Cloning of Hynobius lichenatus (Tohoku hynobiid salamander) p53 and analysis of its expression in response to radiation.

BACKGROUND: Caudata species such as salamanders are easily affected by environmental changes, which can drastically reduce their population. The effects of acute X-rays and chronic γ-irradiation on Hynobius lichenatus, the Japanese Tohoku hynobiid salamander, are known. However, the expression of radiation-inducible genes, such as the DNA-damage checkpoint response gene p53, has not been analyzed in H. lichenatus. This has not occurred because there is no established method for mRNA quantification in H. lichenatus due to a lack of information on available nucleotide sequences corresponding to both radiation-inducible genes and endogenous control genes such as ACTB (ß-actin). RESULTS: In this study, we aimed to evaluate the effects of radiation on gene expression in H. lichenatus. Using RNA extracted from irradiated salamanders, we performed rapid amplification of cDNA ends (RACE) and cloned H. lichenatus ß-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and p53. We confirmed that the cloned cDNAs were able to synthesize salamander proteins by western blotting after transfection into cultured HEK293 cells. Proliferation assays using HEK293 cells stably expressing H. lichenatus p53 protein showed that this protein has antiproliferative effects, similar to that of mammalian p53. Furthermore, RT-qPCR analysis using gene-specific primers revealed that p53 mRNA expression in H. lichenatus was upregulated upon exposure to radiation. CONCLUSION: Our results suggest that H. lichenatus p53 protein take an important role in regulating the cellular responses to various stimuli as mammalian p53 does. Furthermore, our study provides novel data to select appropriate primers to analyze internal control mRNA expression in H. lichenatus and to evaluate p53 expression as a marker of radiation and environmental stimuli.

Functional comparison of the human epidermal growth factor receptor and telomerase reverse transcriptase promoters in canine tumor cells.

We previously showed that the promoter region of the human epidermal growth factor receptor (hEGFR) gene elicits high transduction efficiency, with transgene expression restricted to canine breast tumor cells. However, it was unclear whether this promoter induces tumor cell-specific transgene expression in canine urothelial carcinoma cells. Furthermore, compared with studies in human cancer cells, the utility of the telomerase reverse transcriptase (TERT) gene promoter for therapeutic transgene expression in canine cancer cells has not been evaluated thus far. Here, we compared the activity of these promoters in canine mammary tumor and urothelial carcinoma cells. Our results showed that compared with the TERT promoter, the hEGFR promoter was more useful as a tumor-specific promoter to induce efficient transgene expression in canine tumor cells.

The membrane-type estrogen receptor G-protein-coupled estrogen receptor suppresses lipopolysaccharide-induced interleukin 6 via inhibition of nuclear factor-kappa B pathway in murine macrophage cells.

The female sex hormone estrogen exerts anti-inflammatory effects. The G-protein-coupled estrogen receptor (GPER) has been recently identified as a novel membrane-type estrogen receptor that can mediate non-genomic estrogenic effects on many cell types. We previously demonstrated that GPER inhibits tumor necrosis factor alpha-induced expression of interleukin 6 (IL-6) through repression of nuclear factor-kappa B (NF-κB) promoter activity using human breast cancer cells. Although several reports have indicated that GPER suppresses Toll-like receptor-induced inflammatory cytokine expression in macrophages, the molecular mechanisms of the inhibition of cytokine production via GPER remain poorly understood. In the present study, we examined GPER-mediated inhibition of IL-6 expression induced by lipopolysaccharide (LPS) stimulation in a mouse macrophage cell line. We found that the GPER agonist G-1 inhibited LPS-induced IL-6 expression in macrophage cells, and this inhibition was due to the repression of NF-κB promoter activity by GPER. G-1 treatment also decreased the phosphorylation of inhibitor of κB kinases. Among the mitogen-activated protein kinases, the phosphorylation of c-jun N-terminal kinase (JNK) was increased by G-1. These findings delineate the novel mechanism of the inhibition of LPS-induced IL-6 through GPER-activated JNK-mediated negative regulation of the NF-κB pathway in murine macrophage cells, which links anti-inflammatory effects to estrogen.

Expression of HIF-1α ODD domain fused canine caspase 3 by EGFR promoter-driven adenovirus vector induces cytotoxicity in canine breast tumor cells under hypoxia.

