Relevant Molecular Changes Research Articles

DNA methylation changes in glial cells of the normal-appearing white matter in Multiple Sclerosis patients

ABSTRACT Multiple Sclerosis (MS), the leading cause of non-traumatic neurological disability in young adults, is a chronic inflammatory and neurodegenerative disease of the central nervous system (CNS). Due to the poor accessibility to the target organ, CNS-confined processes underpinning the later progressive form of MS remain elusive thereby limiting treatment options. We aimed to examine DNA methylation, a stable epigenetic mark of genome activity, in glial cells to capture relevant molecular changes underlying MS neuropathology. We profiled DNA methylation in nuclei of non-neuronal cells, isolated from 38 post-mortem normal-appearing white matter (NAWM) specimens of MS patients (n = 8) in comparison to white matter of control individuals (n = 14), using Infinium MethylationEPIC BeadChip. We identified 1,226 significant (genome-wide adjusted P-value < 0.05) differentially methylated positions (DMPs) between MS patients and controls. Functional annotation of the altered DMP-genes uncovered alterations of processes related to cellular motility, cytoskeleton dynamics, metabolic processes, synaptic support, neuroinflammation and signaling, such as Wnt and TGF-β pathways. A fraction of the affected genes displayed transcriptional differences in the brain of MS patients, as reported by publically available transcriptomic data. Cell type-restricted annotation of DMP-genes attributed alterations of cytoskeleton rearrangement and extracellular matrix remodelling to all glial cell types, while some processes, including ion transport, Wnt/TGF-β signaling and immune processes were more specifically linked to oligodendrocytes, astrocytes and microglial cells, respectively. Our findings strongly suggest that NAWM glial cells are highly altered, even in the absence of lesional insult, collectively exhibiting a multicellular reaction in response to diffuse inflammation.

Tumor microenvironment in salivary gland carcinomas: Consequences for new therapeutic concepts

Salivary gland carcinomas (SGCs) are rare tumors which represent achallenge for diagnosis and therapy due to their histological diversity and the different disease courses depending on the respective subtype. Little is known about the composition of the tumor microenvironment in SGCs. Amore comprehensive understanding of the relevant molecular changes and immunological processes of the tumor and surrounding stroma could help to improve therapeutic efficiency, for example by adjuvant immunomodulation. This manuscript highlights recent studies analyzing the composition of the tumor microenvironment in salivary gland carcinomas. The tumor microenvironment displays asignificant diversity in the composition of immune cells among different tumor entities. In one third of the SGCs, an expression of cell surface molecule LAG3 on tumor infiltrating lymphocytes could be observed. LAG3-similar to CTLA‑4 and PD-1-inhibits cellular proliferation, activation, and homeostasis of antitumor-effective Tcells. Especially, prognostically less favorable entities such as salivary duct carcinomas and adenocarcinomas NOS (not otherwise specified) yielded higher expressions. LAG3 is particularly detectable in aggressive entities and advanced tumors. Hence, LAG3 inhibition poses apotential targeted therapy for advanced and metastatic SGCs.

Abstract 336: A recombinant human protein targeting HER2 overcomes drug resistance in HER2-positive breast cancer

