Rapamycin inhibits proliferation of Epstein-Barr virus-positive B-cell lymphomas through modulation of cell-cycle protein expression.
BACKGROUND
Posttransplant lymphoproliferative disease (PTLD) is a serious complication of solid organ and bone marrow transplantation and is closely associated with Epstein-Barr virus (EBV) infection. We have previously shown that rapamycin (RAPA) directly inhibits the in vitro and in vivo proliferation of EBV-infected B lymphoblastoid cell lines (SLCL), derived from patients with PTLD, by arresting cells in the G1 phase of the cell cycle. The aim of this study is to elucidate the mechanism by which RAPA causes cell cycle arrest in EBV+ B cells.


METHODS
SLCL were cultured without or with RAPA (10 ng/ml) and G1-associated cell cycle proteins were analyzed by immunoblot and densitometric analysis. CDK complexes were immunoprecipitated and incubated with retinoblastoma protein (Rb) substrate. Kinase activity of the complex was determined by Western blot with anti-phospho-Rb antibodies.


RESULTS
We show that RAPA decreased both Cyclin D2 and Cyclin D3 protein levels. Furthermore, RAPA decreased the protein levels of cyclin dependent kinase 4 (CDK4) and increased the expression of the CDK inhibitor p27. In contrast, expression of the CDK inhibitor p21 was markedly inhibited by RAPA in the SLCL. Finally, in vitro kinase assays revealed that downstream hyperphosphorylation of Rb by CDK complexes was also decreased by RAPA.


CONCLUSIONS
The results presented here elucidate key targets of RAPA-induced cell cycle arrest, provide insight into the growth pathways of EBV+ B-cell lymphomas, and demonstrate the potential for RAPA as a therapeutic option in the treatment of PTLD and other EBV+ lymphomas.
Vaysberg
Maria
M
Program in Immunology, Division of Transplantation, Stanford University School of Medicine, Stanford, CA, USA.
Balatoni
Cynthia E
CE
Nepomuceno
Ronald R
RR
Krams
Sheri M
SM
Martinez
Olivia M
OM
D018797:Cell Cycle Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004854:Herpesvirus 4, Human / Q000502:physiology*; D006801:Humans; D016393:Lymphoma, B-Cell / Q000378:metabolism* / Q000473:pathology*; D010766:Phosphorylation; D016160:Retinoblastoma Protein / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology*
D018797:Cell Cycle Proteins; D016160:Retinoblastoma Protein; D020123:Sirolimus