P27 Kip1 Research Articles

Adhesion to fibronectin induces p27Kip1 nuclear accumulation through down-regulation of Jab1 and contributes to cell adhesion-mediated drug resistance (CAM-DR) in RPMI 8,226 cells

Mounting evidence has been shown that integrin-mediated cellular adhesion confers resistance to chemotherapy of multiple myeloma. The molecular mechanism underlying cell adhesion-mediated drug resistance (CAM-DR) is, however, poorly understood. In this report, we demonstrated that RPMI 8,226 cells accumulated p27(Kip1) in the nucleus when they were adhered to fibronectin (FN). The adhesion-mediated p27(Kip1) nuclear recruitment was regulated via the down-regulation of Jab1, a negative regulator of cell cycle. Overexpression of Jab1 reversed the elevated p27(Kip1) in the nucleus, which needed phosphorylation of p27(Kip1) on Serine 10, whereas inhibition of Jab1 by siRNA further increased the elevated p27(Kip1). Furthermore, we found overexpression of Jab1 did not affect 8,226 cells adhesion to FN, but reversed doxorubicin or mitoxantrone-induced CAM-DR phenotype. In conclusion, our data suggest that Jab1 plays an important role in CAM-DR, which depends on pSer10-p27(Kip1)-mediated subcellular localization of p27(Kip1). The understanding of this novel molecular mechanism may prove valuable in designing new therapeutic approaches for CAM-DR in Multiple myeloma.

The expression and prognosis of Emi1 and Skp2 in breast carcinoma: associated with PI3K/Akt pathway and cell proliferation

S-phase kinase protein 2 (Skp2) is oncogenic and overexpressed in human breast cancer. The objective of this study was to examine the effect of early mitotic inhibitor-1 (Emi1) over-expression on Skp2 expression and related signaling pathway in breast cancer. Immunohistochemical analysis was performed in 98 human breast carcinoma samples and the data were correlated with clinicopathologic features. Furthermore, Western blot analysis was performed for Emi1 and Skp2 in breast carcinoma samples and cell lines to evaluate their protein levels and molecular interaction. We found that the expression of Emi1 was positively related with Skp2 expression (P<0.01) and Emi1 expression correlated significantly with histologic grade (P=0.005), meanwhile Skp2 expression obtained similar results. Kaplan-Meier analysis revealed that survival curves of low versus high expressers of Emi1 and Skp2 showed a highly significant separation in human breast cancer (P<0.01). While in vitro, following release of breast cancer cell lines from serum starvation, the expression of Emi1, Skp2, phosphor-Akt (p-Akt) was up-regulated, whereas p27(Kip1) was down-regulated. Treatment of phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 could arrest cells growth and diminish Emi1 expression. These results suggested that Emi1's anti-apoptotic and proliferative abilities appear to be triggered at least in part by the modulation of Skp2, combined Emi1 and Skp2 expressions, may be prognostic for patients with invasive breast carcinomas, which also associated with p-Akt and enabled p27(kip1) degradation. Emi1 may serve as a potential therapeutic strategy aimed at PI3K for the management of breast cancer.

The 4 Notch receptors play distinct and antagonistic roles in the proliferation and hepatocytic differentiation of liver progenitors

The Notch signaling pathway is involved in liver development and regeneration. Here, we investigate the role of the 4 mammalian Notch paralogs in the regulation of hepatoblast proliferation and hepatocytic differentiation. Our model is based on bipotential mouse embryonic liver (BMEL) progenitors that can differentiate into hepatocytes or cholangiocytes in vitro and in vivo. BMEL cells were subjected to Notch antagonists or agonists. Blocking Notch activation with a γ-secretase inhibitor, at 50 μM for 48 h, reduced cell growth by 50%. S-phase entry was impaired, but no apoptosis was induced. A systematic paralog-specific strategy was set using lentiviral transduction with constitutively active forms of each Notch receptor along with inhibition of endogenous Notch signaling. This assay demonstrates that proliferation of BMEL cells requires Notch2 and Notch4 activity, resulting in significant down-regulation of p27(Kip1) and p57(Kip2) cyclin-dependent kinase inhibitors. Conversely, Notch3-expressing cells proliferate less and express 3-fold higher levels of p57(Kip2). The Notch3 cells present a hepatocyte-like morphology, enhanced multinucleation, and a ploidy shift. Moreover, Notch3 activity is conducive to hepatocytic differentiation in vitro, while its paralogs impede this fate. Our study provides the first evidence of a functional diversity among the mammalian Notch homologues in the proliferation and hepatocytic-lineage commitment of liver progenitors.

