Gain-of-function mutant p53 downregulates miR-223 contributing to chemoresistance of cultured tumor cells.

Mutant p53 proteins are expressed at high frequency in human tumors and are associated with poor clinical prognosis and resistance to chemotherapeutic treatments. Here we show that mutant p53 proteins downregulate micro-RNA (miR)-223 expression in breast and colon cancer cell lines. Mutant p53 binds the miR-223 promoter and reduces its transcriptional activity. This requires the transcriptional repressor ZEB-1. We found that miR-223 exogenous expression sensitizes breast and colon cancer cell lines expressing mutant p53 to treatment with DNA-damaging drugs. Among the putative miR-223 targets, we focused on stathmin-1 (STMN-1), an oncoprotein known to confer resistance to chemotherapeutic drugs associated with poor clinical prognosis. Mutant p53 silencing or miR-223 exogenous expression lowers the levels of STMN-1 and knockdown of STMN-1 by small interfering RNA increases cell death of mutant p53-expressing cell lines. On the basis of these findings, we propose that one of the pathways affected by mutant p53 to increase cellular resistance to chemotherapeutic agents involves miR-223 downregulation and the consequent upregulation of STMN-1.

Protumorigenic effects of mir-145 loss in malignant pleural mesothelioma.

We identified a discrete number of microRNAs differentially expressed in benign or malignant mesothelial tissues. We focused on mir-145 whose levels were significantly downregulated in malignant mesothelial tissues and malignant pleural mesothelioma (MPM) cell lines as compared to benign tissues (pleura, peritoneum or cysts). We show that promoter hyper-methylation caused very low levels in MPM cell lines and specimens. Treatment of MPM cell lines with mir-145 agonists negatively modulated some protumorigenic properties of MPM cells, such as clonogenicity, cell migration and resistance to pemetrexed treatment. The main effector mechanism of the clonogenic death induced by mir-145 was that of accelerated senescence. We found that mir-145 targeted OCT4 via specific binding to its 3'-UTR. Increased intracellular levels of mir-145 decreased the levels of OCT4 and its target gene ZEB1, thereby counteracting the increase of OCT4 induced by pemetrexed treatment which is known to favor the development of chemoresistant cells. In line with this, reintroduction of OCT4 into mimic-145 treated cells counteracted the effects on clonogenicity and replicative senescence. This further supports the relevance of the mir-145-OCT4 interaction for the survival of MPM cells. The potential use of mir-145 expression levels to classify benign vs malignant mesothelial tissues and the differences between pemetrexed-induced senescence and that induced by the re-expression of mir-145 are discussed.

Expression of TP53 mutation-associated microRNAs predicts clinical outcome in head and neck squamous cell carcinoma patients.

BACKGROUND: TP53 mutation is associated with decreased survival rate in head and neck squamous cell carcinoma (HNSCC) patients. We set out to identify microRNAs (miRNAs) whose expression associates with TP53 mutation and survival in HNSCC. PATIENTS AND METHODS: We analyzed TP53 status by direct sequencing of exons 2 through 11 of a prospective series of 121 HNSCC samples and assessed its association with outcome in 109 followed-up patients. We carried out miRNA expression profiling on 121 HNSCC samples and 66 normal counterparts. miRNA associations with TP53 mutations and outcome were evaluated. RESULTS: A TP53 mutation was present in 58% of the tumors and TP53 mutations were significantly associated with a shorter recurrence-free survival. This association was stronger in the clinical subgroup of patients subjected to adjuvant therapy after surgery. The expression of 49 miRNAs was significantly associated with TP53 status. Among these 49, we identified a group of 12 miRNAs whose expression correlates with recurrence-free survival and a group of 4 miRNAs that correlates with cancer-specific survival. The two groups share three miRNAs. Importantly, miRNAs that correlate with survival are independent prognostic factors either when considered individually or as signatures. CONCLUSIONS: miRNAs expression associates with TP53 status and with reduced survival after surgical treatment of squamous cell carcinoma of the head and neck.

ATM kinase enables the functional axis of YAP, PML and p53 to ameliorate loss of Werner protein-mediated oncogenic senescence.

