Combined effects of a third-generation bisphosphonate, zoledronic acid with other anticancer agents against murine osteosarcoma.

Bisphosphonates (BPs) are widely used to treat bone diseases and also appear to possess direct antitumour activity. We have previously reported that third-generation BPs such as zoledronic acid (ZOL) and minodronic acid (YM529) synergistically augment the effects of anticancer agents in various cancer cells. Recently, we have also reported the antitumour effects of YM529 on murine osteosarcoma cells. As YM529 has not been clinically available, we herein focused on the anti-osteosarcoma effects of ZOL which is clinically available. In addition to ZOL alone, we evaluated the concurrent or sequential combined effects of ZOL with other anticancer agents against murine osteosarcoma cell lines. ZOL showed almost same anti-osteosarcoma activity compared with YM529 and more sensitive growth inhibitory effects against osteosarcoma cells than normal cells. Moreover, ZOL acted synergistically in vitro when administered concurrently with paclitaxel (PAC) or gemcitabine (GEM), not only in wild-type osteosarcoma cells but also in P-glycoprotein (P-gp)-overexpressing osteosarcoma cells, which were much less sensitive against each anticancer agent. Furthermore, 24 h of ZOL pretreatment significantly augmented the sensitivity of doxorubicin (DOX), PAC or GEM against osteosarcoma cells. These findings suggest that combined administration of ZOL with other anticancer agents may improve the osteosarcoma treatment.

Induction of apoptosis in an estrogen-responsive mouse Leydig tumor cell by leukotriene.

For estrogen-responsive B-1F cells, established from estrogen-responsive mouse Leydig cell tumor, it has been reported that the 5-lipoxygenase (5-LOX) metabolic pathway appears to be associated with cell growth. The addition of 5-LOX inhibitor 2-(12-hydroxydodeca-5,10-diyl)-3,5,6-trimethyl-1,4-benzoquinone (AA861) to the medium resulted in a dose-dependent increase in cell yield as described previously. When the growth of the palpable tumors was measured, AA861 had stimulated in vivo tumor growth in adult male mouse inoculated B-1F cells. The effects of AA861 and 17beta-estradiol (E2) on the contents of various arachidonic acid metabolites in B-1F cells and their conditioned medium were examined. Although AA861 and E2 decreased the contents of leukotrienes (LTs), the two did not significantly change those of prostaglandins, thromboxan, prostacyclin, 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE. In immunohistochemical study B-1F cells show positive staining for 5-LOX in the E2-depleted condition, while E2 decreased the expression of 5-LOX. The decrease of the intensities of 79-kDa 5-LOX protein and 403-bp RT-PCR product bands was observed. The growth of Morpholino-anti oligo delivered B-1F cells was higher than that of Standard control oligo delivered cells. The delivery of Morpholino-anti oligo into B-1F cells caused the decrease of contents of LTs and 5-HETE in the cells and medium, and the reduction of 5-LOX activity. When LTD4 was added in the culture medium, the increasing concentrations of LTD4 resulted in a significant inhibition of cell yields of E2-treated B-1F cells. Morphological changes such as nuclear condensation and fragmentation, and DNA ladder pattern were demonstrated in E2-stimulated B-1F cells treated with LTD4 as well as in control cells cultured in the basal medium. These results implicate that 5-LOX at least plays an important role in the growth of B-1F cells and LD4 induces the apoptosis of B-1F cells.

Prenatal and neonatal exposure to bisphenol-A enhances the central dopamine D1 receptor-mediated action in mice: enhancement of the methamphetamine-induced abuse state.

Bisphenol-A (BPA), one of the most common environmental endocrine disrupters, has been extensively evaluated for toxicity in a variety of tests in rodents, including developmental and reproductive toxicity, and carcinogenicity. However, little is known about its action on the CNS. In this report, we show that prenatal and neonatal exposure to BPA in mice leads to the enhancement of the dopamine D1 receptor-dependent rewarding effect induced by a psychostimulant methamphetamine. Furthermore, this treatment with BPA markedly enhanced hyperlocomotion and its sensitization induced by methamphetamine, which reflects extensive abuse associated with sociological and psychiatric problems. We also demonstrated that chronic exposure to BPA produced an up-regulation of dopamine D1 receptor function to activate G-protein in the mouse limbic forebrain, which is thought to be a critical site for the expression of rewarding effects by abuse drugs. Additionally, chronic BPA exposure produced a significant increase in levels of the dopamine D1 receptor mRNA in the whole brain. In contrast, no change in protein levels of methamphetamine-targeted proteins, dopamine transporter or the type 2 vesicle monoamine transporter in the brain was observed by prenatal and neonatal exposure to BPA. The present data provide the first evidence that prenatal and neonatal exposure to BPA can potentiate the central dopamine D1 receptor-dependent neurotransmission, resulting in supersensitivity of methamphetamine-induced pharmacological actions related to psychological dependence on psychostimulants.

