ABSTRACT
INTRODUCTION: Preeclampsia is a hypertensive, gestational disease, which is still the leading cause of pregnancy related morbidity and mortality. The impairment of placental angiogenesis and vascularization is discussed to be of etiopathologic relevance. Deytrosination and tyrosination of α-tubulin is important for the stability and dynamics of microtubules. An increase of α-tubulin detyrosination leads to microtubule stabilization, which is an essential prerequisite for physiologic vascular tube morphogenesis during angiogenesis. So far, little is known about the specific localization of detyrosinated (detyr) and tyrosinated (tyr) tubulin in the placenta and its relevance for preeclampsia. METHODS: Placental expression of detyr- and tyr-tubulin was analyzed by immunohistochemistry, immunofluorescence and western blot. For western blot quantification we used biopsies from healthy placentas (nâ¯=â¯21) and placentas from pregnancies complicated with small for gestational age (nâ¯=â¯5), preeclampsia (nâ¯=â¯5) or both (nâ¯=â¯5). RESULTS: Specific placental localization of detyr-tubulin was detected in the fetal endothelial cells of the placenta. Villous and extravillous trophoblasts as well as villous stroma cells were tyr-tubulin positive. Detyr-tubulin protein expression was significantly decreased in placentas complicated by preeclampsia. CONCLUSIONS: In summary, we report an accumulation of detyr-tubulin in villous vessels of the placenta and a significantly reduced level of detyr-tubulin in placental biopsies of preeclampsia cases. The reduction of placental detyr-tubulin in preeclampsia could suggest a deficit in villous vascular plasticity and might be associated with the impaired arborization of the disease.
Subject(s)
Placenta/metabolism , Pre-Eclampsia/metabolism , Protein Processing, Post-Translational/physiology , Tubulin/metabolism , Tyrosine/metabolism , Chorionic Villi/metabolism , Endothelial Cells/metabolism , Female , Humans , Infant, Small for Gestational Age , Microtubules/metabolism , Pregnancy , Stromal Cells/metabolismABSTRACT
BACKGROUND: Human placental development resembles tumorigenesis, due to the invasive and fusogenic potential of trophoblasts. However, these features are tightly controlled in trophoblasts. Disturbance of this spatial and temporal regulation is thought to contribute to the rare formation of choriocarcinomas. Promoter hypermethylation and loss of the tumor suppressor Retinoic acid receptor responder 1 (RARRES1) were shown to contribute to cancer progression. Our study investigated the epigenetic and transcriptional regulation of RARRES1 in healthy human placenta in comparison to choriocarcinoma cell lines and cases. METHODS: Three choriocarcinoma cell lines (Jeg-3, JAR and BeWo) were treated with three different retinoic acid derivates (Am580, Tazarotene and all-trans retinoic acid) and 5-aza-2'-deoxycytidine. We analyzed RARRES1 promoter methylation by pyrosequencing and performed realtime-PCR quantification to determine RARRES1 expression in placental tissue and trophoblastic cell lines. Additionally, RARRES1 was stained in healthy placentas and in biopsies of choriocarcinoma cases (n = 10) as well as the first trimester trophoblast cell line Swan71 by immunofluorescence and immunohistochemistry. RESULTS: In the choriocarcinoma cell lines, RARRES1 expression could not be induced by sole retinoic acid treatment. Stimulation with 5-aza-2'-deoxycytidine significantly induced RARRES1 expression, which then could be further increased with Am580, Tazarotene and all-trans retinoic acid. In comparison to healthy placenta, choriocarcinoma cell lines showed a hypermethylation of the RARRES1 promoter, which correlated with a reduced RARRES1 expression. In concordance, RARRES1 protein expression was lost in choriocarcinoma tissue. Additionally, in the trophoblastic cell line Swan71, we found a significant induction of RARRES1 expression with increased cell density, during mitosis and in syncytial knots. CONCLUSIONS: Our findings showed that RARRES1 expression is absent in choriocarcinoma due to promoter methylation. Based on our analysis, we hypothesize that RARRES1 might exert tumor suppressive functions in multiple cellular processes (e.g. cell cycle regulation, adhesion, invasion and apoptosis).
