P27 Kip1 Research Articles

Long non-coding RNA LINC-01572:28 inhibits granulosa cell growth via a decrease in p27 (Kip1) degradation in patients with polycystic ovary syndrome.

BackgroundDisordered folliculogenesis is a key feature of polycystic ovary syndrome (PCOS), but the underlying molecular mechanism remains unclear.MethodsLong non-coding RNA (lncRNA) expression in luteinized granulosa cells (hLGCs) derived from women with and without PCOS were analyzed using microarray and qRT-PCR. Immortalized human granulosa cell lines were cultured for proliferation assays after transfection with the LINC-01572:28 over-expression vector in the presence or absence of p27 siRNA. Protein expression analysis, rescue assays, and RNA immunoprecipitation (RIP) were used to confirm the LINC-01572:28 substrate.FindingsLINC-01572:28 and p27 protein were elevated whereas proliferating cell nuclear antigen protein was decreased in the hLGCs of women with PCOS. LINC-01572:28 expression was positively correlated with basal testosterone levels. Over-expression of LINC-01572:28 inhibited cell proliferation and impeded G1/S transition, which were partially reversed by siRNA-mediated p27 knockdown.InterpretationOur findings, therefore, suggest that LINC-01572:28 suppresses cell proliferation and cell cycle progression by reducing the degradation of p27 protein via SKP2 binding.

S100A7, Jab1, and p27kip1 expression in psoriasis and S100A7 CRISPR-activated human keratinocyte cell line.

Psoriasis, a chronic immune-mediated inflammatory skin disease, is characterized by dysregulated keratinocyte proliferation. The EF-hand calcium binding protein S100A7 has been found to be overexpressed in psoriatic keratinocytes. It is know that S100A7 may interact with Jab1, a cofactor that stabilizes c-Jun. Jab1 is known to downregulate the expression of the cell cycle inhibitor p27Kip1 in some cancer models. In this study, we aimed to investigate the possible interaction between S100A7 and Jab1 and the downstream effects on p27 Kip1 expression in normal human keratinocyte cells transfected with S100A7 CRISPR activation plasmid and in archival psoriatic skin samples. Our results showed that the upregulated S100A7 colocalizes with Jab1 at the nuclear level in transfected cells and psoriatic skin samples. We also showed a differential protein expression of Jab1 between cytoplasmic and nuclear compartments, thus suggesting Jab1 translocation from nucleus to cytoplasm. p27 Kip1 protein expression patterns would imply a translocation from nucleus and a subsequent degradation of this protein. The upregulation of S1007 and its interaction with Jab1 would contribute to the p27 Kip1 -dependent impaired proliferation that characterizes psoriatic skin.

Expression of Jun activation domain-binding protein 1 and p27 Kip1 in endometrial polyps and uterine leiomyomas

Expression of Jun activation domain-binding protein 1 and p27 Kip1 in endometrial polyps and uterine leiomyomas

Clinical application of STR genotyping diagnosis for hydatidiform mole and nonmolar gestation

Objective: To investigate the value of short tandem repeat (STR) genotyping in the diagnostic workup of molar and non-molar gestations with correlation of histological characteristics. Methods: Six hundred and fifty-six cases were selected based on clinically suspected hydropic abortion and/or molar pregnancy from July 2015 to September 2017 at Beijing Obstetrics and Gynecology Hospital. DNA was extracted from dissected chorionic villi and paired maternal endometrial FFPE tissue samples by Simplex OUP™ FFPE DNA Tissue Kit. STR genotyping was performed by PowerPlex 16 HS system. Results: DNA genotyping was informative in 649 of 656 cases, leading to identification of 215 hydatidiform mole gestations and 434 non-molar gestations. Most of non-molar gestations (375 cases, 86.4%) were diploid hydropic abortion. Various trisomy syndromes were found (53 cases, 12.2%), including trisomy 2, 3, 4, 7, 8, 13, 16 and 21. Only 2(0.5%) digynic triploid gestations were detected. Moreover, 4 cases (0.9%) of uniparental disomies (homologous or heterologous) were found. There were 196 cases with histologic diagnostic suspicious of hydatidiform moles were accurate sub-classified. Among them, 59 cases hydatidiform moles were under-diagnosed as diploid hydropic abortions, and 28 cases diploid hydropic abortions were over-diagnosed as hydatidiform moles.Compared with partial moles(PHM), there were no specific histomorphological features between the various types of non-molar gestations and partial moles for definitive diagnostic separation. There was no significant difference in the expression of p57(kip2) among PHM, trisomy and diploid hydropic abortions group (P=0.247). Conclusions: STR genotyping can distinguish non-molar gestations from early hydatidiform moles, and efficiently avoid misdiagnosis based only on histological evaluation. Therefore, using STR genotyping, not only can the overdiagnosis of non-molar pregnancy be avoided, but also individualized management can be offered to patients including monitoring of serum hCG.

