Inhibition of ABL tyrosine kinase potentiates radiation-induced terminal growth arrest in anaplastic thyroid cancer cells.

Gleevec, a selective tyrosine kinase inhibitor, retarded the growth of anaplastic thyroid cancer cell lines in vitro and in vivo through selective inhibition of ABL tyrosine kinase activity. In the present study, we investigated the ability of Gleevec to modulate the in vitro and in vivo radiation response of anaplastic thyroid cancer cells. Cell growth assays, colony formation assays and xenograft models were used to quantify the radiosensitizing effect of Gleevec in cells of the anaplastic thyroid cancer cell lines ARO and FRO. FACS, Western blotting and histochemical techniques were employed to study the mechanisms of radiation response after exposure to Gleevec. Gleevec (7.0 microM) increased the anti-proliferative effect of radiation on the growth ARO and FRO cells in vitro. Clonogenic analysis demonstrated that Gleevec reduced cell survival after irradiation. Gleevec combined with radiation produced an increase in tumor growth inhibition compared to treatment with either modality alone in mice bearing anaplastic thyroid cancer xenografts. The drug suppressed radiation-induced ABL activation and promoted CDKN1A (p21(cip1)) accumulation in irradiated anaplastic thyroid cancer cells. Gleevec had an additional effect on radiation-induced apoptosis in cells of both cell lines and potentiated the induction of terminal growth arrest accompanied by the expression of senescence-associated beta-galactosidase. The antitumor effect of Gleevec is potentiated in adjunctive therapy with radiation not only due to inhibition of proliferative cell growth with transient cell cycle arrest and apoptosis, but also due to the terminal growth arrest associated with senescence, suggesting that tumor cell senescence is a mechanism for tumor targeting therapy in combination with ionizing radiation.

Expression in human hepatocellular carcinoma of nucleoside diphosphate kinase, a homologue of the nm23 gene product.

BACKGROUND: Expression of nucleoside diphosphate (NDP) kinase, which is highly homologous to the nm23 gene product in a variety of species, has been found to be inversely associated with metastatic potential in human breast cancer. PURPOSE: The present study was conducted to clarify the association of NDP kinase expression with metastatic potential in human hepatocellular carcinoma. METHODS: The immunohistochemical expression of NDP kinase was analyzed in 30 patients with histopathologically proven hepatocellular carcinoma. These patients included nine with distant metastases and 21 without distant metastases. Tissue specimens were reacted with rabbit anti-rat NDP kinase antibody and stained by the biotin-streptavidin complex method. The relative staining intensities were evaluated by comparing primary tumor sites with adjacent nontumorous liver tissue or with metastatic sites, RESULTS: The expression of NDP kinase in primary sites in patients with distant metastases was significantly less intense than that in patients without distant metastases (P = .018). NDP kinase was expressed significantly less intensely in metastatic sites than in primary sites (P = .005). The intensity of NDP kinase expression did not statistically correlate with tumor size or number of lesions in the liver, histopathological classification of tumor, associated liver diseases, hepatitis virus markers, or tumor markers. CONCLUSION: These results suggest that the reduced expression of NDP kinase is closely associated with distant metastatic potential in hepatocellular carcinoma. IMPLICATIONS: It is possible that both NDP kinase and the nm23 gene product may be active in the progression and differentiation of tumor cells and that their reduced expression induces a high metastatic potential in tumor cells. Studies using Northern blotting or in situ hybridization should be planned to confirm our findings.

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.

Radiation Exposure and Thyroid Cancer Risk After the Fukushima Nuclear Power Plant Accident in Comparison with the Chernobyl Accident.

