Progression through the mammalian cell cycle is regulated by cyclins, cyclin- dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CKIs). The function of these proteins in the irreversible growth arrest associated with terminally differentiated cells is largely unknown. The function of Cip/Kip proteins p21Cip1and p27Kip1 during erythropoietin-induced terminal differentiation of primary erythroblasts isolated from the spleens of mice infected with the anemia-inducing strain of Friend virus was investigated. Both p21Cip1 and p27Kip1 proteins were induced during erythroid differentiation, but only p27Kip1 associated with the principal G1CDKs—cdk4, cdk6, and cdk2. The kinetics of binding of p27Kip1 to CDK complexes was distinct in that p27Kip1 associated primarily with cdk4 (and, to a lesser extent, cdk6) early in differentiation, followed by subsequent association with cdk2. Binding of p27Kip1 to cdk4 had no apparent inhibitory effect on cdk4 kinase activity, whereas inhibition of cdk2 kinase activity was associated with p27Kip1binding, accumulation of hypo-phosphorylated retinoblastoma protein, and G1 growth arrest. Inhibition of cdk4 kinase activity late in differentiation resulted from events other than p27Kip1 binding or loss of cyclin D from the complex. The data demonstrate that p27Kip1 differentially regulates the activity of cdk4 and cdk2 during terminal erythroid differentiation and suggests a switching mechanism whereby cdk4 functions to sequester p27Kip1 until a specified time in differentiation when cdk2 kinase activity is targeted by p27Kip1 to elicit G1 growth arrest. Further, the data imply that p21Cip1 may have a function independent of growth arrest during erythroid differentiation.

Group B streptococci (GBS) are a major cause of severe infection in newborns, pregnant females, and other immunocompromised hosts. Infection often includes septicemia, shock, pneumonia, and respiratory failure. In previous studies, we have reported that GBS induce marked production of tumor necrosis factor alpha (TNF-␣) by human mononuclear cells. The present study was designed to measure the production of TNF-␣ as well as additional cytokines, including interleukin 1␤ (IL-1␤), IL-6, IL-8, IL-12, and gamma interferon (IFN-␥) but also to determine from what cells and at what time point during incubation with GBS that these cytokines are produced. Mixed mononuclear cells were incubated with heat-killed GBS, media alone, or 1 g of Escherichia coli lipopolysaccharide (LPS). Brefeldin A was added to each sample prior to staining, which prevented the export of cytokines by the Golgi apparatus. The cells were then stained with the appropriate conjugated antibodies and analyzed by using a flow cytometer. Results indicate that intracellular cytokines appear, in almost all cases, simultaneous to or before secreted proteins are detected. In contrast to the response to LPS, where TNF-␣, IL-1␤, IL-6, and IL-8 appear almost simultaneously, the human monocyte response to GBS results in the production of TNF-␣ but delayed appearance of IL-1␤, IL-6, and IL-8. The lymphocyte response to GBS was also strikingly different from that to LPS in that both secreted IFN-␥ and IL-12 was detected, while LPS failed to induce production of these critical cytokines. This suggests an important role for TNF-␣, IFN-␥, and IL-12 in GBS pathogenesis and/or immunity.