Musashi1, an evolutionarily conserved neural RNA-binding protein, is a versatile marker of human glioma cells in determining their cellular origin, malignancy, and proliferative activity.

Tumor cells often express phenotypic markers that are specific to the cells from which they originated. A neural RNA-binding protein, Musashil, is an evolutionarily well-conserved marker for neural stem cells/ progenitor cells. To examine the origin of gliomas, we examined the expression of the human Musashil homolog, MSI1, in human glioma tissues and in normal human adult and fetal brains. As we had seen previously in rodents, in the normal human brain, MSI1 was expressed in cells located in the ventricular and subventricular zones, in GFAP-negative glial cells, and in GFAP-positive astrocytes. In glioblastomas, MSI1 was expressed in GFAP-negative tumor cells forming foci that were clearly demarcated and surrounded by GFAP-positive cells. Tumor cells arranged in pseudopalisades were also strongly immunoreactive with MSI1 antibodies. The percentage of MSI1-labeled tumor cells increased in higher-grade astrocytomas and correlated with proliferative activity, as estimated by an MIB-1 staining index. Our results indicate that MSI1 is an excellent marker for neural progenitor cells including neural stem cells in normal human brains. Furthermore, the expression of MSI1 correlates well with the immature nature as well as the malignancy of tumor cells in human gliomas. Thus, we expect the analysis of MSI1 expression to contribute to the understanding of the cellular origin and biology of human gliomas.

Highly efficient gene transfer into murine liver achieved by intravenous administration of naked Epstein-Barr virus (EBV)-based plasmid vectors.

Naked plasmid DNA (pDNA) injection could become an alternative procedure to viral and nonviral gene delivery systems. We have previously shown that Epstein-Barr virus (EBV)-based plasmid vectors containing the EBV nuclear antigen 1 (EBNA1) gene and the oriP sequence enable quite high and long-lasting expression in various in vitro and in vivo transfection systems. The EBV-based plasmids were intravenously injected into mice via their tail vein under high pressure. A large amount of the marker gene product was expressed in the liver; as much as 320 microg of luciferase was demonstrated per gram of liver at 8 to 24 h after a single injection with 10 microg of DNA. More than 70% of liver cells stained with X-gal when beta-gal gene was transferred. The expression level was significantly higher than that obtained by conventional pDNA lacking the EBNA1 gene and oriP. On day 35 after the transfection, the expression from the EBV-based plasmid was approximately 100-fold stronger than the conventional pDNA gene expression. Both the EBNA1 gene and oriP are a prerequisite for the augmentation of the transfection efficiency. These results suggest that the intravascular transfection with naked EBV-based plasmid may provide a quite efficient, simple and convenient means to transduce therapeutic genes in vivo into the liver.

Developmental expression of a unique carbohydrate antigen, Tn antigen, in mouse central nervous tissues.

Using an anti-Tn monoclonal antibody, the Tn antigen was detected immunohistochemically in prenatal and early postnatal central nervous tissues. On embryonic day 9 (E9), the antigen was distributed throughout the single neuroepithelial layer in the neocortex and then became more prominent in the preplate than in the ventricular zone along with formation of the preplate. Following division of the preplate and concomitant formation of the cortical plate, distinct labeling of the neocortex occurred in the marginal, subplate and intermediate zones, whereas in the cortical plate and ventricular zone were virtually not immunostained. It is notable that thalamocortical afferent fibers were also immunostained specifically on E14. After birth, the localization of the antigen became less noticeable and by 3 weeks after birth, the antigen had substantially disappeared. In the developing cerebellum, prominent labeling was also observed in the molecular layer and outskirts of the cerebellar nuclei on early postnatal days. To characterize the glycoprotein bearing the Tn antigen biochemically, immunoblot analysis was performed. The glycoprotein, most of which was extracted with a salt solution, migrated as a broad smeared band corresponding to a molecular weight of about 250 kDa on SDS-PAGE. Among the various tissues examined, this glycoprotein was only detected in the brain and its amount increased until an early postnatal stage with a peak on postnatal day 3 (P3), and then decreased gradually with age. This spatially and developmentally regulated expression of the Tn antigen suggests that this antigen plays a significant role in brain development.

