RESUMO
Metastasis to the breast from extramammary malignancies is rare. This is the third case report of metastatic breast cancer from esophageal cancer. We report the clinical, radiographic, and pathologic findings of a 57-year-old woman who underwent esophagectomy for esophageal cancer and developed metastatic cancer 2 years later. Pathologic examination of a resected specimen of the breast revealed squamous cell carcinoma invading the mammary glands. Estrogen receptor and axillary lymph node metastasis were negative with immunostaining. She is alive 6 months after the modified radical mastectomy.
Assuntos
Neoplasias da Mama/secundário , Neoplasias Esofágicas/patologia , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/terapia , Terapia Combinada , Neoplasias Esofágicas/radioterapia , Neoplasias Esofágicas/cirurgia , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
The properties of 9 delta-aminolevulinate dehydratase (ALAD) mutants from patients with ALAD porphyria (ADP) were examined by bacterial expression of their complementary DNAs and by enzymologic and immunologic assays. ALADs were expressed as glutathione-S-transferase (GST) fusion proteins in Escherichia coli and purified by glutathione-affinity column chromatography. The GST-ALAD fusion proteins were recognized by anti-ALAD antibodies and were enzymatically active as ALAD. The enzymatic activities of 3 ALAD mutants, K59N, A274T, and V153M, were 69.9%, 19.3%, and 41.0% of that of the wild-type ALAD, respectively, whereas 6 mutants, G133R, K59N/G133R, F12L, R240W, V275M, and delTC, showed little activity (< 8%). These variations generally reflect the phenotype of ALAD in vivo in patients with ADP and indicate that GST-ALAD fusion protein is indeed useful for predicting of the phenotype of ALAD mutants. The location of F12L mutation in the enzyme's molecular structure indicates that its disturbance of the quaternary contact of the ALAD dimer appears to have a significant influence on the enzymatic activity. Mouse monoclonal antibodies to human ALAD were developed that specifically recognized a carboxy terminal portion of ALAD, or other regions in the enzyme. This study represents the first complete analysis of 9 mutants of ALAD identified in ADP and indicates the highly heterogeneous nature of mutations in this disorder.
Assuntos
Mutação , Sintase do Porfobilinogênio/deficiência , Sintase do Porfobilinogênio/genética , Porfirias/enzimologia , Adolescente , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Escherichia coli/genética , Feminino , Expressão Gênica , Glutationa Transferase/genética , Humanos , Immunoblotting , Recém-Nascido , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Fenótipo , Sintase do Porfobilinogênio/metabolismo , Porfirias/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Coproporphyrinogen oxidase (CPOX), the sixth enzyme in the heme-biosynthetic pathway, catalyzes oxidative decarboxylation of coproporphyrinogen to protoporphyrinogen and is located in the intermembrane space of mitochondria. To clarify the importance of CPOX in the regulation of heme biosynthesis in erythroid cells, we established human erythroleukemia K562 cells stably expressing mouse CPOX. The CPOX cDNA-transfected cells had sevenfold higher CPOX activity than cells transfected with vector only. Expression of ferrochelatase and heme content in the transfected cells increased slightly compared with the control. When K562 cells overexpressing CPOX were treated with delta-aminolevulinic acid (ALA), most became benzidine-positive without induction of the expression of CPOX or ferrochelatase, and the heme content was about twofold higher than that in ALA-treated control cells. Increases in cellular heme concomitant with a marked induction of the expression of heme-biosynthetic enzymes, including CPOX, ferrochelatase and erythroid-specific delta-aminolevulinic acid synthase, as well as of alpha-globin synthesis, were observed when cells were treated with transforming growth factor (TGF)beta 1. These increases in the transfected cells were twice those in control cells, indicating that overexpression of CPOX enhanced induction of the differentiation of K562 cells mediated by TGF beta 1 or ALA. Conversely, the transfection of antisense oligonucleotide to human CPOX mRNA into untreated and TGF beta 1-treated K562 cells led to a decrease in heme production compared with sense oligonucleotide-transfected cells. These results suggest that CPOX plays an important role in the regulation of heme biosynthesis during erythroid differentiation.
