Scoring System to Predict pt2 in Gallbladder Cancer Based on Carcinoembryonic Antigen and Tumor Diameter.

BACKGROUND AND AIMS: T2 gallbladder cancer requires lymph node dissection for curative resection, whereas simple cholecystectomy is adequate to treat T1 gallbladder cancer. Hence, this study aimed to develop an accurate scoring system to preoperatively predict pT2 in gallbladder cancer. MATERIAL AND METHODS: We retrospectively assessed data from 57 patients with suspected gallbladder cancer who underwent curative resection between September 2003 and May 2017. Six with apparent invasion of adjacent organs on preoperative images were excluded. We evaluated preoperative computed tomography, magnetic resonance and endoscopic ultrasonographic images, blood biochemistry, and the maximum standard uptake value in fluorodeoxyglucose-positron emission tomography images. We analyzed whether correlations between preoperative findings and the depth of tumor invasion could predict pT2. RESULTS: The pathological diagnosis was gallbladder cancer in 30 (58.8%) patients, of whom 21 (69.9%) had pT2 or worse. Multivariate analyses selected carcinoembryonic antigen and tumor diameter as independent predictors of pT2 or worse (odds ratios = 1.741 and 1.098, respectively; 95% confidence intervals = 1.004-3.020 and 1.008-1.197, respectively). A regression formula was created using carcinoembryonic antigen and tumor diameter to calculate pT2 predictive scores. The area under the receiver operating characteristics curve of the pT2 predictive score was 0.873. CONCLUSION: We created a scoring system to predict pT2 in gallbladder cancer using carcinoembryonic antigen and tumor diameter. The present findings suggested that carcinoembryonic antigen is important for the preoperative evaluation of gallbladder cancer.

Integrative genomics identifies YY1AP1 as an oncogenic driver in EpCAM(+) AFP(+) hepatocellular carcinoma.

Identification of key drivers and new therapeutic targets is important given the poor prognosis for hepatocellular carcinoma (HCC) patients, particularly those ineligible for surgical resection or liver transplant. However, the approach to identify such driver genes is facing significant challenges due to the genomically heterogenous nature of HCC. Here we tested whether the integrative genomic profiling of a well-defined HCC subset that is classified by an extreme EpCAM(+) AFP(+) gene expression signature and associated with poor prognosis, all attributes of a stem cell-like phenotype, could uncover survival-related driver genes in HCC. Following transcriptomic analysis of the well-defined HCC cases, a Gene Set Enrichment Analysis coupled with genomic copy number alteration assessment revealed that YY1-associated protein 1 (YY1AP1) is a critical oncoprotein specifically activated in EpCAM(+) AFP(+) HCC. YY1AP1 silencing eliminates oncogene addiction by altering the chromatin landscape and triggering massive apoptosis in vitro and tumor suppression in vivo. YY1AP1 expression promotes HCC proliferation and is required for the maintenance of stem cell features. We revealed that YY1AP1 cooperates with YY1 to alter the chromatin landscape and activate transcription of stemness regulators. Thus YY1AP1 may serve as a key molecular target for EpCAM(+) AFP(+) HCC subtype. Our results demonstrate the feasibility and power of a new strategy by utilizing well-defined patient samples and integrative genomics to uncover critical pathways linked to HCC subtypes with prognostic impact.

Targeting activation-induced cytidine deaminase prevents colon cancer development despite persistent colonic inflammation.

