Cloning and characterization of a specific receptor for mouse oncostatin M

Oncostatin M (OSM) is a member of a family of cytokines that includes ciliary neurotrophic factor, interleukin-6, interleukin-11, cardiotrophin-1, and leukemia inhibitory factor (LIF). The receptors for these cytokines consist of a common signaling subunit, gp130, to which other subunits are added to modify ligand specificity. We report here the isolation and characterization of a cDNA encoding a subunit of the mouse OSM receptor. In NIH 3T3 cells (which endogenously express gp130, LIF receptor beta [LIFRbeta], and the protein product, c12, of the cDNA described here), mouse LIF, human LIF, and human OSM signaled through receptors containing the LIFRbeta and gp130 but not through the mouse OSM receptor. Mouse OSM, however, signaled only through a c12-gp130 complex; it did not use the LIF receptor. Binding studies demonstrated that mouse OSM associated directly with either the c12 protein or gp130. These data highlight the species-specific differences in receptor utilization and signal transduction between mouse and human OSM. In mouse cells, only mouse OSM is capable of activating the mouse OSM receptor; human OSM instead activates the LIF receptor. Therefore, these data suggest that all previous studies with human OSM in mouse systems did not elucidate the biology of OSM but, rather, reflected the biological actions of LIF.     

Differential transactivation by the androgen receptor in prostate cancer cells

BACKGROUND: The purpose of this study was to determine the contribution of different transactivating regions of the androgen receptor (AR) to the induction of androgen-regulated promoters in poorly (PC3 cells) and well-differentiated (LNCaP cells) prostate cancer cell lines. METHODS: PC3 and LNCaP cells were co-transfected with plasmids expressing full-length AR or deletion mutants together with luciferase reporters linked to the probasin (PB) and PSA promoters; as well as to ARR3tk, a PB-derived recombinant promoter. RESULTS: Androgen induction of the ARR3tk promoter in the presence of AR was 8- to 10-fold higher than that seen with the PB promoter. Activation of ARR3tk was greatest with an androgen-independent construct in which the first 231 amino acids and the ligand binding domain had been removed, indicating that this promoter is more responsive to activating functions in the N-terminal domain than in the ligand binding domain. By comparison, induction of the PB promoter was greatest with the full-length AR, which suggests that the ligand binding domain also makes a major contribution to the activation of this promoter. In similar analyses with the PSA promoter, AR regions required for promoter induction was dependent on the host cell type. In PC3 cells, the predominant AR transactivation function was androgen-independent and resided in the N-terminal domain, whereas in LNCaP cells, the highest level of induction was androgen dependent and also required participation of the ligand binding domain. CONCLUSIONS: Our results indicate that the relative utilization of transactivating functions in N-terminal and ligand binding domains of the AR is promoter and cell specific.     

Cloning and characterization of the human galanin GALR2 receptor

We present the molecular cloning and characterization of the human galanin receptor, hGALR2. hGALR2 shares 85%, 39%, and 57% amino acid identities to rGALR2, hGALR1, and hGALR3, respectively. hGALR2, along with rGALR2, can be distinguished from the other cloned galanin receptors by a tolerance for both N-terminal extension and C-terminal deletion of galanin, as well as by a primary signaling mechanism involving phosphatidyl inositol hydrolysis and calcium mobilization. By RT-PCR, GALR2 mRNA was abundant in human hippocampus, hypothalamus, heart, kidney, liver, and small intestine. A weak GALR2 mRNA signal was detected in human retina, and no signal was detected in cerebral cortex, lung, spleen, stomach, or pituitary.     

