Down-Regulation of CK2α Leads toUp-Regulation of the Cyclin-Dependent Kinase Inhibitor p27KIP1 in Conditions Unfavorable for the Growth of Myoblast Cells.

BACKGROUND/AIMS: Compelling evidence indicates that CK2α, which is one of the two catalytic isoforms of protein kinase CK2, is required for cell viability and plays an important role in cell proliferation and differentiation. While much is known on CK2 in the context of disease states, particularly cancer, its critical role in non-cancerous cell growth has not been extensively investigated. METHODS: In the present study, we have employed a cell line derived from rat heart with inducible down-regulation of CK2α and CK2α-knockout mouse tissue to identify CK2-mediated molecular mechanisms regulating cell growth. For this, we have performed Incucyte® live-cell analysis and applied flow cytometry, western blot, immunoprecipitation, immunohistochemistry, RT-qPCR and luciferase-based methods. RESULTS: Here, we show that lack of CK2α results in significantly delayed cell cycle progression through G1, inhibition of cyclin E-CDK2 complex, decreased phosphorylation of Rb protein at S795, and inactivation of E2F transcription factor. These events are accompanied by nuclear accumulation and up-regulation of the cyclin-dependent kinase inhibitor p27KIP1 in cells and CK2α-knockout mouse tissues. We found that increased levels of p27KIP1 are mainly attributable to post-translational modifications, namely phosphorylation at S10 and T197 amino acid residues catalyzed by Dyrk1B and AMPK, respectively, as silencing of FoxO3A transcription factor, which activates CDKN1B the gene coding for p27KIP1, does not result in markedly decreased expression levels of the corresponding protein. Interestingly, simultaneous silencing of CK2α and p27KIP1 significantly impairs cell cycle progression without increasing cell death. CONCLUSION: Taken together, our study sheds light on the molecular mechanisms controlling cell cycle progression through G1 phase when myoblasts proliferation potential is impaired by CK2α depletion. Our results suggest that elevated levels of p27KIP1, which follows CK2α depletion, contribute to delay the G1-to-S phase transition. Effects seen when p27KIP1 is down-regulated are independent of CK2α and reflect the protective role exerted by p27KIP1 under unfavorable cell growth conditions.

ETV4 promotes late development of prostatic intraepithelial neoplasia and cell proliferation through direct and p53-mediated downregulation of p21.

BACKGROUND: ETV4 is one of the ETS proteins overexpressed in prostate cancer (PC) as a result of recurrent chromosomal translocations. In human prostate cell lines, ETV4 promotes migration, invasion, and proliferation; however, its role in PC has been unclear. In this study, we have explored the effects of ETV4 expression in the prostate in a novel transgenic mouse model. METHODS: We have created a mouse model with prostate-specific expression of ETV4 (ETV4 mice). By histochemical and molecular analysis, we have investigated in these engineered mice the expression of p21, p27, and p53. The implications of our in vivo findings have been further investigated in human cells lines by chromatin-immunoprecipitation (ChIP) and luciferase assays. RESULTS: ETV4 mice, from two independent transgenic lines, have increased cell proliferation in their prostate and two-thirds of them, by the age of 10 months, developed mouse prostatic intraepithelial neoplasia (mPIN). In these mice, cdkn1a and its p21 protein product were reduced compared to controls; p27 protein was also reduced. By ChIP assay in human prostate cell lines, we show that ETV4 binds to a specific site (-704/-696 bp upstream of the transcription start) in the CDKN1A promoter that was proven, by luciferase assay, to be functionally competent. ETV4 further controls CDKN1A expression by downregulating p53 protein: this reduction of p53 was confirmed in vivo in ETV4 mice. CONCLUSIONS: ETV4 overexpression results in the development of mPIN but not in progression to cancer. ETV4 increases prostate cell proliferation through multiple mechanisms, including downregulation of CDKN1A and its p21 protein product: this in turn is mediated through direct binding of ETV4 to the CDKN1A promoter and through the ETV4-mediated decrease of p53. This multi-faceted role of ETV4 in prostate cancer makes it a potential target for novel therapeutic approaches that could be explored in this ETV4 transgenic model.

miR-196a-mediated downregulation of p27kip1 protein promotes prostate cancer proliferation and relates to biochemical recurrence after radical prostatectomy.

