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.     

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.     

HIV-1 exploits CCR5 conformational heterogeneity to escape inhibition by chemokines

CC chemokine receptor 5 (CCR5) is a receptor for chemokines and the coreceptor for R5 HIV-1 entry into CD4⁺ T lymphocytes. Chemokines exert anti–HIV-1 activity in vitro, both by displacing the viral envelope glycoprotein gp120 from binding to CCR5 and by promoting CCR5 endocytosis, suggesting that they play a protective role in HIV infection. However, we showed here that different CCR5 conformations at the cell surface are differentially engaged by chemokines and gp120, making chemokines weaker inhibitors of HIV infection than would be expected from their binding affinity constants for CCR5. These distinct CCR5 conformations rely on CCR5 coupling to nucleotide-free G proteins (^NFG proteins). Whereas native CCR5 chemokines bind with subnanomolar affinity to ^NFG protein-coupled CCR5, gp120/HIV-1 does not discriminate between ^NFG protein-coupled and uncoupled CCR5. Interestingly, the antiviral activity of chemokines is G protein independent, suggesting that “low-chemokine affinity” ^NFG protein-uncoupled conformations of CCR5 represent a portal for viral entry. Furthermore, chemokines are weak inducers of CCR5 endocytosis, as is revealed by EC₅₀ values for chemokine-mediated endocytosis reflecting their low-affinity constant value for ^NFG protein-uncoupled CCR5. Abolishing CCR5 interaction with ^NFG proteins eliminates high-affinity binding of CCR5 chemokines but preserves receptor endocytosis, indicating that chemokines preferentially endocytose low-affinity receptors. Finally, we evidenced that chemokine analogs achieve highly potent HIV-1 inhibition due to high-affinity interactions with internalizing and/or gp120-binding receptors. These data are consistent with HIV-1 evading chemokine inhibition by exploiting CCR5 conformational heterogeneity, shed light into the inhibitory mechanisms of anti–HIV-1 chemokine analogs, and provide insights for the development of unique anti–HIV molecules.     

Differential expression of cyclin-dependent kinase inhibitors, p27Kip1 and p57Kip2, by corticotropin in rat adrenal cortex

An important role for the cyclin-dependent kinase inhibitors (CDKIs), p27Kip1 and p57Kip2, in the proliferation and differentiation of adrenal cells has been suggested by their knockout mice, which display adrenal hyperplasia. Adrenal development and function are primarily regulated by ACTH. In the present study, we investigated the effects of ACTH on the expression of p27Kip1, p57Kip2 and proliferating cell nuclear antigen (PCNA) in rat adrenals. Male Wistar rats were treated with dexamethasone (Dex) to inhibit endogenous ACTH secretion. ACTH was then administered to the rats, and the adrenals were examined by Western blot and immunohistochemical analyses. Dex treatment induced shrinkage of adrenals where no PCNA-expressing cells were detected, but most of the cells expressed p27Kip1. Subsequent ACTH treatment resulted in the marked suppression of p27Kip1 expression, specifically in adrenocortical cells at 12 h after the stimulus. At 48 h, the p27Kip1 suppression still continued in the cortex, while the PCNA-expressing cells appeared mainly around the zona glomerulosa and increased at 72 h. At this time, the p27Kip1-expressing cells also appeared in the same zone. In contrast to p27Kip1, the expression of p57Kip2 was not detected in the Dex-treated adrenal. However, its expression was markedly induced by ACTH in the zona glomerulosa at 48 and 72 h. The results demonstrate that the primary site for mitogenic action of ACTH in rat adrenocortex is the zona glomerulosa, and that ACTH modulates proliferation of adrenocortical cells by regulating p27Kip1 and p57Kip2 expression in a time- and site-specific manner.     

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.     

