Quantitative Southern Blot Analysis Research Articles

Psoralen inhibits hepatitis B viral replication by down-regulating the host transcriptional machinery of viral promoters.

The hepatitis B virus (HBV) is a global public health challenge due to its highly contagious nature. It is estimated that almost 300 million people live with chronic HBV infection annually. Although nucleoside analogs markedly reduce the risk of liver disease progression, the analogs do not fully eradicate the virus. As such, new treatment options and drugs are urgently needed. Psoralen is a nourishing monomer of Chinese herb and is known to inhibit virus replication and inactivate viruses. In this study, we evaluated the potential of psoralen as an anti-HBV agent. Quantitative PCR and Southern blot analysis revealed that psoralen inhibited HBV replication in HepG2.2.15 ​cells in a concentration-dependent manner. Moreover, psoralen was also active against the 3TC/ETV-dual-resistant HBV mutant. Further investigations revealed that psoralen suppressed both HBV RNA transcription and core protein expression. The transcription factor FOXO1, a known target for PGC1α co-activation, binds to HBV pre-core/core promoter enhancer II region and activates HBV RNA transcription. Co-immunoprecipitation showed that psoralen suppressed the expression of FOXO1, thereby decreasing the binding of FOXO1 co-activator PGC1α to the HBV promoter. Overall, our results demonstrate that psoralen suppresses HBV RNA transcription by down-regulating the expression of FOXO1 resulting in a reduction of HBV replication.

MiR-29a promotes hepatitis B virus replication and expression by targeting SMARCE1 in hepatoma carcinoma.

AIMTo investigate the functional role and underlying molecular mechanism of miR-29a in hepatitis B virus (HBV) expression and replication.METHODSThe levels of miR-29a and SMARCE1 in HBV-infected HepG2.2.15 cells were measured by quantitative real-time PCR and western blot analysis. HBV DNA replication was measured by quantitative PCR and Southern blot analysis. The relative levels of hepatitis B surface antigen and hepatitis B e antigen were detected by enzyme-linked immunosorbent assay. The Cell Counting Kit-8 (CCK-8) was used to detect the viability of HepG2.2.15 cells. The relationship between miR-29a and SMARCE1 were identified by target prediction and luciferase reporter analysis.RESULTSmiR-29a promoted HBV replication and expression, while SMARCE1 repressed HBV replication and expression. Cell viability detection indicated that miR-29a transfection had no adverse effect on the host cells. Moreover, SMARCE1 was identified and validated to be a functional target of miR-29a. Furthermore, restored expression of SMARCE1 could relieve the increased HBV replication and expression caused by miR-29a overexpression.CONCLUSIONmiR-29a promotes HBV replication and expression through regulating SMARCE1. As a potential regulator of HBV replication and expression, miR-29a could be a promising therapeutic target for patients with HBV infection.

A multi-method approach to the molecular diagnosis of overt and borderline 11p15.5 defects underlying Silver-Russell and Beckwith-Wiedemann syndromes.

BackgroundMultiple (epi)genetic defects affecting the expression of the imprinted genes within the 11p15.5 chromosomal region underlie Silver–Russell (SRS) and Beckwith–Wiedemann (BWS) syndromes. The molecular diagnosis of these opposite growth disorders requires a multi-approach flowchart to disclose known primary and secondary (epi)genetic alterations; however, up to 20 and 30 % of clinically diagnosed BWS and SRS cases remain without molecular diagnosis. The complex structure of the 11p15 region with variable CpG methylation and low-rate mosaicism may account for missed diagnoses. Here, we demonstrate the relevance of complementary techniques for the assessment of different CpGs and the importance of testing multiple tissues to increase the SRS and BWS detection rate.ResultsMolecular testing of 147 and 450 clinically diagnosed SRS and BWS cases provided diagnosis in 34 SRS and 185 BWS patients, with 9 SRS and 21 BWS cases remaining undiagnosed and herein referred to as “borderline.” A flowchart including complementary techniques and, when applicable, the analysis of buccal swabs, allowed confirmation of the molecular diagnosis in all borderline cases. Comparison of methylation levels by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) in borderline and control cases defined an interval of H19/IGF2:IG-DMR loss of methylation that was distinct between “easy to diagnose” and “borderline” cases, which were characterized by values ≤mean −3 standard deviations (SDs) compared to controls. Values ≥mean +1 SD at H19/IGF2: IG-DMR were assigned to borderline hypermethylated BWS cases and those ≤mean −2 SD at KCNQ1OT1: TSS-DMR to hypomethylated BWS cases; these were supported by quantitative pyrosequencing or Southern blot analysis. Six BWS cases suspected to carry mosaic paternal uniparental disomy of chromosome 11 were confirmed by SNP array, which detected mosaicism till 10 %. Regarding the clinical presentation, borderline SRS were representative of the syndromic phenotype, with exception of one patient, whereas BWS cases showed low frequency of the most common features except hemihyperplasia.ConclusionsA conclusive molecular diagnosis was reached in borderline methylation cases, increasing the detection rate by 6 % for SRS and 5 % for BWS cases. The introduction of complementary techniques and additional tissue analyses into routine diagnostic work-up should facilitate the identification of cases undiagnosed because of mosaicism, a distinctive feature of epigenetic disorders.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0183-8) contains supplementary material, which is available to authorized users.

