Lactate Dehydrogenase B Research Articles

Abstract 137: Molecular networks regulated by tumor suppressive microRNA-375 in head and neck squamous cell carcinoma

Abstract BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. In spite of considerable advances in multimodality therapy including surgery, radiotherapy and chemotherapy, the overall five-year survival rate for patients with HNSCC is around 40%. Understanding of the molecular oncogenic pathway underlying HNSCC could significantly improve diagnosis, therapy, and disease prevention. Our microRNA (miRNA) expression signatures of hypopharyngeal squamous cell carcinoma (SCC), maxillary sinus SCC and esophageal SCC revealed that the expression of microRNA-375 (miR-375) was significantly reduced in cancer tissues. In this study, we investigated the functional significance of miR-375 and explored novel molecular networks regulated by miR-375 in HNSCC. METHODS: Cell proliferation assay was performed to investigate the functional significance of miR-375 and its target genes in HNSCC cell lines. Genome-wide gene expression analysis was performed to identify the molecular networks of miR-375 by microarray technique. The expression levels of miR-375 target genes were verified using quantitative real-time RT-PCR in HNSCC clinical specimens. A luciferase reporter assay was used to identify the actual binding site between miR-375 and its candidate target genes. RESULTS: Restoration of miR-375 significantly inhibited cancer cell proliferation in HNSCC cells. Genome-wide molecular targets search and TargetScan database indicated that homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (HERPUD1), lactate dehydrogenase B (LDHB), PC4 and SFRS1 interacting protein 1 (PSIP1), metadherin (MTDH) and solute-carrier family 7, member 11 (SLC7A11) were the candidate genes of miR-375 target. Among them, the expression levels of LDHB and MTDH were significantly up-regulated in clinical HNSCC tumor specimens compared with adjacent normal epithelium. Luciferase reporter assay showed that LDHB and MTDH were directly regulated by miR-375. Silencing of these genes studies demonstrated significant inhibition of cell proliferation in HNSCC cells. CONCLUSIONS: miR-375 functions as a tumor suppressor in HNSCC. LDHB and MTDH are directly regulated by miR-375. These genes may function as oncogenes and contributed to cell proliferation in HNSCC. Tumor suppressive miR-375 and its target oncogenes may provide new insights into the molecular networks of HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 137. doi:1538-7445.AM2012-137

Tumor suppressive microRNA-375 regulates lactate dehydrogenase B in maxillary sinus squamous cell carcinoma

The expression of microRNA-375 (miR-375) is significantly reduced in cancer tissues of maxillary sinus squamous cell carcinoma (MSSCC). The aim of this study was to investigate the functional significance of miR-375 and a possible regulatory role in the MSSCC networks. Restoration of miR-375 significantly inhibited cancer cell proliferation and invasion in IMC-3 cells, suggesting that miR-375 functions as a tumor suppressor in MSSCC. Genome-wide gene expression data and luciferase reporter assays indicated that lactate dehydro-genaseB (LDHB) was directly regulated by miR-375. Cancer cell proliferation and invasion were significantly inhibited by transfection of si-LDHB into IMC-3 cells, suggesting that LDHB may play a role in MSSCC oncogenic function. In clinical MSSCC specimens, LDHB mRNA levels were up-regulated in cancer tissues, which were inversely correlated with the expression of miR-375. In addition, Kaplan-Meier curves and log-rank tests revealed that the high mRNA expression levels of LDHB had a significant adverse effect on survival rate. The identification of a cancer network regulated by the miR-375 tumor suppressor could provide new insights into the molecular mechanisms of MSSCC oncogenesis.

