Intracellular redistribution of Ku immunoreactivity in response to cell-cell contact and growth modulating components in the medium.
Ku is a heterodimeric protein first recognized as a human autoantigen but now known to be widely distributed in mammalian cells. Analysis of repair-deficient mutant cells has shown that Ku is required for DNA repair, and roles in DNA replication and transcription have also been suggested on the basis of in vitro observations. Ku is generally regarded as a nuclear component. However, in the present paper, we show that a quantitatively significant fraction (half or more) of Ku is located in the cytoplasm of cultured primate cells, and that major changes in epitope accessibility of both nuclear and cytoplasmic Ku components are associated with the transition from sparse to confluent cell densities. The same changes in immunoreactivity were seen in HeLa, 293, CV-1 (monkey) and HPV-transformed keratinocyte cell lines, and in primary cultures of human keratinocytes. The immunostaining pattern of sparsely grown cells could be converted to the 'confluent' configuration by re-plating them at the same low density on a monolayer of mouse 3T3 cells. The confluent antigen pattern could also be induced in sparse cells within 15-30 minutes by exposure of the cells to serum- or Ca(2+)-free medium or overnight with 2 mM hydroxyurea. Somatostatin at 0.12 mM blocked the effects of serum/Ca2+ deprivation of Ku p70 antigen distribution in sparse CV-1 cells, and in confluent cultures reversed the usual nuclear concentration of p70 immunoreactivity. However, somatostatin did not alter the expected immunostaining patterns of p86. Preliminary studies indicate that sparse CV-1 cells, but not HeLa cells, respond to as little as 1 pM of TGF-beta 1 in the culture medium by the rapid appearance of nuclear immunoreactivity. TGF-alpha had no apparent effect. These findings are consistent with the participation of Ku in a signal transduction system responsive to the inhibitory effect of cell-cell contact on the one hand and to cytokines and growth-supportive components of the culture medium on the other.
Fewell
J W
JW
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Kuff
E L
EL
D016475:3T3 Cells; D000818:Animals; D034961:Antigens, Nuclear; D002450:Cell Communication; D002455:Cell Division; D002460:Cell Line; D003470:Culture Media; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000276:immunology; D019084:Fluorescent Antibody Technique, Indirect; D006367:HeLa Cells; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D000072200:Ku Autoantigen; D051379:Mice; D009687:Nuclear Proteins / Q000032:analysis* / Q000276:immunology
D034961:Antigens, Nuclear; D003470:Culture Media; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen