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Induction of Cip/Kip and Ink4 Cyclin Dependent Kinase Inhibitors by Interferon-A in Hematopoietic Cell Lines

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Induction of Cip/Kip and Ink4 Cyclin Dependent Kinase Inhibitors by Interferon-A in Hematopoietic Cell Lines Oncogene (1997) 14, 415 ± 423 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 Induction of Cip/Kip and Ink4 cyclin dependent kinase inhibitors by interferon-a in hematopoietic cell lines Olle Sangfelt, Sven Erickson, Stefan Einhorn and Dan Grande r Department of Oncology/Pathology, Radiumhemmet, Building P8, Karolinska Hospital and Institute, S-171 76 Stockholm, Sweden One prominent eect of IFNs is their cell growth exert cell growth inhibitory eects. It has since been inhibitory activity. The exact molecular mechanism found that this antiproliferative action of IFNs can be behind this inhibition of proliferation remains to be observed in many cell types from established cell lines, elucidated. Possible eectors for IFN-induced growth normal tissues and primary tumor cells (Taylor- inhibition are the recently discovered cyclin-dependent Papadimitriou and Rozengurt, 1985). It has been kinase inhibitors. The eect of IFN-a treatment on the postulated that this eect may, at least in part, members of the Ink4 and Cip/Kip families of Cdk mediate the antitumor eects observed in some inhibitors was investigated in three hematopoietic cell malignancies (Brenning et al., 1985). In vitro culture lines Daudi, U-266 and H9. Two of these cell lines, of susceptible cell types with IFN commonly leads to Daudi and U-266, respond to IFN-a by G1 arrest, G1 arrest, but sometimes to a lengthening of S-phase or whereas the H9 cell line is not growth arrested by IFN-a. all phases of the cell cycle (Roos et al., 1984). We show that a p53-independent upregulation of p21 With the discovery of the proteins that normally mRNA occurs following IFN-a treatment in all three cell regulate the cell cycle, some of the molecular eectors lines. In Daudi and U-266 cells, the mRNA induction is responsible for the IFN induced cell growth inhibition accompanied by an increase in p21 protein, followed by have been identi®ed. A number of proteins already an increased binding of p21 to Cdk2 and a subsequent identi®ed as key regulators of the cell cycle such as decrease in Cdk2 activity, temporally coinciding with G1 pRb, cyclin A, E2F, Cdk2 and c-myc are downstream arrest. In both these cell lines, there was also an targets in IFNs growth suppressive action (Kimchi, increased binding of p21 to Cdk4. In contrast, p21 1992; Melamed et al., 1993; Zhang and Kumar, 1994). protein was not expressed in H9 cells, despite high levels However, the exact chain of events responsible for the of p21 mRNA following IFN-a treatment. In U-266 antiproliferative eect of IFN is still unclear. cells, IFN-a increased not only p21 but also p15 mRNA During the last few years a host of knowledge has and protein levels, followed by an increased association been gained in the ®eld of cell cycle regulation. In all of p15 with Cdk4. Furthermore, IFN-a treatment caused organisms studied so far, passage through the cell a four- to sixfold induction of the p16 E1b transcript in cycle is dependent on the sequential formation and U-266 cells. Expression levels of the other Ink4 and Cip/ activation of complexes consisting of cyclin dependent Kip Cdk inhibitors were not induced by IFN treatment in kinases (Cdks) and their cyclin partners. In mamma- any of the cell lines. We conclude that IFN-a can act as lian cells the complexes active in G1 contain cyclin a potent regulator of Cdk-inhibitor expression, correlat- D-Cdk4/6 and cyclin E-Cdk2 (reviewed in GranÄ a and ing with decreased Cdk activity and cell growth Reddy, 1995). Many proteins have been suggested as inhibition. One mechanism for resistance to IFN may targets of dierent cyclin/Cdk complexes (Nigg, 1993). be loss of the ability of cells to upregulate these proteins. One of the most studied substrates is the Rb protein, which upon phosphorylation enables the E2F tran- Keywords: interferon; cell cycle; cyclin-dependent scription factor to activate genes necessary for S phase kinase; cyclin-dependent kinase inhibitors progression (Nevins, 1994). The activity of Cdk complexes have also been shown to be regulated by phosphorylation and dephosphorylation on speci®c residues (Morgan, 1995). Recently a new level of Introduction regulation of these complexes has been elucidated by the indenti®cation of low molecular weight cyclin The interferons (IFNs) are a family of proteins which dependent kinase inhibitors (Sherr and Roberts, 1995). are produced by eukaryotic cells when challenged by In the mammalian system, two families of Cdk- viruses and other agents. Following binding to speci®c inhibitors have been identi®ed. The Ink4 family cell surface receptors, IFNs induce transcription of includes the p15, p16, p18 and p19 proteins, which