Immunohistochemical study of granular cell tumors of the neurohypophysis.

Six granular cell tumors (GCT) of the neurohypophysis were studied by immunohistochemical techniques. They were all labeled by peanut lectin (Arachis hypogaea) and three showed reactivity for S-100 protein. Unlike extracranial GCT, neuron specific enolase (NSE), myelin basic protein (MBP) and vimentin were not detected in the tumor cells. Glial fibrillary acidic protein (GFAP), keratin and desmin were also not observed. On the other hand, some showed reactivity for alpha-1-antitrypsin (AAT), alpha-1-antichymotrypsin (AAC) and cathepsin B. These results suggest that neurohypophysial GCT have some features different from extracranial GCT and that they may not be derived from Schwann cells.     

T cells stimulate catabolic gene expression by the stromal cells from giant cell tumor of bone

The factors that promote the localized bone resorption by giant cell tumor of bone (GCT) are not fully understood. We investigated whether T cells could contribute to bone resorption by stimulating expression of genes for parathyroid hormone-related protein (PTHrP), matrix metalloproteinase (MMP)-13, and the receptor activator of nuclear-factor κB ligand (RANKL). Two cell lines, Jurkat clone E6-1 and D1.1, were co-cultured with isolated GCT stromal cells. Real-time PCR analyses demonstrated a significant increase of all three genes following 48h incubation, and PTHrP and MMP-13 gene expression was also increased at 24h. Further, we examined the expression of CD40 ligand (CD40L), a protein expressed by activated T cells, and its receptor, CD40, in GCT. Immunohistochemistry results revealed expression of the CD40 receptor in both the stromal cells and giant cells of the tumor. RNA collected from whole GCT tissues showed expression of CD40LG, which was absent in cultured stromal cells, and suggests that CD40L is expressed within GCT. Stimulation of GCT stromal cells with CD40L significantly increased expression of the PTHrP and MMP-13 genes. Moreover, we show that inhibition of PTHrP with neutralizing antibodies significantly decreased MMP13 expression by the stromal cells compared to IgG-matched controls, whereas stimulation with PTHrP (1-34) increased MMP-13 gene expression. These results suggest that T cells may potentiate the catabolic effect of GCT.     

Granular cell tumors: evidence for heterogeneous tumor cell differentiation

Eighteen granular cell tumors from various sites were examined with antisera directed against protein S-100, neuron specific enolase (NSE), alpha-1-antichymotrypsin, and alpha-1-antitrypsin, glial fibrillary acidic protein (GFAP), lysozyme, factor VIII-related antigen, myoglobin and vimentin, as well as with a monoclonal antibody (lu-5) directed against a panepithelial marker. The immunocytochemical reaction pattern of the tumors was heterogeneous. The brain and pituitary tumors and one thyroid tumor reacted for alpha-1-antichymotrypsin and alpha-1-antitrypsin, but not for S-100 protein and NSE. However, tumors from other sites showed immunoreactions for S-100 protein and NSE and some also for vimentin. Reactions for alpha-1-antichymotrypsin and alpha-1-antitrypsin were not observed. All other reactions were similarly negative. We conclude that the morphologically homogeneous group of granular cell tumors is biologically heterogeneous.     

Proteomic Profiling of a Mouse Model for Ovarian Granulosa Cell Tumor Identifies VCP as a Highly Sensitive Serum Tumor Marker in Several Human Cancers