Inflammatory bowel disease (IBD) is an important etiologic factor in the development of colorectal cancer. However, the mechanism underlying carcinogenesis through chronic inflammation is still unknown. Activation-induced cytidine deaminase (AID) is induced by the inflammation and involved in various human carcinogenesis via its mutagenic activity. In the current study, we investigated whether the inflammation/AID axis plays an integral role in the development of colitis-associated cancers. Inflammation in the cecum was more severe than that in other colonic regions, and endogenous AID expression was enhanced most prominently in the inflamed cecal mucosa of interleukin (IL)-10(-/-) mice. Blockade of tumor necrosis factor (TNF)-α and IL-12 significantly suppressed AID expression. Although proinflammatory cytokine expression was comparable between IL-10(-/-)AID(+/+) and IL-10(-/-)AID(-/-) mice, sequencing analyses revealed a significantly lower incidence of somatic mutations in Trp53 gene in the colonic mucosa of IL-10(-/-)AID(-/-) than IL-10(-/-)AID(+/+) mice. Colon cancers spontaneously developed in the cecum in 6 of 22 (27.2%) IL-10(-/-)AID(+/+) mice. In contrast, none of the IL-10(-/-)AID(-/-) mice developed cancers except only one case of neoplasia in the distal colon. These findings suggest that the proinflammatory cytokine-induced aberrant production of AID links colonic inflammation to an enhanced genetic susceptibility to oncogenic mutagenesis. Targeting AID could be a novel strategy to prevent colitis-associated colon carcinogenesis irrespective of ongoing colonic inflammation.

A novel mouse model of hepatocarcinogenesis triggered by AID causing deleterious p53 mutations.

Activation-induced cytidine deaminase (AID), the only enzyme that is known to be able to induce mutations in the human genome, is required for somatic hypermutation and class-switch recombination in B lymphocytes. Recently, we showed that AID is implicated in the pathogenesis of human cancers including hepatitis C virus (HCV)-induced human hepatocellular carcinoma (HCC). In this study, we established a new AID transgenic mouse model (TNAP-AID) in which AID is expressed in cells producing tissue-nonspecific alkaline phosphatase (TNAP), which is a marker of primordial germ cells and immature stem cells, including ES cells. High expression of TNAP was found in the liver of the embryos and adults of TNAP-AID mice. HCC developed in 27% of these mice at the age of approximately 90 weeks. The HCC that developed in TNAP-AID mice expressed alpha-fetoprotein and had deleterious mutations in the tumour suppressor gene Trp53, some of which corresponded to those found in human cancer. In conclusion, TNAP-AID is a mouse model that spontaneously develops HCC, sharing genetic and phenotypic features with human HCC, which develops in the inflamed liver as a result of the accumulation of genetic changes.

Examination of signalling pathways involved in muscarinic responses in bovine ciliary muscle using YM-254890, an inhibitor of the Gq/11 protein.

BACKGROUND AND PURPOSE: In the ciliary muscle, the tonic component of the contraction produced by cholinergic agonists is highly dependent on Ca2+ provided by influx through non-selective cation channels (NSCCs) opened by stimulation of M3 muscarinic receptors. We examined effects of YM-254890 (YM), a Gq/11-specific inhibitor, on contraction, NSCC currents and [Ca2+]i elevation induced by carbachol (CCh). EXPERIMENTAL APPROACH: Isometric tension was recorded from ciliary muscle bundles excised from bovine eyes. In ciliary myocytes dispersed with collagenase and cultured for 1-5 days, whole-cell currents were recorded by voltage clamp and the intracellular free Ca2+ concentration [Ca2+]i was monitored using the Fluo-4 fluorophore. Existence and localization of M3 receptors and the alpha subunit of Gq/11 (Galpha(q/11)) were examined by immunofluorescence microscopy using AlexaFluor-conjugated antibodies. KEY RESULTS: Both phasic and tonic components of contractions evoked by 2 microM CCh were inhibited by YM (3-10 microM) in a dose-dependent manner. In the cultured cells, CCh (0.05-10 microM) evoked an NSCC current as well as an elevation of the [Ca2+]i. Both initial and sustained phases of these CCh-evoked responses were abolished by YM (3-10 microM). Immunostaining of the cytoplasmic side of the plasma membrane of ciliary myocytes revealed a dense distribution of M3 receptors and Galpha(q/11). CONCLUSIONS AND IMPLICATIONS: The tonic as well as phasic component of the ciliary muscle contraction appears to be under control of signals conveyed by a G(q/11)-coupled pathway. YM is a useful tool to assess whether Gq/11 is involved in a signal transduction system.