Long Non-Coding RNA LINC-01572:28 Inhibits Granulosa Cell Growth Via a Decrease in p27 (Kip1) Degradation in Patients with Polycystic Ovary Syndrome

Disordered folliculogenesis is a key feature of polycystic ovary syndrome (PCOS), but the underlying molecular mechanism remains unclear. Long non-coding RNA (lncRNA) expression in luteinized granulosa cells (hLGCs) derived from women with and without PCOS were analyzed using microarray and qRT-PCR. Immortalized human granulosa cell lines were cultured for proliferation assays after transfection with the LINC-01572:28 over-expression vector in the presence or absence of p27 siRNA. Protein expression analysis, rescue assays, and RNA immunoprecipitation (RIP) were used to confirm the LINC-01572:28 substrate. LINC-01572:28 and p27 protein were elevated whereas proliferating cell nuclear antigen protein was decreased in the hLGCs of women with PCOS. LINC-01572:28 expression was positively correlated with basal testosterone levels. Over-expression of LINC-01572:28 inhibited cell proliferation and impeded G1/S transition, which were partially reversed by siRNA-mediated p27 knockdown. Our findings, therefore, suggest that LINC-01572:28 suppresses cell proliferation and cell cycle progression by reducing the degradation of p27 protein via SKP2 binding. Funding: This research was supported by grants from the National Key Research and Development Program of China(2017YFC1001002, the National Natural Science Foundation (81490743, 81671413 and 81671414), National Institutes of Health (1R01HD085527), Shanghai Municipal Education Commission--Gaofeng Clinical Medicine (20152510) and Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics (17DZ2271100). Declaration of Interests: Authors have no conflicts of interest to declare. Ethical Approval Statement: The study was approved by the ART Ethnics committee of Ren Ji hospital, school of medicine, shanghai Jiao Tong university (number: 2017041411), and informed consent was obtained from all participants.

Octyl gallate and gallic acid isolated from Terminalia bellarica regulates normal cell cycle in human breast cancer cell lines

Herbal medicines stand unique and effective in treating human diseases. Terminalia bellarica (T. bellarica) is a potent medicinal herb, with a wide range of pharmacological activities. The present study was aimed to evaluate the effect of octyl gallate (OG) and gallic acid (GA) isolated from methanolic fruit extract of T. bellirica to inhibit the survival of breast cancer cells (MCF-7 & MDA-MB-231). Both OG & GA exhibited decreased MCF-7 & MDA-MB-231 survival and induced apoptosis, with IC50 value of OG and GA as 40 μM and 80 μM respectively. No toxic effect was observed on normal breast cells (MCF-10A). The compounds inhibited cell cycle progression by altering the expression of the cell cycle regulators (Cyclin D1, D3, CDK-4, CDK-6, p18 INK4, p21Waf-1 and p27 KIP). Octyl gallate was more effective at low concentrations than GA. In-silico results provided stable interactions between the compounds and target proteins. The present investigation proved the downregulation of positive cell cycle regulators and upregulation of negative cell cycle regulators inducing apoptosis in compound-treated breast cancer cells. Hence, both the compounds may serve as potential anticancer agents and could be developed as breast cancer drugs, with further explorations.

Iron overload induces G1 phase arrest and autophagy in murine preosteoblast cells.