The initial aim of this study was to identify novel serum diagnostic markers for the human ovarian granulosa cell tumor (GCT), a tumor that represents up to 5% of all ovarian cancers. To circumvent the paucity of human tissues available for analyses, we used the Ctnnb1(tm1Mmt/+);Pten(tm1Hwu/tmiHwu);Amhr2(tm3(cre)Bhr/+) transgenic mouse model, which features the constitutive activation of CTNNB1 signaling combined with the loss of Pten in granulosa cells and develops GCTs that mimic aggressive forms of the human disease. Proteomic profiling by mass spectrometry showed that vinculin, enolase 1, several heat shock proteins, and valosin containing protein (VCP) were more abundantly secreted by cultured mouse GCT cells compared to primary cultured GC. Among these proteins, only VCP was present in significantly increased levels in the preoperative serum of GCT cancer patients compared to normal subjects. To determine the specificity of VCP, serum levels were also measured in ovarian carcinoma, non-Hodgkin's lymphoma and breast, colon, pancreatic, lung, and prostate cancer patients. Increased serum VCP levels were observed in the majority of cancer cases, with the exception of patients with lung or prostate cancer. Moreover, serum VCP levels were increased in some GCT, ovarian carcinoma, breast cancer, and colon cancer patients who did not otherwise display increased levels of widely used serum tumor markers for their cancer type (e.g. inhibin A, inhibin B, CA125, CEA, or CA15.3). These results demonstrate the potential use of VCP as highly sensitive serum marker for GCT as well as several other human cancers.     

Interactions of dimethyl sulfoxide and granulocyte-macrophage colony-stimulating factors on the growth and maturation of HL-60 cells

We have studied the interactions of dimethyl sulfoxide (DMSO), Giant Cell Tumor (GCT) cell-conditioned medium (GCT CM), and highly purified granulocyte-macrophage colony-stimulating factors (GM-CSF) on the growth and maturation of a highly passaged population of HL-60 cells. DMSO produced dose-dependent inhibition of HL-60 growth in liquid and semisolid media. Growth was partially to completely restored by the addition of GCT CM to cultures. Experiments in which cell volume, cell cycle kinetics, tritiated thymidine (3HTdr) incorporation, cell number, and nitroblue tetrazolium (NBT) reduction were compared during culture indicated that DMSO inhibited the spontaneous increase in cell volume and flow of cells through the cell cycle which occurred in the first day of culture, the increase in 3HTdr incorporation which was detectable by day 2; and the increment in cell counts which occurred by day 3. These effects were opposed by GCT CM. In contrast, the DMSO-induced increase in NBT reduction which occurred by day 6 was not influenced by GCT CM. The major principle opposing DMSO was GM-CSF, since (1) highly purified GM-CSF from GCT cells and recombinant GM-CSF from COS cells transfected with the Mo cell GM-CSF gene overcame greater than 50% of DMSO inhibition; and (2) conditioned media from cells not producing CSF, G-CSF from GCT cells, and recombinant G-CSF from Escherichia coli transfected with the G-CSF gene from 5,637 cells were inactive. DMSO had little or no effect on the elaboration of autostimulatory activity by HL-60 cells. DMSO is a useful agent for inhibiting the spontaneous growth of HL-60 cells and restoring their dependence on GM-CSF, a property which may be mediated through the effects of DMSO on cell cycle kinetics and/or maturation.     

Isolation and characterization of a multipotent clone of human embryonal carcinoma cells

Histopathological studies suggest that the stem cells of human teratomas may be classified into two major categories: nullipotent stem cells, and multipotent stem cells, capable both of self-renewal and differentiation into a wide range of somatic and extraembryonic cell types. We have isolated a multipotent stem cell clone from the human teratoma cell line GCT 27, and compared its properties to a nullipotent clone derived from the same strain. The multipotent clone GCT 27 X-1 gave rise to colonies of mixed cell morphology in vitro. Analysis of cell surface, cytostructural and extracellular matrix markers in GCT 27 X-1 cells showed that the stem cells of this line were very similar in phenotype to nullipotent cells. The two cell clones were predominantly hypotriploid, and contained several marker chromosomes in common. GCT 27 X-1 was feeder-cell-dependent for continuous growth in vitro; removal of the feeder layer resulted in differentiation of the stem cells into a variety of cell types, some with characteristics of extraembryonic endoderm, others showing neuronal properties. When transplanted into nude mice, GCT 27 X-1 cells gave rise to teratocarcinomas containing embryonal carcinoma stem cells, and many other cell types: yolk sac carcinoma cells; cells producing alphafetoprotein or human chorionic gonadotrophin; glandular, columnar, cuboidal, and squamous epithelium; primitive mesenchyme and cartilage; neuroectodermal cells. Nullipotent GCT 27 C-1 cells could form colonies in the absence of feeder layers, but multipotent GCT 27 X-1 cells could not. While a range of known growth factors and related substances failed to substitute for feeder layers in supporting the growth of GCT 27 X-1 stem cells, supernatants from yolk sac carcinoma cell line GCT 44 could partially replace the feeder cell requirement. Thus, the results revealed a basic difference in growth control between these multipotent and nullipotent human embryonal carcinoma cells, and suggested a possible paracrine regulatory pathway between multipotent stem cells and yolk sac carcinoma cells.     

