Identification of a novel human BCL-X promoter and exon.

BCL-XL is a key anti-apoptotic BCL-2 family protein that is widely expressed in human cancer cells and is induced in response to diverse survival signals. The translation initiation codon for BCL-XL is located in BCL-X exon II and previous analyses have indicated that BCL-XL RNAs initiate close to the start of exon II or additionally contain a non-coding first exon (exon IA) spliced to exon II. Using 5' RACE we have now identified a novel BCL-X non-coding exon (exon IB) which is spliced directly to exon II in place of exon IA. Exon IB-containing RNAs encoded BCL-XL and were detected in non-malignant lymphocytes and lymphoma cells from lymph node biopsies and were expressed at significant levels in cell lines derived from ovarian, colon and breast cancers. We identified two TATA-box sequences upstream of exon IB and demonstrated that surrounding genomic sequences contained strong promoter activity in lymphoma cells (approximately 300-fold active relative to controls). We have therefore identified a powerful new BCL-X promoter and a novel exon that contributes to BCL-XL expression.

Differential effects of estrone and estrone-3-O-sulfamate derivatives on mitotic. Arrest, apoptosis, and microtubule assembly in human breast cancer cells.

There is considerable interest in the potential use of estrogen derivatives for the treatment and prevention of breast cancer. We demonstrated previously that the sulfamoylated estrone derivative 2-methoxyestrone-3-O-sulfamate (2-MeOEMATE) induced G2-M cell cycle arrest and modest levels of apoptosis in breast cancer cells in vitro, whereas the parent estrone derivative, 2-methoxyestrone, did not. 2-MeOEMATE also induced breast tumor regression in vivo in intact rats. To further explore the significance of sulfamoylation on the anticancer activity of estrone derivatives and to elucidate their mechanism of action, we synthesized two additional agents, 2-ethylestrone and 2-ethylestrone-3-O-sulfamate (2EtEMATE). 2-MeOEMATE and 2-EtEMATE inhibited the growth of a panel of estrogen receptor-negative and -positive breast cancer cell lines in vitro, induced mitotic arrest and apoptosis, and suppressed the long-term clonogenic potential of MCF7 and CAL51 breast cancer cells. In each assay, the sulfamoylated estrone derivatives were >10-fold more potent than their parent compounds. The sulfamoylated estrone derivatives were also significantly more potent inhibitors of cell growth than the previously studied endogenous estradiol metabolite 2-methoxyestradiol. 2-MeOEMATE and 2-EtEMATE functioned as antimicrotubule agents and inhibited the ability of paclitaxel to promote tubulin assembly in vitro. Like other antimicrotubule agents, the sulfamoylated estrone derivatives induced BCL-2 and BCL-XL phosphorylation and increased p53 expression. 2-MeOEMATE and 2-EtEMATE are novel antimicrotubule agents that have potent anticancer activity in breast cancer cells in vitro and may be beneficial as anticancer agents in vivo.

The effect of 2-methoxyoestrone-3-O-sulphamate on the growth of breast cancer cells and induced mammary tumours.

2-Methoxyoestrogens are emerging as a new class of drug that can inhibit tumour growth and angiogenesis. As sulphamoylation of oestrogens enhances their potency and bioavailability we have synthesized 2-methoxyoestrone-3-O-sulphamate (2-MeOEMATE) and compared its ability to inhibit the proliferation of breast cancer cells with that of 2-methoxyoestrone (2-MeOE1). 2-MeOEMATE (1 microM) inhibited the growth of oestrogen receptor positive MCF-7 breast cancer cells by 52% whereas 2-MeOE1 had little effect at this concentration. 2-MeOEMATE also inhibited the growth of oestrogen receptor negative MDA-MB-231 breast cancer cells. Exposure of cells to 2-MeOEMATE caused them to round up and become detached suggesting that this compound may induce cells to undergo apoptosis. Cell cycle analysis revealed that 2-MeOEMATE caused cells to arrest in the G(2)/M phase with the increase in G(2)/M arrested cells being detectable by 12 hr. Exposure of MCF-7 cells to 2 L-MeOEMATE for 24 hr followed by culture in drug-free medium for 24 hr did not reverse the arrest of cells in the G(2)/M phase. TUNEL analysis confirmed that 2-MeOEMATE induced apoptosis in a significant proportion of treated MCF-7 cells. In an in vivo study, employing nitrosomethylurea-induced mammary tumours in intact rats, 2-MeOE1 (20mg/kg/d, p.o. for 11 days) had little effect on tumour growth. In contrast, the same dose of 2-MeOEMATE resulted in the almost complete regression of 2/3 tumours over an 11-day period. We conclude that 2-MeOEMATE should have considerable therapeutic potential for the treatment of breast tumours.

