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It is often assumed that genotoxic substances will be detected more easily by using in vitro rather than in vivo genotoxicity tests since higher concentrations, more cytotoxicity and static exposures can be achieved. However, there is a paucity of data demonstrating whether genotoxic substances are detected at lower concentrations in cell culture in vitro than can be reached in the blood of animals treated in vivo. To investigate this issue, we compared the lowest concentration required for induction of chromosomal damage in vitro (lowest observed effective concentration, or LOEC) with the concentration of the test substance in blood at the lowest dose required for biologically relevant induction of micronuclei in vivo (lowest observed effective dose, or LOED). In total, 83 substances were found for which the LOED could be identified or estimated, where concentrations in blood and micronucleus data were available via the same route of administration in the same species, and in vitro chromosomal damage data were available. 39.8 % of substances were positive in vivo at blood concentrations that were lower than the LOEC in vitro, 22.9 % were positive at similar concentrations, and 37.3 % of substances were positive in vivo at higher concentrations. Distribution analysis showed a very wide scatter of > 6 orders of magnitude across these 3 categories. When mode of action was evaluated, the distribution of clastogens and aneugens across the 3 categories was very similar. Thus, the ability to detect induction of micronuclei in bone marrow in vivo regardless of the mechanism for micronucleus induction, is clearly not solely determined by the concentration of test substance which induced chromosomal damage in vitro., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Acetaminophen, a nonmutagenic compound as previously concluded from bacteria, in vitro mammalian cell, and in vivo transgenic rat assays, presented a good profile as a nonmutagenic reference compound for use in the international multilaboratory Pig-a assay validation. Acetaminophen was administered at 250, 500, 1,000, and 2,000 mg·kg -1 ·day -1 to male Sprague Dawley rats once daily in 3 studies (3 days, 2 weeks, and 1 month with a 1-month recovery group). The 3-Day and 1-Month Studies included assessments of the micronucleus endpoint in peripheral blood erythrocytes and the comet endpoint in liver cells and peripheral blood cells in addition to the Pig-a assay; appropriate positive controls were included for each assay. Within these studies, potential toxicity of acetaminophen was evaluated and confirmed by inclusion of liver damage biomarkers and histopathology. Blood was sampled pre-treatment and at multiple time points up to Day 57. Pig-a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as CD59-negative RBC and CD59-negative RET frequencies, respectively. No increases in DNA damage as indicated through Pig-a, micronucleus, or comet endpoints were seen in treated rats. All positive controls responded as appropriate. Data from this series of studies demonstrate that acetaminophen is not mutagenic in the rat Pig-a model. These data are consistent with multiple studies in other nonclinical models, which have shown that acetaminophen is not mutagenic. At 1,000 mg·kg -1 ·day -1 , Cmax values of acetaminophen on Day 28 were 153,600 ng/ml and 131,500 ng/ml after single and repeat dosing, respectively, which were multiples over that of clinical therapeutic exposures (2.6-6.1 fold for single doses of 4,000 mg and 1,000 mg, respectively, and 11.5 fold for multiple dose of 4,000 mg) (FDA 2002). Data generated were of high quality and valid for contribution to the international multilaboratory validation of the in vivo Rat Pig-a Mutation Assay., (© 2020 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.)

