RESUMEN

Gastrointestinal stromal tumours (GISTs) with deletions in KIT exon 11 are characterized by higher proliferation rates and shorter disease-free survival times, compared to GISTs with KIT exon 11 point mutations. Up-regulation of cyclin D is a crucial event for entry into the G1 phase of the cell cycle, and links mitogenic signalling to cell proliferation. Signalling from activated KIT to cyclin D is directed through the RAS/RAF/ERK, PI3K/AKT/mTOR/EIF4E, and JAK/STATs cascades. ERK and STATs initiate mRNA transcription of cyclin D, whereas EIF4E activation leads to increased translation efficiency and reduced degradation of cyclin D protein. The aim of the current study was to analyse the mRNA and protein expression as well as protein phosphorylation of central hubs of these signalling cascades in primary GISTs, to evaluate whether tumours with KIT exon 11 deletions and point mutations differently utilize these pathways. GISTs with KIT exon 11 deletions had significantly higher mitotic counts, higher proliferation rates, and shorter disease-free survival times. In line with this, they had significantly higher expression of cyclin D on the mRNA and protein level. Furthermore, there was a significantly higher amount of phosphorylated ERK1/2, and a higher protein amount of STAT3, mTOR, and EIF4E. PI3K and phosphorylated AKT were also up-regulated, but this was not significant. Ultimately, GISTs with KIT exon 11 deletions had significantly higher phosphorylation of the central negative cell-cycle regulator RB. Phosphorylation of RB is accomplished by activated cyclin D/CDK4/6 complex, and marks a central event in the release of the cell cycle. Altogether, these observations suggest increased KIT signalling with up-regulation of cyclin D as the basis for the unfavourable clinical course in GISTs with KIT exon 11 deletions.

Asunto(s)

Ciclinas/genética , Tumores del Estroma Gastrointestinal/genética , Proteínas Proto-Oncogénicas c-kit/genética , Regulación hacia Arriba , Anciano , Anciano de 80 o más Años , Proliferación Celular , Ciclina D , Ciclinas/metabolismo , Supervivencia sin Enfermedad , Exones , Femenino , Tumores del Estroma Gastrointestinal/mortalidad , Tumores del Estroma Gastrointestinal/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Análisis por Matrices de Proteínas , Proteínas Proto-Oncogénicas c-kit/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Eliminación de Secuencia , Transducción de Señal/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/mortalidad , Neoplasias Gástricas/patología

RESUMEN

Loss of chromosome 9p is a reliable predictor of malignant behaviour in gastrointestinal stromal tumours (GISTs). p16INK4A located at 9p21 inhibits the CDK4/6/cyclin D complex from phosphorylating RB. Phosphorylation of RB through CDK4/6/cyclin D in early G(1) phase frees the transcription factor E2F1 from RB and enables mRNA transcription of genes essential for G(1)/S phase transition. This study aims to determine the impact of 9p loss on mRNA and protein expression of p16INK4A and further key cell cycle regulators in the different phases of the cell cycle. Sixty primary GISTs previously characterized for 9p loss by comparative genomic hybridization were analysed for mRNA expression of p16INK4A, p15INK4B, CDK4, CDK6, cyclin D, p21CIP1p27KIP1, CDK2, cyclin E, cyclin B, RB and E2F1, using quantitative RT-PCR. The protein expression of CDK6, CDK2, p21CIP1, p27KIP1 and phosphorylated RB (S807/S811) was evaluated using protein arrays as a novel and highly sensitive platform for profiling of protein abundance and protein phosphorylation. In parallel, the nuclear percentages of immunohistochemical staining for p16INK4A, cyclin D, E2F1 and RB were quantified on a tissue microarray. GISTs with 9p loss had significantly higher proliferation rates, higher metastatic behaviour and shorter disease-free survival. On the molecular level, GISTs with 9p loss had a significantly reduced mRNA as well as nuclear protein expression of p16INK4A. RB was significantly more phosphorylated in these tumours, together with increased mRNA expression and nuclear staining for E2F1. Furthermore, GISTs with 9p loss had up-regulation of the late G1/S phase promoters CDK2 and cyclin E. We conclude that loss of 9p accompanied by early G1 phase inhibitor p16(INK4A) down-regulation in GISTs facilitates phosphorylation of RB, enabling E2F1-dependent transcription of genes essential for late G1/S phase transition. This study provides a possible basis for the accelerated proliferation and particularly malignant behaviour in GISTs with 9p loss.

