Influence of thyroid hormones and transforming growth factor-ß1 on cystatin C concentrations.

Serum cystatin C concentrations are reported to increase in the hyperthyroid state. Serum concentrations of cystatin C and transforming growth factor-ß1 (TGF-ß1) were measured in patients with thyroid dysfunction, and the effects of 3,5,3'-tri-iodothyronine (T(3)) and TGF-ß1 on cystatin C production in human hepatoblastoma (Hep G2) cells were studied. Serum concentrations of cystatin C and TGF-ß1 were significantly higher in patients with Graves' disease compared with control subjects. Significantly positive correlations were observed between thyroid hormones and cystatin C, thyroid hormones and TGF-ß1, and TGF-ß1 and cystatin C in patients with thyroid dysfunction. Serum concentrations of cystatin C and TGF-ß1 decreased after treatment for hyperthyroidism. Cystatin C mRNA levels and cystatin C secretion were increased by T(3) and TGF-ß1 in cultured Hep G2 cells. These results suggest that serum cystatin C concentrations increase in patients with hyperthyroidism. The mechanisms for this may involve elevation of serum TGF-ß1 levels and the stimulatory effects of T(3) and TGF-ß1 on cystatin C production.

Transfection with a bcl-2 expression vector protects transplanted bone marrow from chemotherapy-induced myelosuppression.

The use of cytokines such as granulocyte-colony-stimulating factor (G-CSF) to ameliorate chemotherapy-induced myelosuppression may not only stimulate the recovery of normal hematopoietic cells but may also enhance the proliferation of the tumor cells with functional receptors for these cytokines. In this study, we show that administration of recombinant human (rh) G-CSF decreased the in vitro and in vivo cytotoxic effects of Adriamycin or etoposide on L1210 murine leukemic cells with receptors for rhG-CSF. Transplantation of bone marrow cells expressing high levels of bcl-2 from a retroviral construct [MPZenNeo(bcl-2)] (bcl-2-BMT) did not decrease the in vivo cytotoxic effect of etoposide on L1210 cells, but enabled recovery of myelopoiesis following etoposide-induced myelosuppression to almost the same extent as did the administration of rhG-CSF. These findings suggest the possibility that bcl-2 transfection could be used to protect transplanted bone marrow from chemotherapy-induced myelosuppression on behalf of administration of rhG-CSF, in case of treatment of tumors with functional receptors for rhG-CSF.

Interleukin-1 beta-converting enzyme mediates cisplatin-induced apoptosis in malignant glioma cells.

Increasing the susceptibility of tumor cells to apoptotic cell death following chemotherapy is of importance to the outcome of cancer treatment. Although the tumor suppressor gene p53 is required for efficient induction of apoptosis by chemotherapeutic agents, it is not the only apoptosis mediator gene. The molecular mechanisms mediating apoptosis following chemotherapy via p53-dependent or p53-independent pathways remain unclear. We show here that cis-diamminedichloroplatinum (cisplatin) induces the expression of interleukin-1 beta-converting enzyme (ICE), a mammalian homologue of the Caenorhabditis elegans cell death gene ced-3, in murine and human malignant glioma cells during apoptosis regardless of their p53 status. Furthermore, overexpression of the murine ICE gene induces apoptosis in these tumor cells. The apoptosis induced by cisplatin treatment or murine ICE overexpression can be suppressed by the tetrapeptide ICE inhibitor Ac-YVAD-CMK or the apoptosis inhibitors bcl-2 or bcl-2-related bcl-XL gene. These findings suggest that ICE may mediate apoptosis induced by chemotherapy, and its induction could represent a novel approach for the effective treatment of malignant glioma.

MDM2 protein confers the resistance of a human glioblastoma cell line to cisplatin-induced apoptosis.

