RESUMO
Climatic changes have played major roles in plants' evolutionary history. Glacial oscillations have been particularly important, but some of their effects on plants' populations are poorly understood, including the numbers and locations of refugia in Asian warm temperate zones. In the present study, we investigated the demographic history of the broadleaved evergreen tree species Castanopsis sieboldii (Fagaceae) during the last glacial period in Japan. We used approximate Bayesian computation (ABC) for model comparison and parameter estimation for the demographic modeling using 27 EST-associated microsatellites. We also performed the species distribution modeling (SDM). The results strongly support a demographic scenario that the Ryukyu Islands and the western parts in the main islands (Kyushu and western Shikoku) were derived from separate refugia and the eastern parts in the main islands and the Japan Sea groups were diverged from the western parts prior to the coldest stage of the Last Glacial Maximum (LGM). Our data indicate that multiple refugia survived at least one in the Ryukyu Islands, and the other three regions of the western and eastern parts and around the Japan Sea of the main islands of Japan during the LGM. The SDM analysis also suggests the potential habitats under LGM climate conditions were mainly located along the Pacific Ocean side of the coastal region. Our ABC-based study helps efforts resolve the demographic history of a dominant species in warm temperate broadleaved forests during and after the last glacial period, which provides a basic model for future phylogeographical studies using this approach.
Assuntos
Teorema de Bayes , Etiquetas de Sequências Expressas , Fagaceae/genética , Genética Populacional , Repetições de Microssatélites , Refúgio de Vida Selvagem , Evolução Biológica , Variação Genética , Japão , Modelos Genéticos , Filogenia , FilogeografiaRESUMO
We investigated the bonding stiffness of individual atoms on substrate surfaces using noncontact atomic force microscopy with frequency modulation. We measured the frequency shift distribution of the (110) plane above buckling-up and buckling-down dimer atoms of the Ge(001)-c(4 × 2) surface using a tungsten-coated atomic force microscopy cantilever. The tip-surface chemical force distribution was reproduced from the frequency shift data using calculations based on Sader's formula. The total harmonic bonding stiffness between the dimer atoms and the substrate was first discovered by fitting the Morse force to the tip-surface chemical force distribution with consideration of the relaxation in the tip-surface gap. By excluding the contribution exerted by the probe tip, we observed that the harmonic bonding compliance of the buckling-up dimer atom was 4.8 × 10(-3) m/N stiffer than that of the buckling-down dimer atom. This technique for probing the elastic bonding states of individual surface atoms at the atomic scale is unique.
RESUMO
Generally, floral characteristics and pollination are important factors enhancing the quality and quantity of reproductive output for regeneration in plant conservation. However, lack of evidence-based management could decrease fitness under ex-situ conservation. We investigated the capitulum and pollination characteristics of Eriocaulon heleocharioides Satake (Eriocaulaceae), which is extinct in the wild, to develop an evidence-based conservation management plan incorporating previously ignored reproductive characteristics. To evaluate the functional characteristics of capitula, pollen-ovule ratio, and reproductive status (maximum pollination success/florivory damage) were investigated along six flowering sequences of capitulum. To evaluate the effect of plant density on pollen transfer, high- and low-density plots were established. Total deposited pollen on stigma, insect visitation, and visit duration per capitulum were observed. A significantly lower pollen-ovule ratio was observed in the first of six capitula, reflecting higher female functionality. The highest pollination success was found in the second-fourth capitula, whereas florivory increased along the terminal capitula position. High plant density affected the pollen deposited on stigmas via insect visitation and low pollinator visit duration. Different capitula in E. heleocharioides could have different effects: different sexual functionality, enhancement of reproductive output both in quality and quantity through active pollen transfer, and escaping from florivores. High plant density could facilitate outcross-pollen transfer in E. heleocharioides. Multiple perspectives are important for determining potential reproductive success in ex-situ conservation. Thus, density management reflecting capitulum characteristics could improve the efficiency of conservation efforts for E. heleocharioides.
