Selective block in erythropoietin-induced differentiation of growth factor-independent retrovirally-infected TF-1 cells

The erythroleukaemic cell line TF-1, infected with either the pBabe neo retrovirus or the retrovirus bearing the human erythropoietin (hEpo) gene, developed three growth factor-independent clones. Erythropoietin (Epo), interleukin-3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) accelerated the proliferation of these clones. Autonomous growth of the clones was independent of Epo because it was not altered by Epo anti-sense oligonucleotides, nor was Epo detectable in culture supernatants. Cells from the mutant clones could not be induced by Epo to express glycophorin A and haemoglobin synthesis was markedly reduced. Haemin reversed the block in Epo-induced haemoglobin synthesis. Acquisition of growth factor-independence appears to be linked with the selective loss of differentiation capacity. These cells may provide a useful model for the study of the mechanisms involved in leukaemic transformation.

Recurrence of Urothelial Carcinoma of the Bladder: A Role for Insulin-Like Growth Factor-II Loss of Imprinting and Cytoplasmic E-Cadherin Immunolocalization

Purpose:This study documents the frequency of insulin-like growth factor-II (IGF-II) loss of imprinting (LOI) in a series of 87 bladder tissues. E-cadherin (CDH1) immunolocalization was also investigated due to the known redistribution of this adherence protein to the cytoplasm following exogenous exposure to IGF-II.
Experimental Design: Informative IGF-II cases were identified following DNA-PCR amplification and subsequent sequencing of the transcribable ApaI RFLP in exon 9 of IGF-II. Similar approaches using primer-specific cDNA templates identified the imprinting status of IGF-II in these informative cases. CDH1cellular localization was assessed on a tissue microarray platform of 114 urothelial carcinoma of the bladder (UCB) cases (70 pTanoninvasive and 44 pT1laminapropria invasive) using the commercially available Novocastra antibody.
Results: IGF-IILOI was evident in 7 of17 (41%) UCB tumors and 4 of11 (36%) tumor-associated normal urothelial samples.Two of four pT1grade 3 tumors, the subject of much debate concerning their suitability for radical cystectomy, showed LOI at the IGF-II locus. In those tumors showing IGF-II LOI, 4 of 7 (57%) displayed concomitant CDH1cytoplasmic staining. In contrast, only 3 of 10 (30%) IGF-IImaintenance ofimprinting tumorshad concomitant CDH1cytoplasmiclocalization. UCB cell lines displaying cytoplasmic CDH1immunolocalization expressed significantly higher levels of IGF-II (CAL29, HT1376, and RT112) compared with RT4, a cell line displaying crisp membranous CDH1staining. Finally, cytoplasmic CDH1staining was an independent predictor of a shorter time to recurrence independent of tumor grade and stage.
Conclusions: We suggest that CDH1 cytoplasmic immunolocalization as a result of increased IGF-II levels identifies those nonmuscle invasive presentations most likely to recur and therefore might benefit from more radical nonconserving bladder surgery

Connective tissue growth factor/CCN2 stimulates actin disassembly through Akt/protein kinase B-mediated phosphorylation and cytoplasmic translocation of p27(Kip-1)