Induction of apoptosis in tumor cells is an important mechanism of chemotherapy-induced cell death. The tumor-suppressor gene p53 is required for the efficient activation of apoptosis following chemotherapy. However, the molecular mechanism regulating p53-associated apoptosis remains controversial. In this study, we show that the expression of both wild-type p53 and MDM2 (murine double minute 2) proteins was induced when cis-diamminedichloroplatinum (cisplatin) caused apoptosis in human glioblastoma U87-MG cells, which expressed neither wild-type p53 nor MDM2 protein prior to treatment. Overexpression of MDM2 in U87-MG cells transfected with human mdm2 expression vector conferred the resistance of tumor cell to cisplatin-induced apoptosis. In contrast, the treatment with mdm2 antisense oligonucleotide targeted against mdm2 mRNA increased the susceptibility of tumor cells to apoptosis. Changes in expression level of MDM2 protein, however, did not affect the expression of wild-type p53 protein. These findings suggest that MDM2 protein may act as a negative regulator of cisplatin-induced apoptosis, and moreover, may play an important role in the development of resistance to cisplatin in human tumors.

The transforming activities of MDM2 in cultured neonatal rat astrocytes.

Although the molecular events regulating the pathogenesis of malignant astrocytomas remains unclear, the inactivation of tumor suppressor genes may be a key factor. The inactivation of p53 by mutation or deletion, however, is not the only obligatory step in astrocytoma genesis. The MDM2 protein has been shown to bind to and downmodulate p53 function, and to have oncogenic capacity. The MDM2 gene is also amplified and overexpressed in a subset of malignant astrocytomas without p53 mutation. Here we show that overexpression of MDM2 promoted the DNA synthesis of cultured neonatal rat astrocytes (RNB cells), abrogated the transcriptional activity of wild-type p53, conferred invasive activity, and subsequently induced the transformation from astrocytes to high-grade astrocytomas. Intriguingly, MDM2 enhanced the expression of angiogenic mitogens; basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) in RNB cells. These results indicate that MDM2 may play an important role in the progression of astrocytomas, by not only conferring invasive activity but also stimulating the expression of angiogenic growth factors.

WAF1/CIP1 increases the susceptibility of p53 non-functional malignant glioma cells to cisplatin-induced apoptosis.

Induction of apoptosis in tumor cells is an important determinant in the outcome of therapy. Molecular details of the apoptosis pathway, however, are still poorly defined. The recently discovered WAF1/CIP1 gene is a potent inhibitor of cyclin-dependent kinases and a mediator of tumor-suppressor p53-dependent apoptosis by DNA damage. In addition, WAF1/CIP1 expression is shown to be triggered through the p53-independent pathway. The relationship between WAF1/CIP1 and p53-independent apoptosis by DNA damage, however, remains unclear. In this study, we show that WAF1/CIP1 was induced in p53-dependent apoptosis of U87-MG glioma cells by cis-diamminedichloroplatinum (cisplatin), and overexpression of WAF1/CIP1 induced apoptosis in U87-MG cells without cisplatin treatment. In contrast, the p53-independent apoptosis of GB-1 glioma cells by cisplatin did not express WAF1/CIP1. Overexpression of WAF1/CIP1 inhibited DNA synthesis in GB-1 cells, but did not induce apoptosis. Interestingly, WAF1/CIP1 increased the susceptibility of GB-1 cells to cisplatin-induced apoptosis. These results suggest that overexpression of WAF1/CIP1 may have potential for the treatment of tumors with non-functional p53.

Apoptosis in peripheral CD4+T cells and thymocytes by Marek's disease virus-infection.

Histological study revealed that Marek's disease virus (MDV) can cause apoptosis in peripheral blood lymphocytes (PBL) in latently infected chickens. Analysis of DNA fragmentation indicated that CD4+T cells but not CD8+T cells underwent apoptosis. These apoptotic changes were also observed in the thymus during the acute phase of the infection. Flow cytometry analysis showed the drastic decrease of CD4+CD8+ thymocytes, indicating that MDV can induce apoptosis in CD4+CD8+ immature thymocytes in acutely infected chickens. These changes might be involved in the immuno-suppression induced by MDV.

Expression and regulation of type II iodothyronine deiodinase in human thyroid gland.

