Expression of Skp2, a p27(Kip1) ubiquitin ligase,in malignant lymphoma: correlation with p27(Kip1) and proliferation index

Reduced levels of p27(Kip1) are frequent in human cancers and have been associated with poor prognosis. Skp2, a component of the Skp1-Cul1-F-box protein (SCF) ubiquitin ligase complex, has been implicated in p27(Kip1) degradation. Increased Skp2 levels are seen in some solid tumors and are associated with reduced p27(Kip1). We examined the expression of these proteins using single and double immunolabeling in a large series of lymphomas to determine if alterations in their relative levels are associated with changes in cell proliferation and lymphoma subgroups. We studied the expression of Skp2 in low-grade and aggressive B-cell lymphomas (n = 86) and compared them with p27(Kip1) and the proliferation index (PI). Fifteen hematopoietic cell lines and peripheral blood lymphocytes were studied by Western blot analysis. In reactive tonsils, Skp2 expression was limited to proliferating germinal center and interfollicular cells. Skp2 expression in small lymphocytic lymphomas (SLLs) and follicular lymphomas (FCLs) was low (mean percentage of positive tumor cells, less than 20%) and was inversely correlated (r = -0.67; P <.0001) with p27(Kip1) and positively correlated with the PI (r = 0.82; P <.005). By contrast, whereas most mantle cell lymphomas (MCLs) demonstrated low expression of p27(Kip1) and Skp2, a subset (n = 6) expressed high Skp2 (exceeding 20%) with a high PI (exceeding 50%). Skp2 expression was highest in diffuse large B-cell lymphomas (DLBCLs) (mean, 22%) and correlated with Ki-67 (r = 0.55; P <.005) but not with p27(Kip1). Cytoplasmic Skp2 was seen in a subset of aggressive lymphomas. Our data provide evidence for p27(Kip1) degradative function of Skp2 in low-grade lymphomas. The absence of this relationship in aggressive lymphomas suggests that other factors contribute to deregulation of p27(Kip1) expression in these tumors.     

Role of p27(Kip1) in human intestinal cell differentiation

BACKGROUND & AIMS: Growth arrest and differentiation are generally considered to be temporally and functionally linked phenomena in the intestinal epithelium. METHODS: To delineate the mechanism(s) responsible for the loss of proliferative potential as committed intestinal cells start to differentiate, we have analyzed the regulation of G(1)-phase regulatory proteins in relation to differentiation in the intact epithelium as well as in well-established intestinal cell models that allow the recapitulation of the crypt-villus axis in vitro. RESULTS: With intestinal cell differentiation, we have observed an induction of the cell cycle inhibitors p21(Cip), p27(Kip1), and p57(Kip2) expression with an increased association of p27(Kip1) and p57(Kip2) with cyclin-dependent kinase 2 (Cdk2). At the same time, there was an accumulation of the hypophosphorylated form of the pRb proteins and a strong decline in Cdk2 activity. Stable expression of a p27(Kip1) antisense complementary DNA in Caco-2/15 cells did not prevent growth arrest induced by confluence, but repressed villin, sucrase-isomaltase, and alkaline phosphatase expression. CONCLUSIONS: Our results indicate that the growth arrest that precedes differentiation involves the activation of Rb proteins and the inhibition of Cdk2. Furthermore, intestinal cell differentiation apparently requires a function of p27(Kip1) other than that which leads to inhibition of Cdks.     

RSK1 drives p27Kip1 phosphorylation at T198 to promote RhoA inhibition and increase cell motility

