Tamoxifen interacts with NEU/C-ERBB-2 receptor and inhibits growth of human malignant glioma cell lines.

The effects of tamoxifen, an antiestrogen, on the inhibition of protein tyrosine phosphorylation in neu/c-erbB-2 receptor, DNA synthesis and proliferation were evaluated using the malignant glioma cell lines U25 IMG and T98G which overexpressing neu/c-erbB-2. Pretreatment of two cell lines with tamoxifen resulted in a dose dependent inhibition of tyrosine phosphorylation as well as DNA synthesis and cell growth in two cell lines correlatively. The results support the hypothesis that activated protein tyrosine kinase receptors are involved in the proliferation of glioma cells. Tamoxifen may be useful in the treatment of malignant glioma.

Overcoming of radioresistance in human gliomas by p21WAF1/CIP1 antisense oligonucleotide.

Malignant gliomas are highly resistant tumors against gamma-irradiation and contained overexpression of p21WAF1/CIP1 (p21). Overexpression of p21 enhanced clonogenic survival and suppressed apoptosis after gamma-irradiation in human brain tumor cell lines with or without p53 protein deficiency. The effect of antisense oligonucleotide to p21 against the gamma-irradiation-induced apoptosis and cytotoxicity in malignant glioma cell lines was examined. Antennapedia homeodomain internalization peptide was used as an insertion vector. The high transfection efficiency of Antennapedia homeodomain internalization peptide joined with antisense oligonucleotide was observed. The pretreatment with antisense oligonucleotide enhanced the gamma-irradiation-induced apoptosis and cytotoxicity in radioresistant glioma cells. p21 may represent an important new target for radiosensitization protocols, possibly involving antisense oligonucleotide directed against p21.

Wild-type p53-dependent etoposide-induced apoptosis mediated by caspase-3 activation in human glioma cells.

OBJECT: In an attempt to understand the roles of several apoptosis-related genes in human glioma cells, the authors investigated the relationship of wild-type p53, interleukin-1beta-converting enzyme (ICE), caspase-3 (CPP32), bax, and bcl-2 to the apoptotic response of three glioma cell lines after treatment with etoposide. METHODS: A human glioma cell line (U-87MG) that expresses wild-type p53, one that expresses mutant p53 (T-98G), and a T-98G derivative (T-98G/p53) that was transfected with a wild-type p53 expression vector (pCDM8-p53/neo) were used. Cell growth inhibition in response to etoposide was quantified using a modified methylthiazol tetrazolium colorimetric assay. Induction of apoptosis was evaluated using Hoechst 33258 staining and a DNA fragmentation assay. To study the expression of the apoptosis-related proteins and messenger RNAs in the three glioma cell lines, Western blotting and polymerase chain reaction were performed. A caspase assay and Western blot analysis were used to assess CPP32 and ICE protease activity. A CPP32 inhibition assay was used to determine whether a specific CPP32 inhibitor, DEVD-CHO, affects the apoptosis induced by etoposide in malignant glioma cells. Etoposide significantly inhibited the growth of U-87MG and T-98G/p53 cells in a dose-dependent manner compared with the growth of the T-98G cells. Treatment with low concentrations of etoposide resulted in the increased expression of wild-type p53; it also initiated CPP32 activity and induced apoptosis in the U-87MG cells. Apoptosis was not induced in T-98G cells at low concentrations of etoposide, although it was induced at high concentrations. Furthermore, low concentrations of etoposide also induced apoptosis in the T-98G/p53 cells by enhancing the expression of transfected wild-type p53, decreasing the expression of bcl-2, and activating CPP32 activity. However, etoposide did not alter the expression of bax and did not initiate ICE activity in these three glioma cell lines. Etoposide-induced apoptosis can be suppressed by the CPP32 inhibitor DEVD-CHO. CONCLUSIONS: These findings indicate that wild-type p53, CPP32, and bcl-2 may mediate apoptosis induced by etoposide. Forced expression of wild-type p53 increases etoposide cytotoxicity in human glioma cells by inducing apoptosis and may have important therapeutic implications.

