Biomedical subjects
A Ohtsuru
Publications and source records attributed to A Ohtsuru.
Antisense inhibition of parathyroid hormone-related peptide gene expression reduces malignant pituitary tumor progression and metastases in the rat.
A newly established metastatic rat pituitary tumor (mGH3) possesses a malignant phenotype that is invasive and hypervascular compared with the original GH3 tumors. mGH3 cells exhibit anchorage independence and expression of elevated levels of parathyroid hormone-related peptide (PTHrP) in vitro. To clarify the role of PTHrP in the development of the malignant phenotype, tumor cells were treated with phosphorothioate antisense PTHrP oligonucleotide. Treatment with antisense PTHrP resulted in a scattering phenomenon in the colony formation assay but did not inhibit cell growth in vitro. Inoculation of mGH3 cells in the cerebral ventricle resulted in a rapid growth of tumor cells within 3 weeks and dissemination throughout the entire ventricular system. Although treatment with sense or mismatched PTHrP oligonucleotide did not influence the subsequent tumor growth, the in vivo coinjection and injection of antisense PTHrP 1 week after tumor cell implantation into the right lateral ventricle markedly reduced tumor size and suppressed metastasis formation. The survival rate of mGH3 tumor-injected rats was prolonged by antisense PTHrP therapy. Our results demonstrated the biological involvement of PTHrP in malignant phenotype in rat pituitary tumors, suggesting that antisense PTHrP may provide a novel antimetastatic therapy for malignant somatotroph tumors.
Expression of parathyroid hormone (PTH)-related peptide (PTHrP) and PTH/PTHrP receptor in giant cell tumour of tendon sheath.
The giant cell tumour of tendon sheath (GCTTS) is mainly composed of mononucleated stromal cells (SC) and multinucleated giant cells (GC), so-called osteoclast-like GC. It is thought that GC are derived from SC, but their precise relationship is not fully understood. Parathyroid hormone (PTH)-related peptide (PTHrP) is now considered to be a cytokine for cell differentiation, which may stimulate osteoclast-like cell formation in haematopoietic cells. Five cases of GCTTS were evaluated immunohistochemically, using a variety of antibodies against PTHrP, PTH/PTHrP receptor, KP-1 as a histiocytic phenotypic antigen, fibronectin as a fibroblastic phenotypic antigen, and proliferating cell nuclear antigen (PCNA). In situ hybridization and immunohistochemistry revealed that in all cases both SC and GC expressed PTHrP. PTH/PTHrP receptor was observed only in histiocytic SC and GC, but not in fibroblastic SC. Almost all GC showed histiocytic features. PCNA immunoreactivity was detected only in the nuclei of SC, and not in GC. Moreover, SC with PTH/PTHrP receptor immunoreactivity were negative for PCNA. These results suggest that GC are derived from histiocytic SC expressing PTH/PTHrP receptor and losing proliferative activity in the process of transition from mononuclear to multinucleated. PTHrP produced by SC and GC may be involved in the formation of osteoclast-like cells in GCTTS by acting in an autocrine/paracrine fashion.
Parathyroid hormone-related peptide in synovial fluid and disease activity of rheumatoid arthritis.
To understand the clinical role of parathyroid hormone-related peptide (PTHrP) in rheumatoid arthritis (RA), we analysed the circulatory and synovial fluid (SF) concentrations of the N- and C-terminal regions of PTHrP (N- and C-PTHrP) in RA (n = 38), osteoarthritis (OA, n = 45) and control (n = 11) subjects. The SF level of C-PTHrP was markedly higher in RA compared with control and OA groups, while no differences in circulatory C-PTHrP were present among the three groups. In contrast, the SF level of N-PTHrP was marginally higher in OA patients. C-PTHrP levels in SF correlated significantly with CRP, ESR and SF IL-1 receptor antagonist. To identify the mechanism of elevated PTHrP levels in SF, immunohistochemistry and in situ hybridization of synovial membrane (SM) were performed in each subject. Overexpression of PTHrP was identified in the sublining cells within papillary proliferated SM of RA patients only. Our results indicate that C-PTHrP produced from SM into SF reflects the disease activity in RA.
Evidence that implicates the parathyroid hormone-related peptide in vascular stenosis. Increased gene expression in the intima of injured carotid arteries and human restenotic coronary lesions.
