Deletion of Cdkn1b in ACI rats leads to increased proliferation and pregnancy-associated changes in the mammary gland due to perturbed systemic endocrine environment.

Mammary epithelial progenitors are the normal cell-of-origin of breast cancer. We previously defined a population of p27+ quiescent hormone-responsive progenitor cells in the normal human breast whose frequency associates with breast cancer risk. Here, we describe that deletion of the Cdkn1b gene encoding the p27 cyclin-dependent kinase inhibitor in the estrogen-induced mammary tumor-susceptible ACI rat strain leads to a decrease in the relative frequencies of Cd49b+ mammary luminal epithelial progenitors and pregnancy-related differentiation. We show by comprehensive gene expression profiling of purified progenitor and differentiated mammary epithelial cell populations that p27 deletion has the most pronounced effects on luminal progenitors. Cdkn1b-/- females have decreased fertility, but rats that are able to get pregnant had normal litter size and were able to nurse their pups implying that loss of p27 in ACI rats does not completely abrogate ovarian function and lactation. Reciprocal mammary gland transplantation experiments indicate that the p27-loss-induced changes in mammary epithelial cells are not only caused by alterations in their intrinsic properties, but are likely due to altered hormonal signaling triggered by the perturbed systemic endocrine environment observed in Cdkn1b-/- females. We also observed a decrease in the frequency of mammary epithelial cells positive for progesterone receptor (Pr) and FoxA1, known direct transcriptional targets of the estrogen receptor (Erα), and an increase in phospho-Stat5 positive cells commonly induced by prolactin (Prl). Characterization of genome-wide Pr chromatin binding revealed distinct binding patterns in mammary epithelial cells of Cdkn1b+/+ and Cdkn1b-/- females and enrichment in genes with known roles in Notch, ErbB, leptin, and Erα signaling and regulation of G1-S transition. Our data support a role for p27 in regulating the pool size of hormone-responsive luminal progenitors that could impact breast cancer risk.

HIV-1 Vpr and p21 restrict LINE-1 mobility.

Long interspersed element-1 (LINE-1, L1) composes ∼17% of the human genome. However, genetic interactions between L1 and human immunodeficiency virus type 1 (HIV-1) remain poorly understood. In this study, we found that HIV-1 suppresses L1 retrotransposition. Notably, HIV-1 Vpr strongly inhibited retrotransposition without inhibiting L1 promoter activity. Since Vpr is known to regulate host cell cycle, we examined the possibility whether Vpr suppresses L1 retrotransposition in a cell cycle dependent manner. We showed that the inhibitory effect of a mutant Vpr (H71R), which is unable to arrest the cell cycle, was significantly relieved compared with that of wild-type Vpr, suggesting that Vpr suppresses L1 mobility in a cell cycle dependent manner. Furthermore, a host cell cycle regulator p21Waf1 strongly suppressed L1 retrotransposition. The N-terminal kinase inhibitory domain (KID) of p21 was required for this inhibitory effect. Another KID-containing host cell cycle regulator p27Kip1 also strongly suppressed L1 retrotransposition. We showed that Vpr and p21 coimmunoprecipitated with L1 ORF2p and they suppressed the L1 reverse transcriptase activity in LEAP assay, suggesting that Vpr and p21 inhibit ORF2p-mediated reverse transcription. Altogether, our results suggest that viral and host cell cycle regulatory machinery limit L1 mobility in cultured cells.

p27Kip1, PCAF and PAX5 cooperate in the transcriptional regulation of specific target genes.

The cyclin-dependent kinase inhibitor p27Kip1 (p27) also behaves as a transcriptional repressor. Data showing that the p300/CBP-associated factor (PCAF) acetylates p27 inducing its degradation suggested that PCAF and p27 could collaborate in the regulation of transcription. However, this possibility remained to be explored. We analyzed here the transcriptional programs regulated by PCAF and p27 in the colon cancer cell line HCT116 by chromatin immunoprecipitation sequencing (ChIP-seq). We identified 269 protein-encoding genes that contain both p27 and PCAF binding sites being the majority of these sites different for PCAF and p27. PCAF or p27 knock down revealed that both regulate the expression of these genes, PCAF as an activator and p27 as a repressor. The double knock down of PCAF and p27 strongly reduced their expression indicating that the activating role of PCAF overrides the repressive effect of p27. We also observed that the transcription factor Pax5 interacts with both p27 and PCAF and that the knock down of Pax5 induces the expression of p27/PCAF target genes indicating that it also participates in the transcriptional regulation mediated by p27/PCAF. In summary, we report here a previously unknown mechanism of transcriptional regulation mediated by p27, Pax5 and PCAF.