Blood-brain barrier formation of grafted human umbilical vein endothelial cells in athymic mouse brain.

Human umbilical vein endothelial cells (HUVECs) were transplanted in athymic mouse brain and neovascularization of grafted endothelial cells was studied. HUVECs were transfected by a reporter gene pEGFPE-N1 in vitro and grafted stereotactically in unilateral striatum of adult nude mice. Histological studies in 4 weeks revealed that grafted HUVECs newly formed microvessels in brain, which were migrated and fused with host vessels. Intravenous injection of Evans blue before sacrificing animals resulted in no extravasation of dye, indicating that a blood-brain barrier (BBB) was formed by the grafted HUVECs. Immunohistochemistry demonstrated that host astrocytes extended glial feet on the grafted endothelial cells and a part of the newly formed vessels was positive with glucose transporter-1. These results indicate that endothelial cells from an ectopic origin have the potential to form a BBB after grafting in the central nervous system.

In vivo gene transfer into the periventricular region by electroporation.

Gene transfer by electroporation was attempted in the normal rat brain. The reporter gene pEGFP-C1 (25 micrograms/5 microliters) was injected into the striatum of young adult rats and various square electrical impulses were applied using a pair of electrodes implanted in the striatum. The brains were removed and sliced after 5 days. Histological examination revealed that the high energy impulses caused extensive tissue damage whereas lower energy impulses (200-400 mJ) resulted in the transfection of more than 300 cells per brain, which were widely distributed in the subependymal region of the lateral ventricle and extended long processes into the striatum.

[A case of metastatic tumor of the pituitary gland presenting as a subarachnoid hemorrhage].

We reported the case of a 55-year-old man with metastatic tumor of the pituitary gland who suffered from symptomatic pituitary apoplexy with subarachnoid hemorrhage. The patient, who had sigmoid colon carcinoma and left parietal metastatic brain tumor, developed severe headache and decrease of right visual acuity. CT showed a pituitary mass with subarachnoid hemorrhage. Transsphenoidal surgery was performed after replacement therapy with corticosteroids. Histological examination revealed metastasis of adenocarcinoma. Pituitary apoplexy is an unusual manifestation of metastatic pituitary tumor. The case of metastatic tumor of the pituitary gland presenting as subarachnoid hemorrhage such as this case is especial rare.

Pineoblastoma showing unusual ventricular extension in a young adult--case report.

A 19-year-old male presented with a 4-week history of headache. Neurological examination showed bilateral papilledema. Computed tomography revealed a pineal region mass with remarkable obstructive hydrocephalus. Magnetic resonance imaging showed a pineal region tumor continuously invading through the tectum into the cerebral aqueduct and the fourth ventricle with the preservation of the adjacent structures. The tumor appeared an iso- to hypointense mass on T1-weighted images, a heterogeneous iso- to hyperintense mass on T2-weighted images, and a heterogeneously enhanced mass after administration of contrast medium. Histological examination after endoscopic biopsy confirmed that the tumor was a pineoblastoma. Radiotherapy was given to the whole brain and the spinal cord, and magnetic resonance imaging showed complete remission of the tumor. Pineoblastomas are highly malignant tumors with seeding potential through the neighboring ventricle or along the meninges, and this type of tumor becomes larger with local extension. We found no previous reports of the continuous extension into the fourth ventricle. The present case showed ventricular extension with minimal mass effect to adjacent structures, and did not disturb ventricular configuration. According to the unusual finding of ventricular extension, this rare case of pineoblastoma requires adjuvant chemotherapy.

Relationship between expression of p21WAF1/CIP1 and radioresistance in human gliomas.