Assuntos
Eriocaulaceae , Polinização , Animais , Flores , Insetos , Pólen , ReproduçãoRESUMO
Coronary collateral vessels reduce damage to ischemic myocardium after coronary obstruction. Factors that stimulate collateral formation are expected to have ameliorating effects on myocardial infarction. In a canine experimental myocardial infarct model, intracoronary injection of basic fibroblast growth factor (bFGF) improved cardiac systolic function and reduced infarct size. Treatment with bFGF increased the number of arterioles and capillaries in the infarct. Thus, the angiogenic action of bFGF might lead to a reduction in infarct size. The application of bFGF might bring about a therapeutic modality for the salvage of infarcted myocardium.
Assuntos
Fator 2 de Crescimento de Fibroblastos/uso terapêutico , Infarto do Miocárdio/tratamento farmacológico , Animais , Arteríolas/patologia , Capilares/patologia , Divisão Celular , Angiografia Coronária , Vasos Coronários/patologia , Cães , Endotélio Vascular/patologia , Fator 2 de Crescimento de Fibroblastos/administração & dosagem , Ventrículos do Coração/patologia , Músculo Liso Vascular/patologia , Infarto do Miocárdio/patologia , Neovascularização Patológica , Tamanho do ÓrgãoRESUMO
We have previously defined a homozygously deleted region at chromosome 1p36.2-p36.3 in human neuroblastoma cell lines, NB-1 and NB-C201, and identified six genes including DFF45/ICAD within this region. In this study, we found that NB-C201 cells are much more resistant to various genotoxic stresses such as cisplatin (CDDP) than CHP134 and SH-SY5Y cells that do not have the homozygous deletion. To examine a role(s) of DFF45 in the regulation of apoptosis in response to CDDP, we have established stably DFF45-expressing NB-C201 cell clones (DFF45-1 and DFF45-3) and a control cell clone (NB-C201-C) using a retrovirus-mediated gene transfer. In contrast to NB-C201-C cells, DFF45-3 cells displayed apoptotic nuclear fragmentation in response to CDDP. Although CDDP-induced proteolytic cleavage of procaspase-3 and DFF45 in DFF45-3 cells, we could not detect a typical apoptotic DNA fragmentation. Additionally, deletion analysis revealed that C-terminal region of DFF45 is required for inducing nuclear fragmentation. Unexpectedly, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays demonstrated that DFF45 has undetectable effect on CDDP sensitivity of NB-C201 cells. Taken together, our present results suggest that DFF45/DFF40 system may be sufficient for CDDP-induced nuclear fragmentation but not DNA cleavage.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Cisplatino/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Deleção de Genes , Antineoplásicos/farmacologia , Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Northern Blotting , Western Blotting , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Clivagem do DNA/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fatores de Tempo , TransfecçãoRESUMO
We examined the kenetics of p15 methylation and expression during myeloid development. We treated human cord blood CD34+ cells with either GM-CSF alone or in combination with stem cell factor and followed methylation at this locus using bisulfite genomic sequencing. CD34+ cells were found to be either fully methylated or completely unmethylated at 27 CpG dinucleotide sites in exon 1 and at 18 CpG sites in the promoter region of the p15 gene. A time-course study showed that the percentage of the allelic methylation of p15 CpG island increased to approximately 50% to 60% until 7 days after cytokine stimulation, then decreased to less than 10% after 21 days. The methylation was also observed in bone marrow CD34+ cells exposed to GM-CSF. p15 expression varied inversely with methylation. Expression was negligible or at low levels until 14 days, after which it increased substantially. The frequency of myeloid colony-forming cells in the progeny decreased and myeloid-specific markers increased in the later stages. Based on our observations on cells grown with GM-CSF and 5-aza-2'-deoxycytidine, DNA methylation of the p15 promoter region CpG island appears to be associated with proliferation rather than differentiation of normal human myeloid progenitors.