We have studied the expression of type II iodothyronine deiodinase (DII) in human thyroid tumors and cultured human thyroid cells to elucidate the mechanisms involved in the regulation of DII expression in human thyroid gland. Three cases with hyperfunctioning thyroid adenoma, including a case that showed an activating mutation of G(s)alpha with a constitutive activation of cAMP production in cultured cells, and six cases with papillary thyroid carcinoma were analyzed in the present study. Free T(3) was increased, whereas free T(4) was within the normal range in all patients with hyperfunctioning thyroid adenoma. Thyroid tumor tissue and surrounding nontumor tissue were obtained at the time of surgery, and DII expression was compared between tumor tissue and nontumor tissue in each case. Northern analysis demonstrated the presence of DII messenger RNA (mRNA) approximately 7.5 kb in size in all of the tumor and nontumor tissues. DII mRNA and DII activity in hyperfunctioning thyroid adenoma were significantly increased compared with those in nontumor tissue in each case. In contrast, DII mRNA and DII activity in papillary thyroid carcinoma were decreased compared with those in nontumor tissue in each case. DII mRNA and DII activity in cultured human thyroid cells were significantly stimulated by TSH in a dose-dependent manner. The promoter activity of the human DII gene including the complete cAMP response element, transfected to cultured human thyroid cells, was stimulated by (Bu)(2)cAMP. In summary, these results suggest that DII expression in human thyroid gland is regulated at the transcriptional level through the TSH receptor-G(s)alpha-cAMP regulatory cascade, which may be related to the increase in circulating T(3) level in patients with Graves' disease and hyperfunctioning thyroid adenoma.

Thyrotropin receptors in brown adipose tissue: thyrotropin stimulates type II iodothyronine deiodinase and uncoupling protein-1 in brown adipocytes.

It has been demonstrated that TSH receptors are expressed not only in thyroid gland but also in extrathyroidal tissues. Brown adipose tissue of guinea pig has been reported to express TSH receptor messenger RNA (mRNA), but the physiological roles of TSH receptors in brown adipose tissue have not been understood. We studied the expression and function of TSH receptors in rat brown adipose tissue and cultured rat brown adipocytes. Northern analysis demonstrated the expression of TSH receptor mRNA in rat brown adipose tissue and cultured rat brown adipocytes. TSH receptor mRNA in rat brown adipose tissue was decreased by cold exposure of the rat, and its mRNA in cultured rat brown adipocytes was also decreased by incubation with TSH or (Bu)(2)cAMP. TSH increased the intracellular cAMP concentration in cultured rat brown adipocytes in a dose dependent manner. Type II iodothyronine deiodinase mRNA, its activity, and uncoupling protein-1 mRNA in cultured rat brown adipocytes were significantly increased by incubation with TSH in a dose-dependent manner. These results suggest the expression of functional TSH receptors in brown adipose tissue, which may be involved in regulation of the expression of type II iodothyronine deiodinase and uncoupling protein-1.

Expression of type II iodothyronine deiodinase in brain tumors.

Type II iodothyronine deiodinase (DII) messenger ribonucleic acid (mRNA) and its activity have been demonstrated in human normal brain. Although DII activity has been demonstrated in brain tumors, expression of DII mRNA has not been studied in these tumors. To investigate the mechanisms involved in the expression of DII activity in brain tumors, we studied DII mRNA and DII activity in astrocytoma (two cases), glioblastoma (three cases), and oligodendroglioma (one case). DII mRNA, the size of which was indistinguishable from that in control cerebral cortical tissue, was demonstrated in all of the brain tumors tested, although the intensity of the hybridization signal showed wide variation among the tumors. DII activity was also detected in all tumors. DII mRNA and DII activity were highest in the tissue from oligodendroglioma. A significantly positive correlation was observed between DII mRNA and DII activity in these tumors (r = 0.94; P < 0.01), suggesting that DII expression in brain tumors is regulated at the pretranslational level. The present results demonstrate, for the first time, that DII mRNA as well as DII activity are expressed in brain tumors, and that DII mRNA is significantly correlated with DII activity in those tissues.

Induction of apoptosis in murine ACTH-secreting pituitary adenoma cells by bromocriptine.

