Regulation of cyclin D1 RNA stability by SNIP1

Cyclin D1 expression represents one of the key mitogen-regulated events during the G1 phase of the cell cycle, whereas Cyclin D1 overexpression is frequently associated with human malignancy. Here, we describe a novel mechanism regulating Cyclin D1 levels. We find that SNIP1, previously identified as a regulator of Cyclin D1 expression, does not, as previously thought, primarily function as a transcriptional coactivator for this gene. Rather, SNIP1 plays a critical role in cotranscriptional or posttranscriptional Cyclin D1 mRNA stability. Moreover, we show that the majority of nucleoplasmic SNIP1 is present within a previously undescribed complex containing SkIP, THRAP3, BCLAF1, and Pinin, all proteins with reported roles in RNA processing and transcriptional regulation. We find that this complex, which we have termed the SNIP1/SkIP–associated RNA-processing complex, is coordinately recruited to both the 3' end of the Cyclin D1 gene and Cyclin D1 RNA. Significantly, SNIP1 is required for the further recruitment of the RNA processing factor U2AF65 to both the Cyclin D1 gene and RNA. This study shows a novel mechanism regulating Cyclin D1 expression and offers new insight into the role of SNIP1 and associated proteins as regulators of proliferation and cancer.

Overexpression of cyclin D1 moderately increases ploidy in megakaryocytes

Background and Objectives. Megakalyocytes undergo a unique cell cycle by which they replicate their complete genome many times in the absence of cytokinesis, In the search for regulators of the endomitotic cell cycle, we previously produced mice transgenic for cyclin D3 to identify this cyclin as able to enhance ploidy and to increase the number of differentiated cells in the megakaryocytic lineage. Of the D-type cyclins, cyclin D3 and to a much lesser extent cyclin D1, are present in megakaryocytes undergoing endomitosis and these cyclins are, respectively, markedly and moderately upregulated following exposure to the ploidy-promoting factor, Mpl-ligand. Our objective was to explore whether cyclin D1 can mimic the effect of cyclin D3 on ploidy in megakalyocytes.

The Cyclin D1 (CCND1) A870G polymorphism predicts clinical outcome to lapatinib and capecitabine in HER2-positive metastatic breast cancer

BACKGROUND: Lapatinib plus capecitabine emerged as an efficacious therapy in metastatic breast cancer (mBC). We aimed to identify germline single-nucleotide polymorphisms (SNPs) in genes involved in capecitabine catabolism and human epidermal receptor signaling that were associated with clinical outcome to assist in selecting patients likely to benefit from this combination.

PATIENTS AND METHODS: DNA was extracted from 240 of 399 patients enrolled in EGF100151 clinical trial (NCT00078572; clinicaltrials.gov) and SNPs were successfully evaluated in 234 patients. The associations between SNPs and clinical outcome were analyzed using Fisher's exact test, Kaplan-Meier curves, log-rank tests, likelihood ratio test within logistic or Cox regression model, as appropriate.

RESULTS: There were significant interactions between CCND1 A870G and clinical outcome. Patients carrying the A-allele were more likely to benefit from lapatinib plus capecitabine versus capecitabine when compared with patients harboring G/G (P = 0.022, 0.024 and 0.04, respectively). In patients with the A-allele, the response rate (RR) was significantly higher with lapatinib plus capecitabine (35%) compared with capecitabine (11%; P = 0.001) but not between treatments in patients with G/G (RR = 24% and 32%, respectively; P = 0.85). Time to tumor progression (TTP) was longer in patients with the A-allele treated with lapatinib plus capecitabine compared with capecitabine (median TTP = 7.9 and 3.4 months; P < 0.001), but not in patients with G/G (median TTP = 6.1 and 6.6 months; P = 0.92).

CONCLUSION: Our findings suggest that CCND1A870G may be useful in predicting clinical outcome in HER2-positive mBC patients treated with lapatinib plus capecitabine.