Subject(s)
Choriocarcinoma/genetics , DNA Methylation , Down-Regulation , Membrane Proteins/genetics , Uterine Neoplasms/genetics , Cell Line, Tumor , Choriocarcinoma/metabolism , Disease Progression , Epigenesis, Genetic , Female , Gene Expression Regulation, Neoplastic , Humans , Membrane Proteins/metabolism , Pregnancy , Promoter Regions, Genetic , Sequence Analysis, DNA , Uterine Neoplasms/metabolismABSTRACT
The placenta is a major barrier that prevents potentially infectious agents from causing fetal diseases or related complications during pregnancy. Therefore, we postulated that the placenta might express a broad repertoire of antimicrobial proteins as well as inflammatory chemokines and cytokines to combat invading microorganisms. Here we demonstrate that placental cells indeed express a wide range of AMPs (antimicrobial peptides and proteins) including bactericidal/permeability-increasing protein (BPI), secretory leukocyte protease inhibitor (SLPI), human ß-defensin 2 (hBD2), acyloxyacyl hydrolase (AOAH), and cathelicidin (CAP18). In addition, these cells also secrete pro-inflammatory cytokines and chemokines upon stimulation with bacterial ligands. Notably, we show that BPI expression by placental cells could be completely attributed to granulocytes while highly purified placental trophoblasts expressed only a subset of the AMPs like SLPI. Unexpectedly, trophoblast AMPs did not exhibit inducible secretion in response to various TLR ligands and further investigations showed that the unresponsiveness of trophoblasts to lipopolysaccharide (LPS) was due to a lack of TLR4 expression. In summary, we have shown that the expression of different AMPs can be allocated to various cells in the placenta and the repertoire of the AMPs expressed by placental cells is a result of a cooperation of leukocytes as well as cells from embryonic origin.
Subject(s)
Antimicrobial Cationic Peptides/genetics , Placenta/cytology , Placenta/physiology , alpha-Defensins/genetics , Antimicrobial Cationic Peptides/metabolism , Cells, Cultured , Cytokines/metabolism , Female , Gene Expression Profiling , Gene Expression Regulation , Humans , Organ Specificity , Placenta/immunology , Placenta/metabolism , Pregnancy , Trophoblasts/cytology , Trophoblasts/metabolism , Trophoblasts/physiology , Up-Regulation , alpha-Defensins/metabolismABSTRACT
The neuron-restrictive silencer factor/RE1-silencing transcription factor (NRSF/REST) is a negative regulator of gene expression restricting the expression of neuronal genes to the nervous system. NRSF/REST is highly expressed in non-neuronal tissues like the lung. In previous work, we identified small-cell lung cancer (SCLC) cell lines with no detectable NRSF/REST expression that, as a consequence, expressed neuronal markers like L1-cell adhesion molecule (L1-CAM) and neural cell adhesion molecule (NCAM). The loss of NRSF/REST expression was linked to malignant progression; however, its mechanistic role remained elusive. Here, we show that NRSF/REST itself, rather than one of its regulated genes, acts like a classic tumour suppressor, being in part regulated by methylation. In SCLCs, NRSF/REST is positively regulated by CREB, with an NRSF/REST promoter fragment showing cell type specificity. Downstream, NRSF/REST directly regulates AKT2, in which NRSF/REST loss leads to an epidermal growth factor-mediated de-regulation of AKT-Serine473 phosphorylation, important for cellular proliferation and survival. Assaying anchorage-independent growth, we observed that with reduced NRSF/REST expression, proliferation was significantly enhanced, whereas NRSF/REST rescue decreased the potential of cells to grow anchorage independently. Our observations support the fact that NRSF/REST may act as an important modulator of malignant progression in SCLC.
Subject(s)
CREB-Binding Protein/genetics , DNA Methylation/genetics , Gene Expression Regulation, Neoplastic/genetics , Lung Neoplasms/genetics , Membrane Proteins/genetics , Small Cell Lung Carcinoma/genetics , Blotting, Western , CREB-Binding Protein/metabolism , Cell Line, Tumor , Disease Progression , Gene Expression , Genes, Tumor Suppressor , Humans , Lung Neoplasms/metabolism , Membrane Proteins/biosynthesis , Phenotype , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics , Small Cell Lung Carcinoma/metabolism , Tumor Suppressor ProteinsABSTRACT
PURPOSE: This study investigated genetic variations in the estrogen pathway and their association with miscarriages. METHODS: A total of 483 patients were recruited from a comprehensive control group for case-control studies. Three variants of the CYP19A1 gene (rs10046, rs4646 and rs700519) and one variant each of the estrogen (ESR1) and progesterone (PGR) receptor genes (rs3020314 and rs1042838) were investigated using polymorphism genotyping. The chi-squared test and one-way analysis of variation (ANOVA) were used for statistical analysis. RESULTS: For rs10046 (CYP19A1), the C/C genotype was associated with a greater frequency of miscarriages (P = 0.017). The other genotypes were not found to be associated with recurrent miscarriage. CONCLUSIONS: This is the first study that has identified a single-nucleotide polymorphism in the aromatase gene that suggests a significant association between genotypes and miscarriage. As aromatase is an essential enzyme in the estrogen pathway, it may be speculated that variations in the aromatase gene in some way give rise to different conditions in the endocrine environment that can lead to impaired fertility.