BACKGROUND: Dysregulation of microRNAs has performed vital gene regulatory functions in the genesis, progression, and prognosis of multiple malignant tumors. This study aimed to elucidate the regulatory mechanism of miR-196a in prostate cancer (PCa) and explore its clinical significance. METHODS: Quantitative real-time polymerase chain reaction was implemented to examine miR-196a and p27kip1 messenger RNA expression in PCa. Cell proliferation was evaluated via Cell Counting Kit-8, colony formation, and nude mouse tumorigenicity assays. Luciferase reporter assay was applied to identify target genes. p27kip1 protein expression in PCa was investigated using Western blot analysis and immunohistochemistry. RESULTS: There was a dramatic upregulation of miR-196a in PCa. Upregulated miR-196a was related to worse Gleason score (GS), later pathological stage, and poor biochemical recurrence (BCR)-free survival. In vivo and in vitro experiments exhibited that miR-196a promoted PCa proliferation and expedited G1/S-phase progression through the downregulation of p27kip1 protein. Additionally, p27kip1 protein was distinctly downregulated in PCa. Low p27kip1 protein expression had a strong correlation with increased GS and was an independent predictor of BCR after radical prostatectomy (RP). CONCLUSIONS: Excessive expression of miR-196a and subsequent downregulation of p27kip1 protein play essential roles in promoting PCa proliferation and leading to BCR after RP. miR-196a and its target p27kip1 may become novel molecular biomarkers and therapeutic targets for PCa.

Robust expression of p27Kip1 induced by viral infection is critical for antiviral innate immunity.

Influenza A virus (IAV) infection regulates the expression of numerous host genes. However, the precise mechanism underlying implication of these genes in IAV pathogenesis remains largely unknown. Here, we employed isobaric tags for relative and absolute quantification (iTRAQ) to identify host proteins regulated by IAV infection. iTRAQ analysis of mouse lungs infected or uninfected with IAV showed a total of 167 differentially upregulated proteins in response to the viral infection. Interestingly, we observed that p27Kip1, a potent cyclin-dependent kinase inhibitor, was markedly induced by IAV both at mRNA and protein levels through in vitro and in vivo studies. Furthermore, it was shown that innate immune signalling positively regulated p27Kip1 expression in response to IAV infection. Ectopic expression of p27Kip1 in A549 cells dramatically inhibited IAV replication, whereas, p27Kip1 knockdown significantly enhanced the virus replication. in vivo experiments demonstrated that p27Kip1 knockout (KO) mice were more susceptible to IAV than wild-type (WT) mice: exhibiting higher viral load in lung tissue, faster body-weight loss, reduced survival rate and more severe organ damage. Moreover, we found that p27Kip1 overexpression facilitated the degradation of viral NS1 protein, caused a dramatic STAT1 activation and promoted the expression of IFN-ß and several critical antiviral interferon-stimulated genes (ISGs). Increased p27Kip1 expression also restricted infections of several other viruses. Conversely, IAV-infected p27Kip1 KO mice exhibited a sharp increase in NS1 protein accumulation, reduced level of STAT1 activation and decreased expression of IFN-ß and the ISGs in the lung compared to WT animals. These findings reveal a key role of p27Kip1 in enhancing antiviral innate immunity.

Steroid resistance in Diamond Blackfan anemia associates with p57Kip2 dysregulation in erythroid progenitors.