Proliferation of vascular smooth muscle cells (VSMCs) is considered to be one key event underlying the pathophysiology of restenosis after angioplasty. The parathyroid hormone-related peptide (PTHrP) and its receptor, a local autocrine and paracrine regulator of cellular growth in a variety of normal cell types, have been reported in the vicinity of VSMCs. To investigate how PTHrP might be involved in the process of neointimal formation after balloon angioplasty, we examined PTHrP expression in balloon-denuded rat carotid arteries and human coronary arteries that had been retrieved by directional atherectomy. In rat carotid arteries, the RNase protection assay and in situ hybridization demonstrated that PTHrP mRNA expression increased fourfold to sixfold 1 to 7 days after denudation and continued for 28 days, coincident with downregulation of PTH/PTHrP receptor mRNA expression. In situ hybridization and immunohistochemistry revealed that PTHrP expression in balloon-denuded carotid arteries was mainly localized to the neointima. To confirm the involvement of the PTHrP in human coronary artery restenotic lesions, immunohistochemical analysis of human coronary atherectomy specimens (23 primary and 10 restenotic lesions) was then performed. The number of intimal cells that expressed PTHrP protein was significantly higher in restenotic (407 +/- 53 cells/mm2; range, 143 to 739) than in stable angina (50 +/- 12 cells/mm2; range, 18 to 132; P<.05) or unstable angina (129 +/- 16 cells/mm2; range, 21 to 232; P<.05) specimens. These data demonstrate that PTHrP gene expression in VSMCs markedly increases during neointimal formation, supporting the hypothesis that PTHrP may play an important role in vascular stenosis as a regulator of VSMC proliferation.
Osteogenic action of parathyroid hormone-related peptide (1-141) in rat ROS cells.
To examine the autocrine/paracrine effect of parathyroid hormone-related peptide (PTHrP) on osteoblast function, the entire coding region of rat PTHrP (1-141) cDNA inserted into the expression vector was stably transfected into the rat clonal strain of the osteoblast-like cell, ROS 17/2.8, and established stable transfectants. Using the PTHrP-overexpressing ROS cells (ROS/PLP/6), we analyzed in vitro cell characterization and in vivo osteogenic properties. As expected, overexpression of endogenous PTHrP in vitro induced PTH/PTHrP receptor down-regulation confirmed by Northern blots, receptor binding assays, and functional analysis. The established transfectants indicated a decreased growth rate compared with the original non-transfected ROS 17/2.8. Although cAMP production induced by exogenous PTH was suppressed in ROS/PLP/6, the stimulatory effects of forskolin and chorela toxin showed no significant difference between the original ROS 17/2.8 and transfected cells, but the in vivo osteogenic properties were histologically potentiated in transfectants with increased bone matrix and acceleration of mineralization within tumors. The levels of osteocalcin and osteopontin mRNAs were also increased in transfectants. The down-regulated in vitro PTH/PTHrP receptor mRNA was restored in in vivo tumor tissues. Our study provides clear evidence that the in vivo osteogenic function in ROS cells is potentiated by PTHrP, through an autocrine/paracrine mode of action.
Characterization of cultured rat embryonic palatal mesenchymal cells.
To investigate developmental palatogenesis, the establishment of palatal cell culture in vitro is preferable to eliminate several complicated biases present in the in vivo environment. We established a primary culture of rat embryonic palatal mesenchymal cells using a special technique to dissect embryonic palatal shelves, and characterized these embryonic cells by immunohistochemical analysis against histiocytic markers. Following preparation of the maxilla of 15.5-day-old rat fetuses, a midline incision of the maxilla was established while the occiput was fixed with microforceps. This procedure allowed eversion of the maxillary process and easy dissection of the palatal shelf. The technique allowed preparation of a large number of palatal shelves with no appendages using a small number of fetuses. Cells cultured with DMEM/F-12 and 10% FBS showed multipotential nature (i.e., not only mere mesenchymal character but also neural, endothelioid, and/or myoblastoid origin were identified by immunostaining with anti-epithelium membrane antigen, keratin, vimentin, S-100 protein, factor VIII, desmin, and lysozyme antibodies, respectively). Our results demonstrated that, during several cell passages, the cultured cell gained myoblastoid characteristics in addition to a neural nature. Further in vitro studies using cultured embryonic palatal mesenchymal cells will assist in characterization of proliferation and differentiation of cells forming the palate.
Parathyroid hormone-related protein (PTHrP) action in rat articular chondrocytes: comparison of PTH(1-34), PTHrP(1-34), PTHrP(1-141), PTHrP(100-114) and antisense oligonucleotides against PTHrP.
