Responsive Reporter Gene Research Articles

Human Mammary Epithelial Cells Express CYP27B1 and Are Growth Inhibited by 25-Hydroxyvitamin D-3, the Major Circulating Form of Vitamin D-3

1alpha,25-dihydroxycholecalciferol [1alpha,25(OH)2D3], the active form of cholecalciferol, is a negative growth regulator of breast cancer cells. CYP27B1 is a cytochrome P450-containing hydroxylase expressed in kidney and other tissues that generates 1alpha,25(OH)2D3 from an inactive vitamin D precursor 25-hydroxycholecalciferol [25(OH)D3]. In these studies, we tested the hypothesis that mammary cells express CYP27B1 and locally produce 1alpha,25(OH)2D3, which acts in an autocrine manner to regulate cell turnover. Using Western blot and quantitative real-time PCR, CYP27B1 mRNA and protein were detected in immortalized, nontumorigenic human mammary epithelial cell (HMEC) cultures. Furthermore, HMEC cultures were dose dependently growth inhibited by physiological concentrations of 25(OH)D3, suggesting that CYP27B1 converts this precursor cholecalciferol metabolite to 1alpha,25(OH)2D3, the ligand for the vitamin D receptor (VDR). In support of this suggestion, both 1alpha,25(OH)2D3 and 25(OH)D3 transactivated VDR in HMEC cultures, as measured by induction of a vitamin D responsive reporter gene and upregulation of CYP24, an endogenous VDR target gene. No induction of CYP24 by 25(OH)D3 was observed in mammary cells derived from CYP27B1 null mice. Similar results were observed in 2 independently derived immortalized HMEC lines as well as in primary cultures derived from human breast epithelium. These are the first studies to demonstrate that nontransformed human mammary cells express CYP27B1, that they are growth inhibited by physiologically relevant concentrations of 25(OH)D3, and that they provide a biological mechanism linking vitamin D status to breast cancer risk.

Activity-Guided Isolation of an Antiandrogenic Compound ofPygeum africanum

Inactivation of the androgen receptor (AR) through androgen ablation and treatment with antiandrogens is a major goal in the therapy for prostate hyperplasia and prostate cancer. Bioactivity-directed fractionation of a selective dichloromethane extract from the stem bark of Pygeum africanum led to the isolation of the antiandrogenic compound atraric acid. Its activity was examined by an androgen receptor responsive reporter gene assay. For lead structure optimization we transformed the natural occurring compound atraric acid into its ethyl, N-propyl and N-butyl esters and their antiandrogenic activities were examined as well. In addition, benzoic acid was isolated. The structures of all compounds were determined and characterized by means of 1H- and 13C-NMR, HR-EI-mass, IR and UV spectroscopy.

Pharmacological characterization of protease activated receptor-1 by a serum responsive element-dependent reporter gene assay: Major role of calmodulin

We studied the protease activated receptor-1 coupling to a serum response element (SRE)-dependent luciferase activity readout in transfected COS-7 cells. Thrombin, with a pEC 50 of 10.5, was 3000-fold more potent than the peptide agonists SFLLR and its derived compound C721-40 in stimulating luciferase activity, although the three agonists exhibited similar efficacy at the maximal concentration tested. Interestingly, SFLLR- and C721-40-induced luciferase activity was biphasic, suggesting that at least two populations of G proteins couple to the receptor. Further pharmacological characterization of this system was performed using selective protease activated receptor-1 antagonists. SCH203099 and ER-112787 blocked SFLLR-induced luciferase activity with similar potencies (p K B of 7), slightly higher than that exhibited by an arylisoxazole derivative compound from Merck (p K B of 6.1). These values correlated with their affinities established by competition binding experiments using [ 3H]-C721-40 as radioligand for protease activated receptor-1. Transduction mechanisms of protease activated receptor-1 coupling to SRE-dependent luciferase activity were examined using specific inhibitors. The Ca 2+ chelator BAPTA-AM, as well as the calmodulin inhibitors W-7 and ophiobolin A, robustly inhibited SFLLR-induced SRE activation. Overexpression of RGS2 and a dominant negative rhoA protein abolished the SFLLR signal in an additive manner, suggesting a major role of Gq and G12/13 proteins. Furthermore, inhibition of phospholipase C, MAP-kinases, phosphatidyl inositol-3 kinase, rho-kinase and Ca 2+/calmodulin-dependent protein kinases, all downstream effectors of Gq and G12/13, partially blocked the SFLLR-induced luciferase signal. Taken together, this SRE-luciferase assay reveals a complex network of transduction pathways of protease activated receptor-1 in accordance with the pleiotrophic action of thrombin.