Despite the effective clinical use of steroids for the treatment of Diamond Blackfan anemia (DBA), the mechanisms through which glucocorticoids regulate human erythropoiesis remain poorly understood. We report that the sensitivity of erythroid differentiation to dexamethasone is dependent on the developmental origin of human CD34+ progenitor cells, specifically increasing the expansion of CD34+ progenitors from peripheral blood (PB) but not cord blood (CB). Dexamethasone treatment of erythroid-differentiated PB, but not CB, CD34+ progenitors resulted in the expansion of a newly defined CD34+CD36+CD71hiCD105med immature colony-forming unit-erythroid (CFU-E) population. Furthermore, proteomics analyses revealed the induction of distinct proteins in dexamethasone-treated PB and CB erythroid progenitors. Dexamethasone treatment of PB progenitors resulted in the specific upregulation of p57Kip2, a Cip/Kip cyclin-dependent kinase inhibitor, and we identified this induction as critical; shRNA-mediated downregulation of p57Kip2, but not the related p27Kip1, significantly attenuated the impact of dexamethasone on erythroid differentiation and inhibited the expansion of the immature CFU-E subset. Notably, in the context of DBA, we found that steroid resistance was associated with dysregulated p57Kip2 expression. Altogether, these data identify a unique glucocorticoid-responsive human erythroid progenitor and provide new insights into glucocorticoid-based therapeutic strategies for the treatment of patients with DBA.

Progesterone up-regulates p27 through an increased binding of the progesterone receptor-A-p53 protein complex onto the non-canonical p53 binding motif in HUVEC.

We previously demonstrated that progesterone (P4) up-regulated p53 expression, which in turn increased p21 and p27 expression, and finally resulted in proliferation inhibition in human umbilical vein endothelial cells (HUVEC). While a direct transcriptional activation of p21 by p53 protein has been clearly elucidated, the mechanism by which p53 induces p27 expression has not been documented. In this study, we identified three putative p53 protein binding domains at the p27 promoter. Luciferase assay showed that the activity of ectopically introduced p27 promoter constructs containing the potential p53 protein binding region was significantly increased by P4. Immunoblotting analysis indicated that P4 increased the level of p53 protein. Treatment with pifithrin-α-HBr (PFTα), a specific blocker of p53-responsive gene transactivation, reduced the P4-increased p27 promoter activity and p27 protein expression. Transfection with dominant-negative mutants of p53 (C135Y, R175H and R248 W) abolished the P4-increased p27 promoter activity. Moreover, deletion or TCCT nucleotide sequence fill-in at the core site of any of p53 protein binding domains led to the irresponsiveness of the p27 promoter to P4 treatment. Interestingly, immunoprecipitation and chromatin-immunoprecipitation analyses demonstrated that P4 increased the complex of p53-P4 receptor (PR) protein in the nucleus and the assembly of PR protein to the p53 protein binding region of the p27 promoter. Ectopic co-overexpression of p53 and PR-A constructs further augmented the P4-increased p27 promoter activity. Taken together, the results from the present study suggest that P4-increased p53 expression might directly up-regulate p27 transactivation, and PR-A protein might promote this effect by forming complex with p53 protein.

Neuroendocrine Breast Carcinomas Share Prognostic Factors with Gastroenteropancreatic Neuroendocrine Tumors: A Putative Prognostic Role of Menin, p27, and SSTR-2A.

OBJECTIVES: Due to the rarity of breast carcinomas with neuroendocrine features (NEBC), the knowledge on their biology is very limited but the identification of their biology and prognostic factors is essential to evaluate both pathogenesis and possible targeted treatment options. We assessed the expression of the well-characterized prognostic factors of gastroenteropancreatic neuroendocrine tumors (GEP-NET) in NEBC. METHODS: We assessed the immunohistochemical expression of neuron-specific enolase (NSE), thymidylate synthase (TS), p27, CD56, menin, and somatostatin receptor type 2A (SSTR-2A) in a series of 36 NEBC and 45 invasive ductal carcinomas (IDC). RESULTS: Nuclear and cytoplasmic TS, nuclear and cytoplasmic NSE, and nuclear p27 had significant overexpression in NEBC compared with IDC (for all, p < 0.01). In NEBC, cytoplasmic SSTR-2A expression was associated with excellent distant disease-free survival (p = 0.013), cytoplasmic menin expression with poorer relapse-free survival (p = 0.022), and nuclear p27 with longer breast cancer-specific survival (p = 0.022). CONCLUSIONS: There is a striking similarity in GEP-NET and NEBC regarding prognostic factors. GEP-NET and NEBC also appear to show similar expression patterns of the studied markers, while there are notable differences compared to IDC. Due to the wide expression of SSTR-2A, the treatment option with somatostatin analogs in NEBC should be evaluated.

