Progression of BRAF-induced thyroid cancer is associated with epithelial-mesenchymal transition requiring concomitant MAP kinase and TGFß signaling.

Mice with thyroid-specific expression of oncogenic BRAF (Tg-Braf) develop papillary thyroid cancers (PTCs) that are locally invasive and have well-defined foci of poorly differentiated thyroid carcinoma (PDTC). To investigate the PTC-PDTC progression, we performed a microarray analysis using RNA from paired samples of PDTC and PTC collected from the same animals by laser capture microdissection. Analysis of eight paired samples revealed a profound deregulation of genes involved in cell adhesion and intracellular junctions, with changes consistent with an epithelial-mesenchymal transition (EMT). This was confirmed by immunohistochemistry, as vimentin expression was increased and E-cadherin lost in PDTC compared with adjacent PTC. Moreover, PDTC stained positively for phospho-Smad2, suggesting a role for transforming growth factor (TGF)ß in mediating this process. Accordingly, TGFß-induced EMT in primary cultures of thyroid cells from Tg-Braf mice, whereas wild-type thyroid cells retained their epithelial features. TGFß-induced Smad2 phosphorylation, transcriptional activity and induction of EMT required mitogen-activated protein kinase (MAPK) pathway activation in Tg-Braf thyrocytes. Hence, tumor initiation by oncogenic BRAF renders thyroid cells susceptible to TGFß-induced EMT, through a MAPK-dependent process.

Comparison of rat olfactory mucosal responses to carcinogenic and non-carcinogenic chloracetanilides.

Alachlor and butachlor are chloracetanilide herbicides that induce olfactory tumors in rats, whereas propachlor does not. The mechanism by which alachlor induces tumors is distinct from many other nasal carcinogens, in that alachlor induces a gradual de-differentiation of the olfactory mucosa (OM) to a more respiratory-like epithelium, in contrast to other agents that induce cytotoxicity, followed by an aberrant regenerative response. We studied biochemical and genomic effects of these compounds to identify processes that occur in common between alachlor- and butachlor-treated rats. Because we have previously shown that matrix metalloproteinase-2 (MMP2) is activated in OM by alachlor, in the present studies we evaluated both MMP2 activation and changes in OM gene expression in response to carcinogenic and non-carcinogenic chloracetanilide treatments. All three chloracetanilides activated MMP2, and >300 genes were significantly up- or downregulated between control and alachlor-treated rats. The most significantly regulated gene was vomeromodulin, which was dramatically upregulated by alachlor and butachlor treatment (>60-fold), but not by propachlor treatment. Except for similar gene responses in alachlor- and butachlor-treated rats, we did not identify clear-cut differences that would predict OM carcinogenicity in this study.

Influence of fatty acid diets on gene expression in rat mammary epithelial cells.

BACKGROUND: This study examines the impact of dietary fatty acids on regulation of gene expression in mammary epithelial cells before and during puberty. METHODS: Diets primarily consisted of n-9 monounsaturated fatty acids (olive oil), n-6 polyunsaturated fatty acids (safflower), saturated acids (butter), and the reference AIN-93G diet (soy oil). The dietary regimen mimics the repetitive nature of fatty acid exposure in Western diets. Diet-induced changes in gene expression were examined in laser capture microdissected mammary ductal epithelial cells at day of weaning and end of puberty. PCNA immunohistochemistry analysis compared proliferation rates between diets. RESULTS: Genes differentially expressed between each test diets and the reference diet were significantly enriched by cell cycle genes. Some of these genes were involved in activation of the cell cycle pathway or the G2/M check point pathway. Although there were some differences in the level of differential expression, all diets showed qualitatively the same pattern of differential expression compared to the reference diet. Cluster analysis identified an expanded set of cell cycle as well as immunity and sterol metabolism related clusters of differentially expressed genes. CONCLUSION: Fatty acid-enriched diets significantly upregulated proliferation above normal physiological levels during puberty. Higher cellular proliferation during puberty caused by enriched fatty acid diets poses a potential increase risk of mammary cancer in later life. The human homologs of 27 of 62 cell cycle rat genes are included in a human breast cancer cluster of 45 cell cycle genes, further emphasizing the importance of our findings in the rat model.

Interphase chromosome folding determines spatial proximity of genes participating in carcinogenic RET/PTC rearrangements.

