Activation of phospholipase D by the LHRH receptor and associated mechanisms

The αT3 -1 gonadotroph- derived cell line was used as a model system to study intracellular signalling systems associated with the LHRH receptor, with particular interest in phospholipase D (PLD) and mechanisms involved in its regulation, such as protein kinase C (PKC) and non -receptor tyrosine kinases.Phospholipase D activation in aT3 -1 cells proceeded after a delay when stimulated by LHRH, or by the phorbol ester, phorbol 12,13 -dibutyrate (PDBu). LHRH -stimulated PLD activity was fully resistant to desensitisation over a 40 min time course. The Cat+ ionophore, ionomycin was unable to stimulate PLD activity. LHRH -stimulated PLD activity was inhibited by PKC downregulation or by bisindolylmaleimide PKC inhibitors at an approximately 8 -fold higher concentration than that seen for the PDBu- stimulated PLD activity. Another PKC inhibitor, H7, inhibited PDBu- stimulated PLD activity in a manner that indicated multiple components to the inhibition and inhibited LHRH -stimulated PLD activity with a low potency. This is consistent with the possibility that one mediator of the LHRH response is a form of PKC which is relatively resistant to certain PKC inhibitors and insensitive to phorbol esters, or that some other unknown kinase is involved. The tyrosine kinase inhibitors, lavendustin A and genistein were able to selectively inhibit the LHRH -stimulated PLD activity. Phospholipase D activation stimulated by LHRH was insensitive to pertussis toxin pretreatment, indicating that the heterotrimeric G- proteins, Gi and Go are not involved.LHRH induced tyrosine phosphorylation of a number of proteins in aT3 -1 cells; a similar phosphorylation profile was also produced by stimulation with PDBu. Tyrosine phosphorylation of proteins in aT3 -1 cells 4 was reduced by ionomycin. Two members of the src- family of non -receptor tyrosine kinases, src and fyn, were detected at significant levels in these cells by immunoblot. Using immunoprecipitation techniques we have shown that the enzymic activity of fyn is increased within 10 min of LHRH stimulation.The LHRH -stimulated PLD activity could not be detectably enhanced by a conditioning preincubation with LHRH, such as brings about the priming effect of LHRH ( a phenomenon of cellular sensitisation elicited by LHRH receptor activation). Removal of steroids from the growth medium severely impaired the PLD activation due to LHRH; this response was largely restored by addition of oestradiol -1713 but not progesterone. This may have implications for the LHRH priming effect in vivo, which is dependent upon the level of circulating steroid hormones.The murine LHRH receptor was transiently expressed in COS 7 cells from the cDNA in a pcDNA vector using a DEAE dextran transfection procedure. It was demonstrated that LHRH -stimulated PLD activation displayed similar sensitivity to the bisindolylmaleimide PKC inhibitor, Ro 31 -8220, to that seen in aT3 -1 cells. It was not however sensitive to tyrosine kinase inhibitors or pertussis toxin. Using site -directed mutagenesis of residues 87 and 318 in the transmembrane 2 and 7 domains of the LHRH receptor (which represents an inversion of the consensus motif in the G- protein coupled receptor family), it was found that mutation of Asp 318 to Asn was alone sufficient to produce a rapidly desensitising PLD response to LHRH, in contrast to the non -desensitising wild type

Next Generation Mapping of Enological Traits in an F2 Interspecific Grapevine Hybrid Family

In winegrapes (Vitis spp.), fruit quality traits such as berry color, total soluble solids content (SS), malic acid content (MA), and yeast assimilable nitrogen (YAN) affect fermentation or wine quality, and are important traits in selecting new hybrid winegrape cultivars. Given the high genetic diversity and heterozygosity of Vitis species and their tendency to exhibit inbreeding depression, linkage map construction and quantitative trait locus (QTL) mapping has relied on F1 families with the use of simple sequence repeat (SSR) and other markers. This study presents the construction of a genetic map by single nucleotide polymorphisms identified through genotyping-by-sequencing (GBS) technology in an F2 mapping family of 424 progeny derived from a cross between the wild species V. riparia Michx. and the interspecific hybrid winegrape cultivar, ‘Seyval’. The resulting map has 1449 markers spanning 2424 cM in genetic length across 19 linkage groups, covering 95% of the genome with an average distance between markers of 1.67 cM. Compared to an SSR map previously developed for this F2 family, these results represent an improved map covering a greater portion of the genome with higher marker density. The accuracy of the map was validated using the well-studied trait berry color. QTL affecting YAN, MA and SS related traits were detected. A joint MA and SS QTL spans a region with candidate genes involved in the malate metabolism pathway. We present an analytical pipeline for calling intercross GBS markers and a high-density linkage map for a large F2 family of the highly heterozygous Vitis genus. This study serves as a model for further genetic investigations of the molecular basis of additional unique characters of North American hybrid wine cultivars and to enhance the breeding process by marker-assisted selection. The GBS protocols for identifying intercross markers developed in this study can be adapted for other heterozygous species