Cloning and characterization of human estrogen receptor beta isoforms

BACKGROUND: The purpose of this study was to describe a new anti-cancer drug regimen for endometrial cancer. METHODS: The cytotoxicities of some anti-cancer drugs regimens against human endometrial cancer xenografted into nude mice (TEI, TET, TEU, TEN) have been studied. The activities of ADM, CDDP, CPM, CPT-11, TXL, CDDP + ADM, CDDP + CPM, CDDP + CPT-11, CDDP + TXL, CPT-11 + TXL were evaluated in comparison with a control group using saline. Three mice were used for each group, and when xenografted tumor reached 6 mm of its diameter, 1/5 LD50 of these drugs was administered into the periotoneal cavity mice once a week for three weeks. RESULTS: The effective regimens were CPT-11 + TXL, CDDP + CPT-11, CDDP + CPM, CDDP + TXL for the endometrial cancer. CONCLUSIONS: It is suggested that these new drugs regimens should be tested in clinical studies.     

Cloning and characterization of a novel promiscuous human beta-chemokine receptor D6

Members of the chemokine family of chemotactic peptides interact with their target cells through heptahelical cell surface receptors. An understanding of the biochemistry and expression patterns of these receptors is therefore central to our overall understanding of the roles played by chemokines in both physiological and pathological processes. To date, eight receptors for the beta-chemokine subfamily have been described. We have recently cloned a novel murine beta-chemokine receptor and report here the identification and characterization of a highly homologous human gene termed human D6 (hD6). This is a promiscuous beta-chemokine receptor and appears to be able to bind the majority of members of the beta-chemokine family. It is, however, specific for this family and shows no detectable affinity for members of the alpha-chemokine or the C or CXXXC chemokines. Unlike the majority of other chemokine receptors, human D6 does not appear to be able to flux calcium following ligand binding, thus it is currently not clear if this novel receptor is indeed a signaling receptor. Human D6 is expressed in a range of tissues including hemopoietic cells although it appears not to be ubiquitously expressed in hemopoietic cells. Human D6, unlike some other beta-chemokine receptors, appears not to be able to function as an entry co-factor for human immunodeficiency virus type 1 (HIV-)1 on CD4-expressing cells.     

Cloning, expression, and characterization of a cDNA encoding a novel human growth factor for primitive hematopoietic progenitor cells

Multiple growth factors synergistically stimulate proliferation of primitive hematopoietic progenitor cells. A human myeloid cell line, KPB-M15, constitutively produces a novel hematopoietic cytokine, termed stem cell growth factor (SCGF), possessing species-specific proliferative activities. Here we report the molecular cloning, expression, and characterization of a cDNA encoding human SCGF using a newly developed lambdaSHDM vector that is more efficient for differential and expression cloning. cDNA for SCGF encodes a 29-kDa polypeptide without N-linked glycosylation. SCGF transiently produced by COS-1 cells supports growth of hematopoietic progenitor cells through a short-term liquid culture of bone marrow cells and exhibits promoting activities on erythroid and granulocyte/macrophage progenitor cells in primary semisolid culture with erythropoietin and granulocyte/macrophage colony-stimulating factor, respectively. Expression of SCGF mRNA is restricted to myeloid cells and fibroblasts, suggesting that SCGF is a growth factor functioning within the hematopoietic microenvironment. SCGF could disclose some human-specific mechanisms as yet unidentified from studies on the murine hematopoietic system.     

Hydrolysis of androgen receptor by cathepsin D: its biological significance in human prostate cancer