Hypoxia- and radiation-inducible, breast cell-specific targeting of retroviral vectors

To facilitate a more efficient radiation and chemotherapy of mammary tumours, synthetic enhancer elements responsive to hypoxia and ionizing radiation were coupled to the mammary-specific minimal promoter of the murine whey acidic protein (WAP) encoding gene. The modified WAP promoter was introduced into a retroviral promoter conversion (ProCon) vector. Expression of a transduced reporter gene in response to hypoxia and radiation was analysed in stably infected mammary cancer cell lines and an up to 9-fold increase in gene expression demonstrated in comparison to the respective basic vector. Expression analyses in vitro, moreover, demonstrated a widely preserved mammary cell-specific promoter activity. For in vivo analyses, xenograft tumours consisting of infected human mammary adenocarcinoma cells were established in SCID/beige mice. Immunohistochemical analyses demonstrated a hypoxia-specific, markedly increased WAP promoter-driven expression in these tumours. Thus, this retroviral vector will facilitate a targeted gene therapeutic approach exploiting the unique environmental condition in solid tumours.

Deregulation of estrogen induced telomerase activity in tamoxifen-resistant breast cancer cells

Telomerase, a ribonucleoprotein enzyme that functions as a reverse transcriptase, is detected exclusively in immortal cells such as germ cells, stem cells and cancer cells. Telomerase activity is present in almost all human cancers. Telomerase activation is considered to be essential to maintain the integrity of the replicating tumor cell and to establish immortality. Based on this concept antiestrogen should initially regulate estrogen-stimulated telomerase but the enzyme would be expected to be constitutive in tamoxifen-resistant tumor cells. We have studied the estrogen regulation of telomerase in T47D:A18 breast cancer cells with a TRAPEZE Telomerase detection kit. Estradiol significantly increased telomerase activity after a 2-day treatment. Telomerase activity induced by estradiol was up to 10-fold higher within 4 days. Antiestrogens 4-hydroxytamoxifen (4-OHT) and ICI 182,780 were inactive alone and significantly blocked estradiol-stimulated increase in telomerase. These effects were correlated with changes in cell replications and changes in the cell cycle. In contrast, 4-OHT resistant T47D:A18 cells (T47D:A18/4-OHT, cultured in 1 microM 4-OHT for 6 months) grew spontaneously and had no changes in the cell cycle with estrogen treatment. The estrogen receptor (ERalpha) was present and still regulated at an estrogen responsive luciferase reporter gene with estrogen despite the fact that progesterone receptor was not increased in response to estradiol in T47D:A18/4-OHT cells. However, telomerase activity was increased about 40-fold in T47D:A18/4-OHT cells and this was not regulated by ICI 182,780. We conclude that the differential regulation of telomerase gene might be an important transition for tamoxifen resistance in T47D:A18 breast cancer cells.

