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Gain of chromosome 21 (Hsa21) is among the most frequent aneuploidies in leukemia. However, it remains unclear how partial or complete amplifications of Hsa21 promote leukemogenesis and why children with Down syndrome (DS) (ie, trisomy 21) are particularly at risk of leukemia development. Here, we propose that RUNX1 isoform disequilibrium with RUNX1A bias is key to DS-associated myeloid leukemia (ML-DS). Starting with Hsa21-focused CRISPR-CRISPR-associated protein 9 screens, we uncovered a strong and specific RUNX1 dependency in ML-DS cells. Expression of the RUNX1A isoform is elevated in patients with ML-DS, and mechanistic studies using murine ML-DS models and patient-derived xenografts revealed that excess RUNX1A synergizes with the pathognomonic Gata1s mutation during leukemogenesis by displacing RUNX1C from its endogenous binding sites and inducing oncogenic programs in complex with the MYC cofactor MAX. These effects were reversed by restoring the RUNX1A:RUNX1C equilibrium in patient-derived xenografts in vitro and in vivo. Moreover, pharmacological interference with MYC:MAX dimerization using MYCi361 exerted strong antileukemic effects. Thus, our study highlights the importance of alternative splicing in leukemogenesis, even on a background of aneuploidy, and paves the way for the development of specific and targeted therapies for ML-DS, as well as for other leukemias with Hsa21 aneuploidy or RUNX1 isoform disequilibrium., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

The high genetic diversity of hepatitis C virus (HCV) complicates effective vaccine development. We screened a cohort of 435 HCV-infected individuals and found that 2%-5% demonstrated outstanding HCV-neutralizing activity. From four of these patients, we isolated 310 HCV antibodies, including neutralizing antibodies with exceptional breadth and potency. High neutralizing activity was enabled by the use of the VH1-69 heavy-chain gene segment, somatic mutations within CDRH1, and CDRH2 hydrophobicity. Structural and mutational analyses revealed an important role for mutations replacing the serines at positions 30 and 31, as well as the presence of neutral and hydrophobic residues at the tip of the CDRH3. Based on these characteristics, we computationally created a de novo antibody with a fully synthetic VH1-69 heavy chain that efficiently neutralized multiple HCV genotypes. Our findings provide a deep understanding of the generation of broadly HCV-neutralizing antibodies that can guide the design of effective vaccine candidates., Competing Interests: Declaration of interests Reported antibodies are in the process of being patented., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Given the plasticity of hematopoietic stem and progenitor cells, multiple routes of differentiation must be blocked in the the pathogenesis of acute myeloid leukemia, the molecular basis of which is incompletely understood. We report that posttranscriptional repression of the transcription factor ARID3A by miR-125b is a key event in the pathogenesis of acute megakaryoblastic leukemia (AMKL). AMKL is frequently associated with trisomy 21 and GATA1 mutations (GATA1s), and children with Down syndrome are at a high risk of developing the disease. The results of our study showed that chromosome 21-encoded miR-125b synergizes with Gata1s to drive leukemogenesis in this context. Leveraging forward and reverse genetics, we uncovered Arid3a as the main miR-125b target behind this synergy. We demonstrated that, during normal hematopoiesis, this transcription factor promotes megakaryocytic differentiation in concert with GATA1 and mediates TGFβ-induced apoptosis and cell cycle arrest in complex with SMAD2/3. Although Gata1s mutations perturb erythroid differentiation and induce hyperproliferation of megakaryocytic progenitors, intact ARID3A expression assures their megakaryocytic differentiation and growth restriction. Upon knockdown, these tumor suppressive functions are revoked, causing a blockade of dual megakaryocytic/erythroid differentiation and subsequently of AMKL. Inversely, restoring ARID3A expression relieves the arrest of megakaryocytic differentiation in AMKL patient-derived xenografts. This work illustrates how mutations in lineage-determining transcription factors and perturbation of posttranscriptional gene regulation can interact to block multiple routes of hematopoietic differentiation and cause leukemia. In AMKL, surmounting this differentiation blockade through restoration of the tumor suppressor ARID3A represents a promising strategy for treating this lethal pediatric disease., (© 2022 by The American Society of Hematology.)

