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- Research article
- Open Access
Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array
© Gardina et al; licensee BioMed Central Ltd. 2006
- Received: 04 October 2006
- Accepted: 27 December 2006
- Published: 27 December 2006
Alternative splicing is a mechanism for increasing protein diversity by excluding or including exons during post-transcriptional processing. Alternatively spliced proteins are particularly relevant in oncology since they may contribute to the etiology of cancer, provide selective drug targets, or serve as a marker set for cancer diagnosis. While conventional identification of splice variants generally targets individual genes, we present here a new exon-centric array (GeneChip Human Exon 1.0 ST) that allows genome-wide identification of differential splice variation, and concurrently provides a flexible and inclusive analysis of gene expression.
We analyzed 20 paired tumor-normal colon cancer samples using a microarray designed to detect over one million putative exons that can be virtually assembled into potential gene-level transcripts according to various levels of prior supporting evidence. Analysis of high confidence (empirically supported) transcripts identified 160 differentially expressed genes, with 42 genes occupying a network impacting cell proliferation and another twenty nine genes with unknown functions. A more speculative analysis, including transcripts based solely on computational prediction, produced another 160 differentially expressed genes, three-fourths of which have no previous annotation. We also present a comparison of gene signal estimations from the Exon 1.0 ST and the U133 Plus 2.0 arrays.
Novel splicing events were predicted by experimental algorithms that compare the relative contribution of each exon to the cognate transcript intensity in each tissue. The resulting candidate splice variants were validated with RT-PCR. We found nine genes that were differentially spliced between colon tumors and normal colon tissues, several of which have not been previously implicated in cancer. Top scoring candidates from our analysis were also found to substantially overlap with EST-based bioinformatic predictions of alternative splicing in cancer.
Differential expression of high confidence transcripts correlated extremely well with known cancer genes and pathways, suggesting that the more speculative transcripts, largely based solely on computational prediction and mostly with no previous annotation, might be novel targets in colon cancer. Five of the identified splicing events affect mediators of cytoskeletal organization (ACTN1, VCL, CALD1, CTTN, TPM1), two affect extracellular matrix proteins (FN1, COL6A3) and another participates in integrin signaling (SLC3A2). Altogether they form a pattern of colon-cancer specific alterations that may particularly impact cell motility.
- Alternative Splice
- Splice Variant
- Splice Event
- Splice Pattern
- Alternative Splice Event
Alternative splicing of mRNA transcripts is one mechanism by which genomic complexity is generated from the surprisingly low number of genes currently estimated from the human genome sequence. The fraction of human genes subject to alternative splicing has risen from 5% in early predictions to at least 75% in a recent genome-wide exon study (Clark et al., in prep.). There are examples of hundreds of alternative splicing events from a single gene, which may affect function by adding or deleting functional domains, changing affinities, and altering mRNA stability. Variable transcripts from a single gene are produced combinatorially through the selection of cassette exons, mutually exclusive exons, retained introns, alternative 3' or 5' splice sites, and alternative promoters or polyA sites [1].
Specific alterations in splicing patterns have been found in association with cancers, many of which may play a functional role in transformation, motility and metastasis of tumor tissue. Alternative splicing appears to affect key aspects of neoplasms by altering hormonal signaling, apoptosis and mediators of cell-cell and cell-matrix interactions. Modifications in functionality may be generated, for example, through the deletion of a signaling domain, increased affinity for messenger ligands, or change in affinity or activity toward extracellular components. The latter frequently results in increased cell migration and invasion (For general reviews, see [2, 3]). Although in most cases the relationship between specific splicing events and the etiology of cancer is largely unproven, alternative splicing presents novel targets for diagnostic and therapeutic measures.
Abnormal splicing of several genes has been observed in tumors of colorectal origin, including CD44 [4, 5], MUC2 [6], SRF [7], NCAM [8], MLH, MSH [9] and members of the Wnt pathway [10]. Some of these alternately spliced gene products may have therapeutic utility as markers of the progression of the disease or as drug targets; however there are insufficient data supporting their relevance in larger clinical populations. Individual patient differences, tissue complexity and lack of tools for comprehensive analysis of splice variation have made the task challenging.
We have analyzed both differential gene expression and alternative splicing in a small collection of colon cancers employing a microarray (GeneChip Human Exon 1.0 ST) targeting over a million putative exons. The exons can be virtually reassembled into over 250,000 transcripts according to a range of annotation sources. The inclusiveness of the array design allowed a flexible gene expression analysis, initially targeting high confidence transcripts and then extending to a more speculative set.
Cancer-specific splice variations were detected with experimental algorithms and candidate events were subsequently validated with RT-PCR. We found several splice variants, some of which have been previously associated with cancer, and several which have not. The majority of the identified splicing events affect mediators of cytoskeletal organization, the extracellular matrix or integrin signaling, and may be involved in cell migration and invasion.
Array Design
The GeneChip Human Exon 1.0 ST array was designed to be as inclusive as possible at the exon level, deriving from annotations ranging from empirically determined, highly curated mRNA sequences to ab-initio computational predictions [11]. The array contains approximately 5.4 million 5-μm features (probes) grouped into 1.4 million probesets interrogating over one million exon clusters (exon annotations from various sources that overlap by genomic location). By comparison, the Affymetrix U133 Plus 2.0 gene expression array contains 1.3 million 11-μm features comprising 54,000 probesets. A Probe Selection Region (PSR) represents a region of the genome (assembly HG16, Build 34) predicted to act as an integral, coherent unit of transcriptional behavior. In many cases, each PSR is an exon; in other cases, due to potentially overlapping exon structures, several PSRs may form contiguous, non-overlapping subsets of a true biological exon. The median size of PSRs is 123 bp with a minimum of 25 bp. Probes were not included for some PSRs because the PSR might be too small or contain highly repeated or otherwise problematic sequence. About 90% of the PSRs are represented by 4 probes (a "probeset"). Such redundancy allows robust statistical algorithms to be used in estimating presence of signal, relative expression, and existence of alternative splicing.
The Exon Array does not include a paired mismatch feature for each perfect match feature, rather surrogate mismatch intensities are derived from 1000 pooled "antigenomic" probes with the same GC content as each perfect match feature [11]. Antigenomic sequences are not present in the human, rat or mouse genomes.
The Exon Array also serves as a gene-level expression array. The median number of probes for each RefSeq gene is 30–40 distributed along the entire length of the transcript, as compared to 11 probes mostly at the 3' end in the GC U133 Plus Array.
The plethora of exon architectures (e.g., cassette exons, mutually exclusive exons, alternative splice sites, alternative transcriptional starts and stops), the variations in confidences of transcript annotations, and the necessity of rapidly incorporating new genomic knowledge has led to a dynamic design for reconstituting exons into genes. A set of rules was created for virtually assembling the probesets (exon-level) into transcript clusters (gene-level) based on the confidence level of the supporting evidence and the juxtapositions of the exon borders [11]. In general, exons were merged into clusters depending on whether and how many higher confidence clusters or exons they overlap, whether they have common exact splice sites, and whether single orphan exons are bounded within another annotation.
The mapping between probesets and transcript clusters is defined by meta-probeset lists (in order of decreasing confidence):
A) "Core" (17,800 transcript clusters): RefSeq and full-length GenBank mRNAs;
B) "Extended" (129K transcript clusters): cDNA transcripts, syntenic rat and mouse mRNA, and Ensembl, microRNA, Mitomap, Vegagene and VegaPseudogene annotations;
C) "Full" (262K transcript clusters): ab-initio predictions from Geneid, Genscan, GENSCAN Suboptimal, Exoniphy, RNAgene, SgpGene and TWINSCAN
The combination of merging rules and metaprobe sets means that some transcript clusters in the Extended and Full sets may contain regions (probesets) that derive from annotations of different confidence levels. Of the 1.4 million probesets, approximately 290,000 are supported by full-length mRNAs (i.e., the Core set).
Gene-level signal comparison between the U133 Plus 2.0 and Exon arrays for normal tissue
Prior to analyzing the colon cancer data, we present a brief comparison of the gene level signals from the exon array and Affymetrix's standard gene expression array, the U133 Plus 2. For this, we utilize a subset of data from a panel of 11 normal tissues that was assayed in parallel on both arrays. The approach was to compare the relative signals from differentially expressed probesets that target the same transcript clusters on both arrays.
Significant genes were determined by pair-wise comparisons between each set of the 11 tissues with p-values at 0.05, 0.001 and 10-8. Genes were accepted into the analysis if they showed significant changes in at least one tissue type on both arrays and could be mapped to the same transcript cluster. At a p-value of 0.05, 7829 probesets from the U133 Plus 2 array and 5507 transcript clusters from the Core meta-probeset of the exon array were accepted and mapped to each other. On average, approximately 1.4 probesets from the U133 Plus 2 array mapped to each transcript cluster on the exon array. This disparity in numbers results because the U133 Plus 2 array frequently has multiple independent probesets targeting the same transcript cluster, whereas the probesets are clustered and summarized into a single gene signal estimate on the exon array. For the 1654 cases of multiple U133 Plus 2 probesets interrogating the same gene, the mean of the individual signals was used as the signal estimate.
Gene-level expression changes in colon cancer for the Core and Full gene sets
The initial analysis of expression changes in colon cancer targeted Core genes, composed of nearly 17,800 transcript clusters based on highly confident supporting evidence. Subsequent to ANOVA analysis, a threshold cutoff was set to p-values less than 0.0015 and at least a 2-fold geometric change in gene-level expression between normal and tumor samples. This yielded 134 significantly up-regulated and 26 down-regulated genes in the tumor samples (see Additional file 1).
The most heavily represented classes of over-expressed genes in the tumor samples encompass mitosis, the cell cycle, apoptosis, cell proliferation, and DNA repair and recombination (34 altogether). One large group involves RNA processing (16 genes), including at least 4 participating in RNA splicing. Components or modulators of the extracellular matrix are also heavily represented (14 genes), many of which have been implicated in cell invasion and metastasis.
Many of the significantly down-regulated genes represent normal colon functions that are lost as the tissue becomes undifferentiated, such as Aquaporin 8, which is normally present only in colon and pancreas [12]. However, the decreased expression of two hormones, Somatostatin and PYY, and HIF3A, a negative regulator of hypoxia-inducible genes, may actively potentiate carcinogenesis [13–15].
The gene-level analysis was repeated with the more speculative Full metaprobe set representing 262,000 transcript clusters, i.e., putative genes generated from exon clusters without strong empirical support. ANOVA analysis produced 290 genes with differential expression between normal and cancerous tissue. Excluding 130 genes that were also found in the Core gene analysis leaves 160 genes found only in the expanded analysis, of which 39 have some annotation (Additional file 2). Of the 121 remaining completely unannotated putative genes, 38 have significant expression (a threshold PLIER signal of 15 in 50% of the samples) in at least one tissue type.
Workflow for the detection of splice variants
RT-PCR validation of differential splicing in colon cancer
Differentially spliced genes in colon cancer detected by the exon microarray and confirmed by PCR.
