F344/NTac Rats Chronically Exposed to Bromodichloroacetic Acid Develop Mammary Adenocarcinomas With Mixed Luminal/Basal Phenotype and Tgfß Dysregulation.

Breast cancer is the most common cancer and the second-leading cause of cancer mortality in women in the United States. A recent 2-year National Toxicology Program carcinogenicity study showed an increased incidence of proliferative mammary lesions (hyperplasia, fibroadenoma, adenocarcinoma) in F344/NTac rats exposed to bromodichloroacetic acid (BDCA), a disinfection by-product in finished drinking water with widespread human exposure. We hypothesized that the increase in mammary tumors observed in BDCA-exposed F344/NTac rats may be due to underlying molecular changes relevant for human breast cancer. The objective of the study was to compare (1) gene and protein expression and (2) mutation spectra of relevant human breast cancer genes between normal untreated mammary gland and mammary tumors from control and BDCA-exposed animals to identify molecular changes relevant for human cancer. Histologically, adenocarcinomas from control and BDCA-exposed animals were morphologically very similar, were estrogen/progesterone receptor positive, and displayed a mixed luminal/basal phenotype. Gene expression analysis showed a positive trend in the number of genes associated with human breast cancer, with proportionally more genes represented in the BDCA-treated tumor group. Additionally, a 5-gene signature representing possible Tgfß pathway activation in BDCA-treated adenocarcinomas was observed, suggesting that this pathway may be involved in the increased incidence of mammary tumors in BDCA-exposed animals.

NAG-1/GDF-15 prevents obesity by increasing thermogenesis, lipolysis and oxidative metabolism.

OBJECTIVE: Obesity is a major health problem associated with high morbidity and mortality. NSAID-activated gene (NAG-1) is a TGF-ß superfamily member reported to alter adipose tissue levels in mice. We investigated whether hNAG-1 acts as a regulator of adiposity and energy metabolism. DESIGN/SUBJECTS: hNAG-1 mice, ubiquitously expressing hNAG-1, were placed on a control or high-fat diet for 12 weeks. hNAG-1-expressing B16/F10 melanoma cells were used in a xenograft model to deliver hNAG-1 to obese C57BL/6 mice. RESULTS: As compared with wild-type littermates, transgenic hNAG-1 mice have less white fat and brown fat despite equivalent food intake, improved glucose tolerance, lower insulin levels and are resistant to dietary- and genetic-induced obesity. hNAG-1 mice are more metabolically active with higher energy expenditure. Obese C57BL/6 mice treated with hNAG-1-expressing xenografts show decreases in adipose tissue and serum insulin levels. hNAG-1 mice and obese mice treated with hNAG-1-expressing xenografts show increased thermogenic gene expression (UCP1, PGC1α, ECH1, Cox8b, Dio2, Cyc1, PGC1ß, PPARα, Elvol3) in brown adipose tissue (BAT) and increased expression of lipolytic genes (Adrb3, ATGL, HSL) in both white adipose tissue (WAT) and BAT, consistent with higher energy metabolism. CONCLUSION: hNAG-1 modulates metabolic activity by increasing the expression of key thermogenic and lipolytic genes in BAT and WAT. hNAG-1 appears to be a novel therapeutic target in preventing and treating obesity and insulin resistance.

Pathology methods for the evaluation of embryonic and perinatal developmental defects and lethality in genetically engineered mice.

The normal embryonic development of organs and other tissues in mice and all species is preprogrammed by genes. Inactivation of a gene involved in any stage of normal embryonic development can have severe consequences leading to embryonic or postnatal developmental defects and lethality. Pathology methods are reviewed for evaluating normal and abnormal placenta and embryo, especially after E12.5. These methods include pathology protocols for necropsy and histopathology in addition to references that will provide additional knowledge for embryo assessment including histology atlases and advanced embryo imaging techniques.

Role of the alveolar type II cell in the development and progression of pulmonary tumors induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in the A/J mouse.

