Differential regulation of LncRNA-SARCC suppresses VHL-mutant RCC cell proliferation yet promotes VHL-normal RCC cell proliferation via modulating androgen receptor/HIF-2α/C-MYC axis under hypoxia.

This corrects the article DOI: 10.1038/sj.gt.3302808.

Differential regulation of LncRNA-SARCC suppresses VHL-mutant RCC cell proliferation yet promotes VHL-normal RCC cell proliferation via modulating androgen receptor/HIF-2α/C-MYC axis under hypoxia.

It is well established that hypoxia contributes to tumor progression in a hypoxia inducible factor-2α (HIF-2α)-dependent manner in renal cell carcinoma (RCC), yet the role of long noncoding RNAs (LncRNAs) involved in hypoxia-mediated RCC progression remains unclear. Here we demonstrate that LncRNA-SARCC (Suppressing Androgen Receptor in Renal Cell Carcinoma) is differentially regulated by hypoxia in a von Hippel-Lindau (VHL)-dependent manner both in RCC cell culture and clinical specimens. LncRNA-SARCC can suppress hypoxic cell cycle progression in the VHL-mutant RCC cells while derepress it in the VHL-restored RCC cells. Mechanism dissection reveals that LncRNA-SARCC can post-transcriptionally regulate androgen receptor (AR) by physically binding and destablizing AR protein to suppress AR/HIF-2α/C-MYC signals. In return, HIF-2α can transcriptionally regulate the LncRNA-SARCC expression via binding to hypoxia-responsive elements on the promoter of LncRNA-SARCC. The negative feedback modulation between LncRNA-SARCC/AR complex and HIF-2α signaling may then lead to differentially modulated RCC progression in a VHL-dependent manner. Together, these results may provide us a new therapeutic approach via targeting this newly identified signal from LncRNA-SARCC to AR-mediated HIF-2α/C-MYC signals against RCC progression.

Molecular determinants of species-specific agonist and antagonist activity of a substituted flavone towards the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) mediates the toxicity of dioxins and related xenobiotics. Other chemicals also bind the AhR to elicit either agonist or antagonist responses. Here we used site-directed mutagenesis within the ligand binding domain of murine AhR to probe for specific residues that might interact differentially with the antagonist 3'-methoxy-4'-nitroflavone (MNF) compared with the prototypical agonist TCDD. Reduced (3)H-TCDD binding, dioxin-response element (DRE) binding, and transcriptional activity were observed for several point mutants. One mutation, R355I, changed the response to MNF from antagonist to agonist. Notably, Ile is the residue found in the guinea pig AhR, towards which MNF has partial agonist activity in contrast to its strong antagonist activity in mouse. A similar reversal of response to MNF was observed in chimeric AhRs in which the C-terminal region of mAhR was replaced with the guinea pig C-terminal region. These data demonstrate that different amino acids can be important in binding of different AhR ligands and can mediate distinct responses. The ultimate response of the AhR also depends on how other portions of the receptor protein are functionally coupled to the initial ligand binding event.

A potential endogenous ligand for the aryl hydrocarbon receptor has potent agonist activity in vitro and in vivo.

The aryl hydrocarbon receptor (AhR) is best known as a mediator of toxicity of a diverse family of xenobiotic chemicals such as dioxins and PCBs. However, many naturally occurring compounds also activate AhR. One such compound, 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), was isolated from tissue and found to be potent in preliminary tests [J. Song, M. Clagett-Dame, R.E. Peterson, M.E. Hahn, W.M. Westler, R.R. Sicinski, H.F. DeLuca, Proc. Natl. Acad. Sci. USA 99 (2002) 14694-14699]. We have synthesized ITE and [(3)H]ITE and further evaluated its AhR activity in several in vitro and in vivo assays in comparison with the toxic ligand, TCDD. AhR in Hepa1c1c7 cell cytosol bound [(3)H]ITE with high affinity and the AhR.ITE complex formed in vitro bound dioxin response element (DRE) oligonucleotide as potently as TCDD.AhR. In cells treated with ITE, nuclear translocation of AhR, and induction of CYP1A1 protein and of a DRE-dependent luciferase reporter gene were observed. ITE administered to pregnant DRE-LacZ transgenic mice activated fetal AhR, observed as X-gal staining in the same sites as in TCDD-treated mice. However, unlike TCDD, ITE did not induce cleft palate or hydronephrosis. TCDD but not ITE induced thymic atrophy in young adult mice, but both ITE and TCDD caused similar loss of cells and alterations of cell profiles in cultured fetal thymi. These data demonstrate that ITE is a potent AhR agonist in cell extracts, cultured cells, and intact animals, but does not cause the toxicity associated with the more stable xenobiotic ligand, TCDD.