Known AS Event2
Preferential Inclusion of Splice Variants4
19a/19b are MEEs
Ex19a in Tumor, 19b in Normal
Ex21 is CE
Ex21 in Normal
Ex19 is CE
Ex19 in Normal
extended Ex5; Ex6 is CE
extended Ex5 + Ex6 in Normal; Ex6 in Normal
Ex2, 3, 4 are CEs
Normal skips all exons
Ex3 and Ex4 are CEs
Ex3 or Ex4 in Tumor
Ex6 is CE
Ex6 in Tumor
Ex11 is CE
Ex11 in Tumor
Ex25 is CE
Ex 25 in Normal
Ex6 Ex6
Ex7 Ex8
Ex7/Ex8 are usually MEEs
Ex7 in Tumor; Ex8 in Normal
Ex20 is CE
Ex20 in Tumor
Ex7 Ex5
Ex9 Ex9
Ex7 alt. start?
Ex7 Alt. Start in Normal
Ex10 Ex11b
Ex12 Ex12
extended Ex11; Ex11b is CE
extended Ex11 + Ex11b in Tumor
Ex11 Alt. 3' end
Alt. 3' end (Ex11b) in Normal
Ex17 appears constitutive
different 3' end of gene
Ex2 and Ex3 are rare CEs
Very Weak
Ex2 + Ex3 in Tumor
Table 1 also includes the gene-level fold change for each of the validated splicing events since strong tissue-specific differences in gene expression may cause misleading results in the RT-PCR validations. However, all of the strongly validated events demonstrated a fold change of less than 2 (two of the ambiguous events were > 2), and none of these genes appeared as significantly differentially expressed in the gene-level analysis.
Validated splicing events
Alpha-actinin 1 (ACTN1) and vinculin (VCL) encode cytoskeletal elements that interact with actin and probably participate in the attachment of the cytoskeleton to the membrane. Exons 19a and 19b of ACTN1 appear to be mutually exclusive exons, with 19a predominant in tumor samples and 19b predominant in normal samples (Fig. 6). Exon 19 of VCL is a cassette exon that tends to be skipped in these tumor samples.
ATP2B4 belongs to the family of ion transporters that exhibits many isoforms with alternatively spliced exons regulated in a tissue-specific manner. Exon 21 is a known cassette-type exon that is found here in normal colon tissue but is relatively reduced in tumor samples. ATP2B4 plays a role in cellular calcium homeostasis that may be altered in tumor cells in parallel to the loss of ion transporters seen at the gene level.
Variants of caldesmon (CALD1), a structural element linking myosin and actin filaments, tend to include exon 6 and an extended exon 5 in normal cells. SLC3A2 encodes the heavy chain of a heterodimeric solute carrier, but also participates in integrin signaling [23, 24]. Transcripts for SLC3A2 are shifted to a higher molecular weight, indicating the inclusion of various combinations of exons 2, 3 and 4 in tumor samples that are much less prevalent in normal tissue. Transcripts for the matrix protein Collagen IV-3A (COL6A3) tend to include the cassette exons 3, 4 (not shown) and 6 (Fig. 6) in tumor samples to a greater extent than in normal samples.
Cortactin (CTTN) also has roles both in cell adhesion and organization of the cytoskeleton. Exon 11 is a cassette exon that appears to be equally likely to be included or skipped in normal tissue, but in the tumor samples, the predominant form is inclusion of Exon 11. Fibronectin (FN1) can be found in the extracellular matrix or at the cell surface, and has a role in cell adhesion and migration. A cassette exon, Exon 25, is more prevalent in normal tissues than in tumor samples.
Tropomyosin 1 (TPM1) is also an actin-binding protein, has a number of known splice variants, and is involved in the contraction of muscle and in the cytoskeleton of non-muscle cells. Since Exons 7 and 8 of TPM1 are mutually exclusive exons of approximately the same size, the alternative amplificates were distinguished by a restriction enzyme that cuts Ex7, but not Ex8. The fragments of Ex7 appear as two faster moving bands that are more prevalent in the tumor samples.
Densitometric scans of the gel bands were used to quantitate the observed splice variants (Additional file 5). The ratio of the intensity of the alternative bands was calculated and compared across the tissue types as a log ratio. The densitometric analysis suggests that the strongest and most consistent changes occurred in ACTN1, ATP2B4, VCL and CALD1. TPM1 and CTTN also showed strong splicing differences between the tissues, but with higher variability. All of the tested events show a p-value of < 0.05 in the t-test across the tissues except for Ex6 of COL6A3, for which the splice variants are more apparent visually than in the scan.
Validation of previously reported splicing events
The relevant exons in CD44 did not score well in our detection analysis, probably because of the inconsistency in the splicing pattern. Densitometric analysis of CD44 (Additional file 5) confirms strong splice variation between the tissues, but with a high variability across samples. Note that the advanced tumor sample in Lane 13, the most predictive for our differential splice patterns, does not display alternative splicing of CD44.
VEGF has a potentially complex splicing pattern, with possible combinations of Ex6a, Ex6b, extended Ex6a and Ex7. McCullough et al. [30] attempted to resolve this complexity with a mass spectrometry-based method that indicated that the majority of the transcripts in normal colon lack Exon 6, whereas we find that about half of the transcripts migrate consistently with inclusion of Exon 6. The results from CD44 and VEGF underscore the difficulty of detecting relatively subtle alterations in small, heterogeneous sample populations.
Pathway analysis of validated splicing events
Comparison to bioinformatic methods of estimating alternative splicing
Several bioinformatic approaches have been developed to identify splice variation between normal tissue types or normal versus tumor tissues. These methods rely on mapping ESTs or mRNAs onto RefSeq transcripts or genomic sequence, making frequency counts of various isoforms, and applying a statistical test to detect differential inclusion of exons in different tissues. The methods vary somewhat in research focus, basic algorithms, statistical methods (e.g., Z-statistics vs. Fischer's exact test), EST source libraries, and mechanisms of normalizing or filtering out differential gene expression, but the essential approach is similar. We compared our empirical data from the exon array to three lists of the cancer-associated splicing events computationally predicted in Wang, et al. [31], the virtual SAGE-filtered list of Kirschbaum-Slager et al. [32] and in the BASD database described in Hui, et al. [33]. When evaluating the results, it must be kept in mind that these bioinformatic approaches assay either tumors generally or a range of tumor types, but not specifically colon cancer (as in our study).
The splicing events derived from the three methods were mapped to Unigene gene symbols where possible and compared to the top 200 candidate splicing events from the exon array, which corresponds to 169 genes (see Additional file 3). Our list of candidates matched approximately an equal proportion of each EST-derived list, between 1.92% and 1.97% of the total genes in each set: 12/625 for Kirschbaum-Slager et al., 33/1733 for BASD and 13/660 for Wang et al. Altogether, 53 of our 169 candidate genes are found in at least one list. A simple chi-squared test indicates that elements of each list are highly overrepresented in each of the other lists (with p-values < 0.01). The strongest association between the exon-array candidates was with the BASD list, most likely because of the increased statistical power of this larger data set. The EST-based prediction sets showed a similar distribution of association to each other as our candidate gene list did to each of them.
Among our 14 validated candidates, three (CALD1, FN1, TPM1) appeared in the BASD list, two (CALD1, GK) appeared on the list of Wang et al., and none appeared on the list of Kirschbaum-Slager et al. Of the six previously reported alternatively spliced genes tested here, two (CD44 and VEGF) are found in BASD, two (MST1R and VEGF) are found in the list of Wang et al. and one (RAC1) appears on the list of Kirschbaum-Slager et al. CD44, SIAHBP1 and VEGF do appear in our list of 169 candidate genes, but none ranked in the top 100.
Discussion and Conclusion
There are many design differences between the exon array and the U133 Plus 2: feature size, the number of probes per probeset, the number of probesets per transcript, background calculation, the assay procedure, etc. The most obvious difference is that the exon array interrogates exons/subexons throughout the transcript, while the U133 Plus 2 generally targets only the 3' end. Alternative splicing and, particularly, alternative polyadenylation sites may account for substantial differences in gene signal estimation. Furthermore, our method of averaging the signal value of multiple probesets targeting the same transcript on the U133 Plus 2 is only a rough compromise. Despite the many differences, the gene-level comparison demonstrated a reasonable correlation in signals for genes that are significantly different between tissue types. The most notable difference in signal estimations is the shift of apparently low expressing genes on the U133 Plus 2 array to higher signals on the exon array. Since the exon array generally contains 3 to 4 times as many probes per transcript as the U133 Plus 2 array and the probesets are distributed throughout the transcript cluster, the exon array may be more sensitive in detecting a subset of low expressing genes, at least in this data set. Resolution of, for example, the portability of results between the arrays types, or the characteristics of transcripts that are differentially detected, will require several dedicated studies, for which data is becoming available.
Among the 160 genes differentially expressed in the Core gene set, 60 have been previously identified as participating in cancers, with 21 specifically in colon or colorectal cancer. Almost one third of the up-regulated genes are part of a tightly interconnected network involved in mitosis, cell cycle control, cell proliferation, invasion, matrix remodeling and Wnt signaling. Constitutive activation within the Wnt signaling pathway has been a prevalent theme in colon cancers, in particular the role of β-catenin [18, 34]. Eight of the over-expressed genes here (BIRC5, MMP7, VEGF, ENC1, CCND1, STRA6, MET and CLDN1) are targets of Wnt/β-catenin regulation and two other Wnt-associated genes, SOX9 and HIG2, are up-regulated in these colon cancer samples.
Twenty nine of the tumor-expressed genes have unknown or weakly annotated functions, but several may be involved in cell proliferation or apoptosis (MCTS1, SIL, HSPA5BP1, WDR18 and IFITM1) and at least eight have previously been associated with various cancers (Additional file 1). Thirteen putative transcriptional regulators and most of the genes classified as signal transducers have unknown biological roles. Another seven genes annotated only as "hypothetical protein" or "open reading frame" (MGC4677, C12orf11, FLJ10726, C6orf167, FLJ31153, FLJ20272, DKFZp762E1312, KIAA1217 and FLJ10719) are strongly expressed above background. The expanded analysis based primarily on ab-initio exon predictions identified 38 more genes with significant expression even though they completely lack previous annotation (Additional file 2). These transcripts with unknown functions may represent a novel set of targets for study in colon cancer oncology and demonstrate the exploratory power of an inclusive array design.
We have identified a number of genes that are differentially spliced between normal and cancerous tissue. Most of the tissue-specific alternative splicing events that were experimentally validated occurred in genes involved in cytoskeletal structure, the extracellular matrix or cell-cell interactions. Some of these events are reported splice variants that occur in a tissue-specific manner and may represent a loss of tissue function as colonic epithelial and smooth muscle cells dedifferentiate, rather than abetting transformation or metastasis. Determination of the role of these splice events requires more detailed study, but in most cases these genes have previously been implicated with active roles in the progression of tumors.
Five of the genes (TPM1, ACTN1, VCL, CTTN and CALD1) that we found to be alternatively spliced in colon cancer have actin-binding domains and play a direct role in the organization or structure of the cytoskeleton. Remodeling of the cytoskeleton is fundamental in proliferation, apoptosis, cell invasion and metastasis [35].
TPM1 appears to act as a tumor suppressor by promoting anoikis (apoptosis induced by cell detachment) [36]. Down-regulation of TPM1 by oncogenic transformation results in a loss of actin stress fibers [37, 38], whereas restoration of TPM1 inhibits cell migration in metastatic cell lines [39]. A splice variant of one of the low molecular weight isoforms of tropomyosin has been found specifically in association with colonic polyps and adenomas, but not normal colon tissue [40].