PCBP1 depletion promotes tumorigenesis through attenuation of p27Kip1 mRNA stability and translation.

BACKGROUND: Poly C Binding Protein 1 (PCBP1) is an RNA-binding protein that binds and regulates translational activity of subsets of cellular mRNAs. Depletion of PCBP1 is implicated in various carcinomas, but the underlying mechanism in tumorigenesis remains elusive. METHODS: We performed a transcriptome-wide screen to identify novel bounding mRNA of PCBP1. The bind regions between PCBP1 with target mRNA were investigated by using point mutation and luciferase assay. Cell proliferation, cell cycle, tumorigenesis and cell apoptosis were also evaluated in ovary and colon cancer cell lines. The mechanism that PCBP1 affects p27 was analyzed by mRNA stability and ribosome profiling assays. We analyzed PCBP1 and p27 expression in ovary, colon and renal tumor samples and adjacent non-tumor tissues using RT-PCR, Western Blotting and immunohistochemistry. The prognostic significance of PCBP1 and p27 also analyzed using online databases. RESULTS: We identified cell cycle inhibitor p27Kip1 (p27) as a novel PCBP1-bound transcript. We then demonstrated that binding of PCBP1 to p27 3'UTR via its KH1 domain mainly stabilizes p27 mRNA, while enhances its translation to fuel p27 expression, prior to p27 protein degradation. The upregulated p27 consequently inhibits cell proliferation, cell cycle progression and tumorigenesis, whereas promotes cell apoptosis under paclitaxel treatment. Conversely, knockdown of PCBP1 in turn compromises p27 mRNA stability, leading to lower p27 level and tumorigenesis in vivo. Moreover, forced depletion of p27 counteracts the tumor suppressive ability of PCBP1 in the same PCBP1 over-expressing cells. Physiologically, we showed that decreases of both p27 mRNA and its protein expressions are well correlated to PCBP1 depletion in ovary, colon and renal tumor samples, independent of the p27 ubiquitin ligase Skp2 level. Correlation of PCBP1 with p27 is also found in the tamoxifen, doxorubincin and lapatinib resistant breast cancer cells of GEO database. CONCLUSION: Our results thereby indicate that loss of PCBP1 expression firstly attenuates p27 expression at post-transcriptional level, and subsequently promotes carcinogenesis. PCBP1 could be used as a diagnostic marker to cancer patients.

ddY Mice Fed 10% Fat Diet Exhibit High p27KIP Expression and Delayed Hepatocyte DNA Synthesis During Liver Regeneration.

BACKGROUND: Excessive intake of a high-calorie diet has been implicated in the development of non-alcoholic fatty liver disease (NAFLD). Several studies have investigated the effect of NAFLD on liver regeneration, but the effects of simple steatosis have been found to be inconsistent. We aimed to assess whether the initial phase of diet-induced lipid accumulation, induced by a diet containing moderate levels of fat, impairs liver regeneration after partial hepatectomy (PHx) of mice. METHODS: Male ddY mice are prone to obesity, even when fed a relatively low-fat diet (FD). A model of early simple steatosis was created by feeding a 10% FD for 6 weeks to male ddY mice. Liver regeneration rate, DNA synthesis in hepatocytes, and DNA damage were then assessed. RESULTS: FD-fed mice had a slightly higher body mass (44.5 ± 2.6 grams vs. 48.1 ± 3.6 grams, P < 0.05), but not liver mass (2.55 ± 0.37 grams vs. 2.69 ± 0.26 grams). Lipid droplets appeared in FD-fed mouse hepatocytes and Oil Red O staining was five times as intense as in control mice. In FD-fed mice, liver regeneration rate after two-thirds PHx was lower and impaired DNA replication was also observed. FD induced the expression of the cyclin-dependent kinase inhibitor p27KIP, but not p21CIP. Moreover, greater histone 2A.X phosphorylation was observed, indicating that FD caused DNA damage. CONCLUSIONS: Even short-term feeding of a moderately high FD to male ddY mice results in lipid accumulation in hepatocytes, DNA damage, and greater expression of p27KIP, implying lower DNA synthesis.

Oncogene-induced senescence: a potential breakpoint mechanism against malignant transformation in plasma cell disorders.