Cryptic sequence features within the disordered protein p27Kip1 regulate cell cycle signaling.

Peptide motifs embedded within intrinsically disordered regions (IDRs) of proteins are often the sites of posttranslational modifications that control cell-signaling pathways. How do IDR sequences modulate the functionalities of motifs? We answer this question using the polyampholytic C-terminal IDR of the cell cycle inhibitory protein p27(Kip1) (p27). Phosphorylation of Thr-187 (T187) within the p27 IDR controls entry into S phase of the cell division cycle. Additionally, the conformational properties of polyampholytic sequences are predicted to be influenced by the linear patterning of oppositely charged residues. Therefore, we designed sequence variants of the p27 IDR to alter charge patterning outside the primary substrate motif containing T187. Computer simulations and biophysical measurements confirm predictions regarding the impact of charge patterning on the global dimensions of IDRs. Through functional studies, we uncover cryptic sequence features within the p27 IDR that influence the efficiency of T187 phosphorylation. Specifically, we find a positive correlation between T187 phosphorylation efficiency and the weighted net charge per residue of an auxiliary motif. We also find that accumulation of positive charges within the auxiliary motif can diminish the efficiency of T187 phosphorylation because this increases the likelihood of long-range intra-IDR interactions that involve both the primary and auxiliary motifs and inhibit their contributions to function. Importantly, our findings suggest that the cryptic sequence features of the WT p27 IDR negatively regulate T187 phosphorylation signaling. Our approaches provide a generalizable strategy for uncovering the influence of sequence contexts on the functionalities of primary motifs in other IDRs.

p27kip1 controls H-Ras/MAPK activation and cell cycle entry via modulation of MT stability.

The cyclin-dependent kinase (CDK) inhibitor p27(kip1) is a critical regulator of the G1/S-phase transition of the cell cycle and also regulates microtubule (MT) stability. This latter function is exerted by modulating the activity of stathmin, an MT-destabilizing protein, and by direct binding to MTs. We recently demonstrated that increased proliferation in p27(kip1)-null mice is reverted by concomitant deletion of stathmin in p27(kip1)/stathmin double-KO mice, suggesting that a CDK-independent function of p27(kip1) contributes to the control of cell proliferation. Whether the regulation of MT stability by p27(kip1) impinges on signaling pathway activation and contributes to the decision to enter the cell cycle is largely unknown. Here, we report that faster cell cycle entry of p27(kip1)-null cells was impaired by the concomitant deletion of stathmin. Using gene expression profiling coupled with bioinformatic analyses, we show that p27(kip1) and stathmin conjunctly control activation of the MAPK pathway. From a molecular point of view, we observed that p27(kip1), by controlling MT stability, impinges on H-Ras trafficking and ubiquitination levels, eventually restraining its full activation. Our study identifies a regulatory axis controlling the G1/S-phase transition, relying on the regulation of MT stability by p27(kip1) and finely controlling the spatiotemporal activation of the Ras-MAPK signaling pathway.

Integrated Cox's model for predicting survival time of glioblastoma multiforme.