Germline CDKN1B/p27Kip1 mutation in multiple endocrine neoplasia

CONTEXT: Germline mutations in the MEN1 gene predispose to multiple endocrine neoplasia type 1 (MEN1) syndrome, but in up to 20-25% of clinical MEN1 cases, no MEN1 mutations can be found. Recently, a germline mutation in the CDKN1B gene, encoding p27(Kip1), was reported in one suspected MEN1 family with two acromegalic patients. OBJECTIVE: Our objective was to evaluate the role of CDKN1B/p27(Kip1) in human tumor predisposition in patients clinically suspected of MEN1 but testing negative for MEN1 germline mutation as well as in familial and sporadic acromegaly/pituitary adenoma patients. DESIGN: Genomic DNA was analyzed for germline mutations in the CDKN1B/p27(Kip1) gene by PCR amplification and direct sequencing. SETTING: The study was conducted at nonprofit academic research and medical centers. PATIENTS: Thirty-six Dutch and one German suspected MEN1 patient, who previously tested negative for germline MEN1 gene mutations, were analyzed. In addition, 19 familial and 50 sporadic acromegaly/pituitary adenoma patients from Europe and the United States were included in the study. MAIN OUTCOME MEASURES: We analyzed germline CDKN1B/p27(Kip1) mutations in individuals with pituitary adenoma and MEN1-like features. RESULTS: A heterozygous 19-bp duplication (c.59_77dup19) leading to a truncated protein product was identified in one Dutch patient with suspected MEN1 phenotype, pituitary adenoma, carcinoid tumor, and hyperparathyroidism (one of 36, 2.8%). No mutations were detected in either familial or sporadic acromegaly/pituitary adenoma patients. CONCLUSIONS: Our results support the previous finding that germline CDKN1B/p27(Kip1) mutations predispose to a human MEN1-like condition. However, such mutations appear uncommon in suspected MEN1 cases and rare or nonexistent in familial or sporadic acromegaly/pituitary adenoma patients.     

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.     

Role of the cyclin-dependent kinase inhibitor p27(Kip1) in androgen-induced inhibition of CAMA-1 breast cancer cell proliferation

Androgens are known to inhibit the growth of breast cancer cells, but the molecular mechanism of androgen-induced growth inhibition remains unknown. To address this question, we examined functional and quantitative alterations in cell cycle regulators in the E-responsive CAMA-1 breast cancer cell line. We report here that the androgen 5 alpha-dihydrotestosterone inhibits the proliferation of CAMA-1 breast cancer cells. This inhibition of cell proliferation was dose dependent, and maximal inhibition of E2-stimulated proliferation was observed at the concentration of 1 nM 5 alpha-dihydrotestosterone. 5 alpha-Dihydrotestosterone-induced growth arrest was accompanied by an increase in the proportion of cells in the G(1) phase of the cell cycle. Compared with control cells, 5 alpha-dihydrotestosterone-treated cells showed an increase in the relative proportion of hypophosphorylated retinoblastoma protein consistent with G(1) arrest. In CAMA-1 cells, 5 alpha-dihydrotestosterone caused an accumulation of the cyclin-dependent kinase inhibitor p27(Kip1). Cyclin E-cyclin-dependent kinase-2-associated kinase activity was strongly inhibited in 5 alpha-dihydrotestosterone-treated cells, and immunoprecipitation-Western blot analysis showed an increase in the amount of p27(Kip1) associated with cyclin E-cyclin-dependent kinase-2 complexes. These results suggest that inhibition of breast cancer cell growth by androgens may be mediated at least in part by inactivation of the cyclin E-cyclin-dependent kinase-2 complexes by p27(Kip1).     

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.     

Cyclin-dependent kinase inhibitor p27Kip1, but not p21WAF1/Cip1, is required for inhibition of hypoxia-induced pulmonary hypertension and remodeling by heparin in mice