Parathyroid hormone-related protein (PTHrP) is thought to be an important autocrine/paracrine factor for chondrocyte metabolism since mice lacking the PTHrP gene exhibit abnormal cartilage development. To determine the biological role of PTHrP in chondrocytes, we first compared the agonist potency of human (h) PTHrP(1-34) with hPTH(1-34) in cultured rat articular chondrocytes. Neither hPTHrP(1-34) nor hPTH(1-34) altered basal DNA synthesis, but attenuated the stimulatory effect of transforming growth factor beta (TGF-beta). Both agents suppressed the expression of alpha(1) type II collagen mRNA in a dose-response fashion with the same potency. In addition, the action of exogenously added hPTHrP(1-34) and hPTH(1-34) on intracellular cAMP and [Ca2+]i levels was similar. We next compared the effect of PTHrP within its entire amino acid sequence (1-141). With regard to thymidine incorporation, alpha(1) type II collagen gene expression and accumulation of cAMP and [Ca2+]i level, there was no significant difference between hPTHrP(1-34) and hPTHrP(1-141). PTHrP C-terminal (100-114) did not show any function. To further investigate PTHrP function, intracellular PTHrP translation was inhibited by a transgene of antisense oligonucleotides against PTHrP. Antisense oligonucleotides decreased PTHrP mRNA translation, specifically inhibited DNA synthesis in control as well as TGF-beta-treated chondrocytes and enhanced alpha(1) type II collagen mRNA expression in TGF-beta-treated chondrocytes. These results suggest that there is no significant difference between exogenously added hPTH(1-34), hPTHrP(1-34) and PTHrP(1-141) with regard to the biological action of these agents, including cell growth, differentiation and second messenger pathway. However, the result of DNA synthesis in the antisense PTHrP-inhibition study suggests that intracellular PTHrP may have an as yet unknown biological role, in addition to a classical PTH/PTHrP receptor-mediated function in the rat articular chondrocyte.
[Viral tumor markers].
Viruses can contribute to the development of human tumors by different mechanisms: directly by altering host cellular gene expression by viral products or by viral DNA integration; indirectly by modifying the host cell genome co-operated with other factors. Human cancer associated with hepatitis B virus (HBV), hepatitis C virus (HCV), human T cell leukemia virus (HTLV-I), papillomavirus (HPV) and Epstein-Barr virus (EBV) infections are responsible for liver cancer (HBV and HCV), adult T cell leukemia (HTLV-I), cervical cancer (HPV) and malignant lymphoma (EBV) respectively. Based on the clinical and experimental knowledge, viral tumor markers are thought of not as diagnostic markers, but as most important risk factors for various tumorigenesis.
[Parathyroid hormone-related peptide (PTHrP) and PTH/PTHrP receptor in the gastrointestinal tract].
Parathyroid hormone-related peptide (PTHrP), originally isolated as a responsible factor for humoral hypercalcemia of malignancy, has been demonstrated to overexpress even in non-hypercalcemic gastrointestinal malignancies. PTHrP and its receptor mRNA expression has been detected in a variety of normal tissues in neonate and adults. PTHrP might be involved in growth and differentiation on fetal gut epithelium, and an important gastrointestinal peptide which regulates gastrointestinal contractile activity in a paracrine/autocrine fashion. Exogenous PTHrP shows a relaxant effect on gastrointestinal muscle strips through PTH/PTHrP receptor in vitro. Increase PTHrP mRNA was induced by distention and contraction in the smooth muscle of the stomach, and stress-induced abnormal contractions was deeply related to PTHrP suppression by steroid.
Expression of the Ets-1 proto-oncogene in human gastric carcinoma: correlation with tumor invasion.
The proto-oncogene Ets-1 is a transcription factor known to control the expression of a number of genes involved in extracellular matrix remodeling and has been postulated to play a role in cell migration and tumor invasion. To elucidate the involvement of Ets-1 in human gastric carcinomas, we examined 11 cases of gastric adenoma and 110 cases of gastric carcinoma by immunohistochemistry and compared the degree of Ets-1 expression with the depth of carcinoma invasion. Ets-1 was not expressed either in the normal gastric epithelium or in gastric adenomas. Among the 110 cases with gastric adenocarcinoma, 70 (63.6%) showed positive staining for the Ets-1 protein. In mucosal carcinomas, only 3 of 26 cases (11.5%) showed positive immunostaining for Ets-1. In contrast, 67 of 84 cases (79.8%) with submucosal or more invasive carcinomas showed immunopositivity and intense staining for Ets-1 in the tumor cells. The pattern of Ets-1 immunostaining in mucosal carcinomas was weak and differed from that of other local invasive carcinomas (P < 0.001). Histologically, signet-ring cell and mucinous carcinomas expressed relatively weak positivity for Ets-1. Ets-1 expression correlated significantly with the presence of lymph node metastasis (P < 0.001). In situ hybridization, using an Ets-1 oligonucleotide probe, also confirmed the presence of Ets-1 mRNA in gastric carcinomas. Expression of Ets-1 mRNA was also detected in four different kinds of cultured human gastric carcinoma cell lines by the reverse transcription polymerase chain reaction method. These findings suggest that Ets-1 is overexpressed in gastric mucosal cells that have undergone malignant conversion and that Ets-1 is one of the factors involved in the penetration of gastric carcinoma beyond the muscularis mucosa.