Assuntos
Proteínas de Ciclo Celular/genética , Ilhas de CpG , Inibidor p16 de Quinase Dependente de Ciclina/genética , Metilação de DNA , Hematopoese/genética , Hematopoese/fisiologia , Proteínas Supressoras de Tumor , Alelos , Antígenos CD34/metabolismo , Sequência de Bases , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Inibidor de Quinase Dependente de Ciclina p15 , DNA/genética , Sangue Fetal/citologia , Sangue Fetal/efeitos dos fármacos , Sangue Fetal/imunologia , Sangue Fetal/metabolismo , Expressão Gênica , Genes Supressores de Tumor , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Granulócitos/efeitos dos fármacos , Granulócitos/imunologia , Granulócitos/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Cinética , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Fator de Células-Tronco/farmacologiaRESUMO
We examined the enzyme protein and biosynthesis of human trifunctional protein harboring enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase activity in cultured skin fibroblasts from two patients with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. The following results were obtained. (a) In cells from patient 1, immunoblot analysis and pulse-chase experiments indicated that the content of trifunctional protein was < 10% of that in control cells, due to a very rapid degradation of protein newly synthesized in the mitochondria. The diminution of trifunctional protein was associated with a decreased activity of enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase, when measured using medium-chain to long-chain substrates. (b) In cells from patient 2, the rate of degradation of newly synthesized trifunctional protein was faster than that in control cells, giving rise to a trifunctional protein amounting to 60% of the control levels. The 3-hydroxy-acyl-CoA dehydrogenase activity with medium-chain to long-chain substrates was decreased drastically, with minor changes in activities of the two other enzymes. These data suggest a subtle abnormality of trifunctional protein in cells from patient 2. Taken together, the results obtained show that in both patients, long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency is caused by an abnormality in the trifunctional protein, even though there is a heterogeneity in both patients.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/deficiência , Acetil-CoA C-Aciltransferase/deficiência , Enoil-CoA Hidratase/deficiência , Mitocôndrias/metabolismo , 3-Hidroxiacil-CoA Desidrogenases/genética , Acetil-CoA C-Aciltransferase/genética , Células Cultivadas , Enoil-CoA Hidratase/genética , Fibroblastos/enzimologia , Humanos , Immunoblotting , Peso Molecular , Mutação , Oxirredução , Ácido Palmítico , Ácidos Palmíticos/metabolismoRESUMO
Mitochondrial very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) was purified from human liver. The molecular masses of the native enzyme and the subunit were estimated to be 154 and 70 kD, respectively. The enzyme was found to catalyze the major part of mitochondrial palmitoylcoenzyme A dehydrogenation in liver, heart, skeletal muscle, and skin fibroblasts (89-97, 86-99, 96-99, and 78-87%, respectively). Skin fibroblasts from 26 patients suspected of having a disorder of mitochondrial beta-oxidation were analyzed for VLCAD protein using immunoblotting, and 7 of them contained undetectable or trace levels of the enzyme. The seven deficient fibroblast lines were characterized by measuring acyl-coenzyme A dehydrogenation activities, overall palmitic acid oxidation, and VLCAD protein synthesis using pulse-chase, further confirming the diagnosis of VLCAD deficiency. These results suggested the heterogenous nature of the mutations causing the deficiency in the seven patients. Clinically, all patients with VLCAD deficiency exhibited cardiac disease. At least four of them presented with hypertrophic cardiomyopathy. This frequency (> 57%) was much higher than that observed in patients with other disorders of mitochondrial long-chain fatty acid oxidation that may be accompanied by cardiac disease in infants.
Assuntos
Ácidos Graxos Dessaturases/isolamento & purificação , Erros Inatos do Metabolismo Lipídico/enzimologia , Mitocôndrias Hepáticas/enzimologia , Acil Coenzima A/metabolismo , Acil-CoA Desidrogenase de Cadeia Longa , Western Blotting , Ácidos Graxos Dessaturases/metabolismo , Feminino , Fibroblastos , Humanos , Masculino , Mitocôndrias Cardíacas/enzimologia , Peso Molecular , Palmitatos/metabolismoRESUMO
Among 11 JMML children, two had an abnormal karyotype, and nine had a normal karyotype at onset. In one patient with trisomy 8 and four patients with a normal karyotype, a new clone with an aberrant karyotype emerged 1-14 months after 6-mercaptopurine (6-MP) therapy as shown by G-banding analyses. Fluorescence in situ hybridization disclosed that an abnormal clone existed in approximately 3-6% of bone marrow cells at onset or before 6-MP therapy in all the four cases examined, and increased to approximately 12-90% during the treatment. In culture with granulocyte-macrophage colony-stimulating factor, cytogenetically abnormal clones that proliferated during 6-MP therapy possessed significantly less sensitivity to the antimetabolite, compared with cells that decreased in numbers after the therapy. A PTPN11 mutation was detected in all of granulocyte-macrophage colonies irrespective of karyotypic aberration in one patient, whereas approximately 80% of erythroid colonies and 20% of mixed colonies possessed neither a PTPN11 mutation nor chromosomal abnormalities. The appearance of chromosomal aberrations shown by G-banding during 6-MP therapy in some JMML cases may result, in part, from the growth of a 6-MP-refractory clone that already exists at onset. It is possible that treatment with 6-MP promotes progression of the disease.