p90 ribosomal S6 kinase (RSK1) is an effector of both Ras/MEK/MAPK and PI3K/PDK1 pathways. We present evidence that RSK1 drives p27 phosphorylation at T198 to increase RhoA-p27 binding and cell motility. RSK1 activation and p27pT198 both increase in early G₁. As for many kinase–substrate pairs, cellular RSK1 coprecipitates with p27. siRNA to RSK1 and RSK1 inhibition both rapidly reduce cellular p27pT198. RSK1 overexpression increases p27pT198, p27-cyclin D1-Cdk4 complexes, and p27 stability. Moreover, RSK1 transfectants show mislocalization of p27 to cytoplasm, increased motility, and reduced RhoA-GTP, phospho-cofilin, and actin stress fibers, all of which were reversed by shRNA to p27. Phosphorylation by RSK1 increased p27pT198 binding to RhoA in vitro, whereas p27T157A/T198A bound poorly to RhoA compared with WTp27 in cells. Coprecipitation of cellular p27-RhoA was increased in cells with constitutive PI3K activation and increased in early G₁. Thus T198 phosphorylation not only stabilizes p27 and mislocalizes p27 to the cytoplasm but also promotes RhoA-p27 interaction and RhoA pathway inhibition. These data link p27 phosphorylation at T198 and cell motility. As for other PI3K effectors, RSK1 phosphorylates p27 at T198. Because RSK1 is also activated by MAPK, the increased cell motility and metastatic potential of cancer cells with PI3K and/or MAPK pathway activation may result in part from RSK1 activation, leading to accumulation of p27T198 in the cytoplasm, p27:RhoA binding, inhibition of RhoA/Rock pathway activation, and loss of actomyosin stability.     

Selective inactivation of p27(Kip1) in hematopoietic progenitor cells increases neointimal macrophage proliferation and accelerates atherosclerosis

Excessive proliferation of immune cells and vascular smooth myocytes (VSMCs) contributes to atherosclerosis. We have previously shown that whole-body inactivation of the growth suppressor p27 exacerbates atherosclerosis in apolipoprotein E-null mice (apoE-/-), and this correlated with increased proliferation of arterial macrophages and VSMCs. In the present study, we postulated that targeted disruption of bone marrow (BM) p27 is sufficient to enhance arterial macrophage proliferation and atherosclerosis. To test this hypothesis, sublethally irradiated apoE-/- mice with an intact p27 gene received a BM transplant from either apoE-/- or p27-/-apoE-/- doubly deficient donor mice and challenged with a high-cholesterol diet. Compared with mice that received an apoE-/- BM transplant, reconstitution with p27-/-apoE-/- doubly deficient marrow increased the expression of proliferating cell nuclear antigen in neointimal macrophages and accelerated aortic atherosclerosis, and this correlated with augmented aortic expression of the inflammatory cytokines CCL2/MCP-1 (monocyte chemoattractant protein 1) and CCL5/RANTES (regulated on activation, normal T-cell expressed and secreted). Overall, these findings provide evidence that p27 deficiency in hematopoietic progenitor cells enhances the inflammatory/proliferative response induced by dietary cholesterol and accelerates atherosclerosis.     

Growth regulation by p27Kip1 is abrogated by multiple mechanisms in aggressive malignant lymphomas

The cyclin-dependent kinase inhibitor p27Kip1 is a key regulator of the G1/S transition, and an inverse relationship between p27Kip1 protein expression and proliferation index has been reported in malignant lymphomas. However, a subset of aggressive B-cell lymphomas demonstrates high p27Kip1 expression despite a high proliferation index. The aim of this study was to determine potential mechanisms by which lymphoma cells abrogate the growth inhibitory effect of high p27Kip1. The effect of transforming growth factor-beta (TGF-beta) and serum stimulation on p27Kip1 expression and cyclin E/cdk2 activity was investigated in four lymphoma cell lines, Jurkat, CEM-6, OCI-Ly1 and Nalm-6. Reactive lymphocytes responded to growth inhibitory TGF-beta by inducing p27Kip1 expression, with subsequent accumulation of cells in G0/G1. In contrast, TGF-beta did not alter the level of p27Kip1 in Jurkat, CEM-6 and OCI-Ly1 cells with no change in cyclin E/cdk2-kinase activity. Serum stimulation also did not result in a significant change in p27Kip1 expression. Western blot analysis of subcellular fractions demonstrated cytoplasmic p27Kip1, corroborated by immunocytochemistry in a subset of the lymphoma cells. Sequestration of p27Kip1 by cyclin D3 was observed in the nuclear and cytoplasmic fractions of Nalm-6, OCI-Ly-1 and NCEB cells. These results indicate that multiple mechanisms contribute to the abrogation of growth regulation by unscheduled high p27Kip1 protein expression including deficient response to TGF-beta and serum, sequestration by cyclin D3 and cytoplasmic displacement.     