Abstract 15768: Mitochondria-Targeted DNA Repair Glycosylase Ogg1 Suppresses Early Stage of Atherogenesis in Ogg1 Deficient Mice

Introduction: Reactive oxygen species (ROS) play a key role in the development of atherosclerosis. Mitochondria are a main source of endogenous ROS in the cell. Mitochondrial DNA (mtDNA) is sensitive to oxidation and our previous results from cultured cell and intact animal models suggest that increasing mtDNA repair prevents both oxidative mtDNA damage and associated cytotoxicity and cellular dysfunction. Involvement of oxidative mtDNA damage in disorders characterized by chronic oxidative stress has been less thoroughly studied. Hypothesis: In the present study we tested the hypothesis that transgenic modulation of Ogg1, a DNA glycosylase mediating the first step in the base excision repair of oxidative mtDNA damage, coordinately regulates atherogenesis in mice fed a high fat diet. Methods: Wild type (WT) mice, Ogg1 knock-out (KO) mice and KO mice transgenically overexpressing mitochondria-targeted Ogg1 (KO-Tg) were fed pro-atherogenic Western type diet for 14 weeks and analyzed for mtDNA damage and signs of atherogenesis. Results: KO mice fed a high fat diet had increased oxidative mtDNA damage in cardiac tissue, whereas KO-Tg animals did not differ from WT mice (WT: 0.18 ± 0.04; KO: 0.35 ± 0.04; KO-Tg: 0.15 ± 0.01 lesions per 10 4 bp; n=3, P<0.05; quantitative Southern blot analysis). We did not observe significant atherosclerotic plaque formation in the aortic valve of animals from any group; however appearance of fatty streaks, indicative of early plaque development, was more evident in KO mice (WT: 179 ± 20; KO: 384 ± 59 pixels, n=4, P<0.05; immunohistochemistry for Fc receptor - general marker of inflammatory cells). This effect was completely blocked in KO-Tg mice. We found increased number of apoptotic cells in the aortic valve of KO, but not KO-Tg mice (WT: 2.00 ± 0.50; KO: 4.25 ± 0.48; KO-Tg: 2.14 ± 0.85 apoptotic cells, n=4, P<0.05; TUNEL assay). Conclusion: Our data demonstrate that Ogg1 deficiency in mice fed a high fat diet leads to increased oxidative mtDNA damage, appearance of fatty streaks and cell apoptosis. In contrast, enhancement of mtDNA repair with mitochondria-targeted Ogg1 reduces fatty streaks formation and apoptosis induced by a high fat diet. These results suggest mtDNA damage and repair could be important targets for atheroprotection.

DNA oxidation in regulatory D‐loop region of the mitochondrial genome is important for hypoxia‐induced mitochondrial biogenesis in rat pulmonary artery endothelial cells (PAECs)