The intestinal epithelium requires self-renewal and differentiation in order to function and adapt to pathological diseases such as inflammatory bowel disease, short gut syndrome, and ulcers. The rodent Slfn3 protein and the human Slfn12 analog are known to regulate intestinal epithelial differentiation. Previous work utilizing a pan-Slfn3 knockout (KO) mouse model revealed sex-dependent gene expression disturbances in intestinal differentiation markers, metabolic pathways, Slfn family member mRNA expression, adaptive immune cell proliferation/functioning genes, and phenotypically less weight gain and sex-dependent changes in villus length and crypt depth. We have now created a Vil-Cre specific Slfn3KO (VC-Slfn3KO) mouse to further evaluate its role in intestinal differentiation. There were increases in Slfn1, Slfn2, Slfn4, and Slfn8 and decreases in Slfn5 and Slfn9 mRNA expression that were intestinal region and sex-specific. Differentiation markers, sucrase isomaltase (SI), villin 1, and dipeptidyl peptidase 4 and glucose transporters, glucose transporter 1 (Glut1), Glut2, and sodium glucose transporter 1 (SGLT1), were increased in expression in VC-Slfn3KO mice based on intestinal region and were also highly female sex-biased, except for SI in the ileum was also increased for male VC-Slfn3KO mice and SGLT1 was decreased for both sexes. Overall, the variations that we observed in these VC-Slfn3KO mice indicate a complex regulation of intestinal gene expression that is sex-dependent., Competing Interests: The authors have declared that no competing interests exist.

Objectives: The present investigation examined the role of factors involved in suicide ideation and suicide attempt using a nationally representative sample of US high school students., Methods: Data were collected from students in Grades 9-12 as part of the Youth Behavioral Survey (n = 11,328). Logistic regression models were used to examine factors associated with suicide ideation and suicide attempt. Variables included demographic characteristics (grade, sex, and race), psychosocial factors (physical fighting, sexual violence, sports participation, bullying, and electronic bullying), and substance use (cigarette usage, marijuana usage, and alcohol usage)., Results: The factor most associated with suicide ideation was bullying (either electronic or physical), whereas the factor most associated with suicide attempt was sexual violence followed closely by physical bullying., Conclusions: The multitude of instigators and commonality among gender, ethnicity, and socio-economic status give an indication of how to prevent suicide and who to target information regarding the hazards of suicide., (Copyright © 2021 The Royal Society for Public Health. Published by Elsevier Ltd. All rights reserved.)