MicroRNA‐212 inhibits proliferation of gastric cancer by directly repressing retinoblastoma binding protein 2

Retinoblastoma binding protein 2 (RBP2), a newly found histone demethylase, is overexpressed in gastric cancer. We examined the upstream regulatory mechanism of RBP2 at the microRNA (miRNA) level and the role in gastric carcinogenesis. We used bioinformatics to predict that microRNA-212 (miR-212) might be a direct upstream regulator of RBP2 and verified the regulation in gastric epithelial-derived cell lines. Overexpression of miR-212 significantly inhibited the expression levels of RBP2, whereas knockdown of miR-212 promoted RBP2 expression. Furthermore, we identified the putative miR-212 targeting sequence in the RBP2 3' UTR by luciferase assay. MiR-212 inhibited the colony formation ability of cells by repressing RBP2 expression and increasing that of P21(CIP1) and P27(kip1), both critical in cell cycle arrest. In addition, the expression of RBP2 and miR-212 in tumor tissue and matched normal tissue from 18 patients further supported the results in vivo. MiR-212 directly regulates the expression of RBP2 and inhibits cell growth in gastric cancer, which may provide new clues to treatment.

Heat Shock Protein B1-Deficient Mice Display Impaired Wound Healing

There is large literature describing in vitro experiments on heat shock protein (hsp)B1 but understanding of its function in vivo is limited to studies in mice overexpressing human hspB1 protein. Experiments in cells have shown that hspB1 has chaperone activity, a cytoprotective role, regulates inflammatory gene expression, and drives cell proliferation. To investigate the function of the protein in vivo we generated hspB1-deficient mice. HspB1-deficient fibroblasts display increased expression of the pro-inflammatory cytokine, interleukin-6, compared to wild-type cells, but reduced proliferation. HspB1-deficient fibroblasts exhibit reduced entry into S phase and increased expression of cyclin-dependent kinase inhibitors p27kip1 and p21waf1. The expression of hspB1 protein and mRNA is also controlled by the cell cycle. To investigate the physiological function of hspB1 in regulating inflammation and cell proliferation we used an excisional cutaneous wound healing model. There was a significant impairment in the rate of healing of wounds in hspB1-deficient mice, characterised by reduced re-epithelialisation and collagen deposition but also increased inflammation. HspB1 deficiency augments neutrophil infiltration in wounds, driven by increased chemokine (C-X-C motif) ligand 1 expression. This appears to be a general mechanism as similar results were obtained in the air-pouch and peritonitis models of acute inflammation.

Phytochemical Linarin Enriched in the Flower of Chrysanthemum indicum Inhibits Proliferation of A549 Human Alveolar Basal Epithelial Cells Through Suppression of the Akt-Dependent Signaling Pathway

In this study, we report the anti-proliferative effect and molecular mechanism of Chrysanthemum indicum (C. indicum) on A549 human alveolar basal epithelial cells. We also analyzed the changes in C. indicum component profiles due to modifications of predrying process, flower size, and extraction method. Among the varieties of modifications tested, high-temperature heat dry (HTD) of small flower biotype followed by the methanolic extraction resulted in the strongest anti-proliferative activity of C. indicum extract in A549 cells. High-performance liquid chromatography of C. indicum revealed that the levels of acacetin 7-O-rutinoside (linarin) are markedly increased by heat treatment, especially HTD. Finally, we showed that linarin-mediated inhibition of cell proliferation is associated with suppression of Akt activation and induction of cyclin-dependent kinase inhibitor p27(Kip1) as evidenced by cell cycle analysis and treatment with LY294002, an inhibitor of phosphatidylinositol 3-kinase/Akt pathway. Taken together, these findings suggest the need for further development and evaluation of linarin from C. indicum for the treatment and prevention of lung cancer.