Asunto(s)

Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Tumores del Estroma Gastrointestinal/genética , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Proteínas de Ciclo Celular/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/análisis , Progresión de la Enfermedad , Factor de Transcripción E2F1/análisis , Factor de Transcripción E2F1/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Genes de Retinoblastoma , Humanos , Inmunohistoquímica , Análisis de Secuencia por Matrices de Oligonucleótidos , Modelos de Riesgos Proporcionales , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos

RESUMEN

We describe the newly established cell line CS-99 derived from a uterine carcinosarcoma retaining features of the sarcomatous phenotype in vitro. CS-99 cells exhibit a mesenchymal morphology with predominantly spindle-shaped cells at nonconfluence turning to pleomorphic appearance at confluence. The mesenchymal phenotype was evidenced immunohistochemically by strong vimentin and moderate SM-actin, which was similar to the sarcomatous component of the primary tumor. P53 was overexpressed in a subset of CS-99 cells. Epithelial membrane antigen was moderately expressed whereas other markers including pan CK, CK 5/6, CK 34, epidermal growth factor receptor, desmin, carcinoembryonic antigen, S100, KIT, ERBB2, and the hormone receptors, estrogen receptor and progesterone receptor revealed either weak or no specific staining in CS-99 cells. High self-renewal capacity corresponded to the population doubling time of 23 h in high passage. CS-99 cells were able to develop three-dimensional tumor spheroids in vitro. Cytogenetic analysis and multicolor fluorescence in situ hybridization of CS-99 demonstrated an almost stable karyotype including numerical changes +8, +18, and +20 and translocations, amongst others der(1)t(1;2), der(1)t(1;7), der(2)t(2;19), der(5)t(5;8), and der(5)t(5;14). Taken together, the cell line CS-99 exhibits strong growths dynamics and a complex but stable karyotype in higher passages, and can be further a useful in vitro model system for studying tumor biology of carcinosarcomas.

Asunto(s)

Carcinosarcoma/patología , Línea Celular Tumoral/patología , Neoplasias Uterinas/patología , Anciano , Carcinosarcoma/genética , Femenino , Humanos , Fenotipo , Neoplasias Uterinas/genética

RESUMEN

To model the cytogenetic evolution in gastrointestinal stromal tumour (GIST), an oncogenetic tree model was reconstructed using comparative genomic hybridization data from 203 primary GISTs (116 gastric and 87 intestinal GISTs, including 151 newly analysed cases), with follow-up available in 173 cases (mean 40 months; maximum 133 months). The oncogenetic tree model identified three major cytogenetic pathways: one initiated by -14q, one by -1p, and another by -22q. The -14q pathway mainly characterized gastric tumours with predominantly stable karyotypes and more favourable clinical course. On the other hand, the -1p pathway was more characteristic of intestinal GISTs, with an increased capacity for cytogenetic complexity and more aggressive clinical course. Loss of 22q, more closely associated with -1p than -14q, appeared to initiate the critical transition to an unfavourable cytogenetic subpathway. This -22q pathway included accumulation of +8q, -9p, and -9q, which could all predict disease-free survival in addition to tumour site. Thus, insights into the cytogenetic evolution obtained from oncogenetic tree models may eventually help to gain a better understanding of the heterogeneous site-dependent biological behaviour of GISTs.

Asunto(s)

Tumores del Estroma Gastrointestinal/genética , Modelos Genéticos , Aberraciones Cromosómicas , Deleción Cromosómica , Cromosomas Humanos Par 1/genética , Cromosomas Humanos Par 14/genética , Cromosomas Humanos Par 22/genética , Análisis Citogenético , Tumores del Estroma Gastrointestinal/cirugía , Humanos , Neoplasias Intestinales/genética , Cariotipificación , Funciones de Verosimilitud , Metástasis de la Neoplasia/genética , Recurrencia Local de Neoplasia/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Pronóstico , Neoplasias Gástricas/genética , Factores de Tiempo

