Expression of CD21 and synthesis of its ligands by HeLa cells after growth in serum-free medium.
We examined the synthesis of C3 and the expression of CD21 (complement receptor type 2) by epithelial cells in serum-free culture to eliminate the effects of competing C3 fragments from serum supplementation. The mean C3 content of HeLa supernatants was twice that of lysates after 4 hours in culture. Immunoprecipitation of metabolically labeled proteins from supernatants and lysates detected the structures of pro-C3 and native C3 in both supernatants and lysates, with C3 fragments iC3b and C3dg detected in supernatants only. Hybridization of total RNA with a human C3 complementary DNA probe revealed bands consistent with the full-length C3 message at 5 to 6 kb, implying that the C3 fragments found in the supernatants were the result of posttranslational processing. Hybridization of HeLa messenger RNA with complementary DNA probes for human complement receptors revealed strong expression of CD21 message but failed to detect message for CD35 (complement receptor type 1) or CD18 (beta chain of complement receptor types 3 and 4). Immunofluorescence with the monoclonal antibodies OKB7 and HB-5 confirmed abundant surface expression of CD21 under serum-free conditions. In a [3H]thymidine uptake assay polyclonal anti-C3 inhibited HeLa proliferation for 4 hours compared with an identical concentration of polyclonal anti-C5 as control. Thus, in contrast to previous reports, we have observed abundant CD21 message at approximately 5 kb and strong surface fluorescence with anti-CD21 monoclonal antibodies in HeLa cells cultured under serum-free conditions. Serum-free conditions may help to promote the interaction of constitutively produced C3 or C3 fragments with CD21 on malignant epithelium.
Michael
E J
EJ
Department of Pediatrics, University of Minnesota, Minneapolis 55455.
McClellan
M
M
Huebner
H
H
Hostetter
M K
MK
D002455:Cell Division; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003176:Complement C3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016895:Culture Media, Serum-Free; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D008024:Ligands; D012333:RNA, Messenger / Q000378:metabolism; D017464:Receptors, Complement 3d / Q000378:metabolism*
D003176:Complement C3; D016895:Culture Media, Serum-Free; D008024:Ligands; D012333:RNA, Messenger; D017464:Receptors, Complement 3d