Cyclin-dependent kinase inhibitors (CKIs) and hematological malignancies

Cell proliferation control is ensured by a group of proteins named cyclin-dependent kinases (CDKs), the activation of which is dependent on phosphorylation and cyclin association. In parallel, these CDKs are negatively controlled by two distinct groups of inhibitory proteins, the cyclin-dependent kinase inhibitors (CKIs). The first group, including p16^Ink4a, p15^Ink4b, p18^Ink4c and p19^Ink4d, is specific for the G1 CDKs, CDK4 and CDK6, inhibiting the kinase activity of cyclin D/CDK4-CDK6 complexes on pRb. p16^Ink4a, down-regulated by pRb, inhibits G1 CDKs by competition with cyclin D; p15^Ink4b, the synthesis of which is induced by TGFß, seems to be a mediator of TGFß-mediated cell cycle arrest. Furthermore, p18^Ink4c inhibits CDK6 phosphorylation and activation by CAK. The second CKIs family is constituted by p21^Waf1, p27^Kip1 and p57^Kip2. Their inhibitory action concerns a large range of cyclin/CDK complexes involved in G1 and S phase. p21^Waf1, induced in part by p53, is up-regulated by senescence, DNA damage and cellular differentiation. p21^Waf1 forms quaternary complexes with CDKs, cyclins and PCNA. Its inhibitory action, preventing CDK from phosphorylation, depends on the stoichiometry of the components. As p15^Ink4d, p27^Kip1 causes late G1 cell cycle arrest after TGFß treatment and contact inhibition. The implications of CKIs in hematological malignancies are function of deletions or mutations of their genes. p16^Ink4a and p15^Ink4b genes, localized on 9p21, present frequent homozygous deletions in ALL T, ATL and lymphoblastic acutisation of CML. The other CKIs present very rare homozygous deletions or mutations, particularly p21^Waf1 and p27^Kip2. However, reduction of inhibitory activity due to hemizygous deletions might favour leukemogenesis.     

Cyclin-dependent kinase inhibitors (CKIs) and hematological malignancies

Cell proliferation control is ensured by a group of proteins named cyclin-dependent kinases (CDKs), the activation of which is dependent on phosphorylation and cyclin association. In parallel, these CDKs are negatively controlled by two distinct groups of inhibitory proteins, the cyclin-dependent kinase inhibitors (CKIs). The first group, including p16^Ink4a, p15^Ink4b, p18^Ink4c and p19^Ink4d, is specific for the G1 CDKs, CDK4 and CDK6, inhibiting the kinase activity of cyclin D/CDK4-CDK6 complexes on pRb. p16^Ink4a, down-regulated by pRb, inhibits G1 CDKs by competition with cyclin D; p15^Ink4b, the synthesis of which is induced by TGFß, seems to be a mediator of TGFß-mediated cell cycle arrest. Furthermore, p18^Ink4c inhibits CDK6 phosphorylation and activation by CAK. The second CKIs family is constituted by p21^Waf1, p27^Kip1 and p57^Kip2. Their inhibitory action concerns a large range of cyclin/CDK complexes involved in G1 and S phase. p21^Waf1, induced in part by p53, is up-regulated by senescence, DNA damage and cellular differentiation. p21^Waf1 forms quaternary complexes with CDKs, cyclins and PCNA. Its inhibitory action, preventing CDK from phosphorylation, depends on the stoichiometry of the components. Asp15^Ink4d, p27^Kip1 causes late G1 cell cycle arrest after TGFß treatment and contact inhibition.The implications of CKIs in hematological malignancies are function of deletions or mutations of their genes. p16^Ink4a and p15^Ink4b genes, localized on 9p21, present frequent homozygous deletions in ALL T, ATL and lymphoblastic acutisation of CML. The other CKIs present very rare homozygous deletions or mutations, particularly p21^Waf1 and p27^Kip2. However, reduction of inhibitory activity due to hemizygous deletions might favour leukemogenesis.     