This study aimed to investigate the cell cycle arrest and autophagy induced by iron overload in MC3T3-E1 cells. MC3T3-E1 cells were cultured in different concentrations of ferric ammonium citrate (FAC), and Perls' Prussian blue reaction was used to detect the iron levels of the cells. CCK-8 assays were used to detect the growth of MC3T3-E1. The level of reactive oxygen species (ROS) within cells was investigated with DCFH-DA. PI staining was used to analyze the cell cycle distribution of MC3T3-E1 cells. Finally, the expression levels of cell cycle related proteins, autophagy related proteins, AKT, p38 MAPK, Stat3, and their downstream proteins were detected with Western blot assays. The results showed that the iron levels of MC3T3-E1 cells increased with increasing concentrations of FAC. High levels of ferric ion inhibited proliferation of MC3T3-E1 cells and increased their ROS levels. Additionally, iron overload induced G1arrest in MC3T3-E1 cells and down-regulated the expression of Cyclin D1 , Cyclin D3 , CDK2, CDK4 and CDK6, but up-regulated p27 Kip1. In addition, the expression levels of Beclin-1 and LC3 II increased, but that of p62 decreased. Further experiments showed that the phosphorylation of AKT and its downstream proteins p-GSK-3β(Ser9) and p-mTOR (Ser2448) were decreased. The levels of p-p38 and p53 were up-regulated while those of cdc25A and p-ERK 1/2 were down-regulated. Phosphorylation of Stat3 and its downstream proteins was all decreased. These results show that iron overload generates ROS, blocks the PI3K/AKT and Jak/Stat3 signal pathways, and activates p38 MAPK, subsequently inducing G1 arrest and autophagy in MC3T3-E1 cells.

Comparison analysis of Baso Cellular Carcinoma and Spino Cellular Carcinoma - Protein Kinase D1, Wnt/β-catenin and Epithelial to Mesenchimal Transition (markers)

Understanding cancer biology is crucial to the successful diagnosis and application of personalized therapies. Skin carcinogenesis is a multi-step process. The first step is the development of potentially malignant disorders (PMDs) known as leukoplakia, erytroplakia, lichen planus, probably trough mutation in the proteins regulated cell cycle (p16INK4A, p21Waf1/Cip1/Sdi1, p27 kip1, Cyclin D1) and, second in p53 (TP53), Ras, hTert, EGFR, will reverse benign phenotype of late precancer lesions (PMDs) into benign cancer lesion. Third mutation probably in PI3K, PKD1 or E-cadherin, will increase malignant potential of SCCs, activating EMTransition, invasion and metastasis, e.g. aggressive phenotype. In BCC mutations of p53 are known to be late events (UV signature), whereas silencing of 14-3-3 takes place early in tumor progression, concomitant with increased activity of Snail. Early increased expression of PKD1 and down-regulation of c-myc mRNA are also events in pathogenesis of BCC. There is no data for detected mutations in PKD1 gene in both cancers, although the kinases is with high expression in BCC and lack of expression in SCC. There is no data concerning PKD1 expression in the PMDs leading to SCCs, nor to BCCs, such data were recently published concerning PKD1 expression in pancreatic oncogenesis. Curently adequate question is whether lack of PKD1 expression in SCCs, despite its PMDs origin, is a consequence of its spinous layer origin, or is a consequense of PKD1 gene mutations (downregulation). Identification of mutations as markers for early malignant transformation could be useful for early diagnosis and treatment of skin head and neck cancers.