Agonist and antagonist sensitivity of non-selective cation channel currents evoked by muscarinic receptor stimulation in bovine ciliary muscle cells.

1 In the bovine ciliary muscle, stimulation of muscarinic receptors with carbachol (CCh) opens two types of non-selective cation channels (NSCCS and NSCCL) with widely different unitary conductances (100 fS and 35 pS). Here we examined the dependence of the activity of NSCCS on the agonist (CCh) concentration by whole-cell voltage clamp in freshly isolated bovine ciliary muscle cells. We also examined the sensitivity of CCh-evoked NSCCS currents to several muscarinic receptor antagonists. 2 The voltage clamp experiments were carried out using Ba2+ as the charge carrier, as this divalent cation is the most permeant for NSCCS of the alkali and alkaline earth metal ions hitherto examined, whereas it is relatively impermeant to NSCCL. For the dose-activation relationship obtained, the apparent dissociation constant K was estimated to be 0.5 +/- 0.2 microm (n = 31), a value of an order of magnitude smaller than the one reported for CCh-evoked NSCCL currents in our previous experiments. 3 In the dose-inhibition experiments we observed that the CCh-evoked NSCCS currents were inhibited by the muscarinic antagonists with the following potency sequence: atropine approximately 4-DAMP >> pirenzepine > AF-DX116, indicating that the activation of NSCCS by CCh is mediated by an M3 muscarinic receptor. 4 We have previously shown by reverse transcriptase-polymerase chain reaction that the bovine ciliary muscle contains mRNAs for several transient receptor potential channel homologues (TRPC1, TRPC3, TRPC4 and TRPC6) which are attracting attention as molecular candidates for receptor-operated NSCCs. In the present experiments, we succeeded in visually identifying these TRPCs in the plasma membrane of cultured bovine ciliary muscle cells by immunofluorescence microscopy.

Regulation of cardiac CFTR Cl(-) channel activity by a Mg(2+)-dependent protein phosphatase.

Dephosphorylation of the CFTR Cl(-) channel is known to be induced by both okadaic-acid- (OA-) sensitive and -insensitive protein phosphatases (PPs). In the present study, the effects of cytosolic free Mg(2+) on the cardiac CFTR Cl(-) current were examined in relation to the latter PP activity in guinea pig ventricular myocytes. Even when maintaining intracellular Mg-ATP at millimolar concentrations under whole-cell patch-clamp mode, cAMP-activated Cl(-) conductance was reversibly suppressed by cytosolic free Mg(2+), with an IC(50) of around 2.5 mmol/l. In contrast, changes in the cytosolic concentration of free Mg(2+) ([Mg(2+)](i)) had no effect on genistein-activated CFTR Cl(-) currents. The Mg(2+) effect on cAMP-activated CFTR Cl(-) conductance was completely reversed by application of anthracene-9-carboxylic acid (9-AC), which was previously shown to inhibit an OA-insensitive PP in cardiac myocytes. A 9-AC-sensitive fraction of endogenous PP activity in the extract of guinea pig ventricle was found to be activated by free Mg(2+) at millimolar concentrations but to be inactive at micromolar concentrations. The intracellular application of OA failed to activate basal Cl(-) conductance at millimolar [Mg(2+)](i). In the presence of OA, however, basal Cl(-) conductance became activated either by reducing [Mg(2+)](i) to micromolar concentrations or by applying 9-AC. Thus, we conclude that a Mg(2+)-dependent PP sensitive to 9-AC plays a key role in the cAMP-mediated regulation of cardiac CFTR Cl(-) channel at physiological [Mg(2+)](i)under both basal and cAMP-activated conditions. Also, it appears that the genistein-activated conformation of the cardiac CFTR channel is not sensitive to the Mg(2+)-dependent PP.

Reliability and validity of Kasahara's scale of melancholic type of personality (Typus melancholicus) in a German sample population.

We explored the reliability and validity of Kasahara's scale of melancholic type of personality (KMT) in a German sample population. Subjects comprised 66 patients diagnosed with an affective disorder (F3, ICD-10) and 94 controls. Concerning reliability, KMT scores showed internal consistency with Cronbach's alpha coefficients of 0.65 for patients and 0.67 for controls. The KMT items, except for number 13 in controls, showed significant item-total correlations. In a test-retest procedure, the KMT total score and individual item scores were statistically similar and correlated. These results indicate reliability of the KMT. Concerning validity, KMT scores were significantly higher in patients than in controls. By controlling the effects of age and sex, partial correlation coefficients in a comparison of KMT and Zerssen's F-List (F-List) scores were 0.40 in patients and 0.53 in controls. These results show both the constructive and concurrent validity of the KMT. Sufficient reliability and validity of the KMT were shown in this German sample population to encourage cross-cultural investigation of Typus melancholicus.

Chromosome evolution involving Robertsonian rearrangements in Xyrichthys fish (Labridae, Perciformes).

Three Xyrichthys fish (Labridae, Perciformes), X. pavo, X. dea, and X. twistii, were cytogenetically studied. X. pavo and X. dea had 2n = 44 chromosomes, which were all acrocentric. X. twistii had 2n = 22 chromosomes consisting of eighteen meta- and submetacentric and four acrocentric chromosomes. The cellular DNA contents of X. pavo and X. twistii measured using flow cytometry were nearly equal. These results suggest that the karyotype of X. twistii evolved by decreasing the number of chromosomes by fusion events, probably Robertsonian fusion. Cytogenetic relationships among the three species were surmized on the basis of features on the karyotypes and the NOR locations. A large gap in the chromosome number between 2n = 44 and 2n = 22 is an interesting feature related to the process of chromosome evolution.

Eysenck's personality and tobacco/nicotine dependence in male ever-smokers in Japan.

To examine the relationship between Eysenck's personality traits and tobacco/nicotine dependence in a male population, a random sample of 200 male ever-smokers aged 35 or older from a community in Japan were interviewed using the World Health Organization (WHO) Composite International Diagnostic Interview (CIDI), which yielded ICD-10, DSM-III-R and DSM-IV diagnoses of tobacco/nicotine dependence. They were also asked to complete the Fagerstrom Tobacco Questionnaire (FTQ) and the short-form Eysenck Personality Questionnaire-Revised. A total of 136 subjects completed both the interview and the questionnaire. Neuroticism scores were significantly higher in those who had lifetime diagnosis of tobacco/nicotine dependence according to ICD-10, DSM-IV, or FTQ criteria than nondependent ever-smokers (p < 0.05). Lie scores were significantly lower in DSM-III-R or DSM-IV tobacco/nicotine dependence than in nondependent ever-smokers (p < 0.05). Multiple logistic regression indicated that neuroticism was significantly associated with a higher risk of ICD-10 tobacco/nicotine dependence (p < 0.05), after controlling for age, education, employment status and smoking behaviors; lie score was significantly associated with a lower risk of DSM-III-R tobacco/nicotine dependence (p < 0.05). It is suggested that neuroticism is associated with a higher risk of tobacco/nicotine dependence in male Japanese ever-smokers. A nonconforming and rebellious attitude or reporting bias represented by higher lie score may be associated with lower rates of tobacco/nicotine dependence.

Effects of modification of the hydrophobic C-1-C-16 segment of tautomycin on its affinity to type-1 and type-2A protein phosphatases.