The actual implementation of the epidemiological study on human health risk from low dose and low-dose rate radiation exposure and the comprehensive long-term radiation health effects survey are important especially after radiological and nuclear accidents because of public fear and concern about the long-term health effects of low-dose radiation exposure have increased considerably. Since the Great East Japan earthquake and the Fukushima Daiichi Nuclear Power Plant accident in Japan, Fukushima Prefecture has started the Fukushima Health Management Survey Project for the purpose of long-term health care administration and medical early diagnosis/treatment for the prefectural residents. Especially on a basis of the lessons learned from the Chernobyl accident, both thyroid examination and mental health care are critically important irrespective of the level of radiation exposure. There are considerable differences between Chernobyl and Fukushima regarding radiation dose to the public, and it is very difficult to estimate retrospectively internal exposure dose from the short-lived radioactive iodines. Therefore, the necessity of thyroid ultrasound examination in Fukushima and the intermediate results of this survey targeting children will be reviewed and discussed in order to avoid any misunderstanding or misinterpretation of the high detection rate of childhood thyroid cancer.

EXTERNAL AND INTERNAL EXPOSURE TO FUKUSHIMA RESIDENTS.

The Great East Japan Earthquake of 11 March 2011, caused the Fukushima Daiichi Nuclear Power Plant Accident, which resulted in the release of a large amount of radioactive materials into the environment, and there is a serious concern about the radiation effects on the health of residents living in the affected areas. The evaluation of exposure dose is fundamental for the estimation of health effects, and whenever possible, the exposure dose should be evaluated by actual measurements as opposed to estimations. Here, the outline of the exposure doses of residents estimated from surveys or obtained by measurements is described. Fukushima Health Management Survey reported the results for 460 408 residents during the first 4 months after the accident; 66.3% received doses <1 mSv, 94.9% received <2 mSv, 99.7% received <5 mSv and the maximum dose was 25 mSv. Thus, it was demonstrated that the results from personal dosemeter measurements were comparable to the estimations. The dose assessment of internal exposure of 184 205 residents conducted by Fukushima Prefecture by using whole body counter showed that 99.986% received <1 mSv, with the maximum dose being 3 mSv. Regarding exposure of the thyroid, there is not enough data for the Fukushima accident, but it is presumed that thyroid doses are much lower than those from Chernobyl. The outline of exposure doses of residents in result of the accident is still being clarified, questions and uncertainties in dose assessment remain and further efforts for more accurate dosimetry are required continuously.

PKC delta mediates ionizing radiation-induced activation of c-Jun NH(2)-terminal kinase through MKK7 in human thyroid cells.

The thyroid gland is one of the most sensitive organs in ionizing radiation (IR)-induced carcinogenesis. To determine, therefore, the specific cascade of IR-induced signal transduction in human thyroid cells, we investigated the functional role of protein kinase C (PKC), especially its interlocking activation of c-Jun NH(2)-terminal kinase (JNK) pathway. In the present study, using adenovirus expression vectors for diverse dominant-negative (DN) types of PKC isoforms (alpha, beta2, delta, epsilon and zeta) expressed in primary cultured human thyroid cells, only DN/PKC delta suppressed IR-induced JNK activation. In addition, Rottlerin, a PKC delta specific inhibitor, inhibited IR-induced JNK activation. IR-induced activation of transcription factor AP-1, downstream target of JNK, was also attenuated by DN/PKC delta. To examine the involvement of upstream kinases of JNK, we performed immune-complex kinase assays of mitogen-activated protein kinase kinase 4 (MKK4) and MKK7. IR activated MKK7 but not MKK4, and this activation was inhibited by Rottlerin. Furthermore, IR-induced JNK activation was suppressed by overexpression of kinase-deficient MKK7. Our results indicate that IR selectively activates the cascade of PKC delta-MKK7-JNK-AP-1 in human thyroid cells, suggesting a not apoptotic but radio-resistant role of PKC delta in human thyroid cells following IR.

Eradication of breast cancer xenografts by hyperthermic suicide gene therapy under the control of the heat shock protein promoter.

