Sulfotransferase 1A3/4 copy number variation is associated with neurodegenerative disease.

The cytosolic aryl sulfotransferase genes SULT1A3 and SULT1A4 are located on chromosome 16p11.2 in a region of chromosomal instability. SULT1A3/4 are important enzymes in the metabolism of catecholamines linked to neurodegenerative diseases such as Parkinson's and Alzheimer's. In the present study, copy number variation of the SULT1A3/4 genes in healthy individuals, as well as a cohort of Parkinson's disease and Alzheimer's disease patients was examined. In all subjects, SULT1A3/4 copy number varied from 1 to 10. In Alzheimer's disease patients, there was a significantly lower copy number compared to controls, and a positive correlation between copy number and age of disease onset. By contrast, there were no differences in Parkinson's disease patients. However, when early-onset Parkinson's disease was evaluated separately, there appeared to be an association with gene copy number and risk. The current study shows that these neurodegenerative diseases may be related to SULT1A3/4 copy number.

E. coli nitroreductase/CB1954 gene-directed enzyme prodrug therapy: role of arylamine N-acetlytransferase 2.

Gene-directed enzyme prodrug therapy is a promising approach to the local management of cancer and a number of gene prodrug combinations have entered clinical trials. The antitumor activity of Escherichia coli nitroreductase (NTR) in combination with the prodrug CB1954 relies on the reduction of the nitro groups to reactive N-hydroxylamine intermediates that are toxic in proliferating and nonproliferating cells. We examined whether secondary metabolic activation of the N-hydroxylamines by sulfotransferases or acetyltransferases altered cell responsiveness to the drug. We evaluated the coexpression of NTR with the human cytosolic sulfotransferases SULT1A1, 1A2, 1A3, 1E1 and 2A1, or the human arylamine N-acetyltransferases NAT1 and NAT2 on SKOV3 cell survival. Only NAT2 significantly altered the toxicity of CB1954, decreasing the IC(50) 16-fold from 0.61 to 0.04 microM. These results suggest that one or more of the N-hydroxyl metabolites are a substrate for O-acetylation by NAT2. We also examined the bystander effect of SKOV3 cells expressing NTR or NTR plus NAT2. Addition of the acetyltransferase resulted in a significant decreased bystander effect (P>0.01), possibly due to a lower concentration of reactive metabolites in the culture medium. These results suggest that a combination of bacterial NTR and NAT2 may provide a greater clinical response at therapeutic concentrations of CB1954 provided the reduction in bystander effect is not clinically significant. Moreover, endogenous NAT2, which is localized predominantly in the liver and gut, may be involved in the dose-limiting hepatic toxicity and gastrointestinal side effects seen in patients treated with the higher doses of CB1954.

Modified influenza virosomes: recent advances and potential in gene delivery.

Influenza virosomes have proven to be effective vehicles for the delivery of antigens in the vaccination of humans against a number of pathogens. However, their potential as a means for gene delivery has yet to be realized. Chemical modification of viruses is emerging as a new strategy for production of safe and efficient gene delivery systems. Influenza virosomes exhibit many of the features of the virus, such as for cell binding, uptake and endosomal escape, which can be easily engineered into designer delivery vehicles capable of safe, efficient and cell-specific cargo delivery. This review focuses on the next generation of influenza virosomes and highlights aspects of their modification that may lead to simple but effective gene delivery vehicles.

Pharmacogenetics of the arylamine N-acetyltransferases.

The arylamine N-acetyltransferases (NATs) are involved in the metabolism of a variety of different compounds that we are exposed to on a daily basis. Many drugs and chemicals found in the environment, such as those in cigarette smoke, car exhaust fumes and in foodstuffs, can be either detoxified by NATs and eliminated from the body or bioactivated to metabolites that have the potential to cause toxicity and/or cancer. NATs have been implicated in some adverse drug reactions and as risk factors for several different types of cancers. As a result, the levels of NATs in the body have important consequences with regard to an individual's susceptibility to certain drug-induced toxicities and cancers. This review focuses on recent advances in the molecular genetics of the human NATs.

Human cyclophilin 40 is a heat shock protein that exhibits altered intracellular localization following heat shock.