The role of the type II cell in the development of pulmonary tumors induced in the adult A/J mouse (6 weeks of age) by treatment with a single dose (100 mg/kg, i.p.) of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was investigated. Twenty-four h following treatment with NNK, the concentration of O6-methylguanine was similar in Clara and type II cells. However, hyperplasias were detected only along the alveolar septa in lungs 14 weeks after carcinogen treatment. Examination of the ultrastructure of several hyperplasias revealed that the proliferating cells resembled type II pneumocytes. The proliferating cells were cuboidal in shape, with centrally localized ovoid nuclei characterized by minor indentations. Lamellar bodies, one of the major hallmarks of the type II cell, were present in the cytoplasm. The progression of pulmonary lesions was followed by sacrificing mice at 4-week intervals from 14 to 54 weeks after treatment with NNK. From 34 to 42 weeks after treatment, progression to neoplasia was demonstrated by a decline in the frequency of hyperplasias and an increase in the frequency of adenomas. Approximately 50% of the adenomas were observed arising within hyperplasias. Carcinomas appeared to increase in frequency 34 weeks after carcinogen treatment and comprised greater than 50% of the pulmonary lesions by 54 weeks. Approximately 30% of the carcinomas were observed arising within adenomas. The growth pattern of carcinomas began to change from solid to mixed (solid and papillary) 42 weeks after NNK. Moreover, electron micrographic analysis demonstrated that, within a hyperplasia, proliferating type II cells could change from cuboidal to columnar in shape and could also exhibit nuclear indentations, both characteristics displayed by the Clara cell. Thus, this divergence of the type II cell from its well characterized morphological features indicates that the selective growth advantage which these initiated cells possess can result in changes to the normal ultrastructure of this cell as it progresses toward malignancy. DNA was isolated from 20 hyperplasias and screened for the presence of an activated K-ras gene. This gene was activated in 17 of 20 lesions, with 85% of the mutations involving a GC to AT transition within codon 12 (GGT to GAT), a mutation consistent with base mispairing produced by the formation of the O6-methylguanine adduct. This specificity for activation of the K-ras gene was identical to that observed previously in adenocarcinomas induced by NNK.(ABSTRACT TRUNCATED AT 400 WORDS)

Dose-response relationship between O6-methylguanine formation in Clara cells and induction of pulmonary neoplasia in the rat by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The relationship between the formation of O6-methylguanine (O6MG) and the induction of lung, liver, and nasal tumors in the Fisher 344 rat by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was examined in a dose-response study. Animals were treated for 20 wk (3 times/wk) with concentrations of NNK ranging from 0.03 to 50 mg/kg to induce tumors. Steady-state concentrations of O6MG were quantitated, and cytotoxicity was assessed in target cells and tissues after 4 wk of treatment with NNK. No cytotoxicity was detected in the lung during treatment with NNK. The formation of O6MG was greatest in Clara cells compared with macrophages, type II cells, small cells, and whole lung at all doses examined. The difference in adduct concentration between the Clara cell and other pulmonary cell types was most pronounced with low doses of carcinogen. The O6MG:dose ratio, an index of alkylation efficiency, increased 29-fold as the dose of NNK was decreased from 50 to 1 mg/kg of carcinogen. In contrast, only a small increase in alkylation efficiency was observed in type II cells and whole lung. A significant number of tumors were induced in the lung at doses of 0.1 to 50 mg/kg with incidences ranging from 10% at the lowest dose up to 87% in the group of animals which received 50 mg/kg of NNK. A linear relationship was observed when the concentration of O6MG in Clara cells as a function of dose was plotted against the corresponding tumor incidence. This relationship was not observed using DNA adduct concentrations in type II cells or whole lung. The development of pulmonary tumors appeared to involve the formation of alveolar hyperplasias which progressed to adenomas and finally to carcinomas. The majority of adenomas were solid, whereas carcinomas were mainly papillary. Examination of the ultrastructure of the hyperplasias, adenomas, and carcinomas revealed morphological structures (e.g., lamellar bodies, tubular myelin) which are associated with type II cells. Thus, these data suggest that the majority of neoplasms in the lung begin as type II cell proliferations with progression to adenomas and carcinomas within the areas of hyperplasia. The lack of agreement between biochemical and morphological findings makes it difficult to hypothesize a cell of origin for the pulmonary neoplasms. In contrast to the lung, tumors were induced in the liver and nasal passages only after exposure to high doses of NNK. Moreover, both the formation of DNA adducts and cytotoxicity appear obligatory for the generation of tumors in these tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of 15-lipoxygenase-1 in human colorectal cancer.