Subject(s)
Abortion, Habitual/genetics , Aromatase/genetics , Estrogen Receptor alpha/genetics , Estrogens/genetics , Estrogens/metabolism , Receptors, Progesterone/genetics , Abortion, Spontaneous/genetics , Case-Control Studies , Female , Humans , Polymorphism, Single Nucleotide , Pregnancy , Risk Factors , Signal Transduction/geneticsABSTRACT
Patients with Mayer-Rokitanski-Kuster-Hauser (MRKH) syndrome have congenital uterine and vaginal aplasia. The main question of this study was, if the absence of a uterus along with other genital and organ malformations could contribute to hormone or other growth factor protein fluctuations involved in communication between the hypothalamus-pituitary axis, ovaries and uterus. Serum from 56 MRKH patients (mean 27.6 years) and 22 female controls (mean 30.7 years) were analyzed using ELISA to determine levels of pituitary and steroid hormones (LH, FSH, estradiol, progesterone), growth factors of the TGF-beta superfamily like activin A, inhibin B, and anti-Müllerian hormone (AMH). All serum levels were analyzed in relation to other organ malformations. Compared to controls, all 56 patients, including 5% with streak ovaries or unilateral ovarian aplasia, were generally similar in hormone and growth factor levels and could be grouped into hormonal phases. However, compared to controls LH/FSH and FSH/LH ratios of patients had significantly higher and lower mean values, of 2.75-fold (p=0.015) and 1.9-fold (p=0.002), respectively. Undetectable inhibin B levels of<10 pg/ml (p=0.05) were noted in 41.1% of MRKH patients, resulting in significantly higher activin A/inhibin B ratios (p<0.001). MRKH patients have hormonal phases supporting ovarian function, but patients with low FSH/LH ratios and undetectable inhibin B levels (<10 pg/ml) could represent cycle phasing irregularities. A model is discussed regarding our findings and the loss of ovarian-uterine communication.
Subject(s)
Genital Diseases, Female/congenital , Genital Diseases, Female/physiopathology , Gonadal Steroid Hormones/blood , Multigene Family , Ovary/abnormalities , Ovary/physiopathology , Pituitary Hormones/blood , Transforming Growth Factor beta/blood , Adult , Case-Control Studies , Female , Genital Diseases, Female/blood , Humans , Syndrome , Young AdultABSTRACT
The aim of this study was to correlate chemotherapy-induced nausea and vomiting (CINV) with commonly occurring single nucleotide polymorphisms (SNP) in the 5-hydroxytryptamine receptor 3 genes (HTR3). Women with breast cancer without previous chemotherapy were eligible for this prospective study. All patients received epirubicin, with or without cyclophosphamide, and preventive medication with ondansetron and dexamethasone. The patients documented every vomiting event on an hourly basis. Real-time polymerase chain reaction (PCR) analysis was performed for the following nonsynonymous SNPs: p.Y129S (HTR3B), p.K163N (HTR3C) and p.A405G (HTR3C). The overall proportion of patients (total n = 110) who reported vomiting in the first 24 h after chemotherapy was 31.8%. The variant genotype of K163N (HTR3C) was associated with vomiting, which occurred in 50.0% (P = 0.009). Polymorphisms in the HTR3C gene could serve as a predictive factor for CINV in patients undergoing moderately emetogenic chemotherapy.
Subject(s)
Antineoplastic Agents/adverse effects , Breast Neoplasms/drug therapy , Polymorphism, Single Nucleotide , Receptors, Serotonin, 5-HT3/genetics , Vomiting/genetics , Anthracyclines/adverse effects , Cyclophosphamide/adverse effects , Dexamethasone/therapeutic use , Epirubicin/adverse effects , Female , Genotype , Humans , Nausea/chemically induced , Nausea/genetics , Nausea/prevention & control , Ondansetron/therapeutic use , Reverse Transcriptase Polymerase Chain Reaction , Risk Factors , Vomiting/chemically induced , Vomiting/prevention & controlABSTRACT
BACKGROUND: Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is a malformation of the female genital tract (vaginal aplasia, rudimentary uterus, normal fallopian tubes and high ovaries). The incidence is one in 4000 female newborns. The aim of the present study was to record genital and associated malformations among siblings and relatives of MRKH patients in order to draw possible conclusions regarding the etiology of the syndrome: heredity (dominant versus recessive) or spontaneous malformation. METHODS: Using a standardized questionnaire, affected MRKH patients were asked about other cases of MRKH and/or associated malformations among siblings and relatives. RESULTS: No other cases of MRKH syndrome had occurred among the siblings or relatives of 73 MRKH patients; however, 13 associated malformations were recorded among a total of 103 siblings. Musculoskeletal malformations were markedly increased (3.27 times higher) in comparison with the prevalence of congenital malformations among newborns in the normal population. CONCLUSIONS: This study shows that dominant inheritance cannot play a role in the etiology of MRKH syndrome, as no further cases of MRKH syndrome occurred among any of the siblings. The study provides support for the view that the syndrome has a multifactorial pathogenesis. Siblings/relatives of MRKH patients should be examined for associated musculoskeletal/urogenital malformations.