The aryl hydrocarbon receptor is a regulator of cigarette smoke induction of the cyclooxygenase and prostaglandin pathways in human lung fibroblasts.

Cigarette smoking can lead to chronic lung inflammation and lung cancer. Chronic inflammation, associated with expression of cyclooxygenase-2 (COX-2) and prostaglandins, predisposes to malignancy. We recently demonstrated that human lung fibroblasts are activated by cigarette smoke to express COX-2 and prostaglandin E(2) (PGE(2)). Little is known about the mechanism whereby smoke activates human lung fibroblasts to produce proinflammatory mediators. Herein, we report the central role of the aryl hydrocarbon receptor (AHR) in cigarette smoke extract (CSE)-induced COX-2, microsomal PGE(2) synthase (mPGES), and PGE(2) production in human lung fibroblasts. Western blot analysis revealed that primary strains of human lung fibroblasts express AHR and aryl hydrocarbon nuclear translocator protein, supporting the possibility that smoke activates lung fibroblasts through this pathway. Experiments were subsequently performed to determine whether the AHR was activated by CSE. Immunocytochemistry and EMSA analysis revealed that CSE induced nuclear translocation of the AHR in human lung fibroblasts. CSE decreased protein levels of the AHR, consistent with AHR ligand-induced proteosome-mediated degradation. CSE also induced mPGES-1 and COX-2 protein and increased PGE(2) production. Treatment of human fibroblasts with AHR antagonists in the presence of CSE inhibited AHR nuclear translocation as well as COX-2, mPGES-1, and PGE(2) production. These data indicate that the AHR pathway plays an important role in cigarette smoke-mediated COX-2 and PG production in human lung fibroblasts and may contribute to tobacco-associated inflammation and lung disease.

Identification of potential aryl hydrocarbon receptor antagonists in green tea.

Previous investigations have implicated green tea to exert chemopreventive effects in animal models of chemical carcinogenesis, including polycyclic aryl hydrocarbon-induced cancers. In an effort to understand the compound(s) responsible for this protection, the effects of green tea extracts (GTE) and individual green tea catechins on aryl hydrocarbon receptor (AhR) gene induction were determined. Green tea (GT) was organically extracted and subsequently fractionated by column chromatography. The chemical composition of each fraction was determined by NMR. Several fractions inhibited tetrachlorodibenzo-p-dioxin-induced transcription of a dioxin responsive element-dependent luciferase reporter in stably transfected mouse hepatoma cells in a concentration-dependent manner. To determine the GT component(s) responsible for the observed effects, individual catechins were tested in the luciferase reporter system at concentrations found within the active fractions. Of the catechins tested, epigallocatechingallate (EGCG) and epigallocatechin (EGC) were the most potent antagonists, with IC(50) values of 60 and 100 microM, respectively. Re-creation of the active fractions using commercially available catechins further confirmed the identification of EGCG and EGC as the active AhR antagonists in green tea. These data suggest that EGCG and EGC are capable of altering AhR transcription and are responsible for most, if not all, of the AhR antagonist activity of GTE.

Agonist but not antagonist ligands induce conformational change in the mouse aryl hydrocarbon receptor as detected by partial proteolysis.

