Faculty Bibliography

BACKGROUND AND AIMS: Epidemiological prospective data on cardiovascular (CV) events in elderly subjects from Mediterranean populations are lacking. We aimed to investigate 15-year incidence of CV events and to evaluate the association with CV risk factors in an elderly Mediterranean population. METHODS AND RESULTS: The population of a small Sicilian village were enrolled, visited and a blood sample was drawn at baseline. CV events were recorded in the 15years of follow-up. From 1351 subjects (75% of the resident population); 315 were in the age range 65-85years; 266 subjects free from CV disease were analysed. Seventy-seven CV events were recorded in 73 out of 266 subjects, with a 19.7% rate (in 10years). Hypertension (HTN) (hazards ratio=2.1) and diabetes mellitus (DM) (hazards ratio=1.8) were independently associated with CV events. Subjects with both DM and HTN showed a lower survival free of CV events compared to those with DM or HTN. CONCLUSIONS: In a 15-year follow-up of an elderly Mediterranean population free from CV disease, diabetes mellitus and hypertension were related to CV events. The control of risk factors in the elderly needs to be reinforced to achieve better results in terms of CV prevention

Background: Skp2, a known oncogene, is overexpressed in several types of tumors and is associated with worse recurrence rate and overall survival in primary melanoma patients. Moreover, the anti-proliferative effects of Skp2 siRNA on various tumor cell lines have prompted the preclinical testing of Skp2 small molecule inhibitors. In this study, we assessed the clinical relevance and molecular mechanism(s) underlying Skp2 overexpression in metastatic melanoma patients. Methods: Skp2 protein levels were measured in 122 metastatic melanoma specimens using immunohistochemistry (IHC), and the association between Skp2 overexpression and post-recurrence survival was examined. Moreover, 22 cell lines (2 normal primary melanocytes, 2 primary immortal melanocytes, 4 primary melanoma cell lines, and 18 metastatic melanoma cell lines) were evaluated for Skp2 genomic amplification using Single Nucleotide Polymorphism (SNP) arrays (Affymetrix 6.0) and Skp2 gene expression using mRNA arrays (Affymetrix U133A 2.0) and quantitative RT-PCR. We also screened 18 cell lines for Skp2 mutation by sequencing. Results: Skp2 overexpression, defined as >25% tumor cells, was associated with shorter 3-yr post-recurrence survival (37%) compared to Skp2 expression <= 25% (55%) (HR=1.89, 95%, CI= 1.04, 3.42, p=0.04). Skp2 overexpression was significantly associated with the site of melanoma metastasis: visceral (n= 12; 89%), lymph node (n=49; 36%), brain (n=15; 14%), and soft-tissue (n=36; 6%) (p<0.001). SNP array revealed genomic amplification at the Skp2 locus in 6 (33%) metastatic cell lines and one primary melanoma cell line. Skp2 genomic amplification was associated with increased transcript expression. No Skp2 mutations were identified. Conclusions: Skp2 protein overexpression is associated with worse prognosis in metastasis in melanoma. Our results also support that gene amplification, rather than a Skp2 gene mutation, may be the major mechanism responsible for Skp2 aberrant expression in metastatic melanoma

The effect of thrombin on tumor cell cycle activation and spontaneous growth was examined in synchronized serum-starved tumor cell lines and a model of spontaneous prostate cancer development in TRAMP mice. BrdUrd incorporation and propidium iodide staining of prostate LNCaP cells arrested in G(0) and treated with thrombin or serum revealed a 48- and 29-fold increase in S phase cells, respectively, at 8 hours. Similar results were obtained with TRAMP cells and a glioblastoma cell line, T98G. Cell cycle kinases and inhibitors in synchronized tumor cells revealed high levels of p27(Kip1) and low levels of Skp2 and cyclins D1 and A. Addition of thrombin, TFLLRN, or serum down-regulated p27(Kip1) with concomitant induction of Skp2, Cyclin D1, and Cyclin A with similar kinetics. LNCaP p27(Kip1)-transfected cells or Skp2 knockdown cells were refractory to thrombin-induced cell cycle activation. MicroRNA 222, an inhibitor of p27(Kip1), was robustly up-regulated by thrombin. The in vitro observations were tested in vivo with transgenic TRAMP mice. Repetitive thrombin injection enhanced prostate tumor volume 6- to 8-fold (P < 0.04). Repetitive hirudin, a specific potent antithrombin, decreased tumor volume 13- to 24-fold (P < 0.04). Thus, thrombin stimulates tumor cell growth in vivo by down-regulation of p27(Kip1)

The BimEL tumor suppressor is a potent proapoptotic BH3-only protein. We found that, in response to survival signals, BimEL was rapidly phosphorylated on three serine residues in a conserved degron, facilitating binding and degradation via the F box protein betaTrCP. Phosphorylation of the BimEL degron was executed by Rsk1/2 and promoted by the Erk1/2-mediated phosphorylation of BimEL on Ser69. Compared to wild-type BimEL, a BimEL phosphorylation mutant unable to bind betaTrCP was stabilized and consequently potent at inducing apoptosis by the intrinsic mitochondrial pathway. Moreover, although non-small cell lung cancer (NSCLC) cells often become resistant to gefitinib (a clinically relevant tyrosine kinase inhibitor that induces apoptosis through BimEL), silencing of either betaTrCP or Rsk1/2 resulted in BimEL-mediated apoptosis of both gefitinib-sensitive and gefitinib-insensitive NSCLC cells. Our findings reveal that betaTrCP promotes cell survival in cooperation with the ERK-RSK pathway by targeting BimEL for degradation