The cyclin D1 (CCND1) rs9344 G>A polymorphism predicts clinical outcome in colon cancer patients treated with adjuvant 5-FU-based chemotherapy

Recent evidence indicates a potential prognostic and predictive value for germline polymorphisms in genes involved in cell cycle control. We investigated the effect of cyclin D1 (CCND1) rs9344 G>A in stage II/III colon cancer patients and validated the findings in an independent study cohort. For evaluation and validation set, a total of 264 and 234 patients were included. Patients treated with 5-fluorouracil-based chemotherapy, carrying the CCND1 rs9344 A/A genotype had significantly decreased time-to-tumor recurrence (TTR) in univariate analysis and multivariate analysis (hazard ratio (HR) 2.47; 95% confidence interval (CI) 1.16-5.29; P=0.019). There was no significant association between CCND1 rs9344 G>A and TTR in patients with curative surgery alone. In the validation set, the A allele of CCND1 rs9344 G>A remained significantly associated with decreased TTR in univariate and multivariate analyses (HR 1.94; 95% CI 1.05-3.58; P=0.035). CCND1 rs9344 G>A may be a predictive and/or prognostic biomarker in stage II/III colon cancer patients, however, prospective trials are warranted to confirm our findings.

Cyclin D3/CDK11p58 complex is involved in the repression of androgen receptor.

Androgen receptor (AR) is essential for the maintenance of the male reproductive systems and is critical for the carcinogenesis of human prostate cancers (PCas). D-type cyclins are closely related to the repression of AR function. It has been well documented that cyclin D1 inhibits AR function through multiple mechanisms, but the mechanism of how cyclin D3 exerts its repressive role in the AR signaling pathway remains to be identified. In the present investigation, we demonstrate that cyclin D3 and the 58-kDa isoform of cyclin-dependent kinase 11 (CDK11p58) repressed AR transcriptional activity as measured by reporter assays of transformed cells and prostate-specific antigen expression in PCa cells. AR, cyclin D3, and CDK11p58 formed a ternary complex in cells and were colocalized in the luminal epithelial layer of the prostate. AR activity is controlled by phosphorylation at specific sites. We found that AR was phosphorylated at Ser-308 by cyclin D3/CDK11p58 in vitro and in vivo, leading to the repressed activity of AR transcriptional activation unit 1 (TAU1). Furthermore, androgen-dependent proliferation of PCa cells was inhibited by cyclin D3/CDK11p58 through AR repression. These data suggest that cyclin D3/CDK11p58 signaling is involved in the negative regulation of AR function.

Regulation of cyclin D1 by the BRCA1-BARD1 complex

Background BRCA1 and cyclin D₁ are both essential for normal breast development and mutation or aberration of their expression is associated with breast cancer [1,2]. Cyclin D₁ is best known as a G₁ cyclin where it regulates the G₁ to S phase transition by acting as a rate-limiting subunit of CDK4/6 kinase activity. More recently, however, Stacey has demonstrated that cyclin D₁ levels in G₂/M determine whether a cell continues to proliferate or exits the cell cycle [3]. The majority of BRCA1 in the cell is bound to BARD1 through their N-terminal RING domains. Heterodimerization is essential for the stability and correct localization of the complex and confers ubiquitin ligase activity to BRCA1. The importance of the ligase activity of BRCA1 to breast cancer development is inferred from the fact that N-terminal diseaseassociated mutations are proposed to reduce ligase activity [4]. Methods Protein–protein interactions were demonstrated using yeast-two-hybrid and coimmunoprecipitation. Protein levels were altered through overexpression, siRNA and antisense technology. The effect of proteasome inhibitors and cycloheximide treatment was also examined. Results We initially identified cyclin D₁ as a binding partner of BARD1 in a yeast-two-hybrid screen and defined the minimal binding region as the N-terminus of BARD1. This interaction was confirmed in vivo by coimmunoprecipitation. The N-terminus of BARD1 also binds BRCA1 and imparts ubiquitin ligase activity to the complex. Covalent modification of proteins with ubiquitin is a common regulatory mechanism in eukaryotic cells. Traditionally polyubiquitin chains linked through lysine 48 target proteins for degradation by the 26 S proteasome. We have demonstrated that cyclin D₁ protein levels are inversely related to BRCA1 and BARD1 levels in several model systems. Furthermore, regulation of cyclin D1 levels occurs through a post-transcriptional mechanism and requires the ligase activity of BRCA1. Interestingly, this phenomenon is cell-cycle regulated, occurring in G₂/M. Conclusion We propose that cyclin D₁ is a potential substrate for BRCA1 ubiquitination and that this targets cyclin D₁ for proteasomal-mediated degradation. Future work will focus on ascertaining the functional consequence of cyclin D₁ regulation by the BRCA1–BARD1 complex; in particular, the impact of BRCA1, mediated through regulation of cyclin D₁, on the proliferation versus differentiation decision.