The cytosolic transcription factor known as the aryl hydrocarbon receptor (AhR) undergoes transformation to a DNA-binding form by a series of processes initiated by binding of ligand. Subsequent steps include dissociation of several proteins that are complexed with the inactive receptor, nuclear translocation, and dimerization with Arnt. We have used limited proteolysis of the in vitro-translated mouse AhR to determine whether this technique can detect conformational change(s) associated with AhR transformation and whether the effect of agonist and antagonist ligands can be distinguished by this assay. Limited digestion of [(35)S]AhR/AhR nuclear translocator (Arnt) by trypsin produced a peptide of approximately 40 kDa that was more resistant to proteolysis in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) than vehicle and was also Arnt-dependent. This trypsin-resistant peptide was also elicited in the presence of other agonist ligands, but not with antagonist ligands that do not form the DNA-binding AhR/Arnt complex. Immunoblot of trypsin-treated AhR/Arnt +/- TCDD indicated that the trypsin-resistant peptide did not include the N-terminal portion of the AhR against which the antibody was made. Truncated AhRs were also subjected to limited trypsinization. From AhR(1-399), a TCDD-dependent peptide of approximately 35 kDa was observed; from the constitutively active AhR(1-348), a band of approximately 30 kDa was produced from vehicle- and TCDD-treated protein. From these observations, we hypothesize that the trypsin-resistant peptide from full-length AhR spans approximately from amino acid 80 to 440. We conclude that agonist ligands initiate structural alteration in AhR that is Arnt-dependent and at least partially involves the ligand-binding/Per-Arnt-Sim domain.

In vivo antagonism of AhR-mediated gene induction by 3'-methoxy-4'-nitroflavone in TCDD-responsive lacZ mice.

The aryl-hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is a member of the bHLH-PAS family of proteins. The highest-affinity ligand of this receptor is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is a potent immunological, reproductive, and developmental toxicant. The mechanism of TCDD-induced toxicity and the gene modulations that result in toxicity have not been fully defined. The majority of work to date exploring AhR function has focused on agonist-activated AhR signaling. However, it is expected that a better understanding of AhR antagonism will lead to an improved understanding of TCDD toxicity and other AhR-mediated events. This study contributes to such investigations by utilizing the AhR antagonist 3'-methoxy-4'-nitroflavone (3'M4'NF) and a dioxin-responsive lacZ transgenic mouse model to characterize antagonism of the receptor system in vivo. The dose-response and time course of TCDD-induced transgene activation were evaluated in transgenic mice to provide information necessary to design 3'M4'NF in vivo studies. TCDD induction of the transgene was noted as early as 8 h after exposure in the lung. 3-miccrog/kg body weight TCDD was the lowest dose found to induce the reporter transgene. Finally, experiments were performed to evaluate the in vivo efficacy of 3'M4'NF. We found that 3'M4'NF inhibits TCDD-mediated reporter gene activation and CYP1A1 induction in vivo. Based on these findings, it is clear that DRE-lacZ animals and the antagonist 3'M4'NF represent important tools which will help in the identification of tissues where AhR is active, and to further characterize AhR-mediated signaling.

Enhanced detection of beta-galactosidase reporter activation is achieved by a reduction of hemoglobin content in tissue lysates.

beta-galactosidase (beta-gal), the product of the E. coli LacZ gene, has been used extensively as a reporter in numerous systems. Until recently, the most commonly used method of detecting beta-gal reporter enzymatic activity was a colormetric assay based on the cleavage of the beta-gal substrate 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) to form a blue precipitate. However, when increased sensitivity is needed, many investigators now turn to alternate substrates that produce fluorescent or luminescent products upon cleavage by beta-gal. These products are much more easily quantified than X-gal. The luminescent and fluorometric assays work very well in cultured cells but are often less sensitive in whole tissue lysates. In this study, we have evaluated the sensitivity of a fluorescent and a luminescent substrate in whole tissue lysates cleared of red blood cells or washed with PBS only. We have found that both assays show increased low-end sensitivity in tissues with reduced levels of hemoglobin (Hb). Hb is apparently able to quench luminescent and, to a lesser degree, fluorescent reporter light emission. Therefore, steps should be taken to reduce Hb levels either by lysis, perfusion, or both to enhance the sensitivity of these assays.

