Use of laser capture microdissection for the assessment of equine lamellar basal epithelial cell signalling in the early stages of laminitis.

REASONS FOR PERFORMING STUDY: Dysadhesion of laminar basal epithelial cells (LBECs) from the underlying dermis is the central event leading to structural failure in equine laminitis. Although many studies of sepsis-related laminitis have reported multiple events occurring throughout the lamellar tissue, there is minimal information regarding signalling events occurring specifically in LBECs. OBJECTIVES: To determine signalling events in the LBECs during the early stages of carbohydrate-induced laminitis. STUDY DESIGN: Experimental study. METHODS: Eight horses were given an overload of carbohydrate (CHO) consisting of corn starch mixture via nasogastric tube. Prior to administration of CHO, lamellar biopsies were taken from the left forefoot (control [CON]). Biopsies were taken from the left hind foot at the onset of fever (developmental [DEV]) and from the right forefoot at the onset of Obel grade 1 lameness (OG1). Laminar basal epithelial cells were isolated from cryosections using a laser capture microdissection (LCM) microscope. Next generation sequencing (RNA-seq) was used to identify transcripts expressed in the LBECs for each time point and bioinformatic analysis was performed with thresholds for between group comparisons set at a greater than 2-fold change and P value ≤0.05. RESULTS: Forty genes (22 increased/18 decreased) were significantly different from DEV time vs. CON and 107 genes (57 increased/50 decreased) were significantly different from OG1 time vs. CON. Significant increases in inflammatory genes were present in addition to significantly altered expression of genes related to extracellular matrix composition, stability and turnover. CONCLUSIONS: Signalling related to inflammatory response and extracellular matrix regulation was strongly represented at the DEV and OG1 times. These results indicate that the LBEC is not only a casualty but also an active participant in lamellar events leading to structural failure of the digital lamellae in equine laminitis.

Aldehyde dehydrogenase-1a1 induces oncogene suppressor genes in B cell populations.

The deregulation of B cell differentiation has been shown to contribute to autoimmune disorders, hematological cancers, and aging. We provide evidence that the retinoic acid-producing enzyme aldehyde dehydrogenase 1a1 (Aldh1a1) is an oncogene suppressor in specific splenic IgG1(+)/CD19(-) and IgG1(+)/CD19(+) B cell populations. Aldh1a1 regulated transcription factors during B cell differentiation in a sequential manner: 1) retinoic acid receptor alpha (Rara) in IgG1(+)/CD19(-) and 2) zinc finger protein Zfp423 and peroxisome proliferator-activated receptor gamma (Pparg) in IgG1(+)/CD19(+) splenocytes. In Aldh1a1(-/-) mice, splenic IgG1(+)/CD19(-) and IgG1(+)/CD19(+) B cells acquired expression of proto-oncogenic genes c-Fos, c-Jun, and Hoxa10 that resulted in splenomegaly. Human multiple myeloma B cell lines also lack Aldh1a1 expression; however, ectopic Aldh1a1 expression rescued Rara and Znf423 expressions in these cells. Our data highlight a mechanism by which an enzyme involved in vitamin A metabolism can improve B cell resistance to oncogenesis.

MicroRNA gene expression during retinoic acid-induced differentiation of human acute promyelocytic leukemia.