Subject(s)
Abnormalities, Multiple , Siblings , Uterus/abnormalities , Vagina/abnormalities , Abnormalities, Multiple/epidemiology , Abnormalities, Multiple/genetics , Adolescent , Adult , Diseases in Twins/genetics , Female , Germany/epidemiology , Heart Defects, Congenital/epidemiology , Humans , Male , Middle Aged , Musculoskeletal Abnormalities/epidemiology , Syndrome , Urogenital Abnormalities/epidemiologyABSTRACT
PURPOSE: Various ATM (ataxia telangiectasia-mutated) mutations and polymorphisms have been reported to be associated with an increased breast cancer risk. Recent studies have produced contradictory results regarding the association between ATM genetic variants and breast cancer risk. MATERIALS AND METHODS: The common ATM polymorphism 5557G>A (p.D1853N) (rs1801516), previously suggested to be associated with bilateral breast cancer, was analyzed using real-time PCR in 514 unselected patients with breast cancer and 511 age-matched healthy control individuals. DNA was obtained from peripheral blood draw. RESULTS: The ATM genotype was weakly associated with the risk for breast cancer (P = 0.04 for the overall test). The odds ratio for women with a heterozygous genotype was 0.70 (95% CI, 0.52-0.94) and for the homozygous variant 0.63 (95% CI, 0.27-1.49). Disease-free survival and overall survival showed no significant association with specific genotypes. CONCLUSIONS: The results of this study might suggest a minor association between polymorphism 5557G>A and a reduced risk of breast cancer.
Subject(s)
Breast Neoplasms/genetics , Cell Cycle Proteins/genetics , DNA-Binding Proteins/genetics , Genetic Predisposition to Disease , Polymorphism, Single Nucleotide , Protein Serine-Threonine Kinases/genetics , Tumor Suppressor Proteins/genetics , Adult , Aged , Ataxia Telangiectasia Mutated Proteins , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Case-Control Studies , Disease-Free Survival , Female , Humans , Kaplan-Meier Estimate , Middle Aged , Reverse Transcriptase Polymerase Chain Reaction , Risk FactorsABSTRACT
OBJECTIVE: The objective of the present study was to evaluate the correlation between anti-müllerian hormone (AMH), inhibin B, and activin A in follicular fluid from patients receiving treatment with in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), to identify a parameter to assess the maturation and developmental potential of oocytes. - MATERIALS AND METHODS: AMH, inhibin B, and activin A were measured in follicular fluid from 27 patients undergoing IVF/ICSI treatment for male-factor infertility, tubal occlusion, endometriosis, or anovulation. The values were correlated with the serum estradiol level, the numbers and maturation of the oocytes, and the outcome of IVF/ICSI. - RESULTS: A positive correlation was found between AMH in follicular fluid and the number of oocytes retrieved. High inhibin B levels in follicular fluid and high serum E subset2 levels indicated a normal ovarian response to stimulation, corresponding to the oocyte numbers, while low inhibin B and 17-beta-estradiol (E subset2) levels indicated poor responders to stimulation. An activin A/inhibin B ratio of less than 1 and very high inhibin B levels correlated with large numbers of oocytes, while a ratio of 1-2 and high inhibin levels correlated with regular numbers of oocytes. An activin/inhibin ratio of more than 3 and low inhibin levels were found in poor responders. Pregnancies occurred predominantly in the group with a normal or high response. Patients with elevated ratios for 17-beta-estradiol/AMH, oocyte numbers/AMH, and metaphase II oocyte numbers/AMH had the best chances of becoming pregnant, indicating an inverse correlation between AMH and the maturation and developmental potential of the oocytes. - CONCLUSIONS: In IVF/ICSI patients, a positive correlation was found between AMH, inhibin B, and the activin A/inhibin B ratio in follicular fluid, on the one hand; and between serum 17-beta-estradiol levels and the numbers of oocytes retrieved, on the other. The activin A/inhibin B ratio correlated with the number of oocytes retrieved. The ratio for 17-beta-estradiol, oocyte numbers, and metaphase II oocytes relative to AMH indicated the best developmental potential, and it can therefore be assumed that there is a negative correlation between AMH levels and the maturation and quality of oocytes.