Connective tissue growth factor (CTGF/CCN2) is a 38-kDa secreted protein, a prototypic member of the CCN family, which is up-regulated in many diseases, including atherosclerosis, pulmonary fibrosis, and diabetic nephropathy. We previously showed that CTGF can cause actin disassembly with concurrent down-regulation of the small GTPase Rho A and proposed an integrated signaling network connecting focal adhesion dissolution and actin disassembly with cell polarization and migration. Here, we further delineate the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The functional response of mesangial cells to treatment with CTGF was associated with the phosphorylation of Akt/protein kinase B (PKB) and resultant phosphorylation of a number of Akt/PKB substrates. Two of these substrates were identified as FKHR and p27(Kip-1). CTGF stimulated the phosphorylation and cytoplasmic translocation of p27(Kip-1) on serine 10. Addition of the PI-3 kinase inhibitor LY294002 abrogated this response; moreover, addition of the Akt/PKB inhibitor interleukin (IL)-6-hydroxymethyl-chiro-inositol-2(R)-2-methyl-3-O-octadecylcarbonate prevented p27(Kip-1) phosphorylation in response to CTGF. Immunocytochemistry revealed that serine 10 phosphorylated p27(Kip-1) colocalized with the ends of actin filaments in cells treated with CTGF. Further investigation of other Akt/PKB sites on p27(Kip-1), revealed that phosphorylation on threonine 157 was necessary for CTGF mediated p27(Kip-1) cytoplasmic localization; mutation of the threonine 157 site prevented cytoplasmic localization, protected against actin disassembly and inhibited cell migration. CTGF also stimulated an increased association between Rho A and p27(Kip-1). Interestingly, this resulted in an increase in phosphorylation of LIM kinase and subsequent phosphorylation of cofilin, suggesting that CTGF mediated p27(Kip-1) activation results in uncoupling of the Rho A/LIM kinase/cofilin pathway. Confirming the central role of Akt/PKB, CTGF-stimulated actin depolymerization only in wild-type mouse embryonic fibroblasts (MEFs) compared to Akt-1/3 (PKB alpha/gamma) knockout MEFs. These data reveal important mechanistic insights into how CTGF may contribute to mesangial cell dysfunction in the diabetic milieu and sheds new light on the proposed role of p27(Kip-1) as a mediator of actin rearrangement.

Connective tissue growth factor [CTGF]/CCN2 stimulates mesangial cell migration through integrated dissolution of focal adhesion complexes and activation of cell polarization

Connective tissue growth factor [CTGF]/CCN2 is a prototypic member of the CCN family of regulatory proteins. CTGF expression is up-regulated in a number of fibrotic diseases, including diabetic nephropathy, where it is believed to act as a downstream mediator of TGF-beta function; however, the exact mechanisms whereby CTGF mediates its effects remain unclear. Here, we describe the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The addition of CTGF to primary mesangial cells induced cell migration and cytoskeletal rearrangement but had no effect on cell proliferation. Cytoskeletal rearrangement was associated with a loss of focal adhesions, involving tyrosine dephosphorylation of focal adhesion kinase and paxillin, increased activity of the protein tyrosine phosphatase SHP-2, with a concomitant decrease in RhoA and Rac1 activity. Conversely, Cdc42 activity was increased by CTGF. These functional responses were associated with the phosphorylation and translocation of protein kinase C-zeta to the leading edge of migrating cells. Inhibition of CTGF-induced protein kinase C-zeta activity with a myristolated PKC-zeta inhibitor prevented cell migration. Moreover, transient transfection of human mesangial cells with a PKC-zeta kinase inactive mutant (dominant negative) expression vector also led to a decrease in CTGF-induced migration compared with wild-type. Furthermore, CTGF stimulated phosphorylation and activation of GSK-3beta. These data highlight for the first time an integrated mechanism whereby CTGF regulates cell migration through facilitative actin cytoskeleton disassembly, which is mediated by dephosphorylation of focal adhesion kinase and paxillin, loss of RhoA activity, activation of Cdc42, and phosphorylation of PKC-zeta and GSK-3beta. These changes indicate that the initial stages of CTGF mediated mesangial cell migration are similar to those involved in the process of cell polarization. These findings begin to shed mechanistic light on the renal diabetic milieu, where increased CTGF expression in the glomerulus contributes to cellular dysfunction.

Sp1-dependent activation of HDAC7 is required for platelet-derived growth factor-BB-induced smooth muscle cell differentiation from stem cells