The role of p21WAF1/CIP1 (p21) in DNA repair and apoptosis following gamma-irradiation remains controversial. In this study the influence of p21 on the radiosensitivity of human brain tumors was investigated. Resected tumors were stained immunohistochemically for p21. Expression of p21 in astrocytic tumors was high, but it was low in medulloblastomas, germinomas, and primary malignant lymphomas. Glioma and medulloblastoma cell lines were transfected with pcDNA/p21 to cause p21 overexpression, then tumor-cell colony formation and apoptosis were assessed following gamma-irradiation of the transfected and nontransfected cells. Overexpression of p21 enhanced clonogenic survival and suppressed apoptosis after gamma-irradiation in human brain tumor cell lines with or without p53 protein deficiency. Radioresistance was acquired when p21 was overproduced in the glioma cell lines irrespective of p53 status.

[A case of primary cerebral neuroblastoma surviving for eight years].

We report a case of a patient with primary cerebral neuroblastoma who has survived for 8 years. A 10-year-old boy was admitted to our hospital because of headache and nausea. CT scan on admission revealed a large cystic tumor on the right frontal lobe. Subtotal tumor resection was carried out. A second operation was performed for the residual tumors which were removed meticulously with confirmation of the absence of tumor cells on each frozen section. After tumor removal, YAG laser was applied at each local area. Histological diagnosis disclosed primary cerebral neuroblastoma. Because of postsurgical meningitis and parent's refusal, neither chemotherapy nor radiation therapy was performed. There have been no findings of the tumor recurrence during the last eight years, and now the patient is enjoying high school life to the full, without any neurological deficits. In reviewing the literature, outcomes of neuroblastoma cases are very poor. Our case seems to be one of the rare long-survival cases.

Differentiation and growth inhibition of glioma cells induced by transfer of trk A proto-oncogene.

The induction of growth inhibition and differentiation of a glioma cell line by transfection of trk A cDNA was examined, and production of endogenous nerve growth factor (NGF) also was studied in these cells. When human trk A cDNA was transfected into a human glioma cell line, U-251MG, which lacks expression of both endogenous trk A and low-affinity NGF receptor, the transfectant expressed the exogenous trk A mRNA and a functional high-affinity NGF receptor. Transfection of trk A cDNA caused a partial induction of cell differentiation, G1 arrest, growth inhibition, tyrosine phosphorylation of the trk A proto-oncogene product, and activation of MAP kinase. Exogenous NGF treatment induced further terminal differentiation and growth inhibition. In summary, our data suggest that endogenous NGF secreted by glioma cells has an important role in the induction of glioma-cell differentiation occuring with transfer of exogenous trk A cDNA.

Bromocriptine-induced apoptosis in pituitary adenoma cells: relationship to p53 and bcl-2 expression.

In an attempt to understand the roles of the tumour suppressor gene p53 and the proto-oncogene bcl-2 in cell death and survival in pituitary adenomas, we investigated the relationship of their expression to the apoptotic response of two pituitary adenoma cell lines (GH3 and AtT-20) to bromocriptine. An MTT (3-4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrasolium bromide) assay was performed after treatment with bromocriptine for various periods of time over a range of concentrations to determine the effect of this drug on cell growth. Bromocriptine inhibited growth of GH3 and AtT-20 cells in a dose dependent manner. DNA fragmentation was assessed in GH3 and AtT-20 cells exposed to 10 ug/ml bromocriptine- for 48 h and 72 h. The DNA of GH3 and AtT-20 cells showed nucleosomal fragmentation, indicative of apoptosis. When assayed 2 days after adding bromocriptine, approximately 60% of GH3 and 58% of AtT-20 cells treated with bromocriptine displayed typical apoptotic morphology, including condensed chromatin and fragmented nuclei. There was a time dependent increase in the proportion of all tumour cells undergoing apoptosis. Decreased expression of bcl-2 and accumulation of wild-type p53 were associated with bromocriptine induced apoptosis in pituitary adenoma cells. DNA analysis confirmed the results obtained by the protein study. Different expression of p53 and bcl-2 genes is consistent with the expression of these gene products. These findings show that bromocriptine activated wild-type p53 and suppressed bcl-2 favouring occurrence of apoptosis in pituitary adenoma cells. Copyright 1999 Harcourt Publishers Ltd.