Important role of energy-dependent mitochondrial pathways in cultured rat cardiac myocyte apoptosis.

Recent studies have suggested that apoptosis and necrosis share common features in their signaling pathway and that apoptosis requires intracellular ATP for its mitochondrial/apoptotic protease-activating factor-1 suicide cascade. The present study was, therefore, designed to examine the role of intracellular energy levels in determining the form of cell death in cardiac myocytes. Neonatal rat cardiac myocytes were first incubated for 1 h in glucose-free medium containing oligomycin to achieve metabolic inhibition. The cells were then incubated for another 4 h in similar medium containing staurosporine and graded concentrations of glucose to manipulate intracellular ATP levels. Under ATP-depleting conditions, the cell death caused by staurosporine was primarily necrotic, as determined by creatine kinase release and nuclear staining with ethidium homodimer-1. However, under ATP-replenishing conditions, staurosporine increased the percentage of apoptotic cells, as determined by nuclear morphology and DNA fragmentation. Caspase-3 activation by staurosporine was also ATP dependent. However, loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bax translocation, and cytochrome c release were observed in both apoptotic and necrotic cells. Moreover, cyclosporin A, an inhibitor of mitochondrial permeability transition, attenuated staurosporine-induced apoptosis and necrosis through the inhibition of DeltaPsi(m) reduction, cytochrome c release, and caspase-3 activation. Our data therefore suggest that staurosporine induces cell demise through a mitochondrial death signaling pathway and that the presence of intracellular ATP favors a shift from necrosis to apoptosis through caspase activation.

Induction of apoptosis in an androgen-dependent mouse mammary carcinoma cell line by methylcobalamin.

SC-3 is a cloned cell line derived from an androgen-dependent mouse mammary tumor (Shionogi Carcinoma 115). A physiological level of androgen stimulates the growth of SC-3 cells through the production of androgen-induced growth factor. Methylcobalamin (MeCbl), one of the active cobalamins, inhibits the growth of SC-3 cells stimulated by androgen. It is known that apoptosis has an important role in tumor growth. The specific aim of this study is to examine the effects of MeCbl, in the presence of androgen, on apoptosis in SC-3 cells. Morphological analysis revealed budding nuclei and chromatin condensation in cells cultured with MeCbl, but few in cells cultured without MeCbl. Low molecular weight DNA extracted from cells cultured with or without MeCbl was analysed by gel electrophoresis. A characteristic nucleosomal size ladder was detected in the culture with MeCbl. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labelling method was also used to evaluate apoptotic cell death in SC-3 cells. Apoptosis was observed more frequently in SC-3 cells treated with MeCbl than in those without MeCbl. These results demonstrate that androgen-dependent SC-3 cells undergo apoptosis by MeCbl even if in the presence of androgen.

Localization of interferon regulatory factor-1 in human endometrium throughout the menstrual cycle.

OBJECTIVE: To determine the expression and localization of IRF-1 in human endometrium throughout the menstrual cycle. DESIGN: A comparative study. SETTING: Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine. PATIENTS: Thirty-eight women aged 33 to 46 years, with regular menstrual cycles and nonpathological endometrium, undergoing hysterectomy. INTERVENTION(S): Endometrial tissues were obtained from operative samples. MAIN OUTCOME MEASURE(S): Expression of IRF-1 mRNA throughout the menstrual cycle was investigated using semiquantitative reverse transcription-polymerase chain reaction. Localization of IRF-1 protein was determined using immunohistochemistry. RESULT(S): IRF-1 mRNA was expressed in the human endometrium at each phase of the menstrual cycle. The immunoreactivity for IRF-1 was observed in the extranuclear compartment of the surface and glandular epithelial cells, both during the proliferative and secretory phases, as well as in the gland secretion during the secretory phase. In contrast, stromal cells were nearly unstained. CONCLUSION(S): IRF-1 was localized in the human endometrium, implying that this nuclear protein plays some role other than as a transcription factor.

