Role of cyclin A and p27 in anti-IgM-induced G(1) growth arrest of murine B-cell lymphomas

Cross-linking surface immunoglobulin (Ig)M on the WEHI-231 B-cell lymphoma results in decreased cell size, G(1)/S growth arrest, and finally DNA cleavage into oligonucleosomal fragments that are the classical features of apoptotic cells. Treatment of WEHI-231 cells with anti-IgM in early G(1) phase prevents phosphorylation of the retinoblastoma gene product (pRb) and inhibits entry into S phase. Using unsynchronized cells, we previously demonstrated that cyclin A-associated and Cdk2-dependent GST-pRb kinase activity were inhibited in WEHI-231 cells treated with anti-IgM. We now show that progression of elutriated early G(1) phase WEHI-231 cells from early into late G(1) phase is accompanied by an increase in the abundance of cyclin A protein and cyclin A-associated kinase activity. Treatment of early G(1) cells with anti-IgM prevented this increase in cyclin A-associated kinase activity at late G(1), despite minimal changes in the overall level of cyclin A and Cdk2 proteins. Late G(1) cells, which already possess high cyclin A-associated kinase activity, were insensitive to anti-IgM treatment and were able to complete the cell cycle. We also found that anti-IgM-treated cells contained increased amounts of the Cdk inhibitor protein p(27Kip1). Essentially all of the cyclin A in treated cells was associated with p27, a result which we propose explains the lack of cyclin A/Cdk2 kinase activity. Accumulation of p27 in cyclin A kinase complexes, however, did not decrease the amount of Cdk2 bound to cyclin A. Thus, cross-Linking IgM on growth-inhibitable B-cell lymphomas affects cyclin A kinase activity by increasing the levels of p27 in this complex, thus preventing productive pRb phosphorylation and leading to cell cycle arrest and subsequent apoptosis. These results are discussed in terms of the cell cycle restriction points that regulate lymphocyte function, as well as the lineage-specific differences in cell cycle control.