Recently, we reported the induction of reticulocyte type 15-lipoxygenase (15-Lox-1) in a human colorectal carcinoma cell line that had been stimulated by butyrate to undergo apoptosis and cell differentiation (H. Kamitani et al., J. Biol. Chem., 273: 21569-21577, 1998). To determine if 15-Lox-1 is expressed in human colorectal cancer tissue, 21 matched pairs of colorectal tumor and adjacent normal tissue were examined by immunoblot analysis using specific antibodies for human 15-Lox-1, prostaglandin H synthase (also called cyclooxygenase, Cox)-1 and Cox-2. Eighteen of the 21 were found to have 15-Lox-1 in both tumor tissue and matched adjacent normal tissue, with the 15-Lox-1 expression being significantly higher in most of the tumor tissue. The expression of Cox-2 was also elevated in most tumors, whereas Cox-1 was frequently expressed at lower levels in the tumor tissue than in the paired normal tissue. Reverse-phase high-performance liquid chromatography analysis of arachidonate metabolites, formed on incubation of arachidonic acid with a crude enzyme preparation from the colon samples, revealed the formation of 15-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid with a much lower level of 12-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (15-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid:12-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid, 6.5:1) which also indicate the presence of 15-Lox-1. Furthermore, reverse transcription-PCR with primers specific for human 15-Lox-1 or 15-Lox-2 cDNA indicated that 15-Lox-1 mRNA was present in the colorectal tumors. The sequence of the PCR product was identical to the human 15-Lox-1. Immunohistochemical studies showed 15-Lox-1 localization in the glandular epithelium of human colorectal tumor tissue. These results suggest that 15-Lox-1 is highly expressed in human colorectal cancer epithelial cells and that its expression may have a role in colorectal carcinogenesis.

High frequency and heterogeneous distribution of p53 mutations in aflatoxin B1-induced mouse lung tumors.

Inactivation of the p53 tumor suppressor gene is one of the most frequent genetic alterations observed in human lung cancers. However, p53 mutations are more rarely detected in chemically induced mouse lung tumors. In this study, 62 female AC3F1 (A/J x C3H/HeJ) mice were treated with aflatoxin B1 (AFB1; 150 mg/kg i.p. divided into 24 doses over 8 weeks). At 6-14 months after dosing, mice were killed, and tumors were collected. A total of 71 AFB1-induced lung tumors were examined for overexpression of p53 protein by immunohistochemical staining. Positive nuclear p53 staining was observed in 79% of the AFB1-induced tumors, but the pattern was highly heterogeneous. In approximately 73% of the positively stained tumors, fewer than 5% of cells demonstrated positive staining; in the other 27%, between 10% and 60% of the cells stained positively, with staining localized to the periphery of the tumors in many cases. Single-strand conformational polymorphism analysis of the evolutionarily conserved regions of the p53 gene (exons 5-8) from AFB1-induced whole lung tumor DNA revealed banding patterns consistent with point mutations in 20 of 76 (26%) tumors, with 85% of the mutations in exon 7 and 15% of the mutations in exon 6. Identification of point mutations could not be confirmed by direct sequence analysis because bands representing putative mutations appeared only weakly on autoradiograms. This was presumably due to the heterogeneous nature of the DNA analyzed. Single-strand conformational polymorphism analysis of DNA from laser capture microdissected cells of paraffin-embedded AFB1-induced tumor tissue sections stained for p53 produced banding patterns consistent with point mutations in 18 of 30 (60%) DNA samples. Direct sequencing of the microdissected samples revealed mutations at numerous different codons in exons 5, 6, and 7. Of 26 mutations found in microdissected regions from adenomas and carcinomas, 9 were G:C-->A:T transitions, 11 were A:T-->G:C transitions, and 5 were transversions (2 G:C-->T:A, 2 T:A-->A:T, and 1 A:T-->C:G), whereas 1 deletion mutation was identified. The concordance between immunostaining and molecular detection of p53 alterations was 72% when laser capture microdissection was used versus 17% based on whole tumor analysis. The high mutation frequency and heterogeneous staining pattern suggest that p53 mutations occur relatively late in AFB1-induced mouse lung tumorigenesis and emphasize the value of analyzing different staining regions from paraffin-embedded mouse lung tumors.

