Immunocytochemical demonstration of the binding and internalization of growth hormone in GERL of Chang hepatoma cells.
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.
Wang
J J
JJ
Department of Biology and Anatomy, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Chang
J P
JP
Teng
C S
CS
D000818:Animals; D000911:Antibodies, Monoclonal; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D005260:Female; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D013006:Growth Hormone / Q000378:metabolism*; D007150:Immunohistochemistry / Q000379:methods; D037102:Lectins; D008114:Liver Neoplasms, Experimental / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D008247:Lysosomes / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D051381:Rats; D011919:Rats, Inbred Strains; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D037102:Lectins; D013006:Growth Hormone