Role of transforming growth factor beta in cancer microenvironment

Transforming growth factor Beta (TGF-Beta) family members are polypeptidic cytokines with pleiotropic physiological properties. In relation to cancer, TGF-Beta exerts a dual tumour-suppressive and oncogenic effect, which is largely dependent on microenvironment stimuli. After activation of TGF-Beta signalling, two pathways can be activated: the canonical one through the mammalian Smad family or the non-canonical one activating, among others, the cellular mitogen-activated protein kinase (MAPK) signalling downstream, which interacts with Smad signalling. During tumorigenesis, cells of many cancer types often lose their response to the tumour-suppressive effects of TGF-Beta, which, in turn, has the opposite effect, acting as an autocrine tumour-promoting factor. In this review, we summarise the current knowledge about this intriguing cytokine, with special emphasis on its immunosuppressive actions (AU)

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Non-genomic action of resveratrol on androgen and oestrogen receptors in prostate cancer: modulation of the phosphoinositide 3-kinase pathway.

Prostate cancer represents a major concern in human oncology and the phytoalexin resveratrol (RES) inhibits growth and proliferation of prostate cancer cells through the induction of apoptosis. In addition, previous data indicate that in oestrogen-responsive human breast cancer cells, RES induces apoptosis by inhibition of the phosphoinositide-3-kinase (PI3K) pathway. Here, using androgen receptor (AR)-positive LNCaP and oestrogen receptor alpha (ERalpha)-expressing PC-3 prostate tumour cells, we have analysed whether the antiproliferative activity of RES takes place by inhibition of the AR- or ERalpha-dependent PI3K pathway. Although RES treatment (up to 150 microM) decreased AR and ERalpha protein levels, it did not affect AR and ERalpha interaction with p85-PI3K. Immunoprecipitation and kinase assays showed that RES inhibited AR- and ERalpha-dependent PI3K activities in LNCaP and PC-3, respectively. Consistently, lower PI3K activities correlated with decreased phosphorylation of downstream targets protein kinase B/AKT (PKB/AKT) and glycogen synthase kinase-3 (GSK-3). GSK-3 dephosphorylation could be responsible for the decreased cyclin D1 levels observed in both cell lines. Importantly, RES markedly decreased PKB/AKT phosphorylation in primary cultures from human prostate tumours, suggesting that the mechanism proposed here could take place in vivo. Thus, RES could have antitumoral activity in androgen-sensitive and androgen-non-sensitive human prostate tumours by inhibiting survival pathways such as that mediated by PI3K.

Rehospitalización de los traumatismos craneoencefálicos tras el alta de una Unidad de Neurorrehabilitación / Traumatic brain injuries rehospitalization after medical dischargefrom the neurorehabilitation unit

Objetivo. Conocer en nuestro medio la incidencia y causas de rehospitalización del traumatismo craneoencefálico (TCE) tras ser dado de alta hospitalaria de una Unidad de Neurorrehabilitación (UNR), identificando factores relacionados con los reingresos y la necesidad de acudir a los servicios de Urgencias tras dicha alta. Pacientes y método. Estudio de cohortes con 209 pacientes dados de alta hospitalaria de una UNR (enero 2000-diciembre 2002) con diagnóstico de TCE, realizando un seguimiento de 2 años. A través de historias clínicas se han obtenido datos demográficos del paciente, gravedad del TCE, lesiones asociadas, imágenes patológicas en tomografía axial computarizada, complicaciones hospitalarias, escalas de valoración, reingresos, necesidad de acudir a Urgencias y seguimiento especializado. Resultados. La incidencia de rehospitalización el primer año fue de un 15,8 %, la mitad programadas, fundamentalmente por malfunción valvular, craneoplastia y retirada de material quirúrgico. En el segundo año los reingresos, en su mayoría programados, descendieron al 5,2 %, preferentemente para la liberación quirúrgica de rigideces articulares. Aunque distintas variables mostraron correlación con los reingresos del TCE, sólo se obtuvo significación estadística con el hematoma subdural. Uno de cada 3 pacientes acudió a Urgencias, al menos una vez, durante los dos años siguientes al alta hospitalaria. Conclusiones. Los motivos principales por los que reingresa un paciente con TCE tras el alta hospitalaria de una UNR son neurológicos, ortopédicos y de cirugía reconstructiva, en la misma línea que las complicaciones por las que se tratan en Urgencias. Se observa un notable descenso de rehospitalización entre el primer y segundo año del alta hospitalaria