Actinin is a component of stress fibers and links the cytoskeleton to adherens-type junctions. It plays a role in cell migration probably by facilitating detachment of focal adhesions distal to the direction of movement [41]. Alternative splicing of actinin-4, which has a high sequence similarity to ACTN1, apparently leads to an abnormal cytoskeleton in small cell lung cancer [42]. ACTN1 also has a binding site for VCL, and the two proteins cooperate to organize the cytoskeleton at adhesion junctions [43].
CALD1 binds actin and responds to calmodulin to promote stress fibers and focal adhesions, and CALD1-defective cells are highly impaired in motility [44]. In cells transformed by Kaposi sarcoma-associated herpes virus (KSHV) or v-erb B, hypermethylation of CALD1 and recruitment of its product into membrane complexes is linked to the loss of cytoskeletal microfilaments [45, 46].
CTTN, frequently overexpressed in breast cancer and squamous cell carcinomas, is highly enriched at tumor invasion fronts [47, 48]. Two conformational forms of CTTN are known, with both forms present in normal cells, but the apparently larger one (p85) is more prevalent in colorectal cancers [47]. Two splice variants that affect cell mobility have been previously identified: SV-1 (lacking Exon 11) and SV-2 (lacking Exons 10 and 11). SV-1 and full-length CTTN were equally abundant in normal cells while SV-2 was barely detectable. SV-1, but not SV-2, can bind and crosslink actin, but overexpression of either form interferes with cell migration [49]. Our results indicate that transcripts with or without Exon 11 are approximately equal (in agreement with previous data), but transcripts carrying Exon 11 (i.e., full length) are relatively more abundant in colon cancer samples, suggesting that these cells may be more competent for motility.
Alternative splicing of FN1, CD44 and COL6A3 may play some role in matrix remodeling and/or cell migration in colon cancer, though COL6A3 has not been previously implicated in this role. Fibronectin was one of the consistently up-regulated genes in an artificial selection for highly metastatic cell lines, which also identified ACTN1 and several collagens [50]. Two splice variants of FN1 have been implicated in the neo-vasculature of a variety of human tumors but not in normal adult tissues, however the role of these species in tumor-related angiogenesis is unclear [51]. Alternatively spliced FN1 containing an extra domain has been found frequently in cancers [3], whereas we find preferential skipping of Exon 25 in tumor tissues. CD44 is involved in both cell-matrix and cell-cell interactions as well as connections to the actin cytoskeleton via ankyrin. The variably spliced region of CD44 (exons 6–15) is preferentially included in many cancer types and appears to affect cell migration, invasion and metastasis [5].
Integrin ITGB4 interacts with the intermediate filament network, stimulates the Ras and PI3-K signaling pathways, and appears to be important for cell invasion in colon cancer [52, 53]. SLC3A2, which functions in transmembrane transport, associates with integrins and appears to participate in integrin-mediated anchorage-independent cell growth and tumorigenesis in 3T3 fibroblasts [23, 54]. The inclusion of several exons of SLC3A2 in colon cancer transcripts may represent a tumor-specific alteration of its role in integrin signaling.
Of the eleven differentially spliced genes we found or confirmed, ten are involved in the organization of the cytoskeleton or interaction with the matrix or other cells. Seven of these genes (TPM1, CALD1, CTTN, FN1, CD44, ITGB4 and SLC3A2) have previously been implicated in cancers, and, in five cases, specific splice variants are correlated with the cancerous state. This grouping may represent a coherent, and possibly coordinated, set of alterations which may impact cell mobility and extracellular interactions. A similar concentration of splice variants was found in mouse brain, where targets of the Nova splicing regulon are clustered into functions affecting synaptic transmission and cell morphology. In fact, the targets appear to act as a modular network that impacts not only signaling functions, but also specifically the actin cytoskeleton, extracellular matrix and cell-cell adhesion [55]. It is possible that a similar splicing network is altered in colon cancer to produce the complex of interacting splice variants seen in this study. Such patterns may be more apparent in a highly parallel genome-wide exon analysis than in traditional methods involving gene-by-gene searches.
One assumption of this type of analysis is that biologically important splicing changes would consistently appear in samples of a particular category (i.e., colon cancer). In some cases, our samples did not reproduce some previously reported events (Rac1, VEGF, SIAHBP1, and MST1R), or else the changes were sporadic (CD44). Furthermore, we find differences even in normal samples with regard to Exon 6 of VEGF compared to the mass spectrometry results of McCullough et al. [30]. In fact, the most relevant splicing change in VEGF for tumorigenesis may be a splice variant involving Exon 8 [56]. In this situation, it may be difficult to resolve what the normal and abnormal states are for VEGF. Inconsistent results may be due to differences in samples, experimental procedures, analyses or interpretations, suggesting that conclusions about subtle changes in splicing may need to be reinforced by multiple sources. On the other hand, many of our validated splicing events have been observed in several other instances. Nearly identical patterns of differential alternative splicing was found in colon tumors by Okumura et al. for TPM1, ACTN1 and ITGB4, and these patterns were even more emphatic in tumor cell lines. Interestingly, differential splicing of CD44 in colon cancer was not observed in that study [57]. ITGB4 and TPM1 were identified by an EST-based analysis, and experimentally validated as differentially spliced in several tumor types [58]. The alternative splicing of CTTN is consistent with protein alterations seen in association with colon cancer [47]. Our sample set ranged from well differentiated to poorly differentiated (i.e., advanced) tumors, yet ACTN1, ATP2B4, VCL and CALD1 show strong and consistent changes across our samples (Additional file 5). The presence of these splice variants in multiple tumor stages argues that they must be both early and persistent events. Finally, differences in splicing patterns may be due to biologically important differences in cancer etiology, and therefore be useful indicators of tumor subtypes or stages.
With the large amount of EST and genomic data available, a great deal of useful information may be gained from in silico prediction of transcript isoforms. Validation rates from such methods have been fairly high and the resulting pattern of predictions are mostly in line with empirical data. There are a number of potential hazards in this approach: EST libraries are highly variable in quality and reliability, it is difficult to account for differential gene expression, and tissues and individual genes are unevenly represented. Almost 45% of EST libraries are from cancer samples [33] and 70% of mRNAs in GenBank are cloned from tumor samples [59], leading to a strong bias against identifying isoforms present in normal tissues. Also, in spite of various methods to remove or normalize differentially expressed genes, this factor may lead to overprediction [32]. The exon array addresses many of the difficulties by providing an empirical platform that is unbiased with regard to tissue or gene representation, and allows for direct normalization of differential gene expression. While a substantial proportion (32%) of our top candidates for alternative splicing matched predictions from three EST-derived methods, only four of our validated splicing events appeared among the 2797 genes with bioinformatically predicted splice variants. This suggests that there are substantial gaps in current EST databases that must be addressed empirically.
The splicing changes seen here are not necessarily as dramatic as seen in a previous study of 16 pure normal tissues with an exon-based microarray (Clark et al., in prep.). This highlights the subtlety of most splicing events, which are typically not an all-or-none phenomenon, so sample size and homogeneity are important considerations. In spite of limitations which are frequently encountered in cancer and other tissue studies in humans, such as a modest sample size, heterogeneous tissues, and multiple categorical variables (tumor stage, gender, and individual patient variation), we were able to identify and validate a number of candidate colon cancer-specific splicing events. Exploration of alternative splicing will promote understanding of cancer etiology and may provide therapeutic targets and diagnostic markers [2, 3].
Sample preparation and array hybridization
Ten matched-pairs (20 total RNA samples) of human colon primary tumor and adjacent normal tissue Total RNA (P/N R8235090-PP-10) were purchased from Biochain Institute, Inc. (Hayward, CA). Tumors were characterized as poorly (2 samples), moderately (4) or well (4) differentiated. Adjacent normal tissue may include both colonic epithelial and smooth muscle tissue. Patient information and associated .CEL files are available from Affymetrix, Inc. [60].
One μg of each sample was processed using a pre-commercial version of the Affymetrix GeneChip Whole Transcript Sense Target Labeling Assay [61]. Pre-commercial versions of the GeneChip WT cDNA Synthesis Kit, WT cDNA Amplification Kit, and WT Terminal Labeling Kit (Affymetrix, Inc., Santa Clara, CA), containing identical formulations to the commercial kits, were used for target preparation. Eight μg of cRNA were input into the second-cycle cDNA reaction. Hybridization cocktails containing 3 to 4 μg of fragmented, end-labeled cDNA were prepared and applied to GeneChip Human Exon 1.0 ST arrays. Hybridization was performed for 16 hours using the MES_EukGE-WS2v5_450-DEV fluidics wash and stain script (pre-commercial FS450_0001 script). Arrays were scanned using the Affymetrix GCS 3000 7G and GeneChip Operating Software v. 1.3 to produce .CEL intensity files.
Data analysis algorithms
Signal estimates were derived from the CEL files of the 20 samples by quantile sketch normalization using the PLIER algorithm for probeset (exon-level) intensities and IterPLIER for gene-level intensities [11]. Presence/absence of exons was determined by "Detection Above Background" (DABG), using surrogate GC mismatch intensities.
Candidate exons for alternative splicing were detected using a Splicing Index and the MIDAS algorithm [11]. The Splicing Index (SI) represents the log ratio of the exon intensities between the two tissues after normalization to the gene intensities in each sample: SIi = log2((e1i/g1j)/(e2i/g2j)), for the i-th exon of the j-th gene in tissue type 1 or 2. The splicing indices are then subjected to a t-test to probe for differential inclusion of the exon into the gene. MIDAS employs the Splicing Index in an ANOVA model to test the hypothesis that no alternative splicing occurs for a particular exon. The ExACT program implements the MIDAS algorithm and is available for download. Default parameters were used in all algorithms.
Data analysis
ANOVA p-values and fold changes for gene expression were calculated using Partek Genomic Suite 6.2 (Partek Inc., St. Louis, MO) and biochemical/regulatory network mapping of significant genes was performed using MetaCore 3.2.1 software (GeneGo Inc., St. Joseph, MI). Patient 3 was removed as an outlier due to aberrant behavior in PCA, which improved the signal-to-noise ratio for discrimination by tissue type. Noise ratios also showed that gender, patient and tumor differentiation categories were relevant as factors in the analysis.
For efficient functioning of the alternative splicing algorithms, exons and genes with low expression were removed from the analysis by the following criteria (see Fig. 4). Exons were accepted if they had a DABG p-value < 0.05 in at least 50% of the samples in either tissue. Genes were accepted if either: a) at least 50% of the core exons in at least 50% of samples in both tissues had a DABG p-value < 0.05 or b) if the IterPLIER gene-level signal intensity exceeded a particular threshold, depending on the filtering version. The gene-level requirement is necessary because alternative splicing is meaningless if the gene is not significantly expressed in both tissues.
Subsequent to filtering, the gene-level and exon-level PLIER intensities were jointly processed by either MIDAS or SI. Exons from the Full meta-probeset that associate with these Core genes were included in the analysis. The results were filtered for a Slicing Index > 0.5, and high scoring hits were taken for further analysis. Candidate exons were visually inspected in genomic context (Integrated Genome Browser, Affymetrix Inc. or BLIS 6.2.1, Biotique Systems Inc., Reno, NV) to ensure that exon-level expression patterns were consistent with potential alternative splicing. For example, exon intensities that strongly and uniformly deviate from that of adjacent well-annotated exons in the same gene may indicate cross hybridization, poorly performing probes or absent target. Visualization at the probeset level is also useful to insure that the signals are consistent within tissue groups, that neighboring constitutive exons are comparably expressed and that multiple probesets interrogating the same exon have similar signals.
