metallothionein and Medulloblastoma

Tumour markers are substances produced by malignant cells or by the organism as a response to cancer development. Determination of their levels can, therefore, be used to monitor the risk, presence and prognosis of a cancer disease or to monitor the therapeutic response or early detection of residual disease. Time-consuming imaging methods, examination of cerebrospinal fluid or tumour tissue and assays for hormones and tumour markers have been used for cancer diagnosis. However, no specific marker for diagnosis of childhood solid tumours has been discovered yet. In this study, metallothionein (MT) was evaluated as a prospective marker for such diseases. Serum metallothionein levels of patients with childhood solid tumours were determined using differential pulse voltammetry - Brdicka reaction. A more than 5-fold increase in the amount of metallothionein was found in sera of patients suffering from cancer disease, compared with those in sera of healthy donors. The average metallothionein level in the sera of healthy volunteers was 0.5 ± 0.2 μmol · dm⁻³ and was significantly different (p<0.05, determined using the Schefe test) from the average MT level found in serum samples of patients suffering from childhood solid tumours (3.4 ± 0.8 μmol · dm⁻³). Results found in this work indicate that the MT level in blood serum can be considered as a promising marker for diagnostics, prognosis and estimation of therapy efficiency of childhood tumours.

Topics: Biomarkers, Tumor; Blotting, Western; Child; Electrochemistry; Humans; Medulloblastoma; Metallothionein; Neoplasms; Neuroblastoma; Osteosarcoma; Risk Factors; Sarcoma, Ewing

Resistance of neoplastic cells to the alkylating drug BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea] has been correlated with expression of O (6)-methylguanine-DNA methyltransferase, which repairs the O (6)-chloroethylguanine produced by the drug. Other possible mechanisms of resistance include raised levels of glutathione or increased repair of the DNA interstrand cross-links formed by BCNU. Transcriptional profiling revealed the upregulation of several metallothionein (MT) genes in a BCNU-resistant medulloblastoma cell line [D341 MED (OBR)] relative to its parental line. Previous studies have shown that MTs, through their reactive thiol groups can quench nitrogen mustard-derived alkylating drugs. In this report, we evaluate whether MTs can also quench BCNU.. To demonstrate the binding of BCNU to MT, we used an assay that measured the release of the MT-bound divalent cations (Zn(2+), Cd(2+)) upon their displacement by the drug. We also measured the decomposition rates of BCNU at those reaction conditions.. The rate of release of the cations was higher in pH 7.4 than at pH 7.0, which is likely a result of more rapid decomposition of BCNU (thus faster release of MT-binding intermediate) at pH 7.4 than at pH 7.0.. We demonstrate that resistance to BCNU may be a result of elevated levels of MTs which act by sequestering the drug's decomposition product(s).

Topics: Carmustine; Cerebellar Neoplasms; Drug Resistance, Neoplasm; Humans; Medulloblastoma; Metallothionein; Tumor Cells, Cultured

Metallothionein (MT) is a low-molecular mass protein playing an essential role in homeostasis of heavy metal ions. Its relation with formation and progression of a tumour disease is discussed in this article. Here, we propose a new methodological approach for visualization of MT on PVDF membranes after dot- and electroblotting by using a commercial mouse monoclonal antibody E9 and polyclonal chicken antibodies. The optimized procedure was as follows. We dotted 1 microL sample volume on PVDF membrane and let it to dry. Then, we blocked the membrane surface with 2% BSA in PBS for 30 min. After that, the membrane was incubated in chicken primary antibody (diluted 1:500), washed, and incubated in rabbit-anti-chicken secondary antibody conjugated with horseradish peroxidase. To visualize the interaction, we used 3-aminoethyl-9-carbazole. Under these conditions, we estimated detection limit as 3 pg of MT per 1 microL. The optimal approach was further utilized for detection of MT level in two human fibroblast cell lines and in blood serum obtained from children with medulloblastoma. The results were in good agreement with differential pulse voltammetry-Brdicka reaction.

Topics: Animals; Antibodies; Blotting, Western; Cadmium; Cell Line, Transformed; Cell Line, Tumor; Chickens; Child; Collodion; Fibroblasts; Humans; Immunoblotting; Medulloblastoma; Metallothionein; Polyvinyls