Adenovirus (Ad) vectors are widely used in cancer gene therapies. However, compared to human patients, relatively limited information is available on gene transduction efficiency or cell-specific cytotoxicity in canine tumor cells transduced with Ad vectors. Since epidermal growth factor receptor (EGFR) is highly expressed on canine breast tumor cells, we sought to develop an Ad vector based on the RGD fiber-mutant adenovirus vector (AdRGD) that expresses canine caspase 3 under the control of EGFR promoter. The aims of this study were to achieve high transduction efficiency with transgene expression restricted to canine breast tumor cells. Using EGFR promoter-driven AdRGD, we were able to restrict transgene expression to canine breast tumor cells with no evidence of expression in normal cells. Canine breast tumor cells transduced with EGFR promoter-driven AdRGD carrying canine caspase 3 gene showed cytotoxic activity. We constructed a second AdRGD vector that expressed oxygen-dependent degradation (ODD)-caspase 3 under the control of the EGFR promoter; the fusion protein contains a core part of the ODD domain of hypoxia inducible factor-1 alpha (HIF-1α) fused to caspase 3. Transduction of canine breast tumor cells with EGFR promoter-driven AdRGD expressing ODD-caspase 3 induced a higher rate of cell death under hypoxic conditions compared with under normoxia. The results indicate that the EGFR promoter-driven AdRGD vectors will be of value for tumor-specific transgene expression and safe cancer gene therapy in dogs.

Detection of Bcl-2 mRNA and its product in the glomerular podocytes of the normal rat kidney.

Podocyte apoptosis underlies podocytepenia leading to glomerulosclerosis. An apoptosis inhibitory protein Bcl-2 is expressed in the podocytes in the early stage of nephrogenesis and downregulated in the maturing stage of human fetal kidneys. Recent studies reported changed localization and expression of Bcl-2 in the renal glomeruli under the pathologic conditions. This study aimed to confirm in situ localization of Bcl-2 mRNA and its product in the glomeruli, and to demonstrate the local expression of Bcl-2 mRNA in normal rat glomeruli. Paraffin sections of the kidneys from normal male Wistar rats were immunostained by anti-Bcl-2 monoclonal antibody. The localization of Bcl-2 mRNA in the glomeruli was evaluated by in situ hybridization. The glomeruli were dissected from frozen sections of the kidneys with the laser microdissection (LMD) system. Total RNA extracted from 10, 100 or 200 dissected glomeruli was used for reverse-transcription polymerase chain reaction (RT-PCR) and real-time PCR. Bcl-2 mRNA and its product were detected in the podocytes but barely in the mesangial cells. In RT-PCR, the specific-sized bands of Bcl-2 from 100 or 200 dissected glomeruli were clearly observed. Real-time PCR for Bcl-2 showed that cDNA from 100 or 200 dissected glomeruli became amplified at 36 or 33cycles, respectively. Bcl-2 is expressed in the glomerular podocytes of the normal rat kidney and quantitative analysis of Bcl-2 mRNA in the renal glomeruli is possible using the LMD technique.

First case report of histoplasmosis in a cat in Japan.

Cytological, histopathological and immunohistochemical examinations were carried out on a presumed 10-year-old Japanese cat showing vomiting and emaciation. On cytologic examination of the mass of the upper abdominal cavity, many yeast-like organisms were detected in the macrophages. At necropsy, the upper part of colon was markedly dilated with a thickened wall. The lung did not show significant changes. Histologically, severe necrotic and granulomatous lesions were observed in the colon. In the colonic lesion, the cytoplasm of the macrophages contained yeast-like organisms with irregularly shaped dots, and the cell walls of these organisms were stained black by Grocott-Gomori methenamine-silver stain. Immunohistochemically, they were found to be positive for anti-histoplasma yeast antibody. This is the first report of feline histoplasmosis in Japan.

Identification of tocopherol-associated protein as an activin/TGF-beta-inducible gene in mast cells.