Assuntos
Coproporfirinogênio Oxidase/metabolismo , Hemoglobinas/biossíntese , Leucemia Eritroblástica Aguda/enzimologia , Ácido Aminolevulínico/farmacologia , Animais , Sequência de Bases , Coproporfirinogênio Oxidase/genética , Primers do DNA , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hemoglobinas/genética , Humanos , Células K562 , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/patologia , Camundongos , Oligonucleotídeos Antissenso/farmacologia , Transfecção , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Coproporphyrinogen oxidase [CPO] gene promoter regulatory element (CPRE) plays an important role in CPO gene regulation. To isolate a CPRE binding protein, we performed Southwestern screening of K562 cDNA expression library using CPRE as a probe and isolated a cDNA clone which encoded a novel protein, Klp1 (K562 cell-derived leucine-zipper-like protein 1). Klp1 mRNA was highly expressed in K562 cells, HeLa cells, and brain as a single transcript (1.4 kb). Gel mobility shift assays revealed that Klp1 specifically binds to CPRE. Computational analysis revealed that Klp1 has a leucine-zipper-like structure, a Leu-X-X-Leu-Leu motif, and a putative nuclear localization signal in the basic amino acid rich region. Transfection of the Klp1 expression vector into THP-1 cells resulted in transcriptional activation of a reporter construct containing CPRE. These results indicate that Klp1 is a DNA sequence-specific transcription factor that regulates gene expression of genes that contain CPRE in their regulatory region.
Assuntos
Coproporfirinogênio Oxidase/genética , Proteínas de Ligação a DNA/genética , Regiões Promotoras Genéticas , Transativadores/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Expressão Gênica , Células HeLa , Humanos , Células K562 , Dados de Sequência Molecular , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Tecidual , Transativadores/metabolismo , Células U937RESUMO
The first and the rate-limiting enzyme of heme biosynthesis is delta-aminolevulinate synthase (ALAS), which is localized in mitochondria. There are 2 tissue-specific isoforms of ALAS, erythroid-specific (ALAS-E) and nonspecific ALAS (ALAS-N). To identify possible mitochondrial factors that modulate ALAS-E function, we screened a human bone marrow cDNA library, using the mitochondrial form of human ALAS-E as a bait protein in the yeast 2-hybrid system. Our screening led to the isolation of the beta subunit of human ATP-specific succinyl CoA synthetase (SCS-betaA). Using transient expression and coimmunoprecipitation, we verified that mitochodrially expressed SCS-betaA associates specifically with ALAS-E and not with ALAS-N. Furthermore, the ALAS-E mutants R411C and M426V associated with SCS-betaA, but the D190V mutant did not. Because the D190V mutant was identified in a patient with pyridoxine-refractory X-linked sideroblastic anemia, our findings suggest that appropriate association of SCS-betaA and ALAS-E promotes efficient use of succinyl CoA by ALAS-E or helps translocate ALAS-E into mitochondria.
