Serum oestrogen receptor alpha and beta bioactivity are independently associated with breast cancer: a proof of principle study.

BACKGROUND: Oestrogens play a crucial role in breast carcinogenesis. Earlier studies have analysed the serum levels of endogenous hormones measured by conventional assays. In this study, we analysed the capacity of serum from breast cancer cases and controls to transactivate the oestrogen receptor alpha (ER-alpha) and beta (ER-beta). METHODS: We used a receptor oestrogen-responsive element (ERE) -- the green fluorescent protein (GFP)-reporter test system in Saccharomyces cerevisiae. Oestrogen receptor-alpha or ER-beta bioactivity was determined in serum from 182 randomly chosen postmenopausal women with breast cancer and from 188 age-matched controls. RESULTS: High serum ER-alpha and ER-beta bioactivity were independently associated with the presence of breast cancer. Women whose levels of serum ER-alpha and ER-beta bioactivity were in the highest quintile among controls had a 7.57-(95% confidence interval (CI): 2.46-23.32; P=0.0004) and a 10.14 (95% CI: 3.19-32.23; P<0.0001)-fold risk for general and oestrogen receptor-positive breast cancer, respectively. CONCLUSION: The use of serum ER-alpha and ER-beta bioactivity assays as clinical tools in the management of breast cancer warrants further research. Future studies will dictate whether surrogate markers of ER-alpha and ER-beta bioactivity will provide a means to monitor the efficacy of anti-endocrine, adjuvant and chemopreventive strategies.

Molecular cloning and functional characterisation of a glucose transporter, CaHGT1, of Candida albicans.

We have cloned the first glucose transporter CaHGT1 (Candida albicans high-affinity glucose transporter) of a pathogenic yeast, Candida albicans. The DNA sequence (GenBank accession number Y16834) analysis revealed an ORF encoding a novel protein of 545 amino acids with a predicted molecular mass of 60.67 kDa. The putative protein with 12 transmembrane domains has 51% identity with Kluyveromyces lactis high-affinity glucose transporter, HGT1. The protein signatures which are conserved and distinctive of the sugar transporters belonging to the major facilitator superfamily (MFS) were also found in CaHgt1p. When heterologously expressed, the ORF functionally complemented a mutant strain of Saccharomyces cerevisiae RE700A which was deleted in seven hexose transporter genes and thus was unable to grow or transport glucose. The expression of CaHGT1 in C. albicans showed a transcript of 1.6 kb which was enhanced in response to the human steroid hormone progesterone. Interestingly, the transcript levels were also enhanced in the presence of drugs, e.g. cycloheximide, chloramphenicol and benomyl. The results suggest that CaHGT1, which encodes a MFS protein, could be linked to the drug resistance phenomenon in C. albicans.

Properties and heterologous expression of the glucose transporter GHT1 from Schizosaccharomyces pombe.

Genomic DNA of the Schizosaccharomyces pombe glucose transporter, GHT1, was obtained by complementation of the glucose transport deficient Sz. pombe strain YGS-5. Here we describe the GHT1 gene that encodes a protein of 565 amino acids with a corresponding molecular mass of 62.5 kDa. This eukaryotic glucose transporter contains 12 putative transmembrane segments and is homologous to the HXT multigene family of S. cerevisiae with several amino acid motifs of this sugar transporter family. It is also homologous to other sugar carriers from human, mouse and Escherichia coli. The function of the Ght1 protein as a glucose transporter was proved both by homologous and heterologous expression in the Sz. pombe mutant YGS-5 and in the S. cerevisiae hxt mutant RE700A, respectively. Both transformed yeast strains transported D-glucose with substrate specificity similar to that in Sz. pombe wild-type cells. Moreover, the cells of the two transformed yeast strains accumulated 2-deoxy-D-glucose, a non-metabolizable D-glucose analogue, with an efficiency similar to Sz. pombe wild-type cells. The ability of the S. cerevisiae mutant RE700A to accumulate 2DG in an delta mu H+ dependent manner after transformation with GHT1 provides evidence that the Sz. pombe transporter catalyses an energy-dependent uptake of glucose.

Incompatibility of connexin 40 and 43 Hemichannels in gap junctions between mammalian cells is determined by intracellular domains.

