Impaired thymopoiesis contributes to immune deficiency following allogeneic stem cell transplantation. Emerging clinical literature suggests that graft-versus-host disease may be exacerbated and graft-versus-leukemia effect compromised in the setting of thymic insufficiency. Data have demonstrated that the thymus is damaged following the transplant preparative regimen, with a depletion of UEA+ thymic epithelial cells. We show that castration of male mice results in thymic renewal, as demonstrated by increased thymocyte number, intrathymic T cell receptor excision circles, and the number of early thymic progenitors (ETP: Lin- CD25- c-kit hi CD44 hi) as early as eight days post-castration. These observations suggested a mechanism of enhanced thymopoieis that occurs through increased immigration of thymocyte precursors. Studies with adoptively transferred congenic marrow progenitors confirm that enhanced ETP immigration is an early event following androgen withdrawal. Data also demonstrate a significant increase in UEA+ thymic epithelial cell proliferation by BRDU+ incorporation in the initial time frame following castration, implicating thymic epithelial cell proliferation in this thymic expansion, and suggesting that there may be an increase in thymic niche regulating ETP uptake. We have further identified an increase in a stromal derived protein important for ETP uptake by Western blot in total thymus lysates consistent with a mechanism of an increased ETP niche. Preliminary data also suggest that the mRNA encoding this protein is increased in sorted thymic epithelial subsets, with the greatest increase in the UEA+ medullary TEC cells, implicating these medullary epithelial cells in ETP entry. Taken together, we show that androgen withdrawal leads to UEA+ medullary cell proliferation with augmented ETP niche and entry. Thus, we identify ETP uptake as a critical and dynamically controlled point of thymic regulation.

Members of the catenin family of proteins are thought to play a major role in the folding and lamination of the cerebral cortex. We have used in situ hybridization to determine the cellular expression patterns of four members of this family, Alpha-E-, Alpha-N-, Beta-, and Gamma-catenins (CTNNA1, CTNNA2, CTNNB1, and JUP respectively) in the adult primate dorsolateral prefrontal cortex (DLPFC) and hippocampus. CTNNA2, CTNNB1, and JUP mRNAs were detected in all layers of the DLPFC and in all neuronal subregions of the hippocampal formation, however CTNNA1 mRNA, coding for an ‘epithelial’ specific catenin, was not detected in any region of the cortex or hippocampus. CTNNA2, a ‘neuronal-specific’ catenin, and CTNNB1 mRNAs were abundant in both DLPFC and hippocampus, with a distinct neuronal localization. CTNNA2 mRNA was concentrated in both granular/stellate cells and large pyramidal cell bodies, while CTNNB1 expression was more strongly associated with granular cell bodies throughout the DLPFC, with expression in pyramidal cells confined mainly to cortical Layers III and VI. CTNNA2 and CTNNB1 mRNAs were also abundant in the granule cells of the dentate gyrus and pyramidal cells of Ammon's horn, apparently co-expressed in the same neurons. JUP mRNA was rather diffusely localized in the DLPFC without the distinct laminar patterns seen for CTNNA2 and CTNNB1 but was distinctly localized in the granule cells of the dentate gyrus and pyramidal cells of Ammon's horn. These studies demonstrate a distinct neuronal pattern of gene expression for catenin family members in primate brain structures characterized by high degrees of folding and strong lamination. The high level expression of these transcripts supports the notion of a major role for catenins even in the adult brain. Such an understanding is also important in view of the multiple interactions that catenins have with many other proteins in the adult and ageing brain. This may also have implications for understanding the pathogenesis of neurodegenerative diseases such as Alzheimer's disease, as well as emerging neuronal stem cell therapies.