Werner syndrome (WS) results from dysfunction of the WRN protein, and is associated with premature aging and early death. Here we report that loss of WRN function elicits accumulation of the Yes-associated protein (YAP protein), a major effector of the Hippo tumor suppressor pathway, both experimentally and in WS-derived fibroblasts. YAP upregulation correlates with slower cell proliferation and accelerated senescence, which are partially mediated by the formation of a complex between YAP and the PML protein, whose activity promotes p53 activation. The ATM kinase is necessary for YAP and PML accumulation in WRN-depleted cells. Notably, the depletion of either YAP or PML partially impairs the induction of senescence following WRN loss. Altogether, our findings reveal that loss of WRN activity triggers the activation of an ATM-YAP-PML-p53 axis, thereby accelerating cellular senescence. The latter has features of SASP (senescence-associated secretory phenotype), whose protumorigenic properties are potentiated by YAP, PML and p53 depletion.

The potential prognostic role of cardiovascular autonomic failure in α-synucleinopathies.

Cardiovascular autonomic failure is the second most common dysautonomic feature of α-synucleinopathies and has significant impact on daily activities and quality of life. Here we provide a systematic review of cardiovascular autonomic failure in α-synucleinopathies, emphasizing its impact on cognitive functions and disease outcomes. Articles spanning the period between January 1985 and April 2012 were identified from the PubMed database using a keyword-based search. Epidemiological studies highlight the negative prognostic effect of cardiovascular autonomic failure on cardiovascular and cerebrovascular outcomes and overall mortality in all α-synucleinopathies. Altered cerebral perfusion, vascular pressure stress, and related disruption of the blood-brain barrier may also contribute to the white matter hyperintensities and cognitive dysfunction frequently found in patients affected by neurocardiovascular instability. These findings support the hypothesis that cardiovascular autonomic failure may play a negative prognostic role in α-synucleinopathies and suggest that precocious screening and therapeutic management of cardiovascular autonomic failure may positively impact disease course.

miR-204 targets Bcl-2 expression and enhances responsiveness of gastric cancer.

Micro RNAs (miRs) are small non-coding RNAs aberrantly expressed in human tumors. Here, we aim to identify miRs whose deregulated expression leads to the activation of oncogenic pathways in human gastric cancers (GCs). Thirty nine out of 123 tumoral and matched uninvolved peritumoral gastric specimens from three independent European subsets of patients were analyzed for the expression of 851 human miRs using Agilent Platform. The remaining 84 samples were used to validate miRs differentially expressed between tumoral and matched peritumoral specimens by qPCR. miR-204 falls into a group of eight miRs differentially expressed between tumoral and peritumoral samples. Downregulation of miR-204 has prognostic value and correlates with increased staining of Bcl-2 protein in tumoral specimens. Ectopic expression of miR-204 inhibited colony forming ability, migration and tumor engraftment of GC cells. miR-204 targeted Bcl-2 messenger RNA and increased responsiveness of GC cells to 5-fluorouracil and oxaliplatin treatment. Ectopic expression of Bcl-2 protein counteracted miR-204 pro-apoptotic activity in response to 5-fluorouracil. Altogether, these findings suggest that modulation of aberrant expression of miR-204, which in turn releases oncogenic Bcl-2 protein activity might hold promise for preventive and therapeutic strategies of GC.

Melatonin triggers p53Ser phosphorylation and prevents DNA damage accumulation.

Several epidemiological studies have shown that high levels of melatonin, an indolic hormone secreted mainly by the pineal gland, reduce the risks of developing cancer, thus suggesting that melatonin triggers the activation of tumor-suppressor pathways that lead to the prevention of malignant transformation. This paper illustrates that melatonin induces phosphorylation of p53 at Ser-15 inhibiting cell proliferation and preventing DNA damage accumulation of both normal and transformed cells. This activity requires p53 and promyelocytic leukemia (PML) expression and efficient phosphorylation of p53 at Ser-15 residue. Melatonin-induced p53 phosphorylation at Ser-15 residue does not require ataxia telangiectasia-mutated activity, whereas it is severely impaired upon chemical inhibition of p38 mitogen-activated protein kinase activity. By and large, these findings imply that the activation of the p53 tumor-suppressor pathway is a critical mediator of melatonin and its anticancer effects. Therefore, it provides molecular insights into increasing observational evidence for the role that melatonin has in cancer prevention.