Objectives. Study the incidence and the reasons of rehospitalization for traumatic brain injury (TBI) after the subject was discharged from the Neurorehabilitation Unit (NRU), identifying those factors related to the rehospitalization and the need of these patients to attend to the emergency department after this discharge. Patients and method. Study of cohort of 209 patients with TBI diagnostic, discharged from the NRU (January 2000-December 2002) with a two year follow-up. We obtained demographic features, severity of the TBI, related injuries, pathological images in a CT, hospital complications, evaluation scales, hospitalization readmissions, need to go to the emergency department and a specialized follow-up from the clinical records. Results. The incidence of rehospitalization in the first year was 15.8 %, half of which were planned, valvular malfunctions, cranioplasty and withdrawal of the surgical equipment were the main reasons. During the second year, readmissions decreased to 5.2 %, most of them planned for the surgical release of the joint stiffness. Although some variables showed correlation with the TBI rehospitalization, only the subdural hematoma was statistically significant. One out of three patients went to the emergency department at least once during the two years following hospital discharge. Conclusions. The main reasons of TBI rehospitalization after hospital discharge are neurological, orthopedic and reconstructive surgery, similar to the complications by which they are treated in the emergency department. There is an important descent of rehospitalization between first and second year after hospital discharge

The dioxin receptor is silenced by promoter hypermethylation in human acute lymphoblastic leukemia through inhibition of Sp1 binding.

The transcription factor aryl hydrocarbon receptor (AhR) has relevant functions in cell proliferation. Interestingly, the AhR can either promote or inhibit proliferation depending on the cell phenotype. Although recent data reveal potential pathways for AhR signaling in cell proliferation, the mechanisms that regulate its activity in tumor cells remain unknown. Here, we have analyzed promoter hypermethylation as a potential mechanism controlling AhR expression in human tumor cells. AhR promoter CpG methylation was sporadic in a panel of 19 tumor cell lines except for the chronic myeloid leukemia (CML) K562 and the acute lymphoblastic leukemia (ALL) REH. When compared with normal lymphocytes, REH had very low constitutive AhR expression that could be attributed to promoter hypermethylation since treatment with the DNA demethylating agent 5-aza-2'-deoxycitidine (AZA) significantly increased AhR mRNA and protein. These results in leukemia-derived cell lines were further confirmed in primary ALL, where 33% of the patients (7/21) had AhR promoter hypermethylation. Chromatin immunoprecipitation (ChIP) showed that methylation impaired binding of the transcription factor Sp1 to the AhR promoter, thus providing a mechanism for AhR downregulation in REH cells. Therefore, promoter hypermethylation represents a novel epigenetic mechanism downregulating AhR activity in hematological malignancies such as ALL.

Estudio de dos casos de trombosis carotídea postraumática / A study of two cases of post-traumatic carotid artery thrombosis

Exponemos dos casos de pacientes jóvenes de sexo masculino que tras sufrir un accidente de tráfico ingresaron politraumatizados en nuestro hospital presentando un traumatismo craneoencefálico grave, diagnosticándose posteriormente en ambos casos una trombosis carotídea postraumática (TCP). En el primer caso se observó una imagen compatible con un ictus extenso al realizarse un escáner craneal de control, confirmándose a continuación la TCP en una angiografía. La evolución después de varios meses ingresado ha sido muy pobre, presentando mutismo, grave afectación motora de miembros derechos y una dependencia total valorada por la medida de la independencia funcional. En el segundo caso se comprobó una hemiparesia derecha franca y una tumefacción en cara lateral del cuello tras revaloración inicial, realizándose una eco-doppler y consecutivamente una angiografía que confirmó la existencia de TCP. Mediante implantación quirúrgica de un stent a nivel de la lesión se consiguió buena revascularización, evolucionando favorablemente en pocas semanas con mínimas secuelas cognitivas y motoras.Los traumatismos craneofaciales graves se asocian con una frecuencia significativa a lesiones carotídeas postraumáticas. Dada la alta mortalidad y el alto índice de secuelas neurológicas que conllevan estas lesiones cuando se complican, es muy importante la revaloración neurológica del paciente, así como la realización de una eco-doppler ante la mínima sospecha, que se complementará con una angiografía o angio-resonancia magnética, si fuera necesario, para confirmar el diagnóstico (AU)

[Complications arising from traumatic brain injuries in a hospital rehabilitation unit: a series of 126 cases]. / Complicaciones de los traumatismos craneoencefálicos en una unidad hospitalaria de rehabilitación: serie de 126 casos.

INTRODUCTION: The consequences of traumatic brain injuries (TBI) are devastating, whether it is in the personal, family, health care or social spheres. Sufferers will have to follow a rehabilitation programme in which we are going to be faced with a large number of medical, neurological and orthopaedic problems that will exert an influence on that programme. AIMS: The aim of this study is to determine the epidemiological data and the gravity of the cases of TBI admitted to our Rehabilitation Unit, to identify medical and orthopaedic problems that occurred during the time patients were in hospital, and also to determine factors and variables that could have an effect on the onset of such complications. PATIENTS AND METHODS: A retrospective descriptive study was conducted in which we surveyed and collected data from 126 case histories chosen at random from the 210 patients admitted to our Rehabilitation Unit between 1999 and 2001. RESULTS: Mean age, 29 years (interval 4 67), ratio of males to females, 4:1. The TBI were due to motorcycle accidents (40%), car accidents (30%) and falls (11.5%). Neurological problems appeared in 63%, the most frequent of which was psychomotor agitation. 14% displayed post traumatic hydrocephalus, and 8% presented post traumatic seizures. Gastro intestinal problems were seen in 41%, the most frequent being constipation. Respiratory problems were found in 36%, and 15.6% of the patients suffered from pneumonia. 8% displayed para articular ossifications. The number of days spent in the ICU and the days in coma were the more highly statistically significant variables associated with the onset of these complications. CONCLUSIONS: The problems presented by TBI patients during their stay in a Hospital Rehabilitation Unit are diverse and complex; the number of days spent in the ICU and the days they are in coma are the variables that can be of most use in predicting the onset of such complications.

Complicaciones de los traumatismos craneoencefálicos en una unidad hospitalaria de rehabilitación: serie de 126 casos / Complications arising from traumatic brain injuries in a hospital rehabilitation unit: a series of 126 cases

Introducción. Las consecuencias de los traumatismos craneoencefálicos (TCE) son devastadoras en diversos ámbitos-personal, familiar, sanitario y social-.Estas personas requerirán un programa de rehabilitación, en el cual vamos a enfrentarnos con una gran cantidad de problemas médicos, neurológicos y ortopédicos que influirán sobre dicho programa. Objetivo. Determinar los datos epidemiológicos y la gravedad de los pacientes con TCE ingresados en nuestra Unidad de Rehabilitación, identificar los problemas médicos y ortopédicos que se presentan durante el ingreso, así como determinar los factores y las variables que pudieran influir en la aparición de dichas complicaciones. Pacientes y métodos. Realizamos un estudio retrospectivo descriptivo, para lo cual revisamos y recogimos los datos de 126 historias clínicas, escogidas aleatoriamente, de los 210 pacientes ingresados en nuestra Unidad de Rehabilitación entre 1999 y 2001. Resultados. La edad media fue de 29 años (intervalo 4-67) y la relación hombre/mujer, 4:1. Las causas del TCE fueron accidentes de moto (40 por ciento), de automóvil (30 por ciento) y precipitaciones (11,5 por ciento). Aparecieron problemas neurológicos en el 63 por ciento de los casos. De ellos, el más frecuente fue la agitación psicomotora. El 14 por ciento de los pacientes presentó hidrocefalia postraumática, y el 8 por ciento, crisis postraumáticas. Aparecieron en el 41 por ciento problemas gastrointestinales, el más frecuente, el estreñimiento. Tuvieron problemas respiratorios el 36 por ciento, y presentaron neumonía el 15,6 por ciento de los pacientes. El 8 por ciento mostraron osificaciones para articulares. Los días en UCI y los días en coma fueron las variables que presentaron una mayor significación estadística con la aparición de dichas complicaciones. Conclusiones. Los problemas que presentan los TCE durante el período de ingreso en una Unidad Hospitalaria de Rehabilitación son múltiples y complejos, y los días en UCI y los días en coma son las variables que más nos pueden ayudar a predecir la aparición de dichas complicaciones (AU)

Traumatismos craneoencefálicos. Escalas de valoración para la medida de resultados en rehabilitación / Cranioencephalic traumatisms. Assessment scales for the measurement of outcome in rehabilitation

La evaluación de la severidad de un traumatismo craneoencefálico, puede contemplarse tanto desde una perspectiva de estudio de las lesiones que el traumatismo determina a nivel intracraneal, como desde la perspectiva de las repercusiones funcionales que dicho impacto mecánico tiene sobre el normal funcionalismo del sistema nervioso central. En este artículo se desarrollan las diferentes escalas que más se utilizan en la actualidad en este proceso. Sin duda, ninguna recoge todos los aspectos que serían deseables para el médico neurorrehabilitador, es por ello que se proponen varias escalas universales para medir aspectos diferentes. (AU)

Manejo terapéutico de la agitación en el paciente con traumatismo craneoencefálico / Therapeutic managementg of agitation in the patient with craneoencephalic traumatism

El objetivo de este trabajo es revisar las posibles terapias atinentes al tratamiento de la conducta agitada que presentan tan a menudo los pacientes afectos de un traumatismo craneoencefálico. La conducta agitada debe ser tratada siguiendo pautas farmacológicas y no farmacológicas. No existe evidencia inequívoca acerca de los fármacos que deben utilizarse o no, aunque sí el consenso suficiente que permite aconsejar unos en vez de otros e incluso optar por los que serían más adecuados ante diferentes situaciones. Respecto a las terapias no farmacológicas, las diversas técnicas de modificación y reorientación de la conducta pueden ser de utilidad, viniendo condicionada la elección de una y otra por las circunstancias del paciente y las capacidades del equipo terapéutico (AU)

Targeted genomic disruption of H-ras and N-ras, individually or in combination, reveals the dispensability of both loci for mouse growth and development.

Mammalian cells harbor three highly homologous and widely expressed members of the ras family (H-ras, N-ras, and K-ras), but it remains unclear whether they play specific or overlapping cellular roles. To gain insight into such functional roles, here we generated and analyzed H-ras null mutant mice, which were then also bred with N-ras knockout animals to ascertain the viability and properties of potential double null mutations in both loci. Mating among heterozygous H-ras(+/-) mice produced H-ras(-/-) offspring with a normal Mendelian pattern of inheritance, indicating that the loss of H-ras did not interfere with embryonic and fetal viability in the uterus. Homozygous mutant H-ras(-/-) mice reached sexual maturity at the same age as their littermates, and both males and females were fertile. Characterization of lymphocyte subsets in the spleen and thymus showed no significant differences between wild-type and H-ras(-/-) mice. Analysis of neuronal markers in the brains of knockout and wild-type H-ras mice showed that disruption of this locus did not impair or alter neuronal development. Breeding between our H-ras mutant animals and previously available N-ras null mutants gave rise to viable double knockout (H-ras(-/-)/N-ras(-/-)) offspring expressing only K-ras genes which grew normally, were fertile, and did not show any obvious phenotype. Interestingly, however, lower-than-expected numbers of adult, double knockout animals were consistently obtained in Mendelian crosses between heterozygous N-ras/H-ras mice. Our results indicate that, as for N-ras, H-ras gene function is dispensable for normal mouse development, growth, fertility, and neuronal development. Additionally, of the three ras genes, K-ras appears to be not only essential but also sufficient for normal mouse development.