Abstract Background &amp; Goals: HER2 is an oncogenic receptor tyrosine kinase (RTK) implicated in several types of human cancer. It is strongly expressed in about 20% of breast cancer (BC), known as HER2-positive BC (HER2-BC), due to gene amplification. HER2 amplification or overexpression is a strong predictor of poor disease prognosis. Several HER2-targeting drugs are available for treating HER2-BC, including monoclonal antibodies trastuzumab (Ttzm) and pertuzumab, T-DM1 (Ttzm coupled to a microtubule inhibitor), and tyrosine kinase inhibitors (TKIs) lapatinib and neratinib. While these agents have greatly improved disease outcomes, primary and acquired drug resistance, including cross-resistance, is common. Overcoming drug resistance remains a major unmet medical need in HER2-BC. Notably, HER2 remains overexpressed in drug-resistant HER2-BC cells. We recently found that recombinant human peptidase D (PEPD), also known as prolidase, strongly downregulates HER2 and EGFR in cancer cells in vitro and in vivo via its binding to HER2 and EGFR. The enzymatic activity of PEPD plays no role in its modulation of HER2 and EGFR, and we subsequently focused on recombinant PEPDG278D, an enzymatically inactive mutant (point mutation at codon 278). Our objective in the present study is to determine whether PEPDG278D is active in preclinical models of HER2-BC resistant to clinically available HER2 inhibitors and if so, its mechanism of action. Results: PEPDG278D was exceedingly effective against HER2-BC cells that are resistant to Ttzm and other HER2 inhibitors. The cells carried activating PIK3CA mutations, low expression of PTEN, and/or cyclin E overexpression. All the cell lines also overexpressed MUC4 which shields HER2 from Ttzm and stabilizes HER2. However, PEPDG278D rapidly bound to and freed HER2 from MUC4, and rapidly disrupted the interaction of HER2 with other RTKs, including MET and IGF1R. PEPDG278D subsequently induced persistent downregulation of HER2 via internalization and lysosomal degradation. PEPDG278D also downregulated EGFR. Thus, PEPDG278D suppresses multiple RTKs either directly or indirectly in HER2-BC cells. In contrast, Ttzm cannot perform any of these functions of PEPDG278D. PEPDG278D was highly active in mouse tumor models of HER2-BC resistant to Ttzm and also enhanced the therapeutic efficacy of paclitaxel. However, adverse effects of PEPDG278D were not detected in the animal studies. Conclusions &amp; Therapeutic Relevance: The therapeutic activity of PEPDG278D highlights that HER2 remains a critical target in drug-resistant HER2-BC. The strong therapeutic activity of PEPDG278D in preclinical models of drug-resistant HER2-BC, which carry clinically relevant molecular changes that confer resistance to current HER2 inhibitors provides the scientific premise for potential evaluation of this recombinant human protein in patients with drug-resistant HER2-BC. Citation Format: Lu Yang, yun Li, Arup Bhattacharya, yuesheng Zhang. A recombinant human protein targeting HER2 overcomes drug resistance in HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 336.

CAFE MOCHA: An Integrated Platform for Discovering Clinically Relevant Molecular Changes in Cancer-An Example of Distant Metastasis- and Recurrence-Linked Classifiers in Head and Neck Squamous Cell Carcinoma.

With large amounts of multidimensional molecular data on cancers generated and deposited into public repositories such as The Cancer Genome Atlas and International Cancer Genome Consortium, a cancer type agnostic and integrative platform will help to identify signatures with clinical relevance. We devised such a platform and showcase it by identifying a molecular signature for patients with metastatic and recurrent (MR) head and neck squamous cell carcinoma (HNSCC). We devised a statistical framework accompanied by a graphical user interface-driven application, Clinical Association of Functionally Established MOlecular CHAnges ( CAFE MOCHA; https://github.com/binaypanda/CAFEMOCHA), to discover molecular signatures linked to a specific clinical attribute in a cancer type. The platform integrates mutations and indels, gene expression, DNA methylation, and copy number variations to discover a classifier first and then to predict an incoming tumor for the same by pulling defined class variables into a single framework that incorporates a coordinate geometry-based algorithm called complete specificity margin-based clustering, which ensures maximum specificity. CAFE MOCHA classifies an incoming tumor sample using either its matched normal or a built-in database of normal tissues. The application is packed and deployed using the install4j multiplatform installer. We tested CAFE MOCHA in HNSCC tumors (n = 513) followed by validation in tumors from an independent cohort (n = 18) for discovering a signature linked to distant MR. CAFE MOCHA identified an integrated signature, MR44, associated with distant MR HNSCC, with 80% sensitivity and 100% specificity in the discovery stage and 100% sensitivity and 100% specificity in the validation stage. CAFE MOCHA is a cancer type and clinical attribute agnostic statistical framework to discover integrated molecular signatures.