The unactivated steroid receptors are chaperoned into a conformation that is optimal for binding hormone by a number of heat shock proteins, including Hsp90, Hsp70, Hsp40, and the immunophilin, FKBP52 (Hsp56). Together with its partner cochaperones, cyclophilin 40 (CyP40) and FKBP51, FKBP52 belongs to a distinct group of structurally related immunophilins that modulate steroid receptor function through their association with Hsp90. Due to the structural similarity between the component immunophilins, FKBP52 and cyclophilin 40, we decided to investigate whether CyP40 is also a heat shock protein. Exposure of MCF-7 breast cancer cells to elevated temperatures (42 degrees C for 3 hours) resulted in a 75-fold increase in CyP40 mRNA levels, but no corresponding increase in CyP40 protein expression, even after 7 hours of heat stress. The use of cycloheximide to inhibit protein synthesis revealed that in comparison to MCF-7 cells cultured at 37 degrees C, those exposed to heat stress (42 degrees C for 3 hours) displayed an elevated rate of degradation of both CyP40 and FKBP52 proteins. Concomitantly, the half-life of the CyP40 protein was reduced from more than 24 hours to just over 8 hours following heat shock. As no alteration in CyP40 protein levels occurred in cells exposed to heat shock, an elevated rate of degradation would imply that CyP40 protein was synthesized at an increased rate, hence the designation of human CyP40 as a heat shock protein. Application of heat stress elicited a marked redistribution of CyP40 protein in MCF-7 cells from a predominantly nucleolar localization, with some nuclear and cytoplasmic staining, to a pattern characterized by a pronounced nuclear accumulation of CyP40, with no distinguishable nucleolar staining. This increase in nuclear CyP40 possibly resulted from a redistribution of cytoplasmic and nucleolar CyP40, as no net increase in CyP40 expression levels occurred in response to stress. Exposure of MCF-7 cells to actinomycin D for 4 hours resulted in the translocation of the nucleolar marker protein, B23, from the nucleolus, with only a small reduction in nucleolar CyP40 levels. Under normal growth conditions, MCF-7 cells exhibited an apparent colocalization of CyP40 and FKBP52 within the nucleolus.

Regulation of the Hsp90-binding immunophilin, cyclophilin 40, is mediated by multiple sites for GA-binding protein (GABP).

Within steroid receptor heterocomplexes the large tetratricopeptide repeat-containing immunophilins, cyclophilin 40 (CyP40), FKBP51, and FKBP52, target a common interaction site in heat shock protein 90 (Hsp90) and act coordinately with Hsp90 to modulate receptor activity. The reversible nature of the interaction between the immunophilins and Hsp90 suggests that relative cellular abundance might be a key determinant of the immunophilin component within steroid receptor complexes. To investigate CyP40 gene regulation, we have isolated a 5-kilobase (kb) 5'-flanking region of the human gene and demonstrated that a approximately 50 base pair (bp) sequence adjacent to the transcription start site is essential for CyP40 basal expression. Three tandemly arranged Ets sites within this critical region were identified as binding elements for the multimeric Ets-related transcription factor, GA binding protein (GABP). Functional studies of this proximal promoter sequence, in combination with mutational analysis, confirmed these sites to be crucial for basal promoter function. Furthermore, overexpression of both GABP alpha and GABP beta subunits in Cos1 cells resulted in increased endogenous CyP40 mRNA levels. Significantly, a parallel increase in FKBP52 mRNA expression was not observed, highlighting an important difference in the mode of regulation of the CyP40 and FKBP52 genes. Our results identify GABP as a key regulator of CyP40 expression. GABP is a common target of mitogen and stress-activated pathways and may integrate these diverse extracellular signals to regulate CyP40 gene expression.

Estradiol-regulated expression of the immunophilins cyclophilin 40 and FKBP52 in MCF-7 breast cancer cells.

The immunophilins, cyclophilin 40 (CyP40) and FKBP52, are associated with the unactivated estrogen receptor in mutually exclusive heterocomplexes and may differentially modulate receptor activity. We have recently shown that CyP40 and FKBP52 mRNA's are differentially elevated in breast carcinomas compared with normal breast tissue. Other studies suggest that such alterations in the ratio of immunophilins might potentially influence steroid receptor function. Studies were therefore initiated to investigate the influence of estradiol on CyP40 and FKBP52 expression in MCF-7 breast cancer cells. Over a 24-h-treatment period with estradiol, CyP40 and FKBP52 mRNA expression was increased approximately five- and 14-fold, respectively. The corresponding protein levels were also elevated in comparison to controls. The antiestrogen, ICI 182,780, was an antagonist for CyP40 and FKBP52 mRNA induction. Cycloheximide treatment did not inhibit this increased immunophilin expression, suggesting that estradiol-mediated activation is independent of de novo protein synthesis. Treatment of MCF-7 cells with estradiol resulted in an increased half-life of both CyP40 and FKBP52 mRNA, as determined by actinomycin D studies. These results suggest that estradiol regulates CyP40 and FKBP52 mRNA expression through both transcriptional and posttranscriptional mechanisms.