Assuntos
5-Aminolevulinato Sintetase/metabolismo , Anemia Sideroblástica/enzimologia , Heme/biossíntese , Isoenzimas/metabolismo , Succinato-CoA Ligases/metabolismo , 5-Aminolevulinato Sintetase/genética , Acil Coenzima A/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Medula Óssea/química , Células CHO , Cricetinae , Cricetulus , DNA Complementar/genética , Indução Enzimática , Células Precursoras Eritroides/enzimologia , Humanos , Isoenzimas/genética , Substâncias Macromoleculares , Mitocôndrias Cardíacas/enzimologia , Dados de Sequência Molecular , Miocárdio/química , Mutação Puntual , RNA Mensageiro/biossíntese , Técnicas do Sistema de Duplo-HíbridoRESUMO
Cloning and expression of the defective gene for delta-aminolevulinate dehydratase (ALAD) from the second of 2 German patients with ALAD deficiency porphyria (ADP), who had been originally reported by Doss et al. in 1979, were performed. Cloning of cDNAs for the defective ALAD were performed using Epstein-Barr virus (EBV)-transformed lymphoblastoid cells of the proband, and nucleotide sequences of cloned cDNA were determined. Two separate mutations of ALAD cDNA were identified in each ALAD allele. One was G457A, termed "H1," resulting in V153M substitution, while the other was a deletion of 2 sequential bases at T(818) and C(819), termed "H2," resulting in a frame shift with a premature stop codon at the amino acid position of 294. Using allele-specific oligonucleotide hybridization, the mother of the proband was shown to have an H1 defect, while using genomic DNA analysis, the father was shown to have an H2 defect. Expression of H1 cDNA in Chinese hamster ovary cells produced an ALAD protein with only a partial activity (10.65% +/- 1.80% of the normal), while H2 cDNA encoded no significant protein. These data thus demonstrate that the proband was associated with 2 novel molecular defects of the ALAD gene, 1 in each allele, and account for the extremely low ALAD activity in his erythrocytes ( approximately 1% of normal).
Assuntos
Mutação , Sintase do Porfobilinogênio/genética , Porfirias Hepáticas/genética , Doença Aguda , Adolescente , Animais , Western Blotting , Células CHO , Clonagem Molecular , Cricetinae , DNA Complementar/biossíntese , Eritrócitos/enzimologia , Humanos , Masculino , Sintase do Porfobilinogênio/deficiência , Porfirias Hepáticas/sangue , Porfirias Hepáticas/enzimologiaAssuntos
Antígenos HLA/genética , Antígenos de Histocompatibilidade Classe I/genética , Proteínas de Membrana , Mutação/genética , Porfiria Cutânea Tardia/genética , Adulto , Idoso , Substituição de Aminoácidos , Análise Mutacional de DNA , Éxons/genética , Feminino , Predisposição Genética para Doença , Proteína da Hemocromatose , Humanos , Íntrons/genética , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Porfiria Cutânea Tardia/epidemiologiaRESUMO
Erythroid 5-aminolevulinate synthase (ALAS-E) catalyzes the first step of heme biosynthesis in erythroid cells. Mutation of human ALAS-E causes the disorder X-linked sideroblastic anemia. To examine the roles of heme during hematopoiesis, we disrupted the mouse ALAS-E gene. ALAS-E-null embryos showed no hemoglobinized cells and died by embryonic day 11.5, indicating that ALAS-E is the principal isozyme contributing to erythroid heme biosynthesis. In the ALAS-E-null mutant embryos, erythroid differentiation was arrested, and an abnormal hematopoietic cell fraction emerged that accumulated a large amount of iron diffusely in the cytoplasm. In contrast, we found typical ring sideroblasts that accumulated iron mostly in mitochondria in adult mice chimeric for ALAS-E-null mutant cells, indicating that the mode of iron accumulation caused by the lack of ALAS-E is different in primitive and definitive erythroid cells. These results demonstrate that ALAS-E, and hence heme supply, is necessary for differentiation and iron metabolism of erythroid cells.
Assuntos
5-Aminolevulinato Sintetase/metabolismo , Células Precursoras Eritroides/citologia , Eritropoese/genética , Heme/deficiência , Ferro/metabolismo , 5-Aminolevulinato Sintetase/deficiência , 5-Aminolevulinato Sintetase/genética , Animais , Diferenciação Celular , Quimera , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Desenvolvimento Embrionário e Fetal , Células Precursoras Eritroides/ultraestrutura , Feminino , Morte Fetal , Citometria de Fluxo , Genótipo , Humanos , Isoenzimas/deficiência , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos KnockoutRESUMO
A novel missense mutation, G663A, in exon 5 of the erythroid-specific delta-aminolevulinate synthase gene (ALAS2) was identified in a Japanese male with pyridoxine-responsive sideroblastic anemia. Activity of the mutant delta-aminolevulinate synthase protein expressed in vitro was 15.1% compared with the normal control, but was increased up to 34.5% by the addition of pyridoxal 5'-phosphate, consistent with the clinical response of the patient to pyridoxine treatment. The same mutation was also detected in genomic DNa from the oral mucosal membrane of the patiet; however, it was not detected in other family member. These findings suggest that this G663A mutation is responsible for sideroblastic anemia in the proband, and may be an index mutation in this pedigree.