Small gastrointestinal stromal tumor in the stomach: identification of precursor for clinical gastrointestinal stromal tumor using c-kit and α-smooth muscle actin expression

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the digestive tract. To find precursors for clinical GISTs of the stomach, small gastric stromal tumors of less than 3 cm were collected and examined immunohistochemically with analysis of the KIT mutation. Sixty-eight of 74 lesions were classified into 4 representative groups according to the expression of c-kit and α-smooth muscle actin (αSMA): group A, c-kit diffusely positive and αSMA negative (18 cases); group B, c-kit diffusely positive and αSMA focally positive (13); group C, c-kit focally positive and αSMA diffusely positive (27); and group D, c-kit negative and αSMA diffusely positive (10). Of the 4 groups, groups A and B of c-kit diffuse expression showed higher cellularity and labeling indices of p27(Kip1) and Ki-67 than did groups C and D of diffuse αSMA expression. Incidence of KIT exon 11 mutation in groups A and B was 86% (25/29), whereas that in groups C and D was 0% (0/20). Small gastric stromal tumors with c-kit diffuse expression were considered precursors for clinical GIST because they were significantly different from c-kit focally positive or negative tumors. The mutation of KIT is considered as an early event in tumorigenesis of GIST.

Increased Cytoplasmic Localization of p27kip1 and Its Modulation of RhoA Activity during Progression of Chronic Myeloid Leukemia

The role of p27kip1 in Chronic Myeloid Leukemia (CML) has been well studied in relation to its function as a cell cycle inhibitor. However, its cytoplasmic function especially in CML remains to be seen. We studied the localization of p27kip1 and its function during the progression of CML from chronic to blast phase. Our investigations revealed an increased localization of p27kip1 in the cytoplasm of CD34+ cells in the blast phase compared to chronic phase. Cytoplasmic p27kip1 was found to modulate RhoA activity in CD34+ stem and progenitor cells. Further, RhoA activity was shown to be dependent on cytoplasmic p27kip1 which in turn was dependent on p210Bcr-Abl kinase activity. Interestingly, RhoA activity was observed to affect cell survival in the presence of imatinib through the SAPK/JNK pathway. Accordingly, inhibition of SAPK/JNK pathway using SP600125 increased apoptosis of K562 cells in presence of imatinib. Our results, for the first time, thus reveal a crucial link between cytoplasmic p27kip1, RhoA activity and SAPK/JNK signalling. To this effect we observed a correlation between increased cytoplasmic p27kip1, increased RhoA protein levels, decreased RhoA-GTP levels and increased SAPK/JNK phosphorylation in blast phase CD34+ cells compared to chronic phase CD34+ cells.

Abstract B65: The mitochondrial antioxidant Superoxide Dismutase 2 is necessary for ovarian cancer spheroid formation

Abstract The formation of cellular spheroid aggregates in the Intraperitoneal Cavity (IP) is considered to be an important step in the metastatic progression of ovarian cancer. Spheroids may represent a unique morphological adaptation that enhances long-term survival of tumor cells in the IP cavity, reminiscent of dormant cell populations. Like other dormant tumor cells, in vivo-isolated and in vitro-cultured spheroids exhibit enhanced chemoresistance and display markers of quiescence. We show that expression of mitochondrial superoxide dismutase (Sod2), which regulates mitochondrial redox balance, is significantly increased during formation of dense spheroids from a number of ovarian cancer cell lines. This is accompanied by increases in the quiescence marker p27(kip1). Importantly, siRNA and shRNA knock-down of Sod2 compromise spheroid formation and integrity, and abrogate increases in p27(kip1) expression in anchorage-independent 3D cultures. Mechanistically, it is likely that increases in Sod2 expression protect cells from build-up of mitochondrial and IP cavity-derived ROS, enhancing their survival as dormant spheroid populations. In addition, bioenergetic data suggest that Sod2 is directly involved in enhancing the mitochondrial integrity of tumor cells within spheroid aggregates. This leads to a metabolic profile that is associated with a slowing in ATP demand and an enhanced respiratory reserve capacity, with Sod2 being intricately involved in manipulating these changes. Enhanced Sod2 expression, a concomitant decrease in mitochondrial ROS and changes in mitochondrial function are likely indicators of a quiescence adaptation of tumor spheroids, necessary for survival in the IP cavity. Manipulating mitochondrial function and redox balance by effectively targeting mitochondrial antioxidant enzymes may provide novel therapeutic strategies to combat recurrence and metastatic spread of ovarian cancer. Citation Format: LP Madhubhani Hemachandra, Usawadee Dier, Larissa M. Uusitalo, Nadine Hempel. The mitochondrial antioxidant Superoxide Dismutase 2 is necessary for ovarian cancer spheroid formation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B65.

