metallothionein and Astrocytoma

Gliomas are heterogeneous, primary brain tumours that originate from glial cells. The main type of gliomas is astrocytomas. There are four grades (I-IV) of astrocytoma malignancy. Astrocytoma grade IV known as glioblastoma multiforme (GBM) is the most common and aggressive type of astrocytic gliomas. Metallothioneins (MT) are low molecular weight, cysteine rich proteins encoded by a family of metallothionein (MT) genes. MT genes play a crucial role in carcinogenesis of diverse malignancies. We proposed MT genes as prognostic markers for malignant astrocytoma. MT1A, MT1E, MT1X, MT2, MT3 gene expression was elevated in grade IV astrocytomas (glioblastomas) as compared to astrocytomas grade I-III. Statistically significant differences were reached for MT1A and MT2 genes (Mann-Whitney test, p < 0.05). High MT1A, MT1X, MT2, MT3 genes expression was associated with shorter patient survival (Log-rank test, p < 0.05). MT1A gene promoter methylation was decreased in glioblastoma (57.6%) while the gene was highly methylated in grade II-III astrocytoma (from 66.7% to 83.3%) and associated with better patient survival (p < 0.05). MT1A gene methylation showed a trend of being associated with higher mRNA expression level in astrocytomas. Increased MT genes expression in grade IV astrocytomas as compared to I-III grade astrocytomas could be associated with malignant tumour behaviour and progression.

Topics: Astrocytoma; Brain Neoplasms; Disease Progression; DNA Methylation; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Metallothionein; Neoplasm Grading; Promoter Regions, Genetic; RNA, Messenger

Astroglia cells structurally and nutritionally support neurons in the central nervous system. They play an important role in guiding the construction of the nervous system and controlling the chemical and ionic environment of neurons. They also represent the major sites for accumulation and immobilisation of toxic metal ions most probably connected with metallothioneins. For this reason astroglia cells possess high cytosolic levels of metallothioneins I, II and III (MT-I,II,III). Our aim was to establish the inducibility and metal binding of MTs in two human astrocytoma cell lines, U87 MG (astrocytoma-glioblastoma, grade IV) and IPDDC-2A (astrocytoma, grade II), on exposure to cadmium chloride (1 microM). MTs were identified by molecular weight (size exclusion chromatography) and their metal content (Cd, Zn and Cu) to follow the interactions between metals. We showed that MTs are constitutively expressed in both human astrocytoma cell lines. In accordance with the higher malignancy grade of U87 MG, the amount of MTs was higher in U87 MG than in IPDDC-2A cells. After 24 hours of exposure to Cd their expression greatly increased in both cell lines and they were capable of immobilising almost all water soluble Cd. Induction of MTs in U87 MG cells was additionally followed up to 48 hours with exposure to different concentrations of CdCl(2) (1, 10 microM). Induction was a time dependent process throughout the period. Isoform III (identified by chromatographic separation of isoform III from I/II) was present at all exposure times, but only in traces with respect to the prevailing amounts of MT-I/II isoforms. So induction can be attributed to isoform I/II only.

Topics: Astrocytoma; Cadmium Chloride; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Humans; Metallothionein; Metals; Protein Binding

Metallothioneins (MT) are common proteins in animal tissues. These proteins take part in the homeostasis of the ions of the metals which are necessary for the proper metabolism of the organism (zinc, copper), biosynthesis regulation and zincprotein activity (for example the activity of the zinc-dependant transcription factors) and they also take part in the detoxication of the tissue from toxic metals. Apart from these, they also protect the tissue from reactive oxygen species, radiation, electrophilic pharmacological agents used in the cancer therapy and the mutagens. The aim of this work was to obtain cellular sub-fractions of brain tumors and to separate these proteins by SDS-polyacrylamide gel electrophoresis and Western Blotting technique and to determine the level of metallothioneins and to determine the level of metallthioneins in the cellular sub-fraction. The experimental materials were the brain neoplastic tissues resected during neurosurgical procedures. The brain tumors were divided into two groups; astrocytoma G-2 and malignant gliomas (astrocytoma GM-4, glioblastoma multiforme). The cellular fractions of tumour tissues were obtained according to Clark and Nicklas and Chauveau et al. methods. The level of the metallothioneins was determined by the cadmium-hemoglobin affinity assay using the cadmium isotope (109Cd). By performing protein dissection on polyacrylamide gel (PAGE) and applying immunoidentifying technique we proved the presence of metallothioneins in all the cell sub-fractions. In GM-4 patients the total MT level increased by 12.06% whereas in the cytosol sub-fraction it increased by 17.02% in comparison with astrocytoma G-2 patients. In the mitochondrial sub-fraction the level increased by 49.09% while in the nuclear and microsomal sub-fractions the increase was by 22.38 and 8.24%, respectively.