To investigate the usefulness of heat shock protein (HSP) promoter for breast cancer gene therapy, hyperthermia and HSV thymidine kinase (tk) suicide gene combination therapy was examined with mouse mammary cancer cell line FM3A. HSP promoter activity was markedly increased after heat shock (41-45 degrees C), with maximum activation (about 400-fold) at 3 hr. An in vitro cytotoxic assay showed that HSP-tk-transduced FM3A cells became more sensitive (more than 50,000 times) to ganciclovir (GCV) with heat shock, but untreated cells showed no increased cytotoxic sensitivity to GCV compared with control FM3A cells. In addition to promoter-oriented selective cell killing, a "chemosensitization effect" as a bystander effect was demonstrated by hyperthermia and suicide gene combination therapy, using a non-heat-inducible promoter. Immunohistochemical analysis revealed that this synergistic killing effect was dependent on apoptotic cell death with upregulation of both Fas and FasL (Fas ligand) expression. We also examined the efficacy of HSP-tk gene therapy in vivo by implanting breast cancer in subcutaneous and intraperitoneal models of BALB/c nude mice targeted by the HVJ-anionic liposome method. Significant tumor regression was observed in HSP-tk-transduced tumors followed by hyperthermia therapy, but no such inhibition was noted in either the mock vector transfection or hyperthermia group compared with control tumor-bearing mice. Our results demonstrate that this combination system is synergistically effective in mediating Fas-dependent apoptosis for a specific gene therapy targeting HSP-expressing mammary carcinomas, even in advanced and heat-resistant breast cancer.

Regression of hepatocellular carcinoma in vitro and in vivo by radiosensitizing suicide gene therapy under the inducible and spatial control of radiation.

To improve the efficacy and selectivity of gene therapy for hepatocellular carcinoma (HCC), we designed a strategy for suicide gene therapy in conjunction with radiation therapy using an HVJ-liposome vector system. The radio-inducible suicide gene was constructed by insertion of the early growth response gene 1 (Egr-1) promoter upstream of the HSV-tk gene (EGF-tk). First, to test the tumor specificity of Egr-1, RT-PCR and immunohistochemistry were performed. The Egr-1 gene was highly expressed in HCC compared with normal liver, where expression was barely detectable. Next, radiation-inducible activity of the Egr-1 promoter was examined in primary cultured normal hepatocytes and human hepatoma cell lines Huh7, HepG2, and PLC/PRF/5 by luciferase assay as a reporter gene system. Egr-1 promoter activity was markedly increased in hepatoma cell lines in a radiation dose-dependent manner, with maximum activation (15- to 28-fold) 12 hr after irradiation. In contrast, only a twofold increase in activation was noted in normal hepatocytes. An in vitro gene therapy experiment showed that EGR-tk-transduced hepatoma cells became highly sensitive to ganciclovir (GCV) after irradiation, but not without irradiation. GCV with or without irradiation did not show any cytotoxic effects against control gene-transfected cells. In addition, a "radiosensitization effect" was also demonstrated by combination therapy with the HSV-tk/GCV system and irradiation. To examine the efficacy of this EGR-tk/GCV gene therapy in vivo, xenografted liver tumors in nude mice were targeted using the HVJ-liposome vector system. EGR-tk-transfected tumors regressed significantly after a combination therapy of irradiation and GCV in all mice (n = 8), and almost disappeared in 3 weeks without any side effects. In comparison, tumors continued to grow in all mice (n = 8 in each group) treated by transfer of EGR-tk followed by either irradiation without GCV or GCV without irradiation. Our data indicate that HSV-tk gene therapy under the control of a radioinducible promoter is effective, and might be selective for hepatoma cells because of its inducible and radiosensitive capacity after radiation exposure as well as its tumor-specific activation.

Keloid fibroblasts resist ceramide-induced apoptosis by overexpression of insulin-like growth factor I receptor.