Glioblastoma multiforme is the most common primary brain tumor and is highly lethal. This study aims to figure out signatures for predicting the survival time of patients with glioblastoma multiforme. Clinical information, messenger RNA expression, microRNA expression, and single-nucleotide polymorphism array data of patients with glioblastoma multiforme were retrieved from The Cancer Genome Atlas. Patients were separated into two groups by using 1 year as a cutoff, and a logistic regression model was used to figure out any variables that can predict whether the patient was able to live longer than 1 year. Furthermore, Cox's model was used to find out features that were correlated with the survival time. Finally, a Cox model integrated the significant clinical variables, messenger RNA expression, microRNA expression, and single-nucleotide polymorphism was built. Although the classification method failed, signatures of clinical features, messenger RNA expression levels, and microRNA expression levels were figured out by using Cox's model. However, no single-nucleotide polymorphisms related to prognosis were found. The selected clinical features were age at initial diagnosis, Karnofsky score, and race, all of which had been suggested to correlate with survival time. Both of the two significant microRNAs, microRNA-221 and microRNA-222, were targeted to p27Kip1 protein, which implied the important role of p27Kip1 on the prognosis of glioblastoma multiforme patients. Our results suggested that survival modeling was more suitable than classification to figure out prognostic biomarkers for patients with glioblastoma multiforme. An integrated model containing clinical features, messenger RNA levels, and microRNA expression levels was built, which has the potential to be used in clinics and thus to improve the survival status of glioblastoma multiforme patients.

p27KIP1 loss promotes proliferation and phagocytosis but prevents epithelial-mesenchymal transition in RPE cells after photoreceptor damage.

PURPOSE: p27KIP1 (p27), originally identified as a cell cycle inhibitor, is now known to have multifaceted roles beyond cell cycle regulation. p27 is required for the normal histogenesis of the RPE, but the role of p27 in the mature RPE remains elusive. To define the role of p27 in the maintenance and function of the RPE, we investigated the effects of p27 deletion on the responses of the RPE after photoreceptor damage. METHODS: Photoreceptor damage was induced in wild-type (WT) and p27 knockout (KO) mice with N-methyl-N-nitrosourea (MNU) treatment. Damage-induced responses of the RPE were investigated with bromodeoxyuridine (BrdU) incorporation assays, immunofluorescence, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays at different stages after MNU treatment. Subcellular localization of p27 in the WT RPE was also analyzed in vivo and in vitro. RESULTS: MNU treatment induced photoreceptor-specific degeneration in the WT and KO retinas. BrdU incorporation assays revealed virtually no proliferation of RPE cells in the WT retinas while, in the KO retinas, approximately 16% of the RPE cells incorporated BrdU at day 2 after MNU treatment. The RPE in the KO retinas developed aberrant protrusions into the outer nuclear layer in response to photoreceptor damage and engulfed outer segment debris, as well as TUNEL-positive photoreceptor cells. Increased phosphorylation of myosin light chains and their association with rhodopsin-positive phagosomes were observed in the mutant RPE, suggesting possible deregulation of cytoskeletal dynamics. In addition, WT RPE cells exhibited evidence of the epithelial-mesenchymal transition (EMT), including morphological changes, induction of α-smooth muscle actin expression, and attenuated expression of tight junction protein ZO-1 while these changes were absent in the KO retinas. In the normal WT retinas, p27 was localized to the nuclei of RPE cells while nuclear and cytoplasmic p27 was detected in RPE cells undergoing EMT, suggesting a role for cytoplasmic p27 in the phenotype changes of RPE cells. CONCLUSIONS: p27 loss promoted proliferation and phagocytic activity of RPE cells while preventing EMT after photoreceptor damage. These findings provide evidence for the role of p27 in the control of RPE responses to retinal damage.

Zinc deficiency impairs the renewal of hippocampal neural stem cells in adult rats: involvement of FoxO3a activation and downstream p27(kip1) expression.