Vitamin E δ-tocotrienol prolongs survival in the LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) transgenic mouse model of pancreatic cancer.

Previous work has shown that vitamin E δ-tocotrienol (VEDT) prolongs survival and delays progression of pancreatic cancer in the LSL-Kras(G12D)(/+);Pdx-1-Cre mouse model of pancreatic cancer. However, the effect of VEDT alone or in combination with gemcitabine in the more aggressive LSL-Kras(G12D)(/+);LSL-Trp53(R172H)(/+);Pdx-1-Cre (KPC) mouse model is unknown. Here, we studied the effects of VEDT and the combination of VEDT and gemcitabine in the KPC mice. KPC mice were randomized into four groups: (i) vehicle [olive oil, 1.0 mL/kg per os twice a day and PBS 1.0 mL/kg intrapertoneally (i.p.) twice a week], (ii) gemcitabine (100 mg/kg i.p. twice a week), (iii) VEDT (200 mg/kg per os twice a day), and (iv) gemcitabine + VEDT. Mice received treatment until they displayed symptoms of impending death from pancreatic cancer, at which point animals were euthanized. At 16 weeks, survival was 10% in the vehicle group, 30% in the gemcitabine group, 70% in the VEDT group (P < 0.01), and 90% in the VEDT combined with gemcitabine group (P < 0.05). VEDT alone and combined with gemcitabine resulted in reversal of epithelial-to-mesenchymal transition in tumors. Biomarkers of apoptosis (plasma CK18), PARP1 cleavage, and Bax expression were more greatly induced in tumors subjected to combined treatment versus individual treatment. Combined treatment induced cell-cycle inhibitors (p27(Kip1) and p21(Cip1)) and inhibited VEGF, vascularity (CD31), and oncogenic signaling (pAKT, pMEK, and pERK) greater than individual drugs. No significant differences in body weight gain between drug treatment and control mice were observed. These results strongly support further investigation of VEDT alone and in combination with gemcitabine for pancreatic cancer prevention and treatment.

PRMT5 Is Upregulated in Malignant and Metastatic Melanoma and Regulates Expression of MITF and p27Kip1

Protein arginine methyltransferase-5 (PRMT5) is a Type II arginine methyltransferase that regulates various cellular functions. We hypothesized that PRMT5 plays a role in regulating the growth of human melanoma cells. Immunohistochemical analysis indicated significant upregulation of PRMT5 in human melanocytic nevi, malignant melanomas and metastatic melanomas as compared to normal epidermis. Furthermore, nuclear PRMT5 was significantly decreased in metastatic melanomas as compared to primary cutaneous melanomas. In human metastatic melanoma cell lines, PRMT5 was predominantly cytoplasmic, and associated with its enzymatic cofactor Mep50, but not STAT3 or cyclin D1. However, histologic examination of tumor xenografts from athymic mice revealed heterogeneous nuclear and cytoplasmic PRMT5 expression. Depletion of PRMT5 via siRNA inhibited proliferation in a subset of melanoma cell lines, while it accelerated growth of others. Loss of PRMT5 also led to reduced expression of MITF (microphthalmia-associated transcription factor), a melanocyte-lineage specific oncogene, and increased expression of the cell cycle regulator p27Kip1. These results are the first to report elevated PRMT5 expression in human melanoma specimens and indicate this protein may regulate MITF and p27Kip1 expression in human melanoma cells.

