Vitamin D arrests thyroid carcinoma cell growth and induces p27 dephosphorylation and accumulation through PTEN/akt-dependent and -independent pathways

We investigated the effects of 1,25-dihydroxycholecalciferol vitamin D(3) (VD) and its noncalciomimetic analog EB1089 on thyroid carcinoma cell growth. VD and EB1089 exhibited anti-proliferative effects in a dose-dependent manner as determined by [(3)H]thymidine incorporation and MIB-1 immunolabeling. VD or EB1089 resulted in similar G(1)-phase arrest. Neither apoptosis nor differentiation was affected. VD and EB1089 induced increased nuclear protein expression of the cyclin-dependent kinase inhibitor, p27(kip1) (p27). VD/EB1089 effects paralleled but were not additive to those of the proteasome inhibitor LLnL, consistent with reduced p27 degradation. As p27 phosphorylation and association with Skp2 is a key step in its degradation, we examined the effects of VD/EB1089 on this reaction. Despite increased total p27, the pThr content of p27 remained unaffected, an effect confirmed by diminished association with Skp2 as well as in situ phosphorylation. Moreover, phosphatase inhibition abrogated the effect of VD/EB1089 on p27 accumulation consistent with a role for phosphatase action in mediating this VD effect. Although VD/EB1089 resulted in comparable increases in p27 in WRO and NPA cells, only WRO but not NPA cells demonstrated a change in the phosphatase PTEN and its downstream target pAkt/PKB in response to VD/EB1089. Transfection of PTEN resulted in p27 accumulation and was partially additive to the effect of VD/EB1089. Moreover, treatment with PI-3 kinase inhibitors decreased pAkt/PKB and increased p27 in both WRO and NPA cells highlighting the potential role of this downstream pathway in regulating p27 in the thyroid. These findings point to a novel mechanism of action for VD/EB1089 inhibition of thyroid carcinoma cell growth by p27 hypophosphorylation, diminished association with Skp2, and consequent accumulation. This effect can be mediated but is not essentially dependent on the phosphatase PTEN/Akt/PKB pathway. These properties support the potential utility of VD analogs in the treatment of thyroid carcinomas irrespective of their PTEN/pAkt status.     

Aberrant DNA methylation of cyclin D2 and p27 genes in rodent pituitary tumor cell lines correlates with specific gene expression

We previously reported that increased DNA methylation was an important mechanism of silencing the p27 gene in some pituitary tumor cell lines [1]. DNA methylation correlated inversely with p27 gene expression. The p27 and cyclin D2 genes are located in the same region of mouse chromosome 6, rat chromosome 4, and human chromosome 12p13. Because both genes are located in the same gene cluster, we investigated whether methylation was a principal mechanism regulating cyclin D2 as well as p27 expression in rodent pituitary cell lines. Bisulfite genomic sequencing showed that the normally unmethylated cytosines of the p27 gene in normal pituitary (NP) were extensively methylated in GH3 and GHRH-CL1 cells, but not in AtT 20, αT₃-1 and LβT₂ cells; but cyclin D2 was extensively inactivated in various pituitary tumor cell lines by increased DNA methylation. These abnormalities of methylation in p27 and cyclin D2 genes occurred with different frequencies in five pituitary tumor cell lines with 100% (5/5) methylation of the cyclin D2 gene and 40% (2/5) methylation of the p27 gene. Treatment with the methyl transferase inhibitor 5′-aza-2′-deoxycytidine (AZAdC) increased expression of cyclin D2 and p27 in GH3 and GHRH-CL1 pituitary tumor cells. There was a correlation between hypermethylation and gene expression. GH₃ tumors implanted into Wistar-Furth rats in vivo did not change the methylation status of the p27 and cyclin D2 genes. These data indicate a coordinately reduced expression of these two linked genes in most rodent pituitary tumor cell lines and suggest that methylation of cyclin D2 and p27 might occur in a “hot spot” in this gene-rich cluster. Supported in part by NIH CA 37231 and CA 42951     