The docking protein p62Dok‐1 (Dok‐1) has a central role in cell signaling mediated by a wide range of protein tyrosine kinases, including intrinsic membrane kinases, such as the insulin‐like growth factor‐1 (IGF‐1) receptor. To elucidate potential IGF signaling mechanisms, we used DNA array technology to investigate novel kinase targets expressed in the primate dorsolateral prefrontal cortex (DLPFC). Dok‐1 transcripts were among the most abundant found in this structure. Because Dok‐1 expression has not been characterized in brain, we evaluated its expression pattern using immunoblotting, in situ hybridization, and immunohistochemistry in the rhesus monkey prefrontal cortex and hippocampal formation. Dok‐1 antibodies identified a 62‐kDa band in lysates from the DLPFC, consistent with the known size for Dok‐1. In situ hybridization showed that Dok‐1 mRNA was expressed in all layers of the DLPFC and in all neuronal subregions of the hippocampal formation. Immunohistochemical analysis showed Dok‐1 immunoreactivity concentrated in pyramidal neurons of cortical layers IV–V and throughout Ammon's horn and in granule neurons of the dentate gyrus. Dok‐1 expression was also identified in endothelial cells of cerebral blood vessels. These expression patterns are very similar to those of the IGF‐1 receptor and suggest that Dok‐1 could be among the downstream targets of IGF signaling in areas of the primate brain involved in learning and memory. Published 2003 Wiley‐Liss, Inc.

Human major histocompatibility (MHC) class I antigen expression is important in controlling the metastatic growth of malignant tumors. Locus-specific down-regulation of MHC class I gene expression is frequently observed in human tumors, leading to decreased susceptibility to cytotoxic T-cell-mediated lysis. The mechanism of this down-regulation is incompletely understood. Here, we describe the identification of human CCAAT displacement protein (CDP/cut) as a locus-specific repressor of HLA-B and C gene expression. Transient and stable transfections in HeLa and K562 cells demonstrated the presence of a repressor element 650 base pairs upstream of the first exon of HLA-B7. A specific binding complex with the HLA-B7 and Cw2 repressor elements was demonstrated by EMSA. Formation of the EMSA complex was inhibited specifically with polyclonal antiserum to human CDP/cut, demonstrating that CDP/cut binds the HLA-B7 repressor element. The corresponding region of the HLA-A2 promoter neither repressed HLA-A2 gene expression nor bound CDP/cut. Overexpression of CDP/cut in cell lines deficient in CDP/cut resulted in a nearly 4-fold repression of reporter constructs containing the HLA-B7 repressor element but not the corresponding region of the HLA-A2 promoter. Repression of HLA-B and C gene expression by CDP/cut does not involve displacement of NF-Y, nor is CDP/cut associated with the histone deacetylase HDAC1 when bound to the HLA-B7 repressor element. To our knowledge, these results identify CDP/cut as the first example of a locus-specific repressor of MHC class I gene transcription in human tumor cells.

RGSZ1 and Ret RGS, members of the regulator of G-protein signaling (RGS) family, are GTPase-activating proteins (GAPs) with high selectivity for Gαz. We show here that RGSZ1 and Ret RGSZ1 are products of two of several splice variants of one gene, RGS20. RGS20 spans ∼107 kb and contains at least seven exons. Five exons account for RGSZ1, including a single exon distinct to RGSZ1 that encodes a newly identified amino-terminal region. The previously described open reading frame (ORF) and 3′ untranslated region are encoded by four downstream exons that also encode about half of Ret RGS. The 5′ end of the RGSZ1 ORF contains several in-frame ATG codons (3–5 depending on the species), and multiple translational start sites may help explain the molecular weight heterogeneity of purified bovine brain RGSZ. Ret RGS replaces the 24 N-terminal amino acid residues of RGSZ1 with a large, N-terminal region that initially distinguished the bovine Ret RGS from human and mouse RGSZ1. This N-terminal domain is encoded by two distinct 5′ exons that are variably combined with the four downstream exons shared with RGSZ1 to produce at least six mRNAs. They encode proteins with N termini that vary in size, hydrophobicity, and the presence of a cysteine string. At least two mRNAs that include the exon that encodes the N-terminal region unique to RGSZ1 were found in brain and a few other tissues, but not retina. RGS20 thus can account for multiple Gz-selective GAPs in different tissues.