Up-regulation of p27Kip1 by progestins is involved in the growth suppression of the normal and malignant human endometrial glandular cells

Progestins are known to suppress the growth of normal human endometrial glands and endometrial carcinomas possessing PRs. To elucidate the molecular mechanisms of progestin-induced growth inhibition, the expression and functional involvement of p27Kip1 (p27), a cyclin-dependent-kinase inhibitor, was investigated using cultured normal endometrial glandular cells and endometrial carcinoma cell lines (Ishikawa; PR-positive, KLE; PR-negative). Growth of the normal endometrial glandular cells and Ishikawa cells was suppressed by treatment with progesterone and medroxyprogesterone acetate, respectively, in association with an increase in p27 protein expression. Immunoprecipitation revealed that progestins accelerated the complex formation of p27 and cdk2 in both types of cells. However, treatment with progestins did not show any marked alterations in the mRNA expression of p27 in either normal glandular cells or Ishikawa cells. On the other hand, p27 protein degradation experiments indicated that treatment with progesterone and medroxyprogesterone acetate prolonged the degradation time of the normal endometrial glandular cells and Ishikawa cells, respectively. Forced expression of the p27 protein using a p27 expression plasmid reduced the growth activity of normal endometrial glandular cells. These findings suggest that p27 is functionally involved in progestin-induced growth suppression of normal and malignant endometrial epithelial cells and that up-regulation of the p27 protein by progestins possibly occurs via posttranslational mechanisms.     

Thyroid hormone regulates the cell cycle inhibitor p27Kip1 in postnatal murine Sertoli cells

Thyroid hormone regulates early postnatal Sertoli cell proliferation. Transient neonatal hypothyroidism allows prolonged postnatal Sertoli cell mitogenesis and doubles adult Sertoli cell numbers, testis weight, and sperm production. The mechanism of this effect is unknown. Cell proliferation is stimulated by cyclins and cyclin-dependent kinases and inhibited by cyclin-dependent kinase inhibitors (CDKIs). T(3) regulates the CDKI p27(Kip1) in other cell types, and mice lacking p27(Kip1) have increased testis size. To test the hypothesis that T(3) regulates Sertoli cell mitogenesis by acting through p27(Kip1), we compared expression of p27(Kip1) in Sertoli cells of testes from euthyroid, hypothyroid, and hyperthyroid mice. At postnatal d 5-25, testes were collected and immunostained for p27(Kip1) expression, or Sertoli cells were isolated enzymatically and used for p27(Kip1) Western blotting. p27(Kip1) immunostaining was low in rapidly proliferating 5-d-old Sertoli cells but had increased strongly in nonproliferating 25-d-old Sertoli cells. p27(Kip1) immunostaining was reduced in Sertoli cells from hypothyroid mice compared with euthyroid controls at 10 and 16 d, consistent with increased Sertoli cell proliferation in these mice. Western blotting corroborated the p27(Kip1) immunostaining, and p27(Kip1) expression was greater in Sertoli cells from control compared with hypothyroid mice at postnatal d 10 and 16, but p27(Kip1) expression was comparable by d 25. Hyperthyroidism increased p27(Kip1) immunostaining relative to controls, and Western analysis indicated that Sertoli cells from 10-d-old hyperthyroid mice expressed more p27(Kip1) than control mice. These results indicate that thyroid hormone status affects p27(Kip1) expression in neonatal Sertoli cells, suggesting that T(3) effects on Sertoli cell proliferation may be mediated through this CDKI.     

GATA-2/estrogen receptor chimera regulates cytokine-dependent growth of hematopoietic cells through accumulation of p21(WAF1) and p27(Kip1) proteins