Design and synthesis of downsized metastin (45–54) analogs with maintenance of high GPR54 agonistic activity

Metastin has been identified as a metastasis suppressor gene product that mediates its function through a G protein coupled receptor, GPR54. To refine insight into the critical pharmacophore for the activation of GPR54, we have conducted alanine and d-amino acid scanning on a biologically active metastin fragment (45–54). Based on these data and structures of peptides previously reported to activate GPR54, a series of shortened metastin (45–54) derivatives were synthesized and tested for the ability to induce GPR54 signaling. These biological experiments were performed in yeast containing human GPR54 that was coupled to the pheromone response pathway and a pheromone responsive lacZ reporter gene. Compounds 32, 33, and 39, which possess an N-terminal basic group and a C-terminal RW-amide motif, were strong agonists, similar to the level of metastin. This may provide an approach to reverse the pro-metastatic effect of metastin deletion in multiple malignant tumors.

Characteristics of a thyroid hormone responsive reporter gene transduced into a Xenopus laevis cell line using lentivirus vector

We introduced a self-inactivation (SIN) lentivirus vector (LV) into Xenopus laevis cell lines and established a permanent cell line expressing a reporter gene in a 3,5,3′- l-triiodothyronine (T 3) dependent manner. The SIN LV contained the luciferase gene downstream from the X. laevis T 3-response elements (TREs) and the SV40 promoter, and the enhanced green fluorescent protein (EGFP) gene downstream from the cytomegalovirus (CMV) promoter. It was integrated into the genome of X. laevis XL58, XTC2, and KR cells. The SIN LV transduced the X. laevis cells as efficiently as mammalian cells; however, the expression of EGFP in the transgene decreased with increasing culture time. A cell clone exhibiting the highest TH-dependent luciferase gene expression (XL58-TRE-Luc clone) was isolated from the EGFP-positive XL58 cell pool and characterized. The minimum effective concentration of T 3 that significantly induced the luciferase gene expression was 10 −11 M in the XL58-TRE-Luc clone. The application of the luciferase gene assay using the permanent XL58-TRE-Luc clone for the screening of thyroid-disrupting chemicals revealed that tetrachlorobisphenol A, at 10 −6 M, had a weak T 3-agonist activity, whereas trichlorobisphenol A, at 10 −8–10 −6 M had a weak T 3-antagonist activity. Our results indicated that the permanent X. laevis cell line containing a T 3-response transgene could be used as a bioassay, with small intra-assay variation, for the rapid screening, identification, and characterization of the thyroid-disrupting chemicals.

Conditional expression of a myocardium‐specific transgene in zebrafish transgenic lines

To develop the first heart-specific tetracycline (Tet)-On system in zebrafish, we constructed plasmids in which the cardiac myosin light chain 2 promoter of zebrafish was used to drive the reverse Tet-controlled transactivator (rtTA) and the green fluorescent protein (GFP) reporter gene was preceded by an rtTA-responsive element. In the zebrafish fibroblast cell-line, rtTA-M2, one of rtTA's derivatives, demonstrated the highest increase in luciferase activity upon doxycycline (Dox) induction. We then generated two germ lines of transgenic zebrafish: line T03 was derived from microinjection of a plasmid containing rtTA-M2 and a plasmid containing a responsive reporter gene, whereas line T21 was derived from microinjection of a single dual plasmid. Results showed that line T21 was superior to line T03 in terms of greater GFP intensity after induction and with of minimal leakiness before induction. The photographic images of induced GFP in the heart of F2 larvae showed that the fluorescent level of GFP was dose-responsive. The level of GFP expressed in the F3 3 days postfertilization larvae that were treated with Dox for 1 hr decreased gradually after the withdrawal of the inducer; and the fluorescent signal disappeared after 5 days. The GFP induction and reduction were also tightly controlled by Dox in the F3 adult fish from line T21. This Tet-On system developed in zebrafish shows much promise for the study of the gene function in a specific tissue at the later developmental stage.