Zinc plays an important role in the development and maintenance of central neural system. Zinc deficiency has been known to alter normal brain function, whose molecular mechanism remains largely elusive. In the present study, we established a zinc deficiency-exposed rat model, and, using western blot and immunohistochemical analyses, found that the expression of FoxO3a and p27(kip1) was remarkably up-regulated in the rat brain hippocampus. Immunofluorescence assay showed that FOXO3a and p27(kip1) were significantly co-localized with nestin, the marker of neural stem cells (NSCs). Furthermore, we identified that the proportion of proliferating NSCs was markedly decreased in zinc-deficient rat hippocampaus. Using C17.2 neural stem cells, it was revealed that exposure to zinc chelator N,N,N',N'-tetrakis-(2-pyridylmethy) ethylenediamine induced the expression of FoxO3a and p27(kip1) , which coincided with reduced NSC proliferation. Furthermore, depletion of FoxO3a inhibited p27(kip1) expression and restored the growth of NSCs. On the basis of these data, we concluded that FoxO3a/p27(kip1) signaling might play a significant role in zinc deficiency-induced growth impairment of NSCs and consequent neurological disorders. We describe here that zinc deficiency induces the proliferative impairment of hippocampal neural stem cells partially through the activation of FOXO3a-p27 axis in rats. Neural progenitor cells exhibited significantly up-regulated expression of FOXO3a and p27 after zinc deficiency in vivo and in vitro. Depletion of FOXO3a ameliorates zinc deficiency-induced expression of p27 and growth impairment of neural stem cells. We provide novel insight into the mechanisms underlying zinc deficiency-induced neurological deficits.

Deficiency of CDKN1A or both CDKN1A and CDKN1B affects the pubertal development of mouse Leydig cells.

Cyclin-dependent kinase inhibitors p21(Cip1) (CDKN1A) and p27(Kip1) (CDKN1B) are expressed in Leydig cells. Previously, we reported that Cdkn1b knockout in the mouse led to increased Leydig cell proliferative capacity and lower steroidogenesis. However, the relative importance of CDKN1A and CDKN1B in these regulations was unclear. In the present study, we examined the relative importance of CDKN1A and CDKN1B in regulation of Leydig cell proliferation and steroidogenesis by whole-body knockout of CDKN1A (Cdkn1a(-/-)) and CDKN1A/CDKN1B double knockout (DBKO). The cell number, 5-bromo-2-deoxyuridine incorporation rate, steroidogenesis, and steroidogenic enzyme mRNA levels and activities of Leydig cells were compared among wild-type (WT), Cdkn1a(-/-), and DBKO mice. Relative to WT mice, Leydig cell number per testis was doubled in the DBKO and unchanged in the Cdkn1a(-/-) mice. Testicular testosterone levels and mRNA levels for luteinizing hormone receptor (Lhcgr), steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage enzyme (Cyp11a1), 17alpha-hydroxylase/17,20-lyase (Cyp17a1), and 17beta-hydroxysteroid dehydrogenase 3 (Hsd17b3) and their respective proteins were significantly lower in the DBKO mice. However, testicular testosterone level was unchanged in the Cdkn1a(-/-) mice, although Lhcgr mRNA levels were significantly lower relative to those in the WT control. We conclude that Cdkn1a(-/-) did not increase Leydig cell numbers (although a defect of Leydig cell function was noted), whereas DBKO caused a significant increase of Leydig cell numbers but a decrease of steroidogenesis.

Connexin 43 controls the multipolar phase of neuronal migration to the cerebral cortex.

The prospective pyramidal neurons, migrating from the proliferative ventricular zone to the overlaying cortical plate, assume multipolar morphology while passing through the transient subventricular zone. Here, we show that this morphogenetic transformation, from the bipolar to the mutipolar and then back to bipolar again, is associated with expression of connexin 43 (Cx43) and, that knockdown of Cx43 retards, whereas its overexpression enhances, this morphogenetic process. In addition, we have observed that knockdown of Cx43 reduces expression of p27, whereas overexpression of p27 rescues the effect of Cx43 knockdown in the multipolar neurons. Furthermore, functional gap junction/hemichannel domain, and the C-terminal domain of Cx43, independently enhance the expression of p27 and promote the morphological transformation and migration of the multipolar neurons in the SVZ/IZ. Collectively, these results indicate that Cx43 regulates the passage of migrating neurons through their multipolar stage via p27 signaling and that interference with this process, by either genetic and/or environmental factors, may cause cortical malformations.

Mechanisms in cardiac fibroblast growth: an obligate role for Skp2 and FOXO3a in ERK1/2 MAPK-dependent regulation of p27kip1.

