Generalizing Diffusion Tensor Imaging of the Physis and Metaphysis.

BACKGROUND: Current methods to predict height potential are inaccurate. Predicting height by using MRI of the physeal cartilage has shown promise but the applicability of this technique in different imaging setups has not been well-evaluated. PURPOSE: To assess variability in diffusion tensor imaging of the physis and metaphysis (DTI-P/M) of the distal femur between different scanners, imaging parameters, tractography software, and resolution. STUDY TYPE: Prospective. POPULATION/SUBJECTS: Eleven healthy subjects (five males and six females ages 10-16.94). FIELD STRENGTH/SEQUENCE: 3 T; DTI single shot echo planar sequences. ASSESSMENT: Physeal DTI tract measurements of the distal femur were compared between different scanners, imaging parameters, tractography settings, interpolation correction, and tractography software. STATISTICAL TESTS: Bland-Altman, Spearman correlation, linear regression, and Shapiro-Wilk tests. Threshold for statistical significance was set at P = 0.05. RESULTS: DTI tract values consistently showed low variability with different imaging and analysis settings. Vendor to vendor comparison exhibited strong correlation (ρ = 0.93) and small but significant bias (bias -5.76, limits of agreement [LOA] -24.31 to 12.78). Strong correlation and no significant difference were seen between technical replicates of the General Electric MRI scanner (ρ = 1, bias 0.17 [LOA -1.5 to 1.2], P = 0.42) and the Siemens MRI scanner (ρ = 0.89, bias = 0.56, P = 0.71). Different voxel sizes (1 × 1 × 2 mm3 vs. 2 × 2 × 3 mm3) did not significantly affect DTI values (bias = 1.4 [LOA -5.7 to 8.4], P = 0.35) but maintained a strong correlation (ρ = 0.82). Gap size (0 mm vs. 0.6 mm) significantly affects tract volume (bias = 1.8 [LOA -5.4 to 1.8]) but maintains a strong correlation (ρ = 0.93). Comparison of tractography algorithms generated significant differences in tract number, length, and volume while maintaining correlation (ρ = 0.86, 0.99, 0.93, respectively). Comparison of interobserver variability between different tractography software also revealed significant differences while maintaining high correlation (ρ = 0.85-0.98). DATA CONCLUSION: DTI of the pediatric physis cartilage shows high reproducibility between different imaging and analytic parameters. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 1.

Pten-NOLC1 fusion promotes cancers involving MET and EGFR signalings.

Inactivation of Pten gene through deletions and mutations leading to excessive pro-growth signaling pathway activations frequently occurs in cancers. Here, we report a Pten derived pro-cancer growth gene fusion Pten-NOLC1 originated from a chr10 genome rearrangement and identified through a transcriptome sequencing analysis of human cancers. Pten-NOLC1 fusion is present in primary human cancer samples and cancer cell lines from different organs. The product of Pten-NOLC1 is a nuclear protein that interacts and activates promoters of EGFR, c-MET, and their signaling molecules. Pten-NOLC1 promotes cancer proliferation, growth, invasion, and metastasis, and reduces the survival of animals xenografted with Pten-NOLC1-expressing cancer cells. Genomic disruption of Pten-NOLC1 induces cancer cell death, while genomic integration of this fusion gene into the liver coupled with somatic Pten deletion produces spontaneous liver cancers in mice. Our studies indicate that Pten-NOLC1 gene fusion is a driver for human cancers.

Positive autoregulation of lag-1 in response to LIN-12 activation in cell fate decisions during C. elegans reproductive system development.

During animal development, ligand binding releases the intracellular domain of LIN-12/Notch by proteolytic cleavage to translocate to the nucleus, where it associates with the DNA-binding protein LAG-1/CSL to activate target gene transcription. We investigated the spatiotemporal regulation of LAG-1/CSL expression in Caenorhabditis elegans and observed that an increase in endogenous LAG-1 levels correlates with LIN-12/Notch activation in different cell contexts during reproductive system development. We show that this increase is via transcriptional upregulation by creating a synthetic endogenous operon, and identified an enhancer region that contains multiple LAG-1 binding sites (LBSs) embedded in a more extensively conserved high occupancy target (HOT) region. We show that these LBSs are necessary for upregulation in response to LIN-12/Notch activity, indicating that lag-1 engages in direct positive autoregulation. Deletion of the HOT region from endogenous lag-1 reduced LAG-1 levels and abrogated positive autoregulation, but did not cause hallmark cell fate transformations associated with loss of lin-12/Notch or lag-1 activity. Instead, later somatic reproductive system defects suggest that proper transcriptional regulation of lag-1 confers robustness to somatic reproductive system development.