Exposing local adaptation: synergistic stressors elicit population-specific lactate dehydrogenase-B (ldh-b) expression profiles in Australian barramundi, Lates calcarifer

The molecular response of fish to independently and/or concurrently applied ecological stressors (e.g. thermal and/or aerobic stress) can be quantified at the level of transcript abundance (i.e. gene expression). In temperate fish, the expression of the metabolic candidate gene lactate dehydrogenase-B (ldh-b) responds to both aerobic swimming challenge and extended acclimation to various ecologically relevant temperatures. We examined hepatic ldh-b expression in juvenile Lates calcarifer from two geographically, genetically and thermally distinct Australian populations to determine if similar environmental stressors also influence the transcription of this locus in a tropical fish. Hepatic ldh-b expression was quantified following 28-day acclimation to ecologically relevant temperatures (20, 25, 30 and 35°C). Expression was also quantified in L. calcarifer subjected to aerobic swimming challenge at these temperatures. Fish from southern (high latitude) and northern (low latitude) populations within this species’ Australian distribution exhibited a significant increase in hepatic ldh-b expression following aerobic swimming challenge at native temperatures of 25 and 30°C, respectively (p < 0.001). Southern and northern fish also exhibited significant increase in hepatic ldh-b expression (p < 0.001 and p < 0.01, respectively) following 28-day acclimation to heat-stress (35°C). However, only southern fish exhibited significant increase in expression (p < 0.001) following 28-day acclimation to cold-stress (20°C). The novel evidence presented herein suggests that (a) transcription of hepatic ldh-b is responsive to both aerobic and thermal stress when applied independently, and (b) southern Australian L. calcarifer populations may be locally adapted to cooler seasonal water temperatures.

Screening the proteins of organophosphoms ester-induced delayed neurotoxicity in the cerebral tissue of hens exposed to tri-ortho-cresyl phosphate

To screen the proteins with differential expression levels in the cerebral tissue of hens exposed to tri-ortho-cresyl phosphate (TOCP), and to provide target proteins for studying the mechanism of organophosphoms ester-induced delayed neurotoxicity (OPIDN). Thirty two adult Roman hens were randomly divided into four groups: TOCP group was exposed to 1000 mg/kg TOCP, PMSF group was exposed to 40 mg/kg PMSF, PMSF plus TOCP group was exposed to 40 mg/kg PMSF and after 24 h exposed to 1000 mg/kg TOCP, control group was exposed to normal saline. All hens exposed to chemicals by gastro-intestine for 5 days were sacrificed, and the cerebral tissue were dissected and homogenized in ice bath. Total proteins extracted from the cerebral tissue were separated by isoelectric focusing as the first dimension and SDS-PAGE as the second dimension. The 2-DE maps were visualized after silver staining and analyzed by Image Master 2D software. At last ,the expressed protein spots were identified by Mass spectrometry. From total proteins in TOCP group, the PMSF plus TOCP group and PMSF group, 1185, 1294 and 1063 spots were detected, respectively. One thousand three hundred thirty two spots from total proteins in control group were detected. The match rates of protein spots in TOCP group, the PMSF plus TOCP group and PMSF group were 78.32 %, 79.56 % and 80.93%, respectively. There were 235 protein spots with differential expression levels between TOCP group and control group, which included 158 up regulation spots and 77 down regulation spots. According to the PMSF features, there were 102 spots with differential expression levels between TOCP group and control group and without differential expression levels between TOCP group and PMSF plus TOCP group, among them there were 13 spots with 4 fold differential expression levels between TOCP group and control group and without differential expression levels between TOCP group and PMSF group. Seven protein spots (homer-1b, Destrin, heat shock protein 70, eukaryotic translation initiation factors, proteasome alpha1 subunit, lactate dehydrogenase B, glutamine synthetase) were detected by Mass spectrometry. There are 112 protein spots with differential expression levels of the cerebral tissue in TOCP group, which may be related to OPIDN, among them 13 protein spots with differential expression levels are associated closely with OPIDN. Seven protein spots detected by Mass spectrometry may be related to the mechanism induced by OPIDN.

Identification of Apo-A1 as a biomarker for early diagnosis of bladder transitional cell carcinoma