Identification of the core protein carrying the Tn antigen in mouse brain: specific expression on syndecan-3.

We isolated glycoproteins carrying the Tn antigen, which was expressed spatiotemporally in the developing mouse brain. The Tn antigen was expressed on two molecular species with a molecular weight from 200 to 350 kDa and 110 to 160 kDa, as judged on SDS-PAGE. Although the two glycoproteins showed different susceptibilities to heparitinase I and solubilities in a salt solution, after treatment with V8 protease they showed the same mobility corresponding to a molecular weight of 90 kDa on SDS-PAGE, suggesting that these two molecules shared a common core protein. Partial N-terminal sequences of the glycoproteins were determined, i.e. AQRXRNENFERPV and ALAAPXAPAMLP, which were identified as the sequences of the N-terminal and central portions of syndecan-3, respectively. Both glycoproteins were reactive to anti-mouse syndecan-3 antibody. These results suggest that one is a soluble syndecan-3 cleaved between mucin-like domain and transmembrane domain, and the other is a membrane-bound syndecan-3 lacking N-terminal glycosaminoglycan attachment sites, and that both glycoproteins have a mucin-like domain characteristic of syndecan-3, in which the Tn antigen may be expressed.

Nitric oxide is an excitatory modulator in the rostral ventrolateral medulla in rats.

Nitric oxide is a messenger molecule having various functions in the brain. Previous studies have reported conflicting results for the roles of nitric oxide in the rostral ventrolateral medulla, a major center that regulates sympathetic and cardiovascular activities. We hypothesized that in this region, nitric oxide may have a biphasic effect on cardiovascular activity. Microinjection of a low dose (1 nmol) of a nitric oxide donor sodium nitroprusside or a cyclic GMP agonist 8-bromocyclic GMP into this area increased arterial pressure, whereas injection of a nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester or a soluble guanylate cyclase inhibitor methylene blue decreased arterial pressure. Microinjection of a high dose (100 nmol) of sodium nitroprusside decreased arterial pressure and inhibited spontaneous respiration with concomitant production of peroxynitrite, a strong cytotoxic oxidant. Increases in arterial pressure caused by microinjection of L-glutamate were inhibited after preinjection of Nomega-nitro-L-arginine methyl ester or methylene blue. Increases in arterial pressure caused by microinjection of sodium nitroprusside (1 nmol) were inhibited after preinjection of a glutamate receptor antagonist kynurenate. These results suggest that low doses of nitric oxide may increase arterial pressure, whereas high doses of nitric oxide may decrease arterial pressure through cytotoxic effects in the rostral ventrolateral medulla. They also indicate that nitric oxide may stimulate neurons both through activation of the nitric oxide cyclic GMP pathway and through modulation of glutamate receptor stimulation, and therefore, increase arterial pressure in rats.

IL-15 expression at human endometrium and decidua.

A large number of natural killer (NK) cells appear in human uterine mucosa during the secretory phase and first trimester pregnancy. We investigated the expression of interleukin (IL)-15, a possible stimulator for these NK cells, in human endometrium and first trimester decidua. Semiquantitative reverse transcriptase-polymerase chain reaction revealed that IL-15 mRNA expression was stronger during the secretory phase and first trimester pregnancy than during the proliferative phase. Immunohistochemistry revealed that immunoreactivity for anti-IL-15 was higher during the secretory phase than it was during the proliferative phase. This was prominent in the perivascular stromal cells around invading spiral arteries during the mid- to late-secretory phase. In first trimester decidua, endothelial cells were also stained as strongly as stromal cells. A membrane-bound IL-15 molecule was detected on the surface of first trimester decidual cells by flow cytometry. Progesterone stimulated the release of soluble IL-15 in the supernatant of cultured decidual cells. These results suggest that IL-15 expression in human uterine mucosa corresponds to the fluctuation of uterine NK cells and that its production is hormonally controlled, especially by progesterone.

