Five Epstein-Barr virus (EBV)-positive human
lymphoma cell lines maintained in
severe combined immune deficiency (SCID) mice were used to investigate the role of G1
cyclins in EBV-induced lymphomagenesis. All the primary
tumors had been negative for EBV but became positive after establishment in SCID mice, with monoclonal immunoglobulin gene rearrangement and EBV monoclonality. To compare the expression status of G1
cyclins, these EBV-associated
lymphoma lines (6 EBV[-] human SCID mouse
lymphoma lines, 13 human
B cell lymphomas and 8 samples of human tonsil tissue) were examined by reverse transcription-polymerase chain reaction-Southern blotting, Western blotting and immunohistochemistry.
mRNA expression of
cyclin D1 (CCND1),
cyclin D2 (CCND2),
cyclin E (CCNE),
cyclin-dependent kinase 2 (CDK2) and 4 (CDK4) was found in all 3 types of
lymphomas. Western blotting demonstrated identical results. Immunohistochemistry revealed CCND1 to be negative in all
lymphomas. CCND2 was positive and restricted to the nuclei in all EBV(+) SCID mouse
lymphoma lines, whereas it was limited to the cytoplasm in half of the EBV(-) counterparts. CCNE was positive in the nuclei in all EBV(+) but negative in all EBV(-) SCID mouse
lymphoma lines. Immunoprecipitation of EBV(+) and (-) SCID mouse
lymphomas for CCND1, CCND2 and CCNE vs. p21,
PCNA and CDK2 or CDK4 demonstrated that, in EBV(+) SCID lines, CCND2/CDK4 complexes were present without binding to p21, suggesting independence from p21 regulation. In EBV(-) SCID mouse
lymphomas, half of the cases showed complex formation of CCND2/CDK4 without binding of p21. In contrast, CCND1/CDK4 and CCNE/CDK2 were under regulation of p21 in both EBV(+) and (-)
lymphomas. These results suggest that differential expression of CCNDs, CCNE and CDKs, as well as variation in their subcellular localization and association with CDK-inhibitor
protein, could explain differences in cell proliferation between EBV(+) and EBV(-)
lymphomas.