BackgroundBladder transitional cell carcinoma (BTCC) is the fourth most frequent neoplasia in men, clinically characterized by high recurrent rates and poor prognosis. Availability of urinary tumor biomarkers represents a convenient alternative for early detection and disease surveillance because of its direct contact with the tumor and sample accessibility.ResultsWe tested urine samples from healthy volunteers and patients with low malignant or aggressive BTCC to identify potential biomarkers for early detection of BTCC by two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS) and bioinformatics analysis. We observed increased expression of five proteins, including fibrinogen (Fb), lactate dehydrogenase B (LDHB), apolipoprotein-A1 (Apo-A1), clusterin (CLU) and haptoglobin (Hp), which were increased in urine samples of patients with low malignant or aggressive bladder cancer. Further analysis of urine samples of aggressive BTCC showed significant increase in Apo-A1 expression compared to low malignant BTCC. Apo-A1 level was measured quantitatively using enzyme-linked immunosorbent assay (ELISA) and was suggested to provide diagnostic utility to distinguish patients with bladder cancer from controls at 18.22 ng/ml, and distinguish patients with low malignant BTCC from patients with aggressive BTCC in two-tie grading system at 29.86 ng/ml respectively. Further validation assay showed that Apo-A1 could be used as a biomarker to diagnosis BTCC with a sensitivity and specificity of 91.6% and 85.7% respectively, and classify BTCC in two-tie grading system with a sensitivity and specificity of 83.7% and 89.7% respectively.ConclusionTaken together, our findings suggest Apo-A1 could be a potential biomarker related with early diagnosis and classification in two-tie grading system for bladder cancer.

Lactate Dehydrogenase B Is Critical for Hyperactive mTOR-Mediated Tumorigenesis

Mammalian target of rapamycin (mTOR) is a major downstream effector of the receptor tyrosine kinase (RTK)-phosphoinositide 3-kinase (PI3K)-v-akt murine thymoma viral oncogene homologue 1 (AKT) signaling pathway. Although this signaling network is frequently altered in cancer, the underlying mechanisms that cause tumorigenesis as a result of activated mTOR remain largely unknown. We report here that expression of lactate dehydrogenase B (LDHB), a critical enzymatic activator of glycolysis, was upregulated in an mTOR-dependent manner in TSC1(-/-), TSC2(-/-), PTEN(-/-), or activated AKT1-expressing mouse embryonic fibroblasts (MEF). LDHB gene expression was transactivated by signal transducer and activator of transcription 3 (STAT3), a key tumorigenic driver in many cancers, acting as a downstream mTOR effector in both mouse MEFs and human cancer cells. LDHB attenuation blunted the tumorigenic potential of oncogenic TSC2-null cells in nude mice. We concluded that LDHB is a downstream target of mTOR that is critical for oncogenic mTOR-mediated tumorigenesis. Our findings offer proof of concept for targeting LDHB as a therapeutic strategy in cancers driven by aberrant activation of the RTK-PI3K-AKT-mTOR signaling cascade.

Serum Autoantibody Profiling Using a Natural Glycoprotein Microarray for the Prognosis of Early Melanoma

The poor prognosis of melanoma and the high cost of lymph node biopsy for melanoma patients have led to an urgent need for the discovery of convenient and accurate prognostic indicators. Here, we have developed a natural glycoprotein microarray to discover serum autoantibodies to distinguish between patients with node negative melanoma and node positive melanoma. Dual-lectin affinity chromatography was used to extract glycoproteins from a melanoma cell line. Liquid-based reverse phase separation and microarray platforms were then applied to separate and spot these natural proteins on nitrocellulose slides. The serum autoantibodies were investigated by exposing these proteins to sera from 43 patients that have already been diagnosed to have different stages of early melanoma. The combination of 9 fractions provides a 55% sensitivity with 100% specificity for the detection of node positive against node negative and a 62% sensitivity with 100% specificity for the detection of node negative against node positive. Recombinant proteins were used to confirm the results using a sample set with 79 patients with diagnosed melanoma. The response of sera against recombinant 94 kD glucose-regulated protein (GRP94), acid ceramidase (ASAH1), cathepsin D (CTSD), and lactate dehydrogenase B (LDHB) shared a similar pattern to the fractions where they were identified. The glycoarray platform provides a convenient and highly reproducible method to profile autoantibodies that could be used as serum biomarkers for prognosis of melanoma.