GATA-2 is considered to be essential for the development, maintenance, and function of hematopoietic stem cells (HSCs). However, it was also reported that GATA-2 inhibits the growth of HSCs. To examine the role of GATA-2 in the growth of hematopoietic cells, we introduced an estradiol-inducible form of GATA-2 (GATA-2/estrogen receptor [ER]) into interleukin 3 (IL-3)-dependent cell lines, Ba/F3, 32D, and FDC-P1. Estradiol-induced GATA-2 suppressed c-myc mRNA expression and inhibited IL-3-dependent growth in these clones. As for this mechanism, GATA-2 was found to inhibit ubiquitin/proteasome-dependent degradation of p21(WAF1) and p27(Kip1) and to induce their accumulation by repressing the expression of Skp2 and Cul1, both of which are components of the ubiquitin ligase for p21(WAF1) and p27(Kip1). Overexpression of c-myc restored the expression of Skp2 and Cul1 mRNA, reduced the amounts of p21(WAF1) and p27(Kip1) proteins, and canceled GATA-2-induced growth suppression, suggesting that down-regulation of c-myc expression may be primarily responsible for GATA-2-induced growth suppression. Next, we transduced retrovirus containing GATA-2/ER into murine bone marrow mononuclear cells (MNCs) and stem/progenitor (Sca-1(+)Lin(-)) cells. GATA-2/ER suppressed cytokine-dependent growth of MNCs and Sca-1(+)Lin(-) cells by about 70%, which was also accompanied by the reduced expression of c-myc, Skp2, and Cul1 mRNA and the accumulation of p21(WAF1) and p27(Kip1) proteins. In addition, the amount of GATA-2 protein was found to decline in hematopoietic stem/progenitor cells that were promoted to enter cell cycle by the stimulation with cytokines. These results suggest that GATA-2 may regulate expression levels of p21(WAF1) and p27(Kip1), thereby contributing to the quiescence of hematopoietic stem/progenitor cells.     

Transforming growth factor beta 1 mediates cell-cycle arrest of primitive hematopoietic cells independent of p21(Cip1/Waf1) or p27(Kip1).

The regulation of stem cell proliferation is a poorly understood process balancing rapid, massive blood cell production in times of stress with maintenance of a multipotent stem cell pool over decades of life. Transforming growth factor beta 1 (TGF-beta 1) has pleiotropic effects on hematopoietic cells, including the inhibition of primitive cell proliferation. It was recently demonstrated that the cyclin-dependent kinase inhibitors, p21(Cip1/Waf1) (p21) and p27(Kip1) (p27), can inhibit the proliferation of hematopoietic stem cells and progenitor cells, respectively. The relation of TGF-beta 1 stimulation to p21 and p27 was examined using a fine-mapping approach to gene expression in individual cells. Abundant TGF-beta 1 expression and p21 expression were documented in quiescent, cytokine-resistant hematopoietic stem cells and in terminally differentiated mature blood cells, but not in proliferating progenitor cell populations. TGF-beta 1 receptor (T beta R II) was expressed ubiquitously without apparent modulation. Cell- cycle-synchronized 32D cells exposed to TGF-beta 1 demonstrated a marked antiproliferative effect of TGF-beta 1, yet neither the level of p21 mRNA nor the protein level of either p21 or p27 was altered. To corroborate these observations in primary cells, bone marrow mononuclear cells derived from mice engineered to be deficient in p21 or p27 were assessed. Progenitor and primitive cell function was inhibited by TGF-beta 1 equivalently in -/- and +/+ littermate controls. These data indicate that TGF-beta 1 exerts its inhibition on cell cycling independent of p21 and p27 in hematopoietic cells. TGF-beta 1 and p21 or p27 participate in independent pathways of stem cell regulation, suggesting that targeting each may provide complementary strategies for enhancing stem or progenitor cell expansion and gene transduction.     

CacyBP/SIP nuclear translocation regulates p27Kip1 stability in gastric cancer cells