Bromocriptine, a dopamine agonist, is now an accepted primary therapeutic agent for patients with prolactinomas and other pituitary adenomas. In this study, we demonstrated that bromocriptine inhibited the proliferation of murine ACTH-secreting pituitary adenoma (AtT-20) cells. In addition, the antitumor activity of bromocriptine was inhibited both by actinomycin D and cycloheximide, suggesting that it was dependent on new RNA and protein synthesis. Interestingly, the results of DNA fragmentation assays and cell cycle analysis clearly demonstrated that bromocriptine induced apoptosis in AtT-20 cells.

Bcl-2 gene prevents induction of apoptosis in l1210 murine leukemia-cells by sn-38, a metabolite of the camptothecin derivative cpt-11.

New camptothecin (CPT) derivatives have recently been synthesized following the finding that CPT has strong antitumor activity due to its inhibition of topoisomerase I through the formation of stable topoisomerase I-DNA cleavable complexes, but has not been clinically used due to its pronounced toxicity. 7-ethyl-10-hydroxy-CPT (SN-38), a metabolite of the CPT derivative 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy-CPT(CPT-11), plays an essential role in mediating the antitumor effect of CPT-11. However, the reasons for the cytotoxicity of SN-38 remain unclear. In this study, we demonstrated using results of DNA fragmentation assay and cell cycle analysis that SN-38 and CPT both induce apoptosis in L1210 murine leukemia cells. We demonstrated in addition that enforced expression of the bcl-2 gene in L1210 cells by MPZenNeo (bcl-2) retroviral gene transfer increased resistance to the apoptosis induced by SN-38 and CPT. These findings suggest the possibility that the bcl-2 gene impedes the activity of a common pathway for apoptosis induced by SN-38 and CPT.

Bcl-2 gene prevents apoptosis in murine acth-secreting adenoma cells induced by bromocriptine.

Bromocriptine, a dopamine agonist, is now an accepted primary therapeutic agent for patients with prolactinomas and other pituitary adenomas. However, the mechanism by which bromocriptine decreases elevated serum levels of prolactin or growth hormone and shrinks tumors by diminishing tumor cell size is not clear. Recently, we obtained bromocriptine induced apoptosis in murine ACTH-secreting pituitary adenoma (AtT-20) cells, based on DNA fragmentation assay and cell cycle analysis (unpublished data). In this study, we demonstrate that enforced expression of bcl-2 gene in AtT-20 cells by the MPZenNeo(bcl-2) retroviral gene transfer increased resistance to apoptosis induced by bromocriptine.

Induction of apoptosis in multi-drug resistant (MDR) human glioblastoma cells by SN-38, a metabolite of the camptothecin derivative CPT-11.

The overexpression of the multidrug resistance (mdr1) gene and its product, P-glycoprotein (P-gp), is thought to limit the successful chemotherapy of human tumors. Recent studies demonstrate that SN-38, a metabolite of the camptothecin (CPT) derivative CPT-11, has antitumor effects on several tumors, but the mechanisms responsible for its cytotoxicity remain unclear. We therefore determined whether SN-38 has cytotoxic effects on MDR human glioblastoma GB-1 cells and non-MDR human glioblastoma U87-MG cells. Furthermore, we determined what role SN-38 plays in the induction of cytotoxicity in these tumor cells. In this study, we demonstrated that SN-38 had significantly stronger antitumor effects on GB-1 and U-87MG cells than did CPT (P < 0.01 and P < 0.05, respectively). In addition, findings obtained using a DNA fragmentation assay, Hoechst 33258 staining, in situ end-labeling and cell cycle analysis demonstrated that SN-38 induced apoptosis in these tumors. Our results suggest that SN-38 has a stronger antitumor effect on malignant glioma cells regardless of MDR expression than does CPT, and therefore can be considered a new chemotherapeutic agent potentially effective in the treatment of human primary or recurrent malignant gliomas resistant to chemotherapy.

Linear focal elastosis. An ultrastructural study.