MicroRNAs (miRNAs) are small non-coding RNAs of 19-25 nucleotides that are involved in the regulation of critical cell processes such as apoptosis, cell proliferation and differentiation. However, little is known about the role of miRNAs in granulopoiesis. Here, we report the expression of miRNAs in acute promyelocytic leukemia patients and cell lines during all-trans-retinoic acid (ATRA) treatment by using a miRNA microarrays platform and quantitative real time-polymerase chain reaction (qRT-PCR). We found upregulation of miR-15a, miR-15b, miR-16-1, let-7a-3, let-7c, let-7d, miR-223, miR-342 and miR-107, whereas miR-181b was downregulated. Among the upregulated miRNAs, miR-107 is predicted to target NFI-A, a gene that has been involved in a regulatory loop involving miR-223 and C/EBPa during granulocytic differentiation. Indeed, we have confirmed that miR-107 targets NF1-A. To get insights about ATRA regulation of miRNAs, we searched for ATRA-modulated transcription factors binding sites in the upstream genomic region of the let-7a-3/let-7b cluster and identified several putative nuclear factor-kappa B (NF-kappaB) consensus elements. The use of reporter gene assays, chromatin immunoprecipitation and site-directed mutagenesis revealed that one proximal NF-kappaB binding site is essential for the transactivation of the let-7a-3/let-7b cluster. Finally, we show that ATRA downregulation of RAS and Bcl2 correlate with the activation of known miRNA regulators of those proteins, let-7a and miR-15a/miR-16-1, respectively.

Relationship between body composition, blood volume and maximal oxygen uptake.

It has long been known that body mass and, more specifically, lean body mass are strongly correlated with maximal oxygen uptake (VO2max) in man and animals. However, there are no data to date describing this phenomenon in the horse. The purpose of this paper is to examine the relationship between body composition and VO2max in the horse. Twenty-three healthy and unfit Standardbred mares performed an incremental exercise test (GXT) to measure VO2max. Rump fat thickness (RTH), a measure of fat covering, was measured using B-mode ultrasound. Plasma volume, total blood volume and red cell volume were determined, using the Evan's Blue dye dilution technique and packed cell volume. VO2max was correlated with body mass (r = 0.541; P<0.01) and exercise haematocrit (exHCT; r = 0.407; P<0.05) but not RTH or the other haematological variables. To eliminate the influence of body mass on the individual variables, a regression analysis was performed on the mass-residuals of VO2max, RTH, plasma volume and exHCT. The residuals of VO2max were correlated negatively with the residuals of RTH (r = -0.687; P = 0.0003) and positively with the residuals of exHCT (r = 0.422; P = 0.045) but not plasma volume. VO2max could be predicted from a linear combination of the residuals of RTH and exHCT (r = 0.767; P<0.0001). These data indicate that VO2max in the horse is significantly related to fat-free mass (FFM), independent of body mass. Red blood cells from the splenic reserve constitute an important factor in the horse's ability to achieve a high VO2max. Therefore, lean body mass may be a more appropriate basis for assessing metabolic function in the athletic horse.

Characterization of the 13q14 tumor suppressor locus in CLL: identification of ALT1, an alternative splice variant of the LEU2 gene.

Chromosome 13q14 deletions constitute the most common genetic abnormality in chronic lymphocytic leukemia (CLL). To identify the putative tumor suppressor gene targeted by 13q14 genomic loss, we completely sequenced and characterized a segment of 790 kb at 13q14 spanning the minimal region of loss in CLL. Transcribed sequences in the region were identified through database homology searches and exon-prediction analysis. Two-hundred kb at the centromeric end of the sequence contain five CpG islands, three previously identified genes LEU5/RFP2, LEU2, and LEU1, seven of seven EST clusters composed of >10 ESTs, and a large number of predicted exons. Homology searches against the mouse EST database have allowed us to identify a highly conserved alternative first exon of the LEU2 gene, giving rise to a novel transcript, ALT1 (GenBank accession no. AF380424), which originates within a G+C region in the vicinity of the D13S272 marker. Two novel 3' exons of LEU2 were also identified and are present in both LEU2 and ALT1 transcripts. However, we have not identified any mutations in leukemia cases, or alterations in expression of mRNAs in the region, that might directly implicate these mRNAs in the pathology of CLL. The centromeric end of the sequence, where all reported genes are located, contains twice the expected amount of ALU repeats, whereas the telomeric end is LINE1 rich and contains four LINE1 elements longer than 4 kb, including two full-length LINE1 sequences. This feature of the sequence may favor the occurrence of chromosomal rearrangements and may confer instability to the region, resulting in deletions that may inactivate an as yet unidentified tumor suppressor.

FEZ1/LZTS1 gene at 8p22 suppresses cancer cell growth and regulates mitosis.