Assuntos
5-Aminolevulinato Sintetase/genética , Anemia Sideroblástica/sangue , Anemia Sideroblástica/genética , Eritrócitos/enzimologia , Mutação de Sentido Incorreto , Piridoxina/uso terapêutico , 5-Aminolevulinato Sintetase/sangue , Anemia Sideroblástica/tratamento farmacológico , Anemia Sideroblástica/enzimologia , Sequência de Bases , Células da Medula Óssea/patologia , Éxons , Humanos , Lactente , Japão , Masculino , Reação em Cadeia da Polimerase , Valores de ReferênciaRESUMO
A 20-year-old man presented with microcytic hypochromic anemia (hemoglobin: 9.3 g/dl, MCV: 82.0 fl, MCHC: 29.5 g/dl) and dimorphism RBCs in circulating blood (RDW: 26.8%). Ringed sideroblasts accounted for 29.5% of bone marrow erythroblasts. Iron overload was also observed. Because the patient had a clear family history of anemia, he was given a diagnosis of X-linked sideroblastic anemia. The activity of delta-aminolevulinic acid synthase (ALAS) in bone marrow erythroblasts was low. However, we did not detect mutation of the gene for ALAS. The patient has responded well to a treatment regimen consisting of oral vitamin B6, Fe-chelation therapy, and phlebotomy.
Assuntos
Anemia Sideroblástica/genética , Ligação Genética , Cromossomo X , Adulto , Humanos , Masculino , LinhagemRESUMO
A novel missense mutation, A1754G, in exon 11 of the erythroid-specific delta-aminolaevulinate synthase gene (ALAS2) was identified in a Japanese male with sideroblastic anaemia. ALAS activity in bone marrow cells of the patient was reduced to 53.3% of the normal control. Consistent with this finding, activity of a bacterially expressed ALAS2 mutant protein harbouring this mutation was 19.5% compared with the normal control, but was increased up to 31.6% by the addition of pyridoxal 5'-phosphate (PLP) in vitro. RFLP analysis with Bsp HI restriction revealed that his mother was a carrier of the mutation. These findings suggest that A1754G mutation was inherited in this family in a manner consistent with X-linked inheritance, and is responsible for sideroblastic anaemia in the patient.
Assuntos
5-Aminolevulinato Sintetase/genética , Anemia Sideroblástica/genética , Mutação de Sentido Incorreto/genética , Adolescente , Humanos , Masculino , Polimorfismo de Fragmento de RestriçãoAssuntos
Anemia Sideroblástica/genética , Hemocromatose/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Éxons , Feminino , Humanos , Lactente , Íntrons , Japão , Masculino , Pessoa de Meia-Idade , MutaçãoRESUMO
Transcription levels of the non-specific delta-aminolevulinate synthase (ALAS-N) and heme oxygenase-1 (HO-1) in the placenta at the terminal stage of pregnancy were comparable to those in the female adult liver and in the spleen, respectively. Immunohistochemical studies demonstrated that both enzymes were exclusively expressed in the trophoblast. During gestation, transcript of ALAS-N slightly increased, while HO-1 mRNA significantly decreased. Induced acute fetal hypoxia resulted in an increase in ALAS-N mRNA and in a decrease in HO-1 mRNA. These findings indicate that placental heme metabolism is influenced by the oxygen supply.