Background: Flubendazole, originally developed to treat infections with intestinal nematodes, has been shown to be efficacious in animal models of filarial infections. For treatment of filarial nematodes, systemic exposure is needed. For this purpose, an orally bioavailable amorphous solid dispersion (ASD) formulation of flubendazole was developed. As this formulation results in improved systemic absorption, the pharmacokinetic and toxicological profile of the flubendazole ASD formulation have been assessed to ensure human safety before clinical trials could be initiated., Methods & Findings: Safety pharmacology, toxicity and genotoxicity studies have been conducted with the flubendazole ASD formulation. In animals, flubendazole has good oral bioavailability from an ASD formulation ranging from 15% in dogs, 27% in rats to more than 100% in jirds. In in vivo toxicity studies with the ASD formulation, high systemic exposure to flubendazole and its main metabolites was reached. Flubendazole, up to high peak plasma concentrations, does not induce Cmax related effects in CNS or cardiovascular system. In repeated dose toxicity studies in rats and dogs, flubendazole-induced changes were observed in haematological, lymphoid and gastrointestinal systems and in testes. In dogs, the liver was an additional target organ. Upon treatment cessation, at least partial recovery was observed for these changes in dogs. In rats, the No Observed Adverse Effect Level (NOAEL) was 5 mg (as base)/kg body weight/day (mg eq./kg/day) in males and 2.5 mg eq./kg/day in females. In dogs, the NOAEL was lower than 20 mg eq./kg/day. Regarding genotoxicity, flubendazole was negative in the Ames test, but positive in the in vivo micronucleus test., Conclusions: Based on these results, in combination with previously described genotoxicity and reproductive toxicity data and the outcome of the preclinical efficacy studies, it was concluded that no flubendazole treatment regimen can be selected that would provide efficacy in humans at safe exposure., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: ownership of stocks or shares of Johnson & Johnson. All authors are employees of Johnson & Johnson.

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiated international validation study of in vivo rat alkaline comet assay (comet assay), p-phenylenediamine dihydrochloride (PPD), o-phenylphenol sodium salt (OPP), and 2,4-diaminotoluene (2,4-DAT), were analyzed in this laboratory as coded test chemicals. Male Sprague-Dawley rats (7-9 weeks of age) were given three oral doses of the test compounds, 24 and 21 h apart and liver and stomach were sampled 3h after the final dose administration. Under the conditions of the test, no increases in DNA damage were observed in liver and stomach with PPD and OPP up to 100 and 1000 mg/kg/day, respectively. 2,4-DAT, a known genotoxic carcinogen, induced a weak but reproducible, dose-related and statistically significant increase in DNA damage in liver cells while no increases were observed in stomach cells., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In genotoxicity testing of pharmaceuticals the rodent alkaline comet assay is being increasingly used as a second in vivo assay in addition to the in vivo micronucleus assay to mitigate in vitro positive results as recommended by the ICH S2(R1) guideline. This paper summarizes a survey suggested by the Safety Working Party of European Medicines Agency (EMA), and conducted by the European Federation of Pharmaceutical Industries and Associations (EFPIA) to investigate the experience among European pharmaceutical companies by conducting the in vivo comet assay for regulatory purpose. A special focus was given on the typology of the obtained results and to identify potential difficulties encountered with the interpretation of study data. The participating companies reported a total of 147 studies (conducted in-house or outsourced) and shared the conclusion on the comet assay response for 136 studies. Most of the studies were negative (118/136). Only about 10% (14/136 studies) of the comet assays showed a positive response. None of the positive comet assay results were clearly associated with organ toxicity indicating that the positive responses are not due to cytotoxic effects of the compound in the tissue examined. The number of comet assays with an equivocal or inconclusive response was rare, respectively <1% (1/147 studies) and 2% (3/147 studies). In case additional information (e.g. repeat assay, organ toxicity, metabolism, tissue exposure) would have been available for evaluation, a final conclusion could most probably have been drawn for most or all of these studies. All (46) negative in vivo comet assays submitted alongside with a negative in vivo micronucleus assay were accepted by the regulatory authorities to mitigate a positive in vitro mammalian cell assay following the current ICH S2 guidance. The survey results demonstrate the robustness of the comet assay and the regulatory acceptance of the current ICH S2 guidance., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Multivariate longitudinal or clustered data are commonly encountered in clinical trials and toxicological studies. Typically, there is no single standard endpoint to assess the toxicity or efficacy of the compound of interest, but co-primary endpoints are available to assess the toxic effects or the working of the compound. Modeling the responses jointly is thus appealing to draw overall inferences using all responses and to capture the association among the responses. Non-Gaussian outcomes are often modeled univariately using exponential family models. To accommodate both the overdispersion and hierarchical structure in the data, Molenberghs et al. A family of generalized linear models for repeated measures with normal and conjugate random effects. Statistical Science 2010; 25:325-347 proposed using two separate sets of random effects. This papers considers a model for multivariate data with hierarchically clustered and overdispersed non-Gaussian data. Gamma random effect for the over-dispersion and normal random effects for the clustering in the data are being used. The two outcomes are jointly analyzed by assuming that the normal random effects for both endpoints are correlated. The association structure between the response is analytically derived. The fit of the joint model to data from a so-called comet assay are compared with the univariate analysis of the two outcomes., (Copyright © 2012 John Wiley & Sons, Ltd.)