Structural and biochemical studies of human proliferating cell nuclear antigen complexes provide a rationale for cyclin association and inhibitor design

The interactions between the tumor suppressor protein p21WAF1 and the cyclin-dependent kinase (CDK) complexes and with proliferating cell nuclear antigen (PCNA) regulate and coordinate the processes of cell-cycle progression and DNA replication. We present the x-ray crystal structure of PCNA complexed with a 16-mer peptide related to p21 that binds with a Kd of 100 nM. Two additional crystal structures of native PCNA provide previously undescribed structures of uncomplexed human PCNA and show that significant changes on ligand binding include rigidification of a number of flexible regions on the surface of PCNA. In the competitive binding experiments described here, we show that a 20-mer sequence from p21 can be associated simultaneously with PCNA and CDK/cyclin complexes. A structural model for this quaternary complex is presented in which the C-terminal sequence of p21 acts like double-sided tape and docks to both the PCNA and cyclin molecules. The quaternary complex shows little direct interaction between PCNA and cyclin, giving p21 the role of an adaptor molecule. Taken together, the biochemical and structural results delineate a druggable inhibitor site on the surface of PCNA that may be exploited in the design of peptidomimetics, which will act independently of cyclin-groove inhibitors.     

Cell cycle exit during terminal erythroid differentiation is associated with accumulation of p27(Kip1) and inactivation of cdk2 kinase

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 p21(Cip1) and p27(Kip1) 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 p21(Cip1) and p27(Kip1) proteins were induced during erythroid differentiation, but only p27(Kip1) associated with the principal G(1) CDKs-cdk4, cdk6, and cdk2. The kinetics of binding of p27(Kip1) to CDK complexes was distinct in that p27(Kip1) associated primarily with cdk4 (and, to a lesser extent, cdk6) early in differentiation, followed by subsequent association with cdk2. Binding of p27(Kip1) to cdk4 had no apparent inhibitory effect on cdk4 kinase activity, whereas inhibition of cdk2 kinase activity was associated with p27(Kip1) binding, accumulation of hypo-phosphorylated retinoblastoma protein, and G(1) growth arrest. Inhibition of cdk4 kinase activity late in differentiation resulted from events other than p27(Kip1) binding or loss of cyclin D from the complex. The data demonstrate that p27(Kip1) differentially regulates the activity of cdk4 and cdk2 during terminal erythroid differentiation and suggests a switching mechanism whereby cdk4 functions to sequester p27(Kip1) until a specified time in differentiation when cdk2 kinase activity is targeted by p27(Kip1) to elicit G(1) growth arrest. Further, the data imply that p21(Cip1) may have a function independent of growth arrest during erythroid differentiation. (Blood. 2000;96:2746-2754)     

Recombinant human prothrombin kringle-2 induces bovine capillary endothelial cell cycle arrest at G0–G1 phase through inhibition of cyclin D1/CDK4 complex: Modulation of reactive oxygen species generation and up-regulation of cyclin-dependent kinase inhibitors

Prothrombin is a plasma glycoprotein involved in blood coagulation and, as we have previously reported, prothrombin kringles inhibit BCE (bovine capillary endothelial) cell proliferation. To reveal the mechanism, we investigated the influence of rk-2 (recombinant human prothrombin kringle-2) on the BCE cell cycle progression and ROS (reactive oxygen species) generation using FACS (fluorescence-activated cell sorter) analysis. Cell cycle analysis showed a decrease of G₁ phase cells in cells treated with bFGF (basic fibroblast growth factor) and an increase in cells treated with rk-2, as compared with the control cells. But, the portion of the S phase was reversed. In Western blot analysis, bFGF induced cytoplasmic translocation of p21^Waf1/Cip1 and p27^Kip1 and phosphorylation of p27^Kip1 but rk-2 treatment inhibited translocation of p21^Waf1/Cip1 and p27^Kip1 from nucleus to cytoplasm and phosphorylation of p27^Kip1. Also, rk-2 induced up-regulation of p53 and nuclear p21^Waf1/Cip1 and inhibited the cyclin D1/CDK4 (cyclin-dependent kinase 4) complex. The ROS level of rk-2-treated BCE cells was increased 2-fold when compared with the control, but treatment with NAC (N-Acetyl-L-cysteine), an anti-oxidant, decreased ROS generation about 55% as compared with the rk-2 treatment. NAC treatment also restored cell cycle progression inhibited by rk-2 and down-regulated p53 and nuclear p21^Waf1/Cip1 expression induced by rk-2.These data suggest that rk-2 induces the BCE cell cycle arrest at G₀–G₁ phase through inhibition of the cyclin D1/CDK4 complex caused by increase of ROS generation and nuclear cyclin-dependent kinase inhibitors.     