Finally, possible splicing patterns were determined by mapping the probesets to publicly available mRNA and EST sequences on the UCSC Genome Browser [62] via BLAT. Candidates consistent with known examples of alternative splicing are more likely to prove to be true positives, but this approach will also suppress the discovery of novel splicing events. In addition, the BLAT results indicate whether there similar sequences elsewhere in the genome that may lead to false signals by crosshybridization.
Array Comparison
For comparison of gene-level expression between the Exon Array and the Human Genome U133 Plus 2.0 Array, total RNA from 11 different tissues were purchased from Ambion, Inc. (Austin, Texas). In brief, three assay replicates were prepared from each tissue with 5 μg (3' IVT) or 1 μg (WTA) of RNA according to the Expression Analysis Technical Manual or the Whole Transcript Sense Target Labeling Assay Manual, and hybridized to the Exon and the U133 Plus 2 arrays. Signal estimations were generated with PLIER, using the Core meta-probeset in the case of the Exon Array. The signals were quantile normalized within tissues and then median normalized across the tissue types. The signal was transformed and variance stabilized by log2(signal +16). The CEL files and signal estimation data are available from Affymetrix [60]. A 1-way ANOVA was performed with the Partek Genomics Suite across all tissue types. P-value cutoffs were determined by Bonferroni multiple test correction with thresholds at 0.05, 0.001 and 10-8. Genes which were significantly different in any pair of tissue types were kept for further analysis. Transcript clusters (Exon) and probesets (U133 Plus 2) were mapped to each other according to the file "HuEx-1_0-st-v2 Transcript Cluster ID Mappings to HG-U133 Plus 2.0" [63]. Multiple U133 Plus 2 probesets which target the same transcript cluster were averaged to obtain the signal estimate. The subsequent comparisons were generated with the Partek Genomics Suite.
RT-PCR validation
Approximately 2 μg of total RNA was reverse transcribed to cDNA using the TaqMan Reverse Transcription Reagents Kit per manufacturer's instructions (Applied Biosciences) with a combination of oligo dT and random hexamers as primers. PCR was carried out for 35 cycles using Taq Polymerase (Promega) per manufacturer's instructions with approximately 50–100 ng of cDNA as template. PCR products were separated on 2.5% agarose gels and stained with ethidium bromide for visualization. All RT-PCR primer sequences are included in Additional file 4.
In order to discriminate between the two mutually exclusive exons (Ex7 & Ex8) of the same size in the TPM1 gene, the resulting PCR products were distinguished with an enzyme that cuts only one of the exons. 30 μl of PCR product was digested with 1 U of PvuII (New England Biolabs) at 37°C for 1 hour per manufacturer's instructions. PvuII cuts a sequence (CAGCTG) present in Ex7, but not in Ex8. Thus, RT-PCR products generated from mRNAs containing Ex7 are cut into two smaller, faster migrating segments when separated on a 2.5% agarose gel.
Densitometric analysis of the RT-PCR gels was carried out using AlphaEaseFC 4.0 (Alpha Innotech Corp., San Leandro, CA) software per manufacturer's recommendations. A ratio of two isoforms was generated using the sum of all pixel intensities within an identically sized region containing each band. In some cases, where appropriate, intensities from multiple bands were added together to generate a single value representing an isoform. In order to determine if the change in isoform expression was statistically significant, a simple two-tailed t-test was carried out on the isoform ratios by grouping the 10 samples from either "tumor" or "normal" tissue. A summary of the results is included in Additional file 5.
We would like to thank Yan Zhang, Gene Tanimoto, Tom Gingeras and John Blume for material support, essential advice and critical reviews of this manuscript. This work was funded by Affymetrix, Inc.
Authors' Affiliations
- Black DL: Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem. 2003, 72: 291-336. 10.1146/annurev.biochem.72.121801.161720.PubMedView ArticleGoogle Scholar
- Faustino NA, Cooper TA: Pre-mRNA splicing and human disease. Genes Dev. 2003, 17 (4): 419-437. 10.1101/gad.1048803.PubMedView ArticleGoogle Scholar
- Venables JP: Unbalanced alternative splicing and its significance in cancer. Bioessays. 2006, 28 (4): 378-386. 10.1002/bies.20390.PubMedView ArticleGoogle Scholar
- Finn L, Dougherty G, Finley G, Meisler A, Becich M, Cooper DL: Alternative splicing of CD44 pre-mRNA in human colorectal tumors. Biochem Biophys Res Commun. 1994, 200 (2): 1015-1022. 10.1006/bbrc.1994.1551.PubMedView ArticleGoogle Scholar
- Goodison S, Urquidi V, Tarin D: CD44 cell adhesion molecules. Mol Pathol. 1999, 52 (4): 189-196.PubMedPubMed CentralView ArticleGoogle Scholar
- Sternberg LR, Byrd JC, Hansson GC, Liu KF, Bresalier RS: Alternative splicing of the human MUC2 gene. Arch Biochem Biophys. 2004, 421 (1): 21-33. 10.1016/j.abb.2003.10.002.PubMedView ArticleGoogle Scholar
- Patten LC, Belaguli NS, Baek MJ, Fagan SP, Awad SS, Berger DH: Serum response factor is alternatively spliced in human colon cancer. J Surg Res. 2004, 121 (1): 92-100. 10.1016/j.jss.2004.02.031.PubMedView ArticleGoogle Scholar
- Huerta S, Srivatsan ES, Venkatesan N, Peters J, Moatamed F, Renner S, Livingston EH: Alternative mRNA splicing in colon cancer causes loss of expression of neural cell adhesion molecule. Surgery. 2001, 130 (5): 834-843. 10.1067/msy.2001.116415.PubMedView ArticleGoogle Scholar
- Genuardi M, Viel A, Bonora D, Capozzi E, Bellacosa A, Leonardi F, Valle R, Ventura A, Pedroni M, Boiocchi M, Neri G: Characterization of MLH1 and MSH2 alternative splicing and its relevance to molecular testing of colorectal cancer susceptibility. Hum Genet. 1998, 102 (1): 15-20. 10.1007/s004390050648.PubMedView ArticleGoogle Scholar
- Pospisil H, Herrmann A, Butherus K, Pirson S, Reich JG, Kemmner W: Verification of predicted alternatively spliced Wnt genes reveals two new splice variants (CTNNB1 and LRP5) and altered Axin-1 expression during tumour progression. BMC Genomics. 2006, 7 (1): 148-10.1186/1471-2164-7-148.PubMedPubMed CentralView ArticleGoogle Scholar
- Affymetrix_Inc.: Affymetrix White Papers: Exon Probeset Annotations and Transcript Cluster Groupings v1.0; Exon Array Background Correction v1.0; Guide to Probe Logarithmic Intensity Error (PLIER) Estimation; Gene Signal Estimates from Exon Arrays v1.0; Statistical Algorithms Reference Guide; Alternative Transcript Analysis Methods for Exon Arrays v1.1. [http://www.affymetrix.com/support/technical/whitepapers.affx]
- Fischer H, Stenling R, Rubio C, Lindblom A: Differential expression of aquaporin 8 in human colonic epithelial cells and colorectal tumors. BMC Physiol. 2001, 1: 1-10.1186/1472-6793-1-1.PubMedPubMed CentralView ArticleGoogle Scholar
- Sereti E, Gavriil A, Agnantis N, Golematis VC, Voloudakis-Baltatzis IE: Immunoelectron study of somatostatin, gastrin and glucagon in human colorectal adenocarcinomas and liver metastases. Anticancer Res. 2002, 22 (4): 2117-2123.PubMedGoogle Scholar
- Tseng WW, Liu CD: Peptide YY and cancer: current findings and potential clinical applications. Peptides. 2002, 23 (2): 389-395. 10.1016/S0196-9781(01)00616-7.PubMedView ArticleGoogle Scholar
- Jang MS, Park JE, Lee JA, Park SG, Myung PK, Lee do H, Park BC, Cho S: Binding and regulation of hypoxia-inducible factor-1 by the inhibitory PAS proteins. Biochem Biophys Res Commun. 2005, 337 (1): 209-215. 10.1016/j.bbrc.2005.09.038.PubMedView ArticleGoogle Scholar
- Bauer TW, Fan F, Liu W, Johnson M, Parikh NU, Parry GC, Callahan J, Mazar AP, Gallick GE, Ellis LM: Insulinlike growth factor-I-mediated migration and invasion of human colon carcinoma cells requires activation of c-Met and urokinase plasminogen activator receptor. Ann Surg. 2005, 241 (5): 748-56; discussion 756-8. 10.1097/01.sla.0000160699.59061.92.PubMedPubMed CentralView ArticleGoogle Scholar
- Wong MP, Cheung N, Yuen ST, Leung SY, Chung LP: Vascular endothelial growth factor is up-regulated in the early pre-malignant stage of colorectal tumour progression. Int J Cancer. 1999, 81 (6): 845-850. 10.1002/(SICI)1097-0215(19990611)81:6<845::AID-IJC1>3.0.CO;2-5.PubMedView ArticleGoogle Scholar
- Brembeck FH, Rosario M, Birchmeier W: Balancing cell adhesion and Wnt signaling, the key role of beta-catenin. Curr Opin Genet Dev. 2006, 16 (1): 51-59. 10.1016/j.gde.2005.12.007.PubMedView ArticleGoogle Scholar
- Clark TA, Sugnet CW, Ares M: Genomewide analysis of mRNA processing in yeast using splicing-specific microarrays. Science. 2002, 296 (5569): 907-910. 10.1126/science.1069415.PubMedView ArticleGoogle Scholar
- Srinivasan K, Shiue L, Hayes JD, Centers R, Fitzwater S, Loewen R, Edmondson LR, Bryant J, Smith M, Rommelfanger C, Welch V, Clark TA, Sugnet CW, Howe KJ, Mandel-Gutfreund Y, Ares M: Detection and measurement of alternative splicing using splicing-sensitive microarrays. Methods. 2005, 37 (4): 345-359. 10.1016/j.ymeth.2005.09.007.PubMedView ArticleGoogle Scholar
- Zacharias DA, Garamszegi N, Strehler EE: Characterization of persistent artifacts resulting from RT-PCR of alternatively spliced mRNAs. Biotechniques. 1994, 17 (4): 652-655.PubMedGoogle Scholar
- Venables JP, Burn J: EASI--enrichment of alternatively spliced isoforms. Nucleic Acids Res. 2006, 34 (15): e103-10.1093/nar/gkl592.PubMedPubMed CentralView ArticleGoogle Scholar
- Feral CC, Nishiya N, Fenczik CA, Stuhlmann H, Slepak M, Ginsberg MH: CD98hc (SLC3A2) mediates integrin signaling. Proc Natl Acad Sci U S A. 2005, 102 (2): 355-360. 10.1073/pnas.0404852102.PubMedPubMed CentralView ArticleGoogle Scholar
- Henderson NC, Collis EA, Mackinnon AC, Simpson KJ, Haslett C, Zent R, Ginsberg M, Sethi T: CD98hc (SLC3A2) interaction with beta 1 integrins is required for transformation. J Biol Chem. 2004, 279 (52): 54731-54741. 10.1074/jbc.M408700200.PubMedView ArticleGoogle Scholar
