Maspin is a marker for early recurrence in primary stage III and IV colorectal cancer.

BACKGROUND: Little is known about the factors that drive metastasis formation in colorectal cancer (CRC). Here, we set out to identify genes and proteins in patients with colorectal liver metastases that correlate with early disease recurrence. Such factors may predict a propensity for metastasis in earlier stages of CRC. METHODS: Gene expression profiling and proteomics were used to identify differentially expressed genes/proteins in resected liver metastases that recurred within 6 months following liver surgery vs those that did not recur for >24 months. Expression of the identified genes/proteins in stage II (n=243) and III (n=176) tumours was analysed by immunohistochemistry on tissue microarrays. Correlation of protein levels with stage-specific outcome was assessed by uni- and multivariable analyses. RESULTS: Both gene expression profiling and proteomics identified Maspin to be differentially expressed in colorectal liver metastases with early (<6 months) and prolonged (>24 months) time to recurrence. Immunohistochemical analysis of Maspin expression on tumour sections revealed that it was an independent predictor of time to recurrence (log-rank P=0.004) and CRC-specific survival (P=0.000) in stage III CRC. High Maspin expression was also correlated with mucinous differentiation. In stage II CRC patients, high Maspin expression did not correlate with survival but was correlated with a right-sided tumour location. CONCLUSION: High Maspin expression correlates with poor outcome in CRC after spread to the local lymph nodes. Therefore, Maspin may have a stage-specific function possibly related to tumour cell dissemination and/or metastatic outgrowth.

Minimum information about a microarray experiment (MIAME)-toward standards for microarray data.

Microarray analysis has become a widely used tool for the generation of gene expression data on a genomic scale. Although many significant results have been derived from microarray studies, one limitation has been the lack of standards for presenting and exchanging such data. Here we present a proposal, the Minimum Information About a Microarray Experiment (MIAME), that describes the minimum information required to ensure that microarray data can be easily interpreted and that results derived from its analysis can be independently verified. The ultimate goal of this work is to establish a standard for recording and reporting microarray-based gene expression data, which will in turn facilitate the establishment of databases and public repositories and enable the development of data analysis tools. With respect to MIAME, we concentrate on defining the content and structure of the necessary information rather than the technical format for capturing it.

cDNA microarray profiles of canine mammary tumour cell lines reveal deregulated pathways pertaining to their phenotype.

Mammary cancer is the most common type of cancer in female dogs with a lifetime risk of over 24% when dogs are not spayed. The elucidation of the complete canine genome opens new areas for development of cancer therapies. These should be tested first by in vitro models such as cell lines. However, to date, no canine mammary cell lines have been characterized by expression profiling. In this study, canine mammary tumour cell lines with histologically distinct primary tumours of origin were characterized using a newly developed canine cDNA microarray. Comparisons of gene expression profiles showed enrichment for distinct biological pathways and were related to biological properties of the cell lines such as growth rate and in vitro tumourigenicity. Additionally, gene expression profiles of cell lines also showed correspondence to their tumour of origin. Major differences were found in Wnt, cell cycle, cytokine/Rho-GTPase, alternative complement and integrin signalling pathways. Because these pathways show an overlap at the molecular level with those found in human breast cancer, the expression profiling of spontaneous canine mammary cancer may also function as a biological sieve to identify conserved gene expression or pathway profiles of evolutionary significance that are involved in tumourigenesis. These results are the basis for further characterization of canine mammary carcinomas and development of new therapies directed towards specific pathways. In addition these cell lines can be used to further investigate identified deregulated pathways and characterize until now unannotated genes.

Promoter-specific activation defects by a novel yeast TBP mutant compromised for TFIIB interaction.