Objective: Neutralising antibodies are key effectors of infection-induced and vaccine-induced immunity. Quantification of antibodies' breadth and potency is critical for understanding the mechanisms of protection and for prioritisation of vaccines. Here, we used a unique collection of human specimens and HCV strains to develop HCV reference viruses for quantification of neutralising antibodies, and to investigate viral functional diversity., Design: We profiled neutralisation potency of polyclonal immunoglobulins from 104 patients infected with HCV genotype (GT) 1-6 across 13 HCV strains representing five viral GTs. Using metric multidimensional scaling, we plotted HCV neutralisation onto neutralisation maps. We employed K-means clustering to guide virus clustering and selecting representative strains., Results: Viruses differed greatly in neutralisation sensitivity, with J6 (GT2a) being most resistant and SA13 (GT5a) being most sensitive. They mapped to six distinct neutralisation clusters, in part composed of viruses from different GTs. There was no correlation between viral neutralisation and genetic distance, indicating functional neutralisation clustering differs from sequence-based clustering. Calibrating reference viruses representing these clusters against purified antibodies from 496 patients infected by GT1 to GT6 viruses readily identified individuals with extraordinary potent and broadly neutralising antibodies. It revealed comparable antibody cross-neutralisation and diversity between specimens from diverse viral GTs, confirming well-balanced reporting of HCV cross-neutralisation across highly diverse human samples., Conclusion: Representative isolates from six neutralisation clusters broadly reconstruct the functional HCV neutralisation space. They enable high resolution profiling of HCV neutralisation and they may reflect viral functional and antigenic properties important to consider in HCV vaccine design., Competing Interests: Competing interests: MC reports personal fees from Abbvie, personal fees from Gilead Sciences, personal fees from Janssen-Cilag, personal fees from Merck, MD, personal fees from Biogen, personal fees from Falk Foundation, personal fees from Siemens, personal fees from Spring Bank, personal fees from GlaxoSmithKline, outside the submitted work., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Although the factors regulating muscle cell differentiation are well described, we know very little about how differentiating muscle fibers are organized into individual muscle tissue bundles. Disruption of these processes leads to muscle hypoplasia or dysplasia, and replicating these events is vital in tissue engineering approaches. We describe the progressive cellular events that orchestrate the formation of individual limb muscle bundles and directly demonstrate the role of the connective tissue cells that surround muscle precursors in controlling these events. We show how disruption of gene activity within or genetic ablation of connective tissue cells impacts muscle precursors causing disruption of muscle bundle formation and subsequent muscle dysplasia and hypoplasia. We identify several markers of the populations of connective tissue cells that surround muscle precursors and provide a model for how matrix-modifying proteoglycans secreted by these cells may influence muscle bundle formation by effects on the local extracellular matrix (ECM) environment., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Genome editing with the CRISPR-Cas9 system has enabled unprecedented efficacy for reverse genetics and gene correction approaches. While off-target effects have been successfully tackled, the effort to eliminate variability in sgRNA efficacies-which affect experimental sensitivity-is in its infancy. To address this issue, studies have analyzed the molecular features of highly active sgRNAs, but independent cross-validation is lacking. Utilizing fluorescent reporter knock-out assays with verification at selected endogenous loci, we experimentally quantified the target efficacies of 430 sgRNAs. Based on this dataset we tested the predictive value of five recently-established prediction algorithms. Our analysis revealed a moderate correlation (r = 0.04 to r = 0.20) between the predicted and measured activity of the sgRNAs, and modest concordance between the different algorithms. We uncovered a strong PAM-distal GC-content-dependent activity, which enabled the exclusion of inactive sgRNAs. By deriving nine additional predictive features we generated a linear model-based discrete system for the efficient selection (r = 0.4) of effective sgRNAs (CRISPRater). We proved our algorithms' efficacy on small and large external datasets, and provide a versatile combined on- and off-target sgRNA scanning platform. Altogether, our study highlights current issues and efforts in sgRNA efficacy prediction, and provides an easily-applicable discrete system for selecting efficient sgRNAs.

The differentiated cell types of the mature ureter arise from the distal ureteric bud epithelium and its surrounding mesenchyme. Uncommitted epithelial cells first become intermediate cells from which both basal and superficial cells develop. Mesenchymal progenitors give rise to separated layers of adventitial fibrocytes, smooth muscle cells and lamina propria fibrocytes. How progenitor expansion and differentiation are balanced is poorly understood. Here, we addressed the role of retinoic acid (RA) signaling in these programs. Using expression analysis of components and target genes, we show that pathway activity is restricted to the mesenchymal and epithelial progenitor pools. Inhibition of RA signaling in ureter explant cultures resulted in tissue hypoplasia with a relative expansion of smooth muscle cells at the expense of lamina propria fibroblasts in the mesenchyme, and of superficial cells at the expense of intermediate cells in the ureteric epithelium. Administration of RA led to a slight reduction of smooth muscle cells, and almost completely prevented differentiation of intermediate cells into basal and superficial cells. We identified cellular programs and transcriptional targets of RA signaling that may account for this activity. We conclude that RA signaling is required and sufficient to maintain mesenchymal and epithelial progenitors in early ureter development.

Chromosomal translocations that generate oncogenic fusion proteins are causative for most pediatric leukemias and frequently affect the MLL/KMT2A gene. In vivo modeling of bona fide chromosomal translocations in human hematopoietic stem and progenitor cells is challenging but essential to determine their actual leukemogenic potential. We therefore developed an advanced lentiviral CRISPR-Cas9 vector that efficiently transduced human CD34 + hematopoietic stem and progenitor cells and induced the t(11;19)/MLL-ENL translocation. Leveraging this system, we could demonstrate that hematopoietic stem and progenitor cells harboring the translocation showed only a transient clonal growth advantage in vitro In contrast, t(11;19)/MLL-ENL-harboring CD34 + hematopoietic stem and progenitor cells not only showed long-term engraftment in primary immunodeficient recipients, but t(11;19)/MLL-ENL also served as a first hit to initiate a monocytic leukemia-like disease. Interestingly, secondary recipients developed acute lymphoblastic leukemia with incomplete penetrance. These findings indicate that environmental cues not only contribute to the disease phenotype, but also to t(11;19)/MLL-ENL-mediated oncogenic transformation itself. Thus, by investigating the true chromosomal t(11;19) rearrangement in its natural genomic context, our study emphasizes the importance of environmental cues for the pathogenesis of pediatric leukemias, opening an avenue for novel treatment options., (Copyright© 2017 Ferrata Storti Foundation.)