A collaborative international trial was conducted to evaluate the reproducibility and transferability of an in vivo mutation assay based on the enumeration of CD59-negative rat erythrocytes, a phenotype that is indicative of Pig-a gene mutation. Fourteen laboratories participated in this study, where anti-CD59-PE, SYTO 13 dye, and flow cytometry were used to determine the frequency of CD59-negative erythrocytes (RBC(CD59-)) and CD59-negative reticulocytes (RET(CD59-)). To provide samples with a range of mutant phenotype cell frequencies, male rats were exposed to N-ethyl-N-nitrosourea (ENU) via oral gavage for three consecutive days (Days 1-3). Each laboratory studied 0, 20, and 40 mg ENU/kg/day (n = 5 per group). Three sites also evaluated 4 mg/kg/day. At a minimum, blood samples were collected three times: predosing and on Days 15 and 30. Blood samples were processed according to a standardized sample processing and data acquisition protocol, and three endpoints were measured: %reticulocytes, frequency of RET(CD59-) , and frequency of RBC(CD59-) . The methodology was found to be reproducible, as the analysis of technical replicates resulted in experimental coefficients of variation that approached theoretical values. Good transferability was evident from the similar kinetics and magnitude of the dose-related responses that were observed among different laboratories. Concordance correlation coefficients showed a high level of agreement between the reference site and the test sites (range: 0.87-0.99). Collectively, these data demonstrate that with adequate training of personnel, flow cytometric analysis is capable of reliably enumerating mutant phenotype erythrocytes, thereby providing a robust in vivo mutation assay that is readily transferable across laboratories., (Copyright © 2011 Wiley-Liss, Inc.)

A collaborative trial was conducted to evaluate the possibility of integrating the rat-liver Comet assay into repeat-dose toxicity studies. Fourteen laboratories from Europe, Japan and the USA tested fifteen chemicals. Two chemicals had been previously shown to induce micronuclei in an acute protocol, but were found negative in a 4-week Micronucleus (MN) Assay (benzo[a]pyrene and 1,2-dimethylhydrazine; Hamada et al., 2001); four genotoxic rat-liver carcinogens that were negative in the MN assay in bone marrow or blood (2,6-dinitrotoluene, dimethylnitrosamine, 1,2-dibromomethane, and 2-amino-3-methylimidazo[4,5-f]quinoline); three compounds used in the ongoing JaCVAM (Japanese Center for the Validation of Alternative Methods) validation study of the acute liver Comet assay (2,4-diaminotoluene, 2,6-diaminotoluene and acrylamide); three pharmaceutical-like compounds (chlordiazepoxide, pyrimethamine and gemifloxacin), and three non-genotoxic rodent liver carcinogens (methapyrilene, clofibrate and phenobarbital). Male rats received oral administrations of the test compounds, daily for two or four weeks. The top dose was meant to be the highest dose producing clinical signs or histopathological effects without causing mortality, i.e. the 28-day maximum tolerated dose. The liver Comet assay was performed according to published recommendations and following the protocol for the ongoing JaCVAM validation trial. Laboratories provided liver Comet assay data obtained at the end of the long-term (2- or 4-week) studies together with an evaluation of liver histology. Most of the test compounds were also investigated in the liver Comet assay after short-term (1-3 daily) administration to compare the sensitivity of the two study designs. MN analyses were conducted in bone marrow or peripheral blood for most of the compounds to determine whether the liver Comet assay could complement the MN assay for the detection of genotoxins after long-term treatment. Most of the liver genotoxins were positive and the three non-genotoxic carcinogens gave negative result in the liver Comet assay after long-term administration. There was a high concordance between short- and long-term Comet assay results. Most compounds when tested up to the maximum tolerated dose were correctly detected in both short- and long-term studies. Discrepant results were obtained with 2,6 diaminotoluene (negative in the short-term, but positive in the long-term study), phenobarbital (positive in the short-term, but negative in the long-term study) and gemifloxacin (positive in the short-term, but negative in the long-term study). The overall results indicate that the liver Comet assay can be integrated within repeat-dose toxicity studies and efficiently complements the MN assay in detecting genotoxins. Practical aspects of integrating genotoxicity endpoints into repeat-dose studies were evaluated, e.g. by investigating the effect of blood sampling, as typically performed during toxicity studies, on the Comet and MN assays. The bleeding protocols used here did not affect the conclusions of the Comet assay or of the MN assays in blood and bone marrow. Although bleeding generally increased reticulocyte frequencies, the sensitivity of the response in the MN assay was not altered. These findings indicate that all animals in a toxicity study (main-study animals as well as toxicokinetic (TK) satellite animals) could be used for evaluating genotoxicity. However, possible logistical issues with scheduling of the necropsies and the need to conduct electrophoresis promptly after tissue sampling suggest that the use of TK animals could be simpler. The data so far do not indicate that liver proliferation or toxicity confound the results of the liver Comet assay. As was also true for other genotoxicity assays, criteria for evaluation of Comet assay results and statistical analyses differed among laboratories. Whereas comprehensive advice on statistical analysis is available in the literature, agreement is needed on applying consistent criteria., (Copyright © 2010 Elsevier B.V. All rights reserved.)