Heparin has growth inhibitory effects on pulmonary artery smooth muscle cell (PASMC) in vitro and in vivo. However, the mechanism has not been fully defined. In this study, we investigated the role of cyclin-dependent kinase inhibitors, p21(WAF1/cip1) (p21) and p27Kip1 (p27), in the inhibitory effect of heparin on PASMC proliferation in vitro and on hypoxia-induced pulmonary hypertension in vivo using p21 and p27-null mice. In vitro, loss of the p27 gene negated the inhibitory effect of heparin on PASMC proliferation, but p21 was not critical for this inhibition. In vivo, heparin significantly inhibited the development of hypoxia-induced pulmonary hypertension and remodeling, as evidenced by decreased right ventricular systolic pressure, ratio of right ventricular weight to left ventricle plus septum weight, and percent wall thickness of pulmonary artery, in p21(+/+), p21(-/-), p27(+/+), and p27(+/-), but not in p27(-/-) mice. We also observed that hypoxia decreased p27 expression significantly in mouse lung, which was restored by heparin. Heparin inhibited Ki67 proliferative index in terminal bronchial vessel walls in p27(+/+) and p27(+/-), but not in p27(-/-) mice exposed to hypoxia. Therefore, we conclude that the cyclin-dependent kinase inhibitor p27, but not p21, is required for the inhibition of hypoxic pulmonary vascular remodeling by heparin.     

NPM/ALK downregulates p27Kip1 in a PI-3K-dependent manner

OBJECTIVES: Anaplastic large-cell lymphomas (ALCL) are frequently associated with the chromosomal translocation t(2;5) (p23;q35) resulting in the NPM/ALK fusion gene that encodes a constitutively activated tyrosine kinase. We showed that NPM/ALK stimulated cell proliferation and that PI-3K/AKT pathway played an important role in this effect. p27Kip1 is a member of the CDK family inhibitory proteins regulating the entry into S phase. It was reported that p27Kip1 function is impaired in many tumors. In this study we investigated the role of PI-3K/AKT in NPM/ALK-dependent downregulation of p27Kip1 protein. MATERIALS AND METHODS: To investigate this phenomenon the pro-B cell line BaF3, BaF3 cell line stably expressing NPM/ALK, and ALCL SUP-M2 cell line were used. The p27Kip1 protein expression before and after LY294002, wortmannin, or epoxomicin treatment and phosphorylation status of AKT were measured in parental and NPM/ALK+ cells by Western analysis. Also, the localization of p27Kip1 protein was analyzed by fractionation and immunoblotting. RESULTS: p27Kip1 was found to be downregulated in NPM/ALK-transformed hematopoietic cells, but inhibition of proteasome-dependent degradation pathway by epoxomicin reversed this effect. In addition, treatment of NPM/ALK+ cells with LY294002, the PI-3K inhibitor, caused elevation of p27Kip1 protein expression and its nuclear localization. CONCLUSIONS: Taken together, we postulate that NPM/ALK-PI-3K pathway stimulates cell proliferation by regulation of the expression and nuclear localization of p27Kip1.     

In utero exposure to dioxin causes neocortical dysgenesis through the actions of p27Kip1

Dioxins have been reported to exert various adverse effects, including cell-cycle dysregulation in vitro and impairment of spatial learning and memory after in utero exposure in rodents. Furthermore, children born to mothers who are exposed to dioxin analogs polychlorinated dibenzofurans or polychlorinated biphenyls have developmental impairments in cognitive functions. Here, we show that in utero exposure to dioxins in mice alters differentiation patterns of neural progenitors and leads to decreased numbers of non-GABAergic neurons and thinner deep neocortical layers. This reduction in number of non-GABAergic neurons is assumed to be caused by accumulation of cyclin-dependent kinase inhibitor p27^Kip1 in nuclei of neural progenitors. Lending support to this presumption, mice lacking p27^Kip1 are not susceptible to in utero dioxin exposure. These results show that environmental pollutants may affect neocortical histogenesis through alterations of functions of specific gene(s)/protein(s) (in our case, dioxins), exerting adverse effects by altering functions of p27^Kip1.     

Rapamycin attenuates atherosclerosis induced by dietary cholesterol in apolipoprotein-deficient mice through a p27 Kip1 -independent pathway