Additive and/or synergistic action (downregulation) of androgens and thyroid hormones on the cellular distribution and localization of a true tissue kallikrein, mK1, in the mouse submandibular gland.

We investigated the effects of 5alpha-dihydrotestosterone (DHT), 3,5,3'-triiodo-l-thyronine (T(3)), and dexamethasone (Dex) on the expression of mK1 in the granular convoluted tubule (GCT) cells of the submandibular gland (SMG) of hypophysectomized (Hypox) male mice by indirect enzyme-labeled antibody and immunogold antibody methods for light and electron microscopy. Hypox resulted in considerable atrophy of the GCT cells, which were always immunoreactive for mK1, and the cells were characterized by apical small dense secretory granules labeled with gold particles suggesting the presence of mK1, small Golgi apparatus, sparse rough endoplasmic reticulum (RER), and developed basal infoldings. Each of the hormones, DHT, T(3), and Dex, enhanced the GCT phenotype to various degrees in Hypox male mice. Both DHT alone and T(3) alone moderately inhibited mK1 synthesis by increasing the number of mK1-immunonegative GCT cells in Hypox males, but Dex alone had no inhibitory effect on mK1 synthesis. A significant trophic effect on GCT cells was induced by combined injection of DHT and T(3) or of all three hormones, and was reflected in the appearance of abundant large secretory granules, well-developed Golgi apparatus and RER, and reduced basal infoldings. Only a few such GCT cells were immunopositive for mK1, and the pattern of immunopositive and immunonegative cells very closely resembled the mosaic pattern seen in normal male GCTs. These findings suggested that the sexual dimorphism of mK1 expression and the morphological appearance of GCT cells can be induced by treatment with two hormones, DHT and T(3), but not by either of them alone. T(3) appears to have a permissive effect on committed GCT cells that results in downregulation of mK1 expression in these cells.     

Developmental and androgenic regulation of the immunocytochemical distribution of mK1, a true tissue kallikrein, in the granular convoluted tubule of the mouse submandibular gland.

The action of androgens on the immunocytochemical distribution of mK1, a true tissue kallikrein, was examined in the submandibular gland (SMG) of developing and adult mice by indirect enzyme-labeled and immunogold-labeled antibody methods for light and electron microscopy, respectively. In both sexes at 3 weeks of age, essentially all of the immature granular convoluted tubule (GCT) cells were uniformly immunostained. At 4 weeks of age (the onset of puberty), morphological differences between the two sexes appeared in the GCTs, in which some cells became immunonegative. Thereafter, the immunonegative GCT cells became more abundant in the SMG of males than of females and considerable intercellular variation in staining intensity for mK1 was seen, especially in males. A few slender GCT cells with strong immunoreactivity appeared in GCT segments only in males. Castration of males resulted in an increase in the number of immunopositive GCT cells, whereas administration of dihydrotestosterone (DHT) decreased the number of immunopositive GCT cells in the SMGs of both sexes. Slender GCT cells immunoreactive for mK1 were seen in females treated with DHT for 6 days. However, there were no immunostained slender GCT cells in female SMGs after injection of DHT for 2 weeks. Immunoelectron microscopy disclosed this type of cell in male SMGs, which closely resembles immature GCT cells of prepubertal mice, with a few small secretory granules uniformly labeled with gold particles, a sparse Golgi apparatus and RER, and basal infoldings. In mature male SMGs and in SMGs of DHT-treated females and castrated males, typical GCT cells had a well-developed Golgi apparatus and a net-like RER but few to no basal infoldings, whereas in the female gland equivalent cells had moderately developed RER and some basal infoldings. These results suggest that mK1 is one of the enzymes characteristically present in immature GCT cells and that its synthesis is inhibited in part by androgens, resulting in decreased numbers of immunopositive cells.     