Keloids are benign dermal tumors, characterized by overgrowth of lesions, invasiveness beyond the original boundary of the insult, and recurrence of lesions. The exact etiology is unknown, however. Our hypothesis is that keloids are acquired as a result of an abnormal or prolonged wound healing process, with persistent proliferation and extracellular matrix production of fibroblasts that should otherwise discontinue in normal wound healing. In this study, we examined the response of keloid fibroblasts to proapoptotic signaling. Cell-permeable ceramide, N-acetyl-D-sphingosine, induced apoptosis of dermal fibroblasts in a dose- and time-dependent manner, which was detected by phase contrast microscopy, fluorescent microscopy, the TUNEL method, flow cytometric analysis, and WST-1 assay. In contrast, keloid fibroblasts resisted apoptosis induced by N-acetyl-D-sphingosine (percent survival with 40 mM ceramide treatment for 12 h, normal versus keloid: 9.6% +/- 6.6% vs 66.8% +/- 5.5%). Western blotting analysis showed insulin-like growth factor I receptor overexpression in keloid fibroblasts, but not in normal fibroblasts. Exogenously added insulin-like growth factor I enhanced the resistance of keloid fibroblasts to ceramide-induced apoptosis. Wort- mannin, a phosphatidylinositol 3 kinase inhibitor, suppressed the antiapoptotic action of insulin-like growth factor I in keloid fibroblasts. Our results suggest that keloid fibroblasts overexpressing insulin-like growth factor I receptor are resistant to apoptosis, thus allowing persistent proliferation and production of excessive extracellular matrix. J Invest Dermatol 115:1065-1071 2000

Correlation between suppression of c-myc and antiproliferative effect of transforming growth factor-beta 1 in thyroid carcinoma cell growth.

The growth regulatory activity of transforming growth factor-beta 1 (TGF beta 1) was studied in a clonal strain of thyroid papillary carcinoma cell (NPA). Despite the presence of TGF beta 1 and its receptor messenger RNA in thyroid carcinoma, the molecular mechanism of TGF beta 1 action on cell growth of thyroid carcinoma has not yet been elucidated. Exogenously added TGF beta 1 inhibited DNA synthesis and cell growth in a dose- and time-dependent manner at concentrations of 0.1-10 ng/ml. TGF beta 1 inhibited not only basal but also fetal bovine serum-stimulated cell proliferation. Steady state levels of c-myc messenger RNA transcripts were inhibited by TGF beta 1 after 0.5-h treatment. Antisense, but not sense, c-myc oligodeoxynucleotides also caused suppression of NPA cell growth in a dose-responsive manner. Transfection studies of the 5'-up-stream flanking region (UFR) of c-myc/chloramphenicol acetyltransferase chimera genes suggest the presence of a TGF beta 1-responsive DNA element in the 2.3-kilobase c-myc 5'-UFR. Deletion mutant studies indicate the element lies between -106 to 70 relative to the P1 transcription start site. Studies with the gel mobility shift assay using 23-basepair double strand DNA showed the presence of at least two nuclear factors in NPA cell. TGF beta 1 treatment did not cause any alteration in TGF beta 1-induced mobility; however, the reduction of a positive band was selectively observed during 30 min to 2 h after treatment with TGF beta 1. In contrast, the position and intensity of another band were not altered by TGF beta 1 treatment. These results demonstrate that the inhibition of a nuclear factor binding to the c-myc 5'-UFR and subsequent suppression of c-myc gene expression are directly involved in the antiproliferative action of TGF beta 1 in NPA cell growth.

Parathyroid hormone-related peptide is a potent tumor angiogenic factor.

