lnterleukin-2-mediated elimination of the p27Kipl cyclin-dependent kinase inhibitor prevented by rapamycin

Abstract

The cyclin-dependent kinase (Cdk) enzymes, when associated with the G1 cyclins D and E, are rate-limiting for entry into the S phase of the cell cycle. During T-cell mitogenesis, antigen-receptor signalling promotes synthesis of cyclin E and its catalytic partner, Cdk2, and interleukin-2 (IL-2) signalling activates cyclin E/Cdk2 complexes. Rapamycin is a potent immunosuppressant which specifically inhibits G1-to-S-phase progression, leading to cell-cycle arrest in yeast and mammals. Here we report that IL-2 allows Cdk activation by causing the elimination of the Cdk inhibitor protein p27Kip1, and that this is prevented by rapamycin. By contrast, the Cdk inhibitor p21 is induced by IL-2 and this induction is blocked by rapamycin. Our results show that p27Kip1 governs Cdk activity during the transition from quiescence to S phase in T lymphocytes and that p21 function may be restricted to cycling cells.

Limiting amounts of p27Kip1 correlates with constitutive activation of cyclin E-CDK2 complex in HTLV-I-transformed T-cells

“…Lysates derived from the G1-arrested PHA-PBMCs were added to the lysates from proliferating PHA-PBMCs and cyclin E-kinase assays were performed. As expected (Nourse et al, 1994), the inhibiting factors present in G1-arrested cell extracts lowered the kinase activity detected in extracts from proliferating cells by 50% (Figure 6a, column 3). Similarly, the addition of G1-arrested PHA-PBMCs extracts, high in p27 Kip1 , to the extracts from N1186-94 serum-deprived cells also lowered the kinase activity detected in N1186-94 cell extracts alone (50% decrease) (Figure 6b, columns 1 and 2), suggesting that the amount of p27 Kip1 in the N1186-94 extract is limiting.…”

“…In fact, IL-2 stimulation of human PBMCs or treatment of the myeloid cell line MO7e with Steel factor (SLF) and granulocyte-macrophage colony stimulating factor (GM-CSF) determines the upregulation of p21 Waf1 and downregulation of p27 Kip1 . Accordingly, when cells are deprived of growth factors, an inverse relationship in the relative amounts of these two CKIs has been observed Mantel et al, 1996;Nourse et al, 1994). Upon IL-2 triggering of normal human PBMCs, the relative stoichiometry of p21 Waf1 and p27 Kip1 is inverted, determining the activation of the cyclin E-CDK2 complex, which, in turn, allows passage through the R point and entry into the S phase Nourse et al, 1994).…”

Interleukin-2 expression in human carcinoma cell lines and its role in cell cycle progression

“…As shown in Table 3, DMFI for IL-2 increased in the S phase, while DMFI for p27 decreased. Taken together, these data indicate that cell cycle associated changes occur in both proteins and suggest that in the S phase, decreased p27 expression is associated with higher expression of IL-2, as reported for lymphoid cells (Nourse et al, 1994). Figure 4 Quantitative competitive reverse transcription-polymerase chain reaction (RT ± PCR) for IL-2 transcripts in total RNA obtained from PCI-1 tumor cells synchronized in the G0/G1, S, or G2/M phase of the cell cycle and from ConA-stimulated normal T lymphocytes.…”

“…In activated T lymphocytes, where IL-2 is known to be responsible for the progression from G1 to S phase Stern and Smith, 1986), and in normal human tissue cells (keratinocytes), we did not detect mitosisassociated up-regulation of IL-2 or IL-2R. The high level of IL-2 and IL-2R expression observed during mitosis in human carcinomas leads to the hypothesis that IL-2 may be involved in the process of cellular division in these cells, analogous to its regulation of CDK activity in G1/S in T lymphocytes (Nourse et al, 1994). To test this hypothesis, expression of CDK inhibitors was compared in carcinoma cells synchronized in G0/G1, S and G2/M phases of the cell cycle, using three di erent methods (Figures 5 and 6) and tumor cells synchronized at G1/S and released from the block (Figure 7).…”

“…We were able to document that a decrease of p27 CDK inhibitor expression coincided with an increase of IL-2 in carcinoma cells during the G1 to S transition (Figures 5 ± 7). Thus, by analogy to T cells (Nourse et al, 1994), IL-2 appears to be able to transiently suppress expression of p27, thus allowing carcinoma cells to pass the G1 restriction point (Pardee, 1989). Using synchronized tumor cells, we observed accumulation of intracellular IL-2 in the G2/ M phase, indicating that IL-2 also plays an important role in the process of cell division.…”