Proteasome inhibition induces nuclear translocation and transcriptional activation of the dioxin receptor in mouse embryo primary fibroblasts in the absence of xenobiotics.

The aryl hydrocarbon receptor (AHR) is a transcription factor that is highly conserved during evolution and shares important structural features with the Drosophila developmental regulators Sim and Per. Although much is known about the mechanism of AHR activation by xenobiotics, little information is available regarding its activation by endogenous stimuli in the absence of exogenous ligand. In this study, using embryonic primary fibroblasts, we have analyzed the role of proteasome inhibition on AHR transcriptional activation in the absence of xenobiotics. Proteasome inhibition markedly reduced cytosolic AHR without affecting its total cellular content. Cytosolic AHR depletion was the result of receptor translocation into the nuclear compartment, as shown by transient transfection of a green fluorescent protein-tagged AHR and by immunoblot analysis of nuclear extracts. Gel retardation experiments showed that proteasome inhibition induced transcriptionally active AHR-ARNT heterodimers able to bind to a consensus xenobiotic-responsive element. Furthermore, nuclear AHR was transcriptionally active in vivo, as shown by the induction of the endogenous target gene CYP1A2. Synchronized to AHR activation, proteasome inhibition also induced a transient increase in AHR nuclear translocator (ARNT) at the protein and mRNA levels. Since nuclear levels of AHR and ARNT are relevant for AHR transcriptional activation, our data suggest that proteasome inhibition, through a transient increase in ARNT expression, could promote AHR stabilization and accumulation into the nuclear compartment. An elevated content of nuclear AHR could favor AHR-ARNT heterodimers able to bind to xenobiotic-responsive elements and to induce gene transcription in the absence of xenobiotics. Thus, depending on the cellular context, physiologically regulated proteasome activity could participate in the control of endogenous AHR functions.

Effect of thioridazine dosage on the debrisoquine hydroxylation phenotype in psychiatric patients with different CYP2D6 genotypes.

Sixteen hospitalized white European Spanish psychiatric patients treated with thioridazine alone were studied with respect to CYP2D6 genotype, debrisoquine metabolic ratio (MR), and the plasma levels of thioridazine and its metabolites mesoridazine and sulforidazine. After decreasing the dose of thioridazine the debrisoquine MR and thioridazine plasma levels were redetermined. At the initial determination (regular clinical doses, 20-300 mg/day), 14 of 16 patients (88%) were classified as poor metabolizers of debrisoquine (PMs). However, after complete withdrawal of thioridazine in 10 patients, all 10 became extensive metabolizers except two who were genotypically PMs (*4/*4). The inhibition of debrisoquine metabolism was genotype dependent. All patients with wt/wt genotype treated with a dose 150 mg/d were phenotypically PMs, all patients with wt/*4 genotype treated with a dose of 50 mg/d or greater were PMs. The debrisoquine MR from all dose changes correlated with the dose (p < 0.001) and plasma level (p < 0.001) of thioridazine. The CYP2D6 hydroxylation capacity was inhibited by thioridazine as determined by the debrisoquine MR. This inhibition was reversible by thioridazine withdrawal, and thus seems to be dose dependent and related to CYP2D6 genotype. One must consider the effects of thioridazine dosage on CYP2D6, because it may influence the metabolism of concomitant drugs or produce clinically important adverse effects such as cardiotoxicity. An awareness of this problem and cautious dosage adjustment of other drugs metabolized by the same enzyme are recommended during treatment with thioridazine.

Altered cell cycle control at the G(2)/M phases in aryl hydrocarbon receptor-null embryo fibroblast.