Recurrent chromosomal rearrangements are common in cancer cells and may be influenced by nonrandom positioning of recombination-prone genetic loci in the nucleus. However, the mechanism responsible for spatial proximity of specific loci is unknown. In this study, we use an 18 Mb region on 10q11.2-21 containing the RET gene and its recombination partners, the H4 and NCOA4 (ELE1) genes, as a model chromosomal region frequently involved in RET/PTC rearrangements in thyroid cancer. RET/PTC is particularly common in tumors from children exposed to ionizing radiation. Using fluorescence in situ hybridization and three-dimensional microscopy, the locations of five different loci in this region were mapped in interphase nuclei of normal human thyroid cells. We show that RET and NCOA4 are much closer to each other than expected based on their genomic separation. Modeling of chromosome folding in this region suggests the presence of chromosome coiling with coils of approximately 8 Mb in length, which positions the RET gene close to both, the NCOA4 and H4, loci. There was no significant variation in gene proximity between adult and pediatric thyroid cells. This study provides evidence for large-scale chromosome folding of the 10q11.2-21 region that offers a structural basis for nonrandom positioning and spatial proximity of potentially recombinogenic intrachromosomal loci.

RET/PTC-induced gene expression in thyroid PCCL3 cells reveals early activation of genes involved in regulation of the immune response.

RET/PTC rearrangements represent key genetic events involved in papillary thyroid carcinoma (PTC) initiation. The aim of the present study was to identify the early changes in gene expression induced by RET/PTC in thyroid cells. For this purpose, microarray analysis was conducted on PCCL3 cells conditionally expressing the RET/PTC3 oncogene. Gene expression profiling 48 h after activation of RET/PTC3 identified a statistically significant modification of expression of 270 genes. Quantitative PCR confirmation of 20 of these demonstrated 90% accuracy of the microarray. Functional clustering of genes with greater than or less than 1.75-fold expression change (86 genes) revealed RET/PTC3-induced regulation of genes with key functions in apoptosis (Ripk3, Tdga), cell-cell signaling (Cdh6, Fn1), cell cycle (Il24), immune and inflammation response (Cxcl10, Scya2, Il6, Gbp2, Oas1, Tap1, RT1Aw2, C2ta, Irf1, Lmp2, Psme2, Prkr), metabolism (Aldob, Ptges, Nd2, Gss, Gstt1), signal transduction (Socs3, Nf1, Jak2, Cpg21, Dusp6, Socs1, Stat1, Stat3, Cish) and transcription (Nr4a1, Junb, Hfh1, Runx1, Foxe1). Genes coding for proteins involved in the immune response and in intracellular signal transduction pathways activated by cytokines and chemokines were strongly represented, indicating a critical role of RET/PTC3 in the early modulation of the immune response.

Bayesian mixture model based clustering of replicated microarray data.

MOTIVATION: Identifying patterns of co-expression in microarray data by cluster analysis has been a productive approach to uncovering molecular mechanisms underlying biological processes under investigation. Using experimental replicates can generally improve the precision of the cluster analysis by reducing the experimental variability of measurements. In such situations, Bayesian mixtures allow for an efficient use of information by precisely modeling between-replicates variability. RESULTS: We developed different variants of Bayesian mixture based clustering procedures for clustering gene expression data with experimental replicates. In this approach, the statistical distribution of microarray data is described by a Bayesian mixture model. Clusters of co-expressed genes are created from the posterior distribution of clusterings, which is estimated by a Gibbs sampler. We define infinite and finite Bayesian mixture models with different between-replicates variance structures and investigate their utility by analyzing synthetic and the real-world datasets. Results of our analyses demonstrate that (1) improvements in precision achieved by performing only two experimental replicates can be dramatic when the between-replicates variability is high, (2) precise modeling of intra-gene variability is important for accurate identification of co-expressed genes and (3) the infinite mixture model with the 'elliptical' between-replicates variance structure performed overall better than any other method tested. We also introduce a heuristic modification to the Gibbs sampler based on the 'reverse annealing' principle. This modification effectively overcomes the tendency of the Gibbs sampler to converge to different modes of the posterior distribution when started from different initial positions. Finally, we demonstrate that the Bayesian infinite mixture model with 'elliptical' variance structure is capable of identifying the underlying structure of the data without knowing the 'correct' number of clusters. AVAILABILITY: The MS Windows based program named Gaussian Infinite Mixture Modeling (GIMM) implementing the Gibbs sampler and corresponding C++ code are available at http://homepages.uc.edu/~medvedm/GIMM.htm SUPPLEMENTAL INFORMATION: http://expression.microslu.washington.edu/expression/kayee/medvedovic2003/medvedovic_bioinf2003.html

XPA protein alters the specificity of ultraviolet light-induced mutagenesis in vitro.

