oxalates and Melanoma

The potent cytotoxicity of reactive oxygen species (ROS) can cause various diseases, however, it may also serve as a powerful chemotherapeutic strategy capable of killing cancer cells. Oxalomalate (OMA, α-hydroxy-β-oxalosuccinic acid), a tricarboxylic acid intermediate, is a well-known competitive inhibitor of two classes of NADP

Topics: Angiogenesis Inhibitors; Animals; Apoptosis; Ataxia Telangiectasia Mutated Proteins; Cell Line, Tumor; Checkpoint Kinase 2; Isocitrate Dehydrogenase; Male; Melanoma; Mice; Neovascularization, Pathologic; Oxalates; Reactive Oxygen Species; Skin Neoplasms; Tumor Suppressor Protein p53

The accumulation of excessive amounts of melanin in melanocytic lesions can obscure cellular morphology and can further hinder immunocytochemical procedures. We have used a modification of the potassium permanganate/oxalic acid melanin-bleaching technique, involving much reduced bleaching times, in order to remove melanin granules prior to incubation with primary antibody. We have assessed a panel of antibodies applicable to the evaluation of melanocytic lesions and in addition have also assessed antibodies that may be more useful in research. The study attempts to determine which antigens may be affected by bleaching and which are not. Antigens S100, HMB 45, NKIC3, CD34, and L26 are relatively unaffected by this procedure. Factor-VIII-related antigen and vimentin and CD68 antigens produced enhanced staining. In contrast, antigens CD3, CD31, and CD45RO were abolished. In addition, smooth muscle actin and desmin antigens demonstrated considerable nonspecific background staining and were not reliable in this study. This technique demonstrates that a fairly wide range of antigens are preserved after bleaching and that distinction between melanocytes and melanophages can reliably be performed using the conventional immunocytochemical chromogen 3,3-diaminobenzidine and without the need for elaborate counterstaining.

Topics: Actins; Antigens, CD; Antigens, CD20; Antigens, CD34; Antigens, Differentiation, Myelomonocytic; Antigens, Neoplasm; CD3 Complex; Desmin; Humans; Immunohistochemistry; Leukocyte Common Antigens; Melanins; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; Oxalates; Oxidation-Reduction; Pigmentation; Platelet Endothelial Cell Adhesion Molecule-1; Potassium Permanganate; Reproducibility of Results; S100 Proteins; Sensitivity and Specificity; Skin; Skin Neoplasms; Vimentin; von Willebrand Factor

This study addresses two questions: i) which antigens can withstand bleaching by 2.5 g/L of potassium permanganate followed by 10 g/L of oxalate, before immunohistochemical staining; and ii) are any other steps in the immunohistochemical staining technique resistant to bleaching? A panel of 10 antigens was stained immunohistochemically and the results compared with staining performed with a bleaching step interpolated at different steps in the procedures. Four antigens (HMB-45, S-100, factor VIII-related antigen and collagen type IV) were unaffected by bleaching; two antigens (CD-20 and CD-45) had their staining enhanced by bleaching; one had the staining reduced (hsp27); and in three it was abolished (CD-45Ro, CD-31 and Ulex/anti-ulex antibody) by bleaching. Two antibodies (UCHL-1 and L-26) showed evidence for altered specificity following bleaching. None of the steps after application of the primary antibody was resistant to bleaching. Three chromagens used for peroxidase demonstration-amino ethyl-carbazole, diaminobenzidine and chloro-naphthol-were also found to be sensitive to bleaching. While some antigens were resistant to the effects of bleaching, some were not, and no other step in the immunohistochemical procedure could withstand bleaching.

Topics: Antigens, Neoplasm; Coloring Agents; Female; Humans; Immunoenzyme Techniques; In Vitro Techniques; Male; Melanins; Melanoma; Oxalates; Oxalic Acid; Potassium Permanganate; Uveal Neoplasms

With the increasing use of immunoperoxidase technics, it may be difficult to differentiate between the dark staining of 3,3'-diaminobenzidine (DAB) compound reaction product and melanin pigment. The latter may be particularly observed in skin. Samples of both normal skin and melanotic malignant melanoma were treated for the removal of melanin by standard technics both before and after a peroxidase-antiperoxidase (PAP) immunohistologic sequence. Many methods for the removal of melanin pigment resulted in diminished DAB staining intensity. Some also caused cellular disruption. However, the method of choice was found to be treatment with 0.25 g/dL potassium permanganate and 1 g/dL oxalic acid before the immunoperoxidase sequence.

Topics: 3,3'-Diaminobenzidine; Humans; Immunoenzyme Techniques; Melanins; Melanoma; Oxalates; Oxalic Acid; Potassium Permanganate; Skin Neoplasms

Topics: Animals; Autoradiography; Bicarbonates; Calcium; Carbon Isotopes; Carcinoma, Ehrlich Tumor; Chemical Phenomena; Chemical Precipitation; Chemistry; Chromium; Citrates; Citric Acid Cycle; Glucose; Glycolysis; Ions; Ketoglutaric Acids; Leukemia, Experimental; Lymphoma, Non-Hodgkin; Melanoma; Mice; Neoplasms, Experimental; Oxalates; Solubility