The FEZ1/LZTS1 gene maps to chromosome 8p22, a region that is frequently deleted in human tumors. Alterations in FEZ1/LZTS1 expression have been observed in esophageal, breast, and prostate cancers. Here, we show that introduction of FEZ1/LZTS1 into Fez1/Lzts1-negative cancer cells results in suppression of tumorigenicity and reduced cell growth with accumulation of cells at late S-G(2)/M stage of the cell cycle. Fez1/Lzts1 protein is hyperphosphorylated by cAMP-dependent kinase during cell-cycle progression. We found that Fez1/Lzts1 is associated with microtubule components and interacts with p34(cdc2) at late S-G(2)/M stage in vivo. Present data show that FEZ1/LZTS1 inhibits cancer cell growth through regulation of mitosis, and that its alterations result in abnormal cell growth.

Fez1/lzts1 alterations in gastric carcinoma.

PURPOSE: Loss of heterozygosity (LOH) involving the short arm of chromosome 8 (8p) is a common feature of the malignant progression of human tumors, including gastric cancer. We have cloned and mapped a candidate tumor suppressor gene, FEZ1/LZTS1, to 8p22. Here we have analyzed whether FEZ1/LZTS1 alterations play a role in the development and progression of gastric carcinoma. EXPERIMENTAL DESIGN: We examined Fez1/Lzts1 expression in 8 gastric carcinoma cell lines by Western blot, and in 88 primary gastric carcinomas by immunohistochemistry. Twenty-six of these 88 primary gastric carcinomas were also microdissected and tested for LOH at the FEZ1/LZTS1 locus and for mutation of the FEZ1/LZTS1 gene. Furthermore, we studied the FEZ1/LZTS1 gene regulation and transcriptional control and the methylation status of the 5' region of the gene in all 8 gastric carcinoma cell lines. RESULTS: Fez1/Lzts1 protein was barely detectable in all of the gastric cancer cell lines tested and was absent or significantly reduced in 39 of the 88 (44.3%) gastric carcinomas analyzed by immunohistochemistry, with a significant correlation (P < 0.001) to diffuse histotype. DNA allelotyping analysis showed allelic loss in 3 of 17 (18%) and microsatellite instability in 4 of 17 (23.5%) cases informative for D8S261 at the FEZ1/LZTS1 locus. When we compared the presence of LOH with Fez1/Lzts1 expression, we found loss of protein expression in all three of the tumors with allelic imbalance at D8S261. A missense mutation was detected in one case that did not express Fez1/Lzts1. Hypermethylation of the CpG island flanking the Fez1/Lzts1 promoter was evident in six of the eight cell lines examined as well as in the normal control. CONCLUSIONS: Our findings support FEZ1/LZTS1 as a candidate tumor suppressor gene at 8p in a subtype of gastric cancer and suggest that its inactivation is attributable to several factors including genomic deletion and methylation.

Sequence conservation at human and mouse orthologous common fragile regions, FRA3B/FHIT and Fra14A2/Fhit.

It has been suggested that delayed DNA replication underlies fragility at common human fragile sites, but specific sequences responsible for expression of these inducible fragile sites have not been identified. One approach to identify such cis-acting sequences within the large nonexonic regions of fragile sites would be to identify conserved functional elements within orthologous fragile sites by interspecies sequence comparison. This study describes a comparison of orthologous fragile regions, the human FRA3B/FHIT and the murine Fra14A2/Fhit locus. We sequenced over 600 kbp of the mouse Fra14A2, covering the region orthologous to the fragile epicenter of FRA3B, and determined the Fhit deletion break points in a mouse kidney cancer cell line (RENCA). The murine Fra14A2 locus, like the human FRA3B, was characterized by a high AT content. Alignment of the two sequences showed that this fragile region was stable in evolution despite its susceptibility to mitotic recombination on inhibition of DNA replication. There were also several unusual highly conserved regions (HCRs). The positions of predicted matrix attachment regions (MARs), possibly related to replication origins, were not conserved. Of known fragile region landmarks, five cancer cell break points, one viral integration site, and one aphidicolin break cluster were located within or near HCRs. Thus, comparison of orthologous fragile regions has identified highly conserved sequences with possible functional roles in maintenance of fragility.