Inhibition of epidermal growth factor binding system by ionizing radiation in A431 human squamous carcinoma cells.
To elucidate the effect of ionizing radiation on the membrane anchored signal transduction, the binding of 125I epidermal growth factor (EGF) to its receptor (EGF-R) and the EGF-dependent EGF-R tyrosine phosphorylation were examined in a human squamous cell carcinoma cell line, A431. The significant suppression of 125I EGF binding to A431 cells was observed from 3-5 h after 10 Gy irradiation, whereas this inhibition was not observed both in non-irradiated and in 5 Gy-irradiated cells. This phenomenon was mediated by the protein kinase C pathway, because the inhibition was not observed in cells which had been pretreated with phorbol ester and treated with an inhibitor of the enzyme, H7. Scatchard analysis showed that the receptor affinity was decreased. In contrast, the level of EGF-dependent EGF-R-tyrosine phosphorylation was not decreased, compared with non-irradiated cells. These results suggest that ionizing radiation may modulate the function of EGF/EGF-R interaction through the direct activation of protein kinase C.
Expression of parathyroid hormone-related peptide in human thyroid tumours.
The purpose of this study was to evaluate the distribution of parathyroid hormone-related peptide (PTHrP) in human thyroid tissues. The presence of PTHrP was studied immunohistochemically in 107 consecutive patients with human thyroid tumours. PTHrP expression was revealed in 97.6 per cent of carcinomas, but not in paranodal normal thyroid epithelial cells. Although there were no differences in the incidence of PTHrP positivity among papillary, follicular, and anaplastic carcinoma cases, PTHrP expression levels were correlated with the growth pattern of thyroid cancer. Strong immunopositivity was detected in 67.3 per cent of papillary growth tissues in papillary carcinomas. A tissue growth pattern consisting of colloid-absent follicles had a high incidence of strong immunopositivity irrespective of the histological type of tumour. Anaplastic carcinoma without colloid production also showed strong immunoreactivity in all cases. In contrast, a growth pattern of colloid-rich follicles did not show strong immunopositivity in either papillary or follicular carcinomas. Follicular adenomas showed positive immunostaining in only one case, and no adenomatous goitres showed PTHrP antigens. In situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR) revealed strong PTHrP mRNA in thyroid cancer tissues, but not in normal thyroid tissues. PTHrP expression was not associated with metastasis, calcification, or hypercalcaemia in thyroid cancers. These results suggest that the expression of PTHrP in human thyroids is closely related to the malignant alteration of normal thyroid epithelial cells, especially in the growth pattern of thyroid carcinoma tissues.
Expression of parathyroid hormone-related protein in rat articular cartilage.
Expression and localization of parathyroid hormone-related protein (PTHrP) in rat articular cartilage during fetal and postnatal periods were investigated by immunohistochemistry and in situ hybridization. PHTrP displayed distinct distribution and intensity of staining at different ages. In fetal (18-day-old) and young (3-week-old) rats, articular chondrocytes expressed abundant PTHrP throughout the entire thickness of cartilage. In contrast, in 60-week-old rats, PTHrP was expressed in a few articular chondrocytes of superficial and middle layers. Regulation of PTHrP and PTH/PTHrP receptor mRNA was also studied in cultured rat articular chondrocytes. Northern blot analysis revealed that both transforming growth factor-beta (TGF-beta), an important stimulator for chondrocyte proliferation and differentiation, and 10% fetal bovine serum (FBS) stimulated the expression of PTHrP mRNA with down-regulation of its receptor mRNA. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA) down-regulated the expression of receptor without changes of PTHrP mRNA level. These results suggest that the changes in abundance and localization of PTHrP and its receptor may be directly involved in the cell growth and differentiation of articular cartilage.
Establishment and characterization of a malignant epithelioid hemangioendothelioma from mouse thyroid tumor.