Studies of ultraviolet (UV) light mutagenesis have demonstrated mutations at common sites in the target genes of shuttle vector plasmids replicated in cultured cells or by cellular extracts. The reasons for the specific pattern of mutagenesis are largely unknown. We have examined the specificity of UV-induced mutagenesis by replicating plasmid pLS189, irradiated with 40 J/m(2) UVC or unirradiated, in either xeroderma pigmentosum group A (XP-A) or HeLa cellular extracts. The XP-A extract displayed slightly lower replication ability, but produced a higher mutant frequency, compared to that of HeLa extract. Use of irradiated plasmid inhibited replication by an average of 63% and increased the mutant frequency by an average of 16.7-fold. Analysis of mutation spectra revealed nonrandom patterns of mutagenesis that differed significantly between HeLa and XP-A extracts. In comparison to HeLa extract, replication in XP-A extract resulted in lower frequencies of GC --> AT transitions and tandem double-base substitutions, and a higher frequency of deletions. Replication in HeLa extract produced hotspots at positions 100, 108, and 156 that were not produced by XP-A extract. Furthermore, XP-A extract produced hotspots at positions 124, 133, and 164, sites not characteristic of previous UV-induced mutagenesis studies using XPA-expressing cells. Addition of purified XPA protein to reactions containing XP-A extract altered each of these parameters, including loss of the hotspots at positions 124 and 133, to yield a more HeLa-like spectrum. These results indicate a previously uncharacterized role of the XPA protein in influencing the specificity of UV-induced mutagenesis during DNA replication.

Expression of ATM in ataxia telangiectasia fibroblasts rescues defects in DNA double-strand break repair in nuclear extracts.

Ataxia telangiectasia (A-T) is a human genetic disorder characterized by progressive cerebellar degeneration, hypersensitivity to ionizing radiation (IR), immunodeficiency, and high cancer risk. At the cellular level, IR sensitivity and increased frequency of spontaneous and IR-induced chromosomal breakage and rearrangements are the hallmarks of A-T. The ATM gene, mutated in this syndrome, has been cloned and codes for a protein sharing homology with DNA-PKcs, a protein kinase involved in DNA double-strand break (DSB) repair and DNA damage responses. The characteristics of the A-T cellular phenotypes and ATM gene suggest that ATM may play a role similar to that of DNA-PKcs in DSB repair and that there is a primary DNA repair defect in A-T cells. In the current study, the function of ATM in DNA DSB repair was evaluated in an in vitro system using two plasmids, carrying either an EcoRI-induced DSB within the lacZalpha gene or various endonuclease-induced DSB in the SupF suppressor tRNA gene. We found that the DSB repair efficiency in A-T nuclear extracts was comparable to, if not higher than, that in normal nuclear extracts. However, the repair fidelity in A-T nuclear extracts was decreased when repairing DSB with short 5' and 3' overhangs (<4 base pairs (bp)) or blunt ends, but not 5' 4-bp overhangs. Sequencing of the mutant plasmids revealed that deletions involving 1-6 nucleotide microhomologies were the major class of mutations in both A-T and normal extracts. However, the size of the deletions in plasmids from A-T nuclear extracts was larger than that from normal nuclear extracts. Expression of the ATM protein in A-T cells corrected the defect in DSB repair in A-T nuclear extracts. These results suggest that ATM plays a role in maintaining genomic stability by preventing the repair of DSB from an error-prone pathway.

Proximity of chromosomal loci that participate in radiation-induced rearrangements in human cells.

Rearrangements involving the RET gene are common in radiation-associated papillary thyroid cancer (PTC). The RET/PTC1 type of rearrangement is an inversion of chromosome 10 mediated by illegitimate recombination between the RET and the H4 genes, which are 30 megabases apart. Here we ask whether despite the great linear distance between them, RET and H4 recombination might be promoted by their proximity in the nucleus. We used two-color fluorescence in situ hybridization and three-dimensional microscopy to map the positions of the RET and H4 loci within interphase nuclei. At least one pair of RET and H4 was juxtaposed in 35% of normal human thyroid cells and in 21% of peripheral blood lymphocytes, but only in 6% of normal mammary epithelial cells. Spatial contiguity of RET and H4 may provide a structural basis for generation of RET/PTC1 rearrangement by allowing a single radiation track to produce a double-strand break in each gene at the same site in the nucleus.

The transcriptional signature of dioxin in human hepatoma HepG2 cells.