MicroRNA-128-2 targets the transcriptional repressor E2F5 enhancing mutant p53 gain of function.

p53 mutations have profound effects on non-small-cell lung cancer (NSCLC) resistance to chemotherapeutic treatments. Mutant p53 proteins are usually expressed at high levels in tumors, where they exert oncogenic functions. Here we show that p53R175H, a hotspot p53 mutant, induces microRNA (miRNA)-128-2 expression. Mutant p53 binds to the putative promoter of miR128-2 host gene, ARPP21, determining a concomitant induction of ARPP21 mRNA and miR-128-2. miR-128-2 expression in lung cancer cells inhibits apoptosis and confers increased resistance to cisplatin, doxorubicin and 5-fluorouracyl treatments. At the molecular level, miR-128-2 post-transcriptionally targets E2F5 and leads to the abrogation of its repressive activity on p21(waf1) transcription. p21(waf1) protein localizes to the cytoplasmic compartment, where it exerts an anti-apoptotic effect by preventing pro-caspase-3 cleavage. This study emphasizes miRNA-128-2 role as a master regulator in NSCLC chemoresistance.

SASP mediates chemoresistance and tumor-initiating-activity of mesothelioma cells.

Here we show that pemetrexed-treated mesothelioma cells undergo accelerated senescence. This is characterized by the secretion of proinflammatory and mitogenic cytokines, reminiscent of an SASP (senescence-associated secretory phenotype). Conditioned media from senescent MPM (malignant pleural mesothelioma) cells trigger the emergence of EMT (epithelial-to-mesenchymal)-like, clonogenic and chemoresistant cell subpopulations, expressing high levels of ALDH (aldehyde dehydrogenase) activity (ALDH(bright) cells). We show by fluorescence-activated cell sorting of purified ALDH(bright) and ALDH(low) cells, that both cell-autonomous and cell-non-autonomous mechanisms converge to maintain the SASP-induced, EMT-like cell subpopulations. Chemoresistant ALDH(bright) cells exist within primary MPM specimens and enrichment for ALDH(bright) cells correlates with an earlier tumor onset into NOD/SCID mice. We show that RAS(v12) expression induces SASP-like changes in untransformed human mesothelial cells, and that p53 ablation increases the effect of RAS(v12) expression. We identify STAT3 activation as a crucial event downstream to SASP signaling. In fact, small hairpin RNA-mediated ablation of STAT3 deeply attenuates the induction of EMT genes and the increase of ALDH(bright) cells induced by SASP-cytokines. This strongly affects the chemoresistance of MPM cells in vitro and leads to anticancer effects in vivo.

[The syncope in Emergency Department: usual management vs guidelines]. / La sincope nel Dipartimento di Emergenza: tra applicazionedelle linee guida e impatto con la realtà dell'urgenza.

INTRODUCTION: The syncope is a common cause of admission to Emergency Departments, representing around 1-3% of all admissions to the service. However, elderly age and important comorbidities often hinder a definite etiologic diagnosis, with increasing requests for diagnostic tests and longer periods of hospitalization. MATERIALS AND METHODS: We analyzed the management of 1,204 patients admitted to our Emergency Department for transient loss of consciousness in the period between 1 June 2009 and 1 June 2010, evaluating the following parameters: average age, gender, triage color code at admittance, performed diagnostic tests, diagnosis at discharge from ED and destination ward. We also studied a subgroup of 93 patients admitted to emergency medicine units evaluating their OESIL score at admittance, comorbidities, performed diagnostic tests and diagnosis at discharge from the ward. RESULTS: In the Emergency Department, 45% of patients were discharged with a diagnosis of syncope of unknown origin; in 21% of patients syncope was excluded; 19% of patients received a diagnosis of cardiogenic syncope; 11% were diagnosed with a presyncope; 3% with orthostatic hypotension and 1% with vasovagal syncope. In emergency medicine units, 51% of patients were discharged with a diagnosis of cardiogenic syncope, 11% were diagnosed with vasovagal syncope, 11% with presyncope, 11% with TIA, 8% with loss of consciousness non-syncope and 8% with syncope of unknown origin. CONCLUSIONS: Management of patients with syncope, elderly people with important comorbidities in particular, is still a serious problem for the emergency physician. The creation of specialized units for the management of syncope, the so-called syncope units, through the implementation of a shared diagnostic and therapeutic protocol, aims at reducing inappropriate hospitalization and average length of stay.