RESUMEN

Residues of pharmaceutical antibiotics are found in the environment, whose fate and effects are governed by sorption. Thus, the extent and mechanisms of the soil sorption of p-aminobenzoic acid and five sulfonamide antibiotics (sulfanilamide, sulfadimidine, sulfadiazine, sulfadimethoxine, and sulfapyridine) were investigated using topsoils of fertilized and unfertilized Chernozem and their organic-mineral particle-size fractions. Freundlich adsorption coefficients (K(f)) ranged from 0.5 to 6.5. Adsorption increased with aromaticity and electronegativity of functional groups attached to the sulfonyl-phenylamine core. Adsorption to soil and particle-size fractions increased in the sequence: coarse silt < whole soil < medium silt < sand < clay < fine silt and was influenced by pH. Sorption nonlinearity (1/n

Asunto(s)

Antiinfecciosos/química , Modelos Teóricos , Contaminantes del Suelo/análisis , Sulfonamidas/química , Adsorción , Antiinfecciosos/análisis , Monitoreo del Ambiente , Tamaño de la Partícula , Sulfonamidas/análisis

RESUMEN

Nongestational choriocarcinomas are rare tumors. In the gastrointestinal tract, they are characterized by a biphasic tumor growth with separated areas of adenocarcinomatous and choriocarcinomatous differentiation. We here report a case of a combined adenocarcinoma-choriocarcinoma of the rectum. The tumor showed an aggressive clinical behavior with metastasis to the liver and lungs. A transient partial remission was achieved after 4 cycles of cisplatinum, etoposide, and ifosfamide chemotherapy, with normalization of serum beta-human chorionic gonadotropin levels. At this time, viable residual choriocarcinoma cells were found in surgically resected lung metastasis. The patient succumbed 8 months after initial diagnosis to a rapid abdominal relapse. We used comparative genomic hybridization (CGH) and fluorescence in situ hybridization to elucidate the genetic relationship of adenocarcinoma and choriocarcinoma in this neoplasm. We found genetic changes characteristic for colorectal adenocarcinomas, a loss of chromosomal regions 8p21-pter as well as 18q21-pter, and a gain of 5p and 20q, in both tumor parts. This provides evidence for the common origin of both components. A differential pattern of additional genetic changes suggests a clonal evolution from a common ancestor cell. In contrast to findings from a comparative study on a choriocarcinoma of the renal pelvis, we did not find an amplification of the germ cell cancer-associated chromosomal region 12p11.2-p12.1 in the areas of choriocarcinoma but found instead a loss of Xp11.3-pter. To our knowledge, this is the first report of a CGH comparison of the adenocarcinomatous and choriocarcinomatous tumor parts in a nongestational choriocarcinoma of the gastrointestinal tract.

Asunto(s)

Adenocarcinoma/secundario , Transformación Celular Neoplásica , Coriocarcinoma no Gestacional/secundario , Neoplasias Primarias Múltiples/patología , Neoplasias del Recto/patología , Adenocarcinoma/genética , Adenocarcinoma/terapia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Coriocarcinoma no Gestacional/genética , Coriocarcinoma no Gestacional/terapia , Aberraciones Cromosómicas , ADN de Neoplasias/análisis , Resultado Fatal , Femenino , Humanos , Histerectomía , Hibridación Fluorescente in Situ , Persona de Mediana Edad , Neoplasias Primarias Múltiples/genética , Hibridación de Ácido Nucleico , Neoplasias del Recto/genética , Neoplasias del Recto/terapia , Recto/cirugía

RESUMEN

Nephroblastomas (Wilms' tumors) are curable with survival rates above 80%. Nevertheless, some tumors fail to respond to therapy and those patients have a poor prognosis. Prognostic factors for nephroblastomas have still not been satisfactorily explored. In an effort to unravel molecular markers for non-responding nephroblastomas, we investigated by means of immunohistochemistty the expression of heat-shock protein 70 (HSP70) in formalin-fixed and paraffin-embedded tissue samples from 32 children afflicted with nephroblastoma. The results were validated using real-time RT-PCR. HSP70 expression was confined to blastemal and epithelial components, while the tumor stroma was negative. HSP70 expression was greater, if the tumors had been chemotherapeutically treated prior to operation, indicating that cytostatic drugs induce HSP70. Furthermore, high HSP70 expression was confined to tumors from children who survived, whereas tumors from dead patients were negative or weakly-positive for HSP70. Though the number of cases analyzed was small, they provide an indication that HSP70 expression may be of prognostic value.