Cloning and characterization of CLLD6, CLLD7, and CLLD8, novel candidate genes for leukemogenesis at chromosome 13q14, a region commonly deleted in B-cell chronic lymphocytic leukemia.

Chromosome 13q14 deletions constitute the most common structural aberration in B-cell chronic lymphocytic leukemia (B-CLL). We constructed a high-resolution physical map covering the critical deleted region in B-CLL at 13q14 and flanking sequences. The order and position of both genomic markers and known genes were determined precisely. Three novel genes, CLLD6, CLLD7, and CLLD8, were isolated and characterized. The predicted protein sequence of CLLD6 revealed no homology with known proteins. However, both CLLD7 and CLLD8 predicted proteins contain known functional domains. CLLD7 has both an RCC1 and a BTB domain, and could thus be involved in cell cycle regulation by chromatin remodeling. CLLD8 contains a methyl-CpG binding, a preSET and a SET domain, suggesting that CLLD8 might be associated with methylation-mediated transcriptional repression. Mutation analysis of hematopoietic tumor cell lines and B-CLL tumor samples revealed no point mutations within the coding region of these three novel genes. The functional domains present within CLLD7 and CLLD8 suggest that the proteins may be involved in critical cellular processes such as cell cycle and transcriptional control and could therefore be directly or indirectly involved in leukemogenesis.

Effect of adenoviral transduction of the fragile histidine triad gene into esophageal cancer cells.

Reintroduction of a tumor suppressor gene product in cancer cells is a promising strategy for cancer gene therapy. The fragile histidine triad (FHIT) gene has been identified in a region at chromosome 3p14.2, which is deleted in many tumors, including esophageal cancer. Previous studies have shown frequent biallelic alterations of the FHIT gene in numerous tumors, and have demonstrated a tumor suppressor function of Fhit. We have studied the biological effects of adenoviral-FHIT transduction in esophageal cancer cell lines. Results showed suppression of cell growth in vitro in three of seven esophageal cancer cell lines, all seven of which showed abundant expression of the transgene. Adenoviral-FHIT expression, but not control adenoviral infections, induced caspase-dependent apoptosis in two esophageal cancer cell lines, TE14 and TE4, which express no or very little Fhit, respectively. Treatment of TE14 cells with adenoviral-FHIT vectors resulted in abrogation of tumorigenicity in nude mice. A third esophageal cancer cell line, TE12, without detectable endogenous Fhit, showed accumulation of cells at S to G2-M and a small apoptotic cell fraction after adenoviral-FHIT transduction. Thus, adenoviral-FHIT expression can inhibit the growth of esophageal cancer cells, at least in part through caspase-dependent apoptosis, suggesting that adenoviral-FHIT infection should be explored as a therapeutic strategy.

Mutation analysis of hBUB1, human mitotic checkpoint gene in multiple carcinomas.

hBUB1 is a human homolog of yeast mitotic check point gene that plays an important role in chromosome segregation. Recently mutations of hBUB1 were reported in colorectal cancer cell lines, indicating that inactivation of this gene could be directly involved in aneuploidy in human carcinoma cells. To obtain information of the magnitude of hBUB1 inactivation in multiple carcinomas, we examined mutations in 59 multiple carcinoma cell lines showing single base alteration, however, there was no mutation of hBUB1 with amino acid change in these carcinomas. There were four silent mutations at codon 93, codon 735, codon 430 and codon 98 in KYSE190, TE8 esophageal carcinoma cells, KATOIII gastric carcinoma cells and 697 B cell leukemia cells, respectively. Two candidates of mutation were identified in TE3 esophageal carcinoma cells and 697 B cell leukemia cell line at codon 9 and codon 285, respectively. This result suggests that the inactivation of hBUB1 may be very rare in human carcinomas, or restricted to certain cell lines of colorectal carcinomas.