Nicotine and caffeine modulate haloperidol-induced changes in postsynaptic density transcripts expression: Translational insights in psychosis therapy and treatment resistance

Caffeine and nicotine are widely used by schizophrenia patients and may worsen psychosis and affect antipsychotic therapies. However, they have also been accounted as augmentation strategies in treatment-resistant schizophrenia. Despite both substances are known to modulate dopamine and glutamate transmission, little is known about the molecular changes induced by these compounds in association to antipsychotics, mostly at the level of the postsynaptic density (PSD), a site of dopamine-glutamate interplay. Here we investigated whether caffeine and nicotine, alone or combined with haloperidol, elicited significant changes in the levels of both transcripts and proteins of the PSD members Homer1 and Arc, which have been implicated in synaptic plasticity, schizophrenia pathophysiology, and antipsychotics molecular action. Homer1a mRNA expression was significantly reduced by caffeine and nicotine, alone or combined with haloperidol, compared to haloperidol. Haloperidol induced significantly higher Arc mRNA levels than both caffeine and caffeine plus haloperidol in the striatum. Arc mRNA expression was significantly higher by nicotine plus haloperidol vs. haloperidol in the cortex, while in striatum gene expression by nicotine was significantly lower than that by both haloperidol and nicotine plus haloperidol. Both Homer1a and Arc protein levels were significantly increased by caffeine, nicotine, and nicotine plus haloperidol. Homer1b mRNA expression was significantly increased by nicotine and nicotine plus haloperidol, while protein levels were unaffected. Locomotor activity was not significantly affected by caffeine, while it was reduced by nicotine. These data indicate that both caffeine and nicotine trigger relevant molecular changes in PSD sites when given in association with haloperidol.

Down-regulation of miR-146b-5p promotes malignant transformation of fusion cells after co-culture of macrophages with glioma stem cells in vitro

Objective: To observe mutual interactions between macrophages(Mφ) and glioma stem cells (GSCs)in dual-color tracing model in vitro, to identify the biological characteristics of fusion cells in multiple levels, and to analysis the relevant molecular mechanisms. Methods: Red fluorescent protein(RFP) gene was stably transfected into human GSCs cell line SU4. Mφ cells were obtained from Balb/c nude mice with enhanced green fluorescent protein (EGFP) expression. Then two cells were co-cultured in dual-color tracing platform. RFP/EGFP double positive cells with high proliferation ability were mono-cloned. The fusion cells were verified by Western blot, fluorescence in situ hybridization, immunocytochemistry and chromosome karyotype analysis.The biological characteristics of fusion cells were further analyzed, together with relevant molecular changes. Results: RFP / EGFP double positive cells were obtained through in vitro co-culture. RFP and EGFP coexpression were proved at transcriptional and translational levels in the fusion cells. They also co-expressed GSCs marker Nestin and Mφ marker CD68, and karyotype analysis showed two types of characteristic chromosomes, which confirmed that the fusion cells originated from spontaneous fusion between SU4-RFP and Mφ.Fusion cell proliferation rate and invasion ability were higher than SU4-RFP, which were relevant with down-regulation of miR-146b-5p and activation of STAT3. Fusion cells transfected with miR-146b-5p showed a higher apoptosis rate(18.83%) and lower tumor formation(4/5). Conclusion: Mφ could fuse with GSCs spontaneously in local tumor micro-environment. The proliferation and invasion abilities of fusion cells were higher than their parent cells, which were relevant with down-regulation of miR-146b-5p and activation of STAT3. It revealed the possible mechanisms of malignant progression of gliomas.

Chronic anosmia induces depressive behavior and reduced anxiety via dysregulation of glucocorticoid receptor and corticotropin-releasing hormone in a mouse model