AIM: To investigate the mechanism of calcyclin binding protein/Siah-1 interacting protein (CacyBP/SIP) nuclear translocation in promoting the proliferation of gastric cancer (GC) cells.METHODS: The effect of CacyBP/SIP nuclear translocation on cell cycle was investigated by cell cycle analysis. Western blot analysis was used to assess the change in expression of cell cycle regulatory proteins and proteasome-mediated degradation of p27Kip1. Co-immunoprecipitation (co-IP) analysis was performed to examine the binding of CacyBP/SIP with Skp1. A CacyBP/SIP truncation mutant which lacked the Skp1 binding site was constructed and fused to a fluorescent protein. Subsequently, the effect on Skp1 binding with the fusion protein was examined by co-IP, while localization of fluorescent fusion protein observed by confocal laser microscopy, and change in p27Kip1 protein expression assessed by Western blot analysis.RESULTS: CacyBP/SIP nuclear translocation induced by gastrin promoted progression of GC cells from G1 phase. However, while CacyBP/SIP nuclear translocation was inhibited using siRNA to suppress CacyBP/SIP expression, cell cycle was clearly inhibited. CacyBP/SIP nuclear translocation significantly decreased the level of cell cycle inhibitor p27Kip1, increased Cyclin E protein expression whereas the levels of Skp1, Skp2, and CDK2 were not affected. Upon inhibition of CacyBP/SIP nuclear translocation, there were no changes in protein levels of p27Kip1 and Cyclin E, while p27Kip1 decrease could be prevented by the proteasome inhibitor MG132. Moreover, CacyBP/SIP was found to bind to Skp1 by immunoprecipitation, an event that was abolished by mutant CacyBP/SIP, which also failed to stimulate p27Kip1 degradation, even though the mutant could still translocate into the nucleus.CONCLUSION: CacyBP/SIP nuclear translocation contributes to the proliferation of GC cells, and CacyBP/SIP exerts this effect, at least in part, by stimulating ubiquitin-mediated degradation of p27Kip1.     

p57(Kip2) cooperates with Nurr1 in developing dopamine cells

Cyclin-dependent kinase inhibitors of the Cip/Kip family play critical roles in regulating cell proliferation during embryogenesis. However, these proteins also influence cell differentiation by mechanisms that have remained unknown. Here we show that p57Kip2 is expressed in postmitotic differentiating midbrain dopamine cells. Induction of p57Kip2 expression depends on Nurr1, an orphan nuclear receptor that is essential for dopamine neuron development. Moreover, analyses of p57Kip2 gene-targeted mice revealed that p57Kip2 is required for the maturation of midbrain dopamine neuronal cells. Additional experiments in a dopaminergic cell line demonstrated that p57Kip2 can promote maturation by a mechanism that does not require p57Kip2-mediated inhibition of cyclin-dependent kinases. Instead, evidence indicates that p57Kip2 functions by a direct protein-protein interaction with Nurr1. Thus, in addition to its established function in control of proliferation, these results reveal a mechanism whereby p57Kip2 influences postmitotic differentiation of dopamine neurons.     

Low expression of the cell cycle inhibitor p27Kip1 in normal corticotroph cells, corticotroph tumors, and malignant pituitary tumors.

The cell cycle is regulated by a number of inhibitors, including p27Kip1 (p27), which belongs to the kip1 family. By binding to the cyclin/cyclin-dependent kinase complexes, it regulates progression of G1 to S phase in the cell cycle. It has been reported that p27 knockout mice develop multiorgan hyperplasia and intermediate lobe pituitary tumors secreting ACTH. Previously, we and others have been unable to show any consistent change in messenger RNA expression or genomic mutations for p27 in human corticotroph adenomas. However, dysregulation at the protein level has been reported in nonendocrine tumors, and we, therefore, investigated the expression of p27 in a range of benign and metastatic pituitary tumors. We studied a total of 107 pituitaries, including normal pituitary (n = 20), Cushing's disease (n = 21), acromegaly (n = 19), nonfunctioning adenomas (n = 18), prolactinomas (n = 7), TSH-omas (n = 2), FSH-omas (n = 6), aggressive tumors showing invasiveness and recurrence (n = 9), and metastatic pituitary carcinomas (n = 5). Using standard immunohistochemical techniques with a highly specific monoclonal antibody, p27 expression was determined quantitatively as the percentage of cells showing strongly positive, weak, or negative staining. In each sample, approximately 500 cells were analyzed. We also analyzed normal pituitaries using double-labeling for p27 and each of the pituitary hormones to characterize the expression of p27 in each cell type. p27 was expressed in normal pituitary cells; in tumors expressing GH, prolactin, TSH, and FSH; and in aggressive tumors, but markedly reduced expression of p27 was seen in corticotroph tumors and pituitary carcinomas. In the normal pituitary, somatotroph, lactotroph, and thyrotroph cells showed strong p27 staining, whereas normal corticotroph cells showed a much lower level of p27 staining (P < 0.001). Somatotroph, lactotroph, gonadotroph, and thyrotroph adenomas showed a lower level of p27 expression compared with normal somatotrophs (P = 0.02), lactotrophs (P = 0.03), gonadotrophs (P = 0.01), and thyrotrophs, respectively, whereas the lower level of p27 expression present in normal corticotrophs virtually disappeared in corticotroph adenomas (P = 0.001). We conclude that pituitary adenomas show a lower level of p27 protein expression than the normal cells from which they are derived, with malignant transformation leading to complete loss of p27 immunoreactivity. Corticotrophs are quite different to other pituitary cell types in terms of p27 immunoreactivity because both normal and tumorous corticotrophs have low p27 staining, and we speculate that this may relate to their inherent control mechanisms.     