A Screen of the Conserved Kinome for Negative Regulators of LIN-12 Negative Regulatory Region ("NRR")-Missense Activity in Caenorhabditis elegans.

Genetic analysis of LIN-12/Notch signaling in C. elegans has provided many insights into human biology. Activating missense mutations in the Negative Regulatory Region (NRR) of the ectodomain of LIN-12/Notch were first described in C. elegans, and similar mutations in human Notch were later found to cause T-cell acute lymphoblastic leukemia (T-ALL). The ubiquitin ligase sel-10/Fbw7 is the prototype of a conserved negative regulator of lin-12/Notch that was first defined by loss-of-function mutations that enhance lin-12 NRR-missense activity in C. elegans, and then demonstrated to regulate Notch activity in mammalian cells and to be a bona fide tumor suppressor in T-ALL. Here, we report the results of an RNAi screen of 248 C. elegans protein kinase-encoding genes with human orthologs for enhancement of a weakly activating NRR-missense mutation of lin-12 in the Vulval Precursor Cells. We identified, and validated, thirteen kinase genes whose loss led to increase lin-12 activity; eleven of these genes have never been implicated previously in regulating Notch activity in any system. Depleting the activity of five kinase genes (cdk-8, wnk-1, kin-3, hpo-11, and mig-15) also significantly enhanced the activity of a transgene in which heterologous sequences drive expression of the untethered intracellular domain of LIN-12, suggesting that they increase the activity or stability of the signal-transducing form of LIN-12/Notch. Precedents set by other regulators of lin-12/Notch defined through genetic interactions in C. elegans suggest that this new set of genes may include negative regulators that are functionally relevant to mammalian development and cancer.

Endoplasmic Reticulum Stress Interacts With Inflammation in Human Diseases.

The endoplasmic reticulum (ER) is a critical organelle for normal cell function and homeostasis. Disturbance in the protein folding process in the ER, termed ER stress, leads to the activation of unfolded protein response (UPR) that encompasses a complex network of intracellular signaling pathways. The UPR can either restore ER homeostasis or activate pro-apoptotic pathways depending on the type of insults, intensity and duration of the stress, and cell types. ER stress and the UPR have recently been linked to inflammation in a variety of human pathologies including autoimmune, infectious, neurodegenerative, and metabolic disorders. In the cell, ER stress and inflammatory signaling share extensive regulators and effectors in a broad spectrum of biological processes. In spite of different etiologies, the two signaling pathways have been shown to form a vicious cycle in exacerbating cellular dysfunction and causing apoptosis in many cells and tissues. However, the interaction between ER stress and inflammation in many of these diseases remains poorly understood. Further understanding of the biochemistry, cell biology, and physiology may enable the development of novel therapies that spontaneously target these pathogenic pathways.

Endoplasmic reticulum stress in intestinal epithelial cell function and inflammatory bowel disease.

In eukaryotic cells, perturbation of protein folding homeostasis in the endoplasmic reticulum (ER) causes accumulation of unfolded and misfolded proteins in the ER lumen, which activates intracellular signaling pathways termed the unfolded protein response (UPR). Recent studies have linked ER stress and the UPR to inflammatory bowel disease (IBD). The microenvironment of the ER is affected by a myriad of intestinal luminal molecules, implicating ER stress and the UPR in proper maintenance of intestinal homeostasis. Several intestinal cell populations, including Paneth and goblet cells, require robust ER function for protein folding, maturation, and secretion. Prolonged ER stress and impaired UPR signaling may cause IBD through: (1) induction of intestinal epithelial cell apoptosis, (2) disruption of mucosal barrier function, and (3) induction of the proinflammatory response in the gut. Based on our increased understanding of ER stress in IBD, new pharmacological approaches can be developed to improve intestinal homeostasis by targeting ER protein-folding in the intestinal epithelial cells (IECs).