Activation of cyclin‐dependent kinases CDC2 and CDK2 in hepatocellular carcinoma

Abstract: Background: The cyclin‐dependent kinases (CDKs) CDC2 and CDK2 are key regulators of the cell cycle. The expression of the CDK alone does not necessary reflect their true activities because they are highly regulated by post‐translational mechanisms. Human hepatocellular carcinoma (HCC) is one of the most common cancers in the world, but the kinase activities of CDKs in HCC have not been examined. Methods: Here we examined the protein expression and kinase activities associated with CDC2 and CDK2 in HCC and the corresponding non‐tumorous liver tissues. Results: CDC2 and CDK2 are activated in HCC in over 70% and 80% of the cases, respectively, but have little correlation with clinical parameters and PCNA expression. Interestingly, PCNA was readily detectable in extracts from non‐tumorous liver, but more than 60% of samples contain higher concentration of PCNA in HCC than the corresponding non‐tumorous tissues. CDC2 and CDK2 are generally activated in the same HCC samples, but the extent of their activation varied significantly, suggesting that the pathways leading to the activation of CDC2 and CDK2 can be regulated independently. Both positive regulators of CDK activity like cyclins and CDKs, and negative regulators of CDK activity like p21^CIP1/WAF1 and Thr14/Tyr15 phosphorylation were up‐regulated in HCC. Conclusion: CDC2 and CDK2 are activated in HCC, and this may be due to a complex interplay between the level of the cyclin, CDK, CDK inhibitors, and inhibitory phosphorylation.     

p12CDK2-AP1 inhibits breast cancer cell proliferation and in vivo tumor growth

Purpose

p12^CDK2-AP1 is a growth suppressor that negatively regulates cyclin-dependent kinase 2 (CDK2) activities and shows to interfere in DNA replication. Here, we aim to elucidate the role of p12^CDK2-AP1 in breast cancer progression.

Methods

Expression of p12^CDK2-AP1 protein was examined in 60 pairs of breast cancer specimens and adjacent non-tumor tissues using immunohistochemistry assay. Loss-of-function and gain-of-function analysis was performed on MCF-7 and MDA-MB-231 breast cancer cells. Routine assays including MTT, colony formation, flow cytometry, and tumorigenesis in nude mice were performed and cell cycle regulators were analyzed.

Results

p12^CDK2-AP1 was found to be significantly downregulated in 60 breast cancer tissues compared to corresponding non-tumorous tissues. The proliferation and colony formation ability was inhibited in cells that transduced with p12^CDK2-AP1 over-expression lentivirus, but enhanced in cells that transduced with p12^CDK2-AP1 RNAi lentivirus. p12^CDK2-AP1 over-expression led to G0/G1 phase arrest in the cell cycle and caused expression changes of cell cycle-related genes (CDK2, CDK4, p16^Ink4A, p21^Cip1/Waf1). Furthermore, p12^CDK2-AP1 over-expression inhibited in vivo tumor growth in immunodeficiency mice, supporting an inhibitory role for p12^CDK2-AP1 in breast cancer development.

Conclusions

As a cell cycle regulator, p12^CDK2-AP1 is involved in the development of breast cancer and maybe a potential therapeutic candidate to suppress tumorigenicity in breast cancer.     

Activation of hPAK65 by caspase cleavage induces some of the morphological and biochemical changes of apoptosis

Apoptosis is a highly regulated form of cell death, characterized by distinctive features such as cellular shrinkage and nuclear condensation. We demonstrate here that proteolytic activation of hPAK65, a p21-activated kinase, induces morphological changes and elicits apoptosis. hPAK65 is cleaved both in vitro and in vivo by caspases at a single site between the N-terminal regulatory p21-binding domain and the C-terminal kinase domain. The C-terminal cleavage product becomes activated, with a kinetic profile that parallels caspase activation during apoptosis. This C-terminal hPAK65 fragment also activates the c-Jun N-terminal kinase pathway in vivo. Microinjection or transfection of this truncated hPAK65 causes striking alterations in cellular and nuclear morphology, which subsequently promotes apoptosis in both CHO and Hela cells. Conversely, apoptosis is delayed in cells expressing a dominant-negative form of hPAK65. These findings provide a direct evidence that the activated form of hPAK65 generated by caspase cleavage is a proapoptotic effector that mediates morphological and biochemical changes seen in apoptosis.     