Among the naturally occurring toxins that are known to have specific inhibitory effects on type-1 and type-2A protein phosphatases (PP1 and PP2A), tautomycin (TM) is unique in that it exhibits significantly higher affinity to PP1 than to PP2A. The ratio of the dissociation constant for the PP1-TM interaction to that for the PP2A-TM interaction (the PP1/PP2A ratio) is 0.01-0.03. The aim of the present study was to evaluate the possible contributions of the C-1-C-16 segment of TM to the affinity characteristics of the toxin. The relatively hydrophobic segment contains a spiroketal motif whose enantiomeric form is present in okadaic acid (OA), which exhibits exceedingly higher affinity to PP2A than to PP1. We therefore synthesized two TM analogues: TM1 in which the side chains of the spiroketal motif of TM were removed but its absolute configuration was retained, and TM2 in which the spiroketal motif of TM1 was replaced with its enantiomeric form. The effects of TM, TM1 and TM2 on the activities of the native catalytic subunits of PP1 (PP1C) and PP2A and a recombinant gamma isoform of PP1 (PP1gamma) were examined. The PP1/PP2A ratio determined thereby was 0.2-0.5 for TM1 and 5-10 for TM2. Both the presence of the side chains and the stereochemistry of the spiroketal moieties may be major determining factors for the affinity characteristics of TM. We also show that a monoclonal antibody raised against OA binds to TM2 albeit with much lower affinity than to OA, whereas it exhibits no measurable affinities to TM and TM1.

Effects of iodoacetate on spontaneous electrical activity, slow wave, in the circular muscle of the guinea-pig gastric antrum.

In the circular muscle of the guinea-pig gastric antrum, the contribution of glycolysis to spontaneous electrical activity, slow wave, was studied. The slow wave could be maintained without a marked change in glucose-free solution for more than 1 h even when treated with iodoacetic acid (IAA, 0.1-0.5 mM). However, reapplication of glucose following the IAA treatment produced clear inhibitory effects on the slow wave. Lactate release from the tissue was reduced to about 10% of the control by IAA (0.1 mM) in the absence of glucose and there was very slow recovery on glucose reapplication. This suggests that IAA did not block glycolysis completely and that the inhibition of slow wave was mainly due to the accumulation of some metabolites. Small electrical activity often remained during the inhibition by IAA and glucose. When the excitability of the smooth muscle was increased by Co(2+) application or Na(+) removal, slow wave-like activity could be generated under the condition in which the slow wave was strongly inhibited by IAA and glucose. These results may be explained by assuming that the accumulation of glycolytic metabolites decreases the excitability of smooth muscle cells and also reduces the driving potential generated in the interstitial cells of Cajal to a subthreshold level for the slow wave in the smooth muscle cells.

Effects of specific modifications of several hydroxyls of tetrodotoxin on its affinity to rat brain membrane.

The widely used sodium channel blocker tetrodotoxin (TTX) is a compound that has six hydroxyl residues at the C-4, C-6, C-8, C-9, C-10, and C-11 positions in addition to a guanidinium group, which is positively charged in biological pH range. Thirteen analogs of this toxin with structural modifications involving one or more of these hydroxyls were examined on their affinity to a rat brain membrane preparation, which is known to contain sodium channels abundantly. The equilibrium dissociation constants associated with the binding of TTX and its analogs to the sodium channels were estimated, from their ability to inhibit the binding of [3H]saxitoxin, as follows (in nM): TTX, 1.8; chiriquitoxin, 1.0; 11-oxoTTX, 1.5; 11-norTTX-6,6-diol, 1.6; 11-norTTX-6(S)-ol, 23; 11-norTTX-6(R)-ol, 31; 11-deoxyTTX, 37; 6-epiTTX, 39; 4-epiTTX, 68; 4,9-anhydroTTX, 180; TTX-8-O-hemisuccinate, >380; TTX-11-carboxylic acid, >2300; tetrodonic acid, >3600; 5,6,11-trideoxyTTX, >5000. The reduction of the affinity observed with the analogs involving reduction or translocation of the hydroxyls at C-6 and C-11 is indicative of the contribution of these residues to the binding to sodium channels as hydrogen bond donors. The especially large value of the dissociation constant for TTX-11-carboxylic acid is consistent with the idea that the C-11-hydroxyl forms a hydrogen bond with a carboxylic acid residue of the channel protein. The markedly low affinity of TTX-8-O-hemisuccinate may possibly be ascribable to intramolecular salt-bridge formation, which neutralizes the positive charge of the guanidinium group.