Assuntos
Antineoplásicos/uso terapêutico , Aberrações Cromossômicas , Leucemia Mielomonocítica Aguda/tratamento farmacológico , Leucemia Mielomonocítica Aguda/genética , Mercaptopurina/uso terapêutico , Bandeamento Cromossômico , Genes ras , Humanos , Hibridização in Situ Fluorescente , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia Mielomonocítica Aguda/patologia , Mutação , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Tirosina Fosfatases/genéticaRESUMO
The p19ARF product of the INK4a/ARF locus is induced in response to potentially oncogenic hyperproliferative signals and activates p53 by interfering with its negative regulator, Mdm2. Mice lacking ARF are highly prone to tumor development, and in this study, 80% of these animals spontaneously developed tumors and died within their first year of life. Mice that were heterozygous for ARF also developed tumors after a longer latency, whereas their wild-type littermates did not. In heterozygotes, tumor formation was accompanied by loss of the residual ARF allele and/or lack of ARF mRNA expression, implying that ARF can act as a canonical "two-hit" tumor suppressor gene. Tumors occurred earlier in life in ARF-null animals that were neonatally irradiated or given dimethylbenzanthrene, and several animals treated with carcinogen simultaneously developed multiple forms of malignancy arising from distinct cell lineages. Although p53-null mice primarily develop lymphomas and fibrosarcomas, the frequency of these two tumor types was inverted in ARF-null animals, with undifferentiated sarcomas predominating in a 3:2 ratio; 28% of ARF-null animals developed carcinomas and tumors of the nervous system, which have been rarely observed in untreated p53-null mice. The longer latency of tumor formation in ARF-null versus p53-null mice, therefore, appears to enable a broader spectrum of tumors to emerge.
Assuntos
Neoplasias Experimentais/genética , Proteínas Nucleares , Proteínas/fisiologia , 9,10-Dimetil-1,2-benzantraceno , Animais , Carcinoma/etiologia , Carcinoma/genética , Linhagem da Célula , Cocarcinogênese , Progressão da Doença , Feminino , Fibrossarcoma/etiologia , Fibrossarcoma/genética , Deleção de Genes , Genes p53 , Genótipo , Perda de Heterozigosidade , Linfoma/etiologia , Linfoma/genética , Masculino , Camundongos , Camundongos Knockout , Neoplasias Experimentais/etiologia , Neoplasias de Tecido Nervoso/etiologia , Neoplasias de Tecido Nervoso/genética , Neoplasias Induzidas por Radiação/genética , Células-Tronco Neoplásicas/patologia , Proteínas/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2 , Fatores de Tempo , Proteína Supressora de Tumor p14ARF , Proteína Supressora de Tumor p53/metabolismoRESUMO
The alternative reading frame product (p19ARF) of the mouse INK4a/ARF locus is induced by oncoproteins such as Myc and E1A as part of a checkpoint response that limits cell cycle progression in response to hyperproliferative signals. ARF binds directly to Mdm2 to prevent down-regulation of p53 and thereby promotes p53-dependent transcription and cell cycle arrest. However, ARF is not required for p53 induction in response to ionizing radiation or other forms of DNA damage. Animals lacking a functional ataxia telangiectasia (Atm) gene are exquisitely sensitive to ionizing radiation; Atm-null mouse embryo fibroblasts (MEFs) undergo premature replicative arrest, which is relieved by the loss of p53. Here we show that the loss of ARF expands the life expectancy of Atm-null MEFs, but alters neither the sensitivity of Atm-null mice to ionizing radiation nor their propensity to develop lymphomas early in life. Therefore, whereas ARF and Atm signal to p53 through distinct pathways, the loss of ARF can modify p53-dependent features of the Atm-null phenotype.