TFIIB is an RNA polymerase II general transcription factor (GTF) that has also been implicated in the mechanism of action of certain promoter-specific activators (see, for examples, [1-11]). TFIIB enters the preinitiation complex (PIC) primarily through contact with the TATA box binding protein (TBP), an interaction mediated by three TBP residues [12-14]. To study the role of TFIIB in transcription activation in vivo, we randomly mutagenized these three residues in yeast TBP and screened for promoter-specific activation mutants. One mutant bearing a single conservative substitution, TBP-E186D, is the focus of this study. As expected, TBP-E186D binds normally to the TATA box but fails to support the entry of TFIIB into the PIC. Cells expressing TBP-E186D are viable but have a severe slow-growth phenotype. Whole-genome expression analysis indicates that transcription of 17% of yeast genes are compromised by this mutation. Chimeric promoter analysis indicates that the region of the gene that confers sensitivity to the TBP-E186D mutation is the UAS (upstream activating sequence), which contains the activator binding sites. Most interestingly, other TBP mutants that interfere with different interactions (TFIIB, TFIIA, or the TATA box) and a TFIIB mutant defective for interaction with TBP all manifest distinct and selective promoter-specific activation defects. Our results implicate the entry of TFIIB into the PIC as a critical step in the activation of certain promoters and reveal diverse mechanisms of transcription activation.

Yeast NC2 associates with the RNA polymerase II preinitiation complex and selectively affects transcription in vivo.

NC2 (Dr1-Drap1 or Bur6-Ydr1) has been characterized in vitro as a general negative regulator of RNA polymerase II (Pol II) transcription that interacts with TATA-binding protein (TBP) and inhibits its function. Here, we show that NC2 associates with promoters in vivo in a manner that correlates with transcriptional activity and with occupancy by basal transcription factors. NC2 rapidly associates with promoters in response to transcriptional activation, and it remains associated under conditions in which transcription is blocked after assembly of the Pol II preinitiation complex. NC2 positively and negatively affects approximately 17% of Saccharomyces cerevisiae genes in a pattern that resembles the response to general environmental stress. Relative to TBP, NC2 occupancy is high at promoters where NC2 is positively required for normal levels of transcription. Thus, NC2 is associated with the Pol II preinitiation complex, and it can play a direct and positive role at certain promoters in vivo.

Instability of human TATA-binding protein CAG triplet repeats during amplification by PCR.

Polymerase chain reaction (PCR) of a TATA-binding protein cDNA that contains CAG triplet repeats results in heterogeneous products. This is caused by a variable loss in the number of CAG triplets. Sequence analysis of PCR products suggests that instability increases with repeat length.

DNA microarrays: raising the profile.

Expression profiling using DNA microarrays is starting to come of age. The past year has seen significant advances in the number, scope and quality of studies that incorporate expression profiling experiments. Attention is starting to move on from making DNA microarrays to appropriate experimental design and sophisticated data analysis techniques.

In vitro comparison of radiofrequency-heated and laser-heated metal probes for angioplasty.

The effects of a metal probe catheter on tissue using radiofrequency (RF) as its energy source is evaluated. The energy dissipation and the temperature increase of this probe was compared with a laser-heated probe. After 15 seconds, the temperature rise of the RF-heated probe at a maximum power setting was 68 degrees C in water and 106 degrees C in plasma. In contrast, the temperature rise of the Nd:YAG laser-heated probe after 10 seconds, 10 watt (W), was 80 degrees C in water and 595 degrees C in plasma. Calorimetric experiments showed that in a 7 to 30 W range of the power setting for the RF generator, only 3.5 to 4.5 W was dissipated at the RF catheter tip. Using axial forces equivalent to 100 g in fatty tissue, the penetration velocity of the RF-heated probe was 0.015 mm/s, with a temperature rise of the tip of 180 degrees C; whereas the velocity of the laser-heated probe was 3.4 mm/s with a temperature rise of the tip of 300 degrees C. These in vitro results suggest that during clinical application, tissue in contact with the front surface of the RF-heated angioplasty probe will be remodeled, whereas with the laser-heated probe tissue will be vaporized circumferentially. The RF-heated probe's risk of vessel wall perforation is probably small.

Gene expression profiles of progestin-induced canine mammary hyperplasia and spontaneous mammary tumors.