- Fiegen D, Haeusler LC, Blumenstein L, Herbrand U, Dvorsky R, Vetter IR, Ahmadian MR: Alternative splicing of Rac1 generates Rac1b, a self-activating GTPase. J Biol Chem. 2004, 279 (6): 4743-4749. 10.1074/jbc.M310281200.PubMedView ArticleGoogle Scholar
- Cheung N, Wong MP, Yuen ST, Leung SY, Chung LP: Tissue-specific expression pattern of vascular endothelial growth factor isoforms in the malignant transformation of lung and colon. Hum Pathol. 1998, 29 (9): 910-914. 10.1016/S0046-8177(98)90195-2.PubMedView ArticleGoogle Scholar
- Collesi C, Santoro MM, Gaudino G, Comoglio PM: A splicing variant of the RON transcript induces constitutive tyrosine kinase activity and an invasive phenotype. Mol Cell Biol. 1996, 16 (10): 5518-5526.PubMedPubMed CentralView ArticleGoogle Scholar
- Matsushita K, Tomonaga T, Shimada H, Shioya A, Higashi M, Matsubara H, Harigaya K, Nomura F, Libutti D, Levens D, Ochiai T: An essential role of alternative splicing of c-myc suppressor FUSE-binding protein-interacting repressor in carcinogenesis. Cancer Res. 2006, 66 (3): 1409-1417. 10.1158/0008-5472.CAN-04-4459.PubMedView ArticleGoogle Scholar
- Clarke AS, Lotz MM, Mercurio AM: A novel structural variant of the human beta 4 integrin cDNA. Cell Adhes Commun. 1994, 2 (1): 1-6.PubMedView ArticleGoogle Scholar
- McCullough RM, Cantor CR, Ding C: High-throughput alternative splicing quantification by primer extension and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Nucleic Acids Res. 2005, 33 (11): e99-10.1093/nar/gni098.PubMedPubMed CentralView ArticleGoogle Scholar
- Wang Z, Lo HS, Yang H, Gere S, Hu Y, Buetow KH, Lee MP: Computational analysis and experimental validation of tumor-associated alternative RNA splicing in human cancer. Cancer Res. 2003, 63 (3): 655-657.PubMedGoogle Scholar
- Kirschbaum-Slager N, Parmigiani RB, Camargo AA, de Souza SJ: Identification of human exons overexpressed in tumors through the use of genome and expressed sequence data. Physiol Genomics. 2005, 21 (3): 423-432. 10.1152/physiolgenomics.00237.2004.PubMedView ArticleGoogle Scholar
- Hui L, Zhang X, Wu X, Lin Z, Wang Q, Li Y, Hu G: Identification of alternatively spliced mRNA variants related to cancers by genome-wide ESTs alignment. Oncogene. 2004, 23 (17): 3013-3023. 10.1038/sj.onc.1207362.PubMedView ArticleGoogle Scholar
- Herynk MH, Tsan R, Radinsky R, Gallick GE: Activation of c-Met in colorectal carcinoma cells leads to constitutive association of tyrosine-phosphorylated beta-catenin. Clin Exp Metastasis. 2003, 20 (4): 291-300. 10.1023/A:1024024218529.PubMedView ArticleGoogle Scholar
- Jaffe AB, Hall A: Rho GTPases in transformation and metastasis. Adv Cancer Res. 2002, 84: 57-80.PubMedView ArticleGoogle Scholar
- Raval GN, Bharadwaj S, Levine EA, Willingham MC, Geary RL, Kute T, Prasad GL: Loss of expression of tropomyosin-1, a novel class II tumor suppressor that induces anoikis, in primary breast tumors. Oncogene. 2003, 22 (40): 6194-6203. 10.1038/sj.onc.1206719.PubMedView ArticleGoogle Scholar
- Leonardi CL, Warren RH, Rubin RW: Lack of tropomyosin correlates with the absence of stress fibers in transformed rat kidney cells. Biochim Biophys Acta. 1982, 720 (2): 154-162. 10.1016/0167-4889(82)90007-6.PubMedView ArticleGoogle Scholar
- Hendricks M, Weintraub H: Multiple tropomyosin polypeptides in chicken embryo fibroblasts: differential repression of transcription by Rous sarcoma virus transformation. Mol Cell Biol. 1984, 4 (9): 1823-1833.PubMedPubMed CentralView ArticleGoogle Scholar
- Varga AE, Stourman NV, Zheng Q, Safina AF, Quan L, Li X, Sossey-Alaoui K, Bakin AV: Silencing of the Tropomyosin-1 gene by DNA methylation alters tumor suppressor function of TGF-beta. Oncogene. 2005, 24 (32): 5043-5052. 10.1038/sj.onc.1208688.PubMedView ArticleGoogle Scholar
- Lin JL, Geng X, Bhattacharya SD, Yu JR, Reiter RS, Sastri B, Glazier KD, Mirza ZK, Wang KK, Amenta PS, Das KM, Lin JJ: Isolation and sequencing of a novel tropomyosin isoform preferentially associated with colon cancer. Gastroenterology. 2002, 123 (1): 152-162. 10.1053/gast.2002.34154.PubMedView ArticleGoogle Scholar
- Otey CA, Carpen O: Alpha-actinin revisited: a fresh look at an old player. Cell Motil Cytoskeleton. 2004, 58 (2): 104-111. 10.1002/cm.20007.PubMedView ArticleGoogle Scholar
- Honda K, Yamada T, Seike M, Hayashida Y, Idogawa M, Kondo T, Ino Y, Hirohashi S: Alternative splice variant of actinin-4 in small cell lung cancer. Oncogene. 2004, 23 (30): 5257-5262. 10.1038/sj.onc.1207652.PubMedView ArticleGoogle Scholar
- Bois PR, Borgon RA, Vonrhein C, Izard T: Structural dynamics of alpha-actinin-vinculin interactions. Mol Cell Biol. 2005, 25 (14): 6112-6122. 10.1128/MCB.25.14.6112-6122.2005.PubMedPubMed CentralView ArticleGoogle Scholar
- Li Y, Lin JL, Reiter RS, Daniels K, Soll DR, Lin JJ: Caldesmon mutant defective in Ca(2+)-calmodulin binding interferes with assembly of stress fibers and affects cell morphology, growth and motility. J Cell Sci. 2004, 117 (Pt 16): 3593-3604. 10.1242/jcs.01216.PubMedView ArticleGoogle Scholar
- Cuomo ME, Knebel A, Platt G, Morrice N, Cohen P, Mittnacht S: Regulation of microfilament organization by Kaposi sarcoma-associated herpes virus-cyclin.CDK6 phosphorylation of caldesmon. J Biol Chem. 2005, 280 (43): 35844-35858. 10.1074/jbc.M503877200.PubMedView ArticleGoogle Scholar
- McManus MJ, Lingle WL, Salisbury JL, Maihle NJ: A transformation-associated complex involving tyrosine kinase signal adapter proteins and caldesmon links v-erbB signaling to actin stress fiber disassembly. Proc Natl Acad Sci U S A. 1997, 94 (21): 11351-11356. 10.1073/pnas.94.21.11351.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhang LH, Tian B, Diao LR, Xiong YY, Tian SF, Zhang BH, Li WM, Ren H, Li Y, Ji JF: Dominant expression of 85-kDa form of cortactin in colorectal cancer. J Cancer Res Clin Oncol. 2006, 132 (2): 113-120. 10.1007/s00432-005-0046-8.PubMedView ArticleGoogle Scholar
- Chuma M, Sakamoto M, Yasuda J, Fujii G, Nakanishi K, Tsuchiya A, Ohta T, Asaka M, Hirohashi S: Overexpression of cortactin is involved in motility and metastasis of hepatocellular carcinoma. J Hepatol. 2004, 41 (4): 629-636. 10.1016/j.jhep.2004.06.018.PubMedView ArticleGoogle Scholar
- van Rossum AG, de Graaf JH, Schuuring-Scholtes E, Kluin PM, Fan YX, Zhan X, Moolenaar WH, Schuuring E: Alternative splicing of the actin binding domain of human cortactin affects cell migration. J Biol Chem. 2003, 278 (46): 45672-45679. 10.1074/jbc.M306688200.PubMedView ArticleGoogle Scholar
- Clark EA, Golub TR, Lander ES, Hynes RO: Genomic analysis of metastasis reveals an essential role for RhoC. Nature. 2000, 406 (6795): 532-535. 10.1038/35020106.PubMedView ArticleGoogle Scholar
- Astrof S, Crowley D, George EL, Fukuda T, Sekiguchi K, Hanahan D, Hynes RO: Direct test of potential roles of EIIIA and EIIIB alternatively spliced segments of fibronectin in physiological and tumor angiogenesis. Mol Cell Biol. 2004, 24 (19): 8662-8670. 10.1128/MCB.24.19.8662-8670.2004.PubMedPubMed CentralView ArticleGoogle Scholar
- Mercurio AM, Rabinovitz I: Towards a mechanistic understanding of tumor invasion--lessons from the alpha6beta 4 integrin. Semin Cancer Biol. 2001, 11 (2): 129-141. 10.1006/scbi.2000.0364.PubMedView ArticleGoogle Scholar
- Mercurio AM, Bachelder RE, Chung J, O'Connor KL, Rabinovitz I, Shaw LM, Tani T: Integrin laminin receptors and breast carcinoma progression. J Mammary Gland Biol Neoplasia. 2001, 6 (3): 299-309. 10.1023/A:1011323608064.PubMedView ArticleGoogle Scholar
- Hara K, Kudoh H, Enomoto T, Hashimoto Y, Masuko T: Malignant transformation of NIH3T3 cells by overexpression of early lymphocyte activation antigen CD98. Biochem Biophys Res Commun. 1999, 262 (3): 720-725. 10.1006/bbrc.1999.1051.PubMedView ArticleGoogle Scholar
- Ule J, Ule A, Spencer J, Williams A, Hu JS, Cline M, Wang H, Clark T, Fraser C, Ruggiu M, Zeeberg BR, Kane D, Weinstein JN, Blume J, Darnell RB: Nova regulates brain-specific splicing to shape the synapse. Nat Genet. 2005, 37 (8): 844-852. 10.1038/ng1610.PubMedView ArticleGoogle Scholar
- Woolard J, Wang WY, Bevan HS, Qiu Y, Morbidelli L, Pritchard-Jones RO, Cui TG, Sugiono M, Waine E, Perrin R, Foster R, Digby-Bell J, Shields JD, Whittles CE, Mushens RE, Gillatt DA, Ziche M, Harper SJ, Bates DO: VEGF165b, an inhibitory vascular endothelial growth factor splice variant: mechanism of action, in vivo effect on angiogenesis and endogenous protein expression. Cancer Res. 2004, 64 (21): 7822-7835. 10.1158/0008-5472.CAN-04-0934.PubMedView ArticleGoogle Scholar
- Okumura M, Kondo S, Ogata M, Kanemoto S, Murakami T, Yanagida K, Saito A, Imaizumi K: Candidates for tumor-specific alternative splicing. Biochem Biophys Res Commun. 2005, 334 (1): 23-29. 10.1016/j.bbrc.2005.06.057.PubMedView ArticleGoogle Scholar
- Xu Q, Lee C: Discovery of novel splice forms and functional analysis of cancer-specific alternative splicing in human expressed sequences. Nucleic Acids Res. 2003, 31 (19): 5635-5643. 10.1093/nar/gkg786.PubMedPubMed CentralView ArticleGoogle Scholar
- Roy M, Xu Q, Lee C: Evidence that public database records for many cancer-associated genes reflect a splice form found in tumors and lack normal splice forms. Nucleic Acids Res. 2005, 33 (16): 5026-5033. 10.1093/nar/gki792.PubMedPubMed CentralView ArticleGoogle Scholar
- Affymetrix_Inc.: HuEx-1_0-st-v2-colon-cancer-data-set. [http://www.affymetrix.com/support/technical/sample_data/exon_array_data.affx]
- Affymetrix_Inc.: Whole Transcript Sense Target Labeling Assay. [http://www.affymetrix.com/support/downloads/manuals]
- UCSC_Genome_Browser: UCSC Genome Browser. [http://www.genome.ucsc.edu]
- Affymetrix_Inc.: HuEx-1_0-st-v2 Transcript Cluster ID Mappings to HG-U133 Plus 2.0. [http://www.affymetrix.com/support/technical/byproduct.affx?product=huexon-st]
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Local VA Hospital Benefits from CCI Employee Generosity
IRON MOUNTAIN, Mich., Dec 15, 2016 – CCI believes in sharing the spirit of giving this holiday season and with that enthusiasm, CCI Systems' management and employees collected inter-office donations December 12th -14th to help benefit the "Paying It Forward" campaign initiated by CJ Graphics.