Asunto(s)

Biomarcadores de Tumor/análisis , Regulación Neoplásica de la Expresión Génica , Proteínas HSP70 de Choque Térmico/biosíntesis , Neoplasias Renales/metabolismo , Proteínas de Neoplasias/biosíntesis , Tumor de Wilms/metabolismo , Adolescente , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Niño , Preescolar , Terapia Combinada , Sistemas de Computación , Dactinomicina/administración & dosificación , Resistencia a Antineoplásicos , Células Epiteliales/química , Proteínas HSP70 de Choque Térmico/análisis , Proteínas HSP70 de Choque Térmico/genética , Humanos , Lactante , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/mortalidad , Neoplasias Renales/patología , Neoplasias Renales/cirugía , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/genética , Estadificación de Neoplasias , Células Madre Neoplásicas/química , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/química , Análisis de Supervivencia , Resultado del Tratamiento , Vincristina/administración & dosificación , Tumor de Wilms/tratamiento farmacológico , Tumor de Wilms/mortalidad , Tumor de Wilms/patología , Tumor de Wilms/cirugía

RESUMEN

The sorption mechanisms and complex formation between humic acid (HA) and a successively increasing number of diethyl phthalate (DEP) molecules have been studied theoretically using molecular mechanics, the number (n) of DEP molecules being varied from 1 to 30. The energy components of the single HA x nDEP complexes have been used as explanatory variables in a principal component analysis for exploring the presence of similarities/dissimilarities in the energetic properties of the individual xenobiotic complexes. The sorption can be explained in terms of a two-step mechanism. Absorption takes place as long as the host humic acid structure offers (a) enough internal docking space and (b) favorable interactions (energy release) with the guest molecule. This takes place for up to 7 DEP molecules. Further increase in the number to 30 DEP molecules will, due to the lack of free available internal voids, lead to surface controlled adsorption. The two-step sorption process apparently results in (a) a linear increase in energy gain by DEP bonds, and similarly (b) a constant incremental rise in molecular properties of the complexes such as volume and surface area. Three outstanding observations emerge: (1) Structural features at the atomic level (nanochemistry), such as partial atomic charges and high aromaticity of the humic acid, are observed to be dominating the intermolecular interactions in the complexes at the specific sorption sites. (2) Torsional relief and favorable changes in bonding energy also prevail for the growing complex. The latter indicates both the structural flexibility of the HA host and the stabilizing effect of DEP on the complex, by filling of the voids within the HA molecule. (3) The intermolecular forces are described mainly by hydrogen bonds (electrostatic energy) and interactions between dipole-dipole, such as carboxylic functions and uncharged moieties such as aromatic rings (van der Waals energy).

Asunto(s)

Sustancias Húmicas/química , Modelos Moleculares , Ácidos Ftálicos/química , Absorción , Adsorción , Contaminantes Ambientales , Cinética , Relación Estructura-Actividad

RESUMEN

The two known estrogen receptors, ERalpha and ERbeta, mediate the effects of estrogen in all target tissues, including blood vessels. We have shown previously that estrogen inhibits vascular injury response to the same extent in female wild-type (WT), ERalpha knockout (ERalphaKO(CH)), and ERbeta knockout (ERbetaKO(CH)) mice. We generated mice harboring disruptions of both ERalpha and ERbeta genes (ERalpha,betaKO(CH)) by breeding and studied the effect of 17beta-estradiol (E2) on vascular injury responses in ovariectomized female ERalpha,betaKO(CH) mice and WT littermates. E2 inhibited increases in vascular medial area following injury in the WT mice but not in the ERalpha,betaKO(CH) mice, demonstrating for the first time that the two known estrogen receptors are necessary and sufficient to mediate estrogen inhibition of a component of the vascular injury response. Surprisingly, as in WT littermates, E2 still significantly increased uterine weight and inhibited vascular smooth muscle cell (VSMC) proliferation following injury in the ERalpha,betaKO(CH) mice. These data support that the role of estrogen receptors differs for specific components of the vascular injury response in the ERalpha,betaKO(CH) mice. The results leave unresolved whether E2 inhibition of VSMC proliferation in ERalpha,betaKO(CH) mice is caused by a receptor-independent mechanism, an unidentified receptor responsive to estrogen, or residual activity of the ERalpha splice variant reported previously in the parental ERalphaKO(CH) mice. These possibilities may be resolved by studies of mice in which ERalpha has been fully disrupted (ERalphaKO(St)), which are in progress.