[Obstructive Sleep Apnea Syndrome (OSAS): The role model of the Occupational Health Physician in specific clinical cases.]. / Sindrome delle Apnee Ostruttive nel Sonno (OSAS): ruolo del Medico Competente di Medicina del Lavoro desunto da specifi ci casi clinici.

Nowadays Sleeping disorders are a very interesting topic in Occupational medicine, they are involved in reduction of working performances and increased risk of work accidents (in work environment or while driving). Medical surveillance made from the Occupational Health Physician can be very helpful in early diagnosis of this kind of disease; during 2008 we fi nd out Obstructive Sleeping Apnea Disease (OSAS) in some Healthcare workers. We reported some clinical cases that show the role model of the occupational health physician in this kind of sickness. Our Experience shows the duty of Occupational health physician it's not limited to medical surveillance, but also to Health Promotion (as wrote in D.Lgs 81/08). This can be obtained by clinical and occupational solutions, like correct work shift planning and lifestyle changes; so the interest of the occupational physician have to be focused on introducing in medical surveillance also measures of health promotion regarding sleep disorders with the aim of preserving health condition in workers.

Combined transcranial Doppler and EEG recording in vasovagal syncope.

BACKGROUND: In neurally mediated syncope a 'typical' EEG pattern during hyperventilation (HV) may be observed. This study aimed to investigate transcranial Doppler (TCD) and EEG variations in response to hyper- and hypocapnia using simultaneous recording. METHODS: Syncope patients with a typical EEG pattern during HV (SEEG+, n = 15) and those without abnormalities (SEEG-, n = 16) were compared with healthy controls (n = 20). Simultaneous TCD and EEG recordings were performed at rest (baseline), during 2 apnea tests and during HV. Cerebrovascular vasoreactivity, index for hypocapnia, total vasomotor reserve and time to flow velocity normalization after HV (t-norm) were recorded. RESULTS: With TCD, a reduction in Vasomotor reserve was observed in SEEG+ compared with the other 2 groups (control: 67 +/- 8%; SEEG-: 67 +/- 10%; SEEG+: 57 +/- 8%; p < 0.0001). t-norm was longer in all syncopal patients and in particular in SEEG+ (control: 20.2 +/- 3 s; SEEG-: 40 +/- 7 s; SEEG+: 123 +/- 45s; p < 0.0001). Quantitative EEG showed an increase in slow bands in all subjects during HV, small and nonsignificant in controls and SEEG-, higher and significant in SEEG+, related with flow reduction. CONCLUSIONS: Changes in the sympathetic modulation of cerebral vasoconstriction may explain both the pathophysiology of vasovagal syncope and the typical paroxysmal EEG findings.

Leopard syndrome.

The L.E.O.P.A.R.D. syndrome is an autosomal, dominant disorder with characteristic features that include: multiple lentigines, café au lait spots, electrocardiographic conduction abnormalities, ocular hypertelorism, obstructive cardiomyopathy, pulmonary stenosis, abnormal (male) genitalia, retardation of growth, and deafness. Patients do not usually present all the clinical features traditionally associated with the disorder. Indeed, several features are not present until late in life and do not become clinically manifest until puberty. It has been observed that this syndrome is caused by a "missense" mutation in PTPN11, a gene encoding the protein tyrosine phosphatase SHP-2 located on chromosome 12q22. A diagnosis of LEOPARD syndrome may be established exclusively on the basis of clinical criteria. In our case, the patient was diagnosed with the syndrome late in his life when he was already exhibiting all its distinctive clinical features. We have reported the case of a LEOPARD syndrome patient exhibiting extremely elongated vertebral and basilar arteries previously undescribed in the literature.

Autonomic cardiovascular function and baroreflex sensitivity in patients with cervical dystonia receiving treatment with botulinum toxin type A.