p27 Kip1 protein expression in Hashimoto's thyroiditis

AIMS: Hashimoto's thyroiditis (HT) is an autoimmune disease in which both proliferation and apoptosis are enhanced. p27(Kip1) protein protects tissues from disease mechanisms that involve excessive cell proliferation and apoptosis. This study investigated whether there is loss of p27(Kip1) expression in HT and whether p27(Kip1) immunoreactivity has any relation to the proliferative indicator Ki-67. Because p27(Kip1) is regulated through either degradation, mediated by the S phase kinase associated protein 2 (Skp2), or sequestration, via D3 cyclin, the expression of these proteins was also investigated. METHODS: Immunohistochemistry was used to assess p27(Kip1), Ki-67, Skp2, and cyclin D3 expression in 19 cases of HT and in 10 normal thyroids. The results were evaluated by image analysis and reported as labelling indices (LIs) in both groups. RESULTS: The p27(Kip1) LI was lower in HT than in normal thyroid (28% v 75%; p < 0.001), whereas Ki-67 (1.13% v 0.13%), Skp2 (0.74% v 0.15%), and cyclin D3 (1.56% v 0.00%) LIs were higher in HT than in normal thyroids (p < 0.001). There was no correlation between p27(Kip1) and the expression of Ki-67, Skp2, and cyclin D3. CONCLUSIONS: p27(Kip1) downregulation is not exclusive to tumours but occurs also in HT, independently of the proliferative status and of changes in Skp2 and cyclin D3 expression. Further investigation is required to understand the mechanisms leading to p27 deregulation because these observations suggest that the regulation of p27(Kip1) expression in epithelial thyroid cells may play a role in HT pathogenesis.     

Downregulation of Skp2 and p27/Kip1 synergistically induces apoptosis in T98G glioblastoma cells

S-Phase kinase associated protein (Skp) 2 is an F-box protein required for substrate recognition of the SCF^Skp2 ubiquitin ligase complex. Skp2 is often overexpressed in transformed cells and in various types of tumors. Downregulation or inhibition of Skp2 inhibits growth of breast cancer cells and small-cell lung carcinoma cells. We downregulated Skp2 in T98G glioblastoma cells using small interfering RNA (siRNA). Downregulation induced p27 and caused growth arrest and apoptosis. Downregulation of both Skp2 and p27 increased apoptosis synergistically. Cyclin E levels and cyclin E-CDK2 kinase activity increased dramatically when both Skp2 and p27 were downregulated. Coincidently, Bcl-2 but not Bcl-xL expression decreased, and caspase-3 was activated. Inhibition of cyclin E-CDK2 kinase activity by forced expression of p21 reversed these effects. Moreover, stable expression of Bcl-2 also abrogated apoptosis induced by downregulation of Skp2 and p27. We suggest that Skp2 in tumor cells suppresses apoptosis through Bcl-2 expression, potentially through regulation of cyclin E-CDK2 activity.     

p16 and p27 are functionally correlated during the progress of hepatocarcinogenesis

The molecular mechanism of the cell-cycle machinery in hepatocellular carcinoma (HCC) has not yet been fully elucidated. Among the various types of cell-cycle regulators, p16 and p27 are now considered to be potent tumor suppressors. p16 is a G1-specific cell-cycle inhibitor that prevents the association of cyclin-dependent kinase (CDK) 4 and CDK6 with cyclin D₁. Many studies have reported that p16 is inactivated not only in aggressive types of HCC but also in preneoplastic liver cirrhosis. In many cases of HCC, p16 is mainly inactivated by extensive CpG methylation, suggesting that epigenetic changes in the p16 gene may be important events during hepatocarcinogenesis. p27, an inhibitor of CDK2, is presently regarded as a potent adverse prognostic factor in many aggressive cancers. It should be noted that some cases of HCC show increased cell proliferation despite the expression of considerable amounts of p27. In these cases, p27 is inactivated by sequestration into cyclin D₁–CDK4-containing complexes. Although the reason for the compositional changes in the p27-containing complexes is unclear, our experimental results indicate that loss of p16 following DNA methylation is closely related to the functional inactivation of p27 in HCC. We suggest that assessment of the p16 status may be useful for a precise prognostic prediction for individuals with HCCs expressing high levels of p27.     

Low expression of p27 protein combined with altered p53 and Rb/p16 expression status is associated with increased expression of cyclin A and cyclin B1 in diffuse large B-cell lymphomas.

