Martin Nau

Frequent overexpression of ETS-related gene-1 (ERG1) in prostate cancer transcriptome.

Transcription factors encoded by the ETS family of genes are central in integrating signals that regulate cell growth and differentiation, stress responses, and tumorigenesis. This study, analysing laser microdissected paired benign and malignant prostate epithelial cells from prostate cancer (CaP) patients (n=114; 228 specimen) by GeneChip and quantitative real-time RT–PCR, identifies ETS-related gene (ERG), a member of the ETS transcription factor family, as the most frequently overexpressed proto-oncogene in the transcriptome of malignant prostate epithelial cells. Combined quantitative expression analysis of ERG with two other genes commonly overexpressed in CaP, AMACR and DD3, revealed overexpression of at least one of these three genes in virtually all CaP specimen (54 of 55). Comprehensive evaluation of quantitative ERG1 expression with clinicopathological features also suggested that ERG1 expression level in prostate tumor cells relative to benign epithelial cells is indicator of disease-free survival after radical prostatectomy.

Androgen-induced expression of endoplasmic reticulum (ER) stress response genes in prostate cancer cells

Evaluations of androgen regulated gene (ARG) repertoire provide new insights into the androgen receptor (AR) mediated signaling at the transcriptional level. Definition of ARGs having critical functions in the biology of normal and malignant prostate should aid in identifying new bio-markers and therapeutic targets for prostate cancer (CaP). Using Affymetrix HuGene FL oligonucleotide arrays, temporal expression profiles of ARGs in widely used hormone responsive LNCaP cells, were analysed by hierarchical clustering methods and functional classification. ARGs in response to different androgen concentrations showed temporal co-regulation of genes involved in specific biochemical pathways. This study focuses on our new observations of the coordinated androgen induction of genes (NDRG1, PDIR, HERPUD1, ORP150) involved in the endoplasmic reticulum (ER) stress response pathway. Expression analysis of the two selected ER stress responsive genes, NDRG1 and HERPUD1 in primary CaPs revealed a significantly reduced tumor associated expression. Intriguing linkage of the androgen signaling to ER stress responsive genes, a protective response to protein unfolding or protein damage resulting from cellular stress signals, suggests that androgens may induce such stress signals in CaP cells. Decreased CaP associated expression of two ER stress responsive genes also suggests that possible abrogation of this pathway in prostate tumorigenesis.

Expression of genes associated with immunoproteasome processing of major histocompatibility complex peptides is indicative of protection with adjuvanted RTS,S malaria vaccine

BACKGROUND Patterns of expressed genes in the peripheral blood mononuclear cells of persons who were receiving RTS,S/AS01 or RTS,S/AS02 malaria vaccine and were undergoing experimental challenge with mosquito-borne falciparum malaria were examined to identify markers associated with protection. METHODS Thirty-nine vaccine recipients were assessed at study entry; on the day of the third vaccination; at 24 h, 72 h, and 2 weeks after vaccination; and on day 5 after challenge. Of 39 vaccine recipients, 13 were protected and 26 were not. Eleven vaccine recipients exhibited delayed onset of parasitemia. All infectivity control subjects developed parasitemia. Prediction analysis of microarrays identified genes corresponding with protection. Gene set enrichment analysis identified sets of genes associated with protection after the third vaccination and before challenge. RESULTS After the third vaccination and before challenge, differential expression of genes in the immunoproteasome pathway distinguished protected and nonprotected persons. At 5 days after challenge, differential expression of genes associated with programmed cell death distinguished between subjects protected and not protected from malaria blood-stage infection. CONCLUSIONS The up-regulation of genes associated with the efficient processing of major histocompatibility complex peptides suggests a potential role of the vaccine in conferring major histocompatibility complex class 1-mediated protection and may represent a useful surrogate marker of vaccine efficacy without the need for challenge.