Assuntos
5-Aminolevulinato Sintetase/genética , Regulação da Expressão Gênica no Desenvolvimento , Heme Oxigenase (Desciclizante)/genética , Placenta/enzimologia , 5-Aminolevulinato Sintetase/metabolismo , Animais , Feminino , Hipóxia Fetal/enzimologia , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1 , Imuno-Histoquímica , Ligadura , Placenta/patologia , Gravidez , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Útero/irrigação sanguíneaRESUMO
A R411C missense mutation of the erythroid-specific delta-aminolaevulinate synthase (ALAS2) gene was identified in a pedigree with X-linked pyridoxine-responsive sideroblastic anaemia (XLSA). The normal and the mutant cDNAs were expressed in E. coli, and the enzyme protein was purified. ALAS activity of the mutant enzyme was 12% and 25%, when incubated in the absence and the presence of pyridoxal 5'-phosphate, respectively, compared with that of the wild-type enzyme. These findings suggest that the R411C mutation accounts for low ALAS activity and a partial pyridoxine-responsiveness of the disease in the patient.
Assuntos
5-Aminolevulinato Sintetase/genética , Anemia Sideroblástica/genética , Mutação de Sentido Incorreto , Adolescente , Anemia Sideroblástica/tratamento farmacológico , Anemia Sideroblástica/enzimologia , Humanos , Masculino , Linhagem , Piridoxina/uso terapêuticoRESUMO
The erythroid transcription factor NF-E2 is an obligate heterodimer composed of two different subunits (p45 and p18), each containing a basic region-leucine zipper DNA binding domain, and it plays a critical role in erythroid differentiation as an enhancer-binding protein for expression of the beta-globin gene. We show here that dimethyl sulfoxide treatment of wild-type murine erythroleukemia cells, but not a mutant clone of dimethyl sulfoxide-resistant cells, increases NF-E2 activity significantly, which involves both up-regulation of DNA binding and transactivation activities. Both activities were reduced markedly by treatment of cells with 2-aminopurine but not by genistein. Activation of the Ras-Raf-MAP kinase signaling cascade increased NF-E2 activity significantly, but this was suppressed when MafK was overexpressed. Domain analysis revealed an activation domain in the NH2-terminal region of p45 and a suppression domain in the basic region-leucine zipper of MafK. These findings indicate that induction of NF-E2 activity is essential for erythroid differentiation of murine erythroleukemia cells, and serine/threonine phosphorylation may be involved in this process. In addition, they also suggest that a MafK homodimer can suppress transcription, not only by competition for the DNA binding site, but also by direct inhibition of transcription. Hence, MafK may function as an active transcription repressor.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Eritropoese/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Leucêmica da Expressão Gênica , Leucemia Eritroblástica Aguda/genética , Fatores de Transcrição/metabolismo , 5-Aminolevulinato Sintetase/biossíntese , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Diferenciação Celular , Dimetil Sulfóxido/farmacologia , Resistência a Medicamentos/genética , Elementos Facilitadores Genéticos , Fatores de Ligação de DNA Eritroide Específicos , Globinas/biossíntese , Heme/análise , Fator de Transcrição MafK , Camundongos , Modelos Genéticos , Fator de Transcrição NF-E2 , Subunidade p45 do Fator de Transcrição NF-E2 , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , RNA Mensageiro/biossíntese , Transdução de Sinais , Ativação Transcricional , Dedos de Zinco , Proteínas ras/metabolismoRESUMO
To elucidate how pyridoxine-refractory X-linked sideroblastic anemia (XLSA) develops, we analyzed the erythroid-specific 5-aminolevulinate synthase (ALAS-E) gene of a patient with the anemia. The activity and amount of the enzyme in bone marrow cells of the patient were found to be approximately 5% of the normal control. We identified a point mutation, which introduces an amino acid substitution from Asp 190 to Val. In transient transfection analyses using quail fibroblasts, accumulation of aberrantly processed proteins, the sizes of which were larger than that of mature ALAS-E, was found in mitochondria. The proteins were reproducibly detected in assays combining in vitro transcription/translation of ALAS-E precursor and import of the precursor into isolated mouse mitochondria. These results suggest that the mutation causing pyridoxine-refractory XLSA affects the processing of the ALAS-E precursor, thus provoking instability of the ALAS-E protein.