Integrins alpha(E)beta(7) and alpha(4)beta(7) are involved in localization of leukocytes at mucosal sites. Although both alpha(E)beta(7) and alpha(4)beta(7) utilize the beta(7) chain, they have distinct binding specificities for E-cadherin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1), respectively. We found that mutation of the metal ion-dependent adhesion site (MIDAS) in the alpha(E) A-domain (D190A) abolished E-cadherin binding, as did mutation F298A on the A-domain surface near the MIDAS cleft. A docking model of the A-domain with E-cadherin domain 1 indicates that coordination of the alpha(E) MIDAS metal ion by E-cadherin Glu(31) and a novel projection of Phe(298) into a hydrophobic pocket on E-cadherin provide the basis for the interaction. The location of the binding site on the alpha(E) A-domain resembles that on other integrins, but its structure appears distinctive and particularly adapted to recognize the tip of E-cadherin, a unique integrin ligand. Additionally, mutation of the beta(7) MIDAS motif (D140A) abolished alpha(E)beta(7) binding to E-cadherin and alpha(4)beta(7)-mediated adhesion to MAdCAM-1, and alpha(4) chain mutations that abrogated binding of alpha(4)beta(1) to vascular cell adhesion molecule-1 and fibronectin similarly reduced alpha(4)beta(7) interaction with MAdCAM-1. Thus, although specificity can be determined by the integrin alpha or beta chain, common structural features of both subunits are required for recognition of dissimilar ligands.

The androgen receptor (AR) is a sequence-specific DNA-binding protein that plays a key role in prostate cancer cellular proliferation by dihydrotestosterone and the induction of secondary sexual characteristics. In this study we demonstrate that the AR can be modified by acetylation in vitro and in vivo. p300 and p300/cAMP-response element-binding protein acetylated the AR at a highly conserved lysine-rich motif carboxyl-terminal to the zinc finger DNA-binding domain. [14C]acetate-labeling experiments demonstrated that AR acetylation by p300 in cultured cells requires the same residues identified in vitro. Point mutation of the AR acetylation site (K632A/K633A) abrogated dihydrotestosterone-dependent transactivation of the AR in cultured cells. Mutation of the p300 CH3 region or the p300/cAMP-response element-binding protein histone acetylase domain reduced ligand-dependent AR function. The identification of the AR as a direct target of histone acetyltransferase co-activators has important implications for targeting inhibitors of AR function.

The discovery that the PTEN tumor suppressor encodes a phosphoinositide 3-phosphatase has raised interest in the effects of constitutive activation of PI 3-kinase. To gain insight into PI 3-kinase function, we have stably expressed a myristoylated form of the catalytic subunit p110α (myr-p110) in cells. The myr-p110 associated with the endogenous p85 regulatory subunit and retained lipid and protein kinase activity. Stable lines expressing myr-p110 had 2- to 4-fold more PI 3-kinase activity than controls. Expression of myr-p110 altered cellular morphology and increased the saturation density in culture. These clones were morphologically transformed but Akt and pp70s6k were not constitutively activated in contrast to transient assays and from tumor cell lines deficient in PTEN. In addition, the ability of PDGF to induce activation of Akt and pp70s6k was diminished. Therefore, expression of a myristoylated PI 3-kinase in murine fibroblasts induces a morphological transformation of the cells.

The RecBCD enzyme of Escherichia coli is an ATP-dependent DNA exonuclease and a helicase. Its exonuclease activity is subject to regulation by an octameric nucleotide sequence called chi. In this study, site-directed mutations were made in the carboxyl-terminal nuclease domain of the RecB subunit, and their effects on RecBCD's enzymatic activities were investigated. Mutation of two amino acid residues, Asp(1067) and Lys(1082), abolished nuclease activity on both single- and double-stranded DNA. Together with Asp(1080), these residues compose a motif that is similar to one shown to form the active site of several restriction endonucleases. The nuclease reactions catalyzed by the RecBCD enzyme should therefore follow the same mechanism as these restriction endonucleases. Furthermore, the mutant enzymes were unable to produce chi-specific fragments that are thought to result from the 3'-5' and 5'-3' single-stranded exonuclease activities of the enzyme during its reaction with chi-containing double-stranded DNA. The results show that the nuclease active site in the RecB C-terminal 30-kDa domain is the universal nuclease active site of RecBCD that is responsible for DNA degradation in both directions during the reaction with double-stranded DNA. A novel explanation for the observed nuclease polarity switch and RecBCD-DNA interaction is offered.

