Hypoxia-inducible factors in stem cells and cancer.

Cellular properties are influenced by complex factors inherent to their microenvironments. While oxygen deprivation (hypoxia) occurs in tumours because of rapid cell proliferation and aberrant blood vessel formation, embryonic cells develop in a naturally occurring hypoxic environment. Cells respond to hypoxia by stabilizing hypoxia-inducible factors (HIFs), which are traditionally viewed to function by altering cellular metabolism and blood vessel architecture. Recently, HIFs have been shown to modulate specific stem cell effectors, such as Notch, Wnt and Oct4 that control stem cell proliferation, differentiation and pluripotency. Direct molecular links have also been established between HIFs and critical cell signalling pathways such as cMyc and p53. These novel links suggest a new role for HIFs in stem cell and tumour regulation.

In silico gene selection for custom oligonucleotide microarray design.

A method for systematically selecting the large number of sequences needed to custom design an oligonucleotide microarray was presented. This approach uses a Perl script to query sequence databases with gene lists obtained from previously designed (and publicly available) microarrays. Homologous sequences passing a user-defined threshold are returned and stored in a candidate gene database. Using this versatile technique, microarrays can be designed for any organism having sequence data. In addition, the ability to select specific input gene lists allows the design of microarrays tailored to address questions pertaining to specific pathways or processes. Given recent concerns about the accuracy of annotation in public sequence databases, it is also necessary to confirm the correct orientation of candidate sequences. This step is performed by a second Perl script that extracts protein similarity information from individual Unigene records, checks for consistency of features, and adds this information to the candidate gene database. Discrepancies between the orientations determined using protein similarities and that predicted by a given sequence's assigned orientation are readily apparent by querying the candidate gene database.

The vesicular glutamate transporter 1 (xVGlut1) is expressed in discrete regions of the developing Xenopus laevis nervous system.

As the major excitatory neurotransmitter in the vertebrate nervous system, glutamate not only plays an essential role in adult neural signaling, but has also been implicated as a trophic factor in neuronal cell maturation, differentiation, and survival. An essential component of the glutamatergic neurotransmission system is the family of glutamate transporters, a multigene family that codes for plasma membrane-bound as well as vesicle-bound proteins responsible for the removal of glutamate from the cleft and its re-uptake into the synaptic vesicle. Here we describe the spatial and temporal expression of the vesicular glutamate transporter (xVGlut1) during the early developmental stages of the amphibian Xenopus laevis. RNAse protection analysis and in situ hybridization reveal that xVGlut1 is first expressed at late neurula stages in the developing spinal cord and trigeminal nerve. By tailbud stages xVGlut1 transcripts are detected in several of the cranial nerves, the pineal gland, and medial forebrain. By hatching stages xVGlut1 expression reappears in localized tracts within the spinal cord. Expression levels increase throughout development into adulthood.

In silico gene selection strategy for custom microarray design.

Microarray technology has become an important tool for studying large-scale gene expression for a diversity of biological applications. However, there are a number of experimental settings for which commercial arrays are either unsuitable or unavailable despite the existence of sequence information. With the increasing availability of custom array manufacturing services, it is now feasible to design high-density arrays for any organism having sequence data. However, there have been relatively few reports discussing gene selection, an important first step in array design. Here we propose an in silico strategy for custom microarray gene selection that is applicable to a wide range of organisms, based on utilizing public domain microarray information to interrogate existing sequence data and to identify a set of homologous genes in any organism of interest. We demonstrate the utility of this approach by applying it to the selection of candidate genes for a custom Xenopus laevis microarray. A significant finding of this study is that 3%-4% of Xenopus expressed sequence tags (ESTs) are in an orientation contrary to that indicated in the public database entry (http://mssaha.people.wm.edu/suppMSS.html).

Integrative genomic analyses of sporadic clear cell renal cell carcinoma define disease subtypes and potential new therapeutic targets.

Sporadic clear cell renal cell carcinoma (ccRCC), the most common type of adult kidney cancer, is often associated with genomic copy number aberrations on chromosomes 3p and 5q. Aberrations on chromosome 3p are associated with inactivation of the tumor suppressor gene von-Hippel Lindau (VHL), which activates the hypoxia-inducible factors HIF1α and HIF2α. In contrast, ccRCC genes on chromosome 5q remain to be defined. In this study, we conducted an integrated analysis of high-density copy number and gene expression data for 54 sporadic ccRCC tumors that identified the secreted glycoprotein STC2 (stanniocalcin 2) and the proteoglycan VCAN (versican) as potential 5q oncogenes in ccRCCs. In functional assays, STC2 and VCAN each promoted tumorigenesis by inhibiting cell death. Using the same approach, we also investigated the two VHL-deficient subtypes of ccRCC, which express both HIF1α and HIF2α (H1H2) or only HIF2α (H2). This analysis revealed a distinct pattern of genomic aberrations in each group, with the H1H2 group displaying, on average, a more aberrant genome than the H2 group. Together our findings provide a significant advance in understanding ccRCCs by offering a molecular definition of two subtypes with distinct characteristics as well as two potential chromosome 5q oncogenes, the overexpression of which is sufficient to promote tumorigenesis by limiting cell death.

HIF-alpha effects on c-Myc distinguish two subtypes of sporadic VHL-deficient clear cell renal carcinoma.

von Hippel-Lindau (VHL) tumor suppressor loss results in hypoxia-inducible factor alpha (HIF-alpha) stabilization and occurs in 70% of sporadic clear cell renal carcinomas (ccRCCs). To determine whether opposing influences of HIF-1alpha and HIF-2alpha on c-Myc activity regulate human ccRCC progression, we analyzed VHL genotype and HIF-alpha expression in 160 primary tumors, which segregated into three groups with distinct molecular characteristics. Interestingly, ccRCCs with intact VHL, as well as pVHL-deficient HIF-1alpha/HIF-2alpha-expressing ccRCCs, exhibited enhanced Akt/mTOR and ERK/MAPK signaling. In contrast, pVHL-deficient ccRCCs expressing only HIF-2alpha displayed elevated c-Myc activity, resulting in enhanced proliferation and resistance to replication stress. These reproducible distinctions in ccRCC behavior delineate HIF-alpha effects on c-Myc in vivo and suggest molecular criteria for selecting targeted therapies.

DBParser: web-based software for shotgun proteomic data analyses.

We describe a web-based program called 'DBParser' for rapidly culling, merging, and comparing sequence search engine results from multiple LC-MS/MS peptide analyses. DBParser employs the principle of parsimony to consolidate redundant protein assignments and derive the most concise set of proteins consistent with all of the assigned peptide sequences observed in an experiment or series of experiments. The resulting reports summarize peptide and protein identifications from multidimensional experiments that may contain a single data set or combine data from a group of data sets, all related to a single analytical sample. Additionally, the results of multiple experiments, each of which may contain several data sets, can be compared in reports that identify features that are common or different. DBParser actively links to the primary mass spectral data and to public online databases such as NCBI, GO, and Swiss-Prot in order to structure contextually specific reports for biologists and biochemists.