Impact of viral infection on the gene expression profiles of proliferating normal human peripheral blood mononuclear cells infected with HIV type 1 RF

Exploiting the power of high-density gene arrays, the simultaneous expression analysis of 5600 cellular genes was executed on proliferating peripheral blood mononuclear cells (PBMCs) from three normal human donors that were infected in vitro with the T cell tropic laboratory strain of HIV-1, RF. Profiles of expressed genes were assessed at 1, 12, 24, 48, and 72 hr postinfection and compared with those of matched uninfected PBMCs. Viral infection resulted in an overall increase in the number of genes expressed with peaks of expression at 1, 12, and 48 hr postinfection. Functional clustering of genes whose expression level in infected PBMCs varied by 2-fold or greater from levels in the controls indicated that cellular activation markers, proteins associated with immune cell function and with transcription and translation, exhibited increased expression subsequent to viral infection. Gene families exhibiting a decline in gene expression were confined to the 72 hr time point and included genes associated with catabolism and a subset of genes involved with cell signaling and synthetic pathways. Self-organizing map (SOM) cluster analysis identified temporal patterns of coordinated gene expression in infected PBMCs including genes associated with the immune response, the cytoskeleton, and ribosomal subunit structural proteins required for protein synthesis.

Patterns of gene expression in peripheral blood mononuclear cells of rhesus macaques infected with SIVmac251 and exhibiting differential rates of disease progression.

Using the Affymetrix HuGeneFL GeneChip, the global expression patterns of genes in the peripheral blood mononuclear cells (PBMCs) of rhesus macaques, infected with SIVmac251 and exhibiting rapid, typical, or slow rates of disease progression, were examined. Assessments of the change in gene expression (fold change), the temporal coordination of gene expression (self-organizing map analysis), and the similarities and significant differences in gene expression across the groups were performed on samples taken before infection and 3 and 7 weeks postinfection. An upregulation of the p27 interferon-inducible gene and of genes associated with cellular activation and immune response was observed in all three groups. Rapidly progressing animals exhibited a modest number of genes with a change in expression of 3-fold or greater, typically progressing animals exhibited the greatest number, and slowly progressing animals exhibited the fewest. Self-organizing map cluster analysis indicated that rapidly progressing animals exhibited the least coordinated gene expression over the three study time points, typically progressing animals exhibited a moderate degree, and animals with slow progression exhibited the most coordinated gene expression. Mann-Whitney U analysis indicated that differences in gene expression were most pronounced between the rapidly and slowly progressing groups and least pronounced between the rapidly and typically progressing animals. These observations elucidate distinct features of gene expression in animals with different rates of disease progression.

HIV DNA Set Point is Rapidly Established in Acute HIV Infection and Dramatically Reduced by Early ART

HIV DNA is a marker of HIV persistence that predicts HIV progression and remission, but its kinetics in early acute HIV infection (AHI) is poorly understood. We longitudinally measured the frequency of peripheral blood mononuclear cells harboring total and integrated HIV DNA in 19 untreated and 71 treated AHI participants, for whom 50 were in the earliest Fiebig I/II (HIV IgM −) stage, that is ≤ 2 weeks from infection. Without antiretroviral therapy (ART), HIV DNA peaked at 2 weeks after enrollment, reaching a set-point 2 weeks later with little change thereafter. There was a marked divergence of HIV DNA values between the untreated and treated groups that occurred within the first 2 weeks of ART and increased with time. ART reduced total HIV DNA levels by 20-fold after 2 weeks and 316-fold after 3 years. Therefore, very early ART offers the opportunity to significantly reduce the frequency of cells harboring HIV DNA.

Relationship between chloroquine toxicity and iron acquisition in Saccharomyces cerevisiae.