[Nation-wide survey on hemimegalencephaly in Japan].

We report here 38 Japanese patients with hemimegalencephaly collected by a national survey study. All the patients were sporadic. There was no familial occurrence or sex difference. Some patients had basic diseases: hypomelanosis of Ito in 3 cases and organic nevus syndromes in 8. Most patients had hemiparesis, and 11 were bed-ridden. All except for 3 patients had mental retardation, being profound in half of them. There was no correlation between the side of hemimegalencephaly and clinical symptoms. All patients had epileptic seizures, which first appeared within 24 hours after birth in 4 cases, within 7 days in 7, within a month in 2, within 6 months in 10, and within a year in 4. Antiepileptic drugs were not very effective for controlling seizures. In 7 patients, however, functional hemispherectomy resulted in seizure control and improved development. The patients whose epileptic symptom occurred earlier tended to be more severe in clinical symptoms.

Gene delivery to human chondrocytes by an adeno associated virus vector.

OBJECTIVE: To investigate the efficiency of gene transduction to human chondrocytes using an adeno associated virus (AAV) vector. METHODS: We transduced green fluorescent protein (GFP) gene using AAV vector to primary human chondrocytes as well as human cartilage organ cultures, in which chondrocytes are surrounded by extracellular matrix. Expression of GFP gene was analyzed at various time points after transduction by fluorescence microscopy and immunohistochemistry. RESULTS: In primary chondrocytes, the percentages of GFP positive cells were 15.9% or 16.0% on Day 1 and 95.0% or 93.7% on Day 7 after gene transduction. In cartilage organ cultures, gene delivery was observed in cells located not only in the superficial layer but also in the deep layer within the cartilage tissue. Up to 45.3+/-7.4% or 46.0+/-3.9% of chondrocytes expressed GFP for at least 28 days. CONCLUSION: AAV vector could be useful for direct gene delivery to chondrocytes in situ.

Age-dependent change in the levels of Abeta40 and Abeta42 in cerebrospinal fluid from control subjects, and a decrease in the ratio of Abeta42 to Abeta40 level in cerebrospinal fluid from Alzheimer's disease patients.

In order to address an age-dependent alteration in the concentration of beta-amyloid polypeptides (Abetas) within the central nervous system and its probable predisposition to amyloidgenesis in Alzheimer's disease (AD), we measured two species of soluble Abetas, Abeta40 and Abeta42, in cerebrospinal fluids (CSF) from randomly selected Japanese control subjects at various ages (n = 33) and then compared these data with those of probable Japanese AD patients (n = 23). CSF concentrations of Abeta40 and Abeta42 peptides were age-dependent (ANOVA, Bonferroni's multiple comparison; p < 0.01 and p < 0.05, respectively) and were lower in the infant than in adults. From mid-20, the Abeta40 concentrations were decreasing while Abeta42 were rather stable. Abetas in CSF from AD patients (n = 23), whose epsilon4 allele frequency of the apolipoprotein E gene was higher than in controls (n = 83, p < 0.03), were not statistically different from those of age-matched controls (n = 13). A linear relationship was detected between the Abeta40 concentration and the Mini-Mental State Examination score (p < 0.05). The ratio of the Abeta42 to the Abeta40 level measured in the AD CSF samples was approximately 38% decreased compared to age-matched controls (p < 0. 05). These data suggest that the physiological metabolism of soluble Abetas in the brain is regulated in an age-dependent manner, and that the ratio of Abeta42 to Abeta40 level in the CSF would be a useful marker for monitoring progression of AD.

Direct intra-cardiomuscular transfer of beta2-adrenergic receptor gene augments cardiac output in cardiomyopathic hamsters.