Loss of KAI1 expression in the progression of colorectal cancer.

The transmembrane 4 superfamily member KAI1 (CD82) has been shown to inhibit pulmonary metastases in experimental metastasis models of prostate cancer and melanoma. KAI1 expression is decreased in the progression of common solid epithelial tumors of adulthood, including lung, prostate, breast, esophageal, gastric, pancreatic, and bladder cancers. The purpose of our study was to investigate KAI1 expression in the progression of human colorectal cancer. We first analyzed 20 colorectal cancer cell lines by immunoblot techniques. KAI1 was expressed heterogeneously, with the tumor cell lines having a more complex degree of glycosylation compared with that of the normal colonic tissue. KAI1 was highly expressed in the primary SW480 colon cancer cell line but was down-regulated 15-fold in the matched metastatic SW620 cell line. We also investigated KAI1 protein expression by immunohistochemistry in tissues from 84 patients with colorectal cancer. Each tissue section was assigned a KAI1 mean score (KMS) from 0 to 300 based on the product of the percentage of cells that stained for KAI1 and the intensity of the stain (1, 2, or 3). In 84 patients with colorectal cancer, KAI1 was expressed at high levels in normal colonic mucosa (KMS 226) but was expressed at lower levels in the primary tumors (KMS 65; P < 0.0001). In a subset of 12 patients with stage IV metastatic disease, we observed a progressive down-regulation of KAI1, from the normal adjacent colonic mucosa (KMS 193) to the primary tumor (KMS 72; P = 0.0001) to the liver metastasis (KMS 25; tumor compared with metastasis, P = 0.0135). We found no correlation between loss of KAI1 expression and stage of disease. In 10 patients, we also noted loss of KAI1 expression in the transition from normal colonic mucosa (KMS 237) to adenoma (KMS 174) to carcinoma (KMS 62; P < 0.0167 for all three comparisons). We conclude that the down-regulation of KAI1 occurs early in the progression of colorectal cancer.

Beta-catenin mutations and protein accumulation in all hepatoblastomas examined from B6C3F1 mice treated with anthraquinone or oxazepam.

The molecular pathogenesis of hepatoblastomas in the B6C3F1 mouse is unclear but may involve alterations in the beta-catenin/Wnt signaling pathway as was recently described for chemically induced hepatocellular neoplasms and human liver cancers. The objective of this study was to characterize the mutation frequency and spectrum of beta-catenin mutations and the intracellular localization of beta-catenin protein accumulation in chemically induced hepatoblastomas. In this study, beta-catenin mutations were identified in all 19 anthraquinone-induced hepatoblastomas and all 8 oxazepam-induced hepatoblastomas examined. Although several hepatoblastomas had multiple deletion and/or point mutations, the pattern of mutations in the hepatoblastomas did not differ from that identified in hepatocellular neoplasms. In a majority of the hepatoblastomas (six of seven) examined by immunohistochemical methods, both nuclear and cytoplasmic localization of beta-catenin protein were detected, whereas in hepatocellular adenomas, carcinomas, and normal liver only membrane staining was observed. Our data suggest that beta-catenin mutations and the subsequent translocation of beta-catenin protein from the cell membrane to the cytoplasm and nucleus may be critical steps in providing hepatocellular proliferative lesions with the growth advantage to progress to hepatoblastoma.

Allelotype analysis of mouse lung carcinomas reveals frequent allelic losses on chromosome 4 and an association between allelic imbalances on chromosome 6 and K-ras activation.

We generated allelotypes of 38 methylene chloride-induced lung carcinomas from female C57BL/6J x C3H/6J F1 (hereafter called B6C3F1) mice. Two or more polymorphic markers per autosome, most of them microsatellites, were examined for loss of heterozygosity. Allelic losses throughout the genome were generally infrequent except for markers on chromosome 4, which were lost in approximately one-half of the carcinomas. Analysis of lung adenomas indicated that chromosome 4 loss was associated with malignant conversion. In addition, chromosome 4 loss were specific for lung carcinomas based on comparison to methylene chloride-induced liver tumors and additional studies of lung tumors from a variety of treatment protocols and different mouse strains. Preferential loss of the maternal chromosome 4 was observed in B6C3F1 carcinomas. Analyses of additional tumors induced in mice from two reciprocal crosses, A/J x C3H/HeJ F1 (hereafter called AC3F1) and C3H/HeJ x A/J F1 (hereafter called C3AF1), provided evidence for the inactivation of one allele of the putative chromosome 4 tumor suppressor gene by parental imprinting. Most B6C3F1 tumors lost all chromosome 4 markers examined, suggesting nondisjunction events. In contrast, several C3AF1 and AC3F1 tumors appeared to have interstitial deletions that defined the smallest region of overlap as a 9-cM interval between Ifa-2 and D4Nds2. The homologous region on human chromosome 9p21-22 is frequently lost in a variety of tumors including lung cancers. A candidate tumor suppressor gene, MTS1, is located in this region, which is homozygously deleted or mutated in cell lines derived from a variety of human tumors. Finally, an association between K-ras gene activation and allelic imbalances on chromosome 6 was observed for B6C3F1 lung tumors.

Carcinogenicity of 2',3'-dideoxycytidine in mice.

2',3'-dideoxycytidine (ddC) is a synthetic pyrimidine nucleoside analogue approved for treatment of HIV-positive patients. Previous studies indicated that ddC has the potential to cause thymic lymphoma in C57BL/6 x C3H F1 (hereafter called B6C3F1) mice. In this study, we evaluated the carcinogenic potential of ddC in two different mouse models. B6C3F1 hybrid mice carry ecotropic endogenous proviral sequences that may be activated to cause lymphoma, whereas NIH Swiss mice lack proviral sequences that can be expressed. The mice were treated with ddC by gavage at 500 and 1000 mg/kg/day for up to 6 months (human dose, 2.25 mg/day) and evaluated for toxicity, plasma levels of ddC, and pathological changes. Lymphocyte cell markers from the thymic lymphomas were assessed by immunophenotyping. Expression of p53 protein was evaluated using immunohistochemical staining. Treatment-related thymic lymphomas were present in both mouse models with a higher incidence in NIH Swiss than in B6C3F1 mice. The lymphomas were more prevalent in females than in males of both mouse models. Most mice with thymic lymphoma died during the course of the study. In addition to the thymus, lymphoma was often present in lymph nodes, spleen, and other organs. Lymphomas arose more frequently in mice that lack endogenous ecotropic retroviral sequences and thus were not due to activation of endogenous provirus. During the third month of the study, a few NIH Swiss mice that died had granulosa cell tumors of the ovary. Treatment-related but reversible thymic atrophy was observed in both mouse models. There was a very high correlation between the internal dose of ddC and the incidence of thymic lymphoma in both mouse models. Most of the lymphocytes from control thymuses and ddC-induced lymphomas were positive for Thy-1.2 (pan-T), heat stable antigen, and CD4 and CD8 markers, with no marked differences in the lymphocyte markers of the tumors between sexes or dose groups. p53 protein was detected in only 20% (23/115) of the ddC-induced lymphomas with mostly minimal expression in scattered cells. Because ddC induced lymphomas in two different mouse models, the potential carcinogenic risk should be considered in long-term treatment of HIV-positive patients, especially children and adolescent patients treated with ddC.

Mutation of beta-catenin is an early event in chemically induced mouse hepatocellular carcinogenesis.