The expression of the cyclin-dependent kinase inhibitor (CDKI) p27 protein was investigated in relation to (1) the expression of the cell cycle regulators p53, Rb and p16 and (2) the proliferation profile as determined by the expression of Ki67, cyclin A, and cyclin B1 in 80 cases of de novo diffuse large B-cell lymphomas (DLBCL). P27 expression was low/null in large tumor cells in 58/80 cases and intermediate/high in 22/80 cases. Increased expression of p53 protein was observed in 39/80 cases. Decreased expression of Rb and p16 proteins was mutually exclusive and was observed in 5/80 and 14/80 cases, respectively. The analysis of the p27 expression status (low/null versus intermediate/high) with respect to the p53 and/or Rb/p16 expression status showed that low/null p27 expression was significantly correlated with increased p53 expression (P =.018) and showed a strong trend for correlation with concurrent increased p53 expression and decreased Rb or p16 expression (P =.050). These findings suggest a tendency for concurrent alterations of the cell cycle regulators p27, p53, and Rb or p16 in DLBCL, which might result in impaired tumor growth control. Indeed, the analysis of the combined p27/p53/Rb/p16 expression status with respect to the proliferation profile showed that (1) three alterations in the combined p27/p53/Rb/p16 status (i.e., low/null P27 expression, increased expression of p53, and decreased expression of Rb or p16) were significantly correlated with increased expression of cyclin B1 (P =.005) and (2) two or three alterations were significantly correlated with increased expression of cyclin A (P =.014). These findings suggest combined impairment of a complex cell-cycle control network involving the CDK inhibitor p27, the P53 pathway, and the Rb1 pathway, which exerts a cooperative effect resulting in enhanced tumor cell proliferation.     

DNA Methylation Regulates p27Kip1 Expression in Rodent Pituitary Cell Lines

We previously reported loss of expression of p27^Kip1 (p27) protein in rat GH₃ and mouse GHRH-CL1 pituitary tumor cells compared with normal pituitary (NP). The molecular basis for the loss of expression of p27 protein in GH₃ and GHRH-CL1 cells is unknown. To determine the role of p27 gene methylation in the regulation of the expression of this cell cycle protein, the methylation patterns of p27 in normal and neoplastic pituitary cells was analyzed. Inhibition of DNA methyltransferase (DNA-MTase) with 5-aza-2′-deoxycytidine (AZAdC) induced expression of both p27 protein and mRNA when GH₃ and GHRH-CL1 cells were treated for 7 days in vitro. DNA methylation correlated inversely with the expression of p27 gene products in NP and pituitary tumor cell lines. Bisulfite genomic sequencing analysis showed that the normally unmethylated cytosines in exon 1 in NP and AtT20 cells were extensively methylated in GH₃ and GHRH-CL1 cells. After treatment of GH₃ and GHRH-CL1 cells with 10 μmol/L AZAdC, there were decreased numbers of methylated cytosines (by 60% to 90%) with variable methylation patterns observed by bisulfite genomic sequencing. Analysis of genomic DNA with methylation-sensitive enzymes showed that all SmaI, HhaI, and AvaI enzyme sites of the p27 gene in exon 1 were methylated in GH₃ cells but not in NP, confirming the bisulfite genomic sequencing results. AtT20 cells and a human pituitary null cell adenoma cell line (HP75), which expressed abundant p27, had a methylation pattern similar to the NP. DNA-MTase activity was elevated fourfold in GH₃ cells and twofold in GHRH-CL1 cells compared with DNA-MTase activity in NP and AtT20 cells. These results suggest that increased DNA methylation is another mechanism of silencing of the p27 gene in some pituitary tumors and possibly in other types of neoplasms.     

Role of p27Kip1 protein in the cell cycle and its appearance in lymphoid tissues, particularly non-Hodgkin's B-cell lymphomas. Review

Cell cycle progression is governed by cyclin dependent kinases (CDK) that are activated by cyclin binding and inhibited by CDK inhibitors. Protein p27Kip1 functions as a CDK inhibitor, which controls the progression from G1 to S phase. Further, p27Kip1 may have a positive regulative influence. In nonneoplastic tissues and in the majority of tumors investigated so far, the immunohistochemical positivity of p27Kip1 showed an inversely proportional relationship to the proliferation index. Among B-cell non-Hodgkin lymphomas, the exceptions to this rule are represented by mantle cell lymphoma, hairy cell leukemia, and the immunoblastic Epstein-Barr virus latent membrane antigen positive diffuse large B-cell lymphoma in AIDS patients. The loss of p27Kip1 expression is a negative prognostic factor in numerous tumors, including the majority of B-cell lymphomas.     