Mental health problems after stroke.

We investigated the mental health of 47 subjects (30 men, mean age 63.8+/-7.7; 17 women, mean age 68.9+/-8.7) with the 60-item General Health Questionnaire (GHQ). All the subjects lived at home in a Japanese rural community and were examined from 2 to 3 years after suffering a stroke. Among the subjects, 18 (38.3%) had GHQ scores of 17 or more, which indicated a mental health problem (MHP). The following variables were included in multiple logistic regression analysis: age, sex (men/women), grade of motor paralysis (no/slight/moderate/severe), side of motor paralysis (no/left side/right side/both sides: in analysis, we used dummy variables), paresthesia (no/yes), rehabilitation (need no rehabilitation or participate in rehabilitation/fail to participate in rehabilitation), social support (not needed or sufficient/insufficient) and overall physical recovery (1/2/3: 1 = 67-100, 2 = 34-66, and 3 = 0-33 on a visual analog scale 100 mm long, 100 meaning full recovery). In univariate analysis all variables except age and sex showed statistically significant associations with MHP. In multivariate analysis, only one variable, overall physical recovery', had a statistically independent association with the status of MHP (Odds ratio 4.39, 95% confidence interval 1.46-13.19). The results of logistic regression analysis indicate that the presence of an MHP is more strongly dependent upon subjective assessment about overall physical recovery after stroke than upon physical impairments and the other psychosocial variables. Therefore, in the community setting, the visual analog scale of overall physical recovery is considered to be a simple, valid method for assessing MHP following stroke.

Life-time prevalence and risk factors of tobacco/nicotine dependence in male ever-smokers in Japan.

UNLABELLED: To estimate the life-time prevalence rate of tobacco/nicotine dependence and demographic variables and smoking habits associated with the disorder in male ever-smokers in Japan. DESIGN: A cross-sectional community-based interview study. SETTING: Takayama city, Gifu Prefecture, Japan. PARTICIPANTS: A total of 170 male ever-smokers aged 35 years or older selected randomly from a community in Japan were interviewed. The response rate was 85%. MEASUREMENTS: The WHO Composite International Diagnostic Interview (CIDI) was used to make diagnoses of tobacco/nicotine dependence according t ICD-10, DSM-III-R and DSM-IV. The Fagerstrom Tolerance Questionnaire (FTQ) was also administered and those who had a FTQ score of 7 or above were identified. FINDINGS: The life-time prevalence rates of tobacco/nicotine dependence in male ever-smokers were 42%, 26% and 32% according to ICD-10, DSM-III-R and DSM-IV criteria, respectively; 19% had a FTQ score of 7 or above. The ICD-10 diagnosis was significantly and negatively associated with quitting smoking (p < 0.05). Multiple logistic regression analyses indicated that number of cigarettes per day when they smoked the most was significantly associated with higher life-time risks of the disorder according to DSM-III-R, DSM-IV and Fagerstrom's classification (p < 0.05). The length of cigarette smoked was associated with higher life-time risks of ICD-10 and DSM-IV diagnoses, and years of smoking were associated with higher life-time risks of ICD-10, DSM-III-R and DSM-IV diagnoses (p < 0.05). Younger birth cohorts had higher cumulative rates of the disorder according to DSM-IV (p for trend < 0.05). CONCLUSIONS: Life-time prevalence rates of tobacco/nicotine dependence according to ICD-10, DSM-III-R and DSM-IV in male ever-smokers in Japan were within the range of rates reported in previous US studies; rates of FTQ score of 7 or above were lower. Fagerstrom scores and diagnostic criteria appear to reflect different aspects of dependence.