beta-catenin activation, and subsequent upregulation of Wnt-signaling, is an important event in the development of certain human and rodent cancers. Recently, mutations in the beta-catenin gene in the region of the serine-threonine glycogen kinase (GSK)-3beta phosphorylation target sites have been identified in hepatocellular neoplasms from humans and transgenic mice. In this study we examined 152 hepatocellular neoplasms from B6C3F1 mice included in five chemical treatment groups and controls for mutations in the beta-catenin gene. Twenty of 29 hepatocellular neoplasms from mice treated with methyleugenol had point mutations at codons 32, 33, 34 or 41, sites which are mutated in colon and other cancers. Likewise, nine of 24 methylene chloride-induced hepatocellular neoplasms and 18 of 42 oxazepam-induced neoplasms exhibited similar mutations. In contrast, only three of 18 vinyl carbamate-induced liver tumors, one of 18 TCDD-induced liver tumors, and two of 22 spontaneous liver neoplasms had mutations in beta-catenin. Thus, there appears to be a chemical specific involvement of beta-catenin activation in mouse hepatocellular carcinogenesis. Expression analyses using Western blot and immunohistochemistry indicate that beta-catenin protein accumulates along cell membranes following mutation. The finding of mutations in both adenomas and carcinomas from diverse chemical treatment groups and the immunostaining of beta-catenin protein in an altered hepatocellular focus suggest that these alterations are early events in mouse hepatocellular carcinogenesis.

Brca1 and Brca2 expression patterns in mitotic and meiotic cells of mice.

The mouse homologues of the breast cancer susceptibility genes, Brca1 and Brca2, are expressed in a cell cycle-dependent fashion in vitro and appear to be regulated by similar or overlapping pathways. Therefore, we compared the non isotopic in situ hybridization expression patterns of Brca1 and Brca2 mRNA in vivo in mitotic and meiotic cells during mouse embryogenesis, mammary gland development, and in adult tissues including testes, ovaries, and hormonally altered ovaries. Brca1 and Brca2 are expressed concordantly in proliferating cells of embryos, and the mammary gland undergoing morphogenesis and in most adult tissues. The expression pattern of Brca1 and Brca2 correlates with the localization of proliferating cell nuclear antigen, an indicator of proliferative activity. In the ovary, Brca1 and Brca2 exhibited a comparable hormone-independent pattern of expression in oocytes, granulosa cells and thecal cells of developing follicles. In the testes, Brca1 and Brca2 were expressed in mitotic spermatogonia and early meiotic prophase spermatocytes. Northern analyses of prepubertal mouse testes revealed that the time course of Brca2 expression was delayed in spermatogonia relative to Brca1. Thus, while Brca1 and Brca2 share concordant cell-specific patterns of expression in most proliferating tissues, these observations suggest that they may have distinct roles during meiosis.

Investigation of the role of the carboxyl-terminal tails of the alpha and beta isoforms of the human thromboxane A(2) receptor (TP) in mediating receptor:effector coupling.

We have investigated the functional coupling of alpha and beta isoforms of the human thromboxane A(2) receptor (TP) to Galpha(16) and Galpha(12) members of the G(q) and G(12) families of heterotrimeric G proteins in human embryonic kidney (HEK) 293 cell lines HEK.alpha10 or HEK.beta3, stably over-expressing TPalpha and TPbeta, respectively. Moreover, using HEK.TP(Delta328) cells which over-express a variant of TP truncated at the point of divergence of TPalpha and TPbeta, we investigated the requirement of the C-tail per se in mediating G protein coupling and effector activation. Both TPalpha and TPbeta couple similarly to Galpha(16) to affect increases in inositol 1,4,5-trisphosphate (IP(3)) and mobilisation of intracellular calcium ([Ca(2+)](i)) in response to the TP agonist U46619. Whilst both TP isoforms mediated [Ca(2+)](i) mobilisation in cells co-transfected with Galpha(12), neither receptor generated corresponding increases in IP(3), indicating that the Galpha(12)-mediated increases in [Ca(2+)](i) do not involve PLC activation. Verapamil, an inhibitor of voltage dependent Ca(2+) channels, reduced [Ca(2+)](i) mobilisation in TPalpha and TPbeta cells co-transfected with Galpha(12) to approximately 40% of that mobilised in its absence, whereas [8-(N,N-diethylamino)-octyl-3,4, 5-trimethoxybenzoate, hydrochloride] (TMB-8), an antagonist of intracellular Ca(2+) release, had no effect on [Ca(2+)](i) mobilisation by either receptor isoform co-transfected with Galpha(12). Despite the lack of differential coupling specificity by TPalpha and TPbeta, TP(Delta328) signalled more efficiently in the absence of a co-transfected G protein compared to the wild type receptors but, on the other hand, displayed an impaired ability to couple to co-transfected Galpha(11), Galpha(12) or Galpha(16) subunits. In studies investigating the role of the C-tail in influencing coupling to the effector adenylyl cyclase, similar to TPalpha but not TPbeta, TP(Delta328) coupled to Galpha(s), leading to increased adenosine 3',5'-cyclic monophosphate (cAMP), rather than to Galpha(i). Whereas TP(Delta328) signalled more efficiently in the absence of co-transfected G protein compared to the wild type TPalpha, co-transfection of Galpha(s) did not augment cAMP generation by TP(Delta328). Hence, from these studies involving the wild type TPalpha, TPbeta and TP(Delta328), we conclude that the C-tail sequences of TP are not a major determinant of G protein coupling specificity to Galpha(11) and Galpha(16) members of the G(q) family or to Galpha(12); it may play a role in determining G(s) versus G(i) coupling and may act as a determinant of coupling efficiency.