Two structures of cyclophilin 40: folding and fidelity in the TPR domains.

BACKGROUND: The "large immunophilin" family consists of domains of cyclophilin or FK506 binding protein linked to a tetratricopeptide (TPR) domain. They are intimately associated with steroid receptor complexes and bind to the C-terminal domain of Hsp90 via the TPR domain. The competitive binding of specific large immunophilins and other TPR-Hsp90 proteins provides a regulatory mechanism for Hsp90 chaperone activity. RESULTS: We have solved the X-ray structures of monoclinic and tetragonal forms of Cyp40. In the monoclinic form, the TPR domain consists of seven helices of variable length incorporating three TPR motifs, which provide a convincing binding surface for the Hsp90 C-terminal MEEVD sequence. The C-terminal residues of Cyp40 protrude out beyond the body of the TPR domain to form a charged helix-the putative calmodulin binding site. However, in the tetragonal form, two of the TPR helices have straightened out to form one extended helix, providing a dramatically different conformation of the molecule. CONCLUSIONS: The X-ray structures are consistent with the role of Cyclophilin 40 as a multifunctional signaling protein involved in a variety of protein-protein interactions. The intermolecular helix-helix interactions in the tetragonal form mimic the intramolecular interactions found in the fully folded monoclinic form. These conserved intra- and intermolecular TPR-TPR interactions are illustrative of a high-fidelity recognition mechanism. The two structures also open up the possibility that partially folded forms of TPR may be important in domain swapping and protein recognition.

Allele and genotype frequencies of polymorphic cytochromes P4502D6, 2C19 and 2E1 in aborigines from western Australia.

The polymorphisms of the important xenobiotic metabolizing enzymes CYP2D6, CYP2C19 and CYP2E1 have been studied extensively in a large number of populations and show significant heterogeneity in the frequency of different alleles/genotypes and in the prevalence of the extensive and poor metabolizer phenotypes. Understanding of inter-ethnic differences in genotypes is important in prediction of either beneficial or adverse effects from therapeutic agents and other xenobiotics. Since no data were available for Australian Aborigines, we investigated the frequencies of alleles and genotypes for CYP2D6, CYP2C19 and CYP2E1 in a population living in the far north of Western Australia. Because of its geographical isolation, this population can serve as a model to study the impact of evolutionary forces on the distribution of different alleles for xenobiotic metabolizing enzymes. Twelve CYP2D6 alleles were analysed. The wild-type allele *1 was the most frequent (85.81%) and the non-functional alleles (*4, * 5, * 16) had an overall frequency of less than 10%. Only one subject (0.4%) was a poor metabolizer for CYP2D6 because of the genotype *5/*5. For CYP2C19, the frequencies of the *1 (wild-type) and the non-functional (*2 and *3) alleles were 50.2%, 35.5% and 14.3%, respectively. The combined CYP2C19 genotypes (*2/*2, *2/*3 or *3/*3) correspond to a predicted frequency of 25.6% for the CYP2C19 poor metabolizer phenotype. For CYP2EI, only one subject had the rare c2 allele giving an overall allele frequency of 0.2%. For CYP2D6 and CYP2C19, allele frequencies and predicted phenotypes differed significantly from those for Caucasians but were similar to those for Orientals indicating a close relationship to East Asian populations. Differences between Aborigines and Orientals in allele frequencies for CYP2D6* 10 and CYP2E1 c2 may have arisen through natural selection, or genetic drift, respectively.

Polyinosinic acid and polycationic liposomes attenuate the hepatic clearance of circulating plasmid DNA.

