Protection of Ewing's sarcoma family tumor (ESFT) cell line SK-N-MC from betulinic acid induced apoptosis by alpha-DL-tocopherol.

Betulinic acid (BA) is known to induce apoptosis in melanoma neuroectodermal and malignant brain cancer cell lines. Present report describes the role of antioxidants on the BA-induced toxicity to human cell line SK-N-MC. Hydrophilic antioxidants viz., L-ascorbic acid (VitC) and N-acetyl-L-cysteine (l-NAC) had no protective effect on BA-induced apoptosis at the maximal concentrations tested. The lipophilic antioxidant, alpha-DL-tocopherol (VitE) showed a concentration and a time dependent effect on the protection of SK-N-MC cells from BA-induced apoptosis. The apoptotic parameters were analyzed using FACS analysis of propidium iodide (PI) stained nuclei, PS externalization using Annexin-V assay and changes in mitochondrial membrane potential. Generation of superoxide radical was monitored by the fluorescent dye hydroethidium (HE). Cells showed Annexin-V positivity and an increase in the propidium iodide (PI) uptake in the early hours of treatment with BA, which was concomitant with the loss of mitochondrial membrane potential. Addition of alpha-DL-tocopherol to the cell cultures 1-h prior to the treatment with BA abolished all the effects of BA-induced apoptosis. These observations suggest that BA initiates events at membrane level leading to induction of apoptosis. The observed ineffectiveness of hydrophilic antioxidants and substantial protection by lipophilic antioxidants indicate involvement of membrane-associated damages that form the basis of BA-induced cytotoxicity.

Electroporation: a novel approach to enhance the radioiodine uptake in a human thyroid cancer cell line.

Radioiodine has been the best choice of treatment for differentiated thyroid cancer. Loss of the ability to concentrate iodide makes thyroid cancer cells refractory to radioiodine therapy. Therefore, the methods that enhance the uptake of radioiodine may have significant therapeutic effect. Electroporation involves the application of short high-voltage electric pulses which transiently permeabilize the plasma membrane, allowing entry of the otherwise impermeable molecules. The aim of our study was to use electroporation for incorporating radioiodine into a non-iodine concentrating thyroid cell line. Cultured WRO thyroid cancer cells that do not incorporate iodine due to the lack of the specific transporter protein incorporated significant amounts of radioiodine after electroporation. The uptake of radioiodine by electroporation showed dependence on the electric field, external concentration of the iodine, time and the temperature of incubation. The incorporated radioiodine was retained over a period of 24 h. The retainability of the incorporated iodine may have a significant effect on the tumoricidal properties if validated in vivo.

Production & evaluation of a monoclonal antibody against human myeloperoxidase.

We describe the production of a mouse monoclonal antibody (H2E1) against human myeloperoxidase antigen. After production and characterisation, this antibody was compared with commercially available monoclonal antibodies, cytochemical myeloperoxidase and previously produced polyclonal antibody. Reaction with various cell lines proved that this monoclonal antibody was specific for myeloid lineage. This monoclonal showed positivity in 81.8 per cent of acute myeloid leukaemias whereas the polyclonal antibody was 100 per cent positive. We found that the polyclonal antibody was more sensitive as compared to the monoclonal. This is probably due to the lack of recognition of individual epitopes on the antigen. We recommend the use of antibodies which have different epitope recognition as most specific for myeloperoxidase.

Measurement of P-glycoprotein expression in multidrug-resistant human neuroblastoma cell lines using self-competitive binding assay.

To date, all reported measurements of multidrug resistance have been semiquantitative. The purpose of the present study is to establish and validate the self-competitive binding assay technique utilizing monoclonal antibody for quantitative estimation of multidrug resistance in tumor cells. This technique is used for P-glycoprotein concentration measurement in BE(2)-C human neuroblastoma cell line and its sublines with primary resistance to colchicine and actinomycin D. Monoclonal antibody MRK-16 was used in this study. It was labeled with iodine-125 (125I) to trace the concentration of antibody-antigen complexes. The binding data were obtained by varying the concentration of the unlabeled antibody. The results were fitted to a model equation to estimate the number of binding sites and antibody-antigen dissociation constant. The P-glycoprotein concentration was significantly higher in the resistant sublines than in the sensitive line. The highest levels were achieved in actinomycin D-resistant cells: 2.1 x 10(6) binding sites/cell versus 5.4 x 10(4) binding sites/cell in the sensitive cells. The consistency of the results was verified by repeating the study three times for each cell line. The binding assay results were confirmed by Western blot experiments performed on the same cell lines.

Monoclonal antibodies to human thyroglobulin: evaluation of immunoreactivity.