Correlation between history of contact with people living with HIV/AIDS (PWAs) and tolerant attitudes toward HIV/AIDS and PWAs in rural Thailand.

This study examines the hypothesis that people who have more contact with PWAs (people living with AIDS) are more tolerant than those who have no contact with them. Four provinces with different incidence of AIDS in 4 different regions of Thailand were selected. Structured questionnaire interviews were conducted with village people, asking about their history of contact with PWAs, and knowledge and attitudes toward HIV/AIDS and PWAs (n = 434). An 'Attitude Score', which indicates an accepting attitude (or tolerance) toward HIV/AIDS and PWAs, was developed using the results of the questionnaire on attitudes. Six factors: sex, education, age, province, knowledge, and history of contact with PWAs were positively correlated with the Attitude Score. After a multiple regression analysis, contact with PWAs was significantly associated with Attitude Score. This study is one of the first analytical studies conducted in a non-Western country to show that people's tolerant attitudes towards HIV/AIDS and PWAs are positively related to their history of contact with HIV/AIDS and PWAs. This findings should have important implications for future educational programmes and preventative intervention.

Development of genotoxicity assay systems that use aquatic organisms.

Our aim is to develop and evaluate monitoring systems that use aquatic organisms to assess the genotoxicity of water in the field and in the laboratory. In a field study, we have shown that the micronucleus assay is applicable to freshwater and marine fishes and that gill cells are more sensitive than hematopoietic cells to micronucleus-inducing agents. Gill cells from Carassius sp. (Funa) and Zacco platypus (Oikawa) collected upstream on the Tomio River (Nara, Japan), tended to have lower micronucleus frequencies than gill cells from fish collected at the midstream of the river. Leiognathus nuchalis (Hiiragi) and Ditrema temmincki (Umitanago), small marine fishes collected periodically at Mochimune Harbor (Shizuoka, Japan), showed seasonal differences in the frequencies of micronucleated gill cells and erythrocytes; they were highest in summer. For laboratory studies, we developed a method for analyzing chromosomal aberrations and micronuclei using Rhodeus ocellatus ocellatus (rose bitterling) embryos. One day after artificial insemination (gastrula stage), we observed structural chromosomal aberrations and micronuclei in the cells of embryos grown in water containing trichloroethylene. Although more work is needed to fully assess their sensitivity, these assays show promise as a means of detecting environmental genotoxins.

Phosphorylated retinoblastoma protein stimulates DNA polymerase alpha.

Human retinoblastoma (Rb) protein, immunopurified from an extract of recombinant baculovirus infected cells, stimulated 10-100-fold the activity of DNA polymerase alpha from calf thymus or human HeLa cells. Purified Rb protein is composed of two electrophoretically distinguishable forms, i.e., partially phosphorylated and under-phosphorylated forms. Dephosphorylation of Rb protein by protein phosphatase 2A largely diminished its stimulatory effect. On the other hand, a hyperphosphorylated Rb protein, obtained from insect cells overexpressing Rb protein, cyclin E and cyclin-dependent kinase 2 simultaneously, stimulated DNA polymerase alpha more strongly than the singly-expressed Rb protein. These results indicate that the phosphorylation is crucial for the stimulation. Rb protein isolated from human Burkitt lymphoma Raji cells also stimulated DNA polymerase alpha. In contrast, Rb protein did not affect eukaryotic DNA primase or Klenow fragment of Escherichia coli DNA polymerase I. By immunoprecipitation using anti-DNA polymerase alpha antibody, Rb protein in nuclear extract of Raji cells was co-precipitated with DNA polymerase alpha. This result indicates that DNA polymerase alpha exists as a complex containing phosphorylated Rb protein in cells. DNA polymerase alpha specifically bound to a purified Rb protein-immobilized Sepharose column. Rb protein also bound to DNA polymerase alpha trapped to anti-DNA polymerase alpha antibody-Sepharose column, suggesting the direct association of these two proteins. These observations suggest a new function of phosphorylated Rb protein in the regulation of DNA replication.