Environmentally relevant exposure to 17α-ethinylestradiol affects the telencephalic proteome of male fathead minnows

Estrogens are key mediators of neuronal processes in vertebrates. As such, xenoestrogens present in the environment have the potential to alter normal central nervous system (CNS) function. The objectives of the present study were (1) to identify proteins with altered abundance in the male fathead minnow telencephalon as a result of low-level exposure to17α-ethinylestradiol (EE 2), and (2) to better understand the underlying mechanisms of 17β-estradiol (E 2) feedback in this important neuroendocrine tissue. Male fathead minnows exposed to a measured concentration of 5.4 ng EE 2/L for 48 h showed decreased plasma E 2 levels of approximately 2-fold. Of 77 proteins that were quantified statistically, 14 proteins were down-regulated after EE 2 exposure, including four histone proteins, ATP synthase, H+ transporting subunits, and metabolic proteins (lactate dehydrogenase B4, malate dehydrogenase 1b). Twelve proteins were significantly induced by EE 2 including microtubule-associated protein tau (Mapt), astrocytic phosphoprotein, ependymin precursor, and calmodulin. Mapt showed an increase in protein abundance but a decrease in mRNA expression after EE 2 exposure , suggesting there may be a negative feedback response in the telencephalon to decreased mRNA transcription with increasing Mapt protein abundance. These results demonstrate that a low, environmentally relevant exposure to EE 2 can rapidly alter the abundance of proteins involved in cell differentiation and proliferation, neuron network morphology, and long-term synaptic potentiation. Together, these findings provide a better understanding of the molecular responses underlying E 2 feedback in the brain and demonstrate that quantitative proteomics can be successfully used in ecotoxicology to characterize affected cellular pathways and endocrine physiology.

Classic approach revitalizes genomics: Complete characterization of a candidate gene for thermal adaptation in two coral reef fishes

Lactate dehydrogenase-B ( ldh-b) encodes a metabolic enzyme (LDH-B) which plays an important role in maintaining aerobic performance and in thermal acclimation and/or adaptation of fish. As the first step in understanding the effect this enzyme has on the ability of tropical coral reef fishes to cope with thermal stress, we characterized both coding and non-coding regions of ldh-b in two congeneric perciformes, Plectropomus leopardus and Plectropomus laevis. Ldh-b was 4666 and 4539 bp in length in P. leopardus and P. laevis, respectively, with coding regions comprising 1005 bp in both species. We report a high level of sequence homology between the coding regions of ldh-b in these two species, with 98.1% identity of nucleotides corresponding to 100% amino acid identity between the deduced protein sequences. Comparison between non-coding (intron) regions of both species revealed the presence of several indels, despite the high level of homology observed (95.9% identity of intron nucleotides). Potential regulatory motifs and elements, including twenty-six simple sequence repeat motifs (mono-, di-, tri- and tetranucleotide) and twenty-three putative microRNA elements are identified within the introns of both species, further supporting recent demonstrations that such short motifs and elements exhibit widespread positioning throughout non-coding regions of the genome. This novel characterization of ldh-b in these two coral reef fishes allows for a wide range of future studies (e.g. analytical comparisons of ldh-b and LDH-B among different fish genera from different thermal environments and habitats).

A novel hypoxia-response element in the lactate dehydrogenase-B gene of the killifish Fundulus heteroclitus

Previous studies have suggested that the lactate dehydrogenase-B gene ( Ldh-B) of the Atlantic killifish, Fundulus heteroclitus, is a hypoxia-responsive gene. Here, we demonstrate that the F. heteroclitus Ldh-B promoter confers hypoxia-dependence upon reporter gene expression in transiently transfected mammalian (Hep3B) and fish (RTG-2 and RTH-149) cells in culture. Mutation and deletion analyses identified a putative hypoxia-response element (HRE) between 109 and 90 nucleotides upstream of the major start site. This HRE is characterized by the sequence 5′-GATGTG-3′ spaced by 8 nucleotides from a perfect inverted repeat, and both sites are necessary for hypoxic induction of reporter gene expression in mammalian and fish cells. This HRE differs from the canonical sequence at one nucleotide position that is invariant among HREs from a wide range of hypoxia-sensitive genes. In fish cells, maximal induction of reporter gene expression driven by this HRE occurred at the lowest oxygen level tested (0.5%), took 48 h to 96 h, and was independent of glucose concentration (between 5.6 and 25 mM). Under all conditions tested, hypoxic induction of gene expression was lower in RTH-149 cells than in RTG-2, suggesting a potential defect in hypoxia signaling in RTH-149 cells. These results demonstrate that the F. heteroclitus Ldh-B promoter contains a novel HRE that is capable of driving reporter gene expression in a sequence-specific and oxygen-, time-, and cell line-dependent manner.