Deciphering the molecular effects of romidepsin on germ cell tumours: DHRS2 is involved in cell cycle arrest but not apoptosis or induction of romidepsin effectors

Testicular germ cell tumours (GCTs) mostly affect young men at age 17‐40. Although high cure rates can be achieved by orchiectomy and chemotherapy, GCTs can still be a lethal threat to young patients with metastases or therapy resistance. Thus, alternative treatment options are needed. Based on studies utilising GCT cell lines, the histone deacetylase inhibitor romidepsin is a promising therapeutic option, showing high toxicity at very low doses towards cisplatin‐resistant GCT cells, but not fibroblasts or Sertoli cells. In this study, we extended our analysis of the molecular effects of romidepsin to deepen our understanding of the underlying mechanisms. Patients will benefit from these analyses, since detailed knowledge of the romidepsin effects allows for a better risk and side‐effect assessment. We screened for changes in histone acetylation of specific lysine residues and analysed changes in the DNA methylation landscape after romidepsin treatment of the GCT cell lines TCam‐2, 2102EP, NCCIT and JAR, while human fibroblasts were used as controls. In addition, we focused on the role of the dehydrogenase/reductase DHRS2, which was strongly up‐regulated in romidepsin treated cells, by generating DHRS2‐deficient TCam‐2 cells using CRISPR/Cas9 gene editing. We show that DHRS2 is dispensable for up‐regulation of romidepsin effectors (GADD45B, DUSP1, ZFP36, ATF3, FOS, CDKN1A, ID2) but contributes to induction of cell cycle arrest. Finally, we show that a combinatory treatment of romidepsin plus the gluccocorticoid dexamethasone further boosts expression of the romidepsin effectors and reduces viability of GCT cells more strongly than under single agent treatment. Thus, romidepsin and dexamethasone might represent a new combinatorial approach for treatment of GCT.     

Androgen regulation of the cellular Distribution of the true tissue kallikrein mK1 in the submandibular gland of the mouse.

The kallikrein gene family encodes for at least four different proteases in the mouse submandibular gland (SMG): mK1 (true tissue kallikrein), mK9, mK13, and mK22. These enzymes and many other biologically active proteins are synthesized by the granular convoluted tubule (GCT), a specialized segment of the SMG duct system. The GCT is under multihormonal regulation by androgens, thyroid hormones, and adrenocortical hormones. Androgens suppress synthesis of mK1 in the SMG but enhance expression of the other three kallikreins. We prepared an antibody with limited immunoreactivity for mK1 and used it to examine the effects of androgen status on the distribution of this isozyme in the SMGs of developing and mature mice by immunoperoxidase staining for the light microscope and immunogold labeling for the electron microscope. In prepubertal mice, every immature GCT cell contains mK1, confined to an accumulation of small granules in the subluminal cytoplasm. In mature mice, not every GCT cell contains mK1, and in those cells that do there is considerable intergranular variation in the intensity of staining for mK1. GCT cells containing mK1 are much more abundant in the glands of females than of males, resulting in a peculiar sexually dimorphic mosaic distribution of this isozyme in the mature SMG. Castration of adult males increases the number of GCT cells expressing mK1. Administration of androgen to intact or castrated males or to intact females reduces the number of cells staining for mK1. In all cases, immunogold labeling for mK1 is confined to secretory granules. No fine structural differences were noted between cells that were positively or negatively stained for mK1. Therefore, although GCT cells appear to be composed of a uniform population of cells on the basis of morphology alone, they are not homogeneous in their content of secretory proteins. These results indicate that androgen regulation of GCT cells is more complex than has been appreciated to date.     