CDK inhibitors p18INK4c and p27Kip1 mediate two separate pathways to collaboratively suppress pituitary tumorigenesis

INK4 and CIP/KIP are two distinct families of cyclin-dependent kinase (CDK) inhibitors implicated in mediating a wide range of cell growth control signals. We have created p18^INK4c-deficient mice. These mice develop gigantism and widespread organomegaly. The pituitary gland, spleen, and thymus are disproportionately enlarged and hyperplastic. T and B lymphocytes develop normally in p18-deficient mice, but both exhibit increased cellularity and a higher proliferative rate upon mitogenic stimulation. Loss of p18, like that of p27, but not other CDK inhibitor genes, leads to a gradual progression from intermediate lobe pituitary hyperplasia in young mice to an adenoma by 10 months of age with a nearly complete penetrance. Mice lacking both p18 and p27, like mice chimeric for Rb deficiency, invariably died from pituitary adenomas by 3 months. Hence, p18 and p27 mediate two separate pathways to collaboratively suppress pituitary tumorigenesis, likely by controlling the function of Rb.     

Immunohistochemical analysis of p27 (Kip1) in human pituitary glands and in various types of pituitary adenomas

p27 (Kip1) plays regulatory roles in the cell cycle by inhibiting the activity of cyclin dependent kinases (CDKs). This immunohistochemical study is aimed at elucidating the expression of p27 in human pituitary and in various types of pituitary adenomas in order to clarify its role in the regulation of proliferation. Sixteen normal pituitary glands and 179 human pituitary adenomas were used for immunohistochemical studies. The tissues were fixed in 10% formalin and embedded in paraffin. Indirect peroxidase method was performed after heat-induced antigen retrieval using a monoclonal antibody against p27 protein. p27 protein was expressed in the nuclei of all 16 normal human pituitary glands. p27 protein was also expressed in 128 of 179 cases of pituitary adenomas (71.5%). A marked decrease of p27 expression was noted in ACTH-secreting adenomas, 8/20 (40.0%), compared with other types of pituitary adenomas—GH-secreting adenomas, 35/46 (76.1%); PRL-secreting adenomas, 22/33 (66.7%); TSH-secreting adenomas, 8/11 (72.7%); and nonfunctioning adenomas, 55/69 (79.7%). These results suggest that p27 may play some role in the regulation of proliferation in all types of pituitary adenomas. The lower levels of p27 in ACTH-secreting adenoma is of particular interest with respect to the intermediate lobe-derived pituitary tumor developed in p27 knockout mice.     

PTEN coordinates G(1) arrest by down-regulating cyclin D1 via its protein phosphatase activity and up-regulating p27 via its lipid phosphatase activity in a breast cancer model

The tumour suppressor gene PTEN/MMAC1/TEP1 encodes a dual-specificity phosphatase that recognizes phosphatidylinositol-3,4,5-triphosphate and protein substrates. We have shown previously that over-expression of PTEN in a tetracycline-controlled inducible system blocks cell cycle progression and induces apoptosis in MCF-7 breast cancer cells. Here, we demonstrate that over-expression of wild-type PTEN leads to the suppression of cell growth through the blockade of cell cycle progression, an increase in the abundance of p27, a decrease in the protein levels of cyclin D1 and the inhibition of Akt phosphorylation. In contrast, expression of the phosphatase-dead mutant, C124S, promotes cell growth and has the opposite effect on the abundance of p27, cyclin D1 levels and the phosphorylation of Akt. The G129E mutant, which does not have lipid phosphatase activity but retains protein phosphatase activity, behaves like C124S except that the former causes decreases in cyclin D1 levels similar to wild-type PTEN. Therefore, PTEN exerts its growth suppression through lipid phosphatase-dependent and independent activities and most likely, via the coordinate effect of both protein phosphatase and lipid phosphatase activities. Addition of either estrogen or insulin abrogates PTEN-mediated up-regulation of p27 and partially blocks PTEN-mediated growth suppression, whereas the combination of estrogen and insulin eliminates the alterations of p27 and cyclin D1 and completely blocks PTEN-mediated growth suppression. Our findings demonstrate that PTEN blocks cell cycle progression differentially through down-regulating the positive cell cycle regulator, cyclin D1, by its protein phosphatase activity, and up-regulating the negative cell cycle regulator, p27, by its lipid phosphatase activity.     

