The tissue and cellular distribution of the DNA repair
protein O6-alkylguanine-DNA-alkyltransferase (ATase) is an important question in relation to the response of tumour and normal tissues to chemotherapeutic regimes employing
alkylating agents such as methyltriazenes and nitrosoureas. In order to examine this issue by immunostaining, we have raised a rabbit antiserum to apparently pure recombinant human
enzyme. The antiserum is highly specific and sensitive, detecting a band at 24 kDa on western blots of
crude extracts of ATase-expressing human lymphoblastoid cells, liver and
melanoma. Adjacent sections of
acetone or
formalin fixed normal human liver and subcutaneous
malignant melanoma were reacted with preimmune serum or antiserum and an immunoperoxidase detection system with
silver enhancement was used to locate binding of the primary antibody to the
antigen. In sections reacted with preimmune serum or with
antigen-preadsorbed antiserum, only faint cytoplasmic and little or no nuclear staining was seen. In contrast, using antiserum, the reaction in positively staining cells was very intense and predominantly nuclear. In the liver, there was interindividual variation in the cellular distribution of reaction with staining present in all discernable cell types in most samples but confined to the hepatocytes and bile duct epithelial cells in others. In the
melanoma sections, all discernable cell types showed mainly nuclear staining: the intensity of staining varied between tissue samples and there was evidence of a range of intermediate staining intensities with some
melanoma cells showing no detectable reaction.