Angiotensin converting-enzyme inhibitor treatment reduces glomerular p16INK4 and p27Kip1 expression in diabetic BBdp rats

Aims/hypothesis. Renal hypertrophy occurs early in diabetes mellitus and precedes the development of glomerulosclerosis and tubulointerstitial fibrosis. We have previously shown that cultured mesangial cells exposed to high glucose are arrested in the G₁-phase of the cell cycle and undergo cellular hypertrophy. High glucose-mediated induction of p27^Kip1, an inhibitor of cyclin-dependent kinases, is essential in this process. Further investigations have also shown that p27^Kip1 and p21^Cip1, other cyclin-dependent kinase inhibitors, are up regulated in the kidneys of mice with Type I (insulin-dependent) as well as Type II (non-insulin-dependent) diabetes mellitus. Our study was undertaken to test a potential effect of short-term treatment with the angiotensin-converting enzyme inhibitor enalapril on the glomerular expression of the cyclin-dependent kinase inhibitors p16^INK4, p21^Cip1, and p27^Kip1 in BBdp rats, an autoimmune model of Type I diabetes.¶Methods. We evaluated p16^INK4, p21^Cip1, and p27^Kip1 protein expression in isolated glomeruli by western blots. We also assessed p27^Kip1 positive glomerular cells by immunohistochemistry.¶Results. Glomerular expression of all three cyclin-dependent kinase inhibitors were stimulated in BBdp rats compared with non-diabetic BBdr animals. Enalapril treatment for 3 weeks, started after the onset of diabetes, reduced the glomerular expression of p16^INK4 and p27^Kip1 but not of p21^Cip1. Enalapril also prevented the increase in kidney weights observed in BBdp rats but had no effect on systolic blood pressure or glucose concentrations.¶Conclusion/interpretation. Our data show that enalapril attenuates the glomerular expression of cyclin-dependent kinase inhibitors in diabetes and suggest a molecular mechanism of how angiotensin-converting enzyme inhibitors prevent renal hypertrophy in diabetes. [Diabetologia (1999) 42: 1425–1432]     

Myosin I heavy chain kinase: Cloning of the full-length gene and acidic lipid-dependent activation by Rac and Cdc42

Acanthamoeba myosin I heavy chain kinase (MIHCK) phosphorylates the heavy chains of amoeba myosins I, increasing their actin-activated ATPase activities. The activity of MIHCK is increased by binding to acidic phospholipids or membranes and by autophosphorylation at multiple sites. Phosphorylation at a single site is necessary and sufficient for full activation of the expressed catalytic domain. The rate of autophosphorylation of native MIHCK is controlled by a region N-terminal to the catalytic domain. By its substrate specificity and the sequence of its C-terminal catalytic domain, MIHCK was identified as a p21-activated kinase (PAK). We have now cloned the full-length genomic DNA and cDNA of MIHCK and have shown it to contain the conserved p21-binding site common to many members of the PAK family. Like some mammalian PAKs, MIHCK is activated by Rac and Cdc42, and this activation is GTP-dependent and accompanied by autophosphorylation. In contrast to mammalian PAKs, activation of MIHCK by Rac and Cdc42 requires the presence of acidic lipids. Also unlike mammalian PAK, MIHCK is not activated by sphingosine or other non-negatively charged lipids. The acidic lipid-binding site is near the N terminus followed by the p21-binding region. The N-terminal regulatory domain of MIHCK contains alternating strongly positive and strongly negative regions. and the extremely Pro-rich middle region of MIHCK has a strongly acidic N-terminal segment and a strongly basic C-terminal segment. We propose that autophosphorylation activates MIHCK by neutralizing the basic segment of the Pro-rich region, thus unfolding the regulatory domain and abolishing its inhibition of the catalytic domain.     