Muscleblind-like 1 (Mbnl1) regulates pre-mRNA alternative splicing during terminal erythropoiesis.

The scope and roles of regulated isoform gene expression during erythroid terminal development are poorly understood. We identified hundreds of differentiation-associated isoform changes during terminal erythropoiesis. Sequences surrounding cassette exons of skipped exon events are enriched for motifs bound by the Muscleblind-like (MBNL) family of splicing factors. Knockdown of Mbnl1 in cultured murine fetal liver erythroid progenitors resulted in a strong block in erythroid differentiation and disrupted the developmentally regulated exon skipping of Ndel1 mRNA, which is bound by MBNL1 and critical for erythroid terminal proliferation. These findings reveal an unanticipated scope of the alternative splicing program and the importance of Mbnl1 during erythroid terminal differentiation.

p53-induced gene 3 mediates cell death induced by glutathione peroxidase 3.

Expression of glutathione peroxidase 3 (GPx3) is down-regulated in a variety of human malignancies. Both methylation and deletion of GPx3 gene underlie the alterations of GPx3 expression in prostate cancer. A strong correlation between the down-regulation of GPx3 expression and progression of prostate cancer and the suppression of prostate cancer xenografts in SCID mice by forced expression of GPx3 suggests a tumor suppression role of GPx3 in prostate cancer. However, the mechanism of GPx3-mediated tumor suppression remains unclear. In this report, GPx3 was found to interact directly with p53-induced gene 3 (PIG3). Forced overexpression of GPx3 in prostate cancer cell lines DU145 and PC3 as well as immortalized prostate epithelial cells RWPE-1 increased apoptotic cell death. Expression of GPx3(x73c), a peroxidase-negative OPAL codon mutant, in DU145 and PC3 cells also increased cell death. The induced expression of GPx3 in DU145 and PC3 cells resulted in an increase in reactive oxygen species and caspase-3 activity. These activities were abrogated by either knocking down PIG3 or mutating the PIG3 binding motif in GPx3 or binding interference from a peptide corresponding to PIG3 binding motif in GPx3. In addition, UV-treated RWPE-1 cells underwent apoptotic death, which was partially prevented by knocking down GPx3 or PIG3, suggesting that GPx3-PIG3 signaling is critical for UV-induced apoptosis. Taken together, these results reveal a novel signaling pathway of GPx3-PIG3 in the regulation of cell death in prostate cancer.

(-)-Epigallocatechin-3-gallate induces Du145 prostate cancer cell death via downregulation of inhibitor of DNA binding 2, a dominant negative helix-loop-helix protein.

(-)-Epigallocatechin-3-gallate (EGCG) is one of the major polyphenol components in green tea. It effectively induces apoptosis in prostate cancer cells. The anticancer effect of this reagent is appealing because it is a natural component of a popular daily beverage that has proven harmless for thousands of years, making it a good candidate chemopreventive agent. EGCG suppresses cell growth and causes cell death, but the mechanisms are not well characterized, especially in androgen-independent prostate cancer cells. In the present study, using Affymetrix genechip Hu133 2.0, we analyzed the gene expression patterns of the androgen-independent prostate cancer cell line Du145, treated with or without EGCG, and found 40 genes whose expression levels were altered (>twofold, either upregulated or downregulated, P < 0.01) upon treatment with EGCG. These gene products are involved in the functions of transcription, RNA processing, protein folding, phosphorylation, protein degradation, cell motility, and ion transport. Among them, inhibitor of DNA binding 2 (ID2), known as a dominant anti-retinoblastoma (Rb) helix-loop-helix protein, was found to be downregulated fourfold by EGCG treatment. Forced expression of ID2 in Du145 cells reduced apoptosis and increased cell survival in the presence of EGCG, and knockdown ID2 expression in Du145 cells using a morpholino oligonucleotide specific for ID2 mimicked the apoptosis effect generated by EGCG treatment, although it was milder. To our knowledge, this is the first report indicating that ID2 is one of the critical factors in the signaling pathway of Du145 cell death induced by EGCG.