Olfactory loss is highly prevalent, and comorbid mood disorders are common. Considering olfactory input is highly interconnected with the limbic system, and that the limbic system manages mood, it is predictable that impairments in the sense of smell may result in mood changes. Chronic olfactory deficits were induced by repeated intranasal irrigation of ZnSO4 for 12 weeks in BALB/c mice. H&E staining, OMP staining, and potato chip finding test were performed to confirm olfactory loss. Tail suspension, forced swim, and splash tests were performed to evaluate depression, as well as open field, elevated plus maze tests were applied to assess anxiety. The mRNA levels of glucocorticoid receptor (GR) and corticotropin releasing hormone (CRH) were measured by real-time PCR to confirm relevant molecular changes. Disruption of the olfactory epithelium and olfactory loss was confirmed in histological studies and potato chip finding test. Behavioral tests show that the chronic anosmic state caused increased depression and reduced anxiety. PCR data showed that mRNA levels of GR in the hypothalamus and CRH in the amygdala were significantly decreased. These results propose that ZnSO4-induced chronic anosmia can cause a depressive and anxiolytic state via decreased hypothalamic GR and amygdalar CRH.

Chronic anosmia induces depressive behavior and reduced anxiety via dysregulation of glucocorticoid receptor and corticotropin-releasing hormone in a mouse model.

Olfactory loss is highly prevalent, and comorbid mood disorders are common. Considering olfactory input is highly interconnected with the limbic system, and that the limbic system manages mood, it is predictable that impairments in the sense of smell may result in mood changes. Chronic olfactory deficits were induced by repeated intranasal irrigation of ZnSO4 for 12 weeks in BALB/c mice. H&E staining, OMP staining, and potato chip finding test were performed to confirm olfactory loss. Tail suspension, forced swim, and splash tests were performed to evaluate depression, as well as open field, elevated plus maze tests were applied to assess anxiety. The mRNA levels of glucocorticoid receptor (GR) and corticotropin releasing hormone (CRH) were measured by real-time PCR to confirm relevant molecular changes. Disruption of the olfactory epithelium and olfactory loss was confirmed in histological studies and potato chip finding test. Behavioral tests show that the chronic anosmic state caused increased depression and reduced anxiety. PCR data showed that mRNA levels of GR in the hypothalamus and CRH in the amygdala were significantly decreased. These results propose that ZnSO4-induced chronic anosmia can cause a depressive and anxiolytic state via decreased hypothalamic GR and amygdalar CRH.

Abstract LB-072: The N-Myc transcriptional program driving the neuroendocrine prostate cancer phenotype

Abstract Although neuroendocrine prostate cancer (NEPC) rarely arises de novo, up to 30% of patients with prostate adenocarcinoma (PCa) develop NEPC features in later stages of their disease as a mechanism of treatment resistance to hormonal therapies including abiraterone and enzalutamide. NEPC is clinically more aggressive than PCa, commonly metastases to visceral organs such as liver and brain, and can be suspected in patients with progressive disease and a disproportionately low serum PSA. There is currently no effective therapy for NEPC, and most patients with NEPC survive less than one year. The development of novel treatment strategies for patients with NEPC represents a significant clinical unmet need. We have previously discovered significant over-expression and gene amplification of AURKA (encoding Aurora-A) and MYCN (encoding N-Myc) in NEPC as compared to prostate adenocarcinoma. As in neuroblastoma, N-Myc interacts with Aurora-A in NEPC which leads to a co-stabilization of both proteins that is targetable with allosteric inhibitors of Aurora A. We have also shown that ectopic expression of N-Myc induces neuroendocrine transformation of prostate adenocarcinoma cells. However, the molecular mechanisms that underlie N-Myc driven NEPC phenotype have yet to be characterized. To address this, we performed RNA-sequencing (RNAseq) and ChIP-sequencing from multiple stable prostate adenocarcinoma cells with and without N-Myc over-expression. We have identified a signature of N-Myc deregulated genes that are both biologically and clinically relevant. Based on RNAseq data from 128 clinical samples (17 NEPC, 10 castrate resistant prostate cancer 68 prostate adenocarcinoma patient tumors and 33 matched benign prostate samples) we found that majority (70%) of the N-Myc deregulated genes identified in our in vitro models distinguish the NEPC from PCa tumor samples. Based on GSEA and pathway analysis we found that N-Myc induces a profile enriched in pro-metastatic, dedifferentiation and Polycomb Repressive Complex deregulated genes and dramatically reduces AR signaling. Furthermore we show that N-Myc is recruited to AR-bound enhancers of AR target genes. To further determine the role of N-Myc in driving the NEPC phenotype we have generated transgenic mice that carry an integrated MYCN gene behind a lox-stop-lox (LSL) cassette at the ROSA26 locus, a floxed Pten locus and a tamoxifen-activated Cre recombinase driven by Tmprss2. N-Myc over-expression in the context of Ptenfl/+ leads to focal high levels of activated AKT pathway that accompany mouse high grade prostatic intraepithelial neoplasia (mHGPIN) at 3 months post-induction and other clinically relevant molecular changes. Littermates that harbor Ptenfl/+ alone do not display mHGPIN or AKT activation. In the context of Pten homozygous loss (Ptenfl/fl), N-Myc is associated with diffuse mHGPIN in the ventral and dorsolateral prostate lobes and irregular gland borders, nuclear atypia and high levels of AKT activity at 3 months (MYCN homozygous) or 6 months (MYCN heterozygous) post-induction. In conclusion, our findings support the role of N-Myc as one of the drivers of the NEPC phenotype and have the potential to ultimately lead to the identification of a new class of disease specific biomarkers and therapeutic alternatives for this aggressive subgroup of prostate cancer. Citation Format: Etienne Dardenne, Kaitlyn Gayvert, Adeline Berger, Andrea Sboner, Brian Robinson, Kenneth Hennrick, Juan Miguel Mosquera, Cynthia Cheung, Martin Eilers, Himisha Beltran, Mark A. Rubin, Olivier Elemento, David S. Rickman. The N-Myc transcriptional program driving the neuroendocrine prostate cancer phenotype. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-072. doi:10.1158/1538-7445.AM2015-LB-072