Loss of heterozygosity (LOH) of the entire chromosome 10 is the most frequent genetic alteration in human glioblastoma (GBM). In addition to PTEN/MMAC1 on 10q23.3, clustering of partial deletion break-points on 10q25.3-26.1 points to a second suppressor locus. The proposed target gene DMBT1 was not confirmed. By somatic deletion mapping of this region, we identified the complementary DNA encoding the human homologue of rat orphan G protein-coupled receptor GPR26. GPR26 is highly expressed in fetal and adult brain, but frequently reduced or absent in glioma cells and biopsies, due to de novo methylation of its 5' CpG island. Silencing of GPR26 was reversed with 5-aza-deoxycytidine and the histone deacetylase inhibitor trichostatin A. Furthermore, overexpression of GPR26 in HEK and in U87 glioma cells increased intracellular cAMP concentration which is considered to induce astrocytic differentiation. Interestingly, we observed concomitant silencing of GPR26 with O6-methylguanine-DNA methyl transferase (MGMT), a DNA repair gene co-localized on 10q25.3-26.1 (p=0.0001). We conclude that epigenetic silencing is a common mechanism in malignant gliomas that simultaneously inactivates MGMT and GPR26. The 10q25.3-26.1 region may contain an important epigenetic pathway in brain tumorigenesis.

Introduction: We investigated whether mRNA levels of E2F1, a key transcription factor involved in proliferation, differentiation and apoptosis, could be used as a surrogate marker for the determination of breast cancer outcome., Methods: E2F1 and other proliferation markers were measured by quantitative RT-PCR in 317 primary breast cancer patients from the Stiftung Tumorbank Basel. Correlations to one another as well as to the estrogen receptor and ERBB2 status and clinical outcome were investigated. Results were validated and further compared with expression-based prognostic profiles using The Netherlands Cancer Institute microarray data set reported by Fan and colleagues., Results: E2F1 mRNA expression levels correlated strongly with the expression of other proliferation markers, and low values were mainly found in estrogen receptor-positive and ERBB2-negative phenotypes. Patients with low E2F1-expressing tumors were associated with favorable outcome (hazard ratio = 4.3 (95% confidence interval = 1.8-9.9), P = 0.001). These results were consistent in univariate and multivariate Cox analyses, and were successfully validated in The Netherlands Cancer Institute data set. Furthermore, E2F1 expression levels correlated well with the 70-gene signature displaying the ability of selecting a common subset of patients at good prognosis. Breast cancer patients' outcome was comparably predictable by E2F1 levels, by the 70-gene signature, by the intrinsic subtype gene classification, by the wound response signature and by the recurrence score., Conclusion: Assessment of E2F1 at the mRNA level in primary breast cancer is a strong determinant of breast cancer patient outcome. E2F1 expression identified patients at low risk of metastasis irrespective of the estrogen receptor and ERBB2 status, and demonstrated similar prognostic performance to different gene expression-based predictors.