We have used a high density microarray hybridization approach to characterize the transcriptional response of human hepatoma HepG2 cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We find that exposure to 10 nM TCDD for 8 hr alters by at least a factor of 2.1 the expression of 310 known genes and of an equivalent number of expressed sequence tags. Treatment with TCDD in the presence of 20 microg/mL of cycloheximide blocked the effect on 202 of these genes, allowing us to distinguish between primary effects of TCDD exposure, which take place whether cycloheximide is present or not, and secondary effects, which are blocked by inhibition of protein synthesis. Of the 310 known genes affected by TCDD, 30 are up-regulated and 78 are down-regulated regardless of cycloheximide treatment, and 84 are up-regulated and 118 are down-regulated only when protein synthesis is not inhibited. Functional clustering of genes regulated by TCDD reveals many potential physiological interactions that might shed light on the multiple biological effects of this compound. Our results, however, suggest that arriving at a sound understanding of the molecular mechanisms governing the biological outcome of TCDD exposure promises to be orders of magnitude more complicated than might have been previously imagined.

What is the potential measurement error in occupational exposure studies?

This paper demonstrates statistical methods that estimate measurement error from available industrial hygiene data. Errors in measuring a continuous exposure variable may arise when all individuals in a work area are assigned the same exposure. An example is when the mean of exposure measurements obtained on a sample of individuals is assigned to all workers with similar jobs. This may lead to inaccurate point and interval estimates in exposure-response modeling. A method of simulating the distribution of true (i.e., unobserved) individual exposures is described in order to estimate the mean and variance of measurement error. The minimum variance unbiased estimator approximates the mean of lognormally distributed exposure measurements. The distribution of true individual exposures is approximated by the distribution of simulated estimates of mean exposure. The methodology is illustrated by exposure data from work areas manufacturing refractory ceramic fiber (RCF) and RCF products. Results show that exposure is slightly underestimated in work areas with between 25 and 113 exposure measurements; measurement error variance averages about 1.3% of the total variance.

Weight change and lung function: implications for workplace surveillance studies.

This study evaluated the relationship between weight change and longitudinal measurement of lung function among 361 men providing at least five pulmonary function tests. The men in this study were participants in a workplace pulmonary surveillance program for subjects with exposure to refractory ceramic fibers (RCFs). Occupational and environmental studies are generally designed to evaluate factors suspected of causing excess decline in lung function. Failure to adequately account for all significant factors may lead to erroneous conclusions regarding change in lung function. This study utilized two different statistical models to evaluate longitudinal changes in a cohort of RCF workers. What was unique to this study was the modeling of longitudinally measured initial weight, weight change, and longitudinal exposure before and during the period of observation. Results showed a strong relationship between weight gain and longitudinal loss in lung function that approximated forced vital capacity declines of 16 mL for every kilogram of weight gain per year in both models. This value is comparable or greater in magnitude and significance to other factors known to be inversely related to lung function, such as age and pack-years smoking to time of initial testing. In conclusion, weight gain was found to have a significant impact on longitudinal change in lung function. Therefore, weight gain becomes a very important variable that requires consideration whenever longitudinal studies of pulmonary function are conducted.

Mutational specificity in a shuttle vector replicating in chromium(VI)-treated mammalian cells.

An SV40-based shuttle vector, pZ189, was used to characterize the mutation specificity and to explore the mechanism of chromium mutagenesis in mammalian cells. We showed previously that mutagenic DNA damage is induced by the treatment of plasmid with chromium(VI) plus glutathione in vitro. The induced mutation pattern suggested that chromium mutagenesis can be induced by the generation of reactive oxygen intermediates. To further investigate the mechanism of chromium mutagenesis, we treated cultured mammalian cells containing normal pZ189 vector with chromium(VI). Mutations were induced by Cr(VI) in a dose-dependent manner. The majority of base substitution mutations were widely distributed across the supF mutation target gene and occurred mainly at GC basepairs. Overall, the mutation spectra were not significantly different from each other except for a mutation hot spot at position 43, observed only in plasmids from Cr(VI)-treated cells. The characteristics of Cr(VI)-induced mutations were similar to those observed in the mutation spectra induced by H2O2 treatment of the pZ189 plasmid or plasmid-containing cells. These results are consistent with the hypothesis that induction of mutations by chromium in cultured cells occurs through the generation of oxidative DNA damage.

Development and evaluation of a microprocessor-based ergonomic dosimeter for evaluating carpentry tasks.

This portable and self-contained lightweight microprocessor based Ergonomic Dosimeter is designed to collect continuously postural angles of the torso and the upper arm in the sagittal plane and the number of kneeling activities. Up to 4 h of task performance data can be stored in a non-volatile memory of the dosimeter, which can then be downloaded to a lap-top computer. The portable dosimeter was tested for test-retest reliability, compared with posture data obtained with a computer-based video analysis system and evaluated at a carpenter's apprentices school and at a construction site. The dosimeter was shown to be suitable for collecting posture and kneeling data for a prolonged period at construction sites.