Cumulative alterations of p27Kip1‐related cell‐cycle regulators in the development of endometriosis‐associated ovarian clear cell adenocarcinoma

Yamamoto S, Tsuda H, Miyai K, Takano M, Tamai S & Matsubara O
(2010) Histopathology 56, 740–749
Cumulative alterations of p27 ^Kip1 ‐related cell‐cycle regulators in the development of endometriosis‐associated ovarian clear cell adenocarcinoma Aims: To identify the key cell‐cycle dysregulations in the development of endometriosis‐associated ovarian clear cell adenocarcinoma (CCA). Methods and results: Expression of p27^Kip1‐interacting cell‐cycle regulators, such as p27^Kip1 itself, Skp2, cyclin‐dependent kinase subunit 1 (Cks1), cyclin A and cyclin E, and Ki67 labelling index (LI), were analysed by immunohistochemistry in 23 CCAs with 36 endometriotic or atypical endometriotic lesions adjacent to CCA from a cohort of 23 patients, and in 31 cases of solitary endometriosis. The cell‐cycle regulators examined were overexpressed (Skp2, Cks1, cyclin A and cyclin E; P < 0.01, each) or down‐regulated (p27^Kip1, P = 0.044) significantly more frequently in the CCAs than in the adjacent endometriosis. The frequency of Skp2 overexpression was significantly higher in atypical endometriosis than in endometriosis, and the frequency of Skp2 and cyclin A overexpression was significantly higher in CCA than in atypical endometriosis (P < 0.01, each). Mean Ki67 LI increased from endometriosis (8.4%) through atypical endometriosis (21.4%) to CCA (46.9%), with statistical significance between each component (P < 0.01, each). The frequency of cell‐cycle regulator expression and mean Ki67 LIs were not significantly different between solitary endometriosis and endometriosis adjacent to CCA. Conclusions: Alteration of the p27^Kip1‐interacting cell‐cycle regulators appeared strongly involved in the progression of endometriosis‐associated ovarian clear cell carcinogenesis through increasing cell proliferative activity.     

Frequent loss of PTEN expression is linked to elevated phosphorylated Akt levels, but not associated with p27 and cyclin D1 expression, in primary epithelial ovarian carcinomas

PTEN (MMAC1/TEP1), a tumor suppressor gene on chromosome subband 10q23.3, is variably mutated and/or deleted in a variety of human cancers. Germline mutations in PTEN, which encode a dual-specificity phosphatase, have been implicated in at least two hamartoma tumor syndromes that exhibit some clinical overlap, Cowden syndrome and Bannayan-Riley-Ruvalcaba syndrome. Among several series of ovarian cancers, the frequency of loss of heterozygosity (LOH) of markers flanking and within PTEN, is approximately 30 to 50%, and the somatic intragenic PTEN mutation frequency is <10%. In this study, we screened primary adenocarcinomas of the ovary for LOH of polymorphic markers within and flanking the PTEN gene and for intragenic mutations of the PTEN gene and compared them to PTEN expression using immunohistochemistry. Furthermore, we sought to detect the expression of the presumed downstream targets of PTEN, such as P-Akt, p27, and cyclin D1 by immunohistochemistry. LOH at 10q23 was observed in 29 of 64 (45%) cases. Of the 117 samples, 6 somatic intragenic PTEN mutations, 1 germline mutation, and 1 novel polymorphism were found in 7 (6%) patients. Immunostaining of 49 ovarian cancer samples revealed that 13 (27%) were PTEN immunostain-negative, 25 (51%) had reduced staining, and the rest (22%) were PTEN expression-positive. Among the 44 informative tumors assessed for 10q23 LOH and PTEN immunostaining, there was an association between 10q23 LOH and decreased or absent staining (P = 0.0317). Of note, there were five (11%) tumors with neither mutation nor deletion that exhibited no PTEN expression and 10 (25%) others without mutation or deletion but had decreased PTEN expression. Among the 49 tumors available for immunohistochemistry, 28 (57%) showed P-Akt-positive staining, 24 (49%) had decreased p27 staining, and cyclin D1 was overexpressed in 35 (79%) cases. In general, P-Akt expression was inversely correlated with PTEN expression (P = 0.0083). These data suggest that disruption of PTEN by several mechanisms, allelic loss, intragenic mutation, or epigenetic silencing, all contribute to epithelial ovarian carcinogenesis, and that epigenetic silencing is a significant mechanism. The Akt pathway is prominently involved, but clearly not in all cases. Surprisingly, despite in vitro demonstration that p27 and cyclin D1 lies downstream of PTEN and Akt, there was no correlation between p27 and cyclin D1 expression and PTEN or P-Akt status. Thus, in vivo, although PTEN and Akt play a prominent role in ovarian carcinogenesis, p27 and cyclin D1 might not be the primary downstream targets. Alternatively, these observations could also suggest that pathways involving other than Akt, p27 and cyclin D1 that lie downstream of PTEN play roles in ovarian carcinogenesis.     

