Culture medium induced morphological changes of melanoma cells associated with change in sensitivity to lysis by lymphokine-activated killer cells.
Three sublines (Clones 1, 2 and 7) of the human melanoma CaCL 73-36 cell line with different cellular morphology, growth patterns, melanin content and surface antigenic profile were maintained in RPMI-1640 medium plus 10% fetal bovine serum (abbreviated as RPMI). Each subline was divided into two groups: one grown in RPMI and the other in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum (abbreviated as DMEM) for 96 h. Phenotypically, Clone 2 expressed Class I and II MHC and ICAM-1 on the surface and in the cytoplasm, while Clones 1 and 7 failed to express these antigens in both the cytoplasm and on the cell surface. Melanotic Clones 1 and 7 cells became even more pigmented, had slower growth rates, and exhibited lower saturation densities when incubated in DMEM than when they were incubated in RPMI. On the other hand, Clone 2 cells maintained in RPMI were grossly amelanotic, contained defective-like melanosomes detected ultrastructurally, and had distinct clusters of microvilli polarly located in most of the cells. Such specialized ultrastructures were not affected by medium conditions. Analysis of sensitivity of the clonal sublines to cytolysis by allogeneic effector cells revealed that in spite of low levels of natural killer (NK) cytotoxicity noted, DMEM produced a 2- to 14-fold increase in sensitivity to NK cells, irrespective of which medium was used. Different levels of lymphokine-activated killer (LAK) cytolytic activity were clearly observed in sublines maintained in RPMI, with Clone 2 being the most sensitive and both Clones 1 and 7 being less sensitive. Cells grown in DMEM exhibited significantly higher levels of sensitivity to LAK cytolysis than cells grown in RPMI as revealed by their differences in lytic units (p < 0.05). This was likely due to the high levels of surface ICAM-1 expression in cells incubated in DMEM vs little expression of this adhesion molecule by cells grown in RPMI. Taken together, these results demonstrate the presence of heterogeneous subpopulations within the CaCL 73-36 melanoma cell line regarding their pigmentary status, antigenic profile, growth pattern and responsiveness to NK/LAK cytolysis. The results also call attention to the importance of utilizing a same medium in short- and long-term cultures of melanomas for biological studies and response evaluations of therapeutic agents such as LAK cells, when multiple cell targets from different patients or multi-metastatic cell lines from individual patients are to be compared. Finally, these melanoma sublines may be valuable for further elucidation of the relationship between MHC expression, and increased sensitivity to LAK cytolysis, and the role of the components of DMEM in the mechanism for the observed induction of cell differentiation and enhanced LAK cytolysis.
Perng
Y P
YP
Tumor Immunology Laboratory, School of Medicine, Chang Gung University, Taoyuan, Taiwan, ROC.
Lin
C C
CC
Perng
I M
IM
Shen
Y C
YC
Chuang
C K
CK
Liao
S K
SK
D000818:Animals; D000951:Antigens, Neoplasm; D003470:Culture Media; D003602:Cytotoxicity, Immunologic; D004273:DNA, Neoplasm / Q000032:analysis; D005260:Female; D006684:HLA-DR Antigens / Q000032:analysis; D015395:Histocompatibility Antigens Class I / Q000032:analysis; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000032:analysis; D015979:Killer Cells, Lymphokine-Activated / Q000276:immunology*; D007694:Killer Cells, Natural / Q000276:immunology; D008297:Male; D008543:Melanins / Q000096:biosynthesis; D008545:Melanoma / Q000276:immunology / Q000473:pathology / Q000628:therapy*; D058950:Melanoma-Specific Antigens; D051379:Mice; D016513:Mice, SCID; D008854:Microscopy, Electron; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000032:analysis; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D003470:Culture Media; D004273:DNA, Neoplasm; D006684:HLA-DR Antigens; D015395:Histocompatibility Antigens Class I; D008543:Melanins; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins; D018799:Intercellular Adhesion Molecule-1