Artemisinin inhibits gastric cancer cell proliferation through upregulation of p53

Recent population studies suggest that the use of artemisinin is associated with reduced incidence and improved prognosis of certain cancers. In the current study, we assessed the effect of artemisinin on gastric cancer cells (AGS and MKN74 cells). We found that artemisinin inhibited growth and modulated expression of cell-cycle regulators in these cells. Treatment with artemisinin was also associated with induction of p27 kip1 and p21 kip1, two negative cell-cycle regulators. Furthermore, we revealed that artemisinin treatment led to an increased expression of p53. Taken together, these results provide evidence for a mechanism that may contribute to the antineoplastic effects of artemisinin suggested by recent population studies and justify further work to explore potential roles for it in gastric cancer prevention and treatment.

Relationship between Expression of p57 KIP2 and cyclin E Proteins and Clinicopathologic Features in Endometrial Adenocarcinoma Tissues

目的 研究子宫内膜样腺癌组织中p57KIP2、cyclinE蛋白的表述与临床病理特征的关系.方法 采用免疫组织化学Elivision法检测100例子宫内膜样腺癌(endometrioid adenocarcinoma,EA)、20例正常子宫内膜、20例子宫内膜增生性病变、20例子宫内膜上皮内瘤变(endometrial intraepithelial neoplasia,EIN)组织中p57KIP2、cyclin E蛋白的表达情况.结果 p57KIP2蛋白在EIN中阳性表达最高,在增生性病变中表达最低,在正常内膜组织中表达稍高于EA,但仅在EIN与在增生性病变中阳性表达比较,差异有统计学意义(P≤0.05);cyclinE蛋白在正常子宫内膜组织中表达最低,在增生性病变、EIN中的阳性表达逐渐增高,在EA中表达最高(P≤0.05,P＜0.01).在EA组织中,p57KIP2、cyclin E蛋白的阳性表达均与组织学分级、肌层浸润深度、手术分期有关(P≤0.05,P＜0.01),cyclin E蛋白的表达还与盆腔淋巴结转移有关(P＜0.01);但两者的阳性表达均与患者年龄无关(P＞0.05).p57KIP2与cyclin E之间的阳性表达呈负相关(P＜0.01).结论 p57KIP2、cyclin E均参与了EA的发生发展过程.联合检测p57KIP2、cyclin E蛋白在子宫内膜癌组织中的表达情况,对评估子宫内膜癌的生物学行为、判断预后有重要的意义.

Cooperative role between p21cip1/waf1 and p27kip1 in premature senescence in glandular proliferative lesions in mice.

Cellular senescence has been considered a novel target for cancer therapy. It has also been pointed out that p21(cip1/waf1) and p27(kip1) cyclin-dependent kinase inhibitors (CKIs) play a role in cellular senescence in some tumor types. Therefore, in order to address the possibility of a cooperative role between p21 and p27 proteins in senescence in vivo we analyzed cellular senescence in spontaneous glandular proliferative lesions (adrenal, thyroid and pituitary glands) in a double-KO mice model, using γH2AX, p53, p16, PTEN and Ki67 as senescence markers. The results obtained showed that p21p27 double-null mice had the lowest number of γH2AX positive cells in glandular hyperplasias and benign tumors. Also, in this group, Ki67 proliferation index correlated with a lower immunohistochemical expression of γH2AX and p53. The expression of p16 and PTEN do not seem to cause synergism of senescence in the benign lesions analyzed in p21p27 double-KO mice. These observations suggest an intrinsic cooperation between p21 and p27 CKIs in the activation of stress-induced cellular senescence and tumor progression in vivo, which would be a physiological mechanism to prevent tumor cell proliferation.

Pax6 is required for normal cell-cycle exit and the differentiation kinetics of retinal progenitor cells.