Oncogene-induced senescence (OIS) is a cellular tumor-suppressive mechanism present in several premalignant conditions. Here, we analyze the possible impact of OIS on malignant transformation in plasma cell disorders. Tumor samples from 125 patients with different disease stages were analyzed immunohistochemically for expression of senescence markers. Protein expression of cyclin-dependent kinase inhibitor p21Cip1/Waf1 was significantly higher in smoldering multiple myeloma (SMM) compared to monoclonal gammopathy of undetermined significance (MGUS) (p = .02) or symptomatic multiple myeloma (MM) (p = .005). SMM plasma cells expressing p21Cip1/Waf1 were negative for Ki67, consistent with senescence. While p27Kip1 was highly expressed in healthy controls, MGUS and SMM, expression decreased significantly in MM (p = .02). SMM plasma cells displayed a mutually exclusive expression of p21Cip1/Waf1/p27Kip1 suggesting compensatory mechanisms of senescence. In conclusion, we found markers of cellular senescence differentially expressed in SMM compared to MGUS and MM supporting the hypothesis of OIS as a breakpoint mechanism against malignant transformation in plasma cell disorders.

Clinical Impact of p27Kip1 and CaSR Expression on Primary Hyperparathyroidism.

We aimed to investigate the expressions of p27 kinase inhibitory protein 1 (p27Kip1) and calcium sensing receptor (CaSR) in adenomas and normal parathyroid tissue and to evaluate the relationship of these molecules with clinical and biochemical parameters in primary hyperparathyroidism (PHPT). Fifty-one patients with histopathologically confirmed parathyroid adenomas and 20 patients with normal parathyroid glands (which were removed incidentally during thyroid resection) were included. Immunohistochemical stainings of CaSR and p27Kip1 were performed in surgical specimens. Clinical features, biochemical parameters, and BMD measurements of patients with PHPT were evaluated retrospectively. Expressions of p27Kip1 and CaSR were decreased in parathyroid adenomas, compared to normal glands (p < 0.05). High intensity of CaSR staining (3+) was more frequent in normal parathyroid tissue (75%) than adenomas (12%) (p < 0.01). Hypertension was not observed in patients with high staining intensity of CaSR (p = 0.032). There was a negative association between CaSR expression and body mass index (BMI) (p = 0.027, r = - 0.313). There was no significant relationship between p27Kip1 and CaSR expressions, serum calcium, plasma parathormone, 25-hydroxy vitamin D levels, and bone density (p > 0.05). The expressions of p27Kip1 and CaSR were decreased in PHPT patients. This reduction may play an important role in the pathogenesis of PHPT. However, neither p27Kip1 nor CaSR expression was found to be useful in predicting prognosis or severity of disease.

Transcriptional and post-transcriptional upregulation of p27 mediates growth inhibition of isorhapontigenin (ISO) on human bladder cancer cells.

There are few approved drugs available for the treatment of muscle-invasive bladder cancer (MIBC). Recently, we have demonstrated that isorhapontigenin (ISO), a new derivative isolated from the Chinese herb Gnetum cleistostachyum, effectively induces cell-cycle arrest at the G0/G1 phase and inhibits anchorage-independent cell growth through the miR-137/Sp1/cyclin D1 axis in human MIBC cells. Herein, we found that treatment of bladder cancer (BC) cells with ISO resulted in a significant upregulation of p27, which was also observed in ISO-treated mouse BCs that were induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Importantly, knockdown of p27 caused a decline in the ISO-induced G0-G1 growth arrest and reversed ISO suppression of anchorage-independent growth in BC cells. Mechanistic studies revealed that ISO promoted p27 expression at mRNA transcription level through increasing direct binding of forkhead box class O1 (FOXO1) to its promoter, while knockdown of FOXO1 attenuated ISO inhibition of BC cell growth. On the other hand, ISO upregulated the 3'-untranslated region (3'-UTR) activity of p27, which was accompanied by a reduction of miR-182 expression. In line with these observations, ectopic expression of miR-182 significantly blocked p27 3'-UTR activity, whereas mutation of the miR-182-binding site at p27 mRNA 3'-UTR effectively reversed this inhibition. Accordingly, ectopic expression of miR-182 also attenuated ISO upregulation of p27 expression and impaired ISO inhibition of BC cell growth. Our results not only provide novel insight into understanding of the underlying mechanism related to regulation of MIBC cell growth but also identify new roles and mechanisms underlying ISO inhibition of BC cell growth.