Longitudinal estimates of pulmonary function in refractory ceramic fiber manufacturing workers.

Refractory ceramic fibers (RCF) are man-made vitreous fibers (MMVF) used in high-temperature industrial applications. Between 1987 and 1994, a prospective study evaluated pulmonary function of 361 male workers currently employed in RCF manufacturing and related operations for plausibility of a causal relationship between RCF exposure and pulmonary function changes. Workers included in the analysis provided at least five pulmonary function tests. The exposure-response relationship was modeled with two exposure variables: years in a production job, and cumulative fiber exposure (fiber-mo/cc). Comparison groups were nonproduction workers and workers with up to 15 fiber-mo/cc cumulative exposure. A statistically significant decrease in FVC was demonstrated among workers employed in production jobs more than 7 yr prior to initial test. A similar but nonstatistically significant result was demonstrated for FVC in workers with greater than 60 fiber-mo/cc cumulative exposure prior to initial pulmonary function test. Similar but nonstatistically significant results were obtained for FEV1. These findings, which primarily reflect workers employed before 1980, did not persist with analysis of follow-up production years and accumulated RCF exposure from initial pulmonary function test. Since longitudinal analyses are sensitive to influences that continue to affect annual decline during the study period, lower RCF exposure levels since the 1980s may be responsible for eliminating any further effect on pulmonary function.

The effect of succimer therapy in lead intoxication using postural balance as a measure: a case study in a nine year old child.

Postural balance testing was used as a measure of the effect of therapy on a 9 year old boy with high lead levels. Following therapy with CaEDTA and succimer, the patient's postural sway responses were comparable to a low-lead (< 10 micrograms/dL) comparison group for 3 out of 4 tests which rely relatively less on the higher centers for balance. This improvement in postural balance may be attributable to the combined influence of pharmacologic and age associated maturational effects. This case study provides suggestive evidence that while chelation therapy can reduce PbB levels quickly, it can also modify gross neuromotor function manifested by postural balance characteristics.

Studies of allelic loss in thyroid tumors reveal major differences in chromosomal instability between papillary and follicular carcinomas.

Loss of heterozygosity (LOH) studies have been used to identify sites harboring tumor suppressor genes involved in tumor initiation or progression. Previous reports have suggested that regions within chromosomes 3p, 11q, 2p, 2q, 10q, and 1p may be frequently deleted in human follicular thyroid cell tumors. We have extended the analysis of these and other selected regions to 65 paired thyroid tumor tissues. Twenty-four were follicular adenomas, 30 were papillary carcinomas, 10 were follicular carcinomas, and 1 was an anaplastic carcinoma. Sixty percent of the follicular carcinomas, 33% of the follicular adenomas, and 23% of the papillary carcinomas presented LOH at least at 1 site. Fifty percent of the follicular carcinomas showed 2 or more chromosome arms affected by deletions, whereas just 1 of the 24 follicular adenomas and none of the papillary carcinomas presented this feature. However, none of the specific loci examined had a rate of LOH greater than 33%, even in follicular carcinomas. This prompted us to place our findings into a broader context, and we, therefore, performed a meta analysis of all published studies of LOH in follicular thyroid neoplasms. There was a phenotype dependency in the overall rate of LOH, with no specific region displaying a particularly high prevalence. Most notably, by contrast to follicular carcinomas, papillary carcinomas had exceedingly low rates of LOH. Thus, there is a sharp distinction between the two major forms of differentiated thyroid cancer in their tendency to lose genetic material. This probably results from a fundamental difference in mechanisms controlling chromosomal stability in these two forms of cancers that in all likelihood has implications for tumor behavior and prognosis.

Effect of chronic low-level exposure to jet fuel on postural balance of US Air Force personnel.

This study used the postural stability technique to investigate the neurological effects of cumulative low-level exposure to raw JP-8 jet fuel vapor on aircraft maintenance personnel. All subjects performed two sets of four 30-second postural sway tests. The results of mean cumulative exposure levels (in parts per million +/- standard error of mean) were the following: naphthas, 1308 +/- 292; benzene, 21.2 +/- 5.7; toluene, 23.8 +/- 6.1; and m-,o-, p-xylene, 22.7 +/- 5.4. Covariate adjusted regression analysis of the exposed group data showed a statistically significant association (P < 0.05) between the solvents (benzene, toluene, and xylene) and increased postural sway response. For all solvent exposures, the "eyes closed, on foam" test provided the strongest association between sway length and JP-8 benzene (r2 range, 0.45 to 0.52), implying subtle influence on vestibular/proprioception functionalities.