Functional and Physical Interaction between Human Lactate Dehydrogenase B and Na+/H+ Exchanger Isoform 1

The ubiquitous plasma membrane Na+/H+ exchanger 1 (NHE1) is rapidly activated in response to various extracellular stimuli and maintains normal cytoplasmic pH. Yeast two‐hybrid screening was used in order to identify proteins interacting with NHE1 using its cytoplasmic domain as a bait from HeLa cDNA library. One of the interacting cDNA clones was human Lactate dehydrogenase B (LDHB). In vitro translated LDHB was pulled down together with GST‐NHE1.cd protein in the GST pull down assay, confirming the interaction in vitro. LDHB antibody immuno‐precipitated endogenous LDHB together with NHE1 from H9c2 cells, validating cellular interaction between NHE1 and LDHB. Subsequent analysis revealed that the overexpression of LDHB increased intracellular PH, implying opening of the NHE1 transporter. Moreover, overexpression of LDHB activated caspase 3 and induced cell death, consistent with the expected phenotype of hyper‐activation of NHE1. Collectively, our data indicate that LDHB modulates NHE1 activity via physical interaction.

Comparative characterization of a temperature responsive gene (lactate dehydrogenase-B, ldh-b) in two congeneric tropical fish, Lates calcarifer and Lates niloticus.

The characterization of candidate loci is a critical step in obtaining insight into adaptation and acclimation of organisms. In this study of two non-model tropical (to sub-tropical) congeneric perciformes (Lates calcarifer and Lates niloticus) we characterized both coding and non-coding regions of lactate dehydrogenase-B (ldh-b), a locus which exhibits temperature-adaptive differences among temperate and sub-tropical populations of the North American killifish Fundulus heteroclitus. Ldh-b was 5,004 and 3,527 bp in length in L. calcarifer and L. niloticus, respectively, with coding regions comprising 1,005 bp in both species. A high level of sequence homology existed between species for both coding and non-coding regions of ldh-b (> 97% homology), corresponding to a 98.5% amino acid sequence homology. All six known functional sites within the encoded protein sequence (LDH-B) were conserved between the two Lates species. Ten simple sequence repeat (SSR) motifs (mono-, di-, tri- and tetranucleotide) and thirty putative microRNA elements (miRNAs) were identified within introns 1, 2, 5 and 6 of both Lates species. Five single nucleotide polymorphisms (SNPs) were also identified within miRNA containing intron regions. Such SNPs are implicated in several complex human conditions and/or diseases (as demonstrated by extensive genome-wide association studies). This novel characterization serves as a platform to further examine how non-model species may respond to changes in their native temperatures, which are expected to increase by up to 6 degrees C over the next century.

Laser microdissection expression profiling of marginal edges of colorectal tumours reveals evidence of increased lactate metabolism in the aggressive phenotype

Background: The morphology of the invasive margin in colorectal cancer can be described as either pushing or infiltrative. These phenotypes carry prognostic significance, particularly in node negative disease, and provide...

Enzyme Sequence and Its Relationship to Hyperbaric Stability of Artificial and Natural Fish Lactate Dehydrogenases

The cDNAs of lactate dehydrogenase b (LDH-b) from both deep-sea and shallow living fish species, Corphaenoides armatus and Gadus morhua respectively, have been isolated, sequenced and their encoded products overproduced as recombinant enzymes in E. coli. The proteins were characterised in terms of their kinetic and physical properties and their ability to withstand high pressures. Although the two proteins are very similar in terms of their primary structure, only 21 differences at the amino acid level exist between them, the enzyme from the deep-sea species has a significantly increased tolerance to pressure and a higher thermostability. It was possible to investigate whether the changes in the N-terminal or C-terminal regions played a greater role in barophilic adaptation by the construction of two chimeric enzymes by use of a common restriction site within the cDNAs. One of these hybrids was found to have even greater pressure stability than the recombinant enzyme from the deep-living fish species. It was possible to conclude that the major adaptive changes to pressure tolerance must be located in the N-terminal region of the protein. The types of changes that are found and their spatial location within the protein structure are discussed. An analysis of the kinetic parameters of the enzymes suggests that there is clearly a trade off between Km and kcat values, which likely reflects the necessity of the deep-sea enzyme to operate at low temperatures.

