Estrogen stimulation of ovarian surface epithelial cell proliferation

Summary Ovarian cancer is the leading cause of gynecological cancer mortality, and 85–90% of this malignancy originates from the ovarian surface epithelium (OSE). The etiology of ovarian epithelial cancer is unknown but a role for estrogens has been suspected. However, the effect of estrogens on OSE cell proliferation remains to be determined. Using the rabbit model, our studies have demonstrated that 17β-estradiol stimulates OSE cell proliferation and the formation of a papillary ovarian surface morphology similar to that seen in human ovarian serous neoplasms of low malignant potential. Immunohistochemical staining of ovarian tissue sections with an antibody to the estrogen receptor α demonstrates its expression in both OSE cells and stromal interstitial cells. In primary ovarian cell cultures, the proliferative response of the epithelial cells to 17β-estradiol depends on the expression of the estrogen receptor α in the epithelial cells. However, when the epithelial cells are grown together with ovarian stromal cells, their proliferative response to this hormone is greatly enhanced, suggesting the involvement of stromal-epithelial interactions. These studies suggest a role for estrogens and the estrogen receptor α in OSE growth.     

Novel oligosaccharide has suppressive activity against human leukemia cell proliferation

Various oligosaccharides containing galactose(s) and one glucosamine (or N-acetylglucosamine) residues with β1–4, α1–6 and β1–6 glycosidic bond were synthesized; Galβ1–4GlcNH₂, Galα1–6GlcNH₂, Galα1–6GlcNAc, Galβ1–6GlcNH₂, Galβ1–4Galβ1–4GlcNH₂ and Galβ1–4Galβ1–4GlcNAc. Galα1–6GlcNH₂ (MelNH₂) and glucosamine (GlcNH₂) had a suppressive effect on the proliferation of K562 cells, but none of the other saccharides tested containing GlcNAc showed this effect. On the other hand, the proliferation of the human normal umbilical cord fibroblast was suppressed by none of the saccharides other than GlcNH₂. Adding Galα1–6GlcNH₂ or glucosamine to the culture of K562 cell, the cell number decreased strikingly after 72 h. Staining the remaining cells with Cellstain Hoechst 33258, chromatin aggregation was found in many cells, indicating the occurrence of cell death. Furthermore, all of the cells were stained with Galα1–6GlcNH-FITC (MelNH-FITC). Neither the control cells nor the cells incubated with glucosamine were stained. On the other hand, when GlcNH-FITC was also added to cell cultures, some of them incubated with Galα1–6GlcNH₂ were stained. The difference in the stainability of the K562 cells by Galα1–6GlcNH-FITC and GlcNH-FITC suggests that the intake of Galα1–6GlcNH₂ and the cell death induced by this saccharide is not same as those of glucosamine. The isolation of the Galα1–6GlcNH₂ binding protein was performed by affinity chromatography (melibiose-agarose) and LC-MS/MS, and we identified the human heterogeneous ribonucleoprotein (hnRNP) A1 (34.3 kDa) isoform protein (30.8 kDa). The hnRNP A1 protein was also detected from the eluate(s) of the MelNH-agarose column by the immunological method (anti-hnRNP-A1 and HRP-labeled anti-mouse IgG (γ) antibodies).     

Phenotypes of HeLa S3 variant cell lines resistant to growth inhibition by sodium butyrate

Summary HeLa cell variants capable of multiplying in the presence of sodium butyrate were used to study the relationship of cell cycle position to human chorionic gonadotropin (hCG) production and regulation of the genes encoding hCG α- and β-subunits. The butyrate-resistant variants exhibit several different stable phenotypes. In wild-type HeLa cells, butyrate arrests cell division and modulates synthesis of α- and β-subunits of glycoprotein hormones by coordinately regulating steady-state levels of their respective mRNAs. Because the variant cell lines replicate, in addition to producing hCG subunits in the presence of butyrate, cell cycle arrest does not seem to be a requirement for expression of glycoprotein hormone genes. Studies of histone modification suggest that neither hyperacetylation of histones H3 and H4 nor dephosphorylation of histones H1 and H2A mediates inhibition of cell replication. In the variants, α-subunit and hCGβ levels are independently regulated, as a consequence of independent regulation of α- and β-hCG mRNA levels. Long-term effects of butyrate include derepression of some genes (hCGβ in the variant AO) and repression of others (hCGα in variant AO). Moreover, hormone production correlates with the steady-state levels of mRNA for each of the subunits, suggesting that regulation occurs before translation. These findings indicate that the butyrate-resistant variant cell lines are valuable for studies of the molecular mechanisms involved in regulation of expression of ectopic hormones.     