Rat pituitary malignant tumor cells; mGH3, show hypervascularization in in vivo xenografts and overexpress parathyroid hormone-related peptide (PTHrP) compared to original GH3 cells. To elucidate whether PTHrP is involved in tumor-derived angiogenesis, we examined the effect of PTHrP on vascular endothelial cells both in vitro and in vivo. Results of in vivo diffusion chamber assay showed a clear hypervascularization on the outer surface of diffusion chambers containing mGH3 tumor cell implants but not in those containing GH3 cells. Co-incubation with antisense PTHrP oligonucleotide (10 microM), but not sense or mismatched PTHrP oligonucleotide, suppressed hypervascularization in diffusion chambers. To further examine the role of PTHrP on endothelial cell function, PTHrP(1-34) was added at various concentrations to cultured bovine endothelial cells (BAECs) harvested from the aorta. PTHrP(1-34) did not alter the proliferation or migration of endothelial cells, but rather dose-dependently increased capillary formation by endothelial cells on the collagen gel matrix. Furthermore, 0.1 mM of 8-bromo-cAMP caused a similar increase in tube formation, which was dose-dependently inhibited by H89, a protein kinase A inhibitor. Our results indicate for the first time that PTHrP is a potential paracrine factor acting via the PKA pathway to enhance angiogenesis through capillary tube formation by endothelial cells in malignant pituitary tumors.

Expression of insulin-like growth factor I messenger ribonucleic acid in developing osteophytes in murine experimental osteoarthritis and in rats inoculated with growth hormone-secreting tumor.

Osteophytes are one of the characteristic features of osteoarthritis and are often found in acromegalic arthropathy. The aim of this study was to investigate insulin-like growth factor I (IGF-I) involvement in osteophyte formation. One percent collagenase solution was injected into murine knee joints as an osteoarthritis model. In a different animal group, GH-secreting tumor cells were inoculated s.c. to the rat thigh as an acromegaly model. A series of osteophyte formation was examined histologically. IGF-I messenger RNA was detected using the in situ hybridization method. Type I IGF receptors were detected immunohistochemically. In the osteoarthritis model, osteophyte formation appeared as synovial or perichondral cell proliferation adjacent to the articular cartilage on day 5, followed by cartilage formation on day 7 and endochondral ossification on day 14. In the acromegaly model, synovial or perichondral cell proliferation was observed 4 weeks after inoculation, followed by osteophyte formation at 8 weeks. In both models, IGF-I messenger RNA and type I IGF receptor were coexpressed by proliferating synovial or perichondral cells, proliferating chondrocytes, and osteoblasts within the developing osteophytes. These results suggest that IGF-I regulated the initiation and development of osteophyte formation in both models in an autocrine and/or paracrine fashion.

Expression of parathyroid hormone-related peptide in relation to perturbations of gastric motility in the rat.

We studied the expression of PTH-related peptide (PTHrP) and its mRNA in rat gastric smooth muscle in relation to various gastric motility states. Male rats were divided into groups subjected to fasting, feeding ad libitum, cold restraint stress, pyloric ligation, and carbachol stimulation. Cold restraint stress induced abnormal contractions. Rhythmic and moderate contractions were produced by carbachol administration, and marked distension was induced by pyloric ligation. PTHrP mRNA expression was weak in the physiological fasting and feeding states, but was markedly increased by pyloric ligation and carbachol stimulation. PTHrP and its mRNA were localized to the proper muscle layer and muscularis mucosa, but not in the mucosa by immunohistochemistry and in situ hybridization. The gene expression of PTHrP receptor in the gastric tissue was confirmed by reverse transcription-polymerase chain reaction, but serum PTHrP levels did not increase in all groups. These findings suggest that PTHrP acts as an autocrine or paracrine factor in gastric smooth muscle that responds to muscle activity caused by distension and cholinergic stimulation. However, PTHrP gene expression was decreased by stress despite the presence of strong contractions, and the sufficient relaxation did not occur. PTHrP suppression by stress is caused by the increase in corticosterone, as pretreatment of metyrapone, an inhibitor of 11 beta-hydroxylation, enhanced PTHrP gene expression in association with serum corticosterone suppression. In conclusion, PTHrP might be an important gastrointestinal peptide that regulates gastric contractile activity and is influenced by the serum corticosterone level.