Emerging evidence suggests that hypoxia‐induced transcription of nuclear genes requires controlled DNA damage and repair (Al‐ Mehdi et al, Sci Signaling, 2012). Whether such a mechanism operates in mitochondria is unknown. Here we explored the hypothesis that hypoxia‐induced mitochondrial (mt) DNA replication requires sequence‐specific oxidative DNA modifications in the mitochondrial genome. Using slot blot and quantitative Southern blot analyses, respectively, we found that increased mtDNA copy number in hypoxic PAECs was accompanied by oxidative modifications in the D‐loop region of the mitochondrial genome. Transfection of rat PAECs with a mitochondria‐targeted DNA repair enzyme OGG1 suppressed hypoxia‐induced DNA oxidation in the D‐loop region and reduced binding of mitochondrial transcription factor TFAM to both regulatory and coding regions of the mitochondrial genome, as assessed by ChIP analysis. OGG1 over‐expression also attenuated hypoxia‐induced mtDNA replication. Interestingly, hypoxia increased number of hyperpolarized mitochondria in wild‐type PAECs, as evidenced by JC‐1 fluorescence. Mitochondria in OGG1‐transfected cells were hyperpolarized under normal conditions and less affected by hypoxia. These observations support the concept that oxidative DNA modifications in the D‐loop region are important for hypoxia‐induced mitochondrial biogenesis. Supported by NIH.

Mitochondrial DNA depletion syndrome presenting with ataxia and external ophthalmoplegia: Case report

The mitochondrial DNA depletion syndromes are autosomal recessive disorders characterized by decreased mitochondrial DNA copy number in affected tissues. Mutations in 2 genes involved in deoxyribonucleotide metabolism, the deoxyguanosine kinase gene and the thymidine kinase 2 gene, had been related to this syndrome. This study aims to describe the clinical, histochemical, biochemical and molecular diagnosis of one Egyptian pediatric patient with the myopathic form of mitochondrial depletion syndrome. The patient presented to Cairo University Pediatric Hospital with the clinical suspicion of mitochondrial encephalomyopathy. Histochemical and biochemical studies of the respiratory chain complexes were performed on the muscle biopsy specimen from the patient. Molecular diagnosis was done by quantitative radioactive Southern blot and sequencing analysis of the whole coding regions of the TK2 gene. Histochemical staining revealed cytochrome oxidase negative fibers and increased staining for succinate dehydrogenase. The activity of complex I was not detected and complex IV activity was about 46% of age matched controls. Southern blot analysis showed reduction of the mitochondrial/nuclear DNA ratio, the degree of depletion was around 30% of aged-matched controls. Sequencing analysis of the TK2 gene revealed no sequence variation. Targeted molecular diagnosis based on the biochemical analysis of the respiratory chain enzymes makes the molecular evaluation of mitochondrial disorders much easier. Involvement of other nuclear genes rather than TK2 gene in the pathogenesis of the myopathic form of mitochondrial depletion syndrome should be considered.

Mitochondrial Biogenesis in Hypoxic Rat Pulmonary Artery Endothelial Cells (PAECs): Possible Involvement of DNA Oxidation

Like many stimuli promoting cell growth and proliferation, hypoxia uses reactive oxygen species (ROS) as signaling molecules. There is emerging evidence that for nuclear genes, controlled ROS‐mediated DNA damage and repair in specific promoter sequences is necessary for normal transcriptional regulation. Mitochondrial (mt) biogenesis in hypoxia requires mtDNA transcription and replication, but the specific mechanisms are unknown. Here we tested the idea that, in PAECs, these key events in hypoxia‐induced mitochondrial biogenesis, similar to hypoxia‐regulated nuclear gene expression, are associated with oxidative base modifications in the mtDNA D‐loop region involved in mtDNA transcriptional control. Using Fpg‐sensitive qPCR and quantitative Southern blot analyses, we found that although hypoxia did not cause alkali‐sensitive lesions in either the D‐loop or coding regions of mtDNA, Fpg‐sensitive modifications were present exclusively in the D‐loop sequence, with the coding region devoid of oxidative base modifications. Slot blot and real time RT‐PCR analyses revealed increased mtDNA copy number and mitochondrial mRNA transcripts after hypoxic exposure. Hypoxia also increased TFAM binding to D‐loop region as shown by ChIP assay. These observations support the concept that controlled oxidative DNA damage in the D‐loop region may be involved in mitochondrial biogenesis. Supported by NIH.