Brock Wallis of M.J. Electric, threw out the challenge to CCI's CEO, John Jamar. John gladly accepted and with the help of CCI's 500+ employees was able to raise $2,270 for the VA medical center in only three days.
CCI chose to "Pay it Forward" to the Oscar G. Johnson VA Medical Center to support the following programs offered by the hospital:
- Butterfly Wish Program – Similar to a Make a Wish Program, the Hospice and Palliative Care team strives
to give Community Living Center and Home Based Primary Care patients an opportunity to make a wish come true.
- Holiday Programs – Supports patient programs for Veterans Day, Holiday Party, Memorial Day and Fourth of July.
The "Pay It Forward" campaign was started by CJ Graphics, a local print company, in the hopes of making a difference this holiday season.
"We want to remind people what this time of year is all about — giving and not receiving," said Sharon Socia, outside salesperson at CJ Graphics.
CJ Graphics Pay it Forward initiative will run Dec. 1 through Dec. 31. Each participant who receives a mug will be challenged to Pay it Forward within 24 hours of receiving it. CJ Graphics will be sharing all those paying it forward on their Facebook page. Those who receive the most likes and comments will receive $250 from CJ's to donate to the charity of their choice.

subset: commoncrawl


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Lithe group embarked on its journey towards the garments industry of Bangladesh in 1993 as a small family run business but today after two decades Lithe group has managed to create a clear image of the Bangladeshi knit industry on the globe. With utmost advise, direction and instruction from our Chairman and Managing Director, Mr. F M Kabir Mohiuddin, the company has attained success. Lithe group is now diversifying the company into eco tourism and also agriculture.
|56 AH Tower, 12th & 13th Floor Road-2 Sector-3, Uttara Dhaka-1230, Bangladesh.
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Emily C. Wheeler, Susan A. Wheeler & Rev. Crosby H. Wheeler: National Armenia & India Relief Association & Euphrates College
The personal papers of Charles L. Huston document his life-long active service in the Presbyterian Church and its missionary work. Included in this collection are 42 photographs of Armenian and Indian children 1916-1933 sent by Miss Emily C. Wheeler < http://
Charles Lukens Huston was born in Coatesville, Pennsylvania on July 8, 1856, the second son of Dr. Charles Huston (1822-1897) and Isabella Pennock Huston (1822-1889). Huston was involved for most of his life in the Young Men's Christian Association and in the organization of the Presbyterian Church. He was a member of the National Association of Evangelicals, vice president of the World's Christian Fundamentals Association (1920-46) and a strong supporter of the Church's foreign missionary work and the Prohibition movement. He died at Coatesville, Pa. on March 14, 1951.
Posted are three of the photographs. The collection may be viewed at Manuscripts and Archives Department, Hagley Museum and Library website: < http://digital.hagley.org/


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If someone had to visualize the world of 'TRON' in real life, it would probably looks suspiciously close to the Electri-City Table. Forgetting about the lights for a second, the table itself is made up of a series of miniature people, streets and more, which is exactly what that digital existence would be like to humans. Encased in acrylic and glass, the Electri-City Table appears to have been sent down by geek heaven.
In reality, the Electri-City Table was created by Benjamin Miall Yates. He used old circuit boards and other electronic components to create a sprawling city diorama that people could get lost in for days. Creating various versions of the Electri-City Table, he has relied on everything from CD players and speaker systems to digital photo frames and PC computers.
Electronic Diorama Furniture
More Stats +/-
Material-Juxtaposed Furniture
Stocking-Inspired Furnishings
Illuminated Resin Tops
Hi-Tech Table Tennis Furniture
Rusty Iron Furniture
The Electri-City Table by Ben Yates Looks Like a Miniaturized 'Tron'
- By: Meghan YoungOct 2, 2012
References: interior-design.wonderhowto & neatorama
References: interior-design.wonderhowto & neatorama


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Hi, Looking at the consensus item: RE: CLIENT SIDE AND SERVER SIDE SOLUTIONS S1 - serving content in different forms is an acceptable way to comply with the guidelines as long as equivalents for all of the information are provided in the different forms and it is all available through the same URI (though it may be linked to it) (server side solutions are acceptable - as specified) it seems that providing multiple versions of web pages is acceptable. It doesn't seem to convey the impression of the approach being a "cop out". It does seem to indicate that using multiple versions of web pages is an adequate design. There was the statement: "You keep saying that this is in the consensus appendix but as I explained before, you have to look at the first and last lines of that excerpt to fully appreciate its impact." Perhaps it would be helpful to clarify what is meant by the first and last lines. Multiple versions of web pages avoid the problems of having to choose whose needs to serve when there are conflicting needs. With the increase in bandwidth and computing power, more visual and interactive technology will be delivered. There will be an increase in conflict of needs. For example, in terms of limited screen real estate, the space could be used to provide some accessibility feature or some feature which will be used by the more general public. Scott > Now that we all understand each other, I have some comments in your > message below marked with dp: > look for designing a more accessible web on trace. > > ----- Original Message ----- > From: "Scott Luebking" <firstname.lastname@example.com> > To: <email@example.com> > Sent: Saturday, December 15, 2001 2:52 PM > Subject: Re: FWD: CHI-WEB: Amazon's version for the Visually Impaired > > > Hi, > > The phrase "multiple serve" is not very clear since there are a number > of interpretations depending on various aspects. > dp: I would not use that in many contexts but we know what we are > talking about here and I have explained it. > > While some people consider multiple versions of a web page to be a cop > out, > other people consider it to be a reasonable use of technology to > accomodate the needs of varying types of users. Since the text that > I've quoted before is a consensus item, it would seem that the > guidelines > group see providing multiple versions of the same page as a reasonable > use to technology in addressing the needs of a variety of people. > dp: let's stick to exact quotes please. I did say that it is often and > most often at that used as a cop out for not providing adaquate design > in the first place. > > You keep saying that this is in the consensus appendix but as I > explained before, you have to look at the first and last lines of that > excerpt to fully appreciate its impact. > > Going back to universal design, a common approach is to provide both > ramps and stairs so people can have their choice. Providing multiple > versions of a web page would be consistant with that approach. > dp: only if it is not possible to fil the bill with one and I mean not > possible and only if those pages were served from the same data set and > there fore had no chance of not being updated when the content was > changed or other changes were made to the site that bear on the > rendering of the data. > > >From a technology point of view, it is probably often easier to > offer different versions of a web page rather than trying to get > everything in one page. Different versions helps to reduce the > conflicts > that can arise in a single version. > dp: I'd like to see you take a typically well designed page and use the > guidelines to fail it. > > The web, like much of technology, is evolving. While some people may > wish > the web was more fixed, other people look at the initial web as being > the seed from which newer aspects have sprung. If guidelines are > developed > with the goal of holding back development of technology, they have a > good chance of being signored or seen as irrelevant, unless there is > some regulatory obligation. > dp: guidelines are not developped to hold back the web. the squabbling > among the players does that well enough. Guidelines are developped in > hopes to ensure that the ramps are there along side the stairs. > > Please refer to my complete remarks when addressing points so that they > don't have to be repeated. We need an approach that allows for the web > to grow and also for it to become more not less accessible such as that > beind developped for pedestrians. > > Scott > > PS It would be helpful if you indicated which particular Trace > documents > you think I should be aware of. I'm familiar with a number of them.Received on Sunday, 16 December 2001 11:00:00 UTC
This archive was generated by hypermail 2.3.1 : Tuesday, 13 October 2015 16:21:15 UTC


text:
JERUSALEM – A leading human rights group on Wednesday criticized Palestinian security forces in the West Bank and Gaza Strip for what it said was an increase in attacks on local journalists, including arbitrary detentions and abuse.
A report by the New York-based Human Rights Watch said that over the past two years, the Palestinian Authority in the West Bank has stepped up harassment of journalists investigating corruption or seen to be supporters of the rival Hamas militant group. The pressure tactics by the Western-backed government have led to more self-censorship among local reporters, the group also said.
The report focused on the West Bank, documenting seven cases of abuse there. But it also included two cases in the Hamas-ruled Gaza Strip — where reported abuses against journalists by Hamas have been less frequent but nevertheless harsh, according to HRW. It said abuse by Hamas, as well as by Israeli security forces, would be the focus of future reporting.
"Palestinian security forces are becoming notorious for assaulting and intimidating journalists who are just trying to do their jobs," said Joe Stork, deputy Middle East director at Human Rights Watch. "Both the Palestinian Authority in the West Bank and Hamas in Gaza need to end these blatant attacks on free expression."
Speaking to reporters in the West Bank town of Ramallah, Stork said the abuses stretched beyond journalists. "Many other citizens in the West Bank, in Gaza, who are critics or who are perceived to be critics of the government or of the authorities are coming to face the same treatment," he said.
The group urged international donors to demand the Palestinian Authority — which receives hundreds of millions of dollars in Western support each year — stop the practice as a condition for receiving aid.
Adnan Dameiri, a spokesman for the security forces in the West Bank, said the cases were aberrations, "individual behavior from certain members in the security forces" and that they do not reflect a "systematic policy of the Palestinian Authority against journalists."
A Hamas official in Gaza said instances of abuse were mistakes, and those responsible have been punished. "The government absolutely rejects any harassment committed against the media," the official said, speaking on condition of anonymity because of the sensitivity of the subject.
HRW cited a 2010 report by the Palestinian Center for Development and Media Freedoms, which found a 45 percent increase over the previous year in physical attacks, arrests, confiscation of equipment and other violations against journalists. The independent, nonprofit group recorded 79 cases of journalist abuse by the Palestinian Authority and Hamas.
In one case documented by Human Rights Watch, freelance journalist and filmmaker Muhannad Salahat was arrested by Palestinian Authority and held without charge on two occasions in early 2010.
During 24 days in custody, he was interrogated about his documentaries and asked to sign a document saying he belonged to a Facebook group that included discussions of governmental corruption. He was then told that was the basis of the case to be brought against him. He was later released.