Differential association of p21Cip1 and p27Kip1 with cyclin E-CDK2 during rat liver regeneration

BACKGROUND/AIMS: The cell cycle inhibitors p21Cip1 and p27Kip1 regulate liver regeneration by modulating the activity of cyclin-dependent kinases (CDKs). However, the specific role of these inhibitors in the regulation of CDK2 activity during liver regeneration remains unknown. The aim of this study was to examine the association of p21Cip1 and p27Kip1 with cyclin E-CDK2 and cyclin A-CDK2 complexes during rat liver regeneration and to correlate the association of both inhibitors with CDK2 activity. METHODS: The association of p21Cip1 or p27Kip1 with cyclin E-CDK2 or cyclin A-CDK2 and the activities of these complexes were analyzed by immunoprecipitation of rat liver homogenates obtained at different times after a partial hepatectomy (PH), followed by Western blotting or kinase assays. RESULTS: High amounts of p27Kip1 bound to cyclin E-CDK2 were observed during the first 13 h after PH, when CDK2 activity was very low. At 24 h, when CDK2 activity was maximal, the amount of bound-p27Kip1 decreased strongly. The amount of p21Cip1 bound to these complexes was low during the first 13 h but subsequently increased. No cyclin A-CDK2 complexes were found during the first 13 h after PH. At 24 h, complexes containing low levels of both inhibitors were detected and at 28 h, a significant increase in p21Cip1 and p27Kip1 associated with cyclin A-CDK2 was observed. CONCLUSIONS: p27Kip1 acts as a brake on cyclin E-CDK2 activity during the first 13 h after a PH. Both p21Cip1 and p27Kip1 down-regulate cyclin A-CDK2 activity at 28 h after PH, after its maximal activation.     

Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase delta holoenzyme.

Cdk-interacting protein 1 (Cip1) is a p53-regulated 21-kDa protein that inhibits several members of the cyclin-dependent kinase (CDK) family. It was initially observed in complexes containing CDK4, cyclin D, and proliferating cell nuclear antigen (PCNA). PCNA, in conjunction with activator 1, acts as a processivity factor for eukaryotic DNA polymerase (pol) delta, and these three proteins constitute the pol delta holoenzyme. In this report, we demonstrate that Cip1 can also directly inhibit DNA synthesis in vitro by binding to PCNA. Cip1 efficiently inhibits simian virus 40 replication dependent upon pol alpha, activator 1, PCNA, and pol delta, and this inhibition can be overcome by additional PCNA. Simian virus 40 DNA replication, catalyzed solely by high levels of pol alpha-primase complex, is unaffected by Cip1. Using the surface plasmon resonance technique, a direct physical interaction of PCNA and Cip1 was detected. We have observed that Cip1 efficiently inhibits synthesis of long (7.2 kb) but not short (10 nt) templates, suggesting that its association with PCNA is likely to impair the processive movement of pol delta during DNA chain elongation, as opposed to blocking assembly of the pol delta holoenzyme. The implications of the Cip1-PCNA interaction with respect to regulation of DNA synthesis, cell cycle checkpoint control, and DNA repair are discussed.     

Coordinate control of proliferation and migration by the p27Kip1/cyclin-dependent kinase/retinoblastoma pathway in vascular smooth muscle cells and fibroblasts

Previous studies have demonstrated a protective effect of the cyclin-dependent kinase (CDK) inhibitor p27Kip1 against atherosclerosis and restenosis, two disorders characterized by abundant proliferation and migration of vascular smooth muscle cells and adventitial fibroblasts. These therapeutic effects might result from p27Kip1-dependent suppression of both cell proliferation and migration. However, the interplay between cell growth and locomotion remains obscure. We show here that p27Kip1 inhibits cellular changes that normally occur during cell locomotion (eg, lamellipodia formation and reorganization of actin filaments and focal adhesions). Importantly, a p27Kip1 mutant lacking CDK inhibitory activity failed to inhibit vascular smooth muscle cell and fibroblast proliferation and migration. Moreover, a constitutively active mutant of the retinoblastoma protein (pRb) insensitive to CDK-dependent hyperphosphorylation inhibited both cell proliferation and migration. In contrast, inactivation of pRb by forced expression of the adenoviral oncogene E1A correlated with high proliferative and migratory activity. Collectively, these results suggest that cellular proliferation and migration are regulated in a coordinated manner by the p27Kip1/CDK/pRb pathway. These findings might have important implications for the development of novel therapeutic strategies targeting the fibroproliferative/migratory component of vascular occlusive disorders.     