Characterization of the inflammatory cell populations in normal colon and colonic carcinomas

Little is known about the nature of the mucosa-associated immune system within the normal colon, or about the immune response to colon carcinoma. In this study inflammatory cells (ICs) in 14 normal colons and 14 carcinomas were characterized. Overall inflammation, lymphocytes, plasma cells, neutrophils, and eosinophils were graded in routine H & E sections. Frozen sections were stained by an immunoperoxidase technique using antibodies to the T cell associated antigens CD2, CD7, CD4, CD8, and T cell receptors αβ and γδ. B cells were identified with CD20, macrophages with CD68, and Class II antigen with anti-HLA DR. Each cell type was semiquantitatively graded in 10 high power fields (HPFs) in the lumenal half (LH) or basal half (BH) of the normal mucosae, and in epithelium or stroma of the carcinomas. In normal colons, ICs were more frequent in LH than in BH. Plasma cells, lymphocytes and monocytes predominated. Subtyping of lymphocytes showed that CD4⁺ TCR αβ⁺ T lymphocytes were most numerous in the lamina propria. Lymphocytes within the epithelium were CD8⁺ T cells. Around carcinomas the overall grade of ICs was 1+ in the majority of cases. Plasma cells, CD4⁺ and CD8⁺ cells with the TCR αβ receptor, and macrophages were most frequent. Lymphoid aggregates of both T and B cells were frequent. Conclusions: 1. Normal colon contains a diffuse lumenally oriented population of TCR αβ⁺ CD4+ T cells, plasma cells, macrophages and class II antigen-expressing cells in the lamina propria. Intraepithelial lymphocytes are of the T suppressor phenotype. CD4+ T cells, macrophages and HLG-DR⁺ cells predominate in the response to colon carcinomas.     

Structural and immunochemical characterization of β-1,2-linked mannobiosyl phosphate residue in the cell wall mannan of Candida glabrata

A mannan of Candida glabrata IFO 0622 digested by Arthrobacter exo-α-mannosidase and a β-1,2-linked mannobiose obtained from the parent mannan by acid treatment was analyzed using ¹³C nuclear magnetic resonance spectroscopy. The results show that the β-1,2-linked mannobiosyl residue is esterified to a phosphate group through position C-1 in the α-configuration, Manβ1– 2Manα1–HPO₃–. The results of immunochemical assays of these mannans using the commercial antigenic factor sera of the genus Candida (Candida Check, Iatron) indicate that the main recognition site of serum no. 6 in this kit is the mannotetraosyl side-chain Manβ1–2Manα1– 2Manα1–2Man in C. glabrata mannan and also suggest that the phosphate-containing unit (such as Manβ1– 2Manα1–HPO₃– in this mannan) behaves as one of the antigenic determinants of serum no. 6, but not of serum no. 5. Therefore, the present and previous findings indicate that serum no. 5 recognizes relatively longer β-1,2-linked oligomannosyl side-chains, Manβ1–[2Manβ1–]_n 2Man (n = 1–6), attached to the phosphate groups previously observed in the cell wall mannans of Candida albicans, Candida stellatoidea, and Candida tropicalis. Received: 18 March 1997 / Accepted: 16 September 1997     

Single chain human chorionic gonadotropin,hCGαβ: Effects of mutations in the α subunit on structure and bioactivity