Altered expression of Fhit in carcinoma and precarcinomatous lesions of the esophagus.

The FHIT gene, located at chromosome 3p14.2, is a tumor suppressor gene often involved in tumors resulting from exposure to environmental carcinogens. We studied 46 pairs of esophageal primary tumors and corresponding normal squamous mucosa specimens by molecular genetic and immunohistochemical methods to investigate the role of the FHIT gene in esophageal carcinoma. In addition, we studied several different types of lesions, such as carcinoma in situ or dysplasia by immunohistochemistry. Loss of heterozygosity at or around the FHIT gene was observed in 35 (76%) primary tumors. Immunohistochemical detection of Fhit protein in the primary tumors demonstrated that 14 (30%) were positive and 32 (70%) were negative. We observed concordance between loss of Fhit protein and loss of heterozygosity and between loss of Fhit protein and RNA abnormalities. Because the FHIT/FRA3B locus is susceptible to damage by environmental carcinogens, we investigated the correlation between Fhit expression and smoking or alcohol habits. In this relatively small study, the patients who were both heavy users of tobacco and alcohol showed a significantly higher frequency of loss of Fhit expression than those who were light users. Noncarcinomatous squamous epithelium showed positive Fhit reactivity in most cases; however, five showed negative Fhit reactivity. Interestingly, all of these five patients had habits of heavy use of tobacco and alcohol. Eight of 12 carcinomas in situ, 2 of 4 severe dysplasias, 4 of 8 moderate dysplasias, and 3 of 9 mild dysplastic lesions showed negative Fhit reactivity. These findings indicated that loss of Fhit expression may be an early event in the development of human esophageal carcinoma and may occur even in normal-appearing squamous epithelium in some patients heavily exposed to environmental carcinogens.

Endocrine disruption: thyroid dysfunction in mummichogs (Fundulus heteroclitus) from a polluted habitat.

Mummichogs (Fundulus heteroclitus) from Piles Creeks (PC), New Jersey (a polluted site), are sluggish and show poorer prey capture and predator avoidance than reference fish from Tuckerton (TK). The behavioral dysfunction of the PC fish may be associated with thyroid impairment due to endocrine disruption. In this study, we compared thyroid histology and thyroid hormones in the two populations and determined experimentally whether the polluted environment could alter thyroid hormone levels. PC fish had larger thyroid follicles, greater follicle cell heights, and contained higher plasma thyroxine (T4) levels than TK fish. However, there were no significant differences in either plasma or tissue triiodothyronine (T3). TK fish held in simulated PC environments had higher plasma T4 and lower plasma T3 than field-sampled fish. PC fish held in clean water had lower plasma T4 and T3 than field-sampled fish. In either case, there was no significant difference in tissue T3 content. The contaminants in PC alter thyroid structure and function, which may relate to the behavioral differences between fish from the polluted and reference sites.

Seasonal specificity of hormonal, behavioral, and coloration responses to within- and between-sex encounters in male lizards (Sceloporus undulatus).

This study reports the gender and seasonal specificity of hormonal, behavioral, and coloration responses displayed by "resident" male lizards (Sceloporus undulatus) exposed to male or female "intruders" during staged encounters in outdoor enclosures. Resident males were engaged in staged encounters with males or females for 1 h per day on 9 consecutive days during the breeding and postbreeding seasons. Male-specific responses occurred during the breeding but not the postbreeding season. These included (1) a transient increase in plasma testosterone (T) that was evident on Day 4 and had subsided by Day 10, (2) behavioral displays of aggression (full shows and chases), and (3) a lightening of dorsal integumental color. Female-specific behavioral responses (nod sets) were displayed in both seasons. Season-specific responses consisted only of a transient increase in plasma corticosterone (B) during the breeding season that was evident on Day 4 and had subsided by Day 10. Pushups were displayed in response to both genders during both seasons, although the frequency of pushups was significantly higher in response to females than to males during the postbreeding season. The coloration of residents did not change in response to male intruders during the postbreeding season or to females during either season. These results define the gender and seasonal specificity of hormonal, behavioral, and coloration responses of resident male S. undulatus in social interactions with conspecifics. Thus, our results clarify the biological significance of these responses in terms of potentially aggressive versus courtship interactions and breeding versus postbreeding contexts.