Risk factors, etiology, and outcome of 180 cases of infective endocarditis (IE) in the Slovak Republic for 5 years were prospectively studied in a national survey. According to the Duke Endocarditis Service Criteria (1994), 169 cases were considered definitive and 21 possible/probable. The aortic valve was infected in 46.7%, mitral in 47.2%, and tricuspidal/pulmonary in 6.1% of cases. The majority of endocarditis cases was caused by Staphylococcus aureus and coagulase-negative staphylococci (CNS) (33.3%); only 12.2% were due to viridans streptococci; 11.7% were due to Enterococcus faecalis; 6.1% due to Haemophilus spp.; 10.1% due to other organisms; and 26.7% were culture negative. Single positive cultures of CNS were not considered clinically significant. More than 25% of 180 patients were older than 60 years. Rheumatic fever was a risk factor in 35.5%, dental surgery in 20.5%, prior cardiosurgery in 7.8%, and neoplasia in 6.7%. All patients were treated with antimicrobials (average length of therapy was 29.5 days) and 33.3% of patients also had surgery (valvular prosthesis replacement). Forty (22.2%) died, and 140 (77.8%) survived at day 60 after the diagnosis of endocarditis was made. All 40 deaths were attributable to infection. Univariate analysis comparing deaths and survivors did not show significant differences in most of the recorded risk factors between both groups, except age > 60 (40.0% versus 21.4%, p < 0.05), staphylococcal etiology (55.0% versus 27.1%, p < 0.04), and antibiotic therapy < 21 days (without surgery) (65.0% versus 3.6%, p < 0.01). These risk factors were significantly more frequently associated with deaths. Viridans streptococcal IE and surgical therapy in addition to antibiotics were associated with lower mortality in comparison to staphylococcal endocarditis (p < 0.045) or to cases treated with antibiotics only (p < 0.05). In comparison to other nationally based surveys in Europe (Greece, Croatia, France), the percentage of culture-negative endocarditis and spectrum of pathogens differed significantly.     

Expression of androgen receptor and growth factors in premalignant lesions of the prostate

To examine whether the expression pattern of fast-muscle type troponin-T (TnT) isoforms was fixed in cell lineage, breast muscle pieces (pectoralis major) from chick embryos and young and adult chickens were grafted on to chorio-allantoic membrane of 9-day-old chick embryos and cultured until the host embryos hatched out. Muscle fibre formation of the grafts was investigated by histological and immunohistochemical methods with anti-fast-muscle type and anti-slow-muscle type TnT sera, and the expression of fast-muscle type TnT in the grafts from chick embryos and young chickens was studied by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional SDS-PAGE, and immunoblotting. In the chorio-allantoic grafting, the breast muscle initially degenerated forming pyknotic nuclei and hyaline cytoplasm. The surviving cells, which were supposed to be satellite cells, regenerated new muscle fibres of the same type as those of the grafted muscle in respect of TnT isoform expression. Therefore, we considered that the ability to express specific isoforms of TnT was fixed in the satellite cells, and that chorio-allantoic grafting was a useful technique for studying muscle differentiation.     

Differential expression and androgen regulation of the human selenium-binding protein gene hSP56 in prostate cancer cells

Low levels of dietary selenium are associated with increased risk of malignancy of several organs, including the prostate. Using a subtractive approach called linker capture subtraction, we have found that the human selenium-binding protein gene hSP56 is differentially expressed by the relatively slow-growing, androgen-sensitive prostate cancer cell line LNCaP but not by the more rapidly growing androgen-insensitive lines PC-3 and DU145. We confirmed this differential expression by Northern blot analysis. Importantly, hSP56 expression by LNCaP cells was reversibly down-regulated by exogenous androgen in a concentration-dependent manner. Marked differences in steady-state hSP56 mRNA levels were found in a variety of normal and neoplastic human cells that were examined. hSP56 expression was especially high in normal tissues that appear to benefit from the cancer-protective action of dietary selenium and was low in many neoplastic cells. The results suggest that hSP56 may play a role in determining the neoplastic phenotype.     

Microsatellite mutation (CAG24-->18) in the androgen receptor gene in human prostate cancer

The androgen receptor (AR) gene contains a polymorphic CAG microsatellite that codes for a variable length of glutamine repeats in the AR protein. Microsatellite DNA sequences may be potential sites of genetic instability. Using the polymerase chain reaction (PCR), we screened 40 human prostate cancer specimens for expansions or deletions of this microsatellite. In one patient, nontumor DNA yielded a single PCR product, as expected for the AR, but the tumor DNA yielded two discrete products, one identical to normal, and a second smaller one. Direct sequencing revealed that the nontumor tissue contained 24 CAGs, whereas the tumor contained one fragment with 24 CAGs (wild-type) and a second fragment with 18 CAGs (mutant), representing a somatic contraction of the AR CAG repeat (CAG24-->CAG18) in the tumor. Interestingly, this patient manifested a paradoxical agonistic response to hormonal therapy with the antiandrogen flutamide.