The common neurotrophin receptor, p75(NTR), has been shown to signal in the absence of Trk tyrosine kinase receptors, including induction of neural apoptosis and activation of NF-kappa B. However, the mechanisms by which p75NTR initiates these intracellular signal transduction pathways are unknown. Here we report interactions between p75(NTR) and the six members of TRAF (tumor necrosis factor receptor-associated factors) family proteins. The binding of different TRAF proteins to p75(NTR) was mapped to distinct regions in p75NTR. Furthermore, TRAF4 interacted with dimeric p75(NTR), whereas TRAF2 interacted preferentially with monomeric p75(NTR). TRAF2-p75(NTR), TRAF4-p75(NTR), and TRAF6-p75(NTR) interactions modulated p75(NTR)-induced cell death and NF-kappa B activation with contrasting effects. Coexpression of TRAF2 with p75NTR enhanced cell death, whereas coexpression of TRAF6 was cytoprotective. Furthermore, overexpression of TRAF4 abrogated the ability of dimerization to prevent the induction of apoptosis normally mediated by monomeric p75NTR. TRAF4 also inhibited the NF-KB response, whereas TRAF2 and TRAF6 enhanced p75NTR-induced NF-kappa B activation. These results demonstrate that TRAF family proteins interact with p75(NTR) and differentially modulate its NF-kappa B activation and cell death induction.

p21-activated protein kinase (PAK)-1 phosphorylated G alpha(z), a member of the G alpha(i) family that is found in time brain, platelets, and adrenal medulla. Phosphorylation approached 1 mol of phosphate/mol of G alpha(z) in vitro. In transfected cells, G alpha(z) was phosphorylated both by wildtype PAK1 when stimulated by the GTP-binding protein Rad and by constitutively active PAK1 mutants. In vitro, phosphorylation occurred only at Ser(16), one of two Ser residues that are the major substrate sites for protein kinase C (PKC). PAK1 did not phosphorylate other G alpha subunits (il, i2, i3, o, s, or q), PAK1-phosphorylated G alpha(z) was resistant both to RGSZ1, a G(z)-selective GTPase-activating protein (GAP), and to RGS4, a relatively nonselective GAP for the G(i) and G(q) families of G proteins. Phosphorylation of Ser(27) by PKC did not alter sensitivity to either GAP. The previously described inhibition of G, GAPs by PKC is therefore mediated lay phosphorylation of Ser(16). Phosphorylation of either Ser(16) by PAK1 or Ser(27) by PKC decreased the affinity of G alpha(z) for G beta gamma; phosphorylation of both residues by PKC caused no further effect. PAK1 thus regulates G alpha(z) function by attenuating the inhibitory effects of both GAPs and G beta gamma. In this context, the kinase activity of PAK1 toward several protein substrates was directly inhibited by G beta gamma, suggesting that PAK1 acts as a G beta gamma-regulated effector protein. This inhibition of mammalian PAK1 by G beta gamma contrasts with the stimulation of the PAK homolog Ste20p in Saccharomyces cerevisiae by the G beta gamma homolog Ste4p/Ste18p.

Two autoimmune murine models—proteoglycan (aggrecan)-induced arthritis (PGIA) and collagen-induced arthritis (CIA)—were developed in parent strains, F1 and F2 hybrids of major histocompatibility complex (MHC)–matched (H-2d) BALB/c × DBA/2 and MHC-unmatched (H-2d/H-2q) BALB/c × DBA/1 intercrosses. The major goal of this comparative study was to identify disease (model)-specific (PGIA or CIA) and shared clinical and immunologic loci in 2 types of genetic intercrosses. Qualitative (binary/susceptibility) and quantitative (severity and onset) clinical trait loci were separated and analyzed independently or together with various pathophysiologic/immunologic traits, such as antigen-specific T- and B-cell responses and cytokine production. The major quantitative trait locus (QTL) was the MHC on chromosome 17, which was especially dominant in CIA. In addition, chromosomes 3, 5, 10, and × contained shared clinical loci in both models, and a total of 8 QTLs (clinical traits together with immunologic traits) were colocalized in PGIA and CIA.