A mouse epithelioid hemangioendothelioma (mEHE) cell line, passagable in vivo without TSH stimulation, was established from a thyroid tumor. Before establishment of the cell line, the primary thyroid tumor which was co-transplanted with a TSH-producing pituitary adenoma showed signs of hyperplasia and then transformed during several in vivo transplantable passages. The cell line which was established from an in vitro culture, was deficient in thyroid-specific differentiation function and had characteristics of endothelial cell origin such as vascular cavity formation and factor VIII expression. The cell growth of mEHE/CH1-5 cell lines was independent of TSH but was inhibited by c-AMP and vitamin D3. c-Myc proto-oncogene expression was also suppressed by vitamin D3 treatment. Both serum depletion and heparin treatment induced morphological changes in mEHE/CH 5 from an epithelial cell-like to an endothelial cell-like shape. Immunohistochemical analysis showed that epithelial membrane antigen expression was decreased and factor VIII expression was increased in relation to the morphological changes. In a further in vivo transplantation experiment, the histology of the mEHE/CH5 cell tumor had an angiosarcoma-like structure. These results indicate that this cell line established from a thyroid tumor possesses both epithelial and endothelial characteristics. The mEHE/CH5 cells might provide a good model for analyzing thyroid tumorigenesis and allow functional characterization of endothelial cells.
Parathyroid hormone-related peptide expression in endocrine tumors.
Parathyroid hormone-related peptide (PTHrP) expression is associated with the histological type in pituitary tumor, but not in thyroid carcinoma. However invasive and metastatic tumors showed intense PTHrP staining in both pituitary and thyroid tumors. Analysis of the established metastatic rat pituitary tumor cell line showed that PTHrP plays a crucial role in in vivo cell proliferation closely related to worsening of malignant transformation and neovascularization in a paracrine fashion.
Coronary atherosclerotic smooth muscle cells overexpress human parathyroid hormone-related peptides.
Parathyroid hormone-related peptide (PTHrP) was originally characterized as a tumor product responsible for hypercalcemia of malignancy and was subsequently found to be produced in many normal tissues. PTHrP is now suggested to play a critical role in the local modulation of vascular smooth muscle function. To elucidate the involvement of PTHrP in coronary atherosclerosis, we immunohistochemically examined coronary arteries obtained from 76 patients with various grades of atherosclerosis and compared the correlation between PTHrP staining and the percent stenosis. Smooth muscle cells at sites of coronary atherosclerosis overexpressed PTHrP, while cells from normal coronary arteries did not. The in situ hybridization using PTHrP riboprobe has also proven the overexpression of PTHrPmRNA in the affected lesions following atherectomy. The intensity of PTHrP expression by smooth muscle cells was significantly correlated with the degree of coronary artery stenosis. Coronary arterial PTHrP overexpression is closely related to the severity and/or progression of coronary atherosclerosis.
Effect of transforming growth factor-beta on the insulin-like growth factor-I autocrine/paracrine axis in cultured rat articular chondrocytes.
Transforming growth factor-beta (TGF-beta) and insulin-like growth factor-I (IGF-I) are essential anabolic factors in articular cartilage. In this study, we concentrated on the elucidation of TGF-beta interaction with IGF-I on cell growth and differentiation in monolayer articular chondrocytes obtained from 5-week-old rats. TGF-beta (1 ng/ml) and IGF-I (25 ng/ml) stimulated DNA synthesis about 6.5- and 2.1-fold over control values, respectively. When TGF-beta and IGF-I were added in combination, DNA synthesis was enhanced about 10.4-fold, indicating that the two peptides act in synergism. This synergistic action was also present in the expression of aggrecan mRNA. To study the mechanism of synergistic action, the effect of TGF-beta on the IGF-I autocrine/paracrine axis was investigated. Administration of increasing concentrations of TGF-beta (0.1-10 ng/ml) resulted in a dose-dependent decrease in medium IGF-I concentration that was reflected by decreased levels of IGF-I mRNA. TGF-beta also inhibited the production of a 41-kDa IGF-binding protein into the culture medium. Pretreatment with TGF-beta (1 ng/ml) for 12 h increased the binding of [125I]IGF-I to 140% of control by increasing the number of receptors without changes of affinity. Immunoprecipitation against phosphorylated tyrosine indicated that IGF-I-dependent autophosphorylation of IGF-I receptor beta-subunit was inhibited by simultaneous TGF-beta stimulation. These observations demonstrate that TGF-beta acts synergistically with IGF-I and regulates the IGF-I autocrine/paracrine axis via a complex regulatory mechanism with decreased production of IGF-I and IGFBPs and dephosphorylation of IGF-I receptor, whereas there is an apparent up-regulation of the binding of [125I]IGF-I.