Invited commentary

Autogenous vein bypass grafting remains an important therapeutic option for patients with peripheral vascular disease. These vein grafts are durable; however, the development of de novo stenosis within the graft occurs in 30% to 50% of patients within the first several years. Despite multiple therapeutic interventions and vein graft manipulations, the incidence of vein graft disease has not changed over the last 30 years. Basic research has focused on endothelial cells, smooth muscle cells, and extracellular matrix as the responsible culprits for the intimal hyperplastic lesion. However, studies have now shown that the adventitial layer may have a more dynamic role in the intimal hyperplastic lesion. Furthermore, recent studies have demonstrated the potential role of genetic variability in the p27 Kip1 gene as a determinant of clinical outcomes in patients with cardiovascular disease and after clinical interventions. The authors present a provocative in vitro study of human venous adventitial and smooth muscle cells that shows that the p27 single nucleotide polymorphism (p27 KIP1-838C>A) is functional and that the levels of p27 messenger RNA and protein were higher in the cells with the AA genotype compared with the CC genotype. Knockdown of p27 increases growth of both cell types with the AA genotype but not those with the CC genotype. What, however, is groundbreaking in their study is the differential effect of the p27 single nucleotide polymorphism on adventitial cell growth. They noted that adventitial cells of the AA genotype grew significantly more slowly than those with the CC genotype. In this study, only the adventitial cells, and not the smooth muscle cells, are responsible to the inhibitory effects of the protective AA genotype in cell growth. The adventitial layer has been noted to have vascular fibroblasts, perivascular adipocytes, and progenitor cells, which have been shown to affect vascular smooth muscle cell phenotype and growth. How these components interact to achieve the complex intimal hyperplastic lesion is still not known. However, using mouse model technology and lineage tracing technology to discover the cells or cells that are really responsible for the intimal hyperplastic lesion and, therefore, more precisely targeting treatment, could one day allow for the treatment of this disease that has been plaguing long-term vascular surgical outcomes for the last 40 years. The opinions or views expressed in this commentary are those of the authors and do not necessarily reflect the opinions or recommendations of the Journal of Vascular Surgery or the Society for Vascular Surgery. A single nucleotide polymorphism of cyclin-dependent kinase inhibitor 1B (p27Kip1) associated with human vein graft failure affects growth of human venous adventitial cells but not smooth muscle cellsJournal of Vascular SurgeryVol. 67Issue 1PreviewCyclin-dependent kinase inhibitor 1B (p27Kip1) is a cell-cycle inhibitor whose -838C>A single nucleotide polymorphism (rs36228499; hereafter called p27 SNP) has been associated with the clinical failure of peripheral vein grafts, but the functional effects of this SNP have not been demonstrated. Full-Text PDF Open Archive

ChIP-Seq analysis identifies p27(Kip1)-target genes involved in cell adhesion and cell signalling in mouse embryonic fibroblasts.

The protein p27Kip1 (p27), a member of the Cip-Kip family of cyclin-dependent kinase inhibitors, is involved in tumorigenesis and a correlation between reduced levels of this protein in human tumours and a worse prognosis has been established. Recent reports revealed that p27 also behaves as a transcriptional regulator. Thus, it has been postulated that the development of tumours with low amounts of p27 could be propitiated by deregulation of transcriptional programs under the control of p27. However, these programs still remain mostly unknown. The aim of this study has been to define the transcriptional programs regulated by p27 by first identifying the p27-binding sites (p27-BSs) on the whole chromatin of quiescent mouse embryonic fibroblasts. The chromatin regions associated to p27 have been annotated to the most proximal genes and it has been considered that the expression of these genes could by regulated by p27. The identification of the chromatin p27-BSs has been performed by Chromatin Immunoprecipitation Sequencing (ChIP-seq). Results revealed that p27 associated with 1839 sites that were annotated to 1417 different genes being 852 of them protein coding genes. Interestingly, most of the p27-BSs were in distal intergenic regions and introns whereas, in contrast, its association with promoter regions was very low. Gene ontology analysis of the protein coding genes revealed a number of relevant transcriptional programs regulated by p27 as cell adhesion, intracellular signalling and neuron differentiation among others. We validated the interaction of p27 with different chromatin regions by ChIP followed by qPCR and demonstrated that the expressions of several genes belonging to these programs are actually regulated by p27. Finally, cell adhesion assays revealed that the adhesion of p27-/- cells to the plates was much higher that controls, revealing a role of p27 in the regulation of a transcriptional program involved in cell adhesion.

Coatomer subunit beta 2 (COPB2), identified by label-free quantitative proteomics, regulates cell proliferation and apoptosis in human prostate carcinoma cells

Label-free quantitative proteomics has broad applications in the identification of differentially expressed proteins. Here, we applied this method to identify differentially expressed proteins (such as coatomer subunit beta 2 [COPB2]) and evaluated the functions and molecular mechanisms of these proteins in prostate cancer (PCA) cell proliferation. Proteins extracted from surgically resected PCA tissues and adjacent tissues of 3 patients were analyzed by label-free quantitative proteomics. The target protein was confirmed by bioinformatics and GEO dataset analyses. To investigate the role of the target protein in PCA, we used lentivirus-mediated small-interfering RNA (siRNA) to knockdown protein expression in the prostate carcinoma cell line, CWR22RV1 cells and assessed gene and protein expression by reverse transcription quantitative polymerase chain reaction and western blotting. CCK8 and colony formation assays were conducted to evaluate cell proliferation. Cell cycle distributions and apoptosis were assayed by flow cytometry. We selected the differentiation-related protein COPB2 as our target protein based on the results of label-free quantitative proteomics. High expression of COPB2 was found in PCA tissue and was related to poor overall survival based on a public dataset. Cell proliferation was significantly inhibited in COPB2-knockdown CWR22RV1 cells, as demonstrated by CCK8 and colony formation assays. Additionally, the apoptosis rate and percentage of cells in the G1 phase were increased in COPB2-knockdown cells compared with those in control cells. CDK2, CDK4, and cyclin D1 were downregulated, whereas p21 Waf1/Cip1 and p27 Kip1 were upregulated, affecting the cell cycle signaling pathway. COPB2 significantly promoted CWR22RV1 cell proliferation through the cell cycle signaling pathway. Thus, silencing of COPB2 may have therapeutic applications in PCA.