Challenges of deciphering gastric cancer heterogeneity.

Gastric cancer is in decline in most developed countries; however, it still accounts for a notable fraction of global mortality and morbidity related to cancer. High-throughput methods are rapidly changing our view and understanding of the molecular basis of gastric carcinogenesis. Today, it is widely accepted that the molecular complexity and heterogeneity, both inter- and intra-tumour, of gastric adenocarcinomas present significant obstacles in elucidating specific biomarkers for early detection of the disease. Although genome-wide sequencing and gene expression studies have revealed the intricate nature of the molecular changes that occur in tumour landscapes, the collected data and results are complex and sometimes contradictory. Several aberrant molecules have already been tested in clinical trials, although their diagnostic and prognostic utilities have not been confirmed thus far. The gold standard for the detection of sporadic gastric cancer is still the gastric endoscopy, which is considered invasive. In addition, genome-wide association studies have confirmed that genetic variations are important contributors to increased cancer risk and could participate in the initiation of malignant transformation. This hypothesis could in part explain the late onset of sporadic gastric cancers. The elaborate interplay of polymorphic low penetrance genes and lifestyle and environmental risk factors requires additional research to decipher their relative impacts on tumorigenesis. The purpose of this article is to present details of the molecular heterogeneity of sporadic gastric cancers at the DNA, RNA, and proteome levels and to discuss issues relevant to the translation of basic research data to clinically valuable tools. The focus of this work is the identification of relevant molecular changes that could be detected non-invasively.

33IN - What are the New Tools Available?

ABSTRACT Extensive sequencing, expression profiling, and recently epigenetic analyses have generated a wealth of data resulting in improved knowledge about molecular pathogenesis of all major tumor entities. Still major challenges remain: How can we translate this knowledge into clinically meaningful diagnostic targets and assays? How can molecular diagnostics contribute to improved personalized tumor therapy? Are we looking at the right collectives? Dramatic improvement has been made in clinical-diagnostic application: a nearly complete arsenal is available for detecting treatment relevant molecular changes in tumor tissue. About half of our assays is based on on-slide analyses (immunohistology, FISH/CISH) and this will remain for the foreseeable future. We have established quality assured conditions for Next Generation Sequencing even on smallest paraffin-embedded tissue specimen (Endris et al., 2014), assembled long-standing diagnostic experience, and performed roll-out and quality assurance measures. These concerted diagnostic activities are essential for recent ‘umbrella concepts’ (such as the ‘Liver Cancer Center Heidelberg’ and ‘STRICT’) joining clinical service and innovative trial concepts to efficiently allocate patients to systemic treatment options and clinical trials. These recent concepts demonstrate that current data from comprehensive profiling efforts, such as ICGC/TCGA are biased by two means: they are derived from resected tumors thus representing a better prognostic group amenable to curative treatment; furthermore DNA-based data are surrogates for therapy relevant protein changes that in frequently show significant deviations. Therefore, profiling of collectives of progressed tumors will provide better data for current and future targeted therapy. A new comprehensive concept integrating exploratory profiling, targeted molecular pathology analyses, clinical trial portefolios including recalling, active patient databases and bedside-bench translational research is necessary to improve personalized treatment approaches and will be outlined. Disclosure: The author has declared no conflicts of interest.