Murine connexin 40 (Cx40) and connexin 43 (Cx43) do not form functional heterotypic gap junction channels. This property may contribute to the preferential propagation of action potentials in murine conductive myocardium (expressing Cx40) which is surrounded by working myocardium, expressing Cx43. When mouse Cx40 and Cx43 were individually expressed in cocultured human HeLa cells, no punctate immunofluorescent signals were detected on apposed plasma membranes between different transfectants, using antibodies specific for each connexin, suggesting that Cx40 and Cx43 hemichannels do not dock to each other. We wanted to identify domains in these connexin proteins which are responsible for the incompatibility. Thus, we expressed in HeLa cells several chimeric gene constructs in which different extracellular and intracellular domains of Cx43 had been spliced into the corresponding regions of Cx40. We found that exchange of both extracellular loops (E1 and E2) in this system (Cx40*43E1,2) was required for formation of homotypic and heterotypic conductive channels, although the electrical properties differed from those of Cx40 or Cx43 channels. Thus, the extracellular domains of Cx43 can be directed to form functional homo- and heterotypic channels. Another chimeric construct in which both extracellular domains and the central cytoplasmic loop (E1, E2, and C2) of Cx43 were spliced into Cx40 (Cx40*43E1,2,C2) led to heterotypic coupling only with Cx43 and not with Cx40 transfectants. Thus, the central cytoplasmic loop of Cx43 contributed to selectivity. A third construct, in which only the C-terminal domain (C3) of Cx43 was spliced into Cx40, i.e., Cx40*43C3, showed neither homotypic nor heterotypic coupling with Cx40 and Cx43 transfectants, suggesting that the C-terminal region of Cx43 determined incompatibility.

The SpTRK gene encodes a potassium-specific transport protein TKHp in Schizosaccharomyces pombe.

Complementary DNAs involved in potassium transport in Schizosaccharomyces pombe were selected by complementation of defective K+ uptake in a trk1 trk2 mutant of Saccharomyces cerevisiae. Here we describe the SpTRK gene that encodes a protein of 833 amino acids. The predicted structure contains 12 putative membrane-spanning domains and resembles various high- and low-affinity systems for K+ transport in yeasts and plants. TKHp, the product of SpTRK exhibits high homology to TRK1 and TRK2 of Saccharomyces cerevisiae as well as to HKT1 of Triticum aestivum, but is not related to HAK1 of another ascomycete, Schwanniomyces occidentalis, suggesting that different routes for potassium uptake evolved independently. This protein is a potassium-specific transporter since functional analysis of the SpTRK complemented mutant strain of Sacch. cerevisiae revealed potassium transport affinities and uptake characteristics similar to those obtained in wild-type Sch. pombe. Patch-clamp analysis in the whole-cell mode confirmed the TKHp-mediated inward current in the complemented strain. The inward current increased by acidification of the extracellular medium thereby suggesting a mechanism of K+H+ cotransport. The inward current is not detectable when external K+ is substituted by Na+, documenting a distinct cation specificity of the protein.

Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells.

DNAs coding for seven murine connexins (Cx) (Cx26, Cx31, Cx32, Cx37, Cx40, Cx43, and Cx45) are functionally expressed in human HeLa cells that were deficient in gap junctional communication. We compare the permeabilities of gap junctions comprised of different connexins to iontophoretically injected tracer molecules. Our results show that Lucifer yellow can pass through all connexin channels analyzed. On the other hand, propidium iodide and ethidium bromide penetrate very poorly or not at all through Cx31 and Cx32 channels, respectively, but pass through channels of other connexins. 4,6 Diamidino-2-phenylindole (DAPI) dihydrochloride shows less transfer among Cx31 or Cx43 transfectants. Neurobiotin is weakly transferred among Cx31 transfectants. Total junctional conductance in Cx31 or Cx45 transfected cells is only about half as high as in other connexin transfectants analyzed and does not correlate exactly with any of the tracer permeabilities. Permeability through different connexin channels appears to be dependent on the molecular structure of each tracer, i.e. size, charge and possibly rigidity. This supports the hypothesis that different connexin channels show different permeabilities to second messenger molecules as well as metabolites and may fulfill in this way their specific role in growth control and differentiation of cell types. In addition, we have investigated the function of heterotypic gap junctions after co-cultivation of two different connexin transfectants, one of which had been prelabeled with fluorescent dextran beads. Analysis of Lucifer yellow transfer reveals that HeLa cells expressing Cx31 (beta-type connexin) do not communicate with any other connexin transfectant tested but only with themselves. Two other beta-type connexin transfectants, HeLa-Cx26 and -Cx32, do not transmit Lucifer yellow to any of the alpha-type connexins analyzed. Among alpha-type connexins, Cx40 does not communicate with Cx43. Thus, connexins differ in their ability to form functional heterotypic gap junctions among mammalian cells.