The spindle assembly checkpoint (SAC) is an important mechanism that prevents the separation of sister chromatids until the microtubules radiating from the spindle poles are correctly attached to the kinetochores. Cdc20, an activator of the Anaphase Promoting Complex/Cyclosome (APC/C), is known as a major downstream target for inhibition by the SAC through the binding of mitotic checkpoint proteins, such as Mad2 and BubR1. Here, we report that the SAC negatively regulates the stability of Cdc20 by targeting it for proteasome-dependent degradation. Once the checkpoint is activated by spindle poisons, a major population of Cdc20 is degraded via APC/C, an event that requires the binding of Cdc20 to Mad2. We propose that the degradation of Cdc20 represents a critical control mechanism to ensure inactivation of APC/C(Cdc20) in response to the SAC

Heterochromatin plays an essential role in the preservation of epigenetic information, the transcriptional repression of repetitive DNA elements and inactive genes, and the proper segregation of chromosomes during mitosis. Here we identify KDM2A, a JmjC-domain containing histone demethylase, as a heterochromatin-associated and HP1-interacting protein that promotes HP1 localization to chromatin. We show that KDM2A is required to maintain the heterochromatic state, as determined using a candidate-based approach coupled to an in vivo epigenetic reporter system. Remarkably, a parallel and independent siRNA screen also detected a role for KDM2A in epigenetic silencing. Moreover, we demonstrate that KDM2A associates with centromeres and represses transcription of small non-coding RNAs that are encoded by the clusters of satellite repeats at the centromere. Dissecting the relationship between heterochromatin and centromeric RNA transcription is the basis of ongoing studies. We demonstrate that forced expression of these satellite RNA transcripts compromise the heterochromatic state and HP1 localization to chromatin. Finally, we show that KDM2A is required to sustain centromeric integrity and genomic stability, particularly during mitosis. Since the disruption of epigenetic control mechanisms contributes to cellular transformation, these results, together with the low levels of KDM2A found in prostate carcinomas, suggest a role for KDM2A in cancer development

The DNA polymerase delta processivity factor Proliferating Cell Nuclear Antigen (PCNA) promotes the DNA damage-induced degradation of the replication initiation factor Cdt1 via the CRL4(Cdt2) E3 ubiquitin ligase complex. Here we demonstrate that PCNA promotes the ubiquitylation and degradation of the CDK inhibitor p21 in cells irradiated with low dose of ultraviolet (UV) by a similar mechanism. Human cells that are depleted of Cul4, DDB1 (damage-specific DNA-binding protein-1), or the DCAF Cdt2, are deficient in the UV-induced ubiquitylation and degradation of p21. Depletion of mammalian cells of PCNA by siRNA, or mutations in p21 that abrogate PCNA binding, prevent UV-induced p21 ubiquitylation and degradation, indicating that physical binding with PCNA is necessary for the efficient ubiquitylation of p21 via the CRL4(Cdt2) ubiquitin ligase. Cdt2 functions as the substrate recruiting factor for p21 to the rest of the CRL4 ubiquitin ligase complex. The CRL4(Cdt2) E3 ubiquitin ligase ubiquitylates p21 both in vivo and in vitro, and its activity is dependent on the interaction of p21 with PCNA. Finally, we show that the CRL4(Cdt2) and the SCF(Skp2) ubiquitin ligases are redundant with each other in promoting the degradation of p21 during an unperturbed S phase of the cell cycle

Apurinic/apyrimidinic endonuclease-1/Redox factor-1, a multifunctional DNA base excision repair and redox regulation enzyme, plays an important role in oxidative signalling, transcription factor regulation, and cell cycle control. Recently, we have demonstrated that following the triggering of CD40 on B cells, APE/Ref-1 translocates from the cytoplasm to the nucleus and regulates the activity of B cell-specific transcription factors. In the present paper we investigate whether APE/Ref-1 plays a role in controlling CD40-mediated B cell proliferation too. We demonstrate a concurrent increase in proliferation and decrease in apoptosis of primary mouse B cells activated by CD40 cross-linking and transfected with functional APE/Ref-1 antisense oligonucleotide. Moreover, we provide evidence that a redox-mediated signalling mechanism is involved in this process and we propose that APE/Ref-1, controlling the intracellular redox state, may also affect the cell cycle by inducing nucleus-cytoplasm redistribution of p21. Together, these findings suggest that APE/Ref-1 could act as a negative regulator in an adaptive response to elevated ROS levels following CD40 cross-linking. Considering the important role of ROS and APE/Ref-1 in CD40-mediated B cell proliferation, our data will contribute to understand the mechanisms of tumor escape and suggest APE/Ref-1 as a novel target for tumor therapeutic approaches

In response to DNA damage in G2, mammalian cells must avoid entry into mitosis and instead initiate DNA repair. Here, we show that, in response to genotoxic stress in G2, the phosphatase Cdc14B translocates from the nucleolus to the nucleoplasm and induces the activation of the ubiquitin ligase APC/C(Cdh1), with the consequent degradation of Plk1, a prominent mitotic kinase. This process induces the stabilization of Claspin, an activator of the DNA-damage checkpoint, and Wee1, an inhibitor of cell-cycle progression, and allows an efficient G2 checkpoint. As a by-product of APC/C(Cdh1) reactivation in DNA-damaged G2 cells, Claspin, which we show to be an APC/C(Cdh1) substrate in G1, is targeted for degradation. However, this process is counteracted by the deubiquitylating enzyme Usp28 to permit Claspin-mediated activation of Chk1 in response to DNA damage. These findings define a novel pathway that is crucial for the G2 DNA-damage-response checkpoint