Pannexin1 Single Nucleotide Polymorphism and Platelet Reactivity in a Cohort of Cardiovascular Patients

Pannexin1 (Panx1), a membrane channel-forming protein permitting the passage of small-sized molecules, such as ATP, is expressed in human platelets. Recently, we showed that inhibiting Panx1 affects collagen-induced platelet aggregation but not aggregation triggered by other agonists. We also found that a single nucleotide polymorphism (SNP; rs1138800) in the Panx1 gene encoded for a gain-of-function channel (Panx1-400C) and was associated with enhanced collagen-induced platelet reactivity. Here, we assessed the association of this SNP with platelet reactivity in a cohort of 758 stable cardiovascular patients from the ADRIE study treated with aspirin and/or clopidogrel. We found that presence of the Panx1-400C allele was not associated with platelet reactivity in stable cardiovascular patients, irrespective of the platelet aggregation agonist used (collagen, ADP or arachidonic acid) or the anti-platelet drug regimen. Moreover, the Panx1-400A?>?C SNP did also not affect the re-occurrence of cardiac ischemic events in the same stable cardiovascular patient cohort.     

Expression and localization of extracellular matrix metalloproteinase inducer in giant cell tumor of bone

Matrix metalloproteinases (MMPs) are regarded as a significant regulator in tumor invasion and metastasis. Previous studies have shown that extracellular matrix metalloproteinase inducer (EMMPRIN) in tumor cells induces the synthesis of MMPs. EMMPRIN is abundantly present on the surface of tumor cells and stimulate adjacent stromal cells to synthesize MMPs to induce tumor progression. Giant cell tumor (GCT) of bone is a benign but locally aggressive primary neoplasm of bone. The spindle-shaped mononuclear stromal cells are considered to be the tumor components of GCT, which are capable of inducing osteoclast formation by recruiting the circulating monocyte and macrophage. In this study, we proposed that EMMPRIN is associated with the biological progression and aggressiveness of GCT. We have conducted semi-quantitative RT-PCR to determine the correlation of EMMPRIN expression with the clinical stage of GCT. We have also examined the cellular localization of EMMPRIN in GCT using in-situ hybridization (ISH) and Immunohistochemistry (IH). The results showed that EMMPRIN was present in GCT and its mRNA levels were associated with the clinical stage of GCT. Higher expression level of EMMPRIN was observed in GCT with advanced stage (stage III). There was a great significance (P < 0.05) of EMMPRIN expression between stage I & II and stage III GCTs. Both ISH and IH demonstrated that EMMPRIN is present at the multinuclear osteoclast-like giant cells of GCT, with strong immunostaining on the cell membrane. The stromal-like tumor cells were also positively stained but the intensity was weaker. Interestingly, the production of EMMPRIN in osteoclast-like cells of GCT seems to be regulated by stromal-like tumor cells. Receptor activator of NF-kappaB ligand (RANKL), which has been previously shown to be produced by the stromal-like tumor cells for the recruitment of osteoclast-like giant cells in GCT, enhanced the expression of EMMPRIN mRNA during the differentiation of macrophage-like RAW(264.7) cells into osteoclasts. In short, our studies suggest that EMMPRIN may be an important regulatory factor involved in the biological behaviors of GCT.     

Erythropoietin expressed in granular convoluted tubule cells of mice submandibular glands under hypoxia, anemia, and nephrectomy

Immunohistochemically detectable erythropoietin-like substance(Epo) in granular convoluted tubule(GCT) cells of submandibular glands (SMG's) was examined in mice in which hemolytic anemia had been induced by phenylhydrazine (ph), and in mice subjected to hypoxia, nephrectomy, or testosterone (TP) injections. Staining for Epo was negative in GCT cells of SMG's in normal mice, while positive staining occurred in GCT cells of the anemic mice and mice subjected to hypoxia or nephrectomy. A positive Epo reaction was also revealed at the luminal borders of distal tubules, and in cells of proximal and distal tubules in the kidney, and in some hepatic and spleen cells, of mice that had received combination regimens producing anemia and hypoxia, or had been nephrectomized. Increased staining of Epo was found in GCT cells of SMG's, and in proximal and distal kidney tubules of mice given the combination treatment plus TP injections. The detection of Epo in GCT cells suggests these extrarenal cells to be sites of accumulation or biosynthesis of the protein under certain specific conditions such as hemolytic anemia and hypoxia.     