Cardiac fibroblast hyperplasia associated with enhanced matrix deposition is a major determinant of tissue remodeling in several disease states of the heart. However, mechanisms controlling cell cycle progression in cardiac fibroblasts remain unexplored. Identification of cell cycle regulatory elements in these cells is important to develop strategies to check adverse cardiac remodeling under pathological conditions. This study sought to probe the mechanisms underlying ERK1/2-mediated p27(Kip1) regulation in mitogenically stimulated cardiac fibroblasts. Addition of 10% fetal calf serum to quiescent cultures of adult rat cardiac fibroblasts promoted ERK1/2 activation, as evidenced by its phosphorylation status. Reduction in [(3)H]thymidine incorporation into DNA increased population doubling time, flow cytometry, and Western blot analysis showing reduced levels of cyclins D and A, p27(Kip1) induction, and retinoblastoma protein (Rb) hypophosphorylation in ERK1/2-inhibited cells indicated ERK1/2 dependence of G1-S transition in cardiac fibroblasts. Lack of p27(Kip1) protein in serum-stimulated, ERK1/2-active cells was associated with increased levels of Skp2, an E3 ubiquitin ligase for p27(Kip1), whose knockdown by RNA interference induced p27(Kip1) expression. Further, forced expression of Skp2 in ERK1/2-inhibited cells downregulated p27(Kip1). Transcriptional upregulation of p27(Kip1) mRNA in ERK1/2-inhibited cells, demonstrated by real-time PCR, correlated with forkhead box O 3a (FOXO3a) transcription factor activation, shown by gel shift assay. FOXO3a knockdown attenuated p27(Kip1) mRNA and protein expression in ERK1/2-inhibited cells. We provide evidence for the first time that, in cardiac fibroblasts, activated ERK1/2 regulates p27(Kip1) expression transcriptionally and posttranslationally via FOXO3a- and Skp2-dependent mechanisms. Additionally, this study uncovers interesting interactions between critical cell cycle regulatory elements that are only beginning to be understood.

Differential expression of Pim-3, c-Myc, and p-p27 proteins in adenocarcinomas of the gastric cardia and distal stomach.

Gastric cardia adenocarcinoma (GCA) is distinct from adenocarcinoma of the distal stomach because of its different etiological factors, tumor characteristics, and biological behavior. However, its pathogenesis is not fully understood. The purpose of this study is to characterize the role of Pim-3, c-Myc, and p-p27 in the tumorigenesis and progression of different sites of gastric adenocarcinoma by determining its pathogenetic significance. The expression of Pim-3, c-Myc, and p-p27 proteins was evaluated by immunohistochemistry in 140 resection specimens of gastric adenocarcinomas (78 GCAs , 62 DGAs and 20 normal gastric tissues). The level of expression of Pim-3, c-Myc, and p-p27 and the co-expression of all three markers (Pim-3+/c-Myc+/p-p27+) in GCA were significantly lower than that in DGA tumors (P < 0.05). Detailed analysis of the immunoreactivity patterns showed that in DGA, Pim-3 immunoreactivity was associated significantly with poor tumor differentiation, advanced tumor stage, and presence of lymph node metastasis. In addition, c-Myc overexpression correlated with tumor stage and lymph node metastasis, and positive p-p27 expression correlated with poor differentiation and tumor stage. The phenotype of Pim-3(+)/c-Myc(+)/p-p27(+) co-expression was closely correlated with tumor stage and lymph node metastasis (P < 0.05). In contrast, GCA only demonstrated a close correlation of Pim-3 overexpression with poor tumor differentiation and tumor stage (P < 0.05). Our results demonstrate the presence of different expression patterns of Pim-3, c-Myc, p-p27, and Pim-3(+)/c-Myc(+)/p-p27(+) and their clinicopathologic significance in GCA and DGA tumors. Our results add support to the notion that distinct molecular mechanisms may be involved in the development and progression of adenocarcinomas from the gastric cardia and distal portion of stomach.

Roles of the Skp2/p27 axis in the progression of chronic nephropathy.