Assuntos
Proteínas Nucleares , Proteínas Serina-Treonina Quinases , Proteínas/genética , Proteínas/fisiologia , Tolerância a Radiação/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular/genética , Proteínas de Ciclo Celular , Células Cultivadas , Dano ao DNA , Reparo do DNA/genética , Proteínas de Ligação a DNA , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/efeitos da radiação , Linfoma/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , Proteína Supressora de Tumor p14ARF , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de TumorRESUMO
Bone is the most common site of metastasis in prostate cancer (PC), and to generate an animal model to investigate the basis of the unique organ tropism of PC cells for bone, we engrafted humanized non-obese diabetic/severe combined immunodeficient (NOD/SCID-hu) mice with human adult bone (HAB) and lung (HAL). Human PC cell lines LNCaP (1 x 10(7)) and PC-3 (5 x 10(6)) were injected into male NOD/SCID-hu mice via the lateral tail vein at 3-4 weeks after implantation. At 8 weeks after the injection, LNCaP and PC-3 cells had metastasized specifically to HAB in 35 and 65%, respectively, of the mice. The tumors formed by LNCaP appeared to be the osteoblastic type, whereas the PC-3 tumors consisted of osteolytic lesions without any surrounding osteogenic response. A feature of experimental metastasis of PC in NOD/SCID-hu mice was its specificity for HAB tissue. Human PC cells had no or very low metastatic potential in regard to implanted HAL, mouse bone, or native mouse bone. These findings indicate that metastasis of PC cells to HAB is both species and tissue specific. The availability of this small animal model could provide a useful tool for identifying and analyzing important features of the human PC metastatic process that cannot be addressed in conventional metastasis models.
Assuntos
Neoplasias Ósseas/secundário , Transplante Ósseo , Modelos Animais de Doenças , Neoplasias Pulmonares/metabolismo , Transplante de Pulmão , Neoplasias da Próstata/patologia , Idoso , Animais , Neoplasias Ósseas/patologia , Humanos , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Transplante de Neoplasias , Especificidade de Órgãos , Especificidade da Espécie , Transplante HeterólogoRESUMO
The cDNA sequence of human liver 70 kDa peroxisomal membrane protein (hPMP70) was determined. The nucleotide sequence contains an open reading frame of 1977 base pairs and encodes an amino acid sequence of 659 residues which exhibits 95.0% identity with that of rat liver PMP70. hPMP70 shares close similarity to the members of a superfamily of ATP-binding transport proteins.
Assuntos
Proteínas de Membrana/genética , Microcorpos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , DNA/genética , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Alinhamento de SequênciaRESUMO
Mitochondrial trifunctional protein is a newly identified enzyme involved in mitochondrial fatty acid beta-oxidation harbouring long-chain enoyl-CoA hydratase, long-chain 3-hydroxyacyl-CoA dehydrogenase and long-chain 3-ketothiolase activity. Over the last few years, we identified more than 26 patients with a deficiency in long-chain 3-hydroxyacyl-CoA dehydrogenase. In order to identify the molecular basis for the deficiency found in these patients, we sequenced the cDNAs encoding the alpha- and beta-subunits which revealed one G-->C mutation at nucleotide position 1528 in the 3-hydroxyacyl-CoA dehydrogenase encoding region of the alpha-subunit. The single base change results in the substitution of a glutamate for a glutamine at amino acid position 510. The base substitution creates a PstI restriction site. Using RFLP, we found that in 24 out of 26 unrelated patients only the C1528 was expressed. The other two patients were heterozygous for this mutation. This mutation was not found in 55 different control subjects. This indicates a high frequency for this mutation in long-chain 3-hydroxyacyl-CoA dehydrogenase deficient patients.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/deficiência , Mitocôndrias/enzimologia , 3-Hidroxiacil-CoA Desidrogenases/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/química , Ácidos Graxos/metabolismo , Humanos , 3-Hidroxiacil-CoA Desidrogenase de Cadeia Longa , Dados de Sequência Molecular , Mutação , Polimorfismo de Fragmento de Restrição , Alinhamento de SequênciaRESUMO