Inhibition of cell cycle progression via p27Kip1 upregulation and apoptosis induction by an ethanol extract of Rhus verniciflua Stokes in AGS gastric cancer cells

Botanical preparations are widely used by patient with cancer in Korea, Japan and China. Rhus verniciflua Stokes (RVS) has traditionally been used as a medicinal ingredient for the therapy of stomach and uterine cancer. In this study, we showed that exposure to an ethanol extract of RVS (50 microg/ml) resulted in a synergistic inhibitory effect on cell growth in AGS cells. Growth inhibition was related with the inhibition of proliferation and induction of apoptosis. The extract induces G1-cell cycle arrest through the regulation of cyclins, the induction of p27Kip1, and decrease the CDK2 kinase activity. The upregulated p27Kip1 level is caused by protein stability increment by the reduction of Skp2, a key molecule related with p27Kip1 ubiquitination and degradation, and de novo protein synthesis. RVS extract induces apoptosis through the expression of Bax, poly(ADP-ribose) polymerase (PARP) and activation of caspase-3. RVS extract induces G1-cell cycle arrest via accumulation of p27Kip1 controlled by Skp2 reduction and apoptosis passing through an intrinsic pathway in human gastric cancer cells but not in normal cells, therefore we suggest that this extract could be a candidate medicine or compound for the development of novel class of anti-cancer drugs.     

Cyclin A correlates with carcinogenesis and metastasis,and p27(kip1) correlates with lymphatic invasion,in colorectal neoplasms

Cyclin A binds to CDK2 and plays critical roles when cells proliferate; staining for Ki67 can monitor the proliferation. The cyclin A expression pattern remains unclear in colorectal carcinogenesis and remote metastasis, however, and no one has reported on the association of its expression with key clinicopathologic factors in primary cancer. p27(kip1) protein-an extremely important inhibitor of CDK2-seems unchanged as colorectal cancers metastasize to the lymph nodes, a result contrary to that seen in gastric and prostatic cancers. To clarify the role of cyclin A in multistage colorectal neoplasms, cyclin A, CDK2, and Ki67 were immunohistochemically stained in 22 normal mucosa, 9 hyperplastic polyps, 61 adenomas, 197 primary carcinomas, 21 lymph node metastases, and 10 hepatic metastases. To clarify the alteration of p27(kip1) during lymphatic invasion, p27(kip1) was also stained in 21 primary cancers and paired lymph node foci. Situated in nuclei, cyclin A expression gradually increased from mild through moderate to severe dysplasia in adenomas and from normal tissue through hyperplasia to adenoma to early carcinoma. Expression was significantly decreased in the hepatic metastases and in the primary cancers showing venous invasion, deep infiltration, lymph node metastasis, mucinous type, advanced stage, or short postoperative survival time. Elevated cyclin A not only was linked with elevated CDK2 in primary cancers, but also was associated with increased Ki67 in both adenomas and primary carcinomas. Lymph node metastases lost more p27(kip1) than primary foci and hepatic lesions. Thus, dysregulation of cyclin A and its control mechanisms may contribute to colorectal carcinogenesis; abatement of overexpression of cyclin A is associated with hepatic metastasis and cancerous invasion. Loss of p27(kip1) may promote lymph node metastasis.     

Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation

The cellular abundance of the cyclin-dependent kinase (Cdk) inhibitor p27 is regulated by the ubiquitin-proteasome system. Activation of p27 degradation is seen in proliferating cells and in many types of aggressive human carcinomas. p27 can be phosphorylated on threonine 187 by Cdks, and cyclin E/Cdk2 overexpression can stimulate the degradation of wild-type p27, but not of a threonine 187-to-alanine p27 mutant [p27(T187A)]. However, whether threonine 187 phosphorylation stimulates p27 degradation through the ubiquitin-proteasome system or an alternative pathway is still not known. Here, we demonstrate that p27 ubiquitination (as assayed in vivo and in an in vitro reconstituted system) is cell-cycle regulated and that Cdk activity is required for the in vitro ubiquitination of p27. Furthermore, ubiquitination of wild-type p27, but not of p27(T187A), can occur in G1-enriched extracts only upon addition of cyclin E/Cdk2 or cyclin A/Cdk2. Using a phosphothreonine 187 site-specific antibody for p27, we show that threonine 187 phosphorylation of p27 is also cell-cycle dependent, being present in proliferating cells but undetectable in G1 cells. Finally, we show that in addition to threonine 187 phosphorylation, efficient p27 ubiquitination requires formation of a trimeric complex with the cyclin and Cdk subunits. In fact, cyclin B/Cdk1 which can phosphorylate p27 efficiently, but cannot form a stable complex with it, is unable to stimulate p27 ubiquitination by G1 extracts. Furthermore, another p27 mutant [p27(CK-)] that can be phosphorylated by cyclin E/Cdk2 but cannot bind this kinase complex, is refractory to ubiquitination. Thus throughout the cell cycle, both phosphorylation and trimeric complex formation act as signals for the ubiquitination of a Cdk inhibitor.     

Skp2 expression is associated with down-regulation of p27 protein and cell proliferation in salivary adenoid cystic carcinoma

Adenoid cystic carcinoma (ACC) is a malignant salivary gland tumor, which shows frequent recurrence and metastasis, ultimately with a poor outcome. We previously demonstrated that p27 down-regulation is frequently found and is due to an enhancement of its degradation in ACC. In this study, we transfected nondegradable p27 mutant (T187A) and wild-type gene into ACC cell line. Transfection of T187A mutant gene was more effective on inhibition of cell growth of ACC cells, suggesting that aberration of p27 degradation may be present in ACC. As F-box protein S-phase kinase-associated protein 2 (Skp2), which is necessary for ubiquitin-mediated degradation of p27, is involved in p27 down-regulation in various cancers, we examined the Skp2 expression and its association with p27 expression in 50 ACC cases. We found Skp2 expression in 36% of ACC cases and inverse association between the expression of Skp2 and p27. Moreover, Skp2 small interfering ribonucleic acid (siRNA) transfection decreased Skp2 protein and accumulation of p27 protein and inhibited the cell growth of ACC cells in vitro. These findings, overall, suggest that Skp2 may play an important role in ACC development through the down-regulation of p27 and that Skp2 siRNA can be a novel modality of cancer gene therapy for suppression of p27 down-regulation in ACC.     

Epigenetic silencing through DNA and histone methylation of fibroblast growth factor receptor 2 in neoplastic pituitary cells

Four members of the fibroblast growth factor receptor (FGFR) family of tyrosine kinases transduce signals of a diverse group of more than 23 fibroblast growth factor (FGF) ligands. Each prototypic receptor is composed of three immunoglobulin-like extracellular domains, two of which are involved in ligand binding. Alternative RNA splicing of one of two exons results in two different forms of the second half of the third immunoglobulin-like domain, the IIIb or IIIc isoforms. The contribution of each receptor and their isoforms in tumorigenesis remains unknown. In the pituitary, FGFR2 is expressed primarily as the IIIb isoform in normal adenohypophysial cells. In contrast, FGFR2 is significantly down-regulated in mouse corticotroph AtT20 tumor cells where the 5' promoter is methylated. Treatment of AtT20 cells with 5'-azacytidine resulted in FGFR2 re-expression, mainly as the FGFR2-IIIb isoform. Chromatin immunoprecipitation revealed evidence of histone methylation, but not of deacetylation, in the silencing of FGFR2 in AtT20 cells. Exposure of these cells to the cognate FGFR2-IIIb ligand FGF-7 resulted in diminished Rb phosphorylation and accumulation of p21 and p27, indicating diminished cell cycle progression. Examination of primary human pituitary adenomas revealed FGFR2 down-regulation in 52% (11 of 21) of samples and FGFR2 promoter DNA methylation in 45% (10 of 22) of samples. These data highlight the contribution from DNA and histone methylation as epigenetic mechanisms responsible for FGFR2 silencing in pituitary neoplasia.