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.     

Gene disruption of p27(Kip1) allows cell proliferation in the postnatal and adult organ of corti

Hearing loss is most often the result of hair-cell degeneration due to genetic abnormalities or ototoxic and traumatic insults. In the postembryonic and adult mammalian auditory sensory epithelium, the organ of Corti, no hair-cell regeneration has ever been observed. However, nonmammalian hair-cell epithelia are capable of regenerating sensory hair cells as a consequence of nonsensory supporting-cell proliferation. The supporting cells of the organ of Corti are highly specialized, terminally differentiated cell types that apparently are incapable of proliferation. At the molecular level terminally differentiated cells have been shown to express high levels of cell-cycle inhibitors, in particular, cyclin-dependent kinase inhibitors [Parker, S. B., et al. (1995) Science 267, 1024-1027], which are thought to be responsible for preventing these cells from reentering the cell cycle. Here we report that the cyclin-dependent kinase inhibitor p27(Kip1) is selectively expressed in the supporting-cell population of the organ of Corti. Effects of p27(Kip1)-gene disruption include ongoing cell proliferation in postnatal and adult mouse organ of Corti at time points well after mitosis normally has ceased during embryonic development. This suggests that release from p27(Kip1)-induced cell-cycle arrest is sufficient to allow supporting-cell proliferation to occur. This finding may provide an important pathway for inducing hair-cell regeneration in the mammalian hearing organ.     

Expression of phosphorylated p27(Kip1) protein and Jun activation domain-binding protein 1 in human pituitary tumors

The cyclin-dependent kinase inhibitor p27(Kip1) (p27) plays a pivotal role in controlling cell proliferation during development and tumorigenesis. p27 has been implicated in pituitary tumorigenesis in studies of knockout mice and in analyses of human pituitary tumor samples. In this study, we further explored the role of p27 in human pituitary tumors by measuring levels of phosphorylated p27 (P-p27), and also Jun activation domain-binding protein 1 (Jab1), which is thought to facilitate the phosphorylation and degradation of p27, in normal pituitary tissue (n = 21), pituitary adenomas (n = 75), and pituitary carcinomas (n = 10). The amount of p27 protein in corticotroph adenomas and pituitary carcinomas was much lower than that in normal pituitary tissue or other types of pituitary adenoma. Nuclear P-p27 protein levels were significantly decreased in the adenomas, compared with the normals, and were much lower in the carcinomas, compared with either normal pituitary tissue or pituitary adenomas. However, P-p27 levels in corticotroph adenomas were similar to normal pituitary tissue, thus demonstrating a greatly increased ratio of P-p27 to p27 specifically in corticotroph tumors. No difference was found in Jab1 protein levels in either corticotroph tumors or other pituitary adenomas, compared with normal tissue, but there was a small but significant increase in Jab1 levels in carcinomas. Corticotroph and metastatic tumors both showed a significantly higher Ki-67 labeling index than normal pituitary or other types of pituitary adenomas, and in general the Ki-67 labeling index was negatively correlated with p27 nuclear staining. The amount of p27 and Jab1 mRNA was positively correlated in all pituitary samples studied but did not correlate with the changes in immunostaining. Our findings suggest that in corticotroph tumors there is an accentuated phosphorylation of p27 into P-p27, possibly related to increased cyclin E expression, whereas both p27 and P-p27 are subject to increased degradation in pituitary carcinomas. Such variations in phosphorylation may play a role in pituitary tumorigenesis, but modulation of Jab1 is unlikely to be important in the pathogenesis of pituitary adenomas.