Functional characterization of MigA and WapR: putative rhamnosyltransferases involved in outer core oligosaccharide biosynthesis of Pseudomonas aeruginosa

Pseudomonas aeruginosa lipopolysaccharide (LPS) contains two glycoforms of core oligosaccharide (OS); one form is capped with O antigen through an alpha-1,3-linked L-rhamnose (L-Rha), while the other is uncapped and contains an alpha-1,6-linked L-Rha. Two genes in strain PAO1, wapR (PA5000) and migA (PA0705), encode putative glycosyltransferases associated with core biosynthesis. We propose that WapR and MigA are the rhamnosyltransferases responsible for the two linkages of L-Rha to the core. Knockout mutants with mutations in both genes were generated. The wapR mutant produced LPS lacking O antigen, and addition of wapR in trans complemented this defect. The migA mutant produced LPS with a truncated outer core and showed no reactivity to outer core-specific monoclonal antibody (MAb) 5C101. Complementation of this mutant with migA restored reactivity of the LPS to MAb 5C101. Interestingly, LPS from the complemented migA strain was not reactive to MAb 18-19 (specific for the core-plus-one O repeat). This was due to overexpression of MigA in the complemented strain that caused an increase in the proportion of the uncapped core OS, thereby decreasing the amount of the core-plus-one O repeat, indicating that MigA has a regulatory role. The structures of LPS from both mutants were elucidated using nuclear magnetic resonance spectroscopy and mass spectrometry. The capped core of the wapR mutant was found to be truncated and lacked alpha-1,3-L-Rha. In contrast, uncapped core OS from the migA mutant lacked alpha-1,6-L-Rha. These results provide evidence that WapR is the alpha-1,3-rhamnosyltransferase, while MigA is the alpha-1,6-rhamnosyltransferase.     

Immunocytochemical localization of renin in the submandibular gland of the mouse during postnatal development.

The localization of renin in the developing mouse submandibular gland was studied immunocytochemically using the unlabelled antibody-enzyme method of Sternberger ('74). Bouin-fixed submandibular glands of mice of both sexes were examined at 5-day-intervals from birth (day 0) to 50 days of age. At all stages studied, only granular convoluted tubule (GCT) cells stained immunocytochemically for renin; such cells were first seen in glands of 30-day-old males and of 30-day-old females. The size and number of renin-containing GCT cells increased rapidly in males, attaining adult status by 50 days of age. In females, differentiation of GCT cells immunoreactive for renin was slower and less regular than in males, and at 50 days of age the GCT segment had not yet reached adult conditions with respect to the distribution of renin. Renin appears in GCT cells at later ages than other GCT cell products (e.g., EGF and amylase), suggesting the existence of independent developmental control for the expression of various biologically active substances in the GCTs.     

Characterization of glycoconjugates found in granular cell tumors, with special reference to keratan sulfate

Nine granular cell tumors (GCTs) were studied using the immunoperoxidase technique with a mouse monoclonal antibody to keratan sulfate and a polyclonal antibody to S-100 protein. Various lectins and basic dye stains were also employed. Schwannomas benign and a malignant, a neurofibroma, a leiomyoma, two examples of nevus pigmentosus and a congenital epulis were similarly examined to compare the histochemical reactivities. Tumor cells of all the GCTs reacted intensely with the antibodies to keratan sulfate and S-100 protein. Peripheral nerve bundles and other neurogenic tumors showed stained for S-100 protein but not for keratan sulfate. Basic dye stain indicated the presence of sulfated glycoconjugates in GCTs. Lectin stains demonstrated that GCTs were rich in glycoconjugates but the reactivity patterns for 14 lectins differed between GCTs and normal tissue components. None of the lectins used in this study was specific for GCTs. These results indicate that GCTs contain abundant glycoconjugates and that the monoclonal antibody to keratan sulfate may be an immunohistochemical marker for GCT.