The intrinsic activity of single-chain pro-urinary-type plasminogen activator (pro-uPA) and whether its receptor (uPAR) potentiates this activity remains controversial. In this report, the pro-uPA/uPAR-(1-281)-peptide complex in solution is shown to have equivalent plasminogen-activator activity to that of active two-chain uPA (tc-uPA). However, the activity of the complex was dependent on a synthetic tripeptide, Spectrozyme plasmin (Spl, H-D-2-aminohexanoic acid(Ahx)-hexatyrosyl-lysine-p-nitroanilide), which can also be used as a chromogenic substrate for plasmin. Furthermore, this activity could be completely suppressed by commonly used carrier proteins and detergents. The pro-uPA/uPAR-(1-281)-peptide complex at 1 nM displayed similar activity to that of tc-uPA for either [Glu1]plasminogen or [Lys77]plasminogen in chromogenic assays with Spl present as the plasmin substrate. When assayed with another plasmin substrate, S2251, the pro-uPA/uPAR-(1-281)-peptide complex was unable to activate plasminogen. The pro-uPA/uPAR-(1-281)-peptide complex and tc-uPA also showed a similar extent of plasminogen activation as measured by SDS/PAGE, when incubated with plasminogen and Spl in the presence of 100 micro M aprotinin, and plasminogen activation by pro-uPA alone was also stimulated in the presence of Spl in this assay. Activation of plasminogen by the pro-uPA/uPAR-(1-281)-peptide strictly required the presence of Spl, and pro-uPA remained in single-chain form during these assays. This activity of the pro-uPA/uPAR-(1-281)-peptide complex but not that of tc-uPA was completely inhibited by human serum albumin, bovine serum albumin, Tween-80, Triton X-100, and Pluronic-F68. Taken together, the data indicates that uPAR-(1-281)-peptide itself is not sufficient to augment pro-uPA activity and the presence of an effector molecule (e.g. Spl) is required to elicit the full plasminogen-activator activity of the pro-uPA/uPAR-(1-281)-peptide complex. It remains to be seen whether there is a physiological counterpart to this phenomenon.

Herein we present multiple lines of evidence which demonstrate that depletion of internal calcium stores is both necessary and sufficient for the activation of calcium-independent phospholipase A2 during arginine vasopressin (AVP)-mediated mobilization of arachidonic acid in A-10 smooth muscle cells. First, AVP-induced [3H]arachidonic acid release was independent of increases in cytosolic calcium yet was decreased by pharmacological inhibition of the release of calcium ion from internal stores. Second, thapsigargin induced the dramatic release of [3H]arachidonic acid from A-10 cells at a similar rate as the AVP-induced release of arachidonic acid, and the release of arachidonic acid by either AVP or thapsigargin was entirely inhibited by (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL). Third, the magnitude of thapsigargin-induced [3H]arachidonic acid release was entirely independent of alterations in cytosolic calcium concentration. Fourth, A23187 resulted in the BEL-inhibitable release of [3H]arachidonic acid from A-10 cells even when ionophore-induced increases in cytosolic calcium were completely prevented by calcium chelators. Fifth, pretreatment of A-10 cells with a calmodulin antagonist (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, HCl) resulted in the time-dependent decrease of subsequent thapsigargin-induced [3H]arachidonic acid release. Collectively, these results identify a novel paradigm which links alterations in calcium homeostasis to the calmodulin-mediated regulation of calcium-independent phospholipase A2 through the depletion of internal calcium stores.

The mechanism by which single-chain urokinase (scuPA) binds to its receptor (uPAR) is incompletely understood. We report that a fragment comprising the first domain of recombinant soluble uPAR (sDI) as well as a fragment comprising the remaining domains (sDII-DIII) competes with the binding of recombinant full-length soluble uPAR (suPAR) to scuPA with an IC50 = 253 nM and an IC50 = 1569, respectively. sDII-III binds directly to scuPA with Kd = 238 nM. Binding of scuPA to each fragment also induces the expression of plasminogen activator activity. sDI and sDII-DIII (200 nM each) induced activity equal to 66 and 36% of the maximum activity induced by full-length suPAR (5 nM), respectively. Each fragment also stimulates the binding of scuPA to cells lacking endogenous uPAR. Although scuPA binds to sDI and to sDII-DIII through its amino-terminal fragment, the fragments act synergistically to inhibit the binding of suPAR and to stimulate plasminogen activator activity. Furthermore, sDII-DIII retards the velocity and alters the pattern of cleavage of sDI by chymotrypsin. These results suggest that binding of scuPA to more than one epitope in suPAR is required for its optimal activation and association with cell membranes.