Increased expression of extracellular matrix metalloproteinase inducer (CD147) in multiple myeloma: role in regulation of myeloma cell proliferation

Multiple myeloma (MM) is preceded by the asymptomatic pre-malignant state, monoclonal gammopathy of undetermined significance (MGUS). Although MGUS patients may remain stable for years, they are at increased risk of progressing to MM. A better understanding of the relevant molecular changes underlying the transition from an asymptomatic to symptomatic disease state is urgently needed. Our studies show for the first time that the CD147 molecule (extracellular matrix metalloproteinase inducer) may be having an important biological role in MM. We first demonstrate that CD147 is overexpressed in MM plasma cells (PCs) vs normal and pre-malignant PCs. Next, functional studies revealed that the natural CD147 ligand, cyclophilin B, stimulates MM cell growth. Moreover, when MM patient PCs displaying bimodal CD147 expression were separated into CD147(bright) and CD147(dim) populations and analyzed for proliferation potential, we discovered that CD147(bright) PCs displayed significantly higher levels of cell proliferation than did CD147(dim) PCs. Lastly, CD147-silencing significantly attenuated MM cell proliferation. Taken together, these data suggest that the CD147 molecule has a key role in MM cell proliferation and may serve as an attractive target for reducing the proliferative compartment of this disease.

Homocysteinylated Albumin Promotes Increased Monocyte-Endothelial Cell Adhesion and Up-Regulation of MCP1, Hsp60 and ADAM17

RationaleThe cardiovascular risk factor homocysteine is mainly bound to proteins in human plasma, and it has been hypothesized that homocysteinylated proteins are important mediators of the toxic effects of hyperhomocysteinemia. It has been recently demonstrated that homocysteinylated proteins are elevated in hemodialysis patients, a high cardiovascular risk population, and that homocysteinylated albumin shows altered properties.ObjectiveAim of this work was to investigate the effects of homocysteinylated albumin - the circulating form of this amino acid, utilized at the concentration present in uremia - on monocyte adhesion to a human endothelial cell culture monolayer and the relevant molecular changes induced at both cell levels.Methods and ResultsTreated endothelial cells showed a significant increase in monocyte adhesion. Endothelial cells showed after treatment a significant, specific and time-dependent increase in ICAM1 and VCAM1. Expression profiling and real time PCR, as well as protein analysis, showed an increase in the expression of genes encoding for chemokines/cytokines regulating the adhesion process and mediators of vascular remodeling (ADAM17, MCP1, and Hsp60). The mature form of ADAM17 was also increased as well as Tnf-α released in the cell medium. At monocyte level, treatment induced up-regulation of ICAM1, MCP1 and its receptor CCR2.ConclusionsTreatment with homocysteinylated albumin specifically increases monocyte adhesion to endothelial cells through up-regulation of effectors involved in vascular remodeling.