Aryl hydrocarbon receptor signaling plays a significant role in mediating benzo[a]pyrene- and cigarette smoke condensate-induced cytogenetic damage in vivo.

This laboratory has previously reported data suggesting that aryl hydrocarbon receptor (AhR) signaling may have a net potentiating effect on the DNA damaging potential of cigarette smoke. The experiments described in this report extend these studies by testing whether the potent AhR antagonist 3'-methoxy-4'-nitroflavone (3'M4'NF) can modify the in vivo genetic toxicity of benzo[a]pyrene (B[a]P) and the complex mixture of chemicals in cigarette smoke condensate (CSC). Initial experiments were designed to determine 3'M4'NF doses which can antagonize AhR in vivo but which have little effect on constitutive cytochrome P4501A (CYP1A) activity. These experiments took three forms: (i) zoxazolamine paralysis tests, a functional assay of cytochrome P450 CYP1A activity in 3'M4'NF-treated C57Bl/6J mice; (ii) co-treatment of AHR: null allele mice with 150 mg/kg B[a]P plus a range of 3'M4'NF concentrations in order to evaluate the potential of the flavone to interact with non-AhR targets which may affect B[a]P toxicity; (iii) an evaluation of the in vivo AhR antagonist activity of 3'M4'NF using transgenic mice which carry a dioxin-responsive element-regulated lacZ reporter. Once an appropriate dose range was determined, C57Bl/6J mice were challenged with B[a]P or CSC with and without 3'M4'NF co-treatment. Chromosome damage was measured by scoring the frequency of micronuclei in peripheral blood reticulocytes. Data presented herein suggest that 3'M4'NF can protect mice from B[a]P-induced bone marrow cytotoxicity and genotoxicity. Furthermore, CSC-associated genotoxicity was abolished by the flavonoid. These data add support to our hypothesis that AhR signaling has a net potentiating effect on the genetic toxicity and, presumably, carcinogenicity of cigarette smoke.

Regulation of DNA binding activity of the ligand-activated aryl hydrocarbon receptor by tyrosine phosphorylation.

Aryl hydrocarbon receptor (AhR), a member of the bHLH-PAS family, is a ligand-activated transcription factor which plays an important role in normal liver development and in mediating the toxicity of polycyclic and halogenated aromatic hydrocarbon pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin. Phosphorylation is known to regulate the transformation process of unliganded AhR into functionally active AhR/ARNT heterodimer that has high affinity for dioxin-responsive elements (DRE) and transactivation activity. Here, we report that DRE binding activity of the AhR is regulated by phosphorylation on the AhR/ARNT complex itself. Studies with specific protein phosphatases indicated that tyrosine phosphorylation is involved in this modulation. In addition, the AhR is phosphorylated at tyrosine residue(s) as determined by anti-phosphotyrosine immunoblot analysis. These results suggest that tyrosine phosphorylation on the AhR is required for its DNA binding activity and may provide mammalian cells with another layer of control mechanism that allows cell type specific and developmental stage specific induction of the AhR target genes.

A single dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin produces a time- and dose-dependent alteration in the murine bone marrow B-lymphocyte maturation profile.