S-phase kinase-associated protein 2 (Skp2) is an F-box protein component of the Skp/Cullin/F-box-type E3 ubiquitin ligase that targets several cell cycle regulatory proteins for degradation through the ubiquitin-dependent pathway. Skp2-mediated degradation of p27, a cyclin-dependent kinase inhibitor, is involved in cell cycle regulation. Tubular epithelial cell proliferation is a characteristic feature of renal damage that is apparent in the early stages of nephropathy. The p27 level is associated with the progression of renal injury, and increased Skp2 expression in progressive nephropathy is implicated in decreases of p27 expression. In Skp2(-/-) mice, renal damage caused by unilateral ureteral obstruction (UUO) was ameliorated by p27 accumulation, mainly in tubular epithelial cells. However, the amelioration of UUO-induced renal injury in Skp2(-/-) mice was prevented by p27 deficiency in Skp2(-/-)/p27(-/-) mice. These results suggest that the Skp2-mediated reduction in p27 is a pathogenic activity that occurs during the progression of nephropathy. Here, we discuss the roles of the Skp2/p27 axis and/or related signaling pathways/components in the progression of chronic nephropathy.

NF-κB hyper-activation by HTLV-1 tax induces cellular senescence, but can be alleviated by the viral anti-sense protein HBZ.

Activation of I-κB kinases (IKKs) and NF-κB by the human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, is thought to promote cell proliferation and transformation. Paradoxically, expression of Tax in most cells leads to drastic up-regulation of cyclin-dependent kinase inhibitors, p21(CIP1/WAF1) and p27(KIP1), which cause p53-/pRb-independent cellular senescence. Here we demonstrate that p21(CIP1/WAF1)-/p27(KIP1)-mediated senescence constitutes a checkpoint against IKK/NF-κB hyper-activation. Senescence induced by Tax in HeLa cells is attenuated by mutations in Tax that reduce IKK/NF-κB activation and prevented by blocking NF-κB using a degradation-resistant mutant of I-κBα despite constitutive IKK activation. Small hairpin RNA-mediated knockdown indicates that RelA induces this senescence program by acting upstream of the anaphase promoting complex and RelB to stabilize p27(KIP1) protein and p21(CIP1/WAF1) mRNA respectively. Finally, we show that down-regulation of NF-κB by the HTLV-1 anti-sense protein, HBZ, delay or prevent the onset of Tax-induced senescence. We propose that the balance between Tax and HBZ expression determines the outcome of HTLV-1 infection. Robust HTLV-1 replication and elevated Tax expression drive IKK/NF-κB hyper-activation and trigger senescence. HBZ, however, modulates Tax-mediated viral replication and NF-κB activation, thus allowing HTLV-1-infected cells to proliferate, persist, and evolve. Finally, inactivation of the senescence checkpoint can facilitate persistent NF-κB activation and leukemogenesis.

Cdk5 phosphorylates and stabilizes p27kip1 contributing to actin organization and cortical neuronal migration.

p27(kip1), a cyclin-dependent kinase (CDK) inhibitor (CKI), generally suppresses CDK activity in proliferating cells. Although another role of p27 in cell migration has been recently suggested in vitro, the physiological importance of p27 in cell migration remains elusive, as p27-deficient mice have not shown any obvious migration-defect-related phenotypes. Here, we show that Cdk5, an unconventional neuronal CDK, phosphorylates and stabilizes p27 as an upstream regulator, maintaining the amount of p27 in post-mitotic neurons. In vivo RNA interference (RNAi) experiments showed that reduced amounts of p27 caused inhibition of cortical neuronal migration and decreased the amount of F-actin in the processes of migrating neurons. The Cdk5-p27 pathway activates an actin-binding protein, cofilin, which is also shown to be involved in cortical neuronal migration in vivo. Our findings shed light on a previously unknown new relationship between CDK and CKI in G0-arrested cells that regulates cytoskeletal reorganization and neuronal migration during corticogenesis.

miR-124 inhibits cell proliferation in gastric cancer through down-regulation of SPHK1.