A novel functional activator of the Drosophila JAK/STAT pathway, unpaired2, is revealed by an in vivo reporter of pathway activation

Striking similarities continue to emerge between the mammalian and Drosophila JAK/STAT signaling pathway. However, until now there has not been the ability to monitor global pathway activity during development. We have generated a transgenic animal with a JAK/STAT responsive reporter gene that can be used to monitor pathway activation in whole Drosophila embryos. Expression of the lacZ reporter regulated by STAT92E binding sites can be detected throughout embryogenesis, and is responsive to the Janus Kinase hopscotch and the ligand upd. The system has enabled us to identify the effect of a predicted gene related to upd, designated upd2, whose expression initiates during germ band extension. The stimulatory effect of upd2 on the JAK/STAT reporter can also be demonstrated in Drosophila tissue culture cells. This reporter system will benefit future investigations of JAK/STAT signaling modulators both in whole animals and tissue culture.

Lysophosphatidic Acid Inhibits Adipocyte Differentiation via Lysophosphatidic Acid 1 Receptor-dependent Down-regulation of Peroxisome Proliferator-activated Receptor γ2

Lysophosphatidic acid (LPA) is a bioactive phospholipid acting via specific G protein-coupled receptors that is synthesized at the extracellular face of adipocytes by a secreted lysophospholipase D (autotaxin). Preadipocytes mainly express the LPA(1) receptor subtype, and LPA increases their proliferation. In monocytes and CV1 cells LPA was recently reported to bind and activate peroxisome proliferator-activated receptor gamma (PPARgamma), a transcription factor also known to play a pivotal role in adipogenesis. Here we show that, unlike the PPARgamma agonist rosiglitazone, LPA was unable to increase transcription of PPARgamma-sensitive genes (PEPCK and ALBP) in the mouse preadipose cell line 3T3F442A. In contrast, treatment with LPA decreased PPARgamma2 expression, impaired the response of PPARgamma-sensitive genes to rosiglitazone, reduced triglyceride accumulation, and reduced the expression of adipocyte mRNA markers. The anti-adipogenic activity of LPA was also observed in the human SGBS (Simpson-Golabi-Behmel syndrome) preadipocyte cell line, as well as in primary preadipocytes isolated from wild type mice. Conversely, the anti-adipogenic activity of LPA was not observed in primary preadipocytes from LPA(1) receptor knock-out mice, which, in parallel, exhibited a higher adiposity than wild type mice. In conclusion, LPA does not behave as a potent PPARgamma agonist in adipocytes but, conversely, inhibits PPARgamma expression and adipogenesis via LPA(1) receptor activation. The local production of LPA may exert a tonic inhibitory effect on the development of adipose tissue.

Selective inhibition of TGF-β responsive genes by Smad-interacting peptide aptamers from FoxH1, Lef1 and CBP

Transforming growth factor beta (TGF-beta) stimulation results in the assembly of Smad-containing protein complexes that mediate activation or repression of TGF-beta responsive genes. To determine if disruption of specific Smad protein-protein interactions would selectively inhibit responses to TGF-beta or generally interfere with Smad-dependent signaling, we developed three Smad-binding peptide aptamers by introducing Smad interaction motifs from Smad-binding proteins CBP, FoxH1 and Lef1 into the scaffold protein E. coli thioredoxin A (Trx). All three classes of aptamers bound to Smads by GST pulldown assays and co-immunoprecipitation from mammalian cells. Expression of the aptamers in HepG2 cells did not generally inhibit Smad-dependent signaling as evaluated using seven TGF-beta responsive luciferase reporter genes. The Trx-xFoxH1b aptamer inhibited TGF-beta-induced expression from a reporter dependent on the Smad-FoxH1 interaction, A3-lux, by 50%. Trx-xFoxH1b also partially inhibited two reporters not dependent on a Smad-FoxH1 interaction, 3TP-lux and Twntop, and endogenous PAI-1 expression. Trx-Lef1 aptamer only inhibited expression of the Smad-Lef1 responsive reporter gene TwnTop. The Trx-CBP aptamer had no significant effect on reporter gene expression. The results suggest that Smad-binding peptide aptamers can be developed to selectively inhibit TGF-beta-induced gene expression.