DNA that enters the circulation is rapidly cleared both by tissue uptake and by DNase-mediated degradation. In this study, we have examined the uptake of linear plasmid DNA in an isolated perfused liver model and following intra-arterial administration to rats. We found that the DNA was rapidly taken up by the isolated perfused liver without degradation. The single-pass extraction ratio was 0.76 +/- 0.05, the mean transit time was 15.3 +/- 3.6 s, and the volume of distribution was 0.29 +/- 0.07 ml/g. Hepatic uptake was saturable and was inhibited by polyinosinic acid or polycationic liposomes but not by condensation of the DNA with polylysine. When the linear plasmid DNA was administered in vivo, plasma half-life was 3.1 +/- 0.2 min, volume of distribution was 670 +/- 85 ml/kg, and clearance was 32 +/- 4 ml/min. Coadministration of cationic liposomes decreased the volume of distribution to 180 +/- 28 ml/kg as well as the half-life (2.6 +/- 0.2 min). By contrast, polyinosinic acid significantly increased the circulating half-life (7.7 +/- 0.5 min), decreased the volume of distribution (95 +/- 17 ml/kg), and partially inhibited DNA degradation. When administered along with the liposomes and the polyinosinic acid, the distribution of plasmid-derived radioactivity decreased in the liver and increased in most other peripheral tissues. This study shows that pharmacological manipulation of the uptake and degradation of DNA can alter its distribution and clearance in vivo. These results may be useful in optimizing gene delivery procedures for in vivo gene therapy.

Allelic loss of cyclophilin 40, an estrogen receptor-associated immunophilin, in breast carcinomas.

PURPOSE: Cyclophilin 40 (CyP40) is an estrogen receptor-associated protein which appears to modify receptor function. The aim of this study was to determine the extent of allelic loss at the CyP40 locus in a panel of breast carcinomas using a newly characterized microsatellite marker located upstream of the CyP40 gene and then to correlate this with losses at chromosomal sites for cancer-associated genes. METHODS: Allelic loss at CyP40 was determined from patients' matched tumor and normal breast tissue using Genescan 672 software analysis of fluorescently labeled, PAGE-separated PCR products incorporating the marker. For each patient, allelic loss at CyP40 was then assessed and compared with losses at markers for various cancer-associated genes. RESULTS: Allelic loss was detected in 30% of breast carcinomas from patients heterozygous for the CyP40 marker. All carcinomas demonstrating allelic loss were grade II or III invasive ductal carcinomas and generally showed multiple losses at other sites near known cancer-associated genes. CONCLUSIONS: The polymorphic marker which we characterized was useful in determining allelic loss at the CyP40 locus in breast cancer patients and when applied in these studies in conjunction with various cancer-associated gene markers, suggests that deletions in the region of the CyP40 gene might be a late event in breast tumor progression.

Inactivation of human arylamine N-acetyltransferase 1 by the hydroxylamine of p-aminobenzoic acid.

Human N-acetyltransferase 1 (NAT1) is a widely distributed enzyme that catalyses the acetylation of arylamine and hydrazine drugs as well as several known carcinogens, and so its levels in the body may have toxicological importance with regard to drug toxicity and cancer risk. Recently, we showed that p-aminobenzoic acid (PABA) was able to down-regulate human NAT1 in cultured cells, but the exact mechanism by which PABA acts remains unclear. In the present study, we investigated the possibility that PABA-induced down-regulation involves its metabolism to N-OH-PABA, since N-OH-AAF functions as an irreversible inhibitor of hamster and rat NAT1. We show here that N-OH-PABA irreversibly inactivates human NAT1 both in cultured cells and cell cytosols in a time- and concentration-dependent manner. Maximal inactivation in cultured cells occurred within 4 hr of treatment, with a concentration of 30 microM reducing activity by 60 +/- 7%. Dialysis studies showed that inactivation was irreversible, and cofactor (acetyl coenzyme A) but not substrate (PABA) completely protected against inactivation, indicating involvement of the cofactor-binding site. In agreement with these data, kinetic studies revealed a 4-fold increase in cofactor K(m), but no change in substrate K(m) for N-OH-PABA-treated cytosols compared to control. We conclude that N-OH-PABA decreases NAT1 activity by a direct interaction with the enzyme and appears to be a result of covalent modification at the cofactor-binding site. This is in contrast to our findings for PABA, which appears to reduce NAT1 activity by down-regulating the enzyme, leading to a decrease in NAT1 protein content.

Characterization of an ATP-dependent pathway of activation for the heterocyclic amine carcinogen N-hydroxy-2-amino-3-methylimidazo[4, 5-f]quinoline.