The strategy of translationally fusing the subunits of heterodimeric proteins into single chain molecules is often used to overcome the mutagenesis-induced defects in subunit interactions. The approach of fusing the α and β subunits of human Chorionic Gonadotropin (hCG) to produce a single chain hormone (phCGαβ) was used to investigate roles of critical residues of the α subunit in hormone receptor interaction and biological activity. The α subunit was mutated using PCR-based site-directed mutagenesis, fused to the wild type β subunit and the fusion protein was expressed using Pichia pastoris expression system. Following partial purification, the mutant proteins were extensively characterized using immunological probes, receptor assays, and in vitro bioassays. The mutation hCGα P38A, which disrupts subunit interaction in the heterodimeric molecule, produced a fusion molecule exhibiting altered subunit interactions as judged by the immunological criteria, but could bind to the receptor with lower affinity and elicit biological response. Mutation of hCGα T54A disrupting the glycosylation at Asparagine 52, believed to be important for bioactivity, also yielded a biologically active molecule suggesting that the glycosylation at this site is not as critical for bioactivity as it is in the case of the heterodimer. The fusion protein approach was also used to generate a superagonist of hormone action. Introduction of four lysine residues in the Loop 1 of the α subunit led to the generation of a mutant having higher affinity for the receptor and enhanced bioactivity. Immunological characterization of single chain molecules revealed that the interactions between the subunits were not identical to those seen in the heterodimeric hormone, and the subunits appeared to retain their isolated conformations, and also retained the ability to bind to the receptors and elicit response. These data suggest the plasticity of the hormone-receptor interactions.     

Studies on the mechanism of the selenite-induced decrease in cell attachment

We previously reported that exposure of HeLa cells to selenite for 2 h results in a decrease in their ability to attach to fibronectin (Yan and Frenkel,Cancer Res. 52, 5803–5807 [1992]), as well as a decrease in the level of fibronectin receptor (α5β1 integrin) at the cell surface (Yan and Frenkel,Biol. Trace Element Res. 46, 79–89 [1994]). We have now found that after exposure to selenite, there was a decrease in the total cellular content of the receptor protein, as well as in the level of the mRNAs for both of the subunits. Exposure of cells to actinomycin D (an inhibitor of RNA synthesis) also resulted in a decrease in the level of these mRNAs, suggesting that the effect of selenite is the result of its known inhibitory effect on RNA synthesis (Frenkel,Toxicol. Lett. 25, 219–223 [1985]). Exposure of cells to actinomycin D for 2 h also resulted in a decrease in the ability of cells to attach to fibronectin. Furthermore, both selenite and actinomycin D caused a decrease in integrin mRNA levels and in cell attachment to fibronectin only when high-density cells were exposed to the agents. In contrast, when low-density cells were exposed,neither agent had any detectable effect on mRNA levels or on cell attachment. These results have suggested the following scheme for the mechanism of the inhibition of cell attachment by selenite: After exposure to selenite for 2 h, there is a significant inhibition of cellular RNA synthesis, which results in a general decrease in the cellular level of those mRNAs with relatively short half-lives, including in particular those of the fibronectin receptor. This leads to a decrease in the intracellular level of the receptor protein and, consequently, in its level at the cell surface, which in turn causes a decrease in the rate of cell attachment to fibronectin.     

T-cell receptor Vβ5 usage defines reactivity to a human T-cell receptor monoclonal antibody

The monoclonal antibody 3D6 reacts with the T-cell receptor (Tcr) on the T-leukemic line HPBALL and with 2–13% of human peripheral blood T lymphocytes. In this study V_α and V_β expression in a panel of T-cell populations and clones expressing the 3D6 epitope was determined by Southern and northern hybridization analysis. The results demonstrate that these 3D6-positive T cells, regardless of CD4/CD8 phenotype or function, express a gene of the V_β5 family, also expressed by HPBALL. No correlation of the HPBALL V_α gene. The results demonstrate that 3D6 recognizes an epitope solely on the Tcr_β chain and that the use of this β chain, together with an appropriate V_α, can impart a diverse pattern of reactivity to a T cell.     

Alpha-Galactosyl trisaccharide epitope: Modification of the 6-primary positions and recognition by human anti-αGal antibody