OBJECTIVE: To investigate possible changes in autonomic cardiovascular regulation and cardiopulmonary baroreflex sensitivity in patients with primary cervical dystonia receiving chronic treatment with botulinum toxin type A. METHODS: Short-term power spectral analysis of heart rate and systolic blood pressure variability, high-frequency and low-frequency oscillations of heart rate variability, low frequency/high frequency ratio and baroreflex sensitivity (alpha index) were measured in 12 patients with cervical dystonia before and 2-4 weeks after botulinum toxin type A injection and compared with normative data. RESULTS: Before treatment, at rest, patients had significantly lower high frequency power than healthy subjects (p < 0.01), whereas no differences were found in low frequency power. Botulinum toxin injection in patients induced no changes in either power frequency. In patients before treatment and healthy subjects the low frequency oscillatory components increased similarly from rest to tilt (p < 0.01), but tilt induced lower low frequency values in patients than in healthy subjects (p < 0.01). In patients before treatment, the high frequency variations from rest to tilt remained unchanged, whereas in healthy subjects they decreased significantly (p < 0.01). Botulinum toxin type A injection in patients induced no changes in low frequency or high frequency powers. In patients before treatment the low frequency/high frequency ratio increased slightly from rest to tilt, but in healthy subjects increased significantly (p < 0.01). Botulinum toxin type A left the pretreatment low frequency/high frequency ratio unchanged. The alpha-index measured at rest in patients before treatment was lower than in healthy subjects (p<0.05), whereas during tilt was similar in both groups. The alpha-index measured after botulinum toxin injection in patients remained unchanged at rest and during tilt. CONCLUSIONS: Patients with cervical dystonia receiving treatment with botulinum toxin type A have mild, subclinical abnormalities in autonomic cardiovascular regulation and cardiopulmonary baroreflex sensitivity. These changes do not worsen after acute botulinum toxin type A injection.

Mutant p53: an oncogenic transcription factor.

Inactivation of tumor-suppressor genes is one of the key hallmarks of a tumor. Unlike other tumor-suppressor genes, p53 is inactivated by missense mutations in half of all human cancers. It has become increasingly clear that the resulting mutant p53 proteins do not represent only the mere loss of wild-type p53 tumor suppressor activity, but gain new oncogenic properties favoring the insurgence, the maintenance, the spreading and the chemoresistance of malignant tumors. The actual challenge is the fine deciphering of the molecular mechanisms underlying the gain of function of mutant p53 proteins. In this review, we will focus mainly on the transcriptional activity of mutant p53 proteins as one of the potential molecular mechanisms. To date, the related knowledge is still quite scarce and many of the raised questions of this review are yet unanswered.

The teriparatide in the treatment of severe senile osteoporosis.

The osteoporosis is a systemic disease of multicausal etiopathogenesis. A progressive bone loss and qualitative alterations in the macro- and micro-architecture of the remaining bones, resulting in a loss of strength of bones to such an extent that even very modest traumas will cause fractures characterize it. Three forms are defined (i) postmenopausal appearing after the menopause, (ii) senile appearing with advancing age, and (iii) the idiopathic forms. Severe osteoporosis is declared when the patients suffer vertebral or femoral fractures without any trauma during a treatment with anti-reabsorptive medicines of at least 1-year. The treatment of osteoporosis is based on various categories of pharmaca, such as bisphosphonates, selective estrogen receptor modulators (SERMs), diaminobutyric acid (DABA), parathyroid hormone (PTH), estrogens and non-hormonal drugs. The teriparatide, the recombinant human (rh)PTH(1-34), is identical in amino acid sequence until the 34th (N-terminal) amino acid of the endogenous, human PTH. It is produced in E. coli using the recombinant DNA technology. It is a pharmacon having a strong trophic-anabolic action on the bone tissue, assuring both the inhibition of the bone loss, and the formation of new bones of good quality. It acts as a stimulant of the osteoblast functions, and at the same time, increases the absorption of calcium from the intestine, and also the renal reabsorption of calcium, and decreases the excretion of phosphates in the kidney. This study summarizes our own experience with the use of rhPTH(1-34) in the treatment of senile patients with severe osteoporosis. Our sample consisted of 40 elderly women of the mean age of 78+/-5 years, having severe osteoporosis. They displayed a columnar T-score>-3.5 and femoral T-score>-2.5, had been under antireabsorptive treatment since at least 12 months. In particular, 15 patients were treated with Alendronate (70 mg/week), 10 of them with Risedronate (35 mg/week), and 15 of them with Raloxifene (60 mg/day). These patients in our study were treated for 1 year with 20 microg/day of rhPTH (1-34), injected subcutaneously, and supplemented also with a daily dose of 1g of calcium and 800 IU of Vitamin D, per os. At start of this treatment (time t(0)), after 6 months (time t(6)) and after 12 months (time t(12)) patients underwent a bone mineral density (BMD) analysis (Dexa-Lunar-DPX-P) on the lumbar vertebral column, (L1-L4 zone), as well as a femoral BMD. We applied also quality of life (QoL) questionnaire of the European Foundation for Osteoporosis (QUALEFFO), and evaluated also the use of non-steroidal anti-inflammatory drugs (NSAIDs). Our final considerations are that the teriparatide therapy increases significantly the bone mass density, expressed in terms of T-Score, reduces the occurrence of new fractures, improves the QoL, and decreases also the consumption of NSAIDs.