The coupling between cell-cycle exit and onset of differentiation is a common feature throughout the developing nervous system, but the mechanisms that link these processes are mostly unknown. Although the transcription factor Pax6 has been implicated in both proliferation and differentiation of multiple regions within the central nervous system (CNS), its contribution to the transition between these successive states remains elusive. To gain insight into the role of Pax6 during the transition from proliferating progenitors to differentiating precursors, we investigated cell-cycle and transcriptomic changes occurring in Pax6 - retinal progenitor cells (RPCs). Our analyses revealed a unique cell-cycle phenotype of the Pax6-deficient RPCs, which included a reduced number of cells in the S phase, an increased number of cells exiting the cell cycle, and delayed differentiation kinetics of Pax6 - precursors. These alterations were accompanied by coexpression of factors that promote (Ccnd1, Ccnd2, Ccnd3) and inhibit (P27 kip1 and P27 kip2) the cell cycle. Further characterization of the changes in transcription profile of the Pax6-deficient RPCs revealed abrogated expression of multiple factors which are known to be involved in regulating proliferation of RPCs, including the transcription factors Vsx2, Nr2e1, Plagl1 and Hedgehog signaling. These findings provide novel insight into the molecular mechanism mediating the pleiotropic activity of Pax6 in RPCs. The results further suggest that rather than conveying a linear effect on RPCs, such as promoting their proliferation and inhibiting their differentiation, Pax6 regulates multiple transcriptional networks that function simultaneously, thereby conferring the capacity to proliferate, assume multiple cell fates and execute the differentiation program into retinal lineages.

Clinicopathology of recurrent hepatocellular carcinomas after radiofrequency ablation treated with salvage surgery

Radiofrequency ablation (RFA) is an effective standard local therapy for small hepatocellular carcinoma (HCC). However, local recurrence and/or tumor seeding after RFA remain major problems. For better understanding of underlying factors, we clarified clinicopathological features of recurrent HCC treated with RFA. This retrospective study included 21 patients who underwent surgical resection for HCC disease recurrence after RFA. Clinicopathological findings, including patterns of recurrence, immunohistochemical expression of proliferation markers (Ki-67 and p27(Kip1) ) and survival outcome were assessed. The median time interval after RFA until the diagnosis of intrahepatic and/or extrahepatic tumor progression was 12 months (range, 3-84). Radical surgical resection was attempted for intrahepatic local recurrence in 16 patients (18 lesions), for peritoneal dissemination in four, for lymph node metastases in three and for adrenal metastasis in two. In 14 of the 21 (67%) patients, the recurrent HCC were histologically diagnosed as of poorly differentiated type. Their average Ki-67 and p27(Kip1) labeling indices were significantly higher (P = 0.020) and lower (P < 0.001), respectively, compared with values for the 108 HCC surgically resected at the initial treatment. Portal involvement was significantly higher (P = 0.01) in recurrent tumors after RFA (72%) than in HCC surgically resected at the initial treatment (43%). The mortality rate of salvage surgery was 0%, with cumulative survival rates at 1 and 3 years of 58.9% and 35.7%, respectively. The recurrent tumors after RFA have characteristics of poor differentiation degree and abnormalities in cell-cycle regulators and are associated with aggressive vascular invasiveness.

Cell adhesion to fibronectin down-regulates the expression of Spy1 and contributes to drug resistance in multiple myeloma cells

Spy1 is a member of the Speedy/Ringo family, which regulates cell proliferation and survival. Spy1 has been demonstrated to promote the cell-cycle progress through p27(Kip1) degradation. Previous investigations have suggested cell adhesion-mediated drug resistance (CAM-DR) in multiple myeloma (MM) is a primary factor for minimal residual disease (MRD) leading to relapse after chemotherapy. However, the precise mechanism remains elusive. In this study, we used MM cell lines to determine whether Spy1 plays a role in CAM-DR. We demonstrated that adhesion of MM cells to fibronectin (FN) decreased Spy1 expression. Overexpression of Spy1 did not affect MM cells adhesion to FN, but did reverse the doxorubicin- or mitoxantrone-induced CAM-DR phenotype. Spy1 protein level was also correlated with reciprocal up-regulation of p27(Kip1) when RPMI 8226 cells bound FN. Spy1 overexpression promoted p27(Kip1) phosphorylation at T187, then induced the p27(Kip1) degradation in the adhesion model. In addition, increasing p27(Kip1) level or disturbing p27(Kip1) phosphorylation at T187 abolished the CAM-DR reversion when Spy1 was overexpressed. Collectively, our data suggest that Spy1 plays an important role in CAM-DR, which depends on the function of p27(Kip1). Our findings provide a rational framework for further development of Spy1 as a novel target for MM therapy.