Purpose: To evaluate and validate mRNA expression markers capable of identifying patients with ErbB2-positive breast cancer associated with distant metastasis and reduced survival., Patients and Methods: Expression of 60 genes involved in breast cancer biology was assessed by quantitative real-time PCR (qrt-PCR) in 317 primary breast cancer patients and correlated with clinical outcome data. Results were validated subsequently using two previously published and publicly available microarray data sets with different patient populations comprising 295 and 286 breast cancer samples, respectively., Results: Of the 60 genes measured by qrt-PCR, urokinase-type plasminogen activator (uPA or PLAU) mRNA expression was the most significant marker associated with distant metastasis-free survival (MFS) by univariate Cox analysis in patients with ErbB2-positive tumors and an independent factor in multivariate analysis. Subsequent validation in two microarray data sets confirmed the prognostic value of uPA in ErbB2-positive tumors by both univariate and multivariate analysis. uPA mRNA expression was not significantly associated with MFS in ErbB2-negative tumors. Kaplan-Meier analysis showed in all three study populations that patients with ErbB2-positive/uPA-positive tumors exhibited significantly reduced MFS (hazard ratios [HR], 4.3; 95% CI, 1.6 to 11.8; HR, 2.7; 95% CI, 1.2 to 6.2; and, HR, 2.8; 95% CI, 1.1 to 7.1; all P < .02) as compared with the group with ErbB2-positive/uPA-negative tumors who exhibited similar outcome to those with ErbB2-negative tumors, irrespective of uPA status., Conclusion: After evaluation of 898 breast cancer patients, uPA mRNA expression emerged as a powerful prognostic indicator in ErbB2-positive tumors. These results were consistent among three independent study populations assayed by different techniques, including qrt-PCR and two microarray platforms.

Objective: Adrenomedullin (AM), a potent vasodilatator and antioxidative peptide, was shown recently to be expressed by adipose tissue. The aim of our study was to investigate the precise localization of AM within human adipose tissue, and to examine AM regulation in obesity., Design: Subcutaneous (SC) and omental (OM) adipose tissues from 9 lean and 13 obese women were profiled for AM expression changes. Preadipocytes from human adipose tissue were isolated and differentiated under defined adipogenic conditions., Methods: AM expression was analyzed by immunohistochemistry, in situ hybridization and quantitative RT-PCR., Results: A strong AM expression was observed in vessel walls, stromal cell clusters and isolated stromal cells, some of them being CD 68 positive, whereas mature adipocytes were not labeled. Calcitonin receptor-like receptor and receptor activity-modifying proteins (RAMP) 2 and RAMP 3 were expressed in vessel walls. In vitro, preadipocytes of early differentiation stages spontaneously secreted AM. No difference in AM localization was found between SC and OM adipose tissue. AM levels in SC tissue did not differ between lean and obese subjects. By contrast, AM levels in OM tissue were significantly higher in obese as compared with lean women. Moreover, we found a positive relationship between OM AM and tumor necrosis factor alpha mRNA levels and AM-immunoreactive area in OM tissue followed the features of the metabolic syndrome., Conclusion: Stromal cells from human adipose tissue, including macrophages, produce AM. Its synthesis increased in the OM territory during obesity and paralleled the features of the metabolic syndrome. Therefore, AM should be considered as a new member of the adipokine family.

Objective: Data from rodents provide evidence for a causal role of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD-1) in the development of obesity and its complications. In humans, 11beta-HSD-1 is increased in subcutaneous adipose tissue (SAT) of obese patients, and higher adipose 11beta-HSD-1 was associated with features of the metabolic syndrome. To date, there is no evidence for an increased expression of 11beta-HSD-1 in human visceral adipose tissue (VAT), although VAT is the major predictor for insulin resistance and the metabolic syndrome., Research Methods and Procedures: 11beta-HSD-1 and hexose-6-phosphate dehydrogenase (the enzyme responsible for the synthesis of nicotinamide adenine dinucleotide phosphate, the cofactor required for 11beta-HSD-1 oxoreductase activity) mRNA levels were measured using real-time quantitative reverse transcriptase-polymerase chain reaction in abdominal SAT and VAT biopsies obtained from 10 normal-weight and 12 obese women. Adiponectin mRNA was used as an internal control., Results: 11beta-HSD-1 mRNA concentrations were significantly increased in both SAT and VAT of obese patients (720% and 450% of controls, respectively; p < 0.05) and correlated with hexose-6-phosphate dehydrogenase mRNA levels. The level of VAT 11beta-HSD-1 mRNA correlated with anthropometric parameters: BMI (r = 0.41, p = 0.05), waist circumference (r = 0.44, p = 0.04), abdominal sagittal diameter (r = 0.51, p = 0.02), and percentage fat (r = 0.51, p = 0.02)., Discussion: Our results demonstrate for the first time that 11beta-HSD-1 mRNA expression is increased in VAT from obese patients. They strengthen the importance of 11beta-HSD-1 in human obesity and its associated complications and suggest the need of clinical studies with specific 11beta-HSD-1 inhibitors.