Oestrogenic activity ofp-hydroxybenzoic acid (common metabolite of paraben esters) and methylparaben in human breast cancer cell lines

This paper addresses the question of whether p-hydroxybenzoic acid, the common metabolite of parabens, possesses oestrogenic activity in human breast cancer cell lines. The alkyl esters of p-hydroxybenzoic acid (parabens) are used widely as preservatives in consumer products to which the human population is exposed and have been shown previously to possess oestrogenic activity and to be present in human breast tumour tissue, which is an oestrogen-responsive tissue. Recent work has shown p-hydroxybenzoic acid to give an oestrogenic response in the rodent uterotrophic assay. We report here that p-hydroxybenzoic acid possesses oestrogenic activity in a panel of assays in human breast cancer cell lines. p-Hydroxybenzoic acid was able to displace [(3)H]oestradiol from cytosolic oestrogen receptor of MCF7 human breast cancer cells by 54% at 5 x 10(6)-fold molar excess and by 99% at 10(7)-fold molar excess. It was able to increase the expression of a stably integrated oestrogen responsive reporter gene (ERE-CAT) at a concentration of 5 x 10(-4) M in MCF7 cells after 24 h and 7 days, which could be inhibited by the anti-oestrogen ICI 182 780 (Faslodex, fulvestrant). Proliferation of two human breast cancer cell lines (MCF7, ZR-75-1) could be increased by 10(-5) M p-hydroxybenzoic acid. Following on from previous studies showing a decrease in oestrogenic activity of parabens with shortening of the linear alkyl chain length, this study has compared the oestrogenic activity of p-hydroxybenzoic acid where the alkyl grouping is no longer present with methylparaben, which has the shortest alkyl group. Intrinsic oestrogenic activity of p-hydroxybenzoic acid was similar to that of methylparaben in terms of relative binding to the oestrogen receptor but its oestrogenic activity on gene expression and cell proliferation was lower than that of methylparaben. It can be concluded that removal of the ester group from parabens does not abrogate its oestrogenic activity and that p-hydroxybenzoic acid can give oestrogenic responses in human breast cancer cells.

The Human Anti-CD30 Antibody 5F11 Activates NF-kB and Sensitizes Lymphoma Cells to Bortezomib Induced Apoptosis.

Purpose: We recently developed a fully human monoclonal antibody (5F11) directed against the CD30 receptor, which is an excellent target for antibody based immunotherapy of malignant lymphoma cells. The antibody is cytotoxic for lymphoma cells in vitro and in vivo, however a partial or even complete resistance of several Hodgkin cell lines has been observed. In this study we analysed, whether the efficacy of 5F11 can be improved by combination with bortezomib.Methods and Results: Bortezomib is an inhibitor of the ubiquitin-proteasomes pathway that is toxic to multiple solid and hemotologic tumor cells. Using XTT viability assays, TUNEL assays and Facs analysis we demonstrate a synergistic toxic effect of 5F11 and bortezomib on the Hodgkin cell lines tested (L540, L1236, L428) and the CD30 expressing ALCL Karpas299. Moreover the growth of subcutaneous L540 derived tumors in SCID mice is inhibited by 5F11 in combination with bortezomib. The synergy depends on the regime of the drugs and is only seen when a pre-incubation with the antibody is followed by exposure to bortezomib. Immunofluorescence analysis demonstrates that the sensitization of the tumor cells correlates with a 5F11 dependent localisation of the survival factor NF-kB into the cell nuclei. A transfected NF-kB responsive reporter gene is 2–3 fold activated in 5F11 treated L428 cells and enhanced NF-kB binding is detected performing EMSA.. We furthermore measured an increased expression of the NF-kB target gene c-flip, that is down regulated after additional incubation of the tumor cells with bortezomib.Conclusion: Our data indicate that initial activation of NF-kB and downstream anti-apoptotic proteins in response to 5F11 via CD30 sensitizes the tumor cells to bortezomib induced cell death. The in vitro and in vivo activity of the combination of 5F11 and bortezomib suggests a therapeutic value for the treatment of HD patients.