MLL3 regulates the CDKN2A tumor suppressor locus in liver cancer

Mutations in genes encoding components of chromatin modifying and remodeling complexes are among the most frequently observed somatic events in human cancers. For example, missense and nonsense mutations targeting the mixed lineage leukemia family member 3 (MLL3, encoded by KMT2C) histone methyltransferase occur in a range of solid tumors, and heterozygous deletions encompassing KMT2C occur in a subset of aggressive leukemias. Although MLL3 loss can promote tumorigenesis in mice, the molecular targets and biological processes by which MLL3 suppresses tumorigenesis remain poorly characterized. Here, we combined genetic, epigenomic, and animal modeling approaches to demonstrate that one of the mechanisms by which MLL3 links chromatin remodeling to tumor suppression is by co-activating the Cdkn2a tumor suppressor locus. Disruption of Kmt2c cooperates with Myc overexpression in the development of murine hepatocellular carcinoma (HCC), in which MLL3 binding to the Cdkn2a locus is blunted, resulting in reduced H3K4 methylation and low expression levels of the locus-encoded tumor suppressors p16/Ink4a and p19/Arf. Conversely, elevated KMT2C expression increases its binding to the CDKN2A locus and co-activates gene transcription. Endogenous Kmt2c restoration reverses these chromatin and transcriptional effects and triggers Ink4a/Arf-dependent apoptosis. Underscoring the human relevance of this epistasis, we found that genomic alterations in KMT2C and CDKN2A were associated with similar transcriptional profiles in human HCC samples. These results collectively point to a new mechanism for disrupting CDKN2A activity during cancer development and, in doing so, link MLL3 to an established tumor suppressor network

Fc-Optimized Anti-CD25 Depletes Tumor-Infiltrating Regulatory T Cells and Synergizes with PD-1 Blockade to Eradicate Established Tumors

CD25 is expressed at high levels on regulatory T (Treg) cells and was initially proposed as a target for cancer immunotherapy. However, anti-CD25 antibodies have displayed limited activity against established tumors. We demonstrated that CD25 expression is largely restricted to tumor-infiltrating Treg cells in mice and humans. While existing anti-CD25 antibodies were observed to deplete Treg cells in the periphery, upregulation of the inhibitory Fc gamma receptor (FcγR) IIb at the tumor site prevented intra-tumoral Treg cell depletion, which may underlie the lack of anti-tumor activity previously observed in pre-clinical models. Use of an anti-CD25 antibody with enhanced binding to activating FcγRs led to effective depletion of tumor-infiltrating Treg cells, increased effector to Treg cell ratios, and improved control of established tumors. Combination with anti-programmed cell death protein-1 antibodies promoted complete tumor rejection, demonstrating the relevance of CD25 as a therapeutic target and promising substrate for future combination approaches in immune-oncology

