UTP suggests that ATP exerted its effects on chloride secretion through the purinergic receptor P2Y, G protein-coupled 2, and the presence of the P2RY2 gene was confirmed by RT-PCR. These results provide strong evidence that purinergic signaling constitutes a key mechanism of regulating chloride secretion and thus fluid formation in the bovine oviduct.

Self-renewal and differentiation of mouse embryonic stem cells as measured by Oct4 expression: the role of the cAMP/PKA pathway.

In this study we examined the role of the cAMP/protein kinase A (PKA) pathway in affecting IOUD2 ES cell self-renewal and differentiation, Oct4 expression, and cell proliferation. Forskolin, the adenylate cyclase agonist, alone had no effect on ES cell self-renewal. However, when cells were treated with the differentiation-inducing agent retinoic acid, forskolin significantly promoted ES cell self-renewal. Effectively, forskolin rescued cells from a pathway of differentiation. Culturing ES cells in the presence of the phosphodiesterase inhibitor IBMX had no effect on ES cell self-renewal but did increase cell proliferation. In the presence of 100 muM IBMX without LIF, 10 muM forskolin significantly increased ES cell self-renewal. The cell permeable cAMP analog 8-Br-cAMP (1 and 5 mM) promoted ES cell differentiation in the presence of LIF, while in the absence of LIF, it promoted ES cell self-renewal. The effect of the PKA specific inhibitors H89 and KT5720 on Oct4 expression was, again, LIF-dependent. In the presence of LIF, these inhibitors decreased Oct4 expression, while they increased Oct4 expression in the absence of LIF. In general, ES cells maintained on a self-renewal pathway through the presence of LIF show little effect from altered cAMP signaling except at higher levels. However, in strict contrast, when ES cell are on a differentiation pathway through exposure to retinoic acid or the removal of LIF, altering cAMP levels can rescue the self-renewal process promoting Oct4 expression. This study clearly shows that the cAMP/PKA pathway plays a role in ES cell self-renewal pathways.