We have previously demonstrated that histone deacetylase 7 (HDAC7) expression and splicing play an important role in smooth muscle cell (SMC) differentiation from embryonic stem (ES) cells, but the molecular mechanisms of increased HDAC7 expression during SMC differentiation are currently unknown. In this study, we found that platelet-derived growth factor-BB (PDGF-BB) induced a 3-fold increase in the transcripts of HDAC7 in differentiating ES cells. Importantly, our data also revealed that PDGF-BB regulated HDAC7 expression not through phosphorylation of HDAC7 but through transcriptional activation. By dissecting its promoters with progressive deletion analysis, we identified the sequence between -343 and -292 bp in the 5'-flanking region of the Hdac7 gene promoter as the minimal PDGF-BB-responsive element, which contains one binding site for the transcription factor, specificity protein 1 (Sp1). Mutation of the Sp1 site within this PDGF-BB-responsive element abolished PDGF-BB-induced HDAC7 activity. PDGF-BB treatment enhanced Sp1 binding to the Hdac7 promoter in differentiated SMCs in vivo as demonstrated by the chromatin immunoprecipitation assay. Moreover, we also demonstrated that knockdown of Sp1 abrogated PDGF-BB-induced HDAC7 up-regulation and SMC differentiation gene expression in differentiating ES cells, although enforced expression of Sp1 alone was sufficient to increase the activity of the Hdac7 promoter and expression levels of SMC differentiation genes. Importantly, we further demonstrated that HDAC7 was required for Sp1-induced SMC differentiation of gene expression. Our data suggest that Sp1 plays an important role in the regulation of Hdac7 gene expression in SMC differentiation from ES cells. These findings provide novel molecular insights into the regulation of HDAC7 and enhance our knowledge in SMC differentiation and vessel formation during embryonic development.

Retinal pigment epithelial atrophy in patients with exudative age-related macular degeneration undergoing anti-vascular endothelial growth factor therapy

PURPOSE:: To evaluate the occurrence of retinal pigment epithelial atrophy in patients with age-related macular degeneration undergoing anti-vascular endothelial growth factor therapy. METHODS:: The study is a retrospective review. Eligible were patients with age-related macular degeneration and choroidal neovascular membranes treated with anti-vascular endothelial growth factor between October 2007 and February 2011; they were followed for >3 months, with fundus photographs and fluorescein angiography at baseline and with autofluorescence and near-infrared autofluorescence images at baseline and follow-up. Demographics, visual acuity, the type of choroidal neovascular membranes, the number of treatments performed, and the length of follow-up were recorded. Autofluorescence and near-infrared autofluorescence images were evaluated for the presence or absence of areas of reduced signal. A multilevel logistic regression model was used to investigate the factors that may be associated with progression of atrophy at follow-up, which was the primary outcome of this study. RESULTS:: Sixty-three patients (72 eyes) were followed for a median of 16 months (range, 3-36 months). Atrophy at baseline was observed in 47% (34/72) of eyes; progression of atrophy occurred in 62% (45/72) of eyes at the last visit. The number of anti-vascular endothelial growth factor injections received was statistically significantly associated with the progression of atrophy at follow-up (odds ratio, 1.35; 95% confidence interval, 1.05-1.73; P = 0.02). CONCLUSION:: Atrophy was frequently observed in patients with age-related macular degeneration and choroidal neovascular membranes undergoing anti-vascular endothelial growth factor therapy.

RUNX3 downregulation in human lung adenocarcinoma is independent of p53, EGFR or KRAS status

RUNX3 aberrations play a pivotal role in the oncogenesis of breast, gastric, colon, skin and lung tissues. The aim of this study was to characterize further the expression of RUNX3 in lung cancers. To achieve this, a lung cancer tissue microarray (TMA), frozen lung cancer tissues and lung cell lines were examined for RUNX3 expression by immunohistochemistry, while the TMA was also examined for EGFR and p53 expression. RUNX3 promoter methylation status, and EGFR and KRAS mutation status were also investigated. Inactivation of RUNX3 was observed in 70% of the adenocarcinoma samples, and this was associated with promoter hypermethylation but not biased to EGFR/KRAS mutations. Our results suggest a central role of RUNX3 downregulation in pulmonary adenocarcinoma, which may not be dependent of other established cancer-causing pathways and may have important diagnostic and screening implications.

