Previous studies suggest that the EpsteinBarr virus nuclear antigen EBNA2 participates in the regulation of the expression of the viral latent membrane protein (LMP). We have used reporter plasmids containing DNA fragments of the 5' flanking region of the LMP gene in cotransfection experiments to analyze the effect of EBNA2 on the activity of the LMP promoter. The results show that the LMP promoter is controlled by positive and negative transcription elements in a DNA fragment that contains the LMP transcription initiation site and 634 base pairs of upstream sequences. The promoter is activated by EBNA2. The region between position -54 and +40 relative to the mRNA cap site contains a positive transcription element that is constitutively active in DG75 cells and independent of EBNA2. The -106 to -54 region contains a negative regulatory element that prevents adjacent positive elements from functioning in the absence of EBNA2. Regulatory sequences between -324 and -144 participate in maintaining a high level of transcription of the LMP promoter after induction with EBNA2. The regulatory elements in the -634 to -54 promoter region have the characteristics of an inducible enhancer, including orientation independence and ability to regulate a heterologous promoter. In this context it might be relevant that transfected EBNA2 could induce the expression of the activation antigen CD23 on the surface of an EBV-negative Burkitt lymphoma (BL) cell line (6). The CD23 antigen is related, to the receptor for a B-cell growth factor (7) and has also been suggested to act as an autocrine growth factor for B cells after shedding from the cell surface (8). Epstein-Barr virus (EBVEBNA2 is a phosphorylated polypeptide with DNAbinding properties, which is encoded by the BYRF1 reading frame of the BamHI WYH region of the EBV genome (reviewed in ref. 1). The protein (the A subtype) has a rather unusual primary structure containing an almost continuous sequence of 40 proline residues, an arginine-and glycine-rich positively charged region, and a negatively charged Cterminal sequence. The overall proline content of the 487-amino acid long polypeptide is 29%. The recent demonstration of a new class of transcriptional activators with a proline-rich domain might provide some clues concerning the action of EBNA2 (9).It is thus conceivable that EBNA2 modulates the cell phenotype by influencing the expression of cellular genes. The effect might be direct, as suggested by the induction of CD23 in EBNA2-transfected cells (6), or indirect and mediated by way of other EBV genes, whose expression is in turn controlled by EBNA2. Evidence for the latter mechanism has been provided recently. The B95-8 virus strain, but not P3HR-1, has been shown to be able to induce expression of LMP in EBV-negative BL cell lines (10). Transfection of the EBNA2 gene into P3HR-1 virus-converted B-lymphoma cell lines induced the expression of LMP and a dramatic change in growth phenotype toward a LCL-like pattern (11). Some, but not all, of these changes could b...

Sex steroid hormones such as estrogens and androgens are involved in the development and differentiation of the breast tissue. The activity and concentration of sex steroids is determined by the availability from the circulation, and on local conversion. This conversion is primarily mediated by aromatase, steroid sulfatase, and 17β-hydroxysteroid dehydrogenases. In postmenopausal women, this is the primary source of estrogens in the breast. Up to 70-80% of all breast cancers express the estrogen receptor-α, responsible for promoting the growth of the tissue. Further, 60-80% express the androgen receptor, which has been shown to have tissue protective effects in estrogen receptor positive breast cancer, and a more ambiguous response in estrogen receptor negative breast cancers. In this review, we summarize the function and clinical relevance in cancer for 17β-hydroxysteroid dehydrogenases 1, which facilitates the reduction of estrone to estradiol, dehydroepiandrosterone to androstendiol and dihydrotestosterone to 3α- and 3β-diol as well as 17β-hydroxysteroid dehydrogenases 2 which mediates the oxidation of estradiol to estrone, testosterone to androstenedione and androstendiol to dehydroepiandrosterone. The expression of 17β-hydroxysteroid dehydrogenases 1 and 2 alone and in combination has been shown to predict patient outcome, and inhibition of 17β-hydroxysteroid dehydrogenases 1 has been proposed to be a prime candidate for inhibition in patients who develop aromatase inhibitor resistance or in combination with aromatase inhibitors as a first line treatment. Here we review the status of inhibitors against 17β-hydroxysteroid dehydrogenases 1. In addition, we review the involvement of 17β-hydroxysteroid dehydrogenases 4, 5, 7, and 14 in breast cancer.

Expression of the Epstein-Ban" virus (EBV) latent membrane protein (LMP1) is regulated by virus-and host cell-specific factors. The EBV nuclear antigen 2 (EBNA2) has been shown to transactivate a number of viral and cellular gene promoters including the promoter for the LMP1 gene. EBNA2 is targeted to at least some of these promoters by interacting with a cellular DNA binding protein, RBP-Jlc. In the present report we confirm and extend our previous observation that the LMP1 promoter can be activated by EBNA2 in the absence of the RBP-Jtc-binding sequence in the LMP1 promoter regulatory region (LRS). We show that two distinct LRS regions, -106 to +40 and -176 to -136, contribute to EBNA2 responsiveness. Site-directed mutagenesis analysis of the upstream -176/-136 EBNA2 responsive element revealed that two critical cis-acting elements are required for full promoter function. These same elements analysed by electrophoretic mobility shift assays define two binding sites recognized by nuclear factors derived from B cells. An octamer-like sequence (-147 to -139) contained overlapping binding sites for an unidentified transcriptional repressor on the one hand and a factor(s) belonging to the POU domain family but distinct from Oct-1 and Oct-2 on the other. An adjacent purine tract (-171 to -155) held a PU.1 binding site, which was also recognized by a related factor. The results suggest that the POU domain protein and either of two PU boxbinding factors bind simultaneously to LRS, creating a ternary complex that might be in part responsible for mediating the transactivation of the LMP1 promoter by EBNA2. There were no qualitative differences between EBV-negative and EBV-positive cells with regard to transcription factor binding to the octamer-like sequence and the PU.1 recognition site, as revealed by electrophoretic mobility shift assays.