E46K Human α-Synuclein Transgenic Mice Develop Lewy-like and Tau Pathology Associated with Age-dependent, Detrimental Motor Impairment

Synucleinopathies are a group of neurodegenerative disorders associated with the formation of aberrant amyloid inclusions composed of the normally soluble presynaptic protein α-synuclein (α-syn). Parkinson disease is the most well known of these disorders because it bears α-syn pathological inclusions known as Lewy bodies (LBs). Mutations in the gene for α-syn, including the E46K missense mutation, are sufficient to cause Parkinson disease as well as other synucleinopathies like dementia with LBs. Herein, we describe transgenic mice expressing E46K human α-syn in CNS neurons that develop detrimental age-dependent motor impairments. These animals accumulate age-dependent intracytoplasmic neuronal α-syn inclusions that parallel disease and recapitulate the biochemical, histological, and morphological properties of LBs. Surprisingly, the morphology of α-syn inclusions in E46K human α-syn transgenic mice more closely resemble LBs than the previously described transgenic mice (line M83) that express neuronal A53T human α-syn. E46K human α-syn mice also develop abundant neuronal tau inclusions that resemble neurofibrillary tangles. Subsequent studies on the ability of E46K α-syn to induce tau inclusions in cellular models suggest that both direct and indirect mechanisms of protein aggregation are probably involved in the formation of the tau inclusions observed here in vivo. Re-evaluation of presymptomatic transgenic mice expressing A53T human α-syn reveals that the formation of α-syn inclusions in mice must be synchronized; however, inclusion formation is diffuse within affected areas of the neuroaxis such that there was no clustering of inclusions. Collectively, these findings provide insights in the mechanisms of formation of these aberrant proteinaceous inclusions and support the notion that α-syn aggregates are involved in the pathogenesis of human diseases.

Mitochondrial DNA integrity may be a determinant of endothelial barrier properties in oxidant-challenged rat lungs

In cultured pulmonary artery endothelial cells and other cell types, overexpression of mt-targeted DNA repair enzymes protects against oxidant-induced mitochondrial DNA (mtDNA) damage and cell death. Whether mtDNA integrity governs functional properties of the endothelium in the intact pulmonary circulation is unknown. Accordingly, the present study used isolated, buffer-perfused rat lungs to determine whether fusion proteins targeting 8-oxoguanine DNA glycosylase 1 (Ogg1) or endonuclease III (Endo III) to mitochondria attenuated mtDNA damage and vascular barrier dysfunction evoked by glucose oxidase (GOX)-generated hydrogen peroxide. We found that both Endo III and Ogg1 fusion proteins accumulated in lung cell mitochondria within 30 min of addition to the perfusion medium. Both constructs prevented GOX-induced increases in the vascular filtration coefficient. Although GOX-induced nuclear DNA damage could not be detected, quantitative Southern blot analysis revealed substantial GOX-induced oxidative mtDNA damage that was prevented by pretreatment with both fusion proteins. The Ogg1 construct also reversed preexisting GOX-induced vascular barrier dysfunction and oxidative mtDNA damage. Collectively, these findings support the ideas that mtDNA is a sentinel molecule governing lung vascular barrier responses to oxidant stress in the intact lung and that the mtDNA repair pathway could be a target for pharmacological intervention in oxidant lung injury.

Oxidative DNA damage in lung tissue from patients with COPD is clustered in functionally significant sequences

Lung tissue from COPD patients displays oxidative DNA damage. The present study determined whether oxidative DNA damage was randomly distributed or whether it was localized in specific sequences in either the nuclear or mitochondrial genomes. The DNA damage-specific histone, gamma-H2AX, was detected immunohistochemically in alveolar wall cells in lung tissue from COPD patients but not control subjects. A PCR-based method was used to search for oxidized purine base products in selected 200 bp sequences in promoters and coding regions of the VEGF, TGF-β1, HO-1, Egr1, and β-actin genes while quantitative Southern blot analysis was used to detect oxidative damage to the mitochondrial genome in lung tissue from control subjects and COPD patients. Among the nuclear genes examined, oxidative damage was detected in only 1 sequence in lung tissue from COPD patients: the hypoxic response element (HRE) of the VEGF promoter. The content of VEGF mRNA also was reduced in COPD lung tissue. Mitochondrial DNA content was unaltered in COPD lung tissue, but there was a substantial increase in mitochondrial DNA strand breaks and/or abasic sites. These findings show that oxidative DNA damage in COPD lungs is prominent in the HRE of the VEGF promoter and in the mitochondrial genome and raise the intriguing possibility that genome and sequence-specific oxidative DNA damage could contribute to transcriptional dysregulation and cell fate decisions in COPD.