SPHK1 expression is elevated in gastric cancer and is associated with shorter survival times for patients. However, the molecular mechanism of SPHK1 up-regulation in gastric cancer remains unclear. In the present study, we report that miR-124 down-regulated SPHK1 expression by directly targeting its 3'-untranslated region (3'-UTR) and that miR-124 expression was inversely correlated with SPHK1 expression in gastric cancer samples. Furthermore, we demonstrated that, similar to the effect of silencing SPHK1, up-regulation of miR-124 markedly inhibited proliferation and tumourigenicity of gastric cancer cells both in vitro and in vivo. This was found to be mechanistically associated with induction of cyclin-dependent kinase inhibitors p21$^{{\rm Cip1}}$ and p27$^{{\rm Kip1}}$, enhancement of the transcriptional activity of FOXO1 and suppression of AKT activity. Moreover, we showed that the re-introduction of SPHK1 (without the 3'-UTR), but not with the 3'-UTR, could abrogate the miR-124-mediated induction of p21$^{{\rm Cip1}}$ and p27$^{{\rm Kip1}}$, as well as rescue the miR-124-induced proliferation inhibition. Together, these results suggest that miR-124 has an important role in the suppression of gastric cancer and presents a novel mechanism of miRNA-mediated SPHK1 expression in cancer cells.

Alcohol suppresses the granulopoietic response to pulmonary Streptococcus pneumoniae infection with enhancement of STAT3 signaling.

Enhanced granulopoietic activity is crucial for host defense against bacterial pneumonia. Alcohol impairs this response. The underlying mechanisms remain obscure. G-CSF produced by infected lung tissue plays a key role in stimulating bone marrow granulopoiesis. This study investigated the effects of alcohol on G-CSF signaling in the regulation of marrow myeloid progenitor cell proliferation in mice with Streptococcus pneumoniae pneumonia. Chronic alcohol consumption plus acute alcohol intoxication suppressed the increase in blood granulocyte counts following intrapulmonary challenge with S. pneumoniae. This suppression was associated with a significant decrease in bone marrow granulopoietic progenitor cell proliferation. Alcohol treatment significantly enhanced STAT3 phosphorylation in bone marrow cells of animals challenged with S. pneumoniae. In vitro experiments showed that G-CSF-induced activation of STAT3-p27(Kip1) pathway in murine myeloid progenitor cell line 32D-G-CSFR cells was markedly enhanced by alcohol exposure. Alcohol dose dependently inhibited G-CSF-stimulated 32D-G-CSFR cell proliferation. This impairment of myeloid progenitor cell proliferation was not attenuated by inhibition of alcohol metabolism through either the alcohol dehydrogenase pathway or the cytochrome P450 system. These data suggest that alcohol enhances G-CSF-associated STAT3-p27(Kip1) signaling, which impairs granulopoietic progenitor cell proliferation by inducing cell cycling arrest and facilitating their terminal differentiation during the granulopoietic response to pulmonary infection.

Activation of the insulin-like growth factor-1 receptor alters p27 regulation by the epidermal growth factor receptor in oral squamous carcinoma cells.

BACKGROUND: Although oral squamous cell carcinomas (OSCCs) commonly overexpress the epidermal growth factor receptor (EGFR), EGFR tyrosine kinase inhibitors (TKIs) exhibit poor efficacy clinically. Activation of the insulin-like growth factor-1 receptor (IGF1R) induces resistance of OSCC cells to EGFR-TKIs in vitro. This study seeks to evaluate the changes in cell cycle status in OSCC cells in response to gefitinib and IGF1R activation. METHODS: SCC-25 OSCC cells were used for in vitro analyses. RESULTS: Gefitinib caused a 50% reduction in S-phase population, and IGF1R activation caused a 2.8-fold increase; combined treatment yielded a baseline S-phase population. Gefitinib treatment increased the cyclin-dependent kinase inhibitor p27, and this was not abrogated by IGF1R activation. pT157-p27 was noted by immunoblot to be decreased on gefitinib treatment, but this was reversed with IGF1R activation. T157 phosphorylation contributes to cytoplasmic localization of p27 where it can promote cell proliferation and cell motility. Using both subcellular fractionation and immunofluorescence microscopy techniques, IGF1R stimulation was noted to increase the relative cytoplasmic localization of p27; this persisted when combined with gefitinib. CONCLUSIONS: IGF1R activation partially reverses the cell cycle arrest caused by gefitinib in OSCC cells. While IGF1R stimulation does not eliminate the gefitinib-induced increase in total p27, its phosphorylation state and subcellular localization are altered. This may contribute to the ability of the IGF1R to rescue OSCC cells from EGFR-TKI treatment and may have important implications for the use of p27 as a biomarker of cell cycle arrest and response to therapy.