S100A2 gene is a direct transcriptional target of p53 homologues during keratinocyte differentiation.

The p53 paralogues p73, p63 and their respective truncated isoforms have been shown to be critical regulators of developmental and differentiation processes. Indeed, both p73- and p63-deficient mice exhibit severe developmental defects. Here, we show that S100A2 gene, whose transcript and protein are induced during keratinocyte differentiation of HaCaT cells, is a direct transcriptional target of p73beta and DeltaNp63alpha and is required for proper keratinocyte differentiation. Transactivation assays reveal that p73beta and DeltaNp63alpha exert opposite transcriptional effects on S100A2 gene. While DeltaNp63alpha is found in vivo onto S100A2 regulatory regions predominantly in proliferating cells, p73beta is recruited in differentiating cells. Silencing of p73 impairs the induction of S100A2 during the differentiation of HaCaT cells. Moreover, silencing of p73 or S100A2 impairs the proper expression of keratinocyte differentiation markers. Of note, p53 family members do not trigger S100A2 gene expression in response to apoptotic doses of cisplatin and doxorubicin.

Mutant p53 gain of function: reduction of tumor malignancy of human cancer cell lines through abrogation of mutant p53 expression.

Mutations in the TP53 tumor suppressor gene are the most frequent genetic alteration in human cancers. These alterations are mostly missense point mutations that cluster in the DNA binding domain. There is growing evidence that many of these mutations generate mutant p53 proteins that have acquired new biochemical and biological properties. Through this gain of function activity, mutant p53 is believed to contribute to tumor malignancy. The purpose of our study was to explore mutant p53 as a target for novel anticancer treatments. To this aim, we inhibited mutant p53 expression by RNA interference in three different cancer cell lines endogenously expressing mutant p53 proteins, and evaluated the effects on the biological activities through which mutant p53 exerts gain of function. We found that depletion of mutant p53 reduces cell proliferation, in vitro and in vivo tumorigenicity, and resistance to anticancer drugs. Our results demonstrate that mutant p53 knocking down weakens the aggressiveness of human cancer cells, and provides further insight into the comprehension of mutant p53 gain of function activity in human tumor.

Multidisciplinary approach for diagnosing syncope: a retrospective study on 521 outpatients.

OBJECTIVES: To describe causes of syncope in outpatients in whom structural heart disease was ruled out as a cause, and to analyse the role of a multidisciplinary approach in a syncope unit for the diagnosis of patients with syncope of unknown origin. METHODS: Cardiovascular autonomic nervous system (ANS) function was evaluated extensively in 521 outpatients by careful history, physical examination including orthostatic blood pressure measurement and standard ECG, and tilt testing. RESULTS: Causes of syncope remained unknown in 29.2% of cases. ANS dysfunction was found in 58.6% of those presenting with either neurally mediated syncope (53.6%) or chronic autonomic failure (5%); 3.8% of the patients suffered from syncope of cardiogenic origin (2.5%) or non-neurogenic hypotension (1.3%), and 8.4% had loss of consciousness of non-syncopal origin. Loss of consciousness was confirmed as being related to seizures in under 30% of patients initially diagnosed as having epilepsy. CONCLUSIONS: Neurally mediated syncope represents the commonest type of syncope. ANS evaluation including tilt testing should be considered as preliminary screening in patients with syncope in the absence of definite heart abnormalities. Neurologists should consider syncope from ANS failure as a comorbid factor in patients with seizures where the clinical characteristics are not straightforward.