2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is one of several mutagenic and carcinogenic heterocyclic amines formed during the cooking process of protein-rich foods. These compounds are highly mutagenic and have been shown to produce tumours in various tissues in rodents and non-human primates. Metabolic activation of IQ is a two-step process involving N-hydroxylation by CYP1A2 followed by esterification to a more reactive species capable of forming adducts with DNA. To date, acetylation and sulphation have been proposed as important pathways in the formation of N-hydroxy esters. In this study we have demonstrated the presence of an ATP-dependent activation pathway for N-hydroxy-IQ (N-OH-IQ) leading to DNA adduct formation measured by covalent binding of [(3)H]N-OH-IQ to DNA. ATP-dependent DNA binding of N-OH-IQ was greatest in the cytosolic fraction of rat liver, although significant activity was also seen in colon, pancreas and lung. ATP was able to activate N-OH-IQ almost 10 times faster than N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (7.7 +/- 0.3 and 0.9 +/- 0.1 pmol/mg protein/min, respectively). Using reported intracellular concentrations of cofactor, the ability of ATP to support DNA binding was similar to that seen with 3'-phosphoadenosine 5'-phosphosulphate and approximately 50% of that seen with acetyl coenzyme A (AcCoA). In addition to DNA binding, HPLC analysis of the reaction mixtures using ATP as co-factor showed the presence of two stable, polar metabolites. With AcCoA, only one metabolite was seen. The kinase inhibitors genistein, tyrphostin A25 and rottlerin significantly inhibited both DNA binding and metabolite formation with ATP. However, inhibition was unlikely to be due to effects on enzyme activity since the broad spectrum kinase inhibitor staurosporine had no effect and the inactive analogue of genistein, daidzein, was as potent as genistein. The effects of genistein and daidzein, which are naturally occurring isoflavones from soy and other food products, on DNA adduct formation may potentially be useful in the prevention of heterocyclic amine-induced carcinogenesis.

Substrate-dependent regulation of human arylamine N-acetyltransferase-1 in cultured cells.

Arylamine N-acetyltransferase-1 (NAT1) is a polymorphically expressed enzyme that is widely distributed throughout the body. In the present study, we provide evidence for substrate-dependent regulation of this enzyme. Human peripheral blood mononuclear cells cultured in medium supplemented with p-aminobenzoic acid (PABA; 6 microM) for 24 h showed a significant decrease (50-80%) in NAT1 activity. The loss of activity was concentration-dependent (EC(50) approximately 2 microM) and selective because PABA had no effect on the activity of constitutively expressed lactate dehydrogenase or aspartate aminotransferase. PABA also induced down-regulation of NAT1 activity in several human cell lines grown at confluence. Substrate-dependent down-regulation was not restricted to PABA. Addition of other NAT1 substrates, such as p-aminosalicylic acid, ethyl-p-aminobenzoate, or p-aminophenol to peripheral blood mononuclear cells in culture also resulted in significant (P <.05) decreases in NAT1 activity. However, addition of the NAT2-selective substrates sulfamethazine, dapsone, or procainamide did not alter NAT1 activity. Western blot analysis using a NAT1-specific antibody showed that the loss of NAT1 activity was associated with a parallel reduction in the amount of NAT1 protein (r(2) = 0.95). Arylamines that did not decrease NAT1 activity did not alter NAT1 protein levels. Semiquantitative reverse transcriptase polymerase chain reaction of mRNA isolated from treated and untreated cells revealed no effect of PABA on NAT1 mRNA levels. We conclude that NAT1 can be down-regulated by arylamines that are themselves NAT1 substrates. Because NAT1 is involved in the detoxification/activation of various drugs and carcinogens, substrate-dependent regulation may have important consequences with regard to drug toxicity and cancer risk.

1998 International Meeting on the Arylamine N-Acetyltransferases: synopsis of the workshop on nomenclature, biochemistry, molecular biology, interspecies comparisons, and role in human disease risk.

On October 22-24, 1998, a workshop was held at Kuranda, Queensland, Australia. The purpose of the meeting was to provide a forum for discussion of a number of diverse research areas of the biochemistry and molecular biology of arylamine N-acetyltransferases and to foster collaboration among several major groups of investigators around the world. In addition, participants were asked to consider how the nomenclature system for arylamine N-acetyltransferases could be strengthened to cope with the burgeoning number of new alleles discovered in the last 3 years. The full text of all meeting abstracts can be viewed at.

The pharmacology of gene therapy.