Nonapical and Cytoplasmic Expression of Interleukin-8, CXCR1, and CXCR2 Correlates with Cell Proliferation and Microvessel Density in Prostate Cancer

Purpose: We characterized interleukin-8 (IL-8) and IL-8 receptor expression (CXCR1 and CXCR2) in prostate cancer to address their significance to this disease. Experimental Design: Immunohistochemistry was conducted on 40 cases of human prostate biopsy containing histologically normal and neoplastic tissue, excised from patients with locally confined or invasive androgen-dependent prostate cancer, and 10 cases of transurethral resection of the prostate material from patients with androgen-independent disease. Results: Weak to moderate IL-8 expression was strictly localized to the apical membrane of normal prostate epithelium. In contrast, membranous expression of IL-8, CXCR1, and CXCR2 was nonapical in cancer cells of Gleason pattern 3 and 4, whereas circumferential expression was present in Gleason pattern 5 and androgen-independent prostate cancer. Each of IL-8, CXCR1, and CXCR2 were also increasingly localized to the cytoplasm of cancer cells in correlation with advancing stage of disease. Cytoplasmic expression (but not apical membrane expression) of IL-8 in Gleason pattern 3 and 4 cancer correlated with Ki-67 expression (R = 0.79; P <0.001), cyclin D1 expression (R = 0.79; P <0.001), and microvessel density (R = 0.81; P <0.001). In vitro studies on androgen-independent PC3 cells confirmed the mitogenic activity of IL-8, increasing the rate of cell proliferation through activation of both CXCR1 and CXCR2 receptors. Conclusions: We propose that the concurrent increase in IL-8 and IL-8 receptor expression in human prostate cancer induces autocrine signaling that may be functionally significant in initiating and promoting the progression of prostate cancer by underpinning cell proliferation and angiogenesis.

Histone deacetylase 7 controls endothelial cell growth through modulation of beta-catenin

Rationale: Histone deacetylase (HDAC)7 is expressed in the early stages of embryonic development and may play a role in endothelial function.

Objective: This study aimed to investigate the role of HDAC7 in endothelial cell (EC) proliferation and growth and the underlying mechanism.

Methods and Results: Overexpression of HDAC7 by adenoviral gene transfer suppressed human umbilical vein endothelial cell (HUVEC) proliferation by preventing nuclear translocation of ß-catenin and downregulation of T-cell factor-1/Id2 (inhibitor of DNA binding 2) and cyclin D1, leading to G1 phase elongation. Further assays with the TOPFLASH reporter and quantitative RT-PCR for other ß-catenin target genes such as Axin2 confirmed that overexpression of HDAC7 decreased ß-catenin activity. Knockdown of HDAC7 by lentiviral short hairpin RNA transfer induced ß-catenin nuclear translocation but downregulated cyclin D1, cyclin E1 and E2F2, causing HUVEC hypertrophy. Immunoprecipitation assay and mass spectrometry analysis revealed that HDAC7 directly binds to ß-catenin and forms a complex with 14-3-3 e, ?, and ? proteins. Vascular endothelial growth factor treatment induced HDAC7 degradation via PLC?-IP3K (phospholipase C?–inositol-1,4,5-trisphosphate kinase) signal pathway and partially rescued HDAC7-mediated suppression of proliferation. Moreover, vascular endothelial growth factor stimulation suppressed the binding of HDAC7 with ß-catenin, disrupting the complex and releasing ß-catenin to translocate into the nucleus.

