Intratumor MAPK and PI3K signaling pathway heterogeneity in glioblastoma tissue correlates with CREB signaling and distinct target gene signatures.

Limitations in discovering useful tumor biomarkers and drug targets is not only due to patient-to-patient differences but also due to intratumor heterogeneity. Heterogeneity arises due to the genetic and epigenetic variation of tumor cells in response to microenvironmental interactions and cytotoxic therapy. We explored specific signaling pathway activation in glioblastoma (GBM) by investigating the intratumor activation of the MAPK and PI3K pathways. We present data demonstrating a striking preponderance for mutual exclusivity of MAPK and PI3K activation in GBM tissue, where MAPK activation correlates with proliferation and transcription factor CREB activation and PI3K activation correlates with CD44 expression. Bioinformatic analysis of signaling and CREB-regulated target genes supports the immunohistochemical data, showing that the MAPK-CREB activation correlates with proliferative regions. In-silico analysis suggests that MAPK-CREB signaling activates a pro-inflammatory molecular signature and correlates with a mesenchymal GBM subtype profile, while PI3K-CREB activation correlates with the proneural GBM subtype and a tumor cell invasive gene signature. Overall, the data suggests the existence of intratumor subtype heterogeneity in GBM and that using combinations of both MAPK and PI3K drug inhibitors is necessary for effective targeted therapy.

Xenome--a tool for classifying reads from xenograft samples.

MOTIVATION: Shotgun sequence read data derived from xenograft material contains a mixture of reads arising from the host and reads arising from the graft. Classifying the read mixture to separate the two allows for more precise analysis to be performed. RESULTS: We present a technique, with an associated tool Xenome, which performs fast, accurate and specific classification of xenograft-derived sequence read data. We have evaluated it on RNA-Seq data from human, mouse and human-in-mouse xenograft datasets. AVAILABILITY: Xenome is available for non-commercial use from http://www.nicta.com.au/bioinformatics.

Human aminopeptidase B (rnpep) on chromosome 1q32.2: complementary DNA, genomic structure and expression.

Aminopeptidase B (APB) is a Zn(2+)-metalloexopeptidase, which selectively removes Arg and/or Lys residues from the N-terminus of several peptide substrates. Several data strongly support the hypothesis that this enzyme could participate in the final stages of precursor processing mechanisms and/or in particular inflammatory processes and tumor developments. Therefore, we have cloned the complementary DNA encoding the human APB, a 658-residues protein, containing the canonical "HEXXH(X(18))E", a signature allowing its classification in the M1 family of metallopeptidases. The genomic structure of the human APB gene (rnpep; 1q32.1-q32.2) was also determined. rnpep is bracketed by pre-protein translocase of the inner mitochondrial membrane gene and ETS family transcription factor ELF3 gene. It spans more than 24 kbp and contains 11 exons ranging from 109 to 574 bp. Finally, expression of the human APB messenger RNA (mRNA) was investigated using a pre-made dot-blot. This mRNA seems to be ubiquitous although its expression level varies depending of the cells or tissues considered.

Expression of the ETS transcription factor ELF3 in the retinal pigment epithelium.

PURPOSE: The ETS family of transcription factors regulate several critical cellular functions. They have also been implicated in invertebrate ocular development. This work was undertaken to determine whether epithelium-specific ETS transcription factors are expressed in the retinal pigment epithelium and to investigate the possible role of these factors in retinal diseases such as age-related macular degeneration. METHODS: The expression of the epithelial ETS transcription factors ELF5, ESE3, and ELF3 was assessed by RT-PCR in the human RPE cell lines D407 and hTERT-RPE1. The full-length coding sequence of rat Elf3 was isolated with 3' rapid amplification of cDNA ends (RACE) and degenerative primers, and its expression was determined in various rat tissues, by RT-PCR and real-time PCR. A polyclonal ELF3 antibody produced from a C-terminal peptide was used to observe the distribution of the transcription factor within the retina. To assess the possible ELF3 regulation of the TIMP3 promoter, transient transfection assays were performed. Promoter activity was determined with a firefly luciferase reporter gene construct. RESULTS: The epithelium-specific ETS transcription factor ELF3 was expressed in the D407 and hTERT-RPE1 cell lines. Neither ESE3 nor ELF5 was detected in the RPE. The cloning of rat Elf3 produced two splice variants, designated Elf3a (1786 bp) and Elf3b (1855 bp). The larger form, Elf3b, contained a 69-bp insert in the coding sequence, which showed high homology to a similar insert previously identified in murine Elf3. Both splice variants were expressed in rat lung, kidney, liver, and retina, but were absent in heart tissue. Real-time PCR analysis showed the retina to contain high levels of Elf3, which was subsequently localized to the RPE. Elf3 upregulated the TIMP3 promoter, with Elf3a and -3b inducing an approximate sixfold increase in activity. CONCLUSIONS: The ELF3 transcription factor is highly expressed in the RPE and can regulate important ocular genes, such as TIMP3, in vitro. The specific expression of ELF3 in the RPE may reflect an important role for this transcription factor in retinal function. Furthermore, its regulation of TIMP3 may have implications for degenerative retinal diseases, such as age-related macular degeneration.

G2/M checkpoint gene expression in developing germ cells.

Cell cycle progression is prevented by signal transduction pathways known as checkpoints which are activated in response to replication interference and DNA damage. We cloned a G2/M cell cycle phase-related checkpoint gene from a neonatal mouse testis cDNA library which was identified as mouse claspin, a proposed adaptor protein for Chk1. As part of a study on germ cell differentiation we examined the expression of the checkpoint gene, Chk1, and claspin at 12.5 and 14.5 days post coitum (dpc) and in the post-natal phase. Chk1 mRNA expression increased from 12.5 to 14.5 dpc in female gonads and was strong in males at both time points. Claspin however, was not detected until 14.5 dpc. This suggests there may be some dissociation of claspin expression from Chk1 in fetal germ cell development. Chk1 and claspin expression was also studied in testis over the first 3 days following birth, when apoptosis regulates germ stem cell number. We modulated checkpoint-related gene expression in testis using the anti-metabolite, 5-fluorouracil, resulting in increased apoptosis and upregulation of Chk1 (P<0.0001) and Cdc2 (P<0.02) mRNA. Although we do not fully understand the role checkpoint gene expression has during mammalian germ cell development this report is the first to show the expression of checkpoint-related genes in early mammalian germ cells.

Sorsby's fundus dystrophy: what does TIMP3 tell us about general mechanisms underlying macular degeneration?

INTRODUCTION: Mutations in tissue inhibitor of metalloproteinases-3 (TIMP3) gene result in the rare autosomal dominant disease Sorsby's fundus dystrophy (SFD), which shows striking similarities to age-related macular degeneration (ARMD). METHODS: Current research is reviewed and suggests that these mutations result in the accumulation of TIMP3 in Bruch's membrane resulting in decreased turnover of the extracellular matrix and consequent thickening of Bruch's membrane. DNA analysis of ARMD patients has failed to show any significant mutations in the coding-regions of TIMP3. However, this should not be taken to imply that TIMP3 is not affected in the disease, as another possibility is that levels of TIMP3 might be elevated by other mechanisms. CONCLUSION: The finding that TIMP3 levels are elevated in simplex retinitis pigmentosa in the absence of coding mutations begs for a revaluation of its role in other retinal conditions such as ARMD.