The objective for human gene therapy is to express exogenous DNA at a site in vivo for long enough, and at sufficient levels to produce a therapeutic response. The obstacles to this objective are numerous and include the formulation or packaging of the DNA, in vivo delivery, penetration of biological barriers, DNA elimination within the cell and from the tissue compartments of the whole body, control of product expression and overt toxicity. The current challenge is to resolve each of these obstacles to produce a practical and efficient gene therapy. In doing so, it is vital to understand the disposition of DNA vectors in vivo, and to know how conventional medicines may be used to modulate this disposition and to enhance the therapeutic effect of these vectors. Many of the general concepts of human gene therapy have been reviewed extensively in the literature. This review discusses some of the pharmacological aspects of gene delivery and the fate of vectors in vivo, and then highlights how drugs are being used to modulate gene therapy.

Purification, characterization and crystallization in two crystal forms of bovine cyclophilin 40.

The purification and crystallization of two different crystal forms of the two-domain protein bovine cyclophilin 40 is reported. Tetragonal crystals grown in methyl pentanediol belong to space group P4222 with unit-cell parameters a = 94.5, c = 118.3 A. Long thin needles grown from PEG belong to space group C2 with unit-cell parameters a = 125.71, b = 47.3, c = 74.6 A, beta = 93.90 degrees. The N-terminal 170 amino acids have significant homology with the well characterized human cyclophilin A. The C-terminal domain is largely made up of three copies of the tetratricopeptide repeat motif thought to be involved in mediating protein-protein interactions. Cyclophilins are frequently found as domains in larger multidomain proteins. To date, only X-ray structures of single-domain cyclophilins have been reported, and this work provides the first example of the purification and crystallization of a larger protein containing a cyclophilin domain.

The common tetratricopeptide repeat acceptor site for steroid receptor-associated immunophilins and hop is located in the dimerization domain of Hsp90.

Structurally related tetratricopeptide repeat motifs in steroid receptor-associated immunophilins and the STI1 homolog, Hop, mediate the interaction with a common cellular target, hsp90. We have identified the binding domain in hsp90 for cyclophilin 40 (CyP40) using a two-hybrid system screen of a mouse cDNA library. All isolated clones encoded the intact carboxyl terminus of hsp90 and overlapped with a common region corresponding to amino acids 558-724 of murine hsp84. The interaction was confirmed in vitro with bacterially expressed CyP40 and deletion mutants of hsp90beta and was delineated further to a 124-residue COOH-terminal segment of hsp90. Deletion of the conserved MEEVD sequence at the extreme carboxyl terminus of hsp90 precludes interaction with CyP40, signifying an important role for this motif in hsp90 function. We show that CyP40 and Hop display similar interaction profiles with hsp90 truncation mutants and present evidence for the direct competition of Hop and FK506-binding protein 52 with CyP40 for binding to the hsp90 COOH-terminal region. Our results are consistent with a common tetratricopeptide repeat interaction site for Hop and steroid receptor-associated immunophilins within a discrete COOH-terminal domain of hsp90. This region of hsp90 mediates ATP-independent chaperone activity, overlaps the hsp90 dimerization domain, and includes structural elements important for steroid receptor interaction.

Expression of the estrogen receptor-associated immunophilins, cyclophilin 40 and FKBP52, in breast cancer.

The estrogen receptor alpha (ER alpha) is implicated in the development of breast cancer. The immunophilins, cyclophilin 40 (CyP40) and FKBP52, are associated with ER alpha and other steroid receptors in mutually exclusive heterocomplexes and may differentially modulate receptor activity. Since previous studies have not assessed the levels of these immunophilins in breast cancer, we examined 10 breast cancer cell lines for mRNA and protein expression of CyP40 and FKBP52 and for amplification of the CyP40 gene. In addition, 26 breast carcinomas, including seven with matched normal breast tissue, were examined for mRNA expression of both immunophilins. CyP40 and FKBP52 were ubiquitously expressed in breast cancer cell lines, but there were significant differences in their pattern of expression. FKBP52 protein levels were generally an order of magnitude greater than those for CyP40. FKBP52 mRNA expression correlated strongly with protein expression and was significantly higher in ER alpha-positive compared with ER alpha-negative cell lines. However, CyP40 mRNA expression did not correlate with protein expression, nor did expression of this immunophilin correlate with ER alpha status. Relatively high expression of CyP40 in one cell line (BT-20) could be attributed to amplification of the CyP40 gene. Both immunophilins were also ubiquitously expressed in breast carcinomas, and we demonstrate for the first time that both CyP40 and FKBP52 mRNA are overexpressed in breast tumors compared to matched normal breast controls. The overexpression of CyP40 and FKBP52, coupled with relative differences in their expression in tumors, may have important functional implications for ER alpha and other steroid receptors in breast cancer.