Asunto(s)

Traumatismos de las Arterias Carótidas/tratamiento farmacológico , Estrógenos/uso terapéutico , Receptores de Estrógenos/genética , Animales , Bromodesoxiuridina/metabolismo , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/genética , División Celular/efectos de los fármacos , Estradiol/uso terapéutico , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Femenino , Genotipo , Ratones , Ratones Noqueados , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Ovariectomía

RESUMEN

To fully characterize the numerous chromosomal aberrations in two human squamous cell carcinomas (SCCs) of the lung, molecular cytogenetic characterization was carried out utilizing conventional banding analysis and multicolor fluorescence in situ hybridization (mFISH), providing simultaneous color discrimination of all 24 human chromosomes. Both tumors displayed complex aneuploid karyotypes with a host of numerical and structural chromosome abnormalities. Structural aberrations common to both SCCs included rearrangements of chromosomes 1, 3p, 7q, and 8q, contributing to net loss of chromosomal sequences on 1p, 3p, and 8p, and a net gain of 8q. The recently introduced mFISH technique enabled the disclosure of cryptic translocations and the chromosomal composition of previously unrecognized marker chromosomes. Furthermore, mFISH greatly enhanced the ability to delineate chromosomal breakpoints when integrating banding information from conventional banding analysis. Eventually, the application of mFISH as a powerful approach to refine complex tumor karyotypes is expected to result in a more detailed and complete picture of cytogenetic events associated with the development and progression of solid tumors.

Asunto(s)

Carcinoma de Células Escamosas/genética , Aberraciones Cromosómicas , Pintura Cromosómica , Neoplasias Pulmonares/genética , Aneuploidia , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/cirugía , Bandeo Cromosómico , Fragilidad Cromosómica , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/cirugía , Masculino

RESUMEN

The testis-specific proacrosin gene encodes for a fertilization-promoting protein. In mouse and rat it is first transcribed in late pachytene spermatocytes and revealed to be translationally regulated. Former proacrosin promoter studies demonstrated that elements necessary for conducting a stage and temporal-specific expression of the gene are located within 0.9 kb upstream of the translational start codon. In the present study we analyzed putative cis-acting elements located in this promoter region for their specific binding properties to nuclear factors assumed to be involved in proacrosin gene regulation. Supplement of specific antibodies in electrophoretic mobility shift assays (EMSA) revealed that two Y-box proteins and the transcription factors CREM and YY1 interact with proacrosin promoter elements. The Y-box proteins, antigenically related to the frog Y-box proteins FRGY1 and FRGY2, bound to the Y-box (55-66 bp upstream of the ATG initiation codon) in brain and testis nuclear extracts, respectively. CREM bound to three elements (30-37, 252-259, and 717-724 bp upstream of ATG). The ubiquitous transcription factor YY1 bound to a conserved element in the central proacrosin promoter (457-473 bp upstream of ATG) and showed almost germ cell-specific truncates in EMSA. These results suggest that the identified factors are involved in proacrosin gene regulation.

Asunto(s)

Acrosina/genética , Precursores Enzimáticos/genética , Células Germinativas/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Represoras , Animales , Secuencia de Bases , Sitios de Unión , Modulador del Elemento de Respuesta al AMP Cíclico , ADN/genética , ADN/metabolismo , Huella de ADN , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Factores de Unión al ADN Específico de las Células Eritroides , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Especificidad de Órganos , Ratas , Elementos de Respuesta/genética , Factores de Transcripción/metabolismo , Factor de Transcripción YY1