Elevated NRD1 metalloprotease expression plays a role in breast cancer growth and proliferation

Understanding the molecular etiology of cancer and increasing the number of drugs and their targets are critical to cancer management. In our attempt to unravel novel breast-cancer associated proteins, we previously conducted protein expression profiling of the MCF10AT model, which comprises a series of isogenic cell lines that mimic different stages of breast cancer progression. NRD1 expression was found to increase during breast cancer progression. Here, we attempted to confirm the relevance of NRD1 in clinical breast cancer and understand the functional role and mechanism of NRD1 in breast cancer cells. Immunohistochemistry data show that NRD1 expression was elevated in ductal carcinoma in situ and invasive ductal carcinomas compared with normal tissues in 30% of the 26 matched cases studied. Examination of NRD1 expression in tissue microarray comprising >100 carcinomas and subsequent correlation with clinical data revealed that NRD1 expression was significantly associated with tumor size, grade, and nodal status (P <0.05). Silencing of NRD1 reduced MCF10CA1h and MDA-MD-231 breast-cancer-cell proliferation and growth. Probing the oncogenic EGF signaling pathways revealed that NRD1 knock down did not affect overall downstream tyrosine phosphorylation cascades including AKT and MAPK activation. Instead, silencing of NRD1 resulted in a reduction of overall cyclin D1 expression, a reduction of EGF-induced increase in cyclin D1 expression and an increase in apoptotic cell population compared with control cells.

Role of vascular endothelial growth factor and placental growth factors during retinal vascular development and hyaloid regression

PURPOSE. Vascular endothelial growth factor (VEGF)-A and placental growth factor (PIGF) are members of a large group of homologous peptides identified as the VEGF family. Although VEGF-A is known to act as a potent angiogenic peptide in the retina, the vasoactive function of PIGF in this tissue is less well defined. This study has sought to elucidate the expression patterns and modulatory role of these growth factors during retinal vascular development and hyaloid regression in the neonatal mouse. METHODS. C57BL6J mice were killed at postnatal days (P)1, P3, P5, P7, P9, and P11. The eyes were enucleated and processed for in situ hybridization and immunocytochemistry and the retinas extracted for total protein or RNA. Separate groups of neonatal mice were also injected intraperitoneally daily from P2 through P9 with either VEGF-neutralizing antibody, PIGF-neutralizing antibody, isotype immunoglobulin (Ig)-G, or phosphate-buffered saline (PBS). The mice were then perfused with fluorescein isothiocyanate (FITC)-dextran, and the eyes were subsequently embedded in paraffin wax or flat mounted. RESULTS. Quantitative (real-time) reverse transcription-polymerase chain reaction (RT-PCR) demonstrated similar expression patterns of VEGF-A and PIGF mRNA during neonatal retinal development, although the fluctuation between time periods was greater overall for VEGF-A. The localization of VEGF-A and PIGF in the retina, as revealed by in situ hybridization and immunohistochemistry, was also similar. Neutralization of VEGF-A caused a significant reduction in the hyaloid and retinal vasculature, whereas PIGF antibody treatment caused a marked persistence of the hyaloid without significantly affecting retinal vascular development. CONCLUSIONS. Although having similar expression patterns in the retina, these growth factors appear to have distinct modulatory influences during normal retinal vascular development and hyaloid regression.

Validation of Next Generation Sequencing Technologies in Comparison to Current Diagnostic Gold Standards for BRAF, EGFR and KRAS Mutational Analysis

Next Generation Sequencing (NGS) has the potential of becoming an important tool in clinical diagnosis and therapeutic decision-making in oncology owing to its enhanced sensitivity in DNA mutation detection, fast-turnaround of samples in comparison to current gold standard methods and the potential to sequence a large number of cancer-driving genes at the one time. We aim to test the diagnostic accuracy of current NGS technology in the analysis of mutations that represent current standard-of-care, and its reliability to generate concomitant information on other key genes in human oncogenesis. Thirteen clinical samples (8 lung adenocarcinomas, 3 colon carcinomas and 2 malignant melanomas) already genotyped for EGFR, KRAS and BRAF mutations by current standard-of-care methods (Sanger Sequencing and q-PCR), were analysed for detection of mutations in the same three genes using two NGS platforms and an additional 43 genes with one of these platforms. The results were analysed using closed platform-specific proprietary bioinformatics software as well as open third party applications. Our results indicate that the existing format of the NGS technology performed well in detecting the clinically relevant mutations stated above but may not be reliable for a broader unsupervised analysis of the wider genome in its current design. Our study represents a diagnostically lead validation of the major strengths and weaknesses of this technology before consideration for diagnostic use.