Spontaneous mammary tumors are the most prevalent type of neoplasms in women as well as in female dogs. Although ovarian hormones estrogen and progesterone are known to play a key role in mammary tumorigenesis, conflicting reports have been obtained from in vivo and in vitro studies concerning the role of especially progesterone in mammary tumorigenesis. Prolonged exposure to high concentrations of progesterone during the unusually long luteal phase of the estrous cycle is suspected to be the key event in canine mammary tumorigenesis. Accordingly, previous studies have shown the development of mammary hyperplasia in dogs upon prolonged progestin administration. In this study, a dog-specific cDNA microarray was used to identify oncogenic determinants in progestin-induced canine hyperplasia (CMH) and spontaneous mammary tumors (CMC) by comparing expression profiles to those obtained from mammary glands of healthy dogs. The CMH profile showed elevated expression of genes involved in cell proliferation such as PCNA, NPY, RAN and also alterations in expression of transcription factors and cell adhesion molecules. Whereas in CMC, major alterations to the expression of genes involved in cell motility, cytoskeletal organization and extra cellular matrix production was evident besides differential expression of cell proliferation inducing genes. The overall gene expression profile of CMH was related to cell proliferation where as that of CMC was associated with both cell proliferation as well as neoplastic transformation. In conclusion, our findings support a strong cell proliferation inducing potential of progestins in the canine mammary gland. Moreover, deregulated genes identified in CMC are potentially involved in their malignant and may serve as prospective therapeutic targets.

High-resolution genetic mapping of mammalian motor activity levels in mice.

The generation of motor activity levels is under tight neural control to execute essential behaviors, such as movement toward food or for social interaction. To identify novel neurobiological mechanisms underlying motor activity levels, we studied a panel of chromosome substitution (CS) strains derived from mice with high (C57BL/6J strain) or low motor activity levels (A/J strain) using automated home cage behavioral registration. In this study, we genetically mapped the expression of baseline motor activity levels (horizontal distance moved) to mouse chromosome 1. Further genetic mapping of this trait revealed an 8.3-Mb quantitative trait locus (QTL) interval. This locus is distinct from the QTL interval for open-field anxiety-related motor behavior on this chromosome. By data mining, an existing phenotypic and genotypic data set of 2445 genetically heterogeneous mice (http://gscan.well.ox.ac.uk/), we confirmed linkage to the peak marker at 79 970 253 bp and refined the QTL to a 312-kb interval containing a single gene (A830043J08Rik). Sequence analysis showed a nucleotide deletion in the 3' untranslated region of the Riken gene. Genome-wide microarray gene expression profiling in brains of discordant F(2) individuals from CS strain 1 showed a significant upregulation of Epha4 in low-active F(2) individuals. Inclusion of a genetic marker for Epha4 confirmed that this gene is located outside of the QTL interval. Both Epha4 and A830043J08Rik are expressed in brain motor circuits, and similar to Epha4 mutants, we found linkage between reduced motor neurons number and A/J chromosome 1. Our findings provide a novel QTL and a potential downstream target underlying motor circuitry development and the expression of physical activity levels.

Integrating functional genomics data.

Functional annotation of fully sequenced genomes is still a major issue. High-throughput data sets could be used to provide more and better functional annotations. However differences in data quality need to be taken into account. For this purpose these high-throughput data sets need to be integrated so that the data quality can be assessed, hypotheses can be prioritized and existing annotations can be improved and extended.

Analysis of open complex formation during RNA polymerase II transcription initiation using heteroduplex templates and potassium permanganate probing.

Open complex formation precedes initiation of transcription by RNA polymerases. In the analysis of transcription initiation from eukaryotic class II promoters, we have used promoter DNA structures that represent intermediates in open complex formation. We describe the preparation and isolation of heteroduplex promoter fragments. Probes containing these DNA structures have a general application in the study of proteins binding to junctions of double- and single-stranded DNA. Such proteins play important roles not only in the regulation of RNA synthesis but also in processes like repair, replication, and recombination of DNA. In addition, a protocol is provided for a rapid and quantitative assay for open complexes and transcription bubbles using potassium permanganate as a chemical probe for single-stranded regions in DNA.

Three transitions in the RNA polymerase II transcription complex during initiation.