Previous studies have demonstrated that treatment with activin A and TGF-beta(1), members of the TGF-beta family, stimulated maturation of mouse bone marrow-derived cultured mast cells (BMMC), which was characterized by morphology and gene expression of mouse mast cell proteases (mmcps). In order to gain a better understanding of activin A- and TGF-beta(1)-induced maturation in mast cells, we investigated the genes that were up-regulated in response to treatment with these two members of the TGF-beta family. The cDNA microarray analyses indicated that in BMMC, five genes were induced by treatment with 4 nM activin A for 2 h. Tocopherol-associated protein (Tap) was one of the induced genes, and the Tap induction in response to activin A treatment was confirmed by real-time RT-PCR analyses. Treatment with TGF-beta(1) at 200 pM but not BMP-2 at 4 nM also increased Tap gene transcript in BMMC. Activin A-induced Tap expression was detected in BMMC but not in RAW264 macrophage-like cells, B16 melanoma cells or P19 embryonic carcinoma cells. Treatment with >1 muM SB431542, an inhibitor of activin and TGF-beta type I receptors ALK4/5, reduced responsiveness of Tap expression to TGF-beta(1), whereas <0.5 microM SB431542 effectively reduced TGF-beta(1)-induced expression of mmcp-1 and mmcp-7. These results suggest that inhibitory effects of SB431542 are different between TGF-beta-induced genes. Reporter assays indicated that Tap expression enhances transcription mediated by the activin/TGF-beta pathway. Thus, the present results suggest that Tap induction in response to activin/TGF-beta occurs predominantly in mast cells and serves as a positive regulator in activin/TGF-beta signaling.

Requirement of Smad3 for mast cell growth.

The involvement of the TGF-beta family in cell growth of bone marrow-derived mast cells (BMMC) cultured with medium containing pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) was examined. Doubling time of BMMC from Smad3-null mice was longer than that from wild-type (WT) mice, and the differences tended to be larger with time of culture. Consistent with the results, uptake and reduction of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] was lower in Smad3-deficient BMMC. Cell cycle analyses revealed no apparent differences between WT BMMC and Smad3-deficient BMMC, suggesting that longer doubling time in Smad3-deficient BMMC resulted from increased cell death. TGF-beta and activin A were supplied by PWM-SCM rather than by self-production by BMMC. Blocking the TGF-beta pathway by anti-TGF-beta neutralizing antibody or an inhibitor for the type I receptors for ligands including TGF-beta and activin, SB431542, inhibited MTS uptake and reduction in WT BMMC, whereas anti-activin A antibody and SB431542 tended to inhibit them in Smad3-deficient BMMC. The present results suggest that TGF-beta-induced and Smad3-mediated signaling is essential for maximal cell growth in mast cells, and that the activin pathway may be required for it when mast cell context is modulated by Smad3 depletion.

Involvement of p38 MAP kinase and Smad3 in TGF-beta-mediated mast cell functions.

Transforming growth factor-beta (TGF-beta) modulates functions of bone marrow-derived cultured mast cells (BMMCs); cell maturation (up-regulation of mouse mast cell proteases (mmcps)), growth arrest and migration. We investigated the roles of p38 MAP kinase and Smad3 in TGF-beta-mediated cell responses in BMMCs. Treating BMMCs with TGF-beta induced the phosphorylation of p38 within 2 h and persisted for 24 h. The involvement of p38 in TGF-beta-induced cell responses depended upon mast cell functions; it was necessary for up-regulation of mmcp-1 and migration, but not for up-regulation of mmcp-7 and inhibition of metabolic activity. New protein synthesis was required for the up-regulation of mmcp-1 but not mmcp-7 in response to TGF-beta treatment, and stabilization of mRNA was partially responsible for the increase in gene transcript of mmcp-1. The decrease in metabolic activity in response to TGF-beta treatment was smaller in Smad3-deficient BMMCs compared to wild-type BMMCs. Maximal migration was detected at a TGF-beta concentration of 40 fM in wild-type BMMCs, whereas TGF-beta-induced migration was absent in Smad3-deficient BMMCs. Thus, the roles of p38 and Smad3 are different among TGF-beta-mediated cell responses in BMMCs.

Transcriptional regulation of mouse mast cell protease-7 by TGF-beta.

Mouse mast cell protease-7 (mmcp-7) is a tryptase predominantly expressed in differentiated connective tissue-type mast cells. Previous study revealed that transforming growth factor-beta (TGF-beta) increases gene transcript of mmcp-7 in mast cells. The present study explored molecular mechanism of the up-regulation of mmcp-7 by TGF-beta. Luciferase-based reporter assays using deletion and point mutations of mmcp-7 promoter showed a critical region spanning nt -126 to -122 relative to the transcriptional start site, a Smad-binding element, for transcriptional activation by the TGF-beta pathway. In addition, a region from nt -104 to -98, a TPA-responsive element, was also necessary for the transactivation. Consistent with the current model for the TGF-beta signaling, Smad4 was required for the transcription of mmcp-7 by Smad3, a signal mediator of TGF-beta. Treatment with TGF-beta in mast cells resulted in the differential gene induction of the AP-1 components, i.e., transient induction of c-fos but not of c-jun and junB. Expression of c-fos further enhanced Smad3 and Smad4-induced transcription of mmcp-7, whereas c-jun expression inhibited the transcription. Our results suggest that TGF-beta stimulates mmcp-7 transcription through the Smad3-Smad4 pathway as well as c-fos induction, and that the AP-1 components distinctly related with the TGF-beta pathway.