Characterization of amplicons that suppress the conditional lethal growth phenotype of a Leishmania donovani mutant lacking normal purine salvage mechanisms

A conditionally lethal mutant of Leishmania donovani that lacks both hypoxanthine-guanine phosphoribosyltransferase and xanthine phosphoribosyltransferase exhibits a strikingly restricted growth phenotype, can only survive as the promastigote under pharmacological constraints, and is profoundly compromised in its ability to infect macrophages and mice. Interestingly, the conditionally lethal growth phenotype displayed by these mutant parasites can be suppressed in vitro by selection of strains that have markedly amplified the adenine phosphoribosyltransferase gene on extrachromosomal elements that are unique to these suppressor strains. Employing pulsed field gel electrophoresis, we have now determined that the amplicons in two of these suppressor lines are linear molecules by: (1) their pulse time-dependent mobility; (2) the failure of γ-irradiation to generate new discrete bands; (3) their susceptibility to λ exonuclease digestion; and (4) the presence of telomeric sequences. Pulsed field gel electrophoresis also shows these amplicons to be approximately 200–275 kb in size. However, quantitative polymerase chain reaction and Southern blot analyses demonstrated that the amplification units are ∼40 kb in length, implying that the formation of these amplicons involved additional chromosomal rearrangements or oligomerization.

Sequence analysis and gene amplification study of the penicillin biosynthesis gene cluster from different strains of Penicillium chrysogenum

Industrial strains of Penicillium chrysogenum possess many genomic changes leading to higher levels of penicillin. In this work several production and wild-type strains of Penicillium chrysogenum were used in comparative nucleotide sequence analysis of the biosynthesis cluster. The alignments confirmed sequence conservation not only in promoter regions of the biosynthesis genes but also throughout the entire 44.7-kbp genomic fragment comprising the whole biosynthesis cluster with 15.5-kbp and 13.1-kbp flanking regions. As another titre-enhancing mechanism we subsequently examined gene dosage in two production strains introduced here, NMU2/40 and B14. Quantitative real-time PCR and Southern blot analysis showed the amplification of the biosynthesis genes in both these strains. Through the real-time PCR method the exact copy number was estimated for each of the pcbAB, pcbC and penDE genes. The equal pool of all three genes per genome was confirmed for the both production strains indicating that in these strains the entire penicillin cluster has been amplified as an intact element. Penicillium chrysogenum NMU2/40 was found to carry four copies of the cluster, while six copies were estimated for B14. This also proves the contribution of the additional titre-enhancing mechanisms in both strains, since the industrial data referred much higher production of these strains compared with the single copy reference strain NRRL 1951.

Mitochondrial DNA Damage Initiates a Cell Cycle Arrest by a Chk2-associated Mechanism in Mammalian Cells

Previous work from our laboratory has focused on mitochondrial DNA (mtDNA) repair and cellular viability. However, other events occur prior to the initiation of apoptosis in cells. Because of the importance of mtDNA in ATP production and of ATP in fuel cell cycle progression, we asked whether mtDNA damage was an upstream signal leading to cell cycle arrest. Using quantitative alkaline Southern blot technology, we found that exposure to menadione produced detectable mtDNA damage in HeLa cells that correlated with an S phase cell cycle arrest. To determine whether mtDNA damage was causatively linked to the observed cell cycle arrest, experiments were performed utilizing a MTS-hOGG1-Tat fusion protein to target the hOGG1 repair enzyme to mitochondria and enhance mtDNA repair. The results revealed that the transduction of MTS-hOGG1-Tat into HeLa cells alleviated the cell cycle block following an oxidative insult. Furthermore, mechanistic studies showed that Chk2 phosphorylation was enhanced following menadione exposure. Treatment of the HeLa cells with the hOGG1 fusion protein prior to menadione exposure resulted in an increase in the rate of Chk2 dephosphorylation. These results strongly support a direct link between mtDNA damage and cell cycle arrest.