Antioxidants attenuate anthralin-induced skin inflammation in BALB/c mice: role of specific proinflammatory cytokines.

Anthralin is the most common therapeutic agent among a small number of pro-oxidant, 9-anthrones effective in the topical treatment of psoriasis. However, the usefulness of this drug is diminished by toxic side effects, including skin irritation and inflammation. The activities of anthralin are believed to be mediated by the generation of reactive oxygen intermediates and anthrone radicals produced in the skin. In this study, the dermal inflammatory response to anthralin was determined using a mouse ear swelling test. Maximum ear swelling induced by anthralin coincided with the elevation of cytokine mRNA expression in the skin, including interleukin-6, granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein-2, and tumor necrosis factor alpha at 24 h post challenge. The role of free radical generation in ear swelling and cytokine modulation were examined by systemic administration of cell permeable and impermeable antioxidants before anthralin challenge. Superoxide dismutase and alpha-tocopherol acetate, but not the glutathione precursor N-acetyl cysteine, were effective inhibitors of anthralin-induced ear swelling and cytokine elevation. Maximum inflammatory cell infiltration occurred 72-96 h post anthralin challenge and was also reduced by antioxidants. These data suggest that oxidative stress, generated at the site of anthralin treatment, alters the expression of dermal chemokines and other cytokines resulting in the recruitment of inflammatory cells. Systemic antioxidant administration may provide opportunities for therapeutic intervention against anthralin-associated toxicities.

White matter hyperintensities and neuropsychological outcome following carbon monoxide poisoning.

BACKGROUND: Carbon monoxide (CO) poisoning may result in white matter hyperintensities (WMH) and neurocognitive impairments. OBJECTIVE: To assess in a prospective study WMH in CO-poisoned patients and their relationship to cognitive functioning. METHODS: Seventy-three consecutive CO-poisoned patients were studied. MR scans and neurocognitive tests were administered on day 1 (within 36 hours after CO poisoning), 2 weeks, and 6 months. Age- and sex-matched control subjects for white matter analyses only were obtained from the authors' normative imaging database. MR scans were rated for WMH in the periventricular and centrum semiovale regions, using a 4-point rating scale. Two independent raters rated the scans, and a consensus was reached. RESULTS: Thirty percent of CO-poisoned patients had cognitive sequelae. Twelve percent of the CO-poisoned patients had WMH, with significantly more periventricular, but not centrum semiovale, WMH than control subjects. The WMH in CO-poisoned patients did not change from day 1 to 6 months. Centrum semiovale hyperintensities were related to worse cognitive performance. Duration of loss of consciousness correlated with cognitive impairment at all three times. Initial carboxyhemoglobin levels correlated with loss of consciousness but not with WMH or cognitive sequelae. CONCLUSIONS: CO poisoning can result in brain injury manifested by WMH and cognitive sequelae. The WMH were not related to CO poisoning severity. The WMH occurred in both the periventricular and the centrum semiovale regions; however, only those in the centrum semiovale were significantly associated with cognitive impairments.

Cutaneous necrotizing granulomatous vasculitis with evolution to T cell lymphoma.

The evolution of unusual cutaneous vasculitis to a systemic T cell lymphoma was observed over a 12-year period. Precise classification of the skin biopsy specimens during the course of this patient's illness was difficult. Different observers suggested malignant hemangioendothelioma, malignant lymphoma, regressing atypical histiocytosis, and granulomatous vasculitis. In retrospect, the biopsy specimens likely represented the spectrum of cutaneous lymphomatoid granulomatosis. This condition is yet another example of a reactive lymphoid proliferation proceeding to a malignant lymphoma.