The halogenated aromatic hydrocarbon, 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), is a ubiquitous, highly toxic environmental contaminant shown to produce immunotoxic effects in mammals. Although its immunotoxicity has been widely reported, little is known regarding its effect upon the development of immune-system cells, especially the B lymphocyte. The present study's purpose was to assess the effect that a single-dose administration of TCDD has, over time, upon bone marrow B-cell progenitors and pro/pre-B-, immature B-, and mature B-cell subpopulations, and to establish a dose-response relationship for these changes. Results showed that the mature B-lymphocyte subpopulation varied in a time-dependent manner, with a significant increase one day following TCDD treatment (30 microg/kg body weight [bw]), followed by a significant decrease at day 9 and a return to near-vehicle levels by day 31. Developing and less mature subpopulations were significantly decreased at days 6 and 9. The earliest B cell-progenitor subpopulation increased until day 9 and then decreased to vehicle-treated levels. Dose response (30, 15, 9, 6, 3, and 0.3 microg TCDD/kg bw) results at 2 days following treatment showed that only the mature-B subpopulation was affected at these doses, and below 6 microg/kg bw no effect was observed. These data suggest that the primary effect of TCDD is on those cells entering, and/or within, the mature B-lymphocyte subpopulation, and the alteration observed in the earlier maturation stages is a compensatory response to the effect on these mature cells.

Differential effects of diethylstilbestrol and 2,3,7,8-tetrachlorodibenzo-p-dioxin on thymocyte differentiation, proliferation, and apoptosis in bcl-2 transgenic mouse fetal thymus organ culture.

Both the estrogenic drug diethylstilbestrol (DES) and the pervasive environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibit thymocyte development. The mechanisms by which either agent induces thymic atrophy are still undetermined. We previously found that TCDD and DES inhibited C57BL/6 murine fetal thymocyte organ cultures (FTOC) at different stages of development. Now, using bcl-2 transgenic (TG) mice, we have further investigated their effects on FTOC proliferation, differentiation, maturation, and apoptosis. As with C57BL/6 mice, thymocyte development in C3H/bcl-2 FTOCs was inhibited by either TCDD (10 nM) or DES (20 microM) in both bcl-2 TG- and TG+ littermates. However, the percentage reduction of cell number induced by DES in bcl-2 TG+ FTOCs was significantly less than the level of inhibition in TG- FTOCs. There was no difference in the level of reduction from TCDD-exposed TG+ or TG- FTOC. Whereas TCDD increased production of mature CD8 cells in either strain, DES mainly yielded cells in the CD4(-)CD8(-)(DN) stage in TG- mice. The anti-apoptotic bcl-2 transgene overcame some DES blocking of DN thymocyte development, allowing more cells to differentiate into CD4 single-positive cells. Analysis of cell cycle showed that TCDD inhibited entry into S phase, whereas DES blocked cell cycling in the G2/M phase. TCDD did not induce detectable apoptosis in FTOC. However, unlike the effects of 17 beta-estradiol (E2) in vivo, DES induced apoptosis in the TG- FTOC, and these apoptotic cells were mainly in the DN subpopulation. This apoptosis could be prevented by the overexpression of bcl-2 in the TG+ mice. Our results demonstrate that, in addition to inhibition of fetal thymocytes at different stages of development by TCDD and DES, DES also induces thymic atrophy by both bcl-2-inhibitable apoptosis and by inducing cell cycle arrest in G2/M in the latest stage in the stem cell compartment. TCDD, on the other hand, does not induce apoptosis, but inhibits entry into cell cycle in the earliest stage in the stem cell compartment.

The aryl hydrocarbon receptor has a role in the in vivo maturation of murine bone marrow B lymphocytes and their response to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The ligand-activated aryl hydrocarbon receptor (AHR) is a cytosolic DNA binding protein. Although no biologic role for AHR has been elucidated, it mediates the immunotoxicity of xenobiotics such as 2, 3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and its targeted inactivation produces abnormal immune system development. While investigators have demonstrated AHR's involvement in TCDD-induced B lymphocyte functional alterations, little is known about the receptor's possible role in early B cell maturation and whether exogenous ligands change this process. The purpose of this study was to determine, (1) whether bone marrow B lymphocyte maturation is affected by AHR presence, (2) if so, its relative importance in hematopoietic and/or nonhematopoietic elements and, (3) whether TCDD alters this process. Radiation chimeras were produced that were AHR positive (Ahr+/+) or negative (Ahr-/-) in either their nonhematopoietic or hematopoietic elements, or both. Marrow cells were analyzed for alterations in B lymphocyte maturation stage cell numbers in both vehicle- and TCDD-treated animals. Our results showed that (1) Ahr-/- animals had significantly higher numbers of pro/pre-B cells than Ahr+/+ animals, (2) TCDD treatment of Ahr+/+ animals produced a decrease in pro/pre-B cell numbers, whereas no effect was observed on Ahr-/- animals, and (3) AHR is required in both hematopoietic and stromal elements for maintenance of B cell subset maturation profiles.