Real-time quantitative RT-PCR assessment of PIM-1 and hK2 mRNA expression in benign prostate hyperplasia and prostate cancer

To investigate the expressions of PIM-1 and hK2 mRNA in normal prostate, benign prostatic glandular hyperplasia (BPH), and prostate cancer (PCa), and to explore the association of PIM-1 and hK2 expressions with PCa progression. The samples were harvested from 37 patients with BPH, 23 patients with PCa, and three with normal prostate tissues. Total RNA was extracted from their prostate tissues and analyzed for PIM-1 and hK2 mRNA levels using SYBR green I-based quantitative real-time RT-PCR (QRT-PCR) assays and Southern blot analysis. The differences of gene expressions were calculated based on standard curve. Quantitative expressions of PIM-1 and hK2 mRNA in normal prostate, BPH, and PCa were 1.05 +/- 0.04, 2.57 +/- 0.74, 4.45 +/- 0.63, and 1.02 +/- 0.03, 2.264 +/- 0.46, 5.905 +/- 0.78, respectively. PIM-1 and hK2 were expressed higher in PCa than those in BPH and normal prostate tissues, the differences among which had statistic significance (P < 0.05). Our results support the hypothesis that PIM-1 and hK2 play a significant role in the growth of PCa and the detection of PIM-1 and hK2 mRNA expressions by QRT-PCR provided more reliable and helpful information on diagnosis, treatment, and prognosis of PCa.

Diminished mitochondrial DNA integrity and repair capacity in OA chondrocytes

Osteoarthritis (OA) is characterized by the failure of chondrocytes to respond to injury and perform the cartilage remodeling process. Human articular chondrocytes actively produce reactive oxygen and nitrogen species (ROS and RNS) capable of causing cellular dysfunction and death. A growing body of evidence indicates that mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage play a causal role in disorders linked to excessive generation of oxygen free radicals. The aim of this study was to determine whether mtDNA damage was present in OA chondrocytes, and whether mtDNA repair capacity is compromised in OA chondrocytes following oxidative stress, leading to chondrocyte death. Human articular cartilage was isolated from knee joints of cadavers available through the Anatomical Gifts Program at the University of South Alabama (normal donors) or OA patients undergoing total knee replacement surgeries (OA patients). Total DNA was isolated from either chondrocytes released following collagenase digestion, or from first passage chondrocytes grown in culture and exposed to ROS or RNS. mtDNA integrity and repair capacity were analyzed by quantitative Southern blot analysis, using a mtDNA-specific radioactive probe. Cell viability was determined by the trypan blue exclusion method. mtDNA damage was found in chondrocytes from OA patients compared to normal donors. It was accompanied with reduced mtDNA repair capacity, cell viability, and increased apoptosis in OA chondrocytes following exposure to ROS and RNS. These results indicate that mtDNA damage and poor mtDNA repair capacity for removing damage caused by oxidative stress may contribute to the pathogenesis of OA.

Gene Targeting of Mutant COL1A2 Alleles in Mesenchymal Stem Cells From Individuals With Osteogenesis Imperfecta

Mesenchymal stem cells (MSCs) are adult cells with the capacity to differentiate into multiple cell types, including bone, fat, cartilage, and muscle cells. In order to effectively utilize autologous MSCs in cell-based therapies, precise genetic manipulations are required to eliminate the effects of disease-causing mutations. We previously used adeno-associated virus (AAV) vectors to target and inactivate mutant COL1A1 genes in MSCs from individuals with the brittle bone disorder, osteogenesis imperfecta (OI). Here we have used AAV vectors to inactivate mutant COL1A2 genes in OI MSCs, thereby demonstrating that both type I collagen genes responsible for OI can be successfully targeted. We incorporated improved vector designs so as to minimize the consequences of random integration, facilitate the removal of potential antigens, and avoid unwanted exon skipping. MSCs targeted at mutant COL1A2 alleles produced normal type I procollagen and formed bone, thereby demonstrating their therapeutic potential.

Array-based comparative genomic hybridization of circulating esophageal tumor cells