Another freelance journalist, Khalid Amayreh, said he has been repeatedly detained by Palestinian Authority security since reporting about torture in West Bank prisons and criticizing the suppression of protests against Israel's 2009 military offensive in Gaza. In one instance, HRW said, he was put in solitary confinement and forced to sleep next to a toilet.
The abuse takes place "in the context of virtual impunity" for security officials, the report said. And while it is not necessarily ordered by Palestinian leaders, "the utter failure of the PA leadership to address the prevailing culture of impunity for such abuses suggests that they reflect government policy," HRW said.
Hamas' security services in the Gaza Strip have also intimidated journalists, calling in reporters to warn them their coverage was slanted and threatening punishment, the report said.
It also noted a recent wave of crackdowns on journalists who cover protests in Gaza that urge the rival Palestinian governments to reconcile. In one instance, Hamas agents threatened to throw a journalist out of a window, HRW said.
Dalia Nammari in Ramallah, West Bank, contributed to this report.


text:
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text:
Class: Antineoplastic Agents
- Antiandrogens
VA Class: AN900
Chemical Name: 2-Methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]-propanamide
Molecular Formula: C11H11F3N2O3
CAS Number: 13311-84-7
Brands: Eulexin
Medically reviewed by Drugs.com. Last updated on Jul 2, 2018.
- Hepatotoxicity
Severe liver injury (i.e., increased serum transaminase concentrations, jaundice, hepatic encephalopathy, acute hepatic failure) reported, sometimes resulting in hospitalization and/or rarely death; manifestations generally occurred within first 3 months and in some patients, were reversible after discontinuance.1
Measure serum transaminase concentrations prior to initiation of therapy, monthly during first 4 months, and periodically thereafter.1
Immediately measure serum transaminase (especially ALT) concentrations if manifestations suggestive of liver dysfunction (e.g., nausea, vomiting, abdominal pain, fatigue, anorexia, flu-like symptoms, hyperbilirubinuria, jaundice, right upper quadrant tenderness) occur.1
Immediately discontinue if jaundice develops or serum ALT concentration is >2 times ULN; monitor liver function closely until resolves.1
Antineoplastic agent; a nonsteroidal antiandrogen.1 2 3 5 6 7 9 11 16 19 23 24 25 26 27
Uses for Flutamide
Prostate Cancer
First-line therapy in combination with a luteinizing hormone-releasing hormone (LHRH) analog (e.g., goserelin, leuprolide acetate, triptorelin) for prostate cancer.1 3 5 6 7 20 22 23
Treatment of locally confined (stage B2 or C)1 20 22 23 and metastatic (stage D2) prostate cancer;1 3 5 6 7 20 should be used in conjunction with an LHRH analog.1 3 5 6 7 20 22 23
Flutamide Dosage and Administration
In patients with stage B2 or C prostate cancer, initiate flutamide and LHRH analog 8 weeks prior to and continue during radiation therapy.1
In patients with stage D2 metastatic prostate cancer, initiate flutamide and LHRH analog concomitantly and continue until disease progression.1
Oral Administration
Administer orally 3 times daily at 8-hour intervals1 2 3 5 6 7 12 14 17 without regard to meals29 .
Prostate Cancer
250 mg 3 times daily, at 8-hour intervals.1 2 3 5 6 7 12 14 17 24 25 27 28
Cautions for Flutamide
Known hypersensitivity to flutamide or any ingredient in the formulation.1
Severe hepatic impairment.1
Hepatic Effects
For warnings regarding hepatotoxicity, see Boxed Warning.
Use in Women
Not intended for use in women, particularly for nonserious or nonlife-threatening conditions.1
Fetal/Neonatal Morbidity and Mortality
May cause fetal harm if used in pregnant women.1
Aniline Toxicity
Metabolized in part to 4-nitro-3-fluoro-methylaniline; toxicities associated with aniline exposure (i.e., methemoglobinemia, hemolytic anemia, cholestatic jaundice) reported.1
Monitor methemoglobin concentrations periodically in susceptible patients (e.g., those with glucose-6-phosphate dehydrogenase deficiency, hemoglobin M disease, smokers).1
Sensitivity Reactions
Photosensitivity reactions (i.e., erythema, ulceration, bullous eruptions, epidermal necrolysis) reported.1
General Precautions
Endocrine Effects
Laboratory Monitoring
Regularly monitor serum PSA to assess response; if PSA increases, evaluate possible disease progression.1
For patients with objective progression of disease and elevated serum PSA, consider temporarily withdrawing flutamide while continuing LHRH analog.1
Specific Populations
Category D.1 (See Fetal/Neonatal Morbidity and Mortality under Warnings.)
Not intended for use in women.1
Pediatric Use
Not studied in pediatric patients.1
Hepatic Impairment
Contraindicated in patients with severe hepatic impairment. (See Boxed Warning.)1
Not intended for use in women, particularly for nonserious or nonlife-threatening conditions.1
Common Adverse Effects
Combined therapy with LHRH analog: hot flashes, loss of libido, impotence, diarrhea, nausea, vomiting, gynecomastia.1
Cystitis, rectal bleeding, proctitis, skin rash, hematuria also frequent when flutamide combined with LHRH analog and radiation therapy.1
Interactions for Flutamide
Specific Drugs
Increased risk of facial flushing31
Avoid alcohol consumption during therapy31
LHRH analog (e.g., goserelin, leuprolide)
Pharmacokinetic interaction unlikely1
Increased PT reported1
Closely monitor PT; adjust anticoagulant dosage as needed1
Flutamide Pharmacokinetics
Rapidly and completely absorbed following oral administration, with peak plasma concentrations of active metabolite (2-hydroxyflutamide) attained within about 2 hours.1 27
Food does not affect bioavailability.1
Special Populations
In patients with chronic renal impairment, there appears to be no correlation between creatinine clearance and maximum plasma concentrations or AUC of flutamide; renal impairment did not affect maximum plasma concentrations or AUC of active metabolite.1
Plasma Protein Binding
Flutamide: 94–96%; 2-hydroxyflutamide: 92–94%.1 27
Rapidly and extensively metabolized to ≥6 metabolites, including an active α-hydroxylated derivative, 2-hydroxyflutamide.1 27
Elimination Route
Excreted principally in urine and to lesser extent in feces (4.2%) as unchanged drug and metabolites.1
2-Hydroxyflutamide: about 6 hours after a single 250-mg dose.1
Special Populations
In patients with renal impairment (Clcr <29 mL/minute), half-life of active metabolite was slightly prolonged; no dosage adjustment necessary in chronic renal impairment.1
In geriatric patients, half-life of active metabolite is about 8 hours after a single 250-mg dose.1
Pharmacokinetics not studied in patients with hepatic impairment, women, or pediatric patients.1
2–30°C.1 Protect unit dose packages from excessive moisture.1
A selective antiandrogen with no estrogenic, antiestrogenic, progestational, antiprogestational, antigonadotropic, or adrenocortical activity in various animal models.3
Competitively blocks nuclear androgen receptors in target tissues (e.g., prostate, seminal vesicles, adrenal cortex).1 3 8 9 10 11 12 24 25
Blockade of androgen receptors in the hormone-sensitive tumor cells may result in growth arrest or transient tumor regression through inhibition of androgen-dependent DNA and protein synthesis.1 3 8 9 10 11 12 24 25
Inhibits initial androgenic stimulation and potential exacerbation of symptoms (e.g., bone pain, urinary obstruction, liver pain, impending spinal cord compression) associated with the first month of LHRH analog therapy.6 10 11 18
Advice to Patients
Risk of liver toxicity.1 Importance of adhering to schedule for laboratory monitoring of serum transaminases and reporting signs or symptoms of hepatic toxicity to clinician immediately.1
Importance of initiating flutamide concomitantly with LHRH analog and of not interrupting or discontinuing therapy without consulting a clinician.1
Risk of facial flushing, particularly if used in conjunction with alcohol.31 Avoidance of alcohol recommended if flushing occurs.31
Not intended for use in women.1
Importance of informing clinicians of existing or contemplated concomitant therapy, including prescription and OTC drugs, as well concomitant illnesses.1
Importance of informing patients of other important precautionary information.1 (See Cautions.)
Excipients in commercially available drug preparations may have clinically important effects in some individuals; consult specific product labeling for details.
Please refer to the ASHP Drug Shortages Resource Center for information on shortages of one or more of these preparations.
* available from one or more manufacturer, distributor, and/or repackager by generic (nonproprietary) name
Dosage Forms
Brand Names
125 mg*
Eulexin (with parabens and povidone;)
Flutamide Capsules
Barr, Genpharm, Sandoz, Teva
AHFS DI Essentials™. © Copyright 2019, Selected Revisions July 1, 2006. American Society of Health-System Pharmacists, Inc., 4500 East-West Highway, Suite 900, Bethesda, Maryland 20814.
1. Schering. Eulexin (flutamide) capsules prescribing information. Kenilworth, NJ; 2000 Dec.
2. Dole EJ, Holdsworth MT. Nilutamide: an antiandrogen for the treatment of prostate cancer. Ann Pharmacother. 1997; 31: 65-75.
3. Brogden RN, Clissold SP. Flutamide: a preliminary review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy in advanced prostatic cancer. Drugs. 1989; 38: 185-203.
4. Harris MG, Coleman SG, Faulds D et al. Nilutamide: a review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy in prostate cancer. Drugs Aging. 1993; 3: 9-25.
5. Schellhammer PF, Sharifi R, Block NL et al. A controlled trial of bicalutamide versus flutamide, each in combination with luteinizing hormone-releasing hormone analogue therapy, in patients with advanced prostatecarcinoma. Cancer. 1996; 78:2164-9. http://www.ncbi.nlm.nih.gov/pubmed/8918410?dopt=AbstractPlus
6. Crawford ED, Eisenberger MA, McLeod DG et al. A controlled trial of leuporlide with and without flutamide in prostatic carcinoma. N Engl J Med. 1989; 321: 419-24. http://www.ncbi.nlm.nih.gov/pubmed/2503724?dopt=AbstractPlus http://www.pubmedcentral.nih.gov/picrender.fcgi?tool=pmcentrez&artid=3091023&blobtype=pdf
7. Schellhammer PF, Venner P, Haas GP et al. Prostate specific antigen decreases after withdrawal of antiandrogen therapy with bicalutamide or flutamide in patients receiving combined androgen blockade. J Urol. 1997; 157: 1731-5. http://www.ncbi.nlm.nih.gov/pubmed/9112515?dopt=AbstractPlus http://www.pubmedcentral.nih.gov/picrender.fcgi?tool=pmcentrez&artid=3461836&blobtype=pdf
8. Geller J. Basis for hormonal management of advanced prostate cancer. Cancer. 1993; 71(Suppl):1039-45. http://www.ncbi.nlm.nih.gov/pubmed/7679038?dopt=AbstractPlus
9. McLeod DG. Antiandrogenic drugs. Cancer. 1993; 71(Suppl):1046-9. http://www.ncbi.nlm.nih.gov/pubmed/8428326?dopt=AbstractPlus
10. Denis L. Prostate cancer: primary hormonal treatment. Cancer. 1993; 71(Suppl):1050-8. http://www.ncbi.nlm.nih.gov/pubmed/8428327?dopt=AbstractPlus
11. Daneshgari F, Crawford ED. Endocrine therapy of advanced carcinoma of the prostate. Cancer. 1993; 71(Suppl):1089-97. http://www.ncbi.nlm.nih.gov/pubmed/8428333?dopt=AbstractPlus
12. Denis L, Murphy GP. Overview of phase II trials on combined androgen treatment in patients with metastatic prostate cancer. Cancer. 1993; 72: 3888-3895.