Role of estrogen receptor alpha in hematopoietic stem cell development and B lymphocyte maturation in the male mouse.

Although estrogens and estrogen receptors (ERs) are known to function in the male brain and reproductive tract, few studies have evaluated their involvement in the male hematopoietic and immune systems. This study was undertaken to determine the role of ERalpha in hematopoietic progenitor and B lymphocyte maturation. ERalpha knockout (ER-/-), wild-type (ER+/+), and radiation chimeric (ERalpha positive or negative in either nonhematopoietic or hematopoietic elements, or both) male mice were used to determine target tissues. ER-/- and ER+/+ animals showed similar hematopoietic progenitor profiles, but the ER-/- animals had fewer cells in all bone marrow B lymphocyte subpopulations. Animals receiving a pharmacological dose (5 mg/kg BW) of 17beta-estradiol (E2) with both elements, ER+/+, had decreased early hematopoietic progenitors and a shift toward a mature B cell subpopulation, whereas animals with both elements, ER-/-, showed changes only in early hematopoietic progenitors. Hematopoietic element ER+/+ animals exhibited greater E2-induced hematopoietic progenitor and B lymphocyte alterations than those having only nonhematopoietic ERalpha. These data indicate that 1) ERalpha is not necessary for regulating male mouse normal hematopoietic progenitor cell proportions, but is involved in B cell regulation; and 2) ERalpha in hematopoietic elements is predominantly responsible for mediating E2-induced hematopoietic and B cell changes.

Effect of 3'-methoxy-4'-nitroflavone on benzo[a]pyrene toxicity. Aryl hydrocarbon receptor-dependent and -independent mechanisms.

This laboratory has studied a number of flavone derivatives for aryl hydrocarbon receptor (AhR) agonist and antagonist potential using cell-free and cell culture systems. The current report extends these investigations by testing the potent AhR antagonist 3'-methoxy-4'-nitroflavone (3'M4'NF) for in vivo activity. Wild-type C57Bl/6 male mice were treated with solvent, benzo[a]pyrene (B[a]P; 150 mg/kg), or concurrently with B[a]P and 3'M4'NF (60 mg/kg; delivered as a split dose). Since B[a]P is bioactivated to genotoxic metabolites by AhR-regulated enzymes, we measured B[a]P-induced chromosomal damage in peripheral blood (i.e. micronuclei) to characterize the antagonistic potential of 3'M4'NF in vivo. The influence of AhR signal transduction was investigated further by challenging wild-type and Ahr null allele mice with B[a]P with and without a 3'M4'NF co-treatment. The micronucleus data obtained from these experiments indicated that 3'M4'NF can attenuate the genotoxicity of B[a]P significantly. Since 3'M4'NF also protected Ahr null allele mice from B[a]P-induced genetic damage, it was apparent that AhR-independent mechanisms contribute to the effects observed. However, as opposed to the protective effects observed with the micronucleus endpoint, histological observations and lethality data indicated that some B[a]P effects are enhanced by 3'M4'NF. Potentiated B[a]P toxicity may be explained by inhibition of basal and induced CYP1A1/2 activities. Both in vitro and in vivo data presented herein support this hypothesis.

Hemopoietic progenitor cells are sensitive targets of 2,3,7,8-tetrachlorodibenzo-p-dioxin in C57BL/6J mice.