Expression of hsa-let-7b-5p, hsa-let-7f-5p, and hsa-miR-222-3p and their putative targets HMGA2 and CDKN1B in typical and atypical carcinoid tumors of the lung.

Typical and atypical carcinoid tumors belong to the neuroendocrine lung tumors. They have low recurrence and proliferation rate, lymph node, and distant metastases. Nevertheless, these tumors have shown a more aggressive behavior. In the last years, microRNAs were screened as new tumor markers for their potential diagnostic and therapeutic relevance. The expression of hsa-let-7b-5p, hsa-let-7f-5p, hsa-miR-222-3p, and their targets HMGA2 (high-mobility group A2) and CDKN1B (cyclin-dependent kynase inhibitor 1B, p27kip1) was evaluated in this rare small group of patients. We analyzed the clinical data of all typical and atypical carcinoid tumors of patients who underwent surgical operation at Marburg University Hospital (n = 18) from 2000. Quantitative reverse transcription polymerase chain reaction was performed in formalin-fixed paraffin-embedded tumor tissue versus four tumor-free lung tissue samples. HMGA2 was stable or downregulated; only one patient showed a significant overexpression. CDKN1B showed a significant overexpression or a stable level; it was downregulated in two samples only. Hsa-miR-222-3p resulted almost stable or overexpressed except for two samples (significantly downregulated). Hsa-let-7f-5p was stable or overexpressed in the majority of analyzed samples, whereas hsa-let-7b-5p was significantly downregulated. HMGA2 and CDKN1B are differently expressed between atypical and typical carcinoid tumors, thus representing valid biomarkers for the classification of the two tumor groups. Hsa-let-7f-5p and HMGA2 are inversely correlated. Hsa-miR-222-3p does not correlate with its predicted target CDKN1B.

PKCζ-dependent upregulation of p27kip1 contributes to oxidative stress induced retinal pigment epithelial cell multinucleation.

Retinal pigment epithelial (RPE) cells increase in size and multinucleate during aging. We have shown using human and mouse cell lines that oxidised photoreceptor outer segments (oxPOS)-induced cytokinesis failure is related to RPE cell multinucleation, although the underlying mechanism remains unknown. This study investigated the role of the PKC pathway in oxPOS-induced RPE multinucleation using ARPE19 cells. oxPOS treatment promoted PKC activity and upregulated the mRNA expression of PKC α, δ, ζ, ι and µ. Inhibition of PKCα with GÓ§6976 resulted in a 33% reduction of multinucleate ARPE19 cells, whereas inhibition of PKCζ with GÓ§6983 led to a 50% reduction in multinucleate ARPE19 cells. Furthermore, oxPOS treatment induced a PKCζ-dependent upregulation of the Cdk inhibitor p27kip1, its inhibition using A2CE reduced oxPOS-induced ARPE19 multinucleation. Our results suggest that oxPOS-induced ARPE19 cytokinesis failure is, at least in part, due to the upregulation of p27kip1 through activating the PKC, particularly PKCζ pathway. Targeting the PKCζ-p27kip1 signalling axis may be a novel approach to restore RPE repair capacity during aging.

Anchimerically Activated ProTides as Inhibitors of Cap-Dependent Translation and Inducers of Chemosensitization in Mantle Cell Lymphoma.

The cellular delivery of nucleotides through various pronucleotide strategies has expanded the utility of nucleosides as a therapeutic class. Although highly successful, the highly popular ProTide system relies on a four-step enzymatic and chemical process to liberate the corresponding monophosphate. To broaden the scope and reduce the number of steps required for monophosphate release, we have developed a strategy that depends on initial chemical activation by a sulfur atom of a methylthioalkyl protecting group, followed by enzymatic hydrolysis of the resulting phosphoramidate monoester. We have employed this ProTide strategy for intracellular delivery of a nucleotide antagonist of eIF4E in mantle cell lymphoma (MCL) cells. Furthermore, we demonstrated that chemical inhibition of cap-dependent translation results in suppression of c-Myc expression, increased p27 expression, and enhanced chemosensitization to doxorubicin, dexamethasone, and ibrutinib. In addition, the new ProTide strategy was shown to enhance oral bioavailability of the corresponding monoester phosphoramidate.