Activation of immune cells and dysregulated growth and motility of vascular smooth muscle cells contribute to neointimal lesion development during the pathogenesis of vascular obstructive disease. Inhibition of these processes by the immunosuppressant rapamycin is associated with reduced neointimal thickening in the setting of balloon angioplasty and chronic graft vessel disease (CGVD). In this study, we show that rapamycin elicits a marked reduction of aortic atherosclerosis in apolipoprotein E (apoE)-null mice fed a high-fat diet despite sustained hypercholesterolemia. This inhibitory effect of rapamycin coincided with diminished aortic expression of the positive cell cycle regulatory proteins proliferating cell nuclear antigen and cyclin-dependent kinase 2. Moreover, rapamycin prevented the normal upregulation of the proatherogenic monocyte chemoattractant protein-1 (MCP-1, CCL2) seen in the aorta of fat-fed mice. Previous studies have implicated the growth suppressor p27(Kip1) in the antiproliferative and antimigratory activities of rapamycin in vitro. However, our studies with fat-fed mice doubly deficient for p27(Kip1) and apoE disclosed an antiatherogenic effect of rapamycin comparable with that found in apoE-null mice with an intact p27(Kip1) gene. Taken together, these findings extend the therapeutic application of rapamycin from the restenosis and CGVD models to the setting of diet-induced atherosclerosis. Our results suggest that rapamycin-dependent atheroprotection occurs through a p27(Kip1)-independent pathway that involves reduced expression of positive cell cycle regulators and MCP-1 within the arterial wall.     

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.     

A Nedd8 conjugation pathway is essential for proteolytic targeting of p27Kip1 by ubiquitination

Temporal control of p27(Kip1) (p27) degradation imposes periodicity in its activity during cell cycle progression and its accumulation during cell cycle exit. Degradation of p27 is initiated by phosphorylation of p27 at Thr-187, which marks the protein for ubiquitination by SCF(Skp2) and subsequent proteolysis by the 26S proteasome. Here we show that the p27 ubiquitination activity in cell extracts depends on the presence of the ubiquitin-like protein Nedd8 and enzymes that catalyze Nedd8 conjugation to proteins. Moreover, we show that reconstitution of the p27 ubiquitination activity of recombinant SCF(Skp2) also requires Nedd8 conjugation pathway components. Inactivation of the Nedd8 conjugation pathway by a dominant negative mutant of the Nedd8-conjugating enzyme Nce1/Ubc12 blocks the ubiquitination and degradation of p27 in cell extracts. Consistent with a role in cell-cycle progression, Nedd8 is expressed in proliferating cells and is itself down-regulated upon cellular differentiation. These results suggest that the Nedd8 conjugation pathway may regulate the turnover of p27(Kip1), independently of p27 phosphorylation, and further establishes the identity of protein components involved in p27 ubiquitination. Finally, these findings provide a direct demonstration of a function for Nedd8 in a biological process.     

Reduced expression levels of the cell-cycle inhibitor p27Kip1 in human pituitary adenomas

The molecular mechanisms leading to increased cellular proliferation rates and, thus, tumor formation in the anterior pituitary gland are poorly understood. The cyclin-dependent kinase inhibitor p27Kip1 is a key molecule regulating the G1 phase of the cell cycle in many cell types. Furthermore, it was shown that p27 knock-out mice develop pro-opiomelanocortin-positive pituitary tumors. In an effort to clarify the role of p27 in the normal and tumorous human pituitary, we studied the expression of p27 by immunohistochemistry, using a highly specific mouse monoclonal anti-human p27 antibody. Normal pituitaries and 54 pituitary adenomas (twelve somatotrope adenomas, nine prolactinomas, twelve corticotrope adenomas, three TSH-producing tumors, six gonadotrope adenomas, six null cell adenomas, and six oncocytomas) were analyzed. p27 expression was determined semiquantitatively with regard to both the percentage of positive cells and the intensity of the staining. Normal human pituitaries showed strong expression of p27 in most nuclei. In contrast, the levels of p27 were reduced in the majority of the tumors analyzed. Twenty-two tumors (six somatotrope adenomas, five prolactinomas, four corticotrope adenomas, two TSH-producing tumors, two gonadotrope adenomas, and three null cell adenomas) were completely p27-negative. In 18 tumors, p27 expression was found in < or = 10% of the cells. In the other ten tumors, 11-80% of the cells were p27-positive. In summary, we were able to demonstrate reduced expression levels of the cell-cycle inhibitor p27 in tumors derived from all pituitary cell types. Our data indicate that p27 may be an important regulator of cellular proliferation in the anterior pituitary, the underexpression of which could play a role in pituitary tumorigenesis.