Integrated genomic analyses of ovarian carcinoma

A catalogue of molecular aberrations that cause ovarian cancer is critical for developing and deploying therapies that will improve patients’ lives. The Cancer Genome Atlas project has analysed messenger RNA expression, microRNA expression, promoter methylation and DNA copy number in 489 high-grade serous ovarian adenocarcinomas and the DNA sequences of exons from coding genes in 316 of these tumours. Here we report that high-grade serous ovarian cancer is characterized by TP53 mutations in almost all tumours (96%); low prevalence but statistically recurrent somatic mutations in nine further genes including NF1, BRCA1, BRCA2, RB1 and CDK12; 113 significant focal DNA copy number aberrations; and promoter methylation events involving 168 genes. Analyses delineated four ovarian cancer transcriptional subtypes, three microRNA subtypes, four promoter methylation subtypes and a transcriptional signature associated with survival duration, and shed new light on the impact that tumours with BRCA1/2 (BRCA1 or BRCA2) and CCNE1 aberrations have on survival. Pathway analyses suggested that homologous recombination is defective in about half of the tumours analysed, and that NOTCH and FOXM1 signalling are involved in serous ovarian cancer pathophysiology.National Institutes of Health (U.S.) (Grant U54HG003067)National Institutes of Health (U.S.) (Grant U54HG003273)National Institutes of Health (U.S.) (Grant U54HG003079)National Institutes of Health (U.S.) (Grant U24CA126543)National Institutes of Health (U.S.) (Grant U24CA126544)National Institutes of Health (U.S.) (Grant U24CA126546)National Institutes of Health (U.S.) (Grant U24CA126551)National Institutes of Health (U.S.) (Grant U24CA126554)National Institutes of Health (U.S.) (Grant U24CA126561)National Institutes of Health (U.S.) (Grant U24CA126563)National Institutes of Health (U.S.) (Grant U24CA143882)National Institutes of Health (U.S.) (Grant U24CA143731)National Institutes of Health (U.S.) (Grant U24CA143835)National Institutes of Health (U.S.) (Grant U24CA143845)National Institutes of Health (U.S.) (Grant U24CA143858)National Institutes of Health (U.S.) (Grant U24CA144025)National Institutes of Health (U.S.) (Grant U24CA143866)National Institutes of Health (U.S.) (Grant U24CA143867)National Institutes of Health (U.S.) (Grant U24CA143848)National Institutes of Health (U.S.) (Grant U24CA143843)National Institutes of Health (U.S.) (Grant R21CA135877

Fc Effector Function Contributes to the Activity of Human Anti-CTLA-4 Antibodies.

With the use of a mouse model expressing human Fc-gamma receptors (FcγRs), we demonstrated that antibodies with isotypes equivalent to ipilimumab and tremelimumab mediate intra-tumoral regulatory T (Treg) cell depletion in vivo, increasing the CD8+ to Treg cell ratio and promoting tumor rejection. Antibodies with improved FcγR binding profiles drove superior anti-tumor responses and survival. In patients with advanced melanoma, response to ipilimumab was associated with the CD16a-V158F high affinity polymorphism. Such activity only appeared relevant in the context of inflamed tumors, explaining the modest response rates observed in the clinical setting. Our data suggest that the activity of anti-CTLA-4 in inflamed tumors may be improved through enhancement of FcγR binding, whereas poorly infiltrated tumors will likely require combination approaches

SBI2 HCS/HCA 3D Imaging: Best Practices and Unmet Needs Colloquium

In this, the third annual SBI2 conference, the afternoon of the opening day was dedicated to short presentations and discussions revolving around HCS/HCA 3D Imaging: Best Practices and Unmet Needs. The growing interest in conducting assays in 3D is driven by the realization that there is often a disconnect between observations made with cells grown in 2D in a tissue culture plate and biology occurring in an organism which occurs in context of other cell types and a complex extracellular matrix. This is apparent when you look at the high failure rate of early candidates in the drug discovery process. The push towards 3D assay systems is an attempt to increase clinical relevance, while at the same time preserving an acceptable level of throughput to evaluate potential clinical candidates. With growing scientific interest in 3D biology imaging-based assays are emerging as an obvious choice to measure complex phenotypes and move towards the realization and quantification of the concept of an “organ in a dish”

A next-generation marker genotyping platform (AmpSeq) in heterozygous crops: a case study for marker-assisted selection in grapevine

Marker-assisted selection (MAS) is often employed in crop breeding programs to accelerate and enhance cultivar development, via selection during the juvenile phase and parental selection prior to crossing. Next-generation sequencing and its derivative technologies have been used for genome-wide molecular marker discovery. To bridge the gap between marker development and MAS implementation, this study developed a novel practical strategy with a semi-automated pipeline that incorporates trait-associated single nucleotide polymorphism marker discovery, low-cost genotyping through amplicon sequencing (AmpSeq) and decision making. The results document the development of a MAS package derived from genotyping-by-sequencing using three traits (flower sex, disease resistance and acylated anthocyanins) in grapevine breeding. The vast majority of sequence reads (⩾99%) were from the targeted regions. Across 380 individuals and up to 31 amplicons sequenced in each lane of MiSeq data, most amplicons (83 to 87%) had <10% missing data, and read depth had a median of 220–244×. Several strengths of the AmpSeq platform that make this approach of broad interest in diverse crop species include accuracy, flexibility, speed, high-throughput, low-cost and easily automated analysis