We studied an 86-year-old Japanese man with linear focal elastosis. The lesions were asymptomatic yellow striae in the lumbar region, histologically composed of massive, well-demarcated basophilic fibers that stained positively with elastic tissue stains. Electron microscopy revealed fine, reticular or granular electron-dense materials, and elastic fiber microfibril-like materials in the matrix, in addition to numerous mature and immature elastic fibers. These findings suggest that active elastogenesis was occurring in the lesions. The four cases reported so far have the three common features of age, sex, and lesion location.

Chondromyxoid fibroma of the frontal bone.

Chondromyxoid fibroma of frontal bone is a rare lesion. Plain skull films showed a round radiolucent mass with a sclerotic margin. It was dense on plain CT scan and showed no convincing contrast enhancement. MR imaging showed low signal intensity relative to gray matter on T1-weighted image (500/20), isointensity on proton-density image (2000/30), high intensity on T2-weighted image, and marked peripheral enhancement on postcontrast (gadopentetate dimeglumine) study.

Induction of apoptosis in rat somatotrophin-secreting pituitary adenoma cells by bromocriptine.

Bromocriptine, a dopamine agonist, is now an accepted primary therapeutic agent for patients with prolactinomas and other pituitary adenomas. In this study, we demonstrated that bromocriptine inhibited the proliferation of rat somatotrophin-secreting pituitary adenoma (GH1) cells. In addition, the antitumor activity of bromocriptine was inhibited both by actinomycin D and cycloheximide, suggesting that it was dependent upon new RNA and protein synthesis. Interestingly, the results of DNA fragmentation assay and cell cycle analysis clearly demonstrated that bromocriptine induced apoptosis in GH1 cells.

Fibrous xanthoma in the gasserian ganglion: case report.

Occurrence of fibrous xanthoma has been reported increasingly in the skull and the central nervous system, but is extremely rare in the gasserian ganglion. We report on the clinical presentation, radiological appearance, surgical treatment, and histological makeup of a fibrous xanthoma arising from the left gasserian ganglion.

Tumor necrosis factor-alpha induces p53-dependent apoptosis in rat glioma cells.

In this study, we demonstrated that tumor necrosis factor (TNF)-alpha inhibited the viability of rat glioma (C6) cells and induced apoptosis but did not affect the viability of rat newborn brain, mainly astroglial cells. The antitumor activity of TNF-alpha against C6 cells was partially inhibited by actinomycin D and cycloheximide, suggesting that it is possibly dependent upon new ribonucleic acid and protein synthesis. The results of immunoblotting assay demonstrated that TNF-alpha decreased the expression of mutant p53 protein but induced the expression of wild-type p53 in C6 cells during apoptosis. We suggest that TNF-alpha may activate the function of wild-type p53 protein by the suppression of mutant p53, at least indirectly, and induce p53-dependent apoptosis in glioma cells.

Interferon-gamma induces a decrease in the susceptibility of human glioma cells to lysis by lymphokine-activated killer cells.

We studied the effect that treating two types of glioblastoma cell lines, U-87 MG and U-251 MG, with interferon (IFN)-gamma had on their susceptibility to lysis by lymphokine-activated killer (LAK) cells. We also examined the participation of cell-adhesion molecules and major histocompatibility complex (MHC) class I and II antigens present on the target cells in lysis by LAK cells. Treatment with IFN-gamma (1000 U/ml) for 48 hours resulted in the increased expression of both intercellular-adhesion molecule 1 and MHC class I antigens on tumor cells. In addition, untreated tumor cells expressed neural-cell-adhesion molecules and MHC class II antigens highly, but their expression was not affected by IFN-gamma treatment. These changes in expression were accompanied by a decreased susceptibility to lysis by LAK cells. Treatment with antisense-intercellular-adhesion molecule-1 oligonucleotide further inhibited LAK lysis of target cells, following treatment with IFN-gamma. In contrast, acid treatment of tumor cells after treatment with IFN-gamma increased their susceptibility to lysis by LAK cells. These findings suggest that treatment of glioblastoma cells with IFN-gamma decreased their susceptibility to lysis by LAK cells, and that this decrease in susceptibility is attributable principally to the increased expression of MHC class I antigen on target cells.