ARID1A in cancer: Friend or foe?

ARID1A belongs to a class of chromatin regulatory proteins that function by maintaining accessibility at most promoters and enhancers, thereby regulating gene expression. The high frequency of ARID1A alterations in human cancers has highlighted its significance in tumorigenesis. The precise role of ARID1A in cancer is highly variable since ARID1A alterations can have a tumor suppressive or oncogenic role, depending on the tumor type and context. ARID1A is mutated in about 10% of all tumor types including endometrial, bladder, gastric, liver, biliopancreatic cancer, some ovarian cancer subtypes, and the extremely aggressive cancers of unknown primary. Its loss is generally associated with disease progression more often than onset. In some cancers, ARID1A loss is associated with worse prognostic features, thus supporting a major tumor suppressive role. However, some exceptions have been reported. Thus, the association of ARID1A genetic alterations with patient prognosis is controversial. However, ARID1A loss of function is considered conducive for the use of inhibitory drugs which are based on synthetic lethality mechanisms. In this review we summarize the current knowledge on the role of ARID1A as tumor suppressor or oncogene in different tumor types and discuss the strategies for treating ARID1A mutated cancers.

Monitoring Somatic Genetic Alterations in Circulating Cell-Free DNA/RNA of Patients with "Oncogene-Addicted" Advanced Lung Adenocarcinoma: A Real-World Clinical Study.

Liquid biopsy has advantages over tissue biopsy, but also some technical limitations that hinder its wide use in clinical applications. In this study, we aimed to evaluate the usefulness of liquid biopsy for the clinical management of patients with advanced-stage oncogene-addicted non-small-cell lung adenocarcinomas. The investigation was conducted on a series of cases-641 plasma samples from 57 patients-collected in a prospective consecutive manner, which allowed us to assess the benefits and limitations of the approach in a real-world clinical context. Thirteen samples were collected at diagnosis, and the additional samples during the periodic follow-up visits. At diagnosis, we detected mutations in ctDNA in 10 of the 13 cases (77%). During follow-up, 36 patients progressed. In this subset of patients, molecular analyses of plasma DNA/RNA at progression revealed the appearance of mutations in 29 patients (80.6%). Mutations in ctDNA/RNA were typically detected an average of 80 days earlier than disease progression assessed by RECIST or clinical evaluations. Among the cases positive for mutations, we observed 13 de novo mutations, responsible for the development of resistance to therapy. This study allowed us to highlight the advantages and disadvantages of liquid biopsy, which led to suggesting algorithms for the use of liquid biopsy analyses at diagnosis and during monitoring of therapy response.

Circulating miR-184 is a potential predictive biomarker of cardiac damage in Anderson-Fabry disease.

Enzyme replacement therapy (ERT) is a mainstay of treatment for Anderson-Fabry disease (AFD), a pathology with negative effects on the heart and kidneys. However, no reliable biomarkers are available to monitor its efficacy. Therefore, we tested a panel of four microRNAs linked with cardiac and renal damage in order to identify a novel biomarker associated with AFD and modulated by ERT. To this end, 60 patients with a definite diagnosis of AFD and on chronic ERT, and 29 age- and sex-matched healthy individuals, were enrolled by two Italian university hospitals. Only miR-184 met both conditions: its level discriminated untreated AFD patients from healthy individuals (c-statistic = 0.7522), and it was upregulated upon ERT (P < 0.001). On multivariable analysis, miR-184 was independently and inversely associated with a higher risk of cardiac damage (odds ratio = 0.86; 95% confidence interval [CI] = 0.76-0.98; P = 0.026). Adding miR-184 to a comprehensive clinical model improved the prediction of cardiac damage in terms of global model fit, calibration, discrimination, and classification accuracy (continuous net reclassification improvement = 0.917, P < 0.001; integrated discrimination improvement [IDI] = 0.105, P = 0.017; relative IDI = 0.221, 95% CI = 0.002-0.356). Thus, miR-184 is a circulating biomarker of AFD that changes after ERT. Assessment of its level in plasma could be clinically valuable in improving the prediction of cardiac damage in AFD patients.

Unraveling the role of microRNA/isomiR network in multiple primary melanoma pathogenesis.