Identification of small molecule inhibitors of p27Kip1 ubiquitination by high‐throughput screening

Dysregulation of p27(Kip1) due to proteolysis that involves the ubiquitin ligase (SCF) complex with S-phase kinase-associated protein 2 (Skp2) as the substrate-recognition component (SCF(Skp2)) frequently results in tumorigenesis. In this report, we developed a high-throughput screening system to identify small-molecule inhibitors of p27(Kip1) degradation. This system was established by tagging Skp2 with fluorescent monomeric Azami Green (mAG) and CDK subunit 1 (Cks1) (mAGSkp2-Cks1) to bind to p27(Kip1) phosphopeptides. We identified two compounds that inhibited the interaction between mAGSkp2-Cks1 and p27(Kip1): linichlorin A and gentian violet. Further studies have shown that the compounds inhibit the ubiquitination of p27(Kip1) in vitro as well as p27(Kip1) degradation in HeLa cells. Notably, both compounds exhibited preferential antiproliferative activity against HeLa and tsFT210 cells compared with NIH3T3 cells and delayed the G1 phase progression in tsFT210 cells. Our approach indicates a potential strategy for restoring p27(Kip1) levels in human cancers.

Transcriptome Analysis Identifies Regulators of Hematopoietic Stem and Progenitor Cells

SummaryHematopoietic stem cells (HSCs) maintain blood homeostasis and are the functional units of bone marrow transplantation. To improve the molecular understanding of HSCs and their proximal progenitors, we performed transcriptome analysis within the context of the ImmGen Consortium data set. Gene sets that define steady-state and mobilized HSCs, as well as hematopoietic stem and progenitor cells (HSPCs), were determined. Genes involved in transcriptional regulation, including a group of putative transcriptional repressors, were identified in multipotent progenitors and HSCs. Proximal promoter analyses combined with ImmGen module analysis identified candidate regulators of HSCs. Enforced expression of one predicted regulator, Hlf, in diverse HSPC subsets led to extensive self-renewal activity ex vivo. These analyses reveal unique insights into the mechanisms that control the core properties of HSPCs.

Airway Smooth Muscle Hyperproliferation Is Regulated by MicroRNA-221 in Severe Asthma

Increased airway smooth muscle (ASM) mass is a feature of asthmatic airways, and could result from augmented proliferation. We determined whether proliferation and IL-6 release are abnormal in ASM cells (ASMCs) from patients with severe asthma, and whether these features could be mediated by microRNA-221 and microRNA-222, through modulation of the cyclin-dependent kinase inhibitors, p21(WAF1) and p27(kip1). ASMCs cultured from bronchial biopsies of healthy subjects and patients with nonsevere or severe asthma were studied. Proliferation was measured by the incorporation of bromodeoxyuridine and IL-6 by ELISA. FCS and transforming growth factor (TGF)-β caused greater proliferation and IL-6 release in patients with severe compared with nonsevere asthma and normal subjects. FCS + TGF-β inhibited p21(WAF1) and p27(kip1) expression, and increased microRNA-221 (miR-221) expression in ASMCs from individuals with severe asthma. miR-221, and not miR-222, mimics the increased proliferation and IL-6 release induced by FCS + TGF in healthy ASM, whereas in patients with severe asthma, the inhibition of miR-221, but not miR-222, inhibited proliferation and IL-6 release. miR-221 inhibition led to the increased expression of FCS + TGF-β-induced p21(WAF1) and p27(kip1). Dexamethasone suppressed proliferation in healthy subjects, but not in subjects with asthma. IL-6 was less suppressible by dexamethasone in patients with nonsevere and severe asthma, compared with healthy subjects. miR-221 did not influence the effects of dexamethasone. ASM from patients with severe asthma shows greater proliferation and IL-6 release than in patients with nonsevere asthma, but both groups show corticosteroid insensitivity. miR-221 regulates p21(WAF1) and p27(kip1) expression levels. Furthermore, miR-221 regulates the hyperproliferation and IL-6 release of ASMCs from patients with severe asthma, but does not regulate corticosteroid insensitivity.