PURPOSE: The tissue oxygenation level, which is theoretically governed by distance from blood vessels, is one of the most important modulators of the radiosensitivity of carcinoma. A computed image analysis system for the detection of tissue oxygenation was developed to establish a method of predicting radiosensitivity in early-stage laryngeal carcinoma treated by curative radiotherapy. EXPERIMENTAL DESIGN: Microvessel structures labeled with CD31 antigen were investigated in 55 patients undergoing curative radiotherapy for T1 and T2 laryngeal carcinoma. We calculated (a) microvessel density [(MVD) vessels/field] under a microscope; (b) the ratio of the total microvessel number (TN):tumor area (TA) [TN:TA; vessels/mm2]; (c) the ratio of the total microvessel perimeter (TP):TA (TP:TA; mm/mm2); and (d) the ratio of tumor tissue area >150 microm from microvessels (hypoxic ratio; %) as parameters of tissue oxygenation in each whole biopsy specimen by using an image analyzer. We compared each of these factors with radiosensitivity. RESULTS: Mann-Whitney's U test revealed that tumors with a high MVD (median, 42 vessels/field), high TN:TA ratio (median=40.9 vessels/mm2), high TP:TA ratio (median, 2.92 mm/mm2), and low hypoxic ratio (median, 30.3%) had significantly greater radiosensitivity than tumors with a low MVD, low TN:TA ratio, low TP:TA ratio or high hypoxic ratio (P = 0.002, P = 0.0004, P < 0.0001, and P = 0.004, respectively). CONCLUSIONS: Prediction of radiosensitivity on the basis of the TP:TA ratio can be used as an efficient means of avoiding ineffective radiation, complications after salvage surgery, and prolonged hospital stays.
Assuntos
Vasos Sanguíneos/patologia , Neoplasias Laríngeas/radioterapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Relação Dose-Resposta à Radiação , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Neoplasias Laríngeas/irrigação sanguínea , Neoplasias Laríngeas/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Dosagem Radioterapêutica , Estatística como AssuntoRESUMO
Curative radiotherapy is the first choice of therapy for T1 and T2 stage laryngeal squamous cell carcinoma (LSCC) patients to preserve their phonation. Patients with recurrent tumors who undergo salvage surgery require prolonged nasal feeding. Therefore, clinical interest has been focused on elucidating a predictive factor indicating which tumors are likely to be radiosensitive before radiotherapy. We analyzed the relations between radiosensitivity and clinicopathological factors (gender, tumor location, histological factors, and clinical tumor-node-metastasis stage), expression of apoptosis-related proteins (p53, bax, bcl-2), apoptotic index using the terminal deoxynucleotidyltransferase-mediated nick end labeling method, expression of cell proliferation-related proteins (Ki-67-labeling index and epidermal growth factor receptor overexpression) and microvessel density (MVD, vessels/field = 0.391 mm2) in biopsy specimens from 31 LSCC patients given radiotherapy (total radiotherapy dose of 52-70 Gy over 4-6.5 weeks). Univariate analysis revealed that tumors with a high MVD (> or =35 vessels/field) showed better radiosensitivity than those with a low MVD (<35 vessels/field, P = 0.008) and that a high Ki-67-labeling index (> or =40%) was weakly associated with radiosensitivity (P = 0.056). Multivariate analysis and Kaplan-Meier analysis showed that MVD alone had significant predictive power for radiosensitivity in T1 and T2 stage LSCCs after radiotherapy (P = 0.012, 0.0003, respectively). No significant association between clinicopathological factors, or of overexpression of p53, bax, bcl-2, epidermal growth factor receptor, or apoptotic index, with radiosensitivity was found. These results indicate that MVD is a potentially useful clinical factor predicting radiosensitivity for patients with early stage LSCCs before treatment.