ERBB2 (HER2/Neu) gene amplification and overexpression is associated with increased risk of metastases and shorter survival in breast cancer. Tyrosine 1248 is a major phosphorylation site of ERBB2 and reflects the activation status of the receptor. The aim of this study was to investigate the relationships between quantitative levels of pY1248-ERBB2 (p-ERBB2) and the expression of epidermal growth factor receptor (EGFR)-family members, and whether p-ERBB2 could provide additional prognostic value compared with established prognostic markers. For this purpose we developed a highly sensitive chemiluminescence-linked immunoassay (CLISA) and detected p-ERBB2 levels in 70 primary breast cancer biopsies. Phosphorylated ERBB2 correlated with EGFR and ERBB2, and inversely with oestrogen receptor (ER), progesterone receptor (PgR) and ERBB4 expression levels. Additionally, p-ERBB2 was associated with poor clinical outcome in univariate and multivariate Cox regression analysis. Further studies are needed to evaluate the predictive value of p-ERBB2.

The assessment of ERa, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERa, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERa, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.

Introduction: Gene expression profiling has been successfully used to classify breast cancer into clinically distinct subtypes, and to predict the risk of recurrence and treatment response. The aim of this study was to investigate whether the gene expression profile (GEP) detected in a core biopsy (CB) is representative for the entire tumor, since CB is an important tool in breast cancer diagnosis. Moreover, we investigated whether performing CBs prior to the surgical excision could influence the GEP of the respective tumor., Methods: We quantified the RNA expression of 60 relevant genes by quantitative real-time PCR in paired CBs and surgical specimens from 22 untreated primary breast cancer patients. Subsequently, expression data were compared with independent GEPs obtained from tumors of 317 patients without preceding CB., Results: In 82% of the cases the GEP detected in the CB correlated very well with the corresponding profile in the surgical sample (rs > or = 0.95, p < 0.001). Gene-by-gene analysis revealed four genes significantly elevated in the surgical sample compared to the CB; these comprised genes mainly involved in inflammation and the wound repair process as well as in tumor invasion and metastasis., Conclusion: A GEP detected in a CB are representative for the entire tumor and is, therefore, of clinical relevance. The observed alterations of individual genes after performance of CB deserve attention since they might impact the clinical interpretation with respect to prognosis and therapy prediction of the GEP as detected in the surgical specimen following CB performance.

Introduction: Akt1, Akt2 and Akt3 kinases are downstream components of phosphoinositol 3-kinase derived signals from receptor tyrosine kinases, which influence cell growth, proliferation and survival. Akt2 overexpression and amplification have been described in breast, ovarian and pancreatic cancers. The present study was designed to investigate the prognostic significance of activated Akt in primary breast cancer and its association with other tumour biomarkers., Methods: Using a two-site chemiluminescence-linked immunosorbent assay, we measured the quantitative expression levels of total phosphorylated (P-S473) Akt (Akt1/Akt2/Akt3) on cytosol fractions obtained from fresh frozen tissue samples of 156 primary breast cancer patients., Results: Akt phosphorylation was not associated with nodal status or ErbB-2 protein expression levels. High levels of phosphorylated Akt correlated (P < 0.01) with poor prognosis, and the significance of this correlation increased (P < 0.001) in the subset of patients with ErbB-2 overexpressing tumours. In addition, phosphorylated Akt was found to be associated with mRNA expression levels of several proliferation markers (e.g. thymidylate synthase), measured using quantitative real-time RT-PCR., Conclusion: Our findings demonstrate that, in breast cancer patients, Akt activation is associated with tumour proliferation and poor prognosis, particularly in the subset of patients with ErbB2-overexpressing tumours.