Subject(s)
Activins/analysis , Anti-Mullerian Hormone/analysis , Follicular Fluid/chemistry , Inhibins/analysis , Oocytes/growth & development , Sperm Injections, Intracytoplasmic , Adult , Estradiol/blood , Female , Fertilization in Vitro , Humans , Male , Middle Aged , Pregnancy , Pregnancy RateABSTRACT
Chemoprevention, prophylactic surgery and intensified screening programs are options which can be offered the patients with an increased lifetime risk (p(life)) for breast cancer (BC). Estimation of p(life) includes BRCA mutation analysis and risk estimation based on individual risk factors and family history. MENDEL and BRCAPRO are models which can estimate mutation carrier status probability (p(mut)), p(life) and p(mut) can be estimated using Cyrillic3 software which incorporates BRCAPRO and MENDEL. To integrate age, hormonal factors and benign breast biopsies in risk assessment the Tyrer-Cuzick model can be used. These models support the decision pro or contra genetic analysis and improve the number of positive gene testing results. Estimations of p(life) and p(mut), based on a mathematical model, should deal with algorithms and penetrance/frequency data adequate to the population counselled. Being the main modulatory factors, reproductive/hormonal data should be incorporated like the Tyrer-Cuzick model does.
Subject(s)
Breast Neoplasms/genetics , Genetic Predisposition to Disease , Models, Biological , Age Factors , Breast Neoplasms/physiopathology , Breast Neoplasms/prevention & control , Chemoprevention , Female , Genes, BRCA1/physiology , Genetic Counseling , Gonadal Steroid Hormones , Humans , Risk AssessmentABSTRACT
Endometriosis is a chronic inflammatory disease, which is especially found in women with subfertility problems with an incidence of up to 30%. The disease is considered an estrogen-dependent disorder, where DNA polymorphisms of the estrogen receptor alpha (ERalpha) in connection with endometriosis are controversially discussed. From a German population of women, clinical data associated with the disease, including the American Fertility Society (AFS) I-IV classification, and non-clinical parameters were evaluated statistically in endometriosis patients (n = 98) and in control women (n = 98) without endometriosis. Using a multivariate statistical analysis, significant associations of endometriosis with dysmenorrhea (P < 0.001) and allergies against medicaments (P = 0.042) were found. A positive trend between first grade family history of endometriosis and allergies against medicaments was also observed, suggesting a genetic relationship. From both collectives, DNA from peripheral blood was analyzed for the frequency of the ERalpha DNA polymorphisms Xba1 (A/G) and PvuII (T/C) in intron 1 and the ERalpha exonic DNA polymorphism (G229A) with an amino acid exchange (Gly77Ser) in the transactivation domain. DNA samples from endometriosis lesions and control tissues from the same collectives were also analyzed for the exonic G229A polymorphism. Only homozygote wild-type alleles for the polymorphism G229A were found, making it a rare polymorphism in mid-European individuals. Allele types for the PvuII and Xba1 polymorphisms were analyzed with the observed statistically significant clinical parameters and showed no significant association with endometriosis; however a trend with AFS IV was noted, which could contribute to lesion severity. In conclusion, the analyzed polymorphisms in the ERalpha do not have a functional role concerning specific clinical parameters associated with endometriosis.
Subject(s)
DNA/analysis , Endometriosis/genetics , Endometrium/metabolism , Estrogen Receptor alpha/genetics , Polymorphism, Genetic , Adolescent , Adult , Case-Control Studies , Drug Hypersensitivity , Estrogen Receptor alpha/metabolism , Female , Genetic Predisposition to Disease , Genome , Humans , Infertility , Middle Aged , Multivariate Analysis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND: The etiology of the Mayer-Rokitanski-Kuster-Hauser (MRKH) syndrome, where congenitally the Mullerian ducts fail to develop into the uterus, cervix and upper vagina, along with other malformations, is unresolved. Anti-Mullerian hormone (AMH) signal transduction inducing the degradation of Mullerian ducts in males is implicated in the MRKH syndrome. This study examined if DNA sequence variations are responsible for the activation of AMH and aberrant hormone levels in MRKH patients. METHODS: The entire AMH promoter and 3' regulatory elements of the constitutively expressed splicing factor SF3a2 were sequenced in 30 MRKH patients and genotyped in 48 control individuals using matrix-assisted laser desorption/ionization-time-of-flight mass spectronomy. Ovarian AMH promoter function was correlated with protein expression in plasma and peritoneal fluid of MRKH patients. RESULTS: Of six identified AMH promoter variations, two at positions -639 (SP1-binding site) and -210 [steroidogenic factor (SF)1-binding site] were homozygote in 73% of patients, and 69% of control individuals, destroying the SP1-binding site. AMH protein levels in plasma and peritoneal fluid from patients were equivalent to control individuals, however in three patients plasma levels were abnormally high. CONCLUSIONS: AMH is an important indicator for ovarian function. AMH promoter sequence variations or the previously proposed SF3a2-AMH fusion co-transcripts cannot be responsible for aberrant AMH expression leading to Mullerian duct degradation.