We have analyzed transcription initiation by RNA polymerase II (pol II) in a highly efficient in vitro transcription system composed of essentially homogeneous protein preparations. The pol II complex was stalled on adenovirus major late promoter templates at defined positions, and the open region and RNA products of these complexes were examined. The first transition is formation of the open complex, which can be reversed by addition of ATPgammaS. The open region is no longer sensitive to ATPgammaS after formation of a four-nucleotide RNA, which constitutes the second transition. This indicates that the ATP-dependent DNA helicase activity of TFIIH is required to maintain the open region only during formation of the first three phosphodiester bonds. The downstream part of the transcription bubble expands in a continuous motion, but the initially opened region (-9/-2 on the non-template strand) recloses abruptly when transcription reaches register 11. This third transition is accompanied by a switch from abortive to productive RNA synthesis, which implies promoter clearance. Our findings provide a framework to analyze regulation of these specific transitions during transcription initiation by pol II.

Mechanism of CW Nd:YAG laser recanalization with modified fiber tips: influence of temperature and axial force on tissue penetration in vitro.

Modified fiber tips are used for laser angioplasty of totally occluded peripheral arteries. It has not been established, however, to what extent the mechanism of action of various laser probes is optical, thermal, or mechanical. We examined transparant contact probes (hemispherical contact probes and ball-shaped fibers) and metal laser probes, coupled to a continuous-wave Nd-YAG laser. By using homogeneous thick porcine fatty tissue samples submerged in blood plasma, tissue penetration was measured in relation to the temperature of the probe and the axial force exerted on the tissue. By using 15 W, 1 s laser pulses, the surface of transparent contact probes had to be first contaminated by carbonized tissue particles to achieve tissue penetration. Penetration increased from 1 to 10 mm per pulse when axial force increased from 20 to 100 g. Metal probes had to be sufficiently insulated from the liquid environment by water vapour entrapped in a denatured protein layer to exceed the threshold temperature of 225 degrees C for tissue penetration. When axial force increased from 20 to 80 g at 10 W continuous exposure, the velocity of tissue penetration increased in the range from 1 to 4 mm/s. Tissue penetration by modified fiber tips is attributed to both remodeling and vaporization of tissue. With transparent contact probes, tissue is heated partly by direct light absorption and partly by a hot probe surface. Axially directed force is necessary to displace lateral non-ablated tissue and to overcome mechanical resistance. We conclude that mechanical dilation due to axial catherization force (Dotter effect) contributes substantially to tissue penetration by transparent contact probes.

Temperature along the surface of modified fiber tips for Nd:YAG laser angioplasty.

For laser angioplasty probes, the thermal properties of the probes will primarily determine their mechanism of action. We examined the absorption, temperature increase, and probe degradation of transparent contact probes (hemispherical contact probe and ball-shaped fibers) and metal laser probes coupled to a continuous-wave Nd-YAG laser. Temperature was recorded by means of thermocouples and the measurements were corrected for direct light absorption by the thermocouple. During 15 W, 1 s exposure, the peak temperature rise of the hemispherical contact probe in contact with tissue dropped from approximately 1,000 degrees C at the front end to below 45 degrees C (95% drop) at the lateral side. In contrast, during continuous exposure the peak temperature rise of metal laser probes in contact with tissue dropped from 560 degrees C at the front end to near 400 degrees C (30% drop) at the 5.5 mm proximal rear end. During exposure in blood or tissue, the transparent contact probes became contaminated. Their absorption increased from 5 to 33% and the probe deteriorated. Repeated use of metal laser probes in blood resulted in a higher temperature at the rear than at the front end due to backburing of the fiber. Owing to the large temperature drop along the surface of transparent contact probes, the area of thermal destruction is limited to the tissue in front of the probe, whereas along the entire surface of metal laser probes the tissue will be affected. The large difference between these temperature distributions should be respected during clinical application of the transparent contact probe and the metal laser probe.

Opening of an RNA polymerase II promoter occurs in two distinct steps and requires the basal transcription factors IIE and IIH.

We have studied promoter opening in assays reconstituted with purified RNA polymerase II and basal transcription factors. We found that creating a region of heteroduplex DNA around the start site of the adenovirus major late (AdML) promoter circumvents the requirement for TFIIE and TFIIH in transcription. The critical size and position of the heteroduplex region that alleviates the requirement for TFIIE and TFIIH is six nucleotides, from -4 to +2. Promoter opening was investigated directly with potassium permanganate (KMnO4), a chemical probe specific for single-stranded thymidines. We found that KMnO4-detectable opening of the AdML promoter requires the presence of the complete pre-initiation complex, DBpolFEH, and that opening occurs in two discrete steps. First, dependent on ATP but prior to initiation, the -9 to +1 region becomes single-stranded. Second, formation of the first phosphodiester bond results in expansion of the open region to the +8 position. Our results lead to a model in which the critical function of the TFIIH-associated DNA helicases is to create a single-stranded region. This gives RNA polymerase II access to the nucleotides of the template strand and allows expansion of the open region upon formation of the first phosphodiester bond.