RESUMEN

SUMMARY: The present study provides evidence that chronic intake of a high-fat diet induces a dramatic extravasation of immunoglobulins, indicating alterations in blood-brain barrier (BBB) functioning, in the brains of apolipoprotein E (apoE)-knockout mice, but not of C57Bl/6 control mice. Using sodium fluorescein as a marker for the permeability of the BBB, we found additional support for age-related disturbances of BBB function in apoE-knockout mice. Behavioral analysis of apoE-knockout mice compared with C57Bl/6 mice indicated that they were also less efficient in acquiring the spatial Morris water maze task. Furthermore, apoE-knockout mice are known to develop severe atherosclerosis, which is exacerbated with a high-fat diet. We therefore compared the apoE-knockout mice with the apoE3-Leiden transgenic mice, which are known to develop atherosclerosis. However, apoE3-Leiden mice that were kept on a high-fat, high-cholesterol diet and that developed atherosclerosis to an extent similar to the apoE-knockout mice, showed no signs of BBB disturbances. These results indicate for the first time that apoE plays an essential role in the maintenance of the integrity of the BBB during aging and that it protects the brain from neuropathology induced by a high-fat diet. We therefore hypothesize that the role of apoE in the maintenance of the integrity of the BBB may be the mechanism by which apoE affects the progression of neurodegeneration, as seen in Alzheimer's disease.

Asunto(s)

Envejecimiento/fisiología , Apolipoproteínas E/fisiología , Barrera Hematoencefálica , Grasas de la Dieta/administración & dosificación , Animales , Apolipoproteínas E/genética , Inmunohistoquímica , Ratones , Ratones Noqueados , Ratones Transgénicos

RESUMEN

Nephroblastomas (Wilms' tumors) are curable with survival rates above 80%. Some tumors, however, fail to respond to therapy and those patients have a poor prognosis. In a search for prognostic markers, we investigated the expression of the multidrug resistance-related protein 1 (MRP1) in 32 nephroblastomas by means of immunohistochemistry. The immunohistochemical results were validated with a real-time RT-PCR technique. MRP1 expression was heterogeneous and predominantly found in the blastemal and epithelial compartments compared to the stromal elements of nephroblastomas. We found significant relationships of MRP1 expression to survival of patients and to expression of p53, HSP70, and LRP/MVP. The relationship between MRP1 and p53 expression is a clue that the transcriptional control of MRP1 by p53 reported for other tumor types may also take place in nephroblastomas. The correlation of MRP1 to other drug resistance genes, e.g. HSP70 and LRP/MVP in nephroblastomas indicates that the co-expression of different drug resistance genes may be under a common regulation of still unknown transcription factors.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/genética , Neoplasias Renales/patología , Tumor de Wilms/patología , Transportadoras de Casetes de Unión a ATP/análisis , Resistencia a Múltiples Medicamentos , Regulación Neoplásica de la Expresión Génica , Proteínas HSP70 de Choque Térmico/análisis , Humanos , Inmunohistoquímica , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Proteínas de Neoplasias/análisis , Estadificación de Neoplasias , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Supervivencia , Proteína p53 Supresora de Tumor/análisis , Partículas Ribonucleoproteicas en Bóveda , Tumor de Wilms/genética , Tumor de Wilms/metabolismo

RESUMEN

Nephroblastomas (Wilms' tumors) are curable with survival rates above 80%. Some tumors, however, fail to respond to therapy and those patients have a poor prognosis. In a search for molecular markers of drug resistance, we investigated the expression of lung resistance protein (LRP) in tissue samples from 32 children with nephroblastoma by means of immunohistochemistry. LRP is a human major vault protein (MVP) and is associated with multidrug resistance of tumors. LRP/MVP expression was found in the blastemal and epithelial compartments but to a significantly lesser extent in the stromal compartment of Wilms' tumors. Expression was generally heterogeneous with respect to staining intensity and percentage of positive cells. We found significant relationships between LRP/MVP expression and chemotherapeutic pre-treatment of tumors and tumor stage. The immunohistochemical results were validated with a real-time RT-PCR technique and a significant association between protein and mRNA expression was observed.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Neoplasias Renales/metabolismo , Proteínas de Neoplasias/metabolismo , Partículas Ribonucleoproteicas en Bóveda/metabolismo , Tumor de Wilms/metabolismo , Antibióticos Antineoplásicos/uso terapéutico , Antineoplásicos Fitogénicos/uso terapéutico , Biomarcadores de Tumor/genética , Niño , Preescolar , Cartilla de ADN/química , Dactinomicina/uso terapéutico , Resistencia a Múltiples Medicamentos , Humanos , Técnicas para Inmunoenzimas , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/patología , Proteínas de Neoplasias/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Partículas Ribonucleoproteicas en Bóveda/genética , Vincristina/uso terapéutico , Tumor de Wilms/tratamiento farmacológico , Tumor de Wilms/patología , Microglobulina beta-2/metabolismo