Dopamine receptor type 2 (DRD2) and somatostatin receptor type 2 (SSTR2) agonists are effective in inhibiting proliferation of progenitor/stem-like cells isolated from nonfunctioning pituitary tumors.

The role of progenitor/stem cells in pituitary tumorigenesis, resistance to pharmacological treatments and tumor recurrence is still unclear. This study investigated the presence of progenitor/stem cells in non-functioning pituitary tumors (NFPTs) and tested the efficacy of dopamine receptor type 2 (DRD2) and somatostatin receptor type 2 (SSTR2) agonists to inhibit in vitro proliferation. They found that 70% of 46 NFPTs formed spheres co-expressing stem cell markers, transcription factors (DAX1, SF1, ERG1) and gonadotropins. Analysis of tumor behavior showed that spheres formation was associated with tumor invasiveness (OR = 3,96; IC: 1.05-14.88, p = 0.036). The in vitro reduction of cell proliferation by DRD2 and SSTR2 agonists (31 ± 17% and 35 ± 13% inhibition, respectively, p < 0.01 vs. basal) occurring in about a half of NFPTs cells was conserved in the corresponding spheres. Accordingly, these drugs increased cyclin-dependent kinase inhibitor p27 and decreased cyclin D3 expression in spheres. In conclusion, they provided further evidence for the existence of cells with a progenitor/stem cells-like phenotype in the majority of NFPTs, particularly in those with invasive behavior, and demonstrated that the antiproliferative effects of dopaminergic and somatostatinergic drugs were maintained in progenitor/stem-like cells.

Cyclin-Dependent Kinase Inhibitor 3 Promotes Cancer Cell Proliferation and Tumorigenesis in Nasopharyngeal Carcinoma by Targeting p27.

Nasopharyngeal carcinoma (NPC) is a common malignancy of the head and neck that arises from the nasopharynx epithelium and is highly invasive. Cyclin-dependent kinase inhibitor 3 (CDKN3) belongs to the dual-specificity protein phosphatase family, which plays a key role in regulating cell division. Abnormal expression of CDKN3 has been found in numerous types of cancer. In the current study, we explored the possible role of CDKN3 in cell proliferation, ability to invade, and radiosensitivity in NPC cells. We reported that CDKN3 was upregulated and p27 was downregulated in NPC tissues and is associated with a worse prognosis for patients. In addition, downregulation of CDKN3 and upregulation of p27 decreased cell proliferation, induced cell cycle arrest, increased apoptosis, decreased cell invasion, and enhanced radiosensitivity. Silencing of p27 significantly inhibited the effects of the knockdown of CDKN3. Moreover, downregulation of CDKN3 and upregulation of p27 inhibited the increase in tumor volume and weight in implanted tumors, decreased the phosphorylation of Akt, and increased the expression of cleaved caspase 3 in tumors. CDKN3 expression was also inversely correlated with p27 expression in NPC patients. Knockdown of CDKN3 increased p27 expression. Silencing of p27 markedly inhibited the effects of CDKN3 on cell proliferation, cell cycle progression, apoptosis, invasion, and radiosensitivity. These results demonstrate that upregulation of p27 is involved in the knockdown of CDKN3-induced decrease in cell proliferation, increase in cell cycle arrest and apoptosis, decrease in invasion, and increase in radiosensitivity. The results demonstrate that the CDKN3/p27 axis may be a novel target in the treatment of NPC.

p27kip1 overexpression regulates IL-1ß in the microenvironment of stem cells and eutopic endometriosis co-cultures.