Ischemia-Reperfusion Injury Leads to Significant Tissue Damage in Free Flap Surgery

Sir:FigureWe read with great interest the article entitled “Expression of HIF-1α in Ischemia and Reperfusion in Human Microsurgical Free Muscle Tissue Transfer” by Dragu et al. (Plast Reconstr Surg. 2011;127:2293–2300). We agree that our current knowledge of the mechanisms of ischemia-reperfusion injury in reconstructive microsurgery is based mainly on animal models, and the evaluation of the related mechanisms in human tissue is a long-neglected necessity. The hypothesis that hypoxia-inducible factor (HIF)-1α levels could be elevated in ischemia-reperfusion in human free flap tissue and could be of pathophysiologic consequence is reasonable, especially considering the regulation of HIF-1α by reactive oxygen species1 that play a pivotal role in ischemia-reperfusion injury.2 Therefore, the results of this well-executed study, at least with regard to the immunohistologic data, are unexpected but will add to our understanding of the ischemia-reperfusion–related molecular changes in free flap surgery that occur at early stages of ischemia-reperfusion injury. However, the data presented by the authors do not allow for the conclusion “that the accepted times of ischemia, generally up to 90 minutes, are not sufficient to induce pathophysiologic processes, which can ultimately lead to flap loss,” which is rebutted by our own data. We showed in our recent work that ischemia-reperfusion injury in human free flap tissue leads not only to significant infiltration of leukocytic cells and expression of the proinflammatory parameters tumor necrosis factor-α and interleukin-1β, but also to a significant increase in interstitial edema and apoptosis,3 thereby opposing the conclusion of Dragu et al. In contrast to the work of Dragu et al., who examined the effects of ischemia-reperfusion on free flap tissue 77 ± 22 minutes after ischemia-reperfusion, we examined free flap tissue before ischemia and 5 days after ischemia-reperfusion to allow enough time for the most relevant morphologic and molecular tissue changes to occur. Interestingly, the authors previously reported no changes of apoptotic parameters in free flap tissue when biopsy specimens were obtained intraoperatively after ischemia-reperfusion4 in an approach very similar to the one presented in the article under discussion. These major differences in our work seem to be related to the different time points of biopsy, and it remains doubtful whether all ischemia-reperfusion–relevant molecular changes can be detected within the short period allowed by Dragu et al. This could potentially also apply to the results obtained for HIF-1α levels and should be taken into account when interpreting the results presented in this elegant study. Yvonne Schmidt, M.D. Holger Bannasch, M.D. Steffen U. Eisenhardt, M.D. Department of Plastic and Hand Surgery, University of Freiburg Medical Center, Freiburg, Germany

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A Cross-Study Transcriptional Analysis of Parkinson's Disease

The study of Parkinson's disease (PD), like other complex neurodegenerative disorders, is limited by access to brain tissue from patients with a confirmed diagnosis. Alternatively the study of peripheral tissues may offer some insight into the molecular basis of disease susceptibility and progression, but this approach still relies on brain tissue to benchmark relevant molecular changes against. Several studies have reported whole-genome expression profiling in post-mortem brain but reported concordance between these analyses is lacking. Here we apply a standardised pathway analysis to seven independent case-control studies, and demonstrate increased concordance between data sets. Moreover data convergence increased when the analysis was limited to the five substantia nigra (SN) data sets; this highlighted the down regulation of dopamine receptor signaling and insulin-like growth factor 1 (IGF1) signaling pathways. We also show that case-control comparisons of affected post mortem brain tissue are more likely to reflect terminal cytoarchitectural differences rather than primary pathogenic mechanisms. The implementation of a correction factor for dopaminergic neuronal loss predictably resulted in the loss of significance of the dopamine signaling pathway while axon guidance pathways increased in significance. Interestingly the IGF1 signaling pathway was also over-represented when data from non-SN areas, unaffected or only terminally affected in PD, were considered. Our findings suggest that there is greater concordance in PD whole-genome expression profiling when standardised pathway membership rather than ranked gene list is used for comparison.