ITRAQ-FACILITATED PROTEOMIC ANALYSIS OF HUMAN PROSTATE CANCER CELLS IDENTIFIES PROTEINS ASSOCIATED WITH PROGRESSION

The unpredictable behavior of prostate cancer presents a major clinical challenge during patient management. In order to gain an insight into the molecular mechanisms associated with prostate cancer progression, we employed the shot-gun proteomic approach of isobaric tags for relative and absolute quantitation (iTRAQ), followed by 2D-LC-MS/MS, using the poorly metastatic LNCaP cell line and its highly metastatic variant LNCaP-LN3 cell line as a model. A total number of 280 unique proteins were identified (≥95% confidence), and relative expression data was obtained for 176 of these. Ten proteins were found to be significantly up-regulated (≥1.50 fold), while 4 proteins were significantly down-regulated (≥−1.50 fold), in LNCaP-LN3 cells. Differential expression of brain creatine kinase (CKBB), soluble catechol- O-methyltransferase (S-COMT), tumor rejection antigen (gp96), and glucose regulated protein, 78 kDa (grp78), was confirmed by Western blotting or independent 2D-PAGE analysis. Additionally, iTRAQ analysis identified absence of the lactate dehydrogenase-B (LDH-B) subunit in LNCaP-LN3 cells, confirming our published data. The clinical relevance of gp96 was assessed by immunohistochemistry using prostate tissues from benign ( n = 95), malignant ( n = 66), and metastatic cases ( n = 3). Benign epithelium showed absent/weak gp96 expression in the basal cells, in contrast to the moderate/strong expression seen in malignant epithelium. Furthermore, there was a statistically significant difference in the intensity of gp96 expression between benign and malignant cases ( p < 0.0005, Mann–Whitney U). Our study is the first to report the application of iTRAQ technology and its potential for the global proteomic profiling of prostate cancer cells, including the identification of absent protein expression.

ITRAQ-Facilitated Proteomic Analysis of Human Prostate Cancer Cells Identifies Proteins Associated with Progression

The unpredictable behavior of prostate cancer presents a major clinical challenge during patient management. In order to gain an insight into the molecular mechanisms associated with prostate cancer progression, we employed the shot-gun proteomic approach of isobaric tags for relative and absolute quantitation (iTRAQ), followed by 2D-LC-MS/MS, using the poorly metastatic LNCaP cell line and its highly metastatic variant LNCaP-LN3 cell line as a model. A total number of 280 unique proteins were identified (> or =95% confidence), and relative expression data was obtained for 176 of these. Ten proteins were found to be significantly up-regulated (> or =1.50 fold), while 4 proteins were significantly down-regulated (> or = -1.50 fold), in LNCaP-LN3 cells. Differential expression of brain creatine kinase (CKBB), soluble catechol-O-methyltransferase (S-COMT), tumor rejection antigen (gp96), and glucose regulated protein, 78 kDa (grp78), was confirmed by Western blotting or independent 2D-PAGE analysis. Additionally, iTRAQ analysis identified absence of the lactate dehydrogenase-B (LDH-B) subunit in LNCaP-LN3 cells, confirming our published data. The clinical relevance of gp96 was assessed by immunohistochemistry using prostate tissues from benign ( n = 95), malignant ( n = 66), and metastatic cases ( n = 3). Benign epithelium showed absent/weak gp96 expression in the basal cells, in contrast to the moderate/strong expression seen in malignant epithelium. Furthermore, there was a statistically significant difference in the intensity of gp96 expression between benign and malignant cases ( p < 0.0005, Mann-Whitney U). Our study is the first to report the application of iTRAQ technology and its potential for the global proteomic profiling of prostate cancer cells, including the identification of absent protein expression.