ABSTRACT Chloroquine is one of the most effective antimalarials, but resistance to it is becoming widespread. However, we do not fully understand either the drugs mode of action or the mechanism of resistance. In an effort to expand our understanding of the mechanism of action and resistance associated with chloroquine, we used Saccharomyces cerevisiae as a model eukaryotic system. To aid in the discovery of potential drug targets we applied the transcriptional profiling method to identify genes transcriptionally responsive to chloroquine treatment in S. cerevisiae. Among the genes that were differentially expressed with chloroquine treatment were a number of metal transporters involved in iron acquisition (SIT1, ARN2, ARN4, and SMF2). These genes exhibit similar expression patterns, and several are known to be regulated by AFT1, a DNA binding protein, which responds to iron levels in the cell. We investigated the role of chloroquine in iron metabolism by using a variety of approaches, including pharmacological, genetic, and biochemical techniques. For these experiments, we utilized yeast lacking the major iron uptake pathways (FET3 and FET4) and yeast deficient in SIT1, encoding the major up-regulated iron siderophore transporter. Our experiments show that yeast genetically or environmentally limited in iron availability has increased sensitivity to chloroquine in pharmacological assays and that the addition of iron rescues these cells from chloroquine killing. 55FeCl3 accumulation was inhibited in the presence of chloroquine, and kinetic analysis demonstrated that inhibition was competitive. These results are consistent with deprivation of iron as a mechanism of chloroquine killing in yeast.

Silencing of Lactotransferrin expression by methylation in prostate cancer progression.

Background: Cancer cells gain selection advantages by the coordinated silencing of protective and by the activation of cell proliferation/cell survival genes. Evaluations of epithelial cell transcriptome of benign and malignant prostate glands by laser capture microdissection (LCM) identified Lactotransferrin (LTF) as the most significantly downregulated gene in prostate cancer (CaP) cells (P

Distinct gene-expression profiles associated with the susceptibility of pathogen-specific CD4 T cells to HIV-1 infection.

In HIV infection, CD4 responses to opportunistic pathogens such as Candida albicans are lost early, but CMV-specific CD4 response persists. Little is currently known about HIV infection of CD4 T cells of different pathogen/antigen specificities. CFSE-labeled PBMCs were stimulated with CMV, tetanus toxoid (TT), and C albicans antigens and subsequently exposed to HIV. HIV infection was monitored by intracellular p24 in CFSE(low) population. We found that although TT- and C albicans-specific CD4 T cells were permissive, CMV-specific CD4 T cells were highly resistant to both R5 and X4 HIV. Quantification of HIV DNA in CFSE(low) cells showed a reduction of strong-stop and full-length DNA in CMV-specific cells compared with TT- and C albicans-specific cells. β-Chemokine neutralization enhanced HIV infection in TT- and C albicans-specific cells, whereas HIV infection in CMV-specific cells remained low despite increased entry by β-chemokine neutralization, suggesting postentry HIV restriction by CMV-specific cells. Microarray analysis (Gene Expression Omnibus accession number: GSE42853) revealed distinct transcriptional profiles that involved selective up-regulation of comprehensive innate antiviral genes in CMV-specific cells, whereas TT- and C albicans-specific cells mainly up-regulated Th17 inflammatory response. Our data suggest a mechanism for the persistence of CMV-specific CD4 response and earlier loss of mucosal Th17-associated TT- and C albicans-specific CD4 response in AIDS.

Transcriptome analyses of benign and malignant prostate epithelial cells in formalin-fixed paraffin-embedded whole-mounted radical prostatectomy specimens

Formalin-fixed paraffin-embedded (FFPE) prostate specimens are rich sources of molecular pathological information. However, FFPE-based microarray analysis of tissue samples may be hampered by the degradation and chemical alteration of RNA molecules due to the preservation procedure. In this report, we employed a probe analyses of Affymetrix oligonucleotide arrays at individual probe level to compensate for the potential loss of gene identifications associated with compromised mRNA quality in FFPE preparations. Furthermore, to increase the sample quality, we utilized laser capture microdissection of prostate tumor and benign epithelial cells. Remarkably, combination of these approaches recapitulated the common prostate cancer-associated gene expression alteration. Identification of prostate cancer associated-gene expression alterations such as AMACR, Kallikrein gene family and genes associated with androgen signaling such as PDEF and STEAP were consistent with previous findings reported in prostate cancer. These data suggest that combination of laser capture dissection with computational enhancement of microarray data may be useful for the assessment of gene expression changes in FFPE prostate cancer specimens.