Molecular Highlighting Analysis of Mutational P27 Gene Products in Association with Human T-lymphotropic (HTLV-1) Infection in Tissues from Iraqi Patients with Non-Hodgkin’s Lymphoma

T-cell lymphoma is a rare disease in which T lymphocyte cells become cancerous. These lymphomas account between 10 percent and 15 percent of all cases of Non-Hodgkin lymphoma in the United States. Like the B-cell lymphomas, T-cell lymphomas are classified into two broad categories: aggressive (fast-growing) or indolent (slow-growing) (1). The G1/S checkpoint of the cell cycle is controlled by pRb protein, which functions in its hypophosphorylated form as a negative regulator of growth. p27 (Kip1), a member of CIP/KIP family of cyclin inhibitory proteins, participates in inhibition of forming complexes that allow pRb to phosphorylate and lead the cell into mitosis (2). To analyze the impact of concordant expression of p27 and HTLV-1 infection on a group of tissues with Non-Hodgkin’s lymphoma (NHL). Eighty formalin-fixed, paraffin- embedded lymph node tissues were enrolled in this study; (40) biopsies from Non-Hodgkin’s lymphoma (NHL), and (40) lymph nodes with (unremarkable pathological changes) as apparently healthy controls. Detection of HTLV-1was done by ultra-sensitive version of In situ hybridization method where as immunohisto chemistry detection system was used to demonstrate the expression of P27 gene expression. The HBZ gene of HTLV-1positive –CISH reaction was detected in (45%: 18 out of 40 cases) of Non-Hodgkin lymphoma tissues. No HTLV-1 positive – CISH reaction was detected in healthy lymph nodes tissues of the control group. The differences between the percentages of HTLV-1 detection in NHL tissues and control groups were statistically highly significant (P value = < 0.05). The positive P27-IHC reactions wase detected in 42.5% (17 out of 40 cases) of Non-Hodgkin lymphoma cases. A strong positive correlation was found between the detection, scores and intensity of p27 marker. Significant expressions of both p27 markers as well as HTLV-1 genes in Non-Hodgkin’s lymphoma could indicate for their possible roles both in lymph node pathogenesis and carcinogenesis

Euptox A Induces G1 Arrest and Autophagy via p38 MAPK- and PI3K/Akt/mTOR-Mediated Pathways in Mouse Splenocytes.

Euptox A (9-oxo-10, 11-dehydroageraphorone), the main toxin isolated from Eupatorium adenophorum, is known to induce immunotoxicity in animals. However, the precise mechanism underlying the effects of Euptox A on splenocytes is unclear. Here, we aimed to investigate the molecular mechanisms underlying the effect of Euptox A in mouse spleens after its intragastric administration and found that Euptox A exhibits proautophagic effects in splenocytes. Euptox A markedly arrested the splenocytes in the G0/G1 phase, which was accompanied by inhibition of the expression of the positive regulators CDK4, CDK2, cyclin D1, PCNA, and E2F1, and promotion of the expression of the negative regulators p53, p21 Waf1/Cip1, p27 Kip1, and Chk1. We also found that Euptox A did not markedly induce splenocyte apoptosis, but induced autophagy while increasing the subcellular localization of punctate LC3, ratio of LC3-II/LC3-I, and Beclin 1 levels, and decreasing p62 levels. Euptox A also significantly inhibited p-PI3K, p-p38 MAPK, p-Akt, and p-mTOR expression, but increased PTEN and p-AMPK expression. These results indicated that Euptox A induced splenocyte autophagy by inhibiting the PI3K/Akt/mTOR pathway, suppressing p38 MAPK expression, and activating AMPK. These findings provide new insights into the mechanisms involved in spleen toxicity caused by Euptox A in mice.