RESUMEN

Adsorption and desorption isotherms of the herbicides imazapyr, imazethapyr and imazaquin on a soil humic acid have been performed at pH 2.8 and 4.0 (below and above the pKa of the herbicides). At both pH, adsorption increased according to the lipophilic character of the molecules (imazapyr < imazethapyr << imazaquin). The extent of adsorption was higher at pH 2.8 than at pH 4.0 because of the partial ionization of the carboxylic groups of both herbicides and humic acids at increasing pH. Desorption of imazapyr and imazethapyr was nearly complete at pH 4 and higher than 60% at pH 2.8 while desorption of imazaquin was 45 and 8% at pH 4 and 2.8, respectively. No differences between adsorption isotherms at 10 degrees C and 25 degrees C were observed a pH 4.0 indicating that adsorption involved very weak bonds while at pH 2.8, adsorption was higher at 10 degrees C than at 25 degrees C indicating an exothermic process. The isosteric enthalpy of adsorption of each herbicide was low (about -1 kJoule mole(-1)) suggesting that low energetic bonds were involved. Adsorption on different humic acids has indicated that for each herbicide, the extent of adsorption expressed as Kd was correlated with the amount of carboxylic and aromatic groups of humic acids suggesting that hydrogen bonding and/or charge-transfer complexes formation could take place. Molecular modeling and geometry optimization of humic acid and soil organic matter (SOM) herbicide complexes were also performed. The results obtained with this theoretical approach gave a consistent chemical interpretation of the experimental results. To the best of our knowledge this is the first report to contribute to a better understanding of site-specific bonding of herbicides in SOM complexes by nanochemical modeling and distinct energy descriptors.

Asunto(s)

Herbicidas/química , Sustancias Húmicas/metabolismo , Imidazoles/química , Niacina/análogos & derivados , Niacina/química , Ácidos Nicotínicos/química , Quinolinas/química , Contaminantes del Suelo , Suelo , Adsorción , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Suelo/análisis , Temperatura

RESUMEN

Clear-cell odontogenic carcinoma (CCOC) is a rare neoplasm with malignant potential and unknown cytogenetic alterations. We describe the case of a 43-year-old woman who presented with an unusual odontogenic epithelial tumor. Histologically, the tumor was composed of clear-cell areas and exhibited a squamous pattern with little nuclear pleomorphism similar to benign squamous odontogenic tumor. Multiple small pulmonary nodules occurring 3 years after primary surgical treatment histologically closely resembled benign minute pulmonary meningothelial-like nodules (MPMN) with clear-cell features. Comparative genomic hybridization (CGH) and immunohistochemistry, performed as diagnostic adjuncts, revealed in the odontogenic tumor and the pulmonary lesions a very similar pattern of chromosomal aberrations (loss of 9, gains of 14q, 19 and 20 in both, and additional loss of 6 in the odontogenic tumor) and the same pattern of expression (positive for cytokeratin 5, 6, 8, 19 and negative for cytokeratin 18, epithelial membrane antigen, and vimentin), differing from that of MPMN. These findings confirmed the final diagnosis of metastasizing CCOC with partial squamous differentiation, substantiated the unfavorable prognosis of the clear-cell component, and highlighted the diagnostic impact of CGH and immunohistochemistry for classification of these morphologically peculiar pulmonary CCOC metastases.

Asunto(s)

Adenocarcinoma de Células Claras/secundario , Ameloblastoma/secundario , Neoplasias Maxilomandibulares/patología , Neoplasias Pulmonares/secundario , Paraganglioma Extraadrenal/patología , Adenocarcinoma de Células Claras/química , Adenocarcinoma de Células Claras/genética , Adulto , Ameloblastoma/química , Ameloblastoma/genética , Aneuploidia , Biomarcadores de Tumor/química , ADN de Neoplasias/análisis , Diagnóstico Diferencial , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Neoplasias Maxilomandibulares/química , Neoplasias Maxilomandibulares/genética , Proteínas de Neoplasias/análisis , Hibridación de Ácido Nucleico , Radiografía Torácica , Tomografía Computarizada por Rayos X