We have earlier reported production and characterization of monoclonal antibodies (MAbs) to human thyroglobulin (h-tg). In the present study H10 I MAb was evaluated for its immunoreactivity towards different forms of tg and various human thyroid tumours. The specificity of H10 I MAb was validated by the absence of cross reaction with tri-iodothyronine (T3) Thyroxine (T4) and human gamma globulins. Sodium-dodicyl-sulphate polyacrylamide gel electrophoresed (SDS-PAGE) immunoblot of h-tg on the nitrocellulose membrane revealed multiple immunoreactive bands on reaction with polyclonal antibody (PAb) in comparison with total lack of reactivity with H10 I MAb. The absence of immunoreactivity of H10 I MAb was demonstrated with SDS treated, Dithiothreitol (DT) treated and heat denatured tg using dot immunobinding technique. However, the H10 I MAb was able to react with tg treated with unfolding agents such as urea and guanidine hydrochloride. All the treated forms of tg were equally recognized by PAb. The immunoreactivity of the oxidized/reduced tg towards H10 I MAb was markedly reduced (60.0%) as compared to that obtained with native tg. It appears that H10 I MAb is directed towards conformational epitope involving sulphydryl bonds. Immunohistochemically, a comparable immunoreactivity between PAb and MAb was observed with normal thyroid tissues, follicular thyroid tissues, Hurthle cell carcinoma tissues and poorly differentiated thyroid tumor tissues using immunoperoxidase staining. The sections from papillary carcinoma tissue (thyroid as well as metastatic lymph node) exhibited intense immunoreactivity with PAb. Thyroglobulin present on these sections was not recognized by H10 I MAb. Nonetheless, H10 I MAb was able to detect tg in follicular differentiation wherever present. The absence of immunoreactivity of H10 I MAb in papillary carcinoma strongly suggests that this neoplasm produces tg which is antigenically different from the protein present in the normal tissue. The reactivity of H10 I MAb with metastatic lymph node of an unknown primary origin suggests its usefulness in the identification of prevalent metastasis of differentiated thyroid carcinoma other than papillary type.

TSH receptors in human thyroid tumours.

To evaluate the binding of human TSH (h-TSH) to various human thyroid tumours using radio receptor assay technique, 26 thyroid tumour specimens were examined. Five specimens did not show displacement by stable h-TSH. A wide variation was observed in B0, non specific binding, affinity and capacity of TSH in all the tumours examined. The Scatchard analysis of the binding of h-TSH to thyroid membranes suggested the presence of the receptors in 57.7 per cent (15 of 26, Ka much greater than 10(9)) and more than one component in 46 per cent (12 of 26) of the tumours studied. There was no consistent pattern of the binding of TSH for thyroid tissue with respect to its pathology. However, with 35 pairs of observations log affinity appeared to be linearly related to log capacity with a slope -0.95, intercept 9.96 and r value -0.93.

Monoclonal antibodies to human thyroglobulin: production and characterization.

Human thyroglobulin was used as an antigen for the development of monoclonal antibodies by the hybridoma technique. Spleen cells of BALB/c mice immunized with human thyroglobulin were fused with SP2/0 mouse myeloma cells. Seven clones secreting specific monoclonal antibodies to thyroglobulin were established. Two of these monoclonal antibodies have been purified and characterized. Their equilibrium association constants (Ka) as determined by Scatchard analysis were 0.24 X 10(11) L/M and 1.4 X 10(11) L/M respectively. The specificity of both these antibodies was validated by immunohistochemical staining of human tissues (normal human thyroid, brain, salivary gland, skeletal and smooth muscle, mucous membrane, parathyroid, adrenal) obtained from autopsy material. Only follicles and follicular cells of thyroid tissue were stained by both the monoclonal antibodies. H10 I monoclonal antibody was used for constructing a standard curve for in vitro immunoassay using a solid phase ELISA technique. The minimum amount detectable was 7.8 ng/ml. Thirty six sera from patients of various thyroid disorders were evaluated using ELISA and compared with conventional RIA. A good agreement was seen (r = 0.92) between the two techniques. These specific monoclonal antibodies may prove to be valuable for in vitro immunoassays and in vivo immunoscintigraphy.

Modifications in biphasic liquid-scintillation vial system for radiometry.

Several modifications of the biphasic liquid-scintillation vial system for radiometry have been tried in order to improve the counting efficiency. The biphasic system consisted of an inner sterile vial containing medium and substrate, and an outer liquid-scintillation vial lined on the inside with filter paper impregnated with scintillation fluors and alkali. The system gave an overall counting efficiency of 14.6%. Substitution of methanolic NaOH for impregnation of the paper raised the counting efficiency to 29.1%. This could be further enhanced to 33.8% by lining only half of the outer vial with filter paper, thereby allowing improved optical transmission of scintillation light. Increasing the amount of fluor did not change the efficiency significantly. A complete interchange in the system, whereby half of the inner vial was lined with filter paper and was otherwise empty, while the outer vial contained the medium and substrate, gave the highest efficiency (36.9%). This also allowed the use of larger amounts of medium and the inoculum.