Degradation of p57Kip2 mediated by SCFSkp2-dependent ubiquitylation

The abundance of the cyclin-dependent kinase (CDK) inhibitor p57Kip2, an important regulator of cell cycle progression, is thought to be controlled by the ubiquitin-proteasome pathway. The Skp1/Cul1/F-box (SCF)-type E3 ubiquitin ligase complex SCFSkp2 has now been shown to be responsible for regulating the cellular level of p57Kip2 by targeting it for ubiquitylation and proteolysis. The elimination of p57Kip2 was impaired in Skp2-/- cells, resulting in abnormal accumulation of the protein. Coimmunoprecipitation analysis also revealed that Skp2 interacts with p57Kip2 in vivo. Overexpression of WT Skp2 promoted degradation of p57Kip2, whereas expression of a dominant negative mutant of Skp2 prolonged the half-life of p57Kip2. Mutation of the threonine residue (Thr-310) of human p57Kip2 that is conserved between the COOH-terminal QT domains of p57Kip2 and p27Kip1 prevented the effect of Skp2 on the stability of p57Kip2, suggesting that phosphorylation at this site is required for SCFSkp2-mediated ubiquitylation. Finally, the purified recombinant SCFSkp2 complex mediated p57Kip2 ubiquitylation in vitro in a manner dependent on the presence of the cyclin E-CDK2 complex. These observations thus demonstrate that the SCFSkp2 complex plays an important role in cell-cycle progression by determining the abundance of p57Kip2 and that of the related CDK inhibitor p27Kip1.     

Functional analysis of an Orc6 mutant in Drosophila

The origin recognition complex (ORC) is a 6-subunit complex required for the initiation of DNA replication in eukaryotic organisms. ORC is also involved in other cell functions. The smallest Drosophila ORC subunit, Orc6, is important for both DNA replication and cytokinesis. To study the role of Orc6 in vivo, the orc6 gene was deleted by imprecise excision of P element. Lethal alleles of orc6 are defective in DNA replication and also show abnormal chromosome condensation and segregation. The analysis of cells containing the orc6 deletion revealed that they arrest in both the G₁ and mitotic stages of the cell cycle. Orc6 deletion can be rescued to viability by a full-length Orc6 transgene. The expression of mutant transgenes of Orc6 with deleted or mutated C-terminal domain results in a release of mutant cells from G₁ arrest and restoration of DNA replication, indicating that the DNA replication function of Orc6 is associated with its N-terminal domain. However, these mutant cells accumulate at mitosis, suggesting that the C-terminal domain of Orc6 is important for the passage through the M phase. In a cross-species complementation experiment, the expression of human Orc6 in Drosophila Orc6 mutant cells rescued DNA replication, suggesting that this function of the protein is conserved among metazoans.     

Inhibition of Akt increases p27Kip1 levels and induces cell cycle arrest in anaplastic large cell lymphoma

Anaplastic large cell lymphoma (ALCL) is a highly proliferative neoplasm that frequently carries the t(2;5)(p23;q35) and aberrantly expresses nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). Previously, NPM-ALK had been shown to activate the phosphatidylinositol 3 kinase (PI3K)/Akt pathway. As the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) (p27) is usually not expressed in ALCL, we hypothesized that activated Akt (pAkt) phosphorylates p27 resulting in increased p27 proteolysis and cell cycle progression. Here we demonstrate that inhibition of pAkt activity in ALCL decreases p27 phosphorylation and degradation, resulting in increased p27 levels and cell cycle arrest. Using immunohistochemistry, pAkt was detected in 24 (57%) of 42 ALCL tumors, including 8 (44%) of 18 ALK-positive tumors and 16 (67%) of 24 ALK-negative tumors, and was inversely correlated with p27 levels. The mean percentage of p27-positive tumor cells was 5% in the pAkt-positive group compared with 26% in the pAkt-negative group (P = .0076). These findings implicate that Akt activation promotes cell cycle progression through inactivation of p27 in ALCL.