Molecular study on differentiation-associated genes involved in both malignant progression of glioma and differentiation of human fetal neural stem cells

OBJECTIVE It is unclear whether differentiation disturbances or deregulation of neural stem cells (NSCs) are the early key steps for gliomagenesis and tumor development. Furthermore, relevant molecular changes and gene-regulation pathways are unknown. This study focused on screening and validating differentiation-associated genes from both human NSCs and glioma cells with malignant progression, for the purpose of offering an experimental basis for the cellular origin of gilomas and molecular pathology of gliomagenesis. METHODS The differential-gene expression profiles of malignant progression of gliomas were established, then the differentiation related genes were screened out with a bioinformatics analysis. Expression levels of these genes was further analyzed in cultured human fetal NSCs undergoing differentiation processes with a semi-quantitative RT-PCR assay. RESULTS Eight genes were screened out from the gene-expression profiling of which the expression levels were associated with the differentiation processes of NSCs, namely CXCR4, TN-C, GLT1, IL1-RI, EGFR-8, CDC2, Ndr3 and MAPKK4. Three of them, ie., GLT1, CDC2 and MAPKK4, were further analyzed, showing that expression levels decreased with the differentiation processes of NSCs, and increased with the malignant progression of ganglioglioma. CONCLUSION Three differentiation associated genes were found negatively associated with NSCs differentiation and positively associated with malignant progression of gliomas, suggesting that differentiation disturbances of neural stem cells may be involved in oncogenesis, and that further studies on their roles in gliomagenesis should be conducted.

Induction of colon cancer cell death by 7-hydroxystaurosporine (UCN-01) is associated with increased p38 MAPK and decreased Bcl-xL.

UCN-01, a selective inhibitor of protein kinase C, is known to inhibit the growth of cancer cells. Although it is currently undergoing clinical evaluation, information about its effect on human colon cancer is limited and the mechanism responsible is lacking. The objective of this study was to examine the cytotoxicity of UCN-01 to human colon cancer cells in vitro and its effect on the apoptotic molecules. HT-29, a radiation- and chemotherapy-resistant human colon cancer cell, was used in the study. Cell death/apoptosis was determined by the MTT assay and DNA fragmentation measurement. NF-kappaB activity was measured by an enzyme immunoassay method. Western blot was employed to examine the expression of relevant apoptotic molecules. The result showed that UCN-01 could induce apoptosis of human colon cancer cells in a time- and dose-dependent manner. It markedly reduced the expression of Bcl-xL, but enhanced the level of p38 MAPK. In addition to Bcl-xL and p38 MAPK, UCN-01 also increased both caspase-3 and peroxisome proliferator activated receptor gamma protein levels. HT-29 cells transfected with exogenous Bcl-xL showed a significant increase in NF-kappaB activity and prevented apoptosis induced by UCN-01. The overexpression of Bcl-xL also reversed other relevant molecular changes observed in UCN-01-treated cells. In conclusion, UCN-01 exerted an antitumor effect in human colon cancer cells by inducing apoptosis. The mechanism responsible appeared to be related to reduction of Bcl-xL and increased p38 MAPK. The overexpression of Bcl-xL can significantly prevent apoptosis induced by UCN-01.

High-Throughput Tissue Microarray Analysis to Evaluate Genes Uncovered by cDNA Microarray Screening in Renal Cell Carcinoma

Many genes and signaling pathways are involved in renal cell carcinoma (RCC) development. However, genetic tumor markers have not gained use in RCC diagnostics and prognosis prediction. Identification and evaluation of new molecular parameters are of utmost importance in cancer research and cancer treatment. Here we present a novel approach to rapidly identify clinically relevant molecular changes in cancer. To identify genes with relevance to RCC, a cDNA array analysis was first performed on 5184 cDNA clones on a filter to screen for genes with differential expression between the renal cancer cell line CRL-1933 and normal kidney tissue. There were 89 differentially expressed genes in the cancer cell line, one of them coding for vimentin, a cytoplasmic intermediate filament. In a second step, a renal cancer tissue microarray containing 532 RCC specimen was used to determine vimentin expression by immunohistochemistry. Vimentin expression was seen frequently in clear cell (51%) and papillary RCC (61%), but rarely in chromophobe RCC (4%) and oncocytomas (12%). Furthermore, vimentin expression was significantly associated with poor patient prognosis (P < 0.007) independent of grade and stage. These results obtained from minute arrayed tumor samples match well with previous findings on vimentin expression in renal tumors. It is concluded that the combination of tumor arrays and cDNA arrays is a powerful approach to rapidly identify and further evaluate genes that play a role in tumor biology.