13. Vogelzang NJ, Kennealey GT. Recent developments in endocrine treatment of prostate cancer. Cancer. 1992; 70:966-76. http://www.ncbi.nlm.nih.gov/pubmed/1386283?dopt=AbstractPlus
14. Kennealey GT, Furr BJA. Use of the nonsteroidal anti-androgen Casodex in advanced prostatic carcinoma. Urol Clin North Am. 1991; 18:99-110. http://www.ncbi.nlm.nih.gov/pubmed/1992575?dopt=AbstractPlus
15. Blackledge G. Casodexmechanisms of action and opportunities for usage. Cancer. 1993; 72(Suppl):3830-3. http://www.ncbi.nlm.nih.gov/pubmed/7504578?dopt=AbstractPlus
16. Tyrrell CJ, Altwein JE, Klippel F et al et al. A multicenter randomized trial comparing the luteinizing hormone-releasing hormone analogue goserelin acetate alone and with flutamide in the treatment of advanced prostate cancer. J Urol. 1991; 146:1321-6. http://www.ncbi.nlm.nih.gov/pubmed/1834864?dopt=AbstractPlus
17. Santen RJ. Endocrine treatment of prostate cancer. J Clin Endocrinol Metab. 1992; 75:685-9. http://www.ncbi.nlm.nih.gov/pubmed/1517354?dopt=AbstractPlus
18. Chrisp P, Goa KL. Goserelin: a review of its pharmacodynamic and pharmacokinetic properties, and clinical use in sex hormone-related conditions. Drugs. 1991; 41:254-88. http://www.ncbi.nlm.nih.gov/pubmed/1709853?dopt=AbstractPlus
19. Soloway MS, Matzkin H. Antiandrogenic agents as monotherapy in advanced prostatic carcinoma. Cancer. 1993; 71(Suppl):1083-8. http://www.ncbi.nlm.nih.gov/pubmed/8428332?dopt=AbstractPlus
20. Prostate cancer. From: CancerNet/PDQ. Physician data query (database). Bethesda, MD: National Cancer Institute; 2001 Oct.
21. Smith PH. Deferred therapy in patients with advanced disease. Cancer. 1993; 71(Suppl):1074-7. http://www.ncbi.nlm.nih.gov/pubmed/8428330?dopt=AbstractPlus
22. Anon. Drugs of choice for cancer. Treat Guide Med Lett. 2003; 1:41-52.
23. Cersosimo RJ, Carr D. Prostate cancer: current and evolving strategies. Am J Health-Syst Pharm. 1996; 53:381-96. http://www.ncbi.nlm.nih.gov/pubmed/8673658?dopt=AbstractPlus
24. Erlichman C, Loprinzi CL. Hormonal therapies. In: DeVita VT Jr, Hellman S, Rosenberg SA, eds. Cancer: principles and practice of oncology. 5th ed. Philadelphia, PA: J. B. Lippincott; 1997:395-405.
25. Wilson JD. Androgens. In: Hardman JG, Limbird LE, Molinoff PB et al, eds. Goodman and Gilman's the pharmacological basis of therapeutics. 9th ed. New York: McGraw-Hill; 1995:1441-57.
26. McLeod DG, Kolvenbag GJ. Defining the role of antiadnrogens in the treatment of prostate cancer. Urology. 1996: 47; 85-9.
27. Goldspiel BR, Kohler DR. Flutamide: an antiandrogen for advanced prostate cancer. DICP. 1990: 24; 616-23. (IDIS 266817)
28. Anon. Flutamide for prostate cancer. Med Lett Drugs Ther. 1989: 31; 72. (IDIS 257670)
29. Schering, Kenilworth, NJ: Personal communication.
30. Wysowski DK, Fourcroy JL. Flutamide hepatotoxicity. J Urol. 1996; 155: 209-12. http://www.ncbi.nlm.nih.gov/pubmed/7490837?dopt=AbstractPlus
31. Kirschenbaum A. Management of hormonal treatment effects. Cancer. 1995; 75:1983-6.
More about flutamide
- Side Effects
- During Pregnancy
- Dosage Information
- Drug Images
- Drug Interactions
- Pricing & Coupons
- En Español
- Drug class: antiandrogens
Consumer resources
Professional resources
Other brands: Eulexin


text:
Understanding Power
The Indispensable ChomskyBook - 2002
An introduction to Noam Chomsky's views on the politics of power discusses third-party politics in the United States, the suppression of dissent, U.S. foreign and domestic policy, and the role of the media.
Norton Pub
In a series of enlightening and wide-ranging discussions, all published here for the first time, Chomsky radically reinterprets the events of the past three decades, covering topics from foreign policy during Vietnam to the decline of welfare under the Clinton administration. And as he elucidates the connection between America's imperialistic foreign policy and the decline of domestic social services, Chomsky also discerns the necessary steps to take toward social change. With an eye to political activism and the media's role in popular struggle, as well as U.S. foreign and domestic policy, Understanding Power offers a sweeping critique of the world around us and is definitive Chomsky.
Characterized by Chomsky's accessible and informative style, this is the ideal book for those new to his work as well as for those who have been listening for years.
From the critics
Community Activity
CommentAdd a Comment
Understanding Power is a great primer for Chomsky's political thought.
A previous reviewer suggests that Chomsky "never names names, just uses generalities." One wonders if he read the book at all, because, quite to the contrary, Chomsky is sometimes specific to a fault, consistently drawing on historical source documents --both government and media. Citations abound in this book.
Indeed, there are SO many citations--449 pages worth, for a 401 page book--that the publishers chose to put them on a website, rather than in the book itself.
Unfortunately, the website has since went down. You can, however, find all the citations at archive.org
Chomsky claims: /// Kennedy substantially increased the level of violence in Latin America \\\ HUH???? JFK renewed the promise of FDR, that he would not dispatch the US military in support of the multinationals. It was JFK who halted foreign aid to no less than 7 dictators who refused to move towards democracy. It was JFK who warned the Dutch off of Indonesia and worked out a compromise between [Rockefeller-owned] Stanvac and CalTex with the Indonesian government - - it was Eisenhower and his CIA who dispatched mercenaries to kill thousands of Indonesians, just as it was Lyndon Johnson and his CIA who led the coup in the overthrow of Sukarno, with over one-half million Indonesians killed! Chomsky lies and lies and lies about the Kennedy administration and one wonders what paymaster is Chomsky lying for? And Operation Brother Sam occurred under Johnson, the military and foreign aid in the overthrow of the democratically elected government of Brazil! Suggest anyone who doubts me read: Donald Gibson's book, Battling Wall Street: the Kennedy presidency /// Andrew Cohen's book, Two Days in June, and all the executive orders and legislation signed by JFK.
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text:
Last week I watched a group of anti-Wall Street protestors as they paraded down the street. One of their signs said, "Corporate America is Guilty." A sweeping generalization if there ever was one. If capitalism is guilty, what can we say about anti-capitalism? In judging the free market, we often forget who the enemies of the free market have been. We forget one of the foremost voices of anti-capitalism, which sounded a clarion call. "I have the gift of reducing all problems to their simplest foundations," said Adolf Hitler. "War is the origin of all things."
Such were the words of history's most famous anti-capitalist politician. "There is a broadened strategy, a war with intellectual weapons," Hitler explained. "When I wage war, in the midst of peace, troops will suddenly appear…. No one will stop them." Hitler's plan was to overthrow all governments, to override the free market and install his own lackeys. "We shall find such men," he explained, "[and] we shall find them in every country. We shall not need to bribe them. They will come of their own accord. Ambition and delusion, party squabbles and self-seeking arrogance will drive them. Peace will be negotiated before the war has begun."
The capitalists want peace; so let them think it is "peace in our time." Attack them when they least expect it; for the anti-capitalist program has always been predicated on war. "Our strategy," said Hitler, "is to destroy the enemy from within, to conquer him through himself." Instead of artillery barrages against entrenched enemy troops, the Nazi leader envisioned revolutionary propaganda used to psychologically break down entire countries. "The enemy people must be demoralized and ready to capitulate, driven into moral passivity, before military action can even be thought of," Hitler declared.
How was this moral breakdown to be achieved? "We shall not shrink from plotting revolutions," Hitler explained. "We shall have friends who will help us in all the enemy countries. We shall know how to obtain such friends. Mental confusion, contradiction of feeling, indecisiveness, and panic: these are our weapons." The history of revolutions are always the same, he mused. "The ruling classes capitulate. Why? They no longer have the will to conquer. The lessons of revolution … I have learnt from the Bolsheviks. I do not hesitate to say so."
The program for victory in modern times is simple. "When the enemy is demoralized from within, when he stands on the brink of revolution, when social unrest threatens, that is the right moment. A single blow must destroy him. Aerial attacks, stupendous in their mass effect, surprise, terror, sabotage, assassination from within, the murder of leading men, overwhelming attacks … sudden attacks, all in the same second, without regard for reserves or losses: that is the war of the future. A gigantic, all-destroying blow. I do not consider the consequences; I think only of this one thing," Hitler admitted.
The dictator's words were set down by Hermann Rauschning, and published in a book, Voice of Destruction (which predicted Hitler's future trajectory with remarkable accuracy). Besides Hitler, there have been other voices of destruction. Consider a secret speech of Joseph Stalin to the Central Committee of the Communist Party Soviet Union on 19 August 1939:
We have before us a vast field of action to develop the World Revolution. Comrades! It is in the interest of the USSR – the Fatherland of the Workers – that war should break out between the Reich [Nazi Germany] and the Franco-British capitalist bloc. We must do everything so that the war should last as long as possible with the aim of weakening both sides. It is for these reasons that we must give priority to the approval of the conclusion of the pact proposed by Germany, and to work so that this war, which will be declared within a few days, shall last as long as possible. It is therefore necessary to strengthen the work of propaganda in the countries that will have entered the war….
When we look around today, and hear the "necessary work of propaganda" on all sides, we should remember that someone who is not a capitalist wants today's wars to continue so that ordered society will be weakened; so that revolution can be advanced; and the secret of revolution is to target capitalism, to malign business. As Hitler said again and again, "We are socialists, we are enemies of today's capitalistic economic system … and we are all determined to destroy this system under all conditions."
Why are they determined to destroy capitalism?
The reason is simple. Capitalism stands in the way of dictatorship. Capitalism represents liberty, and property rights, and democratic government. Therefore, if you are like Hitler, and seeking total power, you must oppose capitalism in principle as well as in practice. For you will never achieve total power unless you eradicate the capitalist system and the culture of freedom it engenders. Such is the thinking that lies behind the North Korean regime, the ruling Iranian clerics, and the "Caliphate" of the al Qaeda terrorists. They all hate capitalism, they all want nuclear weapons, and they all claim that corporate America is "guilty."
One needs only a little common sense to realize that capitalism is dedicated to producing goods and services. It is capitalism that has raised the world's overall standard of living, while anti-capitalism has left nothing but a vast trail of destruction in its wake. One has only to study the leading exemplars of anti-capitalism – today's "voices of destruction" – to grasp this fact.