Assuntos
Carcinoma de Células Escamosas/irrigação sanguínea , Carcinoma de Células Escamosas/radioterapia , Neoplasias Laríngeas/irrigação sanguínea , Neoplasias Laríngeas/radioterapia , Neovascularização Patológica/fisiopatologia , Tolerância a Radiação/fisiologia , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/biossíntese , Antígenos de Diferenciação Mielomonocítica/biossíntese , Apoptose/fisiologia , Carcinoma de Células Escamosas/metabolismo , Receptores ErbB/biossíntese , Feminino , Humanos , Antígeno Ki-67/biossíntese , Neoplasias Laríngeas/metabolismo , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Análise de Sobrevida , Proteína Supressora de Tumor p53/biossíntese , Proteína X Associada a bcl-2RESUMO
We examined the effects of granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), and thrombopoietin (TPO), alone or in combination, on the generation of neutrophils by bone marrow (BM) cells from three patients with severe congenital neutropenia (SCN) through the use of a serum-deprived liquid culture system. Synergistic effects of G-CSF and SCF on the neutrophil production by BM CD34+CD38+c-kit+ cells were observed in SCN patients as well as in normal controls. The addition of TPO to the culture containing G-CSF and SCF further augmented the growth of neutrophils in the two groups. Single-cell culture experiments revealed that the three-factor combination caused increases in both the number and size of neutrophil colonies compared with G-CSF + SCF in normal BM cells, whereas only a significant increment in the colony size was observed in SCN patients. Even in the presence of SCF or SCF + TPO, the concentrations of G-CSF necessary for the substantial production of neutrophils by CD34+CD38+c-kit+ cells were higher in two patients compared with the levels obtained by normal control cells. In addition, TPO did not accelerate the maturation of neutrophilic cells supported by G-CSF + SCF. When BM CD34+CD38-c-kit+ cells were targeted, the addition of TPO to the culture containing G-CSF and SCF was required for significant neutrophil colony growth in the two groups. These results suggest that TPO enhances the G-CSF-dependent neutrophil production with the aid of SCF in this disorder.
Assuntos
Antígenos CD , Medula Óssea/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Neutropenia/patologia , Neutrófilos/patologia , Fator de Células-Tronco/farmacologia , Trombopoetina/farmacologia , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Antígenos CD34/análise , Antígenos de Diferenciação/análise , Apoptose , Medula Óssea/patologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Pré-Escolar , Ensaio de Unidades Formadoras de Colônias , Meios de Cultura Livres de Soro , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Células-Tronco Hematopoéticas/patologia , Humanos , Lactente , Masculino , Glicoproteínas de Membrana , NAD+ Nucleosidase/análise , Neutropenia/tratamento farmacológico , Proteínas Proto-Oncogênicas c-kit/análise , Proteínas Proto-Oncogênicas c-kit/efeitos dos fármacos , Fator de Células-Tronco/administração & dosagem , Trombopoetina/administração & dosagemRESUMO
We investigated the properties of granulocyte-macrophage (GM) progenitors obtained from patients with juvenile chronic myelogenous leukemia (JCML). CD34+ bone marrow cells from a patient with JCML, unlike normal bone marrow cells, generated a large number of cells in serum-containing liquid culture without additional hematopoietic factors. In serum-deprived culture, only granulocyte colony-stimulating factor (G-CSF) had a modest stimulatory effect on GM colony growth in normal controls. In contrast, stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3), as well as G-CSF, when tested individually, generated significant numbers of GM colonies in some JCML patients. All two-factor combinations generated significantly more GM colonies in JCML compared with normal controls. In particular, GM-CSF plus SCF exerted an interaction equivalent to the all-factor combination in most patients. Significant differences in the size and constituent cells of GM colonies stimulated by GM-CSF plus SCF were also observed. These results suggest that one possible mechanism for the excessive cell production in JCML is the strong proliferation of GM progenitors induced by hematopoietic factors, especially SCF. According to immunofluorescent analysis, however, it is unlikely that this multiplication is due to an increase in the cell surface expression of c-kit receptors on JCML progenitors.