Galactose oxidase (EC 1.1.3.9, GAO) was used to convert the C-6′ OH of Galβ(1 → 4)Glcβ–OBn (5) to the corresponding hydrated aldehyde (7). Chemical modification, through dehydratative coupling and reductive amination, gave rise to a small library of Galβ(1 → 4)Glcβ–OBn analogues (9a–f, 10, 11). UDP-[6-³H]Gal studies indicated that α1,3-galactosyltransferase recognized the C-6′ modified Galβ(1 → 4)Glcβ–OBn analogues (9a–f, 10, 11). Preparative scale reactions ensued, utilizing a single enzyme UDP-Gal conversion as well as a dual enzymatic system (GalE and α1,3GalT), taking full advantage of the more economical UDP-Glc, giving rise to compounds 6, 15–22. Galα(1 → 3)Galβ(1 → 4)Glcβ–OBn trisaccharide (6) was produced on a large scale (2 g) and subjected to the same chemoenzymatic modification as stated above to produce C-6″ modified derivatives (23–30). An ELISA bioassay was performed utilizing human anti-αGal antibodies to study the binding affinity of the derivatized epitopes (6, 15–30). Modifications made at the C-6′ position did not alter the IgG antibody's ability to recognize the unnatural epitopes. Modifications made at the C-6″ position resulted in significant or complete abrogation of recognition. The results indicate that the C-6′ OH of the αGal trisaccharide epitope is not mandatory for antibody recognition. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Diethylstilbestrol increases the density of prolactin cells in male mouse pituitary by inducing proliferation of prolactin cells and transdifferentiation of gonadotropic cells

Diethylstilbestrol (DES) has been implicated in mammalian abnormalities. We examined the effects of DES on follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) cells in the pituitaries of male mice treated with various doses of DES for 20 days. DES reduced the density of FSH and LH cells in a dose-dependent manner, but increased that of PRL cells. When the expression of estrogen receptor (ER) α and β was assessed, an induction of ERβ by DES was found predominantly in PRL cells. However, since these effects were abolished in ERα knockout mice, DES appears to act primarily through ERα. When the expression of Ki-67 and Pit-1 in PRL cells was examined at various time-points after DES treatment, some PRL cells became Ki-67 positive at 10–15 days, and Pit-1-positive cells were increased at 5–15 days. Furthermore, some FSH and LH cells became Pit-1 positive, and co-localized with PRL at 5–10 days. Our results indicate that DES increases PRL cells by inducing proliferation of PRL cells and transdifferentiation of FSH/LH cells to PRL cells.     

Relative ability of ovine follicle stimulating hormone and itsβ-subunit to generate antibodies having bioneutralization potential in nonhuman primates

The relative ability of ovine follicle stimulating hormone and itsβ-subunit, two potential candidates for male contraceptive vaccine, to generate antibodies in monkeys capable of bioneutralizing follicle stimulating hormone was assessed usingin vitro model systems. Antiserum against native ovine follicle stimulating hormone was found to be highly specific to the intact form with no cross-reactivity with either of the two subunits while the antiserum againstβ-subunit of follicle stimulating hormone could bind to theβ-subunit in its free form as well as when it is combined withα-subunit to form the intact hormone. Both antisera could block the binding of the hormone to the receptor if the hormone was preincubated with the antibody. However, the follicle stimulating hormoneβ-antisera could only inhibit the binding of the hormone partially (33% inhibition) if the antibody and receptor were mixed prior to the addition of the hormone, while antisera to the native follicle stimulating hormone could block the binding completely (100% inhibition) in the same experiment. Similarly antisera to the native follicle stimulating hormone was significantly effective in blocking (100%) response to follicle stimulating hormone but not theβ-subunit antisera (0%) as checked using anin vitro granulosa cell system. Thus the probability of obtaining antibodies of greater bioneutralization potential is much higher if intact hormone is used as an antigen rather than itsβ-subunit as a vaccine. Majority of the work reported here was carried out during the tenure of Visiting Scientist fellowship awarded by the MRC Canada to the first author.     

CD40-ligation-mediated protection from apoptosis of a Fas-sensitive Hodgkin's-disease-derived cell line