In chronic heart failure, down-regulation of beta-adrenergic receptor (beta-AR) occurs in cardiomyocytes, resulting in low catecholamine response and impaired cardiac function. To correct the irregularity in the beta-AR system, beta-AR gene was transduced in vivo into failing cardiomyocytes. The Epstein-Barr virus (EBV)-based plasmid vector carrying human beta2-AR gene was injected into the left ventricular muscle of Bio14.6 cardiomyopathic hamsters whose beta-AR is down-regulated in the cardiomyocytes. The echocardiographic examinations revealed that stroke volume (SV) and cardiac output (CO) were significantly elevated at 2 to 4 days after the beta2-AR gene transfer. Systemic loading of isoproterenol increased the cardiac parameters more significantly on day 2 to day 7, indicating that the adrenergic response was augmented by the genetic transduction. The same procedure did not affect the cardiac function of normal hamsters. Immunohistochemical examinations demonstrated human beta2-AR expression in failing cardiomyocytes transduced with the gene. RT-PCR analysis detected mRNA for the transgene in the heart but not in the liver, spleen, or kidney. The procedures may provide a feasible strategy for gene therapy of severe heart failure. Gene Therapy (2000) 7, 2087-2093.

Detection of aromatase cytochrome P-450 in endometrial biopsy specimens as a diagnostic test for endometriosis.

OBJECTIVE: To evaluate the clinical usefulness of examining endometrial biopsy specimens for aromatase cytochrome P-450 as a diagnostic test for endometriosis. DESIGN: Retrospective, case-controlled study. SETTING: Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, Kyoto, Japan. PATIENT(S): One hundred five women of reproductive age with normal menstrual cycles underwent endometrial biopsy laparotomy or laparoscopy, and examination of their tissue revealed endometriosis, adenomyosis, and/or leiomyomas. Patients who had cervical carcinoma in situ but no other gynecologic disease were considered to be disease-free. INTERVENTION(S): Endometrial biopsy specimens were collected. MAIN OUTCOME MEASURE(S): The expression of aromatase cytochrome P-450 was examined by reverse transcription-polymerase chain reaction and immunohistochemical analysis. The distribution and intensity of the immunostaining was assessed using a semiquantitative index designed H-score. RESULT(S): Immunostaining for aromatase cytochrome P-450 was detected in biopsy specimens obtained from patients with endometriosis, adenomyosis, and/or leiomyomas but not in specimens obtained from disease-free patients (H-score <20), with a sensitivity and specificity of 91% and 100%, respectively. CONCLUSION(S): The expression of aromatase cytochrome P-450 in biopsy specimens of eutopic endometrium distinguishes between disease-free women and women with endometriosis, adenomyosis, and/or leiomyomas. This technique can be used at outpatient infertility clinics as an initial screening procedure to rule out the presence of estrogen-dependent disease.

Mutation of the sterol 27-hydroxylase gene (CYP27) results in truncation of mRNA expressed in leucocytes in a Japanese family with cerebrotendinous xanthomatosis.

OBJECTIVES: A Japanese family with cerebrotendinous xanthomatosis (CTX) was investigated for a sequence alteration in the sterol 27-hydroxylase gene (CYP27). The expression of CYP27 has been mostly explored using cultured fibroblasts, prompting the examination of the transcripts from blood leucocytes as a simple and rapid technique. METHODS: An alteration in CYP27 of the proband was searched for by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and subsequent sequencing. Samples of RNA were subjected to reverse transcription PCR (RT-PCR) and the product of the proband was amplified with nested primers and sequenced. RESULTS: A homozygous G to A transition at the 5' end of intron 7 was detected in the patient. In RT-PCR analysis, only a truncated transcript was detected in the patient, whereas both normal and truncated transcripts were detected in the siblings. The sequencing of the patient's cDNA fragment disclosed a direct conjuction of exon 6 and exon 8. CONCLUSION: The mutation at splice donor site and the truncation of mRNA were identical with those of a recently reported Italian patient, although different in symptomatology. The application of blood leucocytes can be a simple technique on analysing a constructive abnormality of CYP27 mRNA.

Isolation and expression analysis of a novel human homologue of the Drosophila glial cells missing (gcm) gene.

A novel human homologue (GCMB) of the Drosophila glial cells missing gene (dGCM) was isolated using RACE. GCMB contained a gcm motif sequence and a nuclear targeting sequence similar to that of dGCM and mouse GCMb. Homology searches indicated that GCMB was located within chromosome 6p24.2. Transcripts of GCMB were detected by means of RT-PCR in fetal brain, normal adult kidney, 3/3 medulloblastomas, 1/3 gliomas and 4/8 non-neuroepithelial tumor cell lines. Our data suggest that humans have two homologues of gcm like mice and that human gcm genes form a novel family which may function not only during fetal development but also in the postnatal or pathological stage.