Transcriptional regulation of plasminogen activator inhibitor-1 by transforming growth factor-beta, activin A and microphthalmia-associated transcription factor.

Plasminogen activator inhibitor-1 (PAI-1) is a key molecule that regulates turnover of the extracellular matrix. In the present study, we characterized PAI-1 gene expression in mast cells and melanocytes. In bone marrow-derived cultured mast cells, up-regulation of the PAI-1 gene was observed upon treatment with TGF-beta1, and was regulated at the transcriptional level. Microphthalmia-associated transcription factor (MITF), a member of the basic helix-loop-helix leucine zipper family of tissue-specific transcription factors predominantly expressed in mast cells, melanocytes and osteoclasts, also stimulated PAI-1 gene transcription, and TGF-beta1 did not increase PAI-1 mRNA levels in mast cells from mi/mi mice expressing dominant-negative MITF. MITF isoforms regulated TGF-beta1-induced transcription of PAI-1 differently; MITF-E-mediated transcription was further increased by TGF-beta1, whereas transcriptional activation by TGF-beta1 was blocked by MITF-M or MITF-mc expression. In contrast, activin A, another member of the TGF-beta family, enhanced transcription induced by MITF-M, as well as by MITF-E, although MITF-mc blocked activin A-induced transcription of PAI-1. Different regulation of PAI-1 gene expression upon TGF-beta1 and activin A treatment was also detected in B16 melanocytes; TGF-beta1 transiently increased the PAI-1 mRNA level, whereas activin A had prolonged effects on up-regulation of PAI-1. Our results on the control of PAI-1 gene expression by MITF isoforms, TGF-beta1 and activin A suggest that discrete regulation of the plasminogen activator system occurs in a cell type-specific manner.

Responses during cell preparation for functional analyses in mouse bone marrow-derived cultured mast cells.

Murine bone marrow-derived cultured mast cells (BMMCs) are most widely used in in vitro experiments for evaluation of mast cell functions. The present study has shown that cell preparation procedure, i.e., cell collection by centrifugation and the subsequent adjustment and culture of cell density at the desired concentrations, transiently induced gene expression of plasminogen activator inhibitor-1 (PAI-1) and the AP-1 components (c-fos, c-jun, and junB). The level of PAI-1 gene transcript was closely related to the cell density and the gene expression was enhanced by pretreatment with okadaic acid, an inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A). The cell preparation procedure also caused dephosphorylation of MAP kinases, i.e., ERK, p38, and JNK, resulting from PP1/PP2A activation. In view of the cell responses to the cell preparation procedure itself, care is needed in the interpretation of in vitro data using BMMCs.

Up-regulation of mouse mast cell protease-6 gene by transforming growth factor-beta and activin in mast cell progenitors.

Previous studies have revealed that members of the transforming growth factor-beta (TGF-beta) including TGF-beta1 and activin A modulate the function of mast cells. Here we show the up-regulation of mouse mast cell protease-6 (mMCP-6), which is expressed in differentiated mast cells, by TGF-beta1 and activin A in bone marrow-derived cultured mast cell progenitors (BMCMCs). Quantitative real time RT-PCR analyses revealed that the mRNA level of mMCP-6 was slightly but reproducibly increased by treatment with TGF-beta1 or activin A, which was regulated at the transcription level. Reporter assays showed that Smad3, a signal mediator of the TGF-beta/activin pathway, was responsible for the transcription. The TGF-beta response element is located at -153 bp relative to the transcription initiation site, CAGA. Microphthalmia-associated transcription factor (MITF), a tissue-specific transcription factor predominantly expressed in mast cells, melanocytes, the heart and skeletal muscle, also stimulated the transcription of mMCP-6. The region at -166 bp, GACCTG, was responsible for MITF-induced transcription. Mutations of the CAGA motif and the MITF responsive site indicated that the MITF site of mMCP-6 promoter is indispensable for the transcriptional activation by a constitutively active TGF-beta receptor (ALK5-TD), whereas the CAGA motif is dispensable for transcription by MITF. Transcriptional activation of mMCP-6 by the TGF-beta pathway was differently interacted with that by MITF isoform; ALK5-TD further enhanced MITF-E-induced transcription, whereas MITF-M-induced transcription abolished responsiveness to ALK5-TD. The positive regulation of mMCP-6 by the TGF-beta/activin pathway and the differential regulation by the MITF isoform suggest a rigorous regulation of mast cell function as effector cells of immune response.