We have expressed the RecD subunit of the RecBCD enzyme from Escherichia coli as a fusion protein with a 31-amino acid NH2-terminal extension including 6 consecutive histidine residues (HisRecD). The overexpressed fusion protein can be purified in urea-denatured form by metal chelate affinity chromatography. The mixture of renatured HisRecD protein and the RecB and RecC proteins has a high level of ATP-dependent nuclease activity with either single- or double-stranded DNA, enhanced DNA unwinding activity, enhanced ATP hydrolysis activity in the presence of a small DNA oligomer cosubstrate, and χ-cutting activity. These are all characteristics of the RecBCD holoenzyme. The HisRecD protein by itself hydrolyzes ATP in the presence of high concentrations of single-stranded DNA (polydeoxythymidine). The activity is unstable at 37°C, but is measurable at room temperature (about 23°C). The HisRecD has very little ATPase activity in the presence of a much shorter single-stranded DNA (oligodeoxy(thymidine)12). HisRecD hydrolyzes ATP more efficiently than GTP and UTP, and has very little activity with CTP. We also purified a fusion protein containing a Lys to Gln mutation in the putative ATP-binding site of RecD. This mutant protein has no ATPase activity, indicating that the observed ATP hydrolysis activity is intrinsic to the RecD protein itself.

G-proteins convey information by traversing a cycle of controlled guanosine triphosphatase (GTP) binding and hydrolysis. Receptors initiate a signal by promoting the binding of GTP to the G-protein a subunit, thereby activating the G-protein and causing it to activate its effector. Activation is terminated when bound GTP is hydrolyzed. Hydrolysis of Gα-bound GTP is an unusually and anomalously slow enzymatic reaction. Although the rate of deactivation of adenylyl cyclase is generally about equal to the rate of hydrolysis of Gs-bound GTP, physiological rates of termination of most G-protein signals in cells are much faster than the measured rate of hydrolysis of GTP by the relevant isolated G-protein. In such cases, hydrolysis of bound GTP is accelerated by GTPase-activating proteins (GAPs). G-protein GAPs perform one or more important regulatory functions. The first GAPs for heterotrimeric G-proteins to be identified were the G-protein-regulated effectors phospholipase C-β (PLC-β) and the cyclic GMP phosphodiesterase of photoreceptor cells. The GAP activity of effectors is thought to enhance the temporal resolution of signaling, as discussed earlier, or to increase the selectivity of the G-protein among different receptors. The recently recognized RGS proteins, whose prototypes are involved in desensitization or antagonism of G-protein signaling, are also G-protein GAPs. Their primary function is assumed to be attenuation of signaling, although the regulation of RGS proteins is only being recently investigated. GAPs for small monomeric GTP-binding proteins such as p21ras also are negative signaling elements. Lastly, GAPs for some of the monomeric GTP-binding proteins involved in organelle trafficking are thought to terminate G-protein-mediated assembly, fusion, or transit functions, although this role has not been conclusively demonstrated. This chapter describes the activities of two GAPS for heterotrimeric G-proteins, one G-protein is an effector and the other a newly identified GAP whose physiological role is still unknown.

Aromatase, a cytochrome P450, catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogens. In this study, the regulatory properties of a 696-base pair region, that contains the promoter II and is situated immediately upstream of exon II of the human aromatase gene, were investigated. Chloramphenicol acetyltransferase (CAT) functional studies with DNA segments derived from this genomic region and primer-extension analysis revealed the presence of a second promoter which is functional in adipose stromal cells and in breast cancer cells. Detailed DNase-1 footprinting analysis, DNA mobility shift assays, and CAT functional studies of this genomic region were performed and led to the identification of a segment (B1) that could act as a promoter (probably promoter I.3) in adipose stromal and breast cancer cells. The study revealed further that the B1 region could be divided into two domains which were designated RE1 and RE2. RE1 was found to have the promoter activity, and RE2 was found to regulate the promoter activity of RE1, but in different manners in MCF-7 cells (as an example of breast cancer cells) and in adipose stromal cells. RE2 was found to function as a positive regulatory element in MCF-7 cells and as a negative regulatory element in adipose stromal cells, respectively. DNA mobility shift and UV-cross-linking experiments with BrUrd-substituted B1 fragment and nuclear extracts isolated from two types of cells were performed. The experiments identified DNA-bound proteins with molecular masses around 50 kDa. These findings serve as the basis for further examination of the regulatory mechanism of aromatase expression in human breast cancer and adipose stromal cells.