Cancer-specific chromosome alterations in the constitutive fragile region FRA3B.

We have sequenced 870 kilobases of the FHIT/FRA3B locus, from FHIT intron 3 to intron 7. The locus is AT rich (61.5%) and Alu poor (6. 2%), and it apparently does not harbor other genes. In a detailed analysis of the 308-kilobase region between FHIT exon 5 and the telomeric end of intron 3, a region known to encompass a human papillomavirus-16 integration site and two clusters of aphidicolin-induced chromosome 3p14.2 breakpoints, we have precisely mapped 10 deletion and translocation endpoints in cancer-derived cell lines relative to positions of specific repetitive elements, regions of high genome flexibility and aphidicolin-induced breakpoints. Conclusions are (i) that aphidicolin-induced breakpoint clusters fall close to high-flexibility sequences, suggesting that these sequences contribute directly to aphidicolin-induced fragility; (ii) that 9 of the 10 FHIT allelic deletions in cancer cell lines resulted in loss of exons, with 7 deletion endpoints near long interspersed nuclear elements or long terminal repeat elements; and (iii) that cancer-specific deletions encompass multiple high-flexibility genomic regions, suggesting that fragile breaks may occur at these regions, whereas repair of the breaks involves homologous pairing of flanking sequences with concomitant deletion of the damaged fragile sequence.

The FEZ1 gene at chromosome 8p22 encodes a leucine-zipper protein, and its expression is altered in multiple human tumors.

Alterations of human chromosome 8p occur frequently in many tumors. We identified a 1.5-Mb common region of allelic loss on 8p22 by allelotype analysis. cDNA selection allowed isolation of several genes, including FEZ1. The predicted Fez1 protein contained a leucine-zipper region with similarity to the DNA-binding domain of the cAMP-responsive activating-transcription factor 5. RNA blot analysis revealed that FEZ1 gene expression was undetectable in more than 60% of epithelial tumors. Mutations were found in primary esophageal cancers and in a prostate cancer cell line. Transcript analysis from several FEZ1-expressing tumors revealed truncated mRNAs, including a frameshift. Alteration and inactivation of the FEZ1 gene may play a role in various human tumors.

Large scale screening of the mitochondrial DNA reveals no pathogenic mutations but a haplotype associated with multiple sclerosis in Caucasians.

We report the first large-scale screening of mitochondrial (mt) DNA in 77 Caucasian patients with relapsing-remitting or secondary progressive form of multiple sclerosis (MS) and in 84 Caucasian controls by using the method of restriction site polymorphism and haplotype analysis. No pathogenic mtDNA mutation was found in association with MS. However, mtDNA haplotypes K* and J* defined by the simultaneous presence of Ddel restriction sites at nucleotides 10,394 and 14,798 of the mtDNA in haplogroups K and J showed association with MS at a P-value of 0.001. A relative increase of MS patients compared to controls either with the J* or with the K* haplotype (+10,394Ddel/+14,798Ddel in haplogroup J or K) also was detected (each with a P<0.05). No distinct phenotypic characteristics of MS were observed when clinical data of patients with haplotypes K* or J* were analyzed. In addition to previous complete sequencing in several MS patients, the population screening of mtDNA presented here suggests that mtDNA point mutations are not likely to be involved in the pathogenesis of typical forms of MS. However, the mitochondrial genetic background (haplotype K* and J*) may moderately contribute to MS susceptibility. The reported association between MS and Leber's hereditary optic nerve atrophy, a disease caused by mtDNA point mutations preferentially occurring in haplogroup J, may be at least in part related to the overlapping mitochondrial genetic background of the two diseases.