In many human cancers, the INK4A locus is frequently mutated by homozygous deletions. By alternative splicing this locus encodes two non-related tumor suppressor genes, p16(INK4A) and p14(ARF) (p19(ARF) in mice), which regulate cell cycle and cell survival in the retinoblastoma protein (pRb) and p53 pathways, respectively. In mice, the role of p16(INK4A) as the critical tumor suppressor gene at the INK4A locus was challenged when it was found that p19(ARF) only knock-out mice developed tumors, including gliomas. We have analysed the genetic status of the INK4A locus in 105 primary gliomas using both microsatellite mapping (MSM) and quantitative real-time PCR (QRT-PCR). Comparison of the results of the two methods revealed agreement in 67% of the tumors examined. In discordant cases, fluorescence in situ hybridization (FISH) analysis was always found to support QRT-PCR classification. Direct assessment of p14(ARF) exon 1beta, p16(INK4A) exon 1alpha and exon 2 by QRT-PCR revealed 43 (41%) homozygous and eight (7%) hemizygous deletions at the INK4A locus. In 49 (47%) gliomas, both alleles were retained. In addition, QRT-PCR, but not MSM, detected hyperploidy in five (5%) tumors. Deletion of p14(ARF) was always associated with co-deletion of p16(INK4A) and increased in frequency upon progression from low to high grade gliomas. Shorter survival was associated with homozygous deletions of INK4A in the subgroup of glioblastoma patients older than 50 years of age (P=0.025, Anova test single factor, alpha=0.05).

P16 and P14ARF are two tumor suppressors encoded by the locus ink4a-arf which is frequently deleted in human tumors. Recent experiments performed with mouse embryonic fibroblasts have shown that P14ARF is an upstream regulator of the P53 pathway. This raises the question as to whether in human tumors the loss of p14arf and mutation of p53 are mutually exclusive events which segregate with genetic alterations at other loci. To examine this question we performed a multigenic analysis on 29 gliomas. We analysed p53 and p14arf in relation with five other genetic loci encoding the most frequently mutated genes in human gliomas: cdkn2a, mdm2, egfr, pten and the chromosomal regions 10q23.3 and 10q25-26. Our study shows for the first time that p53 mutations and p14arf deletions appear mutually exclusive in human glioblastoma, suggesting that they may be functionally redundant in glioma tumorigenesis. The P53 pathway is, therefore, disrupted in 81.8% of malignant gliomas (WHO grades III and IV), either by mutation of the p53 gene (31.8%) or by p14arf deletion (54.5%). These tumors further showed MDM2 overexpression (9.1%), egfr oncogene amplification/egfr overexpression (50%), pten mutations (27.3%) and loss of heterozygosity (LOH) at the chromosomal regions 10q23.3 (86.4%) and 10q25-26 (100%). These alterations did not segregate with p53 mutations or p14arf deletions, while p14arf and cdkn2a were always deleted.

Ageing can be defined as the time-dependent decline of physiological functions of an organism. The molecular causes for the ageing process are multiple, involving both genetic and environmental factors. It has been proposed that antioxidants may positively influence the ageing process, protecting the organism against free radical-induced damage. Here we show that the antioxidant N-acetylcysteine (NAC) has a life-extending effect on Drosophila melanogaster. Dietary uptake of NAC results in a dose-dependent increase in median and maximum life span. Flies fed on 1 mg/ml NAC food live 16.6% longer; at 10 mg/ml, life span increases by 26.6%. We have examined the effect of NAC treatment on protein and RNA levels: we observe an NAC-dependent increase in absolute amounts of total RNA and ribosomal RNA, but no differences in protein levels. The NAC effect on longevity may involve differential expression of specific mRNA genes, as suggested by RNA finger-printing experiments.

In all organisms the process of aging is characterized by a gradual loss of homeostatic maintenance functions and physiological fitness. This might be caused-in part-by a down-regulation of important housekeeping and maintenance genes. In our previous work we have shown that in Drosophila melanogaster aging is accompanied by a decrease in steady-state levels of most RNA species. This includes structural genes and housekeeping genes, though at a different level. Here we have examined age-related changes in the mRNA levels of the gene family encoding the six Drosophila actin genes. Two actin isoforms code for cytoskeletal actins and represent housekeeping genes, whereas the four muscle actin genes are expressed tissue-specifically in differentiated muscles. The results suggest that the six actin genes are differentially regulated during aging as well. The two cytoskeletal actin genes show little changes in their steady-state mRNA levels. Drastic changes are observed, however, in the mRNAs coding for muscle actins, particularly the mRNAs coding for the jump and flight muscle actins.