Analysis of F ST outliers at allozyme loci in Pacific salmon: implications for natural selection

Natural selection has been invoked to explain the observed geographic distribution of allozyme allele frequencies for a number of teleost species. The effects of selection on allozyme loci in three species of Pacific salmon were tested. A simulation-based approach to estimate the null distribution of population differentiation (F ST) and test for F ST outliers was used. This approach showed that a majority of allozyme loci conform to neutral expectations predicted by the simulation model, with relatively few F ST outliers found. No consistent F ST outlier loci were found across species. Analysis of population sub-groups based on geography and genetic identity reduced the number of outlier loci for some species, indicating that large geographic groups may include genetically divergent populations and/or that there is geographic heterogeneity in selection pressure upon allozyme loci. Two outlier allozyme loci found in this analysis, lactate dehydrogenase-B and malic enzyme, have been shown to be influenced by selection in other teleost species. This approach is also useful in identifying allozyme loci (or other genetic markers) that meet assumptions for population genetic study.

Lactate dehydrogenase-B is silenced by promoter hypermethylation in human prostate cancer

In order to identify novel candidates associated with prostate cancer metastasis, we compared the proteomic profile of the poorly metastatic human prostate cancer cell line LNCaP, with its highly metastatic variant LNCaP-LN3, by two-dimensional gel electrophoresis. A major protein spot (pI of 5.9 and molecular weight of 37 kDa) was seen in LNCaP cells, but not in LNCaP-LN3 cells and was identified as lactate dehydrogenase-B (LDHB), by tandem mass spectrometry. Furthermore, enzyme kinetic assays and zymography showed a higher LDH enzyme activity in LNCaP cells compared with LNCaP-LN3. Bisulphite-modified DNA sequencing showed promoter hypermethylation in LNCaP-LN3 cells but not in LNCaP, Du145, PC3, CWR22 or BPH45 cells. Treatment of LNCaP-LN3 cells with 5'-azacytidine caused re-expression of LDHB transcripts. In tissues, LDHB promoter hypermethylation occurred at a higher frequency in prostate cancer, 14/ 31 (45%), compared to adjacent nonmalignant or benign tissue, 2/19 (11%) (P < 0.025). Immunohistochemistry showed a higher frequency of LDHB expression in benign or non-malignant tissues, 59/ 73 (81%), compared to cancer cases, 3/53 (6%) (P < 0.001). Absent LDHB expression was also seen in 7/7 (100%) cases of metastatic cancer in bone. Our data are the first to show loss of LDHB expression in prostate cancer, the mechanism of which appears to involve promoter hypermethylation.

21 Lactate dehydrogenase‐B is silenced by promoter hypermethylation in human prostate cancer

In order to identify novel candidates associated with prostate cancer metastasis, we compared the proteomic profile of the poorly metastatic human prostate cancer cell line LNCaP, with its highly metastatic variant LNCaP‐LN3, by two‐dimensional gel electrophoresis. A major protein spot (pI of 5.9 and molecular weight of 37 KDa) was seen in LNCaP cells, but not in LNCaP‐LN3 cells and was identified as Lactate dehydrogenase‐B (LDHB), by tandem mass spectrometry. Furthermore, enzyme kinetic assays and Zymography showed a higher LDH enzyme activity in LNCaP cells compared with LNCaP‐LN3. Bisulfite modified DNA sequencing showed promoter hypermethylation in LNCaP‐LN3 cells but not in LNCaP, Du145, PC3, CWR22 or BPH45 cells. Treatment of LNCaP‐LN3 cells with 5’‐azacytidine caused re‐expression of LDHB transcripts. In tissues, LDHB promoter hypermethylation occurred at a higher frequency in prostate cancer 14/31 (45%), compared to adjacent non‐malignant or benign tissue, 2/19 (11%), (P &lt; 0.025). Immunohistochemistry showed a higher frequency of LDHB expression in benign or non‐malignant tissues, 59/ 73 (81%), compared to cancer cases, 3/53 (6%), (P &lt; 0.001). Absent LDHB expression was also seen in 7/7 (100%) cases of metastatic cancer in bone. Our data are the first to show loss of LDHB expression in prostate cancer, the mechanism of which appears to involve promoter hypermethylation.

621Promoter hyper-methylation of lactate dehydrogenase-B is associated with prostate cancer development

621Promoter hyper-methylation of lactate dehydrogenase-B is associated with prostate cancer development