Effect of dexamethasone on cell proliferation of neuroepithelial tumor cell lines.

The effect of glucocorticoid on cell proliferation, the expression of glucocorticoid receptor, and the relationship between inhibition of cell growth and apoptosis were investigated in four established neuroepithelial tumor cell lines (KNS42, T98G, A172, and U251MG). Glucocorticoid receptor expression was located in the cytoplasm of untreated cells, but translocated into nuclei after treatment with dexamethasone in KNS42, T98G, and A172 cells. U251MG did not express glucocorticoid receptors. Dexamethasone significantly inhibited the growth of KNS42 and T98G cell lines, at high concentrations in contrast to growth stimulation at low concentration. Dexamethasone inhibited proliferation of A172 cell line at all concentrations from 10(-4) M to 10(-7) M. These were prevented by RU38486, a specific glucocorticoid antagonist. Apoptosis did not occur in any cell lines after dexamethasone treatment. There was no response to glucocorticoid by U251MG cells. Dexamethasone treatment of neuroepithelial tumor cells expressing glucocorticoid receptors causes translocation into the nucleus to modulate cell proliferation upon binding of different concentrations of dexamethasone in vitro. Dexamethasone inhibits proliferation of some neuroepithelial cell lines, not by glucocorticoid-induced apoptosis. The bimodal potential of glucocorticoid to stimulate or suppress proliferation of neuroepithelial tumor cells expressing glucocorticoid receptor must be considered in clinical trials.

Overexpression of p21WAF1/CIP1 induces cell differentiation and growth inhibition in a human glioma cell line.

p21WAF1/CIP1 is a downstream mediator of p53 and mediates growth arrest by inhibiting the action of G1 cyclin-dependent kinases. Since cellular differentiation is frequently characterized by G1 arrest, we examined whether p21WAF1/CIP1 overexpression would induce growth suppression and differentiation in p53-defective human glioma cells. Overexpression of p21WAF1/CIP1 resulted in an accumulation of cells in G1, altered morphology, growth arrest and cell differentiation. The extent of cell differentiation correlated with the level of p21WAF1/CIP1 as well as of proliferating cell nuclear antigen, cyclin E, and cdk 2, which associates with p21WAF1/CIP1. Our data suggest that gene transfer of p21WAF1/CIP1 may arrest glioma cell growth in vivo by committing malignant glioma cells to a pathway of terminal differentiation.

Clinical features and growth fractions of pituitary adenomas.

BACKGROUND: The Ki-67 monoclonal antibody is expressed by proliferating and dividing cells, but not by resting cells. The specificity of the monoclonal antibody, MIB-1, against the Ki-67 antigen has been established by immunostaining of formalin-fixed paraffin-embedded tissue in a microwave oven. METHODS: The growth fraction of 85 pituitary adenomas was studied retrospectively by immunohistochemical analysis using the monoclonal antibody MIB-1. The adenomas were classified into three types: microadenoma, expansive type, and invasive type, based on findings on Gd DTPA enhanced magnetic resonance imaging. RESULTS: The mean MIB-1 index in nonfunctioning microadenomas was higher than in expansive and invasive adenomas, but this difference was not significant. The MIB-1 index in younger patients (under 30 years) with nonfunctioning adenomas was significantly higher than in patients over 40 years of age. One of 14 patients with recurrent disease had an elevated MIB-1 index, but generally patients with an MIB-1 index over 2.0% did not suffer recurrence. The mean MIB-1 index was higher in expansive and invasive functioning adenomas than microadenomas, but not significantly. No correlation between the MIB-1 index and the serum GH or PRL concentration was established. No MIB-1 positive nuclei were observed in two GH-producing adenomas treated with the somatostatin analog SMS 201-995. CONCLUSIONS: No significant relationship was identified between growth fraction and the invasiveness or recurrence of pituitary adenomas. The growth fraction of nonfunctioning pituitary adenomas was higher in patients under 30 years than over 40 years of age.