Prop-1 gene expression in human pituitary tumors.

A novel type of pituitary-specific transcription factor, Prophet of Pit-1 (Prop-1) gene (PROP1), expresses in just early embryonic stage in mouse and closely related as a causative gene in combined pituitary hormone deficiency. We studied PROP1 expression to further clarify its correlation with tumorigenesis and biological behavior in human pituitary adenomas. Eighteen pituitary adenomas and three normal pituitary glands were examined for the expression of PROP1 and POU1F1 by using RT-PCR Pituitary adenomas were diagnosed as non-functioning adenomas (n = 11), prolactinomas (n=5), GH-producing adenoma (n = 1) and ACTH-producing adenoma (n = 1). One of non-functioning adenomas was pituitary carcinoma with cerebellar metastasis and CSF dissemination. Our results demonstrated PROP1 expression in all pituitary tumors examined, in contrast, POU1F1 was detected in 14 of pituitary tumors. PROP1 was also expressed in normal pituitary gland, however, it was not in normal brain tissue, glioblastomas (cell lines and tumor tissues) and meningioma. Our data indicated that PROP1 expression was observed constantly both in the pituitary tumors and normal human adult pituitary tissues, suggesting that PROP1 is an essential transcriptional factor for pituitary specific gene expression in human. Therefore, detection of PROP1 might be a useful indicator for differentiating pituitary adenomas, regardless of their hormonal phenotypes, from other brain tumors.

Therapeutic usefulness of wild-type p53 gene introduction in a p53-null anaplastic thyroid carcinoma cell line.

Anaplastic thyroid carcinomas very often harbor the mutations in the tumor suppressor gene p53. We have previously shown that wild-type (wt) p53 gene introduction led to cell growth arrest, but not apoptosis, in p53-null anaplastic thyroid carcinoma cells. The present studies were designed to evaluate other therapeutic effects of wt-p53 gene introduction on p53-null thyroid carcinoma cells, as chemo- and radiosensitization and inhibition of angiogenesis have also been described recently as additional therapeutic advantages of wt-p53 gene introduction in tumor cells with p53 mutations. A p53-null anaplastic thyroid carcinoma cell line, FRO, and a FRO subline stably expressing a temperature-sensitive (ts) mutant of p53 (p53Val138), tsFRO, were used. ts-p53 functions as mutant and wt at nonpermissive (37 C) and permissive (32 C) temperatures, respectively. tsFRO showed a prolonged cell doubling time compared to parental FRO when cultured at 32 C, but the cell growth rate was similar between FRO and tsFRO at 37 C. The cytotoxic and clonogenic assays demonstrated that although the sensitivity to three different anticancer agents (cisplatin, 5-fluorocytosine, and doxorubicin) was unaltered, radiosensitivity was enhanced in tsFRO compared to FRO at 32 C. Unexpectedly, in studies on angiogenesis, expression levels of vascular endothelial growth factor (an angiogenic factor) messenger ribonucleic acid were similar between FRO and tsFRO, and thrombospondin-1 (an antiangiogenic factor) messenger ribonucleic acid and protein levels were about 2.5-fold lower in tsFRO than FRO at 32 C, although any difference could not be detected in their ability to inhibit in vitro angiogenesis with the culture medium conditioned by tsFRO and FRO at 32 C. These results suggest that p53-defective thyroid carcinomas may benefit from the combination of p53 gene therapy and radiotherapy. However, further study will be necessary to clarify the pathological significance of thrombospondin-1 in angiogenesis and thyroid tumor growth.

Involvement of receptor-type tyrosine kinase gene families in cardiac hypertrophy.