The requirement for the basal transcription factor IIE is determined by the helical stability of promoter DNA.

The role of the basal transcription factor TFIIE was investigated in RNA polymerase II transcription reactions reconstituted with purified proteins. Using negatively supercoiled templates, which circumvent the requirement for TFIIH, we observed that transcription from the adenovirus major-late (ML) core promoter is more dependent on TFIIE than transcription from the adenovirus E4 (E4) or mouse mammary tumor virus (MMTV) promoters. For all three promoters, an increase in the ionic strength of the reaction mixtures led to an increased dependence on TFIIE. Analysis of hybrid ML/MMTV promoters showed that the region encompassing the start site, from -10 to +10, dictates this dependence. Transcription from a relaxed E4 template with a pre-melted -8 to +2 region was completely independent of both TFIIE and TFIIH. We propose that on negatively supercoiled templates TFIIE can facilitate promoter melting.

Broad, but not universal, transcriptional requirement for yTAFII17, a histone H3-like TAFII present in TFIID and SAGA.

The RNA polymerase II general transcription factor TFIID is a multisubunit complex comprising TATA box-binding protein (TBP) and associated factors (TAFIIs). Experiments in yeast have shown that although most TAFIIs are required for viability, many genes are transcribed normally upon inactivation of individual and even multiple yTAFIIs. Here we analyze yTAFII17, recently found to be present in both the SAGA HAT complex as well as TFIID. Functional inactivation of yTAFII17 by temperature-sensitive mutation or depletion results in loss of transcription of many, but not all, genes. The upstream activating sequence (UAS), which contains the activator binding sites, is the region that renders a gene yTAFII17 dependent. In conjunction with previous studies, our results reveal that different TAFIIs have remarkably distinct properties.

TCF-1, a T cell-specific transcription factor of the HMG box family, interacts with sequence motifs in the TCR beta and TCR delta enhancers.

We have recently identified and cloned TCF-1, a T cell-specific transcription factor with specificity for the AACAAAG motif in the CD3 epsilon enhancer and for the TTCAAAG motif in the TCR alpha enhancer. TCF-1 belongs to the family of transcription-regulating proteins which share a region of homology termed the HMG-box. Here, we show by gel retardation analysis that TCF-1 specifically recognizes the T beta 5 element of the TCR beta enhancer and the T delta 7 element of the TCR delta enhancer. Comparison of the sequences of all elements recognized by TCF-1 defines a consensus motif A/T A/T C A A/G A G. These observations imply that TCF-1 is involved in the control of several T cell-specific genes and might thus play an important role in the establishment and maintenance of the mature T cell phenotype.

Redundant roles for the TFIID and SAGA complexes in global transcription.

The transcription factors TFIID and SAGA are multi-subunit complexes involved in transcription by RNA polymerase II. TFIID and SAGA contain common TATA-binding protein (TBP)-associated factor (TAF(II)) subunits and each complex contains a subunit with histone acetyltransferase activity. These observations have raised questions about whether the functions of the two complexes in vivo are unique or overlapping. Here we use genome-wide expression analysis to investigate how expression of the yeast genome depends on both shared and unique subunits of these two complexes. We find that expression of most genes requires one or more of the common TAF(II) subunits, indicating that the functions of TFIID and SAGA are widely required for gene expression. Among the subunits shared by TFIID and SAGA are three histone-like TAF(II)s, which have been proposed to form a sub-complex and mediate a common function in global transcription. Unexpectedly, we find that the histone-like TAF(II)s have distinct roles in expression of the yeast genome. Most importantly, we show that the histone acetylase components of TFIID and SAGA (TAF(II)145 and Gcn5) are functionally redundant, indicating that expression of a large fraction of yeast genes can be regulated through the action of either complex.