Conclusions: These findings demonstrate that HDAC7 interacts with ß-catenin keeping ECs in a low proliferation stage and provides a novel insight into the mechanism of HDAC7-mediated signal pathways leading to endothelial growth

Elevated NRD1 metalloprotease expression plays a role in breast cancer growth and proliferation

Understanding the molecular etiology of cancer and increasing the number of drugs and their targets are critical to cancer management. In our attempt to unravel novel breast-cancer associated proteins, we previously conducted protein expression profiling of the MCF10AT model, which comprises a series of isogenic cell lines that mimic different stages of breast cancer progression. NRD1 expression was found to increase during breast cancer progression. Here, we attempted to confirm the relevance of NRD1 in clinical breast cancer and understand the functional role and mechanism of NRD1 in breast cancer cells. Immunohistochemistry data show that NRD1 expression was elevated in ductal carcinoma in situ and invasive ductal carcinomas compared with normal tissues in 30% of the 26 matched cases studied. Examination of NRD1 expression in tissue microarray comprising >100 carcinomas and subsequent correlation with clinical data revealed that NRD1 expression was significantly associated with tumor size, grade, and nodal status (P <0.05). Silencing of NRD1 reduced MCF10CA1h and MDA-MD-231 breast-cancer-cell proliferation and growth. Probing the oncogenic EGF signaling pathways revealed that NRD1 knock down did not affect overall downstream tyrosine phosphorylation cascades including AKT and MAPK activation. Instead, silencing of NRD1 resulted in a reduction of overall cyclin D1 expression, a reduction of EGF-induced increase in cyclin D1 expression and an increase in apoptotic cell population compared with control cells.

Cyclin G associated kinase interacts with interleukin 12 receptor beta2 and suppresses interleukin 12 induced IFN-gamma production.

BACKGROUND: Although severe encephalopathy has been proposed as a possible contraindication to the use of noninvasive positive-pressure ventilation (NPPV), increasing clinical reports showed it was effective in patients with impaired consciousness and even coma secondary to acute respiratory failure, especially hypercapnic acute respiratory failure (HARF). To further evaluate the effectiveness and safety of NPPV for severe hypercapnic encephalopathy, a prospective case-control study was conducted at a university respiratory intensive care unit (RICU) in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) during the past 3 years. METHODS: Forty-three of 68 consecutive AECOPD patients requiring ventilatory support for HARF were divided into 2 groups, which were carefully matched for age, sex, COPD course, tobacco use and previous hospitalization history, according to the severity of encephalopathy, 22 patients with Glasgow coma scale (GCS) <10 served as group A and 21 with GCS = 10 as group B. RESULTS: Compared with group B, group A had a higher level of baseline arterial partial CO2 pressure ((102 +/- 27) mmHg vs (74 +/- 17) mmHg, P <0.01), lower levels of GCS (7.5 +/- 1.9 vs 12.2 +/- 1.8, P <0.01), arterial pH value (7.18 +/- 0.06 vs 7.28 +/- 0.07, P <0.01) and partial O(2) pressure/fraction of inspired O(2) ratio (168 +/- 39 vs 189 +/- 33, P <0.05). The NPPV success rate and hospital mortality were 73% (16/22) and 14% (3/22) respectively in group A, which were comparable to those in group B (68% (15/21) and 14% (3/21) respectively, all P > 0.05), but group A needed an average of 7 cm H2O higher of maximal pressure support during NPPV, and 4, 4 and 7 days longer of NPPV time, RICU stay and hospital stay respectively than group B (P <0.05 or P <0.01). NPPV therapy failed in 12 patients (6 in each group) because of excessive airway secretions (7 patients), hemodynamic instability (2), worsening of dyspnea and deterioration of gas exchange (2), and gastric content aspiration (1). CONCLUSIONS: Selected patients with severe hypercapnic encephalopathy secondary to HARF can be treated as effectively and safely with NPPV as awake patients with HARF due to AECOPD; a trial of NPPV should be instituted to reduce the need of endotracheal intubation in patients with severe hypercapnic encephalopathy who are otherwise good candidates for NPPV due to AECOPD.