Abstract 305: TET blocks cell cycle progression in pancreatic cancer cells at G1-S boundary by decreasing cyclin D1 and increasing CDK inhibitors (CKIs), p21 (WAF1, Cip1) and p27 (Kip1)

Abstract Introduction: Pancreatic cancer (PaCa) is the fourth leading cause of cancer related deaths in the United States with an overall five-year survival rate of less than five percent. The current standard treatment/s for PaCa are largely ineffective thus there is an urgent need for development of therapeutic agents for the treatment of PaCa. Owing to its mutational landscape, which involves mutated KRAS and p53 genes these cancer cells have a deregulated G1 checkpoint of cell cycle control and therefore confer high rate of proliferation and therapeutic resistance. Recently we observed that Tetrandrine derivative (TET), a bis-benzylisoquinoline alkaloid halts cell cycle progression at G0/G1 boundary in vitro and in vivo. In the present study we evaluated potential mechanism of TET induced cell cycle arrest in two pancreatic cancer cells with either wild type KRAS or mutant KRAS, and mutant p53 by evaluating critical cell cycle regulatory proteins involved in G0/G1 to S transition. Methods: Pancreatic Cancer cell lines: BxPC3 (Pancreatic Ductal Adeno-Carcinoma) and MiaPaCa2 (pancreatic carcinoma) were used in the study. Cells were treated with various concentrations of TET for different time points. Cell proliferation was evaluated using the MTT survival assay. Cell cycle was analyzed following PI staining via Flow cytometry. Changes in protein expression were analyzed by Western Blot analysis in cells. Changes in mRNA were evaluated by real time PCR. Results: Treatment of pancreatic cancer cells with TET resulted in decreased levels cyclin D1 and phospho-Rb, two critical proteins required for G0/G1 to S transition. Pretreatment of the cells with proteasomal inhibitor MG132 was able to increase the levels of cyclin D1 in control cells but was unable to prevent its loss upon TET treatment. Our results also show that levels of CKIs, p21WAF1/Cip1 and p27Kip1 were increased at the protein level. Gene expression analysis revealed that cyclin D1 mRNA was decreased and p21WAF1/Cip1 mRNA was increased while there was no significant change in p27 mRNA levels. The cycloheximide chase revealed that TET stabilizes p27Kip1 and to some extent p21WAF1/Cip1 as well, suggesting that TET increased p27Kip1 and also p21WAF1/Cip1 levels in part by increasing protein stability. These data suggest a complex mechanism by which TET regulates expression of critical cell cycle regulatory molecules at both transcriptional, translational and post-translational level. Such a mechanism would provide for cell cycle arrest in pancreatic cancer cells following TET treatment. Conclusion: These results show that TET halts cell cycle at G0/G1 boundary in PaCa cells by modulating the cell cycle regulatory proteins and stabilizing the CKIs by altering their protein stability, suggesting that TET can not only halt deregulated cell cycle but also overcome the therapeutic resistance in pancreatic cancer cells. Citation Format: Karnika Singh, Qin Dong, Sweaty Koul, Hari K. Koul. TET blocks cell cycle progression in pancreatic cancer cells at G1-S boundary by decreasing cyclin D1 and increasing CDK inhibitors (CKIs), p21 (WAF1, Cip1) and p27 (Kip1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 305. doi:10.1158/1538-7445.AM2017-305

FLT3 and FLT3-ITD phosphorylate and inactivate the cyclin-dependent kinase inhibitor p27Kip1 in acute myeloid leukemia.