Subject(s)
DNA/genetics , Genitalia, Female/abnormalities , Glycoproteins/genetics , Glycoproteins/metabolism , Testicular Hormones/genetics , Testicular Hormones/metabolism , Abnormalities, Multiple/blood , Abnormalities, Multiple/genetics , Abnormalities, Multiple/metabolism , Adolescent , Adult , Alleles , Anti-Mullerian Hormone , Ascitic Fluid/metabolism , Base Sequence , Case-Control Studies , Exons , Female , Gene Expression , Genetic Variation , Glycoproteins/blood , Humans , Middle Aged , Mullerian Ducts/embryology , Nucleoproteins/genetics , Phenotype , Promoter Regions, Genetic , RNA Splicing Factors , RNA-Binding Proteins/genetics , Syndrome , Testicular Hormones/bloodABSTRACT
Mammalian interphase and mitotic cells were analyzed for their cation composition using a three-dimensional high resolution scanning ion microprobe. This instrument maps the distribution of bound and unbound cations by secondary ion mass spectrometry (SIMS). SIMS analysis of cryofractured interphase and mitotic cells revealed a cell cycle dynamics of Ca2+, Mg2+, Na+, and K+. Direct analytical images showed that all four, but no other cations, were detected on mitotic chromosomes. SIMS measurements of the total cation content for diploid chromosomes imply that one Ca2+ binds to every 12.5-20 nucleotides and one Mg2+ to every 20-30 nucleotides. Only Ca2+ was enriched at the chromosomal DNA axis and colocalized with topoisomerase IIalpha (Topo II) and scaffold protein II (ScII). Cells depleted of Ca2+ and Mg2+ showed partially decondensed chromosomes and a loss of Topo II and ScII, but not hCAP-C and histones. The Ca2+-induced inhibition of Topo II catalytic activity and direct binding of Ca2+ to Topo II by a fluorescent filter-binding assay supports a regulatory role of Ca2+ during mitosis in promoting solely the structural function of Topo II. Our study directly implicates Ca2+, Mg2+, Na+, and K+ in higher order chromosome structure through electrostatic neutralization and a functional interaction with nonhistone proteins.
Subject(s)
Calcium/metabolism , Chromatin/metabolism , Chromosomes/metabolism , Magnesium/metabolism , Mitosis/physiology , Animals , Calcium/analysis , Cations/analysis , Cations/metabolism , Cell Line , Cell Nucleus/chemistry , Cell Nucleus/enzymology , Cryopreservation , DNA Topoisomerases, Type II/metabolism , Deer , Fibroblasts/cytology , Humans , Magnesium/analysis , Male , Metaphase/physiology , Potassium/analysis , Potassium/metabolism , Sodium/analysis , Sodium/metabolism , Spectrometry, Mass, Secondary IonABSTRACT
The human AF9 gene at 9p22 is one of the most common fusion partner genes with the MLL gene at 11q23, resulting in the t(9;11)(p22;q23). The MLL-AF9 fusion gene is associated with de novo acute myelo-genous leukemia (AML), rarely with acute lymphocytic leukemia (ALL) and with therapy related leukemia (t-AML). The AF9 gene is >100 kb and two patient breakpoint cluster regions (BCRs) have been identified; BCR1 is within intron 4, previously called site A, whereas BCR2 or site B spans introns 7 and 8. Patient breakpoint locations were determined previously by RT-PCR and by genomic DNA cloning. In this study, we defined the exon-intron boundaries and identified several different structural elements in AF9 including a co-localizing in vivo DNA topo II cleavage site and an in vitro DNase I hypersensitive (DNase 1 HS) site in intron 7 in BCR2. Reversibility experiments demonstrated a religation of the topo II cleavage sites. The location of the in vivo topo II cleavage site was confirmed in vitro using a topo II cleavage assay. In addition, two scaffold associated regions (SARs) are located centromeric to the topo II and DNase I HS cleavage sites and border both patient breakpoint regions: SAR1 is located in intron 4, whereas SAR2 encompasses parts of exons 5-7. This study demonstrates that the patient breakpoint regions of AF9 share the same structural elements as the MLL BCR. We describe a DNA breakage and repair model for non-homologous recombination between MLL and its partner genes, particularly AF9.