T-box binding protein type two (TBX2) is an immediate early gene target in retinoic-acid-treated B16 murine melanoma cells

Retinoic acid induces growth arrest and differentiation in B16 mouse melanoma cells. Using gene arrays, we identified several early response genes whose expression is altered by retinoic acid. One of the genes, tbx2, is a member of T-box nuclear binding proteins that are important morphogens in developing embryos. Increased TBX2 mRNA is seen within 2 h after addition of retinoic acid to B16 cells. The effect of retinoic acid on gene expression is direct since it does not require any new protein synthesis. We identified a degenerate retinoic acid response element (RARE) between −186 and −163 in the promoter region of the tbx2 gene. A synthetic oligonucleotide spanning this region was able to drive increased expression of a luciferase reporter gene in response to retinoic acid; however, this induction was lost when a point mutation was introduced into the RARE. This oligonucleotide also specifically bound RAR in nuclear extracts from B16 cells. TBX2 expression and its induction by retinoic acid was also observed in normal human and nonmalignant mouse melanocytes.

The suppression of Crk inhibits the metastatic profile of breast cancer cells lines

The suppression of Crk inhibits the metastatic profile of breast cancer cells lines

AtchitIV gene expression is stimulated under abiotic stresses and is spatially and temporally regulated during embryo development

The expression of AtchitIV gene was analysed in Arabidopsis plants submitted to abiotic stresses. Transcript accumulation was detected in leaves in response to UV light exposure, exogenous salicylic acid administration and wounding. Transgenic Arabidopsis plants carrying AtchitIV promoter::gus fusion also showed differential expression of the reporter gene in response to these treatments. The AtchitIV expression was also analysed during Arabidopsis embryo development. GUS assay demonstrated AtchitIV promoter activation in zygotic embryos from torpedo stage up to full maturation. Promoter deletion analysis indicated that all the 5' cis-acting elements responsible for the specific tissue expression are located in a region of 1083 bp, adjacent to the start of transcription. A negative regulatory region located between portions -1083 and -600 was also observed.

The C protein of measles virus inhibits the type I interferon response

Type I interferons (IFNα/β) are an important part of innate immunity to viral infections because they induce an antiviral response and limit viral replication until the adaptive response clears the infection. Since the nonstructural proteins of several paramyxoviruses inhibit the IFNα/β response, we chose to explore the role of the C protein of measles virus (MV) in such inhibition. Previous studies have suggested that the MV C protein may serve as a virulence factor, but its role in the pathogenesis of MV remains undefined. In the present study, a recombinant MV strain that does not express the C protein (MV C−) and its parental strain (Ed Tag) were used. Growth of MV C− was restricted in human peripheral blood mononuclear cells and HeLa cells, but in the presence of neutralizing antibodies to IFNα/β, MV C− produced titers that were equivalent to those of Ed Tag. In addition, expression of the MV C protein from plasmid DNA inhibited the production of an IFNα/β responsive reporter gene and, to a lesser extent, inhibited an IFNγ responsive reporter gene. The ability of the MV C protein to suppress the IFNα/β response was confirmed using a biologic assay. After IFNβ stimulation, HeLa cells infected with Ed Tag produced five-fold less IFNα/β than cells infected with MV C−. While the mechanism of inhibition remains unclear, these data suggest that the MV C protein plays an important role in the pathogenesis of MV by inhibiting IFNα/β signaling.

The FORKED genes are essential for distal vein meeting in Arabidopsis.