Cyclin-dependent kinase 11(p58) interacts with HBO1 and enhances its histone acetyltransferase activity.

CDK11(p58), a 58kDa protein of the PITSLRE kinase family, plays an important role in cell cycle progression, and is closely related to cell apoptosis. To gain further insight into the function of CDK11(p58), we screened a human fetal liver cDNA library for its interacting proteins using the yeast two-hybrid system. Here we report that histone acetyltransferase (HAT) HBO1, a MYST family protein, interacts with CDK11(p58) in vitro and in vivo. CDK11(p58) and HBO1 colocalize in the cell nucleus. Recombinant CDK11(p58) enhances the HAT activity of HBO1 significantly in vitro. Meanwhile, overexpression of CDK11(p58) in mammalian cells leads to the enhanced HAT activity of HBO1 towards free histones. Thus, we conclude that CDK11(p58) is a new interacting protein and a novel regulator of HBO1. Both of the proteins may be involved in the regulation of eukaryotic transcription.

Identification of the p28 subunit of eukaryotic initiation factor 3(eIF3k) as a new interaction partner of cyclin D3.

Cyclin D3 is found to play a crucial role not only in progression through the G1 phase as a regulatory subunit of cyclin-dependent kinase 4 (CDK 4) and CDK 6, but also in many other aspects such as cell cycle, cell differentiation, transcriptional regulation and apoptosis. In this work, we screened a human fetal liver cDNA library using human cyclin D3 as bait and identified human eukaryotic initiation factor 3 p28 protein (eIF3k) as a partner of cyclin D3. The association of cyclin D3 with eIF3k was further confirmed by in vitro binding assay, in vivo coimmunoprecipitation, and confocal microscopic analysis. We found that cyclin D3 specifically interacted with eIF3k through its C-terminal domain. Immunofluorescence experiments showed that eIF3k distributed both in nucleus and cytoplasm and colocalized with cyclin D3. In addition, the cellular translation activity in HeLa cells was upregulated by cyclin D3 overexpression and the mRNA levels are constant. These data provide a new clue to our understanding of the cellular function of cyclin D3.

A study of magnetic bright points in the Na I D1 line.

High-cadence, multiwavelength, optical observations of solar magnetic bright points (MBPs), captured at the disk center using the ROSA and IBIS imaging systems on the Dunn Solar Telescope, are presented. MBPs manifesting in the Na I D-1 core are found to preferentially exist in regions containing strong downflows, in addition to cospatial underlying photospheric magnetic field concentrations. Downdrafts within Na I D-1 bright points exhibit speeds of up to 7 km s(-1), with preferred structural symmetry in intensity, magnetic field, and velocity profiles about the bright point center. Excess intensities associated with G-band and Ca II K observations of MBPs reveal a power-law trend when plotted as a function of the magnetic flux density. However, Na I D-1 observations of the same magnetic features indicate an intensity plateau at weak magnetic field strengths below approximate to 150 G, suggesting the presence of a two-component heating process: one which is primarily acoustic and the other predominantly magnetic. We suggest that this finding is related to the physical expansion of magnetic flux tubes, with weak field strengths (approximate to 50 G) expanding by similar to 76%, compared to a similar to 44% expansion when higher field strengths (approximate to 150 G) are present. These observations provide the first experimental evidence of rapid downdrafts in Na I D-1 MBPs and reveal the nature of a previously unresolved intensity plateau associated with these structures.