In order to support drug research in the selection process for non-embryotoxic pharmaceutical compounds, a screening method for embryotoxicity is needed. The murine embryonic stem cell test (EST) is a validated in vitro test based on two permanent mouse cell lines and delivering results in 10-days. Implementation of this test within our laboratory, revealed variability in the differentiation potential of the embryonic stem cells and, as a consequence, a lot of assays needed to be rejected due the fact the acceptance criteria were not reached. In order to gain a better yield of contracting myocardial cells, we used (1) a stringent control of the cell growth during subcultivation and a standardised hanging drop culture method and (2) a non-enzymatic cell harvest instead of a trypsin/EDTA cell harvest. Implementing of these cell culture modifications resulted in a decreased variability in the size of embryonic bodies, an increase of the number of acceptable tests and a significant increase of the differentiation potential of embryonic cells into strong beating myocardium, which made scoring less time consuming. Testing of 6 reference compounds in the optimized EST showed that the cell culture modifications did not changed the in vitro classification.

The objective of the current study was to develop cellular delivery approaches for catalytic DNA enzymes (DNAzymes) which cleave targeted messenger RNA, using vectors based on colloidal gold. The model DNAzyme was a 32mer oligonucleotide designed to specifically interact with and cleave c-myc mRNA. Colloidal gold particles were prepared by reduction of tetrachlororauric [III] acid with sodium citrate. Particles could be produced in the 1-90 nm range. A cationic substrate linked to transferrin was electrostatically/hydrophobically bound to the gold particle. These vectors were then treated with the DNAzyme to yield the condensed DNA-cationic polymer-particulate product. The pH (4-11.5), the quantity of the DNAzymes (0.079-0.567 microg/probe), the cationic polymer (polylysine (PL) or polyethylenimine (PEI)) as well as the surfactant (PVP) concentration (0-0.5%) were varied to give stable constructs which decomplexed under the desired conditions (i.e., in lysosomes and at lower pH values). Cellular uptake of the FITC-labelled c-myc DNAzyme incorporated in this vector was measured using FACS analysis in human HT29 colon carcinoma cells. Data suggested that PEI gave better delivery efficiencies than PL. The use of PVP to stabilize the formed dispersions was detrimental to DNAzyme delivery when PL was used but had little effect in the PEI systems. In the best cases, delivery to 77% of the cells was possible using PEI with the PVP stabilizer and completing the DNA condensation at pH 5.5 with 0.118 microg of DNAzyme/probe. In contrast, the best conditions for PL gave only transfection to 43% of the cells (no PVP, condensed at pH 5.7 and with a loading of 0.079 microg DNAzyme/probe). The PL probe tended to be more toxic than the PEI-based systems (65% cell death in PL transfected cells compared to 22% for PEI). These results suggest that cellular targeting using colloidal gold appears feasible for DNAzyme delivery.