Endometriosis is a gynecological benign chronic disease defined as the growth of endometrial glands and stroma in extra-uterine sites, most commonly implanted over visceral and peritoneal surfaces within the female pelvis causing inflammatory lesions. It affects around 10% of the female population and is often accompanied by chronic pelvic pain, adhesion formation and infertility. Therefore, endometriosis could be considered a "social disease", since it affects the quality of life, reproductivity and also has a socio-economic impact. The expression of cell cycle and inflammatory proteins is modified in the endometriotic tissues. Immunostaining of glandular and stromal cells in endometrial biopsies obtained from patients with endometriosis compared with those of healthy control demonstrated that endometriotic tissues have lower levels of p27kip1 protein. Endometriosis endometrial cells cultures have also lower levels of p27kip1 compared to health endometrial cells cultures and restore the cell cycle balance when transduced with an adenoviral vector carring the p27kip1 coding gene (Adp27EGFP). The low levels of p27kip1 are related to the S phase in the cell cycle, whereas higher levels lead to a G1 cell cycle arrest. The inflammatory cytokine IL-1ß was recently identified as another key protein in the endometriosis proliferation. This cytokine has elevated levels during the proliferative and secretory phases of the menstrual cycle. In endometriosis endometrial cells cultures the IL-1ß stimulates the production of IL-6 and IL-8, increasing the cell proliferation and reducing the apoptosis and Bax expression in these cells. According to these remarks, this work aims to evaluate the inflammatory effects in vitro, but more next to what happens in a woman's body, associating endometrial cells with stem cells, thus mimicking the endometrial microenvironment, with gene therapy using Adp27, notoriously known as controller cell cycle, apoptosis and potent modulator of VEGF expression.

Roscovitine Protects From Arterial Injury by Regulating the Expressions of c-Jun and p27 and Inhibiting Vascular Smooth Muscle Cell Proliferation.

PURPOSE: Roscovitine (Rosc) is a selective inhibitor of cyclin-dependent kinases (CDKs) and a promising therapy for various cancers. However, limited information is available on the biological significance of Rosc in vascular smooth muscle cells (VSMCs), the cell type critical for the development of proliferative vascular diseases. In this study, we address the effects of Rosc in regulating VSMC proliferation, both in vitro and in vivo, exploring the underlying molecular mechanisms. METHODS: The proliferations and cell-cycle distributions of in vitro cultured VSMCs, as well as several other cancer cell lines, were examined by cell-counting assay and flow cytometry, respectively. Molecular changes in various CDKs, cyclins, and other regulatory molecules were examined by reverse transcription polymerase chain reaction, Western blot, or immunocytochemistry. The in vivo effects of Rosc were examined on a carotid arterial balloon-injury model. RESULTS: Rosc significantly inhibited VSMC proliferation in response to serum or angiotensin II and arrested these cells at the G0/G1 phase. These changes were associated with a specific and robust decrease in CDK4, cyclin E, c-Jun, and a dramatic increase in p27kip1 in VSMCs, which was also translated in vivo and correlated with the protection of Rosc on injury-induced neointimal hyperplasia. CONCLUSIONS: Acting on distinct molecular targets in VSMCs versus cancer cells, Rosc inhibits VSMC proliferation and protects from proliferative vascular diseases.

Growth arrest of vascular smooth muscle cells in suspension culture using low-acyl gellan gum.

The proliferation of vascular smooth muscle cells (SMCs) causes restenosis in biomaterial vascular grafts. The purposes of this study were to establish a suspension culture system for SMCs by using a novel substrate, low-acyl gellan gum (GG) and to maintain SMCs in a state of growth inhibition. When SMCs were cultured in suspension with GG, their proliferation was inhibited. Their viability was 70% at day 2, which was maintained at more than 50% until day 5. In contrast, the viability of cells cultured in suspension without GG was 5.6% at day 2. By cell cycle analysis, the ratio of SMCs in the S phase when cultured in suspension with GG was lower than when cultured on plastic plates. In SMCs cultured in suspension with GG, the ratio of phosphorylated retinoblastoma (Rb) protein to Rb protein was decreased and p27Kip1 expression was unchanged in comparison with SMCs cultured on plastic plates. In addition, SMCs could be induced to proliferate again by changing the culture condition from suspension with GG to plastic plates. These results suggest that our established culturing method for SMCs is useful to maintain SMCs in a state of growth inhibition with high viability.