Esophageal squamous cell carcinoma (ESCC) shows a high frequency of lymphatic and/or systemic metastasis, even when the tumor invades only the submucosa. To investigate the genetic alterations in circulating esophageal tumor cells, we performed array-based comparative genomic hybridization (CGH) analysis of 8 DNA samples of xenografts, which were previously established from the thoracic duct lymph of 13 ESCC patients. A total of 5 loci (or genes), 10q21.3 (EGR2), 11q13.3 (CCND1/CyclinD1, FGF4, and EMS1), 11q14 (PAK1), and 22qtel (ARSA) were found to be candidate amplified loci in the xenograft. In contrast, a total of 24 loci including 9p21 (p16 and MTAP) were found to be homozygously deleted candidates in the xenograft. Both p16 homozygous deletion and CCND1 amplification were detected in 6 (75%) and 5 (62.5%) of the 8 xenografts. Furthermore, by quantitative Southern blot analysis, we found p16 homozygous deletion in 30.8% (8/26) of the primary tumors and in 50% (4/8) of the metastasized lymph nodes. The frequency of CCND1 amplification and p16 homozygous deletion is suggested to be associated with ESCC progression. Matrigel invasion assays of p16-deleted ESCC cells showed that restoring wild-type p16 activity into the cells significantly inhibits tumor-cell invasion, suggesting that p16 inactivation could be involved in ESCC invasion. This is the first report showing the genetic alteration of concealed tumor cells in the thoracic duct lymph. The present gene list should be helpful for identifying new amplified and deleted genes in primary ESCCs as well as in metastasized lymph nodes.

Molecular Characterization of Large Deletions in the von Hippel-Lindau (VHL) Gene by Quantitative Real-Time PCR

Mutations of the von Hippel-Lindau (VHL) gene are responsible for VHL disease. This is a familial autosomal-dominant syndrome, predisposing to the development of benign and malignant tumors, including CNS and retinal hemangioblastomas, pheochromocytomas, and clear cell renal carcinomas. At least 30% of the disease-causing mutations in the VHL gene involve large alterations. Identification of these mutations is not possible using PCR-based mutational scanning methods. Quantitative Southern blot analysis has been traditionally employed for the detection of complete or partial deletions and more complex rearrangements of the gene. An alternative quantitative method was developed using a combination of quantitative Southern blot analysis and real-time PCR. With this approach, we studied 24 large VHL gene alterations to determine the exact nature of the mutations and to possibly characterize the boundaries of the deleted regions. This combined molecular approach showed that all the VHL alterations studied were due to deletions, from which the position in the gene could be more precisely mapped. One of the samples that was completely characterized was found to carry an intragenic 2.2kb deletion with both 5' and 3' breakpoints located within Alu-repeat sequences. This is the first report on the molecular analysis of large VHL alterations. The results of our study and the complete characterization of a large deletion lead to the hypothesis that an Alu-mediated mechanism may be responsible for the common occurrence of large alterations in the VHL gene.

Thrombin induces oxidative mitochondrial (mt) DNA damage through a membrane NADPH oxidase in pulmonary microvascular endothelial cells

Recent evidence suggests that mtDNA mutations may play a role in the pathogenesis of atherosclerosis (Mutat. Res., 570: 80, 2005). Though oxidative damage to mtDNA is a precursor to mtDNA mutations, the source of oxidants leading to mtDNA damage in ECs is unclear. Thrombin and other inflammatory mediators incriminated in development of atherosclerosis are known to utilize reactive oxygen species (ROS) generated from a membrane-bound NADPH oxidase as second messengers. In this study, we tested the hypothesis that thrombin-induced ROS production causes mtDNA damage in pulmonary microvascular endothelial cells. Fluorescence microscopy with 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red) and 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) showed that thrombin caused ROS production that was sensitive to diphenyliodonium (DPI), thus supporting involvement of NADPH oxidase. In addition, quantitative Southern blot analyses indicated that thrombin increased the equilibrium lesion density of oxidative lesions within the mitochondrial genome. Like the thrombin-induced ROS production, the increase in mtDNA lesion density also was suppressed by DPI. These findings raise the possibility that ROS used as second messengers in inflammatory signaling damage mtDNA in ECs thus leading to formation of mtDNA mutations that contribute to atherogenesis. Supported by NIH

Panel of aneuploid cell lines for physical mapping of the proximal long arm of human chromosome 21

Some forms of familial Alzheimer disease (FAD) have shown linkage to DNA probes for the loci D21S1 and D21S16 on chromosome 21. To investigate the physical location of these DNA probes, we have constructed a physical map of this region of chromosome 21 by using quantitative Southern blot analysis of DNA sequences unique to chromosome 21 and a series of cell lines aneuploid for parts of chromosome 21. We now show that both D21S16 and D21S1/S11 are located in the same region of chromosome 21, in q11.2-q21.05. This new panel of aneuploid cell lines will allow the rapid mapping of new DNA probes in the vicinity of the FAD gene.