26 research outputs found
Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation
There is overwhelming evidence that in vitro three-dimensional tumor cell cultures more accurately
reflect the complex in vivo microenvironment than simple two-dimensional cell monolayers, not least with respect
to gene expression profiles, signaling pathway activity and drug sensitivity. However, most currently available threedimensional
techniques are time consuming and/or lack reproducibility; thus standardized and rapid protocols are
urgently needed. To address this requirement, we have developed a versatile toolkit of reproducible three-dimensional
tumor spheroid models for dynamic, automated, quantitative imaging and analysis that are compatible with
routine high-throughput preclinical studies. Not only do these microplate methods measure three-dimensional
tumor growth, but they have also been significantly enhanced to facilitate a range of functional assays
exemplifying additional key hallmarks of cancer, namely cell motility and matrix invasion. Moreover, mutual tissue
invasion and angiogenesis is accommodated by coculturing tumor spheroids with murine embryoid bodies within
which angiogenic differentiation occurs. Highly malignant human tumor cells were selected to exemplify
therapeutic effects of three specific molecularly-targeted agents: PI-103 (phosphatidylinositol-3-kinase (PI3K)-
mammalian target of rapamycin (mTOR) inhibitor), 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) (heat
shock protein 90 (HSP90) inhibitor) and CCT130234 (in-house phospholipase C (PLC)g inhibitor). Fully automated
analysis using a Celigo cytometer was validated for tumor spheroid growth and invasion against standard image
analysis techniques, with excellent reproducibility and significantly increased throughput. In addition, we
discovered key differential sensitivities to targeted agents between two-dimensional and three-dimensional
cultures, and also demonstrated enhanced potency of some agents against cell migration/invasion compared with
proliferation, suggesting their preferential utility in metastatic disease.: We have established and validated a suite of highly reproducible tumor microplate threedimensional
functional assays to enhance the biological relevance of early preclinical cancer studies. We believe
these assays will increase the translational predictive value of in vitro drug evaluation studies and reduce the need
for in vivo studies by more effective triaging of compounds.This work was
funded by The National Centre for the Replacement, Refinement and
Reduction of Animals in Research (G1000121 ID no. 94513), Cancer Research
UK (grant number C309/A8274), and by Red Tematica de Investigación
Cooperativa en Cancer (RD06/0020/1022). We acknowledge NHS funding to
the NIHR Biomedical Research Centre. MM is supported by a postdoctoral
research contract (FIS, Program ‘Sara Borrell’, Instituto de Salud Carlos III),
Ministerio de Ciencia e Innovación, Spain
Decreased levels of BAG3 in a family with a rare variant and in idiopathic dilated cardiomyopathy.
The most common cause of dilated cardiomyopathy and heart failure (HF) is ischemic heart disease; however, in a third of all patients the cause remains undefined and patients are diagnosed as having idiopathic dilated cardiomyopathy (IDC). Recent studies suggest that many patients with IDC have a family history of HF and rare genetic variants in over 35 genes have been shown to be causative of disease. We employed whole-exome sequencing to identify the causative variant in a large family with autosomal dominant transmission of dilated cardiomyopathy. Sequencing and subsequent informatics revealed a novel 10-nucleotide deletion in the BCL2-associated athanogene 3 (BAG3) gene (Ch10:del 121436332_12143641: del. 1266_1275 [NM 004281]) that segregated with all affected individuals. The deletion predicted a shift in the reading frame with the resultant deletion of 135 amino acids from the C-terminal end of the protein. Consistent with genetic variants in genes encoding other sarcomeric proteins there was a considerable amount of genetic heterogeneity in the affected family members. Interestingly, we also found that the levels of BAG3 protein were significantly reduced in the hearts from unrelated patients with end-stage HF undergoing cardiac transplantation when compared with non-failing controls. Diminished levels of BAG3 protein may be associated with both familial and non-familial forms of dilated cardiomyopathy
FLNC Gene Splice Mutations Cause Dilated\ua0Cardiomyopathy
OBJECTIVE:
To identify novel dilated cardiomyopathy (DCM) causing genes, and to elucidate the pathological mechanism leading to DCM by utilizing zebrafish as a model organism.
BACKGROUND:
DCM, a major cause of heart failure, is frequently familial and caused by a genetic defect. However, only 50% of DCM cases can be attributed to a known DCM gene variant, motivating the ongoing search for novel disease genes.
METHODS:
We performed whole exome sequencing (WES) in two multigenerational Italian families and one US family with arrhythmogenic DCM without skeletal muscle defects, in whom prior genetic testing had been unrevealing. Pathogenic variants were sought by a combination of bioinformatic filtering and cosegregation testing among affected individuals within the families. We performed function assays and generated a zebrafish morpholino knockdown model.
RESULTS:
A novel filamin C gene splicing variant (FLNC c.7251+1 G>A) was identified by WES in all affected family members in the two Italian families. A separate novel splicing mutation (FLNC c.5669-1delG) was identified in the US family. Western blot analysis of cardiac heart tissue from an affected individual showed decreased FLNC protein, supporting a haploinsufficiency model of pathogenesis. To further analyze this model, a morpholino knockdown of the ortholog filamin Cb in zebrafish was created which resulted in abnormal cardiac function and ultrastructure.
CONCLUSIONS:
Using WES, we identified two novel FLNC splicing variants as the likely cause of DCM in three families. We provided protein expression and in vivo zebrafish data supporting haploinsufficiency as the pathogenic mechanism leading to DCM
A G-quadruplex-interactive potent small-molecule inhibitor of telomerase exhibiting in vitro and in vivo antitumor activity
ABSTRACT The telomerase complex is responsible for telomere maintenance and represents a promising cancer therapeutic target. We describe herein the antitelomerase and antitumor properties of a small-molecule compound designed by computer modeling to interact with and stabilize human G-quadruplex DNA, a structure that may form with telomeric DNA, thereby inhibiting access to telomerase. The 3,6,9-trisubstituted acridine 9-[4-(N,N-dimethylamino)phenylamino]-3,6-bis(3-pyrrolodinopropionamido) acridine (BRACO19) represents one of the most potent cell-free inhibitors of human telomerase yet described (50% inhibitory concentration of 115 Ϯ 18 nM). Moreover, in contrast to G-quadruplex interactive agents described previously, BRACO19 did not cause nonspecific acute cytotoxicity at similar concentrations to those required to completely inhibit telomerase activity. There exists a 90-fold differential (mean 50% inhibitory concentration for acute cell kill across seven human tumor cell lines of 10.6 Ϯ 0.7 M). The exposure of 21NT human breast cancer cells, which possess relatively short telomeres, to nonacute cytotoxic concentrations of BRACO19 (2 M) resulted in a marked reduction in cell growth after only 15 days. This was concomitant with a reduction in intracellular telomerase activity and onset of senescence as indicated by an increase in the number of -galactosidase positive-staining cells. Intraperitoneal administration of nontoxic doses of BRACO19 (2 mg/kg) to mice bearing advanced stage A431 human vulval carcinoma subcutaneous xenografts and previously treated with paclitaxel induced a significant increase in antitumor effect compared with that observed with paclitaxel alone. BRACO19 thus represents the first of a "second generation" of G-quadruplex-mediated telomerase/telomere-interactive compounds. It possesses nanomolar potency against telomerase but low nonspecific cytotoxicity, growth inhibitory effects, and induction of senescence in a human breast cancer cell line and, moreover, significant antitumor activity in vivo when administered post paclitaxel to mice bearing a human tumor xenograft carcinoma