According to the current Organization of Economic Cooperation and Development (OECD) and International Committee on Harmonization (ICH) guidelines for the mammalian erythrocyte micronucleus (MN) test, analysis of peripheral blood reticulocytes (RETs) for the presence of micronuclei can be performed using flow cytometry. The MicroFlow PLUS method (Litron Laboratories, Rochester, NY) for MN analysis by flow cytometry is based on the binding of FITC-labeled antibodies to the CD71 transferrin receptor of immature RETs, on parallel RNA degradation, and on propidium iodide staining of DNA present as micronuclei. The objective of this study was to assess the sensitivity of this flow cytometry method to detect time- and dose-dependent induction of micronuclei in mouse peripheral blood RETs after treatment with nine chemical agents. Five known clastogens, two known aneugens, and two compounds previously reported to be inactive in the mouse bone marrow MN test were evaluated at three dose levels. Multiple blood sampling of the same animal before and at two time points after treatment was conducted. All known mutagens produced a dose-dependent increase in micronucleated reticulocytes (MN-RETs); the compounds previously shown to be inactive in the in vivo MN test were also negative using the present methodology. The highest frequency of MN-RETs was observed at 48 hr after treatment, except for 5-fluorouracil, which had its peak response at 72 hr. The results indicate that micronuclei can be measured by multiple blood sampling of the same animal before and after treatment without altering the sensitivity of the assay. The results confirm that the flow cytometric assessment of MN-RETs in mouse peripheral blood using MicroFlow PLUS is a sensitive method with high analysis throughput, and robust quality control., ((c) 2005 Wiley-Liss, Inc.)

An interlaboratory study was performed to validate an anti-CD71/flow cytometry-based technique for enumerating micronucleated reticulocytes (MN-RETs) in mouse peripheral blood. These experiments were designed to address International Workshop on Genotoxicity Test Procedures validation criteria by evaluating the degree of correspondence between MN-RET measurements generated by flow cytometry (FCM) with those obtained using traditional microscopy-based methods. In addition to these cross-methods data, flow cytometric MN-RET measurements for each blood sample were performed at two separate sites in order to evaluate the reproducibility of data between laboratories. In these studies, groups of male CD-1 mice were treated with vehicle (saline or vegetable oil), a negative control (saline or vegetable oil), or four dose levels of five known genotoxicants (clastogens: cyclophosphamide, benzo[a]pyrene, 5-fluorouracil, methotrexate; aneugen: vincristine sulfate). Exposure occurred on 3 consecutive days via intraperitoneal injection, and blood samples were obtained approximately 24 hr after the final treatment. MN-RET frequencies were determined for each sample based on the analysis of 2,000 (microscopy) and 20,000 (FCM) reticulocytes. Regardless of the method utilized, each genotoxic agent was observed to cause statistically significant increases in the frequency of MN-RETs, and each response occurred in a dose-dependent manner. Spearman's correlation coefficient (rs) for FCM versus microscopy-based MN-RET measurements (nine experiments, 252 paired measurements) was 0.740, indicating a high degree of correspondence between methods. The rs value for all flow cytometric MN-RET measurements performed at the two independent sites was 0.857 (n = 248), suggesting that the automated method is highly transferable between laboratories. Additionally, the flow cytometric system offered advantages relative to microscopy-based scoring, including a greater number of cells analyzed, much faster analysis times, and a greater degree of objectivity. Collectively, data presented in this report suggest that the overall performance of mouse peripheral blood micronucleus tests is enhanced by the use of the flow cytometric scoring procedure., (2004 Wiley-Liss, Inc.)