RESUMEN

The human organic anion transporter 1 (hOAT1) plays a key role in the secretion of an array of potentially toxic organic anions including many clinically important drugs. Here we report on the genomic cloning of hOAT1. A human genomic library was used for screening of a PAC (P1 artificial chromosome) clone applying PCR techniques. Sequencing of several restriction subclones and of a PCR-generated clone revealed that the hOAT1 gene spans 8.2 kb and is composed of 10 exons divided by 9 introns. RT-PCR studies in a human kidney specimen led to the detection of two new splice variants, hOAT1-3 and hOAT1-4, showing a 132-bp in-frame deletion. Using fluorescence in situ hybridization (FISH) we mapped the hOAT1 gene as a single signal to chromosome 11q13.1-q13.2. Additionally, 600 bp of the 5' flanking region was analyzed, illustrating the probable transcription start site at nt -280, a NF-kappaB-site at nt -397 and several putative transcription factor binding sites.

Asunto(s)

Proteínas Portadoras/genética , Proteínas de Transporte de Anión , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 11 , ADN , Cartilla de ADN , Exones , Humanos , Hibridación Fluorescente in Situ , Intrones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

We report the successful fusion of human choriocarcinoma cells with normal human trophoblast cells to a choriocarcinoma/trophoblast hybrid. The hybrid cells ACH1P were derived from fusion of primary male trophoblast cells with the HGPRT-defective choriocarcinoma cell line AC1-1. The karyotypes of the parental choriocarcinoma cell line JEG-3, its HGPRT-defective mutant clones AC1-1, AC1-5, and AC1-9, and the choriocarcinoma/trophoblast hybrid ACH1P are presented, together with a detailed characterization of the AC1-specific chromosomal marker add(X)(q26) using conventional cytogenetic banding techniques and multiplex-fluorescence in situ hybridization (M-FISH). To our knowledge, this is the first report of a stably proliferating human cell hybrid of trophoblastic origin, providing a unique cell culture model to study trophoblast-related invasion and its underlying genetic mechanisms.

Asunto(s)

Coriocarcinoma/genética , Dermatoglifia del ADN , Células Híbridas , Cariotipificación , Trofoblastos , Neoplasias Uterinas/genética , Fusión Celular , Bandeo Cromosómico , Femenino , Humanos , Hibridación Fluorescente in Situ , Embarazo , Células Tumorales Cultivadas

RESUMEN

The proacrosin gene is specifically expressed in the testis and encodes an acrosomal enzyme. Previously, footprint analyses have shown binding of nuclear extracts from testis and brain to a highly conserved 17 bp motif (F1 element: 5'-AACTTCAAAATGGCTCC/T-3') located in the proacrosin promoter. By using this DNA-element as a target in a yeast one-hybrid assay, a cDNA fragment coding for the C-terminal part of the transcription factor YY1 was isolated. The binding of YY1 to this F1 element was confirmed by immunocompetition in EMSA. Because putative YY1 binding sites were also found in the promoters of other testis-specific genes, the YY1 transcription factor could play an important role in testicular gene expression.

Asunto(s)

Acrosina/genética , Proteínas de Unión al ADN/metabolismo , Precursores Enzimáticos/genética , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismo , Levaduras/genética , Animales , Anticuerpos/farmacología , Secuencia de Bases , Unión Competitiva/efectos de los fármacos , Secuencia de Consenso/genética , Sondas de ADN/genética , Sondas de ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Factores de Unión al ADN Específico de las Células Eritroides , Regulación de la Expresión Génica , Biblioteca de Genes , Masculino , Ratones , Proteínas Nucleares/metabolismo , Fragmentos de Péptidos/metabolismo , Análisis de Secuencia de ADN , Testículo/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/inmunología , Factor de Transcripción YY1

RESUMEN

The combination of pyrolysis (Py) with gas chromatography/mass spectrometry (GC/MS) is already well established for polymer analysis. A first approach is reported using this method for detailed quality monitoring of a complex technical polymer system. Six similar solvent-based paints (one standard and five modifications) have been used for evaluation. The major pyrolysis products were identified and qualitative and quantitative modifications were detected and specified. Principal component analysis (PCA) was applied for visualization of differences and similarities.