Contribution of mast cells to bacterial clearance and their proliferation during experimental cystitis induced by type 1 fimbriated E. coli.

Bacterial infections of the urinary bladder are very common, and the role of mast cells in these infections is invariably thought of as a detrimental one. However, recent studies have shown that mast cells play a key role in host defense against various enterobacterial infections. In this manuscript, using mast cell-deficient (WBB6F1 - W/Wv) and mast cell-sufficient (WBB6F1 - +/+) mice we have investigated the protective role of mast cells in urinary bladder infections in vivo. Our findings show that (i) the mast cells are activated by FimH-expressing E. coli, and release large amount of histamine in the urinary bladder; (ii) the number of surviving bacteria in the urine is dependent on the presence of mast cells, and (iii) mast cell number in the bladder increases following uropathogenic infection in mice which is likely due to an increase in the mast cell growth-promoting cytokine IL-3 in bacteria-activated mast cells. Taken together, these observations suggest a beneficial role of mast cells in urinary bladder infections in mice.

Transcriptional activation of mouse mast cell protease-9 by microphthalmia-associated transcription factor.

We explored transcriptional regulation of mouse mast cell protease-9 (mMCP-9), which is implicated in inflammation of the jejunum during helminth infections and tissue remodeling of the uterus during pregnancy. Transcription was positively regulated by microphthalmia-associated transcription factor (MITF), a member of the basic helix-loop-helix-leucine zipper family that binds to the E-box, a CANNTG sequence. The most significant segment for positive regulation by MITF was nt -183 to -177 of the mMCP-9 promoter, CATCATG, which bound MITF-M. In addition, not only other MITF isoforms but also TFE3, another member of the family, activated mMCP-9 transcription through this nucleotide sequence inserted one base within the E-box.

Degranulation in RBL-2H3 cells: regulation by calmodulin pathway.

Involvement of the calmodulin pathway in Ca2+-induced degranulation was evaluated in RBL-2H3 mast cells. Pretreatment of RBL-2H3 cells with a calmodulin antagonist, W-13, blocked ionomycin-dependent release of beta-hexosaminidase into the supernatant, although W-13 treatment alone slightly but significantly increased the release. Ca2+/calmodulin activates various protein kinases and phosphatases including myosin-light chain kinase (MLCK), calmodulin-dependent protein kinases (CaMKs), and calcineurin. When RBL-2H3 cells were pretreated with a MLCK inhibitor, ML-7, or a CaMKs inhibitor, KN-93, the ionomycin-dependent release of beta-hexosaminidase into the supernatant was inhibited. In addition, pretreatment with calcineurin inhibitors, cyclosporin A and FR901725, resulted in blockage of the ionomycin-dependent release of beta-hexosaminidase into the supernatant. Our results indicate that Ca2+/calmodulin, activated calmodulin, is indispensable for Ca2+-induced degranulation, and that within the calmodulin pathways, at least MLCK, CaMKs and calcineurin positively regulate the release of granules initiated by increasing cytosolic Ca2+ concentrations in RBL-2H3 cells.

Altered function of murine mast cells in response to lipopolysaccharide and peptidoglycan.

Toll-like receptors (TLRs) recognize and signal the presence of bacterial components such as lipopolysaccharide (LPS) and peptidoglycan (PG) as a part of innate immunity. Our previous studies revealed that mast cells function as effector cells in the protection of mice against lethal enterobacterial infections. In this study, we examined both the gene expression of molecules involved in TLR signaling and the effects of LPS and PG in bone marrow-derived cultured mast cells (BMCMCs). The mRNA expression of TLR2, TLR4 and TLR6 was detected in BMCMCs. CD14, MD-2 and MyD88, which are also involved in TLR pathway, were also expressed. Neither LPS nor PG affected degranulation in BMCMCs, but release of tumor necrosis factor increased slightly in response to LPS and PG. Both LPS and PG enhanced expression of pro-matrix metalloproteinase 9 (pro-MMP-9) in a dose-dependent manner, and DNA fragmentation was induced by LPS, but not by PG. These results suggest that mast cells are the targets of LPS and PG, and that the functions of these molecules produced exclusively by bacteria partly overlap, but are distinct.