P27 as a prognostic factor of early cervical carcinoma.

OBJECTIVE: The aim of this study was to clarify whether the evaluation of cell-cycle regulatory protein p27 can serve as a prognostic factor in patients with International Federation of Gynecology and Obstetrics (FIGO) stage IB cervical carcinoma. PATIENTS AND METHODS: A retrospective study was performed on 130 surgically treated patients with FIGO stage IB cervical carcinoma with at least a 5-year follow-up. The expression of p27 was investigated independently by 2 experienced pathologists using immunohistochemistry. The prognostic significance of established prognostic factors and p27 expression were analyzed using univariate and multivariate analyses. RESULTS: In a univariate analysis, lymph node status, tumor diameter, Gynecologic Oncology Group (GOG) score, lymph vascular space invasion, and p27 expression were significant prognostic factors for overall survival (OS). We found a correlation between p27 expression and lymph node status, tumor diameter, invasion, and GOG score. The p27 expression was a statistically significant prognostic factor for OS in a univariate analysis (log-rank test, P = 0.03). In a multivariate analysis, only lymph node status and tumor diameter were statistically significant prognostic factors for OS. CONCLUSIONS: This study demonstrated that a low p27 expression is associated with lymph node metastasis, deep stromal invasion, tumor diameter more than 20 mm, and high GOG score and had a prognostic influence on OS in a univariate analysis in a series of 130 women with FIGO stage IB cervical carcinoma. Lymph node status and the diameter of the tumor were the only statistically significant prognostic factors in multivariate analysis.

17ß-Estradiol attenuates hypoxic pulmonary hypertension via estrogen receptor-mediated effects.

RATIONALE: 17ß-Estradiol (E2) attenuates hypoxic pulmonary vasoconstriction and hypoxic pulmonary hypertension (HPH) through an unknown mechanism that may involve estrogen receptors (ER) or E2 conversion to catecholestradiols and methoxyestradiols with previously unrecognized effects on cardiopulmonary vascular remodeling. OBJECTIVES: To determine the mechanism by which E2 exerts protective effects in HPH. METHODS: Male rats were exposed to hypobaric hypoxia while treated with E2 (75 µg/kg/d) or vehicle. Subgroups were cotreated with pharmacologic ER-antagonist or with inhibitors of E2-metabolite conversion. Complementary studies were performed in rats cotreated with selective ERα- or ERß-antagonist. Hemodynamic and pulmonary artery (PA) and right ventricular (RV) remodeling parameters, including cell proliferation, cell cycle, and autophagy, were measured in vivo and in cultured primary rat PA endothelial cells. MEASUREMENTS AND MAIN RESULTS: E2 significantly attenuated HPH endpoints. Hypoxia increased ERß but not ERα lung vascular expression. Co-treatment with nonselective ER inhibitor or ERα-specific antagonist rendered hypoxic animals resistant to the beneficial effects of E2 on cardiopulmonary hemodynamics, whereas ERα- and ERß-specific antagonists opposed the remodeling effects of E2. In contrast, inhibition of E2-metabolite conversion did not abolish E2 protection. E2-treated hypoxic animals exhibited reduced ERK1/2 activation and increased expression of cell-cycle inhibitor p27(Kip1) in lungs and RV, with up-regulation of lung autophagy. E2-induced signaling was recapitulated in hypoxic but not normoxic endothelial cells, and was associated with decreased vascular endothelial growth factor secretion and cell proliferation. CONCLUSIONS: E2 attenuates hemodynamic and remodeling parameters in HPH in an ER-dependent manner, through direct antiproliferative mechanisms on vascular cells, which may provide novel nonhormonal therapeutic targets for HPH.