Treatment of adult C57BL6J mice with tetrachlorodibenzo-p-dioxin (TCDD) elicits altered bone marrow hemopoietic cellular potentials and markedly reduced T-lymphoid-reconstituting activity. The latter has been hypothesized to play a role in TCDD-induced thymic atrophy. To investigate cellular targets responsible for reduced prothymocyte capacity, bone marrow cells from TCDD-treated C57BL/6J mice were assessed for hemopoietic alterations within the lineage-negative (lin-) compartment by the examination of Sca-1 and c-Kit levels. Lin- hemopoietic cells from C57BL/6J mice, treated with 30 microg/kg of TCDD, were assessed for phenotypic alterations following 24 h through 31 days. The responses of lin- cells to TCDD doses ranging from 0.3 to 30 microg/kg were also assessed at 2 days following TCDD treatment. The data reveal increases in the number of bone marrow lin- Sca-1+ c-Kit+ cells, relative to control, over 24 h through 31 days following treatment, as well as dose-dependent increases in this population when examined at 2 days. Increases in lin- Sca-1+ c-Kit- cells occurred on a more transient basis and were also dependent upon TCDD dose. These data suggest that proliferation and/or differentiation processes of hemopoietic stem cells are affected by TCDD and that these effects contribute to a reduced capacity of bone marrow to generate pro-T lymphocytes.

Malaria-infected erythrocytes serve as biological standards to ensure reliable and consistent scoring of micronucleated erythrocytes by flow cytometry.

A procedure for optimizing the configuration of flow cytometers for enumerating micronucleated erythrocytes is described. The method is based on the use of a biological model for micronucleated erythrocytes, the malaria parasite Plasmodium berghei. P. berghei endows target cells of interest (erythrocytes) with a micronucleus-like DNA content. Unlike micronuclei, parasitized red blood cells have a homogenous DNA content, and can be very prevalent in circulation. These characteristics make malaria-infected erythrocytes extremely well suited for optimizing instrument setup on a daily basis. The experiment described herein was designed to test the hypothesis that malaria-infected erythrocytes can greatly enhance the consistency with which flow cytometers are configured for micronucleus analyses, and thereby minimize intra- and interexperimental variation. Data collected over the course of several months, on two different flow cytometers, supports the premise that malaria-infected blood represents a useful biological standard which helps ensure reliable and consistent flow cytometric enumeration of rare micronucleated erythrocytes.

Function of estrogen receptor tyrosine 537 in hormone binding, DNA binding, and transactivation.

The human estrogen receptor (hER) is a ligand-activated transcription factor which, like many other members of the nuclear receptor protein family, exhibits a dimerization-dependent transcriptional activation. Several previous reports have provided evidence of the phosphorylation of the hER at tyrosine 537 (Y537). However, the exact function of a putative phosphorylation at this site remains controversial. Using a yeast transactivation assay, and in vitro biochemical approaches, we show that phosphorylation of tyrosine 537 is not required for the hER to bind hormone, or to activate transcription. An hER tyrosine 537 to phenylalanine (Y537F) mutant retains 70-75% of the transactivation potential of wild type hER in a yeast reporter system. Furthermore, the mutated receptor exhibits wild type hormone and DNA binding affinities. However, this mutation results in a decrease in receptor stability as measured by a decrease in the extent of hormone binding over time. The most striking difference between the wild type and Y537F hER is in the estradiol binding kinetics. Whereas the off-rate for estradiol exhibits a two-state binding mechanism, the Y537F mutant hER exhibits a monophasic estradiol off-rate. On the basis of these data and other reports describing the structure and activity of Y537 mutations, as well as knowledge of the three-dimensional structure of the hER ligand binding domain, we propose an alternate model wherein Y537F mutation favors an "open" pocket conformation, affecting the estrogen binding kinetics and stability of the hormone-bound, transcriptionally active "closed" pocket conformation. Although its phosphorylation is not essential for function of the hER, Y537 is nevertheless a critical residue intricately involved with the conformational changes of the hER and its ability to activate transcription.