Malignant cutaneous melanoma (CM) is a potentially lethal form of skin cancer whose worldwide incidence has been constantly increasing over the past decades. During their lifetime, about 8% of CM patients will develop multiple primary melanomas (MPMs), usually at a young age and within 3 years from the first tumor/diagnosis. With the aim of improving our knowledge on MPM biology and pathogenesis, we explored the miRNome of 24 single and multiple primary melanomas, including multiple tumors from the same patient, using a small RNA-sequencing approach. From a supervised analysis, 22 miRNAs were differentially expressed in MPM compared to single CM, including key miRNAs involved in epithelial-mesenchymal transition. The first and second melanoma from the same patient presented a different miRNA profile. Ten miRNAs, including miR-25-3p, 149-5p, 92b-3p, 211-5p, 125a-5p, 125b-5p, 205-5p, 200b-3p, 21-5p, and 146a-5p, were further validated in 47 single and multiple melanoma samples. Pathway enrichment analysis of miRNA target genes revealed a more differentiated and less invasive status of MPMs compared to CMs. Bioinformatic analyses at the miRNA isoform (isomiR) level detected a panel of highly expressed isomiRs belonging to miRNA families implicated in human tumorigenesis, including miR-200, miR-30, and miR-10 family. Moreover, we identified hsa-miR-125a-5p|0|-2 isoform as tenfold over-represented in melanoma than the canonical form and differentially expressed in MPMs arising in the same patient. Target prediction analysis revealed that the miRNA shortening could change the pattern of target gene regulation, specifically in genes implicated in cell adhesion and neuronal differentiation. Overall, we provided a putative and comprehensive characterization of the miRNA/isomiR regulatory network of MPMs, highlighting mechanisms of tumor development and molecular features differentiating this subtype from single melanomas.

Molecular testing on bronchial washings for the diagnosis and predictive assessment of lung cancer.

Cytopathological analyses of bronchial washings (BWs) collected during fibre-optic bronchoscopy are often inconclusive for lung cancer diagnosis. To address this issue, we assessed the suitability of conducting molecular analyses on BWs, with the aim to improve the diagnosis and outcome prediction of lung cancer. The methylation status of RASSF1A, CDH1, DLC1 and PRPH was analysed in BW samples from 91 lung cancer patients and 31 controls, using a novel two-colour droplet digital methylation-specific PCR (ddMSP) technique. Mutations in ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, ROS1 and TP53 and gene fusions of ALK, RET and ROS1 were also investigated, using next-generation sequencing on 73 lung cancer patients and 14 tumour-free individuals. Our four-gene methylation panel had significant diagnostic power, with 97% sensitivity and 74% specificity (relative risk, 7.3; odds ratio, 6.1; 95% confidence interval, 12.7-127). In contrast, gene mutation analysis had a remarkable value for predictive, but not for diagnostic, purposes. Actionable mutations in EGFR, HER2 and ROS1 as well as in other cancer genes (KRAS, PIK3CA and TP53) were detected. Concordance with gene mutations uncovered in tumour biopsies was higher than 90%. In addition, bronchial-washing analyses permitted complete patient coverage and the detection of additional actionable mutations. In conclusion, BWs are a useful material on which to perform molecular tests based on gene panels: aberrant gene methylation and mutation analyses could be performed as approaches accompanying current diagnostic and predictive assays during the initial workup phase. This study establishes the grounds for further prospective investigation.

The Importance of microRNAs in RAS Oncogenic Activation in Human Cancer.

microRNAs (miRNAs) regulate gene expression by modulating the translation of protein-coding RNAs. Their aberrant expression is involved in various human diseases, including cancer. Here, we summarize the experimental pieces of evidence that proved how dysregulated miRNA expression can lead to RAS (HRAS, KRAS, or NRAS) activation irrespective of their oncogenic mutations. These findings revealed relevant pathogenic mechanisms as well as mechanisms of resistance to target therapies. Based on this knowledge, potential approaches for the control of RAS oncogenic activation can be envisioned.

High-sensitivity assay for monitoring ESR1 mutations in circulating cell-free DNA of breast cancer patients receiving endocrine therapy.

Approximately 70% of breast cancers (BCs) express estrogen receptor alpha (ERα) and are treated with endocrine therapy. However, the effectiveness of this therapy is limited by innate or acquired resistance in approximately one-third of patients. Activating mutations in the ESR1 gene that encodes ERα promote critical resistance mechanisms. Here, we developed a high sensitivity approach based on enhanced-ice-COLD-PCR for detecting ESR1 mutations. The method produced an enrichment up to 100-fold and allowed the unambiguous detection of ESR1 mutations even when they consisted of only 0.01% of the total ESR1 allelic fraction. After COLD-PCR enrichment, methods based on next-generation sequencing or droplet-digital PCR were employed to detect and quantify ESR1 mutations. We applied the method to detect ESR1 mutations in circulating free DNA from the plasma of 56 patients with metastatic ER-positive BC. Fifteen of these patients were found to have ESR1 mutations at codons 536-538. This study demonstrates the utility of the enhanced-ice-COLD-PCR approach for simplifying and improving the detection of ESR1 tumor mutations in liquid biopsies. Because of its high sensitivity, the approach may potentially be applicable to patients with non-metastatic disease.

Circulating miR-29a, among other up-regulated microRNAs, is the only biomarker for both hypertrophy and fibrosis in patients with hypertrophic cardiomyopathy.