All-trans-retinoic acid is a potent inhibitor of cell proliferation and inducer of differentiation. However, the clinical use of all-trans-retinoic acid in the treatment of cancer is significantly hampered by its toxicity and the prompt emergence of resistance, believed to be caused by increased all-trans-retinoic acid metabolism. Inhibitors of all-trans-retinoic acid metabolism may therefore prove valuable in the treatment of cancer. In this study, we characterize R116010 as a new anticancer drug that is a potent inhibitor of all-trans-retinoic acid metabolism. In vitro, R116010 potently inhibits all-trans-retinoic acid metabolism in intact T47D cells with an IC(50)-value of 8.7 nM. In addition, R116010 is a selective inhibitor as indicated by its inhibition profile for several other cytochrome P450-mediated reactions. In T47D cell proliferation assays, R116010 by itself has no effect on cell proliferation. However, in combination with all-trans-retinoic acid, R116010 enhances the all-trans-retinoic acid-mediated antiproliferative activity in a concentration-dependent manner. In vivo, the growth of murine oestrogen-independent TA3-Ha mammary tumours is significantly inhibited by R116010 at doses as low as 0.16 mg kg(-1). In conclusion, R116010 is a highly potent and selective inhibitor of all-trans-retinoic acid metabolism, which is able to enhance the biological activity of all-trans-retinoic acid, thereby exhibiting antitumour activity. R116010 represents a novel and promising anticancer drug with an unique mechanism of action.

Embryonal carcinoma cell lines (F9 EC and P19 EC) were stably transfected with 1.8 kb promoter sequence of RARbeta2 coupled to the lacZ gene as a system for measuring active retinoids. These stable transfectants, designated F9-1.8 and P19-1.8, were used as reporter cell lines to investigate different retinoids for their ability to activate the reporter gene. F9-1.8 cells showed similar EC(50) values for the acidic retinoids all-trans retinoic acid (RA), 4-oxo RA, 9-cis RA, and 13-cis RA, in the range of 1-7 nM, while P19-1.8 cells were less sensitive. Retinal showed decreased activity compared to the RA isomers in both lines. However, P19-1.8 cells hardly showed beta-gal activity after treatment with retinol, while the lacZ reporter in F9-1.8 cells was still inducible by this retinoid. In addition, the reporter system was used to investigate RA metabolism and its inhibition by P450 inhibitors. A combination of RA and liarozole showed a 10 times greater induction of the RARbeta2-lacZ reporter in P19-1.8 cells, but not in F9-1.8 cells. The EC(50) value for 4-oxo RA, however, was not altered, indicating that metabolic conversion of RA to 4-oxo RA is the target for inhibition by liarozole in P19-1.8 cells. HPLC analysis revealed nearly complete inhibition of RA metabolism after liarozole treatment in P19-1.8 cells, resulting in higher levels of RA. Finally, the F9-1.8 cells were used to detect active retinoids during different stages of chick limb bud development, demonstrating that it is the limb bud mesenchyme which generates RA and not the epidermis, with a twofold higher level of RA in the posterior half than in the anterior half., (Copyright 1999 Academic Press.)

In order to characterize the gene encoding the ligand binding (1(st); alpha) chain of the human IFN-gamma receptor, two overlapping cosmid clones were analyzed. The gene spans over 25 kilobases (kb) of the genomic DNA and has seven exons. The extracellular domain is encoded by exons 1 to 5 and by part of exon 6. The transmembrane region is also encoded by exon 6. Exon 7 encodes the intracellular domain and the 3' untranslated portion. The gene was located on chromosome 6q23.1, as determined by in situ hybridization. The 4 kb region upstream (5') of the gene was sequenced and analyzed for promoter activity. No consensus-matching TATA or CAAT boxes in the 5' region were found. Potential binding sites for Sp1, AP-1, AP-2, and CREB nuclear factors were identified. Compatible with the presence of the Sp1/AP-2 sites and the lack of TATA box, S1-nuclease mapping experiments showed multiple transcription initiation sites. Promoter activity of the 5' flanking region was analyzed with two different reporter genes: the Escherichia coli chloramphenicol acetyltransferase and human growth hormone. The smallest 5' region of the gene that still had full promoter activity was 692 base pairs in length. In addition, we found sequences belonging to the oldest family of Alu repeats, 2 - 3 kb upstream of the gene, which could be useful for genetic studies.