The aryl hydrocarbon receptor (AHR) is known to mediate the toxic and carcinogenic effects of polycyclic aromatic hydrocarbons and dioxins. High-affinity AHR ligands, such as 2,3,7, 8-tetrachlorodibenzeno-p-dioxin, have been shown to modify cell proliferation and differentiation. However, the mechanisms by which AHR affects cell proliferation and differentiation are not fully understood. To investigate the role of AHR in cell proliferation, mouse embryonic fibroblasts (MEFs) derived from AHR-null mice were obtained and characterized. Compared with wild-type MEFs, AHR-null cells exhibited a lower proliferation rate with an accumulation of 4N DNA content and increased apoptosis. The expression levels of Cdc2 and Plk, two kinases important for G(2)/M phase of cell cycle, were down-regulated in AHR-null MEFs. In contrast, transforming growth factor-beta (TGF-beta), a proliferation inhibitor in several cell lines, was present at high levels in conditioned medium from AHR-null MEFs. Concomitant with G(2)/M cell accumulation, treatment of wild-type MEFs with TGF-beta3 also resulted in down-regulation of both Cdc2 and Plk. Thus, overproduction of TGF-beta in AHR-deficient cells appears to be the primary factor that causes low proliferation rates and increased apoptosis. Taken together, these results suggest that AHR influences TGF-beta production, leading to an alteration in cell cycle control.

Hepatic fibrosis and cytochrome P450: experimental models of fibrosis compared to AHR knockout mice.

Hepatic fibrosis is characterized by abnormal collagen deposition resulting from increased collagen synthesis and decreased collagen degradation. Cytochrome P450 mediates major drug metabolizing enzyme activity in the liver and this activity is reduced in hepatic fibrosis. In this study we assess cytochrome P450 and CYP 1A mRNA in livers of animals that have been induced to hepatic fibrosis using the heterologous serum induced model of fibrosis in rats compared to controls. Fibrosis was confirmed by assessing the collagen in liver sections as quantified by Sirius red/Fast green staining, a quantitative measure of fibrosis as well as by visualization of the hepatic fibrosis. Collagen levels in the liver sections of heterologous serum induced fibrotic rats was increased by 33% compared to controls and a typical fibrotic pattern was seen. Messenger RNA was prepared from heterologous serum induced fibrotic rats and compared to controls. CYP 1A2 was assessed using a specific probe and the CYP 1A2 level was significantly reduced in the heterologous serum induced fibrotic rats compared to controls. These results further suggest that cytochrome P450 is reduced in the presence of hepatic fibrosis. Thus, in three well established experimental models of hepatic fibrosis which had clearly developed hepatic fibrosis (as shown by Sirius red/Fast green staining), cytochrome P450 mediated enzyme activity, or specifically, CYP 1A messenger RNA is decreased. We then investigated a transgenic mouse, deficient in the arylhydrocarbon hydroxylase receptor (AHR), which has undetectable levels of CYP 1A messenger RNA. We quantitated the collagen in liver sections obtained from AHR knockout mice compared to controls, as an indication of the presence of hepatic fibrosis. Collagen concentration was significantly increased by 53% (P<0.0005) in sections from Ahr-/- (knockout) mice compared to wild-type controls. Collagen in livers of the Ahr+/- heterozygous mice was not different from wild-type controls. The increase in collagen concentration in liver sections is an indication of fibrosis in Ahr-/- mice. Collagen protein deposition was also elevated in liver sections from bile duct ligated rats (by 44%) compared to sham operated controls, was elevated in liver sections from heterologous serum induced fibrosis in rats (33%) compared to controls, and was elevated in liver sections from yellow phosphorous induced hepatic fibrosis (74%) compared to vehicle treated controls. In conclusion, these results indicate that cytochrome P450 and specific subtypes of P450, the CYP 1A subgroup, are significantly reduced in three experimental models of hepatic fibrosis when there is evidence of increased collagen deposition in the livers. These results also indicate that mice that are deficient in CYP 1A have elevated levels of hepatic collagen protein, an indication of hepatic fibrosis.

Potassium-induced apoptosis in rat cerebellar granule cells involves cell-cycle blockade at the G1/S transition.