Immunochemical and electrophysiological characterization of murine connexin40 and -43 in mouse tissues and transfected human cells.

Human HeLa or SkHep1 cells, defective in intercellular communication through gap junctions, were transfected with coding sequences of murine connexin40 (Cx40) and -43. The transfected cells were restored in gap junctional coupling as shown by 100-fold increased electrical conductance. When studied by the double whole-cell patch-clamp technique, Cx40 HeLa transfectants exhibited single channel conductances of gamma = 121 +/- 7 pS and gamma = 153 +/- 5 pS. They were voltage gated with an equivalent gating charge of z = 4.0 +/- 0.5 for a voltage of half-maximal inactivation U0 = 44 +/- 7 mV. The corresponding values of connexin43 (Cx43) HeLa transfectants are: gamma = 60 +/- 4 pS and gamma = 40 +/- 2 pS as well as z = 3.7 +/- 0.8 and U0 = 73 +/- 7 mV. Transfer of the dye Lucifer Yellow was always considerably lower in Cx40- than in Cx43-transfectants though their total junctional conductance was similar or even higher than for Cx43-transfectants. In order to characterize cell and tissue distribution as well as phosphorylation of connexin40 and -43 proteins, antibodies to C-terminal oligopeptides of these proteins were prepared and used for immunoblotting, immunoprecipitation, and immunofluorescence analysis of transfected cells where they exhibited the punctate pattern characteristic of gap junctions on contacting membranes. Phosphorylation of connexin40 was shown by immunoprecipitation from 32P-labeled, transfected SkHep1 cells. Analyses of protein distribution in tissues revealed that the amount of connexin40 detected in heart was higher than in lung which is the inverse of the level of connexin40 mRNA in these tissues, suggesting posttranscriptional control of expression. Connexin40 protein in adult mouse heart and skin is about 20-fold more abundant than in the corresponding embryonic tissue. Connexin43 in adult mouse heart appears to be more highly phosphorylated than in embryonic heart or in transfected human cells.

Molecular cloning and functional expression of mouse connexin40, a second gap junction gene preferentially expressed in lung.

From a mouse genomic library, a clone has been isolated that codes for a connexin-homologous sequence of 358 amino acids. Because of its theoretical molecular mass of 40.418 kD it is named connexin40 (Cx40). Based on both protein and nucleotide sequence, mouse Cx40 is more closely related to mouse Cx43 (alpha subgroup of connexins) than to mouse Cx32 (beta subgroup). The highest overall homology detected, however, was to chick Cx42 (67% amino acid and 86% nucleotide identity), raising the possibility that Cx40 may be the mouse analogue. The coding region of Cx40 is uninterrupted by introns and is detected as a single copy gene in the mouse genome. High stringency hybridization of Northern blots with the coding sequence of Cx40 identified a single transcript of 3.5 kb that is at least 16-fold more abundant in lung-similar to mouse Cx37-than in other adult tissues (kidney, heart, and skin). In embryonic kidney, skin, and liver the level of the Cx40 transcript is two- to fourfold higher than in the corresponding adult tissues. Microinjection of Cx40 cRNA into Xenopus oocytes induced functional cell-to-cell channels between pairs. These channels show a symmetrical and markedly cooperative closure in response to transjunctional voltage (Boltzmann parameters of Vo = +/- 35 mV; A = 0.32) which is also fast relative to other connexin channels recorded similarly (tau = 580 ms at Vj of +/- 50 mV). Although Cx40-expressing oocytes did not couple efficiently with oocytes expressing endogenous connexins, they did couple well to Cx37-expressing oocytes. The heterotypic channels which formed had voltage-gating properties modified from those of the original homotypic forms. Transfection of mouse Cx40 DNA, under control of the SV-40 early promoter, into coupling-deficient human HeLa or SK-Hep-1 cells resulted in expression of the expected transcript and restoration of fluorescent dye transfer in transfected clones.