Role of activin A in murine mast cells: modulation of cell growth, differentiation, and migration.

Activins, members of the transforming growth factor-beta (TGF-beta) superfamily, are potent growth and differentiation factors. Our previous studies revealed that activin A, a homodimer of inhibin/activin beta(A), was induced in mast cells and peritoneal macrophages in response to their activation. In the present study, we examined the roles of activin A in murine bone marrow-derived, cultured mast cell progenitors (BMCMCs), which expressed gene transcripts for molecules involved in activin signaling, suggesting that BMCMCs could be target cells of activin A. Treatment of activin A inhibited 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide uptake into BMCMCs in a dose-dependent manner. The IC(50) concentration was 2.1 nM, which was less potent than 185 pM TGF-beta(1). Activin A treatment caused morphological changes toward the differentiated cells at 2 nM and up-regulated mRNA of mouse mast cell protease-1 (mMCP-1), a marker enzyme of mature mucosal mast cells, at 1 nM. Activin A also showed activity in inducing migration of BMCMCs; the optimal concentration for maximal migration was 10 pM, which was much lower than the concentrations to inhibit cell growth and to activate the mMCP-1 gene. Taking the present results together with our previous results, it is suggested that activin A secreted from activated immune cells recruits mast cell progenitors to sites of inflammation and that with increasing activin A concentration, the progenitors differentiate into mature mast cells. Thus, activin A may positively regulate the functions of mast cells as effector cells of the immune system.

Calcium-regulated expression of activin A in RBL-2H3 mast cells.

The present study examined the regulatory expression of activin A, a potent growth and differentiation factor, in rat basophilic leukemia (RBL-2H3) mast cells. Treatment of RBL-2H3 cells sensitized with anti-dinitrophenyl IgE with multivalent dinitrophenyl led to a clear increase in RT-PCR products of inhibin/activin beta(A). The steady-state mRNA of inhibin/activin beta(A) was also induced by increasing cytosolic Ca(2+) concentration with ionomycin, which required de novo protein synthesis, and was regulated at the transcriptional level. Pretreatment of RBL-2H3 cells with antagonists or inhibitors for the calmodulin pathway blocked ionomycin-dependent inhibin/activin beta(A) transcription and mRNA induction, suggesting the involvement of calmodulin-dependent kinase (CaMK) and calcineurin. The ionomycin-dependent inhibin/activin beta(A) induction was also partially blocked by preincubation with c-Jun NH(2)-terminal kinase (JNK) and p38 kinase inhibitors, but not with MEK1 inhibitor. These results suggest that inhibin/activin beta(A) gene activation is achieved by the JNK and p38 kinase activation through the calmodulin pathway in mast cells.

Transepithelial transport and cellular accumulation of steroid hormones and polychlorobiphenyl in porcine kidney cells expressed with human P-glycoprotein.

Endocrine disrupters such as sex hormone-like chemicals and the non-physiological ligands for aryl hydrocarbon receptor (AhR) exert many adverse biological effects. The ligands for AhR disturb gene expression downstream of the gene induced by estrogen receptor at a very low concentration. Thus, transepithelial transport and cellular accumulation of cortisol (COR) and estrogen as congeners of sex hormone-like chemicals, and 3,3',4,4'-tetrachlorobiphenyl (TeCB) as one of the ligands for AhR were examined in a monolayer of porcine kidney cells transfected with human P-glycoprotein (LLC-COL). The net basal-to-apical transport of COR increased in LLC-COL compared to that in the wild type cells (LLC-PKI) the same as in vinblastine, whereas the net transport of estradiol (EST) was not detected in either cell group. Though the diffusion transports of EST for both directions, basal-to-apical and apical-to-basal, were higher than that of COR, cellular accumulation of EST was higher than that of COR. Transepithelial transport of TeCB was very low and the net basal-to-apical transport was not detected, while it was highly accumulated in the epithelial cells. The accumulation was slightly higher in LLC-COL than in LLC-PKI at high dose.