Assuntos
Fatores de Crescimento de Células Hematopoéticas/farmacologia , Células-Tronco Hematopoéticas/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Antígenos CD34 , Medula Óssea/patologia , Divisão Celular/efeitos dos fármacos , Pré-Escolar , Meios de Cultura Livres de Soro/farmacologia , Sinergismo Farmacológico , Feminino , Sangue Fetal/fisiologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Lactente , Interleucina-3/farmacologia , Masculino , Proteínas de Neoplasias/biossíntese , Proteínas Proto-Oncogênicas c-kit/biossíntese , Proteínas Recombinantes/farmacologia , Fator de Células-Tronco/farmacologia , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
Although CD133 has been considered to be a molecular marker for cancer stem cells, its functional roles in tumorigenesis remain unclear. We here examined the molecular basis behind CD133-mediated signaling. Knockdown of CD133 resulted in the retardation of xenograft tumor growth of colon cancer-derived HT-29 and LoVo cells accompanied by hypophosphorylation of AKT, which diminished ß-catenin/T-cell factor-mediated CD44 expression. As tyrosine residues of CD133 at positions 828 and 852 were phosphorylated in HT-29 and SW480 cells, we further addressed the significance of this phosphorylation in the tumorigenesis of SW480 cells expressing mutant CD133, with substitution of these tyrosine residues by glutamate (CD133-EE) or phenylalanine (CD133-FF). Forced expression of CD133-EE promoted much more aggressive xenograft tumor growth relative to wild-type CD133-expressing cells accompanied by hyperphosphorylation of AKT; however, CD133-FF expression had negligible effects on AKT phosphorylation and xenograft tumor formation. Intriguingly, the tyrosine phosphorylation status of CD133 was closely linked to the growth of SW480-derived spheroids. Using yeast two-hybrid screening, we finally identified receptor-type protein tyrosine phosphatase κ (PTPRK) as a binding partner of CD133. In vitro studies demonstrated that PTPRK associates with the carboxyl-terminal region of CD133 through its intracellular phosphatase domains and also catalyzes dephosphorylation of CD133 at tyrosine-828/tyrosine-852. Silencing of PTPRK elevated the tyrosine phosphorylation of CD133, whereas forced expression of PTPRK reduced its phosphorylation level markedly and abrogated CD133-mediated AKT phosphorylation. Endogenous CD133 expression was also closely associated with higher AKT phosphorylation in primary colon cancer cells, and ectopic expression of CD133 enhanced AKT phosphorylation. Furthermore, lower PTPRK expression significantly correlated with the poor prognosis of colon cancer patients with high expression of CD133. Thus, our present findings strongly indicate that the tyrosine phosphorylation of CD133, which is dephosphorylated by PTPRK, regulates AKT signaling and has a critical role in colon cancer progression.
Assuntos
Antígenos CD/metabolismo , Neoplasias do Colo/metabolismo , Glicoproteínas/metabolismo , Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Antígeno AC133 , Animais , Células CACO-2 , Proliferação de Células/fisiologia , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Células HT29 , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fosforilação , Transdução de Sinais , beta Catenina/metabolismoRESUMO
At least three different sizes of GH-1 gene deletions (approximately 6.7, 7.0 and 7.6 kilobases) have been detected by Southern blot analysis of DNA from individuals with familial isolated GH deficiency type IA (IGHD1A). It is likely that these deletions result from unequal crossing over events between homologous regions that flank the GH-1 gene. Heterogeneity in clinical phenotypes is suggested by reports of good responses to exogenous GH treatment in most IGHD1A subjects with 7.6 kilobase deletions as opposed to poor responses in many subjects with smaller deletions. To determine if characteristic differences in gene deletions could be detected that correlate with response to treatment we analyzed the DNA sequences that normally flank the GH-1 gene. Digestion patterns of the PCR amplification products of these sequences from DNA of IGHD type IA patients with the restriction endonucleases BglI, HaeII, or SmaI showed characteristic differences for each of the three deletion sizes studied. The location and size of all deletions agreed with previous size estimates based on Southern blot analysis. Interestingly, clinical differences observed in the development of high titers of anti-GH antibodies and poor growth responses after GH treatment are unexplained, since discordant outcomes were observed in patients who had deletions of the same size and approximate location.