Topics: Astrocytoma; Blotting, Western; Brain Neoplasms; Electrophoresis, Polyacrylamide Gel; Glioma; Humans; Metallothionein; Subcellular Fractions

Metallothioneins take part in the homeostasis of the ions of the metals which are necessary for the proper metabolism of the organism (zinc, copper), biosynthesis regulation of the zinc containing proteins. They also take part in the detoxication of metals from the tissues. Besides,they protect the tissue from the effects of free radicals, radiation, electrophilic pharmacologic agents used in the cancer therapy and from mutagens. The experimental materials were brain astrocytomas, benign gliomas (astrocytoma G-2) and malignant gliomas (glioblastoma multiforme GM-4). The levels of the metallothionein were determined by cadmium-hemoglobin affinity assay using the cadmium isotope (109Cd). The value of zinc and copper were determined by means of atomic absorption spectrophotometry. In our studies, the level of metallothioneins in the group of malignant neoplasms was slightly higher than the level of these proteins in the group of benign neoplasms. There was a statistical difference, but there is no significant difference in the levels of copper between malignant and benign groups.

Topics: Astrocytoma; Brain Neoplasms; Copper; Culture Techniques; Female; Glioblastoma; Humans; Male; Metallothionein; Spectrophotometry, Atomic; Zinc

Metallothionein-I (MT-I) was expressed in neonatal rat primary astrocyte cultures and an astrocytoma cell line by pGFAP-MT-I plasmid transfection under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. Following transient transfection of the pGFAP-MT-I plasmid, MT-I mRNA and MT-I protein levels were determined by northern blot and immunoprecipitation analyses, respectively. The ability of cells over-expressing MT-I to withstand acute methylmercury (MeHg) treatment was measured by the release of preloaded Na251CrO4, an indicator of membrane integrity. Transfection with the pGFAP-MT-I plasmid led to increased mRNA (2. 5-fold in astrocytes and 7.4-fold in astrocytomas) and MT-I protein (2.4-fold in astrocytes and 4.0-fold in astrocytomas) levels compared with their respective controls. Increased expression of MT-I was associated with attenuated release of Na251CrO4 upon MeHg (5 microM) treatment. These results demonstrate that MT-I can be highly expressed both in primary astrocyte cultures and astrocytomas by pGFAP-MT-I plasmid transfection, and lend credence to the hypothesis that increased expression of MT-I affords protection against the cytotoxic effects of MeHg. Taken together, the data suggest that MT offer effective cellular adaptation to MeHg cytotoxicity.

Topics: Animals; Animals, Newborn; Astrocytes; Astrocytoma; Cell Survival; Cells, Cultured; Drug Resistance; Drug Resistance, Neoplasm; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Glial Fibrillary Acidic Protein; Metallothionein; Methylmercury Compounds; Promoter Regions, Genetic; Rats; Rats, Sprague-Dawley; Transfection