P27Kip1 (p27) can prevent cell proliferation by inactivating cyclin-dependent kinases. This function is impaired upon phosphorylation of p27 at tyrosine residue 88. We observed that FLT3 and FLT3-ITD can directly bind and selectively phosphorylate p27 on this residue. Inhibition of FLT3-ITD in cell lines strongly reduced p27 tyrosine 88 phosphorylation and resulted in increased p27 levels and cell cycle arrest. Subsequent analysis revealed the presence of tyrosine 88 phosphorylated p27 in primary patient samples. Inhibition of FLT3 kinase activity with AC220 significantly reduced p27 tyrosine 88 phosphorylation in cells isolated from FLT3 wild type expressing acute myeloid leukemia (AML) patients. In FLT3-ITD positive AML patients, p27 tyrosine 88 phosphorylation was reduced in 5 out of 9 subjects, but, surprisingly, was increased in 4 patients. This indicated that other tyrosine kinases such as Src family kinases might contribute to p27 tyrosine 88 phosphorylation in FLT3-ITD positive AML cells. In fact, incubation with the Src family kinase inhibitor dasatinib could decrease p27 tyrosine 88 phosphorylation in these patient samples, indicating that p27 phosphorylated on tyrosine 88 may be a therapeutic marker for the treatment of AML patients with tyrosine kinase inhibitors.

Expression and clinical significance of Ser10 phosphorylated p27(kip1) protein in human breast carcinoma

Objective: To explore the expression and clinicopathological significance of Ser10 phosphorylated p27(kip1)(P-p27Ser10) protein in human breast carcinoma tissue. Methods: Tissue specimens were obtained from 111 cases with breast carcinoma from January 2004 to December 2005. And the expression of P-p27Ser10 protein for each specimen was detected by immunohistochemical (EnVision) analysis. Six representative paired samples of cancerous and paired adjacent normal tissues were collected and detected by Western blot. The relationships between the expression levels of P-p27Ser10 protein and its clinicopathological characteristics in primary breast carcinoma were retrospectively analyzed.The association of P-p27Ser10 and Jab1 protein was detected by co-immunoprecipitation method. Results: A high-level expression of P-p27Ser10 was present in cancerous tissues but not in paired adjacent normal tissues. The positive expression rate of P-p27Ser10 protein was as high as 88.3% (98/111) and 7.2% (8/111) in cancerous and paired adjacent normal tissues respectively (P<0.05). P-p27Ser10 expression was correlated with tumor size, lymph node metastasis and TNM stage (all P<0.05). P-p27Ser10 expression was significantly correlated with Ki-67 (r=0.582, P<0.05), and inversely correlated with p27 (r=-0.426, P<0.05). Co-immunoprecipitation revealed that the association of P-p27Ser10 and Jab1 protein was increased in breast carcinoma. Conclusion: Over-expression of P-p27Ser10 protein might play an important role in the pathogenesis of breast carcinoma. P-p27Ser10 protein therefore represents a novel diagnostic marker and therapeutic target in patients with breast carcinoma.

The miR-106a~363Xpcl1 miRNA cluster induces murine T cell lymphoma despite transcriptional activation of the p27Kip1 cell cycle inhibitor.

The miR-106a~363 cluster encodes 6 miRNAs on the X-chromosome which are abundant in blood cells and overexpressed in a variety of malignancies. The constituent miRNA of miR-106a~363 have functional activities in vitro that are predicted to be both oncogenic and tumor suppressive, yet little is known about their physiological functions in vivo. Mature miR-106a~363 (Mirc2) miRNAs are processed from an intragenic, non-protein encoding gene referred to as Xpcl1 (or Kis2), situated at an X-chromosomal locus frequently targeted by retroviruses in murine lymphomas. The oncogenic potential of miR-106a~363Xpcl1 has not been proven, nor its potential role in T cell development. We show that miR106a~363 levels normally drop at the CD4+/CD8+ double positive (DP) stage of thymocyte development. Forced expression of Xpcl1 at this stage impairs thymocyte maturation and induces T-cell lymphomas. Surprisingly, miR-106a~363Xpcl1 also induces p27 transcription via Foxo3/4 transcription factors. As a haploinsufficient tumor suppressor, elevated p27 is expected to inhibit lymphomagenesis. Consistent with this, concurrent p27Kip1 deletion dramatically accelerated lymphomagenesis, indicating that p27 is rate limiting for tumor development by Xpcl1. Whereas down-regulation of miR-106a~363 is important for normal T cell differentiation and for the prevention of lymphomas, eliminating p27 reveals Xpcl1's full oncogenic potential.

Ponatinib promotes a G1 cell-cycle arrest of merlin/NF2-deficient human schwann cells.