Cerebellopontine angle ependymoma with internal auditory canal enlargement and pineal extension--case report.

A 38-year-old male presented with a posterior fossa ependymoma with unusual extension from the cerebellopontine angle to the pineal region. Magnetic resonance imaging clearly demonstrated that these two components were continuous through the right ambient cistern. Computed tomography using a bone algorithm revealed enlargement of the right internal auditory canal. This case suggests that ependymoma can extend anywhere within the subarachnoid space along the path of least resistance.

Induction of differentiation by wild-type p53 gene in a human glioma cell line.

Wild-type human p53 gene was transfected into the human glioma cell line T-98G. Transfectants were then isolated and characterized for growth potential and differentiation phenotype. Growth suppression, overexpression of GFAP, and accumulation in G1 phase were more commonly observed in transfectants than in T-98G cells. p21WAF1/CIP1 was overexpressed in transfectants, and the binding of PCNA and CDK 2 to p21WAF1/CIP1 were increased in transfectants. These results suggested the roles of p21WAF1/CIP1, PCNA, and CDK2 in regulation of differentiation in glioma cells and the gene transfer of wild-type p53 may be effective for the control of glial differentiation in glioma cells.

Intrasellar pituitary gangliocyto-adenoma presenting with acromegaly: case report.

OBJECTIVE AND IMPORTANCE: A gangliocytoma in the sellar region is extremely rare. We describe a rare case of intraseller gangliocytoma coexisting with a growth hormone-producing pituitary adenoma, which presented with acromegaly. CLINICAL PRESENTATION AND INTERVENTION: A 64-year-old woman was admitted to our hospital with headache and acromegaly. Endocrinological studies revealed an elevated serum level of growth hormone (GH). Magnetic resonance imaging showed a tumor at the intrasellar and suprasellar regions. The tumor was totally removed via a transsphenoidal approach. RESULTS: A histological examination of the resected specimen showed areas of ganglionic cells and adenomatous cells. Immunohistochemical examination demonstrated GH-releasing hormone-positive ganglionic cells and GH-positive pituitary adenoma. CONCLUSION: Based on these immunohistochemical findings, we hypothesized that the intrasellar gangliocytoma promoted the growth of the pituitary adenoma, which had been transformed from a region of pituitary hyperplasia by chronic overstimulation from excess GH-releasing hormone produced by the intrasellar gangliocytoma.

Effects of bromocriptine and terguride on cell proliferation and apoptosis in the estrogen-stimulated anterior pituitary gland of the rat.

The effects of bromocriptine and terguride on the estrogen-stimulated anterior pituitary gland of the female Wistar rat were investigated. Pituitary weight and serum prolactin (PRL) levels were reduced by treatment with bromocriptine or terguride. Immunohistological staining for proliferating cell nuclear antigen (PCNA) revealed that the PCNA labeling index of PRL-producing cells was significantly decreased by treatment with bromocriptine or terguride compared with untreated cells. The number of apoptotic cells analyzed by the terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate-biotin nick end labeling method was significantly increased in rats treated with bromocriptine or terguride. Suppression of cell proliferation and induction of apoptosis are important effects of bromocriptine and terguride in the treatment of prolactinomas and other hyperprolactinemias.

Functional analysis of trk proto-oncogene product in medulloblastoma cells.

MED-L cells with the 75 kd low-affinity nerve growth factor receptor (p75NGFR) and MED-H cells with the proto-oncogene tropomyosin receptor kinase product (p140trk) were isolated selectively from a parent MED-3 cell line derived from cerebellar medulloblastoma by panning, and the interaction of nerve growth factor (NGF) with these cell lines was analyzed. NGF treatment induced neuronal differentiation, growth inhibition, and tyrosine phosphorylation of p140trk in MED-H cells, but not in MED-L cells. Medulloblastoma cells express the functional NGFR, p140trk, which regulates their differentiation and growth.