[Effect of different patterns activation forms of CD40 on cloning of CD40 mutant and its proliferation and phenotype in RPMI8226 cell line].
OBJECTIVE
To analyze the cloning result of CD40 mutant from RPMI8226 cells, a multiple myeloma (MM) cell line, and study the change of the expressions of costimulatory molecules and the apoptosis of RPMI8226 cells after activated with CD40.


METHODS
CD40 gene mutant in RPMI8226 cell was detected by RT-PCR and DNA sequencing. The cell lines were cultured with sCD40L, L929/CD40L, soluble 5C11 (an anti-CD40 mAb) plate-bound 5C11 and their respective controls. Their growth curves, change of phenotypes and cell cycles were detected. The signalosome of CD40 on RPMI8226 cells were analyzed with laser scanning confocal microscope.


RESULTS
There was a single base substitution (TCA-->TTA) in the open reading frame of CD40 from RPMI8226 cells, resulting in the conversion of a amino acid (Ser124Leu). Only plate-bound antibody could inhibit RPMI8226 cell proliferation [(2.5 +/- 0.6) x 10(5) vs (7.8 +/- 1.2) x 10(5), P <0.05] and cause G1 arrested [(58.0 +/- 3.6)% vs (42.0 +/- 2.3)%, P <0.05]. muCD40 was translocated to CD40 signalosome while CD40 activated.


CONCLUSIONS
The mutated CD40 in RPMI8226 cell might decrease its affinity to CD40L, leading to the disorder of CD40 signal.
Zheng
Lu
L
Bio-technology Institute, Suzhou University, Suzhou 215007, China.
Ma
Hong-Bing
HB
Qi
Chun-Jian
CJ
Dong
Qiu-Ming
QM
Zhang
Xue-Guang
XG
D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D019013:CD40 Antigens / Q000235:genetics / Q000276:immunology*; D023201:CD40 Ligand / Q000235:genetics / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004252:DNA Mutational Analysis; D006801:Humans; D009101:Multiple Myeloma / Q000235:genetics / Q000473:pathology*; D009154:Mutation; D010641:Phenotype; D019076:Transgenes
D000911:Antibodies, Monoclonal; D019013:CD40 Antigens; D023201:CD40 Ligand