Subject(s)
DNA, Neoplasm/genetics , DNA-Binding Proteins/genetics , Leukemia/genetics , Nuclear Proteins/genetics , Oncogene Proteins, Fusion/genetics , Proto-Oncogenes , Transcription Factors , Binding Sites , Cell Line , Chromatin/genetics , DNA Topoisomerases, Type II/metabolism , DNA, Neoplasm/chemistry , Deoxyribonuclease I/metabolism , Histone-Lysine N-Methyltransferase , Humans , Introns , Jurkat Cells , K562 Cells , Molecular Sequence Data , Myeloid-Lymphoid Leukemia Protein , Nuclear Proteins/metabolism , Recombination, Genetic , Sequence Analysis, DNA , Translocation, Genetic , Tumor Cells, CulturedABSTRACT
Chromosomal translocations involving the MLL gene occur in about 80% of infant leukemia. In the search for possible agents inducing infant leukemia, we identified bioflavonoids, natural substances in food as well as in dietary supplements, that cause site-specific DNA cleavage in the MLL breakpoint cluster region (BCR) in vivo. The MLL BCR DNA cleavage was shown in primary progenitor hematopoietic cells from healthy newborns and adults as well as in cell lines; it colocalized with the MLL BCR cleavage site induced by chemotherapeutic agents, such as etoposide (VP16) and doxorubicin (Dox). Both in vivo and additional in vitro experiments demonstrated topoisomerase II (topo II) as the target of bioflavonoids similar to VP16 and Dox. Based on 20 bioflavonoids tested, we identified a common structure essential for topo II-induced DNA cleavage. Reversibility experiments demonstrated a religation of the bioflavonoid as well as the VP16-induced MLL cleavage site. Our observations support a two-stage model of cellular processing of topo II inhibitors: The first and reversible stage of topo II-induced DNA cleavage results in DNA repair, but also rarely in chromosome translocations; whereas the second, nonreversible stage leads to cell death because of an accumulation of DNA damage. These results suggest that maternal ingestion of bioflavonoids may induce MLL breaks and potentially translocations in utero leading to infant and early childhood leukemia.
Subject(s)
DNA-Binding Proteins/genetics , Diet , Flavonoids/adverse effects , Hematopoietic Stem Cells/cytology , Leukemia, Myeloid, Acute/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Proto-Oncogenes , Transcription Factors , Translocation, Genetic , Adult , Cell Differentiation , Cells, Cultured , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/drug effects , Exons , Fetal Blood/cytology , Flavonoids/pharmacology , Histone-Lysine N-Methyltransferase , Humans , Infant , Infant, Newborn , Leukemia, Myeloid, Acute/epidemiology , Myeloid-Lymphoid Leukemia Protein , Precursor Cell Lymphoblastic Leukemia-Lymphoma/epidemiologyABSTRACT
A damaged nucleus has long been regarded simply as a "bag of broken chromosomes," with the DNA free ends moving around and forming connections with randomly encountered partners. Recent evidence shows this picture to be fundamentally wrong. Chromosomes occupy specific nuclear domains within which only limited movement is possible. In a human diploid nucleus, 6.6 x 10(9) base pairs (bp) of DNA are compartmentalized into chromosomes in a way that allows stringent control of replication, differential gene expression, recombination and repair. Most of the chromatin is further organized into looped domains by the dynamic binding of tethered bases to a network of intranuclear proteins, the so-called nuclear scaffold or matrix. Thus, DNA movement is severely curtailed, which limits the number of sites where interchanges can occur. This intricate organizational arrangement may render the genome vulnerable to processes that interfere with DNA repair. Both lower and higher eukaryotic cells perform homologous recombination (HR) and illegitimate recombination (IR) as part of their survival strategies. The repair processes comprising IR must be understood in the context of DNA structural organization, which is fundamentally different in prokaryotic and eukaryotic genomes. In this paper we first review important cellular processes including recombination, DNA repair, and apoptosis, and describe the central elements involved. Then we review the different DNA targets of recombination, and present recent evidence implicating the nuclear matrix in processes which can induce either repair, translocation, deletion, or apoptosis. J. Cell. Biochem. Suppl. 35:3-22, 2000.
Subject(s)
Cell Nucleus/metabolism , DNA/chemistry , DNA/metabolism , Animals , Apoptosis , Chromosomes/chemistry , DNA Repair , Humans , Models, Genetic , Mutation , Recombination, GeneticABSTRACT
The DNA binding properties of herpes simplex virus type 1 DNA polymerase (HSV pol), an alpha-like DNA polymerase, were investigated using an optimized band-shift assay. With linear double-stranded DNA (dsDNA), HSV pol formed two complexes. The favored DNA template was dsDNA with protruding 5'-phosphoryl termini. Stable binding of HSV pol was observed with a DNA hairpin containing a primer region of 9 bp of dsDNA, a 6-base loop and a 12-base 5'-terminal single-stranded extension. For the polymerization activity of HSV pol on poly(dT) an optimal primer length of 8 to 10 nucleotides was determined. The DNA binding event could be clearly separated from the enzymatic activities by its unique response to divalent cations and salt. Under ionic strength conditions where HSV pol exerts optimal polymerization activity in vitro, novel polymerase-DNA complexes were detected by band-shift analysis. These new complexes were similar while either in DNA polymerase or 3',5' exonuclease mode. Using a polymerase trap method and high-resolution polyacrylamide gel electrophoresis, HSV pol demonstrated internal switching from 3',5' exonuclease to polymerase-active mode during one DNA binding event. These results support the role of HSV pol as a true replicase, which proofreads without dissociating from the DNA template.
Subject(s)
DNA-Binding Proteins/genetics , DNA-Directed DNA Polymerase/metabolism , DNA/metabolism , Exodeoxyribonucleases/metabolism , Viral Proteins , Cell Line , DNA Polymerase I/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli/enzymology , Heparin/pharmacology , Poly A/metabolism , Poly T/metabolism , Protein Conformation , Recombinant Proteins/genetics , Salts/pharmacology , Substrate Specificity/geneticsABSTRACT
The human myeloid-lymphoid leukemia gene, MLL (also called ALL-1, Htrx, or HRX ), maps to chromosomal band 11q23. MLL is involved in translocations that result in de novo acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), mixed lineage leukemia, and also in therapy AML (t-AML) and therapy ALL (t-ALL) resulting from treatment with DNA topoisomerase II (topo II) targeting drugs. MLL can recombine with more than 30 other chromosomal bands, of which 16 of the partner genes have been cloned. Breaks in MLL occur in an 8. 3-kb breakpoint cluster region (BCR) encompassing exons 5 through 11. We recently demonstrated that 75% of de novo patient breakpoints in MLL mapped in the centromeric half of the BCR between two scaffold-associated regions (SAR), whereas 75% of the t-AML patient breakpoints mapped to the telomeric half of the BCR within a strong SAR. We have mapped additional structural elements in the BCR. An in vivo DNA topo II cleavage site (induced with several different drugs that target topo II) mapped near exon 9 in three leukemia cell lines. A strong DNase I hypersensitive site (HS) also mapped near exon 9 in four leukemia cell lines, including two in which MLL was rearranged [a t(6;11) and a t(9;11)], and in two lymphoblastoid cell lines with normal MLL. Two of the leukemia cell lines also showed an in vivo topo II cleavage site. Our results suggest that the chromatin structure of the MLL BCR may influence the location of DNA breaks in both de novo and therapy-related leukemias. We propose that topo II is enriched in the MLL telomeric SAR and that it cleaves the DNase I HS site after treatment with topo II inhibitors. These events may be involved in recombination associated with t-AML/t-ALL breakpoints mapping in the MLL SAR.
Subject(s)
Chromosome Aberrations , Chromosomes, Human, Pair 11/genetics , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/genetics , Deoxyribonuclease I/metabolism , Leukemia/genetics , Proto-Oncogenes , Transcription Factors , Cell Transformation, Neoplastic/genetics , Chromosome Mapping , Chromosomes, Human, Pair 11/ultrastructure , Histone-Lysine N-Methyltransferase , Humans , Myeloid-Lymphoid Leukemia Protein , Recombination, Genetic , Substrate Specificity , Translocation, Genetic , Tumor Cells, CulturedABSTRACT
Though acne is not generally cured, treatment can reduce the severity and frequency of outbreaks, lessen discomfort from inflamed lesions, improve appearance, and prevent or minimize scarring, thereby averting or ameliorating potentially serious adverse psychosocial effects. A number of new agents are available; combination therapies that achieve synergism are described.