Neurofibromatosis type 2 (NF2) is a genetic syndrome that predisposes individuals to multiple benign tumors of the central and peripheral nervous systems, including vestibular schwannomas. Currently, there are no FDA approved drug therapies for NF2. Loss of function of merlin encoded by the NF2 tumor suppressor gene leads to activation of multiple mitogenic signaling cascades, including platelet-derived growth factor receptor (PDGFR) and SRC in Schwann cells. The goal of this study was to determine whether ponatinib, an FDA-approved ABL/SRC inhibitor, reduced proliferation and/or survival of merlin-deficient human Schwann cells (HSC). Merlin-deficient HSC had higher levels of phosphorylated PDGFRα/β, and SRC than merlin-expressing HSC. A similar phosphorylation pattern was observed in phospho-protein arrays of human vestibular schwannoma samples compared to normal HSC. Ponatinib reduced merlin-deficient HSC viability in a dose-dependent manner by decreasing phosphorylation of PDGFRα/β, AKT, p70S6K, MEK1/2, ERK1/2 and STAT3. These changes were associated with decreased cyclin D1 and increased p27Kip1levels, leading to a G1 cell-cycle arrest as assessed by Western blotting and flow cytometry. Ponatinib did not modulate ABL, SRC, focal adhesion kinase (FAK), or paxillin phosphorylation levels. These results suggest that ponatinib is a potential therapeutic agent for NF2-associated schwannomas and warrants further in vivo investigation.

Capsaicinoids are silent estrogens, a class of estrogenic chemicals without cell-proliferation activity

Abstract Although environmental estrogens, including phytoestrogens, play important biological roles, their mechanisms of action are often not explored due to the lack of appropriate biological tools. For example, while capsaicin has been shown to exhibit various biological activities, its association with estrogenic cell signaling is mostly not well understood. We used an oligodeoxynucleotide-based DNA microarray containing estrogen-responsive genes to examine the gene expression profiles of capsaicinoids (capsaicin, dihydrocapsaicin, nordihydrocapsaicin and nonivamide) in human breast cancer MCF-7 cells to understand estrogenic signaling pathways at the transcription level, which was followed by Western blot analysis for signaling pathways at the protein level. While these capsaicinoids lacked cell-proliferation activity in MCF-7 cells, they showed expression profiles with high or moderate correlations (correlation coefficient or R = 0.66 to 0.83) with that of 17 β -estradiol ( E 2 ). Capsaicin seems to regulate the activation of Erk1/2 and PI3K/Akt through the pathways associated with both the epidermal growth factor receptor (EGFR) and the capsaicin receptor (TRPV1). In addition, capsaicin and E 2 differed in the regulation of cell cycle-related factors, such as cyclin D1 and p27 kip1 . These results indicate that, while these capsaicinoids have estrogenic activity and are functionally similar to E 2 , at both the transcriptional and protein levels, they lack the signaling for cell proliferation, and thus belong to silent estrogens, a class of estrogenic chemicals without cell-proliferation activity.

Role and regulation of p27 in neuronal apoptosis.

It is necessary for the cell-cycle machinery of neurons to be suppressed to promote differentiation and maintenance of their terminally differentiated state. Reactivation of the cell cycle in response to neurotoxic insults leads to neuronal cell death and some cell-cycle-related proteins contribute to the process. p27 kip1 (p27), an inhibitor of cyclin-dependent kinases, prevents unwarranted cyclin-dependent kinase activation. In this study, we have elucidated a novel mechanism via which p27 promotes apoptosis of neurons stimulated by neurotoxic amyloid peptide Aβ42 (Amyloid β1-42 peptide). Co-immunoprecipitation analysis revealed that p27 promotes interaction between Cyclin-dependent kinase 5 (Cdk5) and cyclin D1, which is induced by Aβ42 in cortical neurons. As a result, Cdk5 is sequestered from its neuronal activator p35 resulting in kinase deactivation. The depletion of p27, which was achieved by specific siRNA, restored Cdk5/p35 interaction by preventing association between Cdk5 and cyclin D1 and also abrogated Aβ42 induced apoptosis of cortical neurons. Furthermore, analysis of cell cycle markers suggested that p27 may play a role in Aβ42 induced aberrant cell cycle progression of neurons, which may result in apoptosis. These findings provide novel insights into how p27, which otherwise performs important neuronal functions, may become deleterious to neurons under neurotoxic conditions.