Evaluation of external-beam radiation therapy plus 5-fluorouracil (5-FU) versus external-beam radiation therapy plus hycanthone (HYC) in confined, unresectable pancreatic cancer.

From March 1981 to November 1987, 87 patients with histologically confirmed pancreatic adenocarcinoma, unresectable but confined to the pancreatic region, were randomized to two treatments. The standard treatment was 40-50 Gy external-beam radiation therapy (RT) to gross tumor plus potential microscopic tumor with a 5 Gy boost to gross tumor plus a 1.5-2.0 cm margin, using multiple fields and 5-fluorouracil (5-FU) 500 mg/m2/d intravenously by rapid infusion. The 5-FU was given each of the initial 3 days of each of three 20 Gy radiation courses. The experimental treatment used identical radiation fields, but the two Gy daily radiation fractions were administered in a continuous course to a total dose of 50 Gy. Hycanthone was administered 60 mg/m2 intravenously within 2 to 4 hr during each day of the 5-day course of infusions during the first and fifth weeks of radiation therapy. There was no statistically significant difference between treatment arms in survival (p = 0.82) or disease-free survival (p = 0.27). Seven percent of hycanthone-treated patients demonstrated hepatic toxicity which was usually mild in nature. There was, however, one death due to hepatic toxicity.

An enhancement method for immunohistochemical staining of proliferating cell nuclear antigen in archival rodent tissues.

An enhanced immunohistochemical procedure to detect proliferating cell nuclear antigen (PCNA), an endogenous cell replication marker, has been successfully applied to formalin-fixed, paraffin-embedded archival rat and mouse tissues. The procedure involves microwave oven heating of tissue sections in a commercially available antigen retrieval solution of heavy metal salts. Successful immunohistochemical staining of PCNA can be consistently obtained in tissues fixed for over 24 months in formalin and in sections made from paraffin blocks stored in our tissue archives for up to 19 months. Use of this technique will allow retrospective staining of rodent tissues for identification of S phase cells as an indication of DNA replicative activity in previously conducted toxicity and carcinogenicity studies.

Prostaglandin D(2) receptor-mediated desensitization of the alpha isoform of the human thromboxane A(2) receptor.

Thromboxane (TX) A(2) and prostaglandin (PG) D(2) mediate opposing actions in platelets and in vascular and non-vascular smooth muscle. Here, we investigated the effects of stimulation of the PGD(2) receptor (DP) on signaling by the TXA(2) receptor (TP) expressed in human platelets and in human embryonic kidney (HEK) 293 cells over-expressing the individual TP alpha and TP beta isoforms. In platelets, the selective DP agonist BW245C abolished TP-mediated mobilization of intracellular calcium ([Ca(2+)](i)) and inhibited platelet aggregation in response to the TXA(2) mimetic U46619. DP-mediated desensitization of TP signaling in platelets was prevented by pretreatment with the cAMP-dependent PKA inhibitor, H-89, but was unaffected by the PKC inhibitor GF 109203X. In HEK 293 cells, signaling by TP alpha, but not TP beta, was subject to DP-mediated desensitization in a PKA-dependent, PKC-independent manner. U46619-induced signaling by TP(Delta 328), a truncated variant of TP containing only those residues common to TP alpha and TP beta, was insensitive to prior DP stimulation, indicating that the carboxyl terminal tail of TPalpha contains the target site(s) for DP-mediated desensitization. Mutation of Ser(329) to Ala(329) within a consensus PKA site in TP alpha rendered the mutant TP alpha(S329A) insensitive to BW245C-mediated desensitization. Whole cell phosphorylation assays established that TP alpha, but not TP beta or TP alpha(S329A), was subject to DP-mediated phosphorylation and that TP alpha phosphorylation was blocked by the PKA inhibitor H-89. These data establish that TP alpha, but not TP beta, is subject to DP-mediated cross desensitization, which occurs through direct PKA-mediated phosphorylation of TP alpha at Ser(329).