Retinoic acid (RA) inhibits proliferation of estrogen receptor (ER)-positive human breast cancer cells, but not the growth of ER-negative cells. We have shown previously that ER-positive cells express higher levels of retinoic acid receptor (RAR) alpha, suggesting that RAR alpha gene expression may be regulated in breast cancer cells by estrogens. We here report that estradiol (E2) increases RAR alpha mRNA in a time- and concentration-dependent manner resulting in a marked increase in RAR alpha protein expression, and present evidence that RAR alpha 1 is the only known isoform of RAR alpha regulated by E2 in breast cancer cells. In parallel we demonstrate that ER-positive cells exhibit greater RA sensitivity in the presence of E2, suggesting that E2-induced expression of RAR alpha 1 is involved in growth inhibition by RA. To directly investigate the role of RAR alpha 1 in RA-mediated growth inhibition, we introduced RAR alpha 1 expression vectors into RA-resistant and ER-negative MDA-MB-231 cells. The RAR alpha 1-transfected cells were growth inhibited by RA, while mock- and untransfected cells were unresponsive. Together, our data indicate that adequate levels of RAR alpha 1, either generated by introduction of expression vectors or endogenously induced by estrogens, are required for growth inhibition of breast cancer cells by RA.

The retinoic acid (RA) receptor (RAR) beta 2 promoter is strongly activated by RA in embryonal carcinoma (EC) cells. We examined this activation in the P19 EC-derived END-2 cell line and in E1A-expressing counterparts and found strong RA-dependent RAR beta 2 promoter activation in the E1A-expressing cells, which was not observed in the parental cell line, indicating a possible role for E1A in RAR beta 2 activation. In transient transfection assays, E1A functioned as a coactivator of RA-dependent RAR beta 2 promoter activation and, moreover, was able to restore this activation in cells lacking RAR beta 2 activation. By deletion analysis, two regions in the RAR beta 2 promoter were identified that mediate the stimulatory effect of E1A: the RA response element and TATA box-containing region and a more up-stream region between -180 and -63, in which a cAMP response element-related motif was identified as a target element for E1A. In addition, determination of endogenous E1A-like activity by measuring E2A promoter activity in transient transfection assays in EC and differentiated cells revealed a correlation between RA-dependent RAR beta 2 promoter activation and the presence of this activity, suggesting an important role for the cellular equivalent of E1A in regulation of the RAR beta 2 promoter.

In contrast to other members of the steroid/thyroid hormone superfamily, not much is known about the regions involved in transactivation of the receptors for retinoic acid. To determine the transactivation function of RARs, fusion proteins between the DNA-binding domain of the yeast transcription factor GAL4 and retinoic acid receptor-alpha (RAR alpha) or RAR beta were made. Transfection of these constructs resulted in RA-induced activation of a GAL4-responsive element-containing promoter. Deletion analysis revealed that RAR beta-2 has two transcription activation functions (TAFs). TAF-1 activates transcription constitutively and was mapped to the first 32 amino acids of the A-region. TAF-2 is located in the ligand-binding domain between amino acids 137 and 410 and activated transcription only in the presence of RA. The presence of two TAFs was confirmed by cotransfection of RAR beta deletion constructs with the human RAR beta-2 promoter as reporter, showing that the absence of RAR beta TAF-1 causes a decrease in transactivation, whereas truncation of TAF-2 completely blocks this function. Internal deletions in the ligand-binding domain in both GAL-RAR beta and RAR beta expression constructs resulted in a nonfunctional receptor, indicating that the complete ligand-binding domain is required for its transactivation function. Furthermore, we have shown that the contribution of the two TAFs in transcription activation varies among different cell lines, suggesting that they act in a cell-specific manner.