Metallothionein-I (MT-I) was expressed in neonatal rat primary astrocyte cultures and an astrocytoma cell line by pGFAP-MT-I plasmid transfection under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. Following transient transfection of the pGFAP-MT-I plasmid, MT-I mRNA and MT-I protein levels were determined by Northern blot and immunoprecipitation analyses, respectively. The ability of cells overexpressing MT-I to withstand acute methylmercury (MeHg) treatment was measured by the release of preloaded Na2(51)CrO4, an indicator of membrane integrity. Transfection with the pGFAP-MT-I plasmid led to increased mRNA (2.5-fold in astrocytes and 7.4-fold in astrocytomas) and MT-I protein (2.4-fold in astrocytes and 4.0-fold in astrocytomas) levels compared with their respective controls. Increased expression of MT-I was associated with attenuated release of Na2(51)CrO4 upon MeHg (5 microM) treatment. These results demonstrate that MT-I can be highly expressed both in primary astrocyte cultures and astrocytomas by pGFAP-MT-I plasmid transfection, and lend credence to the hypothesis that increased expression of MT-I affords protection against the cytotoxic effects of MeHg. Taken together, the data suggest that MTs offer effective cellular adaptation of MeHg cytotoxicity.

Topics: Animals; Animals, Newborn; Astrocytes; Astrocytoma; Cells, Cultured; Glial Fibrillary Acidic Protein; Metallothionein; Methylmercury Compounds; Plasmids; Rats; Transfection

Metallothionein (MT) is the name for a family of predominantly intracellular protein thiol compounds involved in anticancer drug resistance. For certain tumors, MT is related to grade of tumor malignancy and prognosis. The authors evaluated the expression of MT in 114 astrocytic tumors in relation to the proliferative potential of tumors and the survival of patients.. Paraffin embedded tissue sections were stained with monoclonal anti-metallothionein and MIB-1 Ki-67 antibodies by avidin-biotin complex immunohistochemistry.. MT expression was observed in 2 of 6 pilocytic astrocytomas, in 10 of 24 Grade 2 astrocytomas, in 16 of 25 anaplastic astrocytomas, and in 47 of 59 glioblastomas. In addition to the tumor cells, microvascular endothelial proliferation and smooth muscle of tumor vessel walls were frequently MT positive. The glioblastomas had a significantly higher percentage of MT positive cells compared with low grade (P < 0.0001) and anaplastic (P < 0.04) astrocytomas. MT expression in astrocytomas had no correlation with tumor recurrence. The mean Ki-67 labeling index (LI) was significantly higher in the high grade (3-4) compared with the low grade (1-2) astrocytomas. MT positive astrocytic tumors had statistically significantly higher mean Ki-67 LI compared with MT negative tumors, irrespective of histologic grade. Although the levels of MT and Ki-67 LI varied in individual tumors, the mean Ki-67 LI increased in parallel to the increasing MT staining grade, and this difference attained statistical significance only for glioblastoma. MT positive anaplastic astrocytoma and glioblastoma patients did not survive as long as the MT negative patients, although this difference attained statistical significance only for anaplastic astrocytoma.. The current study suggests that MT might play a significant role in the growth of astrocytic tumors, with an acquired enhanced ability to produce MT as the malignant potential of a tumor increases.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Astrocytoma; Cell Division; Child; Child, Preschool; Female; Glioblastoma; Humans; Immunohistochemistry; Infant; Ki-67 Antigen; Male; Metallothionein; Middle Aged; Survival Rate

The effects of cytokines and heavy metals on the expression and localization of metallothioneins (MTs) within U373MG astrocytoma cells were analyzed by using indirect immunofluorescence using a monoclonal anti-MT antibody (MT45). IL-1, CdCl2 (50 microM) or ZnCl2 (500 microM) remarkably augmented intracellular MT levels, whereas IL-6 or 10 microM of ZnCl2 showed no inducing activity. From 24 to 48 h after the addition of CdCl2 or IL-1, immunoreactive MTs were found in the cytoplasm and the nucleus. After 72 h, immunoreactive MTs accumulated in a granular form near the cell surfaces in the presence of CdCl2 (50 microM) or IL-1 plus ZnCl2 (10 microM). However, this accumulation was not observed when only IL-1 was added. Thus, Zn2+ facilitated the appearance of the granular form of immunoreactive MTs at a concentration where they do not induce MTs by themselves.

Topics: Astrocytoma; Cadmium; Cadmium Chloride; Chlorides; Humans; Interleukin-1; Metallothionein; Metals; RNA, Messenger; Tumor Cells, Cultured; Zinc; Zinc Compounds