Immunocytochemical demonstration of the binding and internalization of growth hormone in GERL of Chang hepatoma cells

Abstract

The binding and internalization of endogenous growth hormone in Chang hepatoma cells were localized on the cell surface and in the Golgi-endoplasmic reticulum-lysosome (GERL) area by various indirect immunocytochemical labeling techniques, namely, peroxidase or colloidal gold conjugated to secondary antibody, and avidin-biotin complex methods. Rabbit antiserum and monoclonal antibodies raised against HPLC-purified porcine growth hormone were used in this study. In fixed material, antigen-antibody complexes were found to be homogeneously distributed along the cell membrane. Control groups showed negative binding on the cell surface. Trypsin treatment before immunolabeling removed antibody binding completely, but hyaluronidase was ineffective. Pretreatment of lectins did not block the recognition of primary antibody to antigen molecules on cell surface. Internalization of the antigen-antibody peroxidase or gold complexes was demonstrated in the cells, which were immunolabeled at 4 degrees C, and then reincubated for 0-30 min at 37 degrees C before fixation. After reincubation, the internalized ligand complexes were found in vesicles near the cell surface or in the GERL area near the Golgi apparatus which, however, did not label for peroxidase. These findings suggest that the trypsin-sensitive growth hormone, specifically bound and internalized into Chang hepatoma cells, is localized in the GERL instead of the Golgi apparatus and might be involved in the mechanism of tumor cell growth.

Ultrastructural and enzyme-cytochemical studies of hepatocytes in rat and human hepatoma and aflatoxin-B1-treated rat liver.

“…However, light cells are still found among dark cells, which are the major cell population in Chang hepatoma (4,26). It is obvious that the phenomena of dark cells and light cells in ascites cells are not caused by the methods of fixation.…”

“…Chang hepatoma cells, ascites and solid form Chang hepatoma ascites cells, induced by feeding 3'-methyl-4-dimethyl-aminoazobenzene, have been routinely maintained (4,26). The In Vivo and In Vitro ascites cells were collected and washed with 0.1 M phosphate-buffered saline (PBS) at pH 7.2, and then diluted to 5 x 106 cells/ml (16).…”

“…The cytochemical and ultrastructural changes have been reported mostly in the light cells of human hepatoma (19,23), rat hepatoma ascites cells (16,17,26), and rat livers after AFB,-treatment (13). The diverse morphological and histochemical characteristics of dark and light cells indicate that there are two different cell populations in the liver (10).…”