Absence of expression of the Wiskott-Aldrich syndrome protein in peripheral blood cells of Wiskott-Aldrich syndrome patients.

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency that is usually associated with thrombocytopenia and eczema. The very variable phenotype of WAS results from defects in the WAS protein (WASP), the function of which is not well understood. In many cases causative mutations have now been identified in the WAS gene. Attempts have been made to correlate the nature of the mutations with the severity of the disease. In this study we investigated mutations in 13 patients with WAS and analyzed the expression of WASP in patient blood samples by immunoblot analysis. We found that despite extensive variation in the nature of the mutations in patients with severe WAS symptoms, none express the protein. However, in 1 patient with a mild clinical phenotype WASP expression was detected. Such an analysis could be used as an initial screening procedure for the diagnosis of WAS prior to genotypic analysis.

Evidence that the Wiskott-Aldrich syndrome protein may be involved in lymphoid cell signaling pathways.

Wiskott-Aldrich syndrome is an X-linked combined immunodeficiency affecting cells of several different hemopoietic lineages. The Wiskott-Aldrich syndrome protein (WASP), which has no homology with any other known protein families, is rich in proline motifs known to contribute to Src homology 3 binding sites. However, its function has not been determined. The Tec family of cytoplasmic tyrosine kinases, which include Btk (the X-linked agammaglobulinemia gene), Itk, and Tec, is thought to be involved in lymphoid cell signaling pathways. In this work, we show binding of WASP to the Src homology 3 domains of Btk, Itk, Tec, Grb2, and phospholipase C-gamma, which suggests a function for WASP in lymphoid cell signaling.

The protein product of the c-cbl protooncogene is phosphorylated after B cell receptor stimulation and binds the SH3 domain of Bruton's tyrosine kinase.

X-linked agammaglobulinemia, a B cell immunodeficiency, is caused by mutations in the Bruton's tyrosine kinase (Btk) gene. The absence of a functional Btk protein leads to a failure of B cell differentiation and antibody production. B cell receptor stimulation leads to the phosphorylation of the Btk protein and it is, therefore, likely that Btk is involved in B cell receptor signaling. As a nonreceptor tyrosine kinase, Btk is likely to interact with several proteins within the context of a signal transduction pathway. To understand such interactions, we have generated glutathione S-transferase fusion proteins corresponding to different domains of the human Btk protein. We have identified a 120-kD protein present in human B cells as being bound by the SH3 domain of Btk and which, after B cell receptor stimulation, is one of the major substrates of tyrosine phosphorylation. We have shown that this 120-kD protein is the protein product of c-cbl, a protooncogene, which is known to be phosphorylated in response to T cell receptor stimulation and to interact with several other tyrosine kinases. Association of the SH3 domain of Btk with p120cbl provides evidence for an analogous role for p120cbl in B cell signaling pathways. The p120cbl protein is the first identified ligand of the Btk SH3 domain.

Discrimination between major histocompatibility complex class II DQ and DR locus products in cattle.

We have used a panel of anti-major histocompatibility complex (MHC) class II monoclonal antibodies (mAbs) and have assessed their specificity for the products of the individual bovine MHC (BoLA) class II subregions. The mAbs identified two distinct class II molecules by affinity purification and ELISA. Two-dimensional immunoblotting confirmed these data and NH2-terminal sequencing of the purified class II alpha chains of one member of each group identified the subregion specificity of the mAbs. The mAbs VPM36, TH22A and TH81A are specific for BoLA DQ, whereas VPM54, TH14B and J11 are specific for BoLA DR. SW73.2 reacts with both MHC subgroups of all cattle tested.

Improved discrimination of bovine class II DR beta-chains polymorphisms using immunoblotting.

An immunoblotting technique is reported that reveals electrophoretic variants in the beta-chains of class II antigens of the bovine major histocompatibility complex. One monoclonal antibody, mAb VPM57, reacted on immunoblots with an epitope present in approximately half of the haplotypes investigated. This reagent is especially useful in discriminating electrophoretic variants that have similar isoelectric points.