The role of regulators controlling the G1/S transition of the cell cycle was analyzed during neuronal apoptosis in post-mitotic cerebellar granule cells in an attempt to identify common mechanisms of control with transformed cells. Cyclin D1 and its associated kinase activity CDK4 (cyclin-dependent kinase 4) are major regulators of the G1/S transition. Whereas cyclin D1 is the regulatory subunit of the complex, CDK4 represents the catalytic domain that, once activated, will phosphorylate downstream targets such as the retinoblastoma protein, allowing cell-cycle progression. Apoptosis was induced in rat cerebellar granule cells by depleting potassium in presence of serum. Western-blot analyses were performed and protein kinase activities were measured. As apoptosis proceeded, loss in cell viability was coincident with a significant increase in cyclin D1 protein levels, whereas CDK4 expression remained essentially constant. Synchronized to cyclin D1 accumulation, cyclin-dependent kinase inhibitor p27Kip1 drastically dropped to 20% normal values. Cyclin D1/CDK4-dependent kinase activity increased early during apoptosis, reaching a maximum at 9-12 h and decreasing to very low levels by 48 h. Cyclin E, a major downstream target of cyclin D1, decreased concomitantly to the reduction in cyclin D1/CDK4-dependent kinase activity. We suggest that neuronal apoptosis takes place through functional alteration of proteins involved in the control of the G1/S transition of the cell cycle. Thus, apoptosis in post-mitotic neurons could result from a failed attempt to re-enter cell cycle in response to extracellular conditions affecting cell viability and it could involve mechanisms similar to those that promote proliferation in transformed cells.

Genomic instability in Gadd45a-deficient mice.

Gadd45a-null mice generated by gene targeting exhibited several of the phenotypes characteristic of p53-deficient mice, including genomic instability, increased radiation carcinogenesis and a low frequency of exencephaly. Genomic instability was exemplified by aneuploidy, chromosome aberrations, gene amplification and centrosome amplification, and was accompanied by abnormalities in mitosis, cytokinesis and growth control. Unequal segregation of chromosomes due to multiple spindle poles during mitosis occurred in several Gadd45a -/- cell lineages and may contribute to the aneuploidy. Our results indicate that Gadd45a is one component of the p53 pathway that contributes to the maintenance of genomic stability.

CYP1A2 is not the primary enzyme responsible for 4-aminobiphenyl-induced hepatocarcinogenesis in mice.

4-Aminobiphenyl (4-ABP), a potent carcinogen in rodents (liver cancer) and human (bladder cancer), is found as an environmental contaminant and in tobacco smoke. Hemoglobin adducts and lung DNA adducts of 4-ABP are found in tobacco smokers. In vitro metabolism studies with human and rat liver microsomes have shown that CYP1A2 is primarily responsible for catalyzing N-hydroxylation, the initial step in the metabolic activation of 4-ABP. To determine whether this P450 is a rate limiting pathway for hepatocarcinogenesis, CYP1A2-null mice were analyzed at 16 months of age and were compared with wild-type mice in their response to 4-ABP using the neonatal mouse bioassay and two different doses of the carcinogen. Overall differences in incidences of hepatocellular adenoma, carcinoma and preneoplastic foci were not significant between either genotypes or 4-ABP doses used, whereas small, but significant, differences were found for specific types of foci. These results suggest that while CYP1A2 levels may not be rate limiting for 4-ABP metabolism to produce tumors and foci, it may modulate the induction process of some types of liver foci in either a positive or negative manner. In vitro studies using CYP1A2-null and wild-type mouse liver microsomes revealed that CYP1A2 is not the sole P450 required for 4-ABP N-hydroxylation and that another, yet to be identified, P450 is likely to be involved.

Targeted disruption of the microsomal epoxide hydrolase gene. Microsomal epoxide hydrolase is required for the carcinogenic activity of 7,12-dimethylbenz[a]anthracene.