 Paclitaxel or Taxol has attracted a great deal of attention in recent years because of its immense success as a chemotherapeutic agent for numerous types of cancer. It is known that paclitaxel stabilizes microtubules, and this characteristic is the presumed primary mechanism for its antitumor activity. Recently, however, paclitaxel’s ability to regulate gene expression, particularly in the murine system, has been reported by several groups. Here, we present research examining paclitaxel’s ability to alter expression of the interleukin-1β (IL-1β) and IL-8 cytokines in primary human monocytes, T lymphocytes, and four human breast cancer cell lines: MCF-7, ZR-75-1, MDA-MB-468, and MDA-MB-231. This report shows for the first time that treatment with 5–50 μM paclitaxel increases steady-state levels of IL-1β mRNA in unprimed human monocytes, MCF-7, and ZR-75-1 cells. Monocytes from eight donors in 16 experiments showed increased IL-1β secretion upon treatment; however, the increase in IL-1β production by monocytes was predicated on culturing in the absence of fetal bovine serum or in the presence of autologous human serum. In contrast to the IL-1β results, paclitaxel did not have significant effects on IL-8 expression by monocytes, T lymphocytes, or the breast cancer cells. These data show a specific effect of paclitaxel on cytokine synthesis by both immune cells and cancer cells. Received: 8 September 1997 / Accepted: 26 November 1997     

Serum immunoglobulins in patients with carcinoma of the oral cavity,uterine cervix and breast

Summary Serum immunoglobulin levels (IgG, IgA, IgM, IgD and IgE) were estimated in 196 patients with carcinoma of the oral cavity, 172 patients with cervical cancer and 166 patients with breast cancer. The values were compared with those of 50 patients with benign lesions of the breast and cervix and 100 healthy adult controls. Only the serum IgE levels were found to be elevated in the benign group. Serum IgA, IgD and IgE levels were found to be elevated in all the three types of cancers and the levels were found to increase with clinical stage. In carcinoma of uterine cervix IgG levels were also found to be elevated. Immunoglobulins A and D returned to normal after clinical cure whereas IgE remained slightly elevated. IgD and IgE remained high in patients who had residual cancer.     

Cytokine production by a megakaryocytic cell line

Summary The regulation of megakaryopoeisis by cytokines is not yet well understood. It is possible that autocrine loops are established during megakaryocyte growth and differentiation, aiding in the maturation of these cells. The CHRF-288-11 human megakaryoblastic cell line has been examined for cytokine production in growing cells and cells stimulated to differentiate by the addition of phorbol esters. It has been demonstrated that these cells produce RNA corresponding to the interleukins IL-1α, 1β, 3, 7, 8, and 11, granulocyte-macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), interferon-α (INF-α), and basic fibroblast growth factor (bFGF). Additionaly, RNA corresponding to the receptors for IL-6, GM-CSF, SCF, INF-α,β, bFGF, and monocyte colony stimulating factor (M-CSF) were also expressed by the cells. The receptor for TNF-α was detected immunologically. Analysis at the protein level demonstrated that significant amounts of INF-α, TNF-α, GM-CSF, SCF, IL-1α, and a soluble form of the IL-6 receptor were produced by the cells. Addition of phorbol esters to CHRF-288-11 cells enhances their megakaryocytic phenotype; such treatment also results in increased secretion of INF-α, TNF-α, and GM-CSF. These results suggest that potential autocrine loops are established during the differentiation of CHRF-288-11 cells, which may alter the capability of the cell to differentiate. These findings are similar to those recently obtained for marrow-derived megakaryocytes (Jiang et al.) suggesting that CHRF-288-11 cells provide a useful model system for the study of cytokine release during megakaryocyte differentiation.     

The helper T-cell repertoire of mice expressing class II major histocompatibility complex β chains in the absence of α chains

 Mutant mice generated by disrupting the H2-Aa ^b major histocompatibility complex (Mhc) gene are demonstrated here to express Aβ^b chains in the absence of α chains. These mice possess a CD4⁺ helper T cell (Th) repertoire that uses predominantly the Vβ7 T-cell antigen receptor (Tcr) segment for recognition of any protein antigen presented by the α-free Aβ molecule. As an alloantigen, the Aα-free Aβ molecule is recognized very poorly by T cells from a series of class II disparate mouse strains, indicating that it is grossly different from normal α/β heterodimers. Indeed, molecular modeling suggests a β/β homodimer arrangement with an altered geometry of the Tcr contact area. Interestingly, the mutant mice exhibit normal alloreactivity, without a restricted Vβ usage, toward a series of foreign α/β class II heterodimers, although their T cells developed in the absence of such heterodimers. Thus, the complementarity of Tcr to normal α/β heterodimers, and thereby also alloreactivity, appears to be an ontogeny independent (i. e., germline-encoded) feature. Received: 30 September 1996 / Revised: 18 October 1996