Recently three isoforms of the mouse retinoic acid receptor (mRAR beta 1, mRAR beta 2, mRAR beta 3) have been described, generated from the same gene (Zelent et al., 1991). The isoforms differ in their 5'-untranslated (5'-UTR) and A region, but have identical B to F regions. The N-terminal variability of mRAR beta 1/beta 3 is encoded in the first two exons (E1 and E2), while exon E3 includes N-terminal sequences of the mRAR beta 2 isoform. We have determined the structure of the human RAR beta 2 gene, using a genomic library from K562 cells. The open reading frame is split into eight exons: E3 contains sequences for the N-terminal A region and E4 to E10 encode the common part of the receptor, including the DNA-binding domain and ligand-binding domain. Corresponding to other nuclear receptors, both 'zinc-fingers' of the DNA-binding domain are encoded separately in two exons and the ligand-binding domain is assembled from five exons.

The int-1 mammary oncogene is frequently activated by proviral insertion in mouse mammary tumors. To characterize the target cell for the oncogenic action of int-1, we have isolated permanent cell lines with distinct morphologies and differentiation characteristics, starting from a tumor with a rearranged int-1 gene. Polygonal cells had retained many differentiation markers of epithelial cells and produced adenocarcinomas upon transplantation in syngenic mice. Sphere-forming-cuboidal cells are poorly differentiated and produced anaplastic tumors. Cuboidal and elongated cells were negative for epithelial markers. Cuboidal cells were poorly tumorigenic, but elongated cells produced highly malignant sarcoma-like tumors. In all lines, the int-1 gene was identically rearranged due to insertion of proviral DNA of the Mouse Mammary Tumor Virus, but the expression of int-1 varied with the state of differentiation of the cells. Polygonal cells contained relatively high levels of int-1 RNA, which were not influenced by steroid hormones. In the sphere-forming-cuboidal cells, expression of int-1 was low but inducible by dexamethasone. In the cuboidal and elongated cells no expression of int-1 was detectable, showing that the continued expression of int-1 was not required for progression to more malignant cells. By immunoprecipitation, two int-1 protein species, of 42 and 40 kD were identified in polygonal and in sphere-forming-cells but not in the culture media.

The transport of reactive intermediates through gap junctions was studied in a co-cultivation system consisting of primary chick-embryo liver cells and V79 Chinese hamster cells. The formation of gap junctions was studied by measurement of the incorporation of [3H]hypoxanthine in HGPRT deficient V79 mutant cells after co-cultivation with the hepatocytes. Under control conditions the heterologous gap junctions allowed for the transfer of [3H]hypoxanthine resulting in an average value of 13 grains/cell. Addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), inhibited this transfer in a dose related way to background values. The transfer of reactive intermediates was measured as the number of sister chromatid exchanges (SCEs) induced in the V79 cells. For both compounds tested, benzo[a]pyrene and dimethylnitrosamine, transport of reactive intermediates through gap junctions was observed. When the inhibitory effects of TPA were investigated at different time points after start of the co-cultivation, inhibition was measurable after 6 h and increased to a maximal inhibition of 50%, observed after 24 h. No effect of TPA was observed on the number of SCEs. When the metabolic cooperation deficient V79 mutant cell line MC--27, was used the effects were completely comparable to those of TPA for both compounds. The lower mutagenic effects in the MC--27 cells cannot be attributed to a lower intrinsic sensitivity for mutagens of these cells.

Antibody affinity is of major significance in immunoassays. Since affinity may be influenced by the immunoassay methodology it is important to determine this parameter under the conditions of the assay used. Here a method is described for the determination of binding constants (K) in a direct ELISA with the use of the computer program LIGAND. Five of the antibodies studied bound to their antigen with two classes of antigen binding site, while all the other antibodies studied reacted with only a single class of antigen binding site. The accuracy of the method and the implications for antigen-antibody reactions are discussed.