Microsomal epoxide hydrolase (mEH) is a conserved enzyme that is known to hydrolyze many drugs and carcinogens, and a few endogenous steroids and bile acids. mEH-null mice were produced and found to be fertile and have no phenotypic abnormalities thus indicating that mEH is not critical for reproduction and physiological homeostasis. mEH has also been implicated in participating in the metabolic activation of polycyclic aromatic hydrocarbon carcinogens. Embryonic fibroblast derived from the mEH-null mice were unable to produce the proximate carcinogenic metabolite of 7,12-dimethylbenz[a]anthracene (DMBA), a widely studied experimental prototype for the polycylic aromatic hydrocarbon class of chemical carcinogens. They were also resistant to DMBA-mediated toxicity. Using the two-stage initiation-promotion skin cancer bioassay, the mEH-null mice were found to be highly resistant to DMBA-induced carcinogenesis. In a complete carcinogenesis bioassay, the mEH mice were totally resistant to tumorigenesis. These data establish in an intact animal model that mEH is a key genetic determinant in DMBA carcinogenesis through its role in production of the ultimate carcinogenic metabolite of DMBA, the 3,4-diol-1,2-epoxide.

Expression of CYP2A genes in human liver and extrahepatic tissues.

Members of the human cytochrome P450 2A (CYP2A) subfamily are known to metabolize several promutagens, procarcinogens, and pharmaceuticals. In this study, the expression of the three genes found in the human CYP2A gene cluster was investigated in the liver and several extrahepatic tissues by gene-specific reverse transcriptase-polymerase chain reaction (RT-PCR). All three transcripts (CYP2A6, CYP2A7, and CYP2A13) were found to be present in liver. Quantitative RT-PCR analysis showed that CYP2A6 and CYP2A7 mRNAs were present at roughly equal levels in the liver, while CYP2A13 was expressed at very low levels. Two putative splicing variants of CYP2A7 were found in the liver. Nasal mucosa contained a low level of CYP2A6 and a relatively high level of CYP2A13 transcripts. Kidney, duodenum, lung, alveolar macrophages, peripheral lymphocytes, placenta, and uterine endometrium were negative for all transcripts. This survey gives a comprehensive picture of the expression pattern of CYP2A genes in liver and extrahepatic tissues and constitutes a basis for a search for functional CYP2A forms and their roles in chemical toxicity in liver and nasal mucosa.

Amelioration of TCDD-induced teratogenesis in aryl hydrocarbon receptor (AhR)-null mice.

The aryl hydrocarbon receptor (AhR) mediates many of the biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and transcriptional activation of genes encoding a number of xenobiotic metabolizing enzymes. Prenatal exposure of mice to TCDD causes severe alterations in embryo and fetal development, including hydronephrosis and cleft palate. However, the mechanisms underlying these effects are unclear. In this work, the teratogenicity of TCDD in AhR-null mice was evaluated to determine if this effect is mediated by the AhR. Homozygous wild-type (+/+) or AhR-null (-/-) female mice were mated with males of the same genotype overnight. On gestation day (GD)-10, mice were intubated orally with either corn oil (vehicle control) or 25 micrograms/kg TCDD. Fetuses were examined on GD18 for visceral and skeletal alterations. For non-TCDD-exposed litters, all developmental endpoints were comparable between genotypes, with the exception of a lower incidence of large interfrontal bones in (-/-) mice. For TCDD-exposed litters, (+/+) fetuses had a significantly greater incidence of cleft palate, hydronephrosis, small kidneys, tortuous ureters and greater dilation of the renal pelves and ureters compared to (-/-) fetuses. Interestingly, an increased resorption rate was observed in (-/-) fetuses exposed to TCDD. Results from this work demonstrate that fetal development per se is generally unaffected by the absence of the AhR or that other genes may have compensated for the loss of the AhR. More importantly, these data indicate that the AhR mediates TCDD-induced teratogenicity. Further, since a higher percentage of resorptions was observed in (-/-) litters from TCDD-treated dams, it is possible that AhR-independent mechanisms contribute to TCDD-induced developmental toxicity.