Mutations that inactivate the transactivational ability of TP53 are more frequent in advanced CRC and are associated with worse prognosis in this stage of disease.

A polymorphism consisting of a deletion near the 5' splice site of exon 18 on the alpha2-macroglobulin (A2M) gene (A2M-2) has been suggested to be associated with Alzheimer's disease (AD) in family-based studies. We studied the A2M-2 allele together with the ApoE alleles in a large series on patients with AD (n = 449) and age-matched controls (n = 349). Neuropathologically confirmed diagnoses were available in 199 cases (94 AD and 107 control cases). We found no increase in A2M-2 genotype or allele frequencies in AD (27.5% and 14.6%) versus controls (26.4% and 14.9%). In contrast, a marked increase (p < 0.0001) in ApoE epsilon4 genotype or allele frequencies was found in AD (66.6% and 41.2%) as compared with controls (29.8% and 16.5%), suggesting sufficient statistical power in our sample. No relation was found between the A2M-2 and the ApoE epsilon4 allele. No change in A2M exon 17-18 mRNA size or sequence or A2M protein size was found in cases carrying the A2M-2 deletion, suggesting that there is no biological consequences of the A2M intronic deletion. No change in A2M protein level in cerebrospinal fluid was found in AD, suggesting that the A2M-2 allele does not effect the A2M protein expression in the brain. The lack of an association between the A2M-2 allele and AD in the present study, and the lack of abnormalities in the A2M mRNA or protein suggest that the A2M-2 allele is not associated with AD.

Estrogens have an important role in the progression of breast cancer. The 17B-hydroxysteroid dehydrogenase (17HSD) family has been identified to be of significance in hormonedependent tissues. 17HSD1 and 17HSD2 are the main 17HSD enzymes involved in breast cancer investigated this far, but it is possible that other hormone-regulating enzymes have a similar role. 17HSD5 and 17HSD12 are associated with sex steroid metabolism, and 17HSD14 is a newly discovered enzyme that may be involved in the estrogen balance. The mRNA expression of 17HSD5, 17HSD12, and 17HSD14 were analyzed in 131 breast cancer specimens by semiquantitative real-time PCR. The results were compared with recurrencefree survival and breast cancer-specific survival of the patients. The breast cancer cell lines MCF7, SKBR3, and ZR75-1 were transiently transfected with 17HSD14 to investigate any possible effect on estradiol levels. We found that high 17HSD5 was related to significantly higher risk of late relapse in estrogen receptor (ER)-positive patients remaining recurrence-free later than 5 years after diagnosis (P = 0.02). No relation to 17HSD12 expression was found, indicating that 17HSD12 is of minor importance in breast cancer. Patients with ER-positive tumors with high expression levels of 17HSD14 showed a significantly better prognosis about recurrence-free survival (P = 0.008) as well as breast cancerspecific survival (P = 0.01), confirmed by multivariate analysis (P = 0.04). Transfection of 17HSD14 in the human breast cancer cells MCF7 and SKBR3 significantly decreased the levels of estradiol, presenting an effect of high expression

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The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) is one of the first EBV-encoded gene products expressed after infection of primary B lymphocytes. EBNA2 is essential for the growth-transforming potential of the virus and it functions as a transcriptional activator of a set of viral and cellular genes. Sequencespecific DNA-binding by EBNA2 has not been demonstrated but the molecule is targeted to specific DNA regions by a cellular protein, RBP-JK, which recognizes the GTGGGAA sequence present in the regulatory region of all EBNA2-responsive promoters defined so far. We have determined the contribution of a RBP-Jx recognition sequence, an adjacent interferon-stimulated response element (ISRE) motif and a PU.l-binding site in the LMP1 regulatory sequence (LRS) to EBNA2-induced transactivation of the promoter by sitedirected mutagenesis of LRS-carrying reporter plasmids. EBNA2 responsiveness was reduced by approximately twofold when either or both of the RBP-JK-binding and ISRE sequences were mutated. ISRE seemed to function as an EBNA2-independent positive element. On the other hand, mutation of the PU box resulted in a drastic reduction of EBNA2 responsiveness, irrespective of whether the RBP-JK site or the ISRE motif was present. A comparative study by deletion mutation identified regions of EBV B95-8 EBNA2 involved in the transactivation of the LMP1 and the EBNA Cp promoters. Two domains of EBNA2 defined by deletion of amino acids 247-337 and 437-476 were found to be important for the activation of both promoters, while two different domains corresponding to residues 4-18 and 118-198 were required solely for the LMP1 promoter. Thus, EBNA2 must activate the LMP1 and Cp promoters by different mechanisms. All deletions involved in transcriptional activation of the two promoters contained regions that are conserved in EBNA2 of B95-8 EBV (type 1), AG876 EBV (type 2) and herpesvirus papio origin.