IFN-stimulated genes through activation of the Jak/ all contain several ankyrin repeat like sequences. STAT signaling pathway (Shuai, 1994), whereby IFNs These inhibitors bind speci®cally to Cdk4 and 6, elicit a number of physiological responses in cells, with thereby interfering with cyclin D binding to the the common denominator of inhibiting viral replication kinases. The p16 gene is commonly mutated or (reviewed in De Mayer and De Mayer-Guiginard, deleted in human malignancies (Kamb et al., 1994; 1988). It was shown in the early 60s that IFNs also Nobori et al., 1994). Recently, an alternative ®rst exon (E1b) of the p16 gene has been described. When the E1b exon is spliced onto exon 2 of the p16 gene, the Correspondence: D Grande r original reading frame is shifted, and translation of Received 21 June 1996; revised 16 September 1996; accepted 17 this so called b transcript creates a protein completely September 1996 unrelated to p16. Interestingly this protein seems to Induction of Cdk-inhibitors by interferon O Sangfelt et al 416 have cell growth inhibitory properties, although it has [3H]thymidine incorporation. U-266 as well as Daudi not been shown to be a direct Cdk-inhibitor (Quelle et cells responded to IFN-a with an accumulation of cells al., 1995). in the G1 phase after 16 ± 24 h of incubation with The Cip/Kip family include the sequence, p21, p27 IFN-a (Figure 1). In contrast, no change in cell cycle and p57 proteins, which share no homology with the distribution was observed in H9 cells after IFN-a Ink4 family (Sherr and Roberts, 1995). The latter treatment (Figure 1). Both U-266 and H9 cells were group of Cdk-inhibitors associate with and inhibit the found to undergo apoptosis following longer times of cyclin E-Cdk2 and cyclin A-Cdk2 complexes, in exposure to IFN-a (Sangfelt et al., submitted). addition to inhibiting cyclin D-Cdk4 and 6 com- Since the Rb protein is implicated in the control of plexes. The p21 gene is transcriptionally regulated by G1 progression and has previously been shown to be an the p53 tumor suppressor gene, thereby mediating cell important target for IFN's cell growth inhibitory cycle arrest. However, p53 independent induction of action, we examined the status and eects of IFN-a p21 has recently been described (El-Deiry et al., 1993; treatment on this protein in the three cell lines. In Elbendary et al., 1994; Datto et al., 1995; Parker et al., cycling Daudi cells the major portion of the Rb protein 1995; Reynisdo ttir et al., 1995). In addition to appeared as slowly-migrating hyperphosphorylated associating with dierent cyclin/Cdk complexes, the p21 protein (but not p27 or p57) can bind to proliferating cell nuclear antigen (PCNA), and inhibit DNA replication (Waga et al., 1994). However this activity maps to a dierent region of the p21 protein than the one interacting with cyclin/Cdk complexes (Chen et al., 1995). Moreover, p21 has also been shown to disrupt the interaction between Cdk2 and the E2F- p107/p130 complexes (Zhu et al., 1995; Shianov et al., 1995). A few previous observations prompted our interest in examining whether Cdk-inhibitors may be targets for regulation by IFNs. Firstly, other extracellular signals for growth arrest such as TGF-b, contact inhibition and serum starvation have been shown to act, at least in part, through the modulation of Cdk- inhibitors. TGF-b induces p15 and p21 at the transcriptional level. It also increases the amount of p27 bound to Cdk2, and reduces Cdk4 levels (Elbendary et al., 1994; Hannon and Beach, 1994; Polyak et al., 1994; Datto et al., 1995., Reynisdo ttir et al., 1995). Other growth inhibitory cytokines may conceivably work in a similar fashion. Secondly, the previously described eects on cell cycle regulating proteins by IFN, such as reduced pRb phosphorylation and the inhibition of Cdk2 kinase activity are all compatible with an induction of Cdk-inhibitor activity. We have therefore analysed the eects of IFN-a on the expression of the Ink4- and Cip/Kip-families of Cdk- inhibitors in three hematopoietic cell lines. Results Figure 1 Flow cytometry analysis of asynchronous U-266 (a), As IFN under physiological circumstances is likely to Daudi (b) and H9 cells (c) cultured in the absence or presence of act by arresting actively proliferating cells, we 5000 U/ml of IFN-a for 24 h investigated the eects of IFN-a treatment of proliferating cell cultures, rather than on cells released from quiescence. time (hours): 0 4 8 12 24 48 IFN-a eects on cell cycle progression in Daudi, U-266 and H9 cells The integrity of the IFN signal transduction pathway Rbphos was shown by demonstrating that all three cell lines were Rb sensitive to IFN-a, as measured by induction of the IFN stimulated gene, 2',5'oligoadenylate synthetase. A 11 ± 40-fold induction of enzyme activity was detected after Figure 2 pRb protein levels in Daudi cells at various timepoints incubation with 5000 U/ml of IFN-a for 24 h.
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