OBJECTIVE: The activation of protein tyrosine kinases (PTKs) has been postulated to be involved in cell differentiation and proliferation. To elucidate the involvement of tyrosine kinase genes in normal and pathological conditions, we analysed the expression patterns of receptor-type PTKs in the normal and hypertensive hypertrophied heart in rats. MATERIALS AND METHODS: Hypertrophied and normal rat hearts were obtained from hypertensive rats; deoxycorticosterone acetate (DOCA)-salt and 2 kidney-1 clip (2K-1C), and their sham-operated rats, respectively. A reverse transcription-polymerase chain reaction (RT-PCR) was performed using degenerated primers which were designed from highly conserved regions in the catalytic domains of receptor-type PTKs. The PCR products were ligated into a sequence vector, and subcloned by transforming bacteria. To compare the expression level of these PTK mRNAs in the normal and hypertrophied heart, we performed semi-competetive RT-PCR and immunohistochemical and Western blot analyses. RESULTS: Nucleotide sequencing of approximately 80 clones of PTKs revealed 10 receptor-type, five nonreceptor-type and two unknown types in the rat heart. Tie-2/Tek, Ryk, insulin-like growth factor-I receptor were abundantly expressed in the rat heart as members of receptor-type PTKs. Immunohistochemistry and RT-PCR demonstrated the presence of platelet-derived growth factor (PDGF)-alpha receptor, PDGF-beta receptor and fibroblast growth factor-3 receptor in both normal and hypertrophied hearts. We also confirmed the presence of Flt-1, KDR/FIk-1, and their ligand vascular endothelial growth factor, c-Met and its ligand hepatocyte growth factor (HGF), and Tie-1, Tie-2/Tek by immunohistochemistry and RT-PCR. The coexpression of cardiac HGF and c-Met in hypertrophied hearts, especially in 2K-1 C rats, was induced more intensively than that in DOCA-salt rats. CONCLUSION: These findings suggest that HGF/c-Met interactions may play an important role in cardiac hypertrophy and remodeling, probably as a result of the activation of the local renin-angiotensin system.

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.

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.

Expression of parathyroid hormone related peptide in human pituitary tumours.

The presence of parathyroid hormone related peptide (PTHrP) was studied in 20 patients with pituitary adenomas and one patient with pituitary adenocarcinoma. PTHrP expression was shown in almost all of the pituitary adenomas (95%) and in 100% (n = 7) growth hormone producing pituitary adenomas. A metastatic lesion from a pituitary growth hormone producing adenocarcinoma revealed strongly expressed PTHrP. It was weakly detected in normal pituitary cells in all of the specimens (n = 10). There was no significant correlation, however, between PTHrP expression and the clinical or pathological features of growth hormone producing tumours. Apart from an important role in the physiological function of the pituitary gland, PTHrP may be closely related to somatotroph tumorigenicity.

Expression of parathyroid hormone-related peptide in human osteoarthritis.

To evaluate the involvement of the expression of parathyroid hormone-related peptide gene in human articular cartilage pathology, we performed immunohistochemical staining and in situ hybridization on specimens of femoral head cartilage obtained from 15 patients with osteoarthritis, 11 with rheumatoid arthritis, and 12 control subjects. Parathyroid hormone-related peptide-positive chondrocytes were observed predominantly in degenerated lesions of osteoarthritic tissue and were less evident in rheumatoid arthritic samples, while the normal cartilage expressed little parathyroid hormone-related peptide. In addition, the level of parathyroid hormone-related peptide expression was clearly dependent on the degree of cartilage degeneration; cartilage tissues with moderate degenerative changes contained more positive chondrocytes compared with mildly or severely degenerated cartilage. In situ hybridization confirmed the localization of parathyroid hormone-related peptide protein and demonstrated intense expression of mRNA of the peptide in osteoarthritic samples. This is the first demonstration of parathyroid hormone-related peptide expression in chondrocytes from pathologic articular cartilage of humans. Our results suggest that parathyroid hormone-related peptide may be involved in the pathophysiology of osteoarthritis.