As in most dicotyledonous plants, the leaves and cotyledons of Arabidopsis have a closed, reticulate venation pattern. This pattern is proposed to be generated through canalization of the hormone auxin. We have identified two genes, FORKED 1 (FKD1) and FORKED 2 (FKD2), that are necessary for the closed venation pattern: mutations in either gene result in an open venation pattern that lacks distal meeting. In fkd1 leaves and cotyledons, the defect is first evident in the provascular tissue, such that the distal end of the newly forming vein does not connect to the previously formed, more distal vein. Plants doubly mutant for both genes have widespread defects in leaf venation, suggesting that the genes function in an overlapping manner at the distal junctions, but act redundantly throughout leaf veins. Expression of an auxin responsive reporter gene is reduced in fkd1 leaves, suggesting that FKD1 is necessary for the auxin response that directs vascular tissue development. The reduction in reporter gene expression and the fkd1 phenotype are relieved in the presence of auxin transport inhibition. The restoration of vein junctions in situations where auxin concentrations are increased indicates that distal vein junctions are sites of low auxin concentration and are particularly sensitive to reduced FKD1 and FKD2 activity.

SARA and Hgs attenuate susceptibility to TGF-beta1-mediated T cell suppression.

Transforming growth factor-beta1 (TGF-beta1) is a pluripotent cytokine that controls peripheral T cell tolerance mainly in mucosal immunity. It is secreted by regulatory T cells (Tr /Th3) but also by other immununologically active cells. Smad anchor for receptor activation (SARA) and hepatic growth factor-regulated tyrosine kinase substrate (Hgs) are involved in TGF-beta1 signaling. Both molecules are known to present Smad2 and Smad3 to the TGF-beta receptor complex. The role of SARA and Hgs in TGF-beta1 susceptibility of human CD4+ T cells is unclear. We demonstrate here that TGF-beta1 up-regulates SARA mRNA expression in CD4+ T cells similar to that of Smad7. However, the increase in SARA expression was lower (6.1+/-0.3-fold vs. 25+/-4.1-fold) compared with Smad7 and delayed, with a maximum at 12 h compared with 2 h. Th1 and Th2 cell subsets expressed the same levels of SARA and Hgs. Compared with resting cells, significantly lower levels of the two molecules were found in antigen/allergen- or anti-CD3/CD28-stimulated cells. Down-regulation of SARA and Hgs mRNA in preactivated CD4+ T cells was accompanied by a twofold increase in a TGF-beta1 responsive reporter gene assay. Overexpression of SARA and Hgs in T cells yielded a dose-dependent decrease in cotransfected reporter gene expression, indicating an inhibitory function of both molecules. Thus, SARA and Hgs are regulators of TGF-beta1 susceptibility in T cells and integrate regulatory signals into the influence of TGF-beta1-mediated suppression of human T cells.

Upregulation of cellular NF-kappa B activity by alkylating carcinogens in human epidermal keratinocytes.

Effect of alkylating carcinogens, i.e., N-nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU), as well as the simpler alkylating agents, methyl iodide and ethyl iodide, on the activation of NF-kappaB was evaluated in human epidermal squamous cell carcinoma (SCC-13) keratinocytes in order to investigate the possible correlation of cellular NF-kappaB activity with chemical carcinogenesis. The activities of NF-kappaB induced by chemical carcinogens were determined in human SCC-13 keratinocytes transfected with pNF-kappaB-SEAP-NPT plasmid, permitting expression of the secretory alkaline phosphatase (SEAP) reporter gene in response to the NF-kappaB activity and contains the neomycin phosphotransferase (NPT) gene conferring resistance to the geneticin. In this cell-based assay system, all alkylating carcinogens significantly upregulated the cellular NF-kappaB activations in a time- and dose-dependent manner until 72 h, at concentrations of 0.5-5 microM. These results suggest that carcinogenicity by alkylating chemicals may be associated with the modulation of cellular NF-kappaB activity in human skin cells.