OBJECTIVES: The purpose of this paper was to determine whether microRNAs (miRNAs) involved in myocardial remodeling were differentially expressed in the blood of hypertrophic cardiomyopathy (HCM) patients, and whether circulating miRNAs correlated with the degree of left ventricular hypertrophy and fibrosis. BACKGROUND: miRNAs-small, noncoding ribonucleic acids (RNAs) that regulate gene expression by inhibiting RNA translation-modulate cellular function. Myocardial miRNAs modulate processes such as cardiomyocyte (CM) hypertrophy, excitation-contraction coupling, and apoptosis; non-CM-specific miRNAs regulate myocardial vascularization and fibrosis. Recently, the possibility that circulating miRNAs may be biomarkers of cardiovascular disease has been raised. METHODS: Forty-one HCM patients were characterized with conventional transthoracic echocardiography and cardiac magnetic resonance. Peripheral plasma levels of 21 miRNAs were assessed by quantitative real-time polymerase chain reaction and were compared with levels in a control group of 41 age- and sex-matched blood donors. RESULTS: Twelve miRNAs (miR-27a, -199a-5p, -26a, -145, -133a, -143, -199a-3p, -126-3p, -29a, -155, -30a, and -21) were significantly increased in HCM plasma. However, only 3 miRNAs (miR-199a-5p, -27a, and -29a) correlated with hypertrophy; more importantly, only miR-29a correlated also with fibrosis. CONCLUSIONS: Our data suggest that cardiac remodeling associated with HCM determines a significant release of miRNAs into the bloodstream: the circulating levels of both cardiac- and non-cardiac-specific miRNAs are significantly increased in the plasma of HCM patients. However, correlation with left ventricular hypertrophy parameters holds true for only a few miRNAs (i.e., miR-199a-5p, -27a, and -29a), whereas only miR-29a is significantly associated with both hypertrophy and fibrosis, identifying it as a potential biomarker for myocardial remodeling assessment in HCM.

Routine assessment of on-clopidogrel platelet reactivity and gene polymorphisms in predicting clinical outcome following drug-eluting stent implantation in patients with stable coronary artery disease.

OBJECTIVES: This study sought to assess the usefulness of clopidogrel-pathway genotyping and on-treatment platelet reactivity (OTR) testing in predicting major adverse cardiac events (MACE) in stable coronary artery disease (CAD) patients receiving drug-eluting stents (DES) under dual antiplatelet (clopidogrel plus aspirin) therapy. BACKGROUND: The role of pharmacogenetics and OTR in predicting MACE-death, myocardial infarction, or stent thrombosis-in stable CAD patients scheduled for DES implantation is still debated. METHODS: Patients with stable CAD treated by DES implantation (n = 1,432) were genotyped with a TaqMan OpenArray (Applied Biosystems, Carlsbad, California) and assessed for OTR with the VerifyNow P2Y12 test (Accumetrics Inc., San Diego, California). Genes tested were ABCB1, CYP1A2, CYP2B6*9, CYP2C8*3, CYP2C9*2, CYP2C19, CYP3A4, CYP3A5*3, P2RY12, and PON1CYP2C19. High OTR was defined as P2Y12 reaction units ≥230. The endpoint at 12-month follow-up was MACE occurring during antiplatelet therapy. RESULTS: All groups that were stratified for loss-of-function variants of the cytochrome P450 gene CYP2C19 had significant hazard ratios (HR) for MACE (genotypic HR: 1.41, 95% confidence interval [CI]: 1.06 to 1.89, p = 0.01; allelic HR: 1.56, 95% CI: 2.26 to 1.2, p = 0.01). Variants of other clopidogrel-pathway genes were not significantly associated with MACE. When OTR was assessed, clinical significance was found only in high-risk diabetic (HR: 2.11, 95% CI: 1.29 to 3.45, p < 0.001) and chronic kidney disease (HR: 2.03, 95% CI: 1.03 to 4.02, p = 0.04) patients. CONCLUSIONS: CYP2C19 metabolizer status is an independent predictor of MACE after DES implantation and can be used for prognostication in all stable CAD patients. High OTR, as assessed by the VerifyNow P2Y12 test, is an independent predictor of MACE only for high-risk subsets, that is, patients with diabetes or chronic kidney disease.

Doubly heterozygous LMNA and TTN mutations revealed by exome sequencing in a severe form of dilated cardiomyopathy.

Familial dilated cardiomyopathy (DCM) is a heterogeneous disease; although 30 disease genes have been discovered, they explain only no more than half of all cases; in addition, the causes of intra-familial variability in DCM have remained largely unknown. In this study, we exploited the use of whole-exome sequencing (WES) to investigate the causes of clinical variability in an extended family with 14 affected subjects, four of whom showed particular severe manifestations of cardiomyopathy requiring heart transplantation in early adulthood. This analysis, followed by confirmative conventional sequencing, identified the mutation p.K219T in the lamin A/C gene in all 14 affected patients. An additional variant in the gene for titin, p.L4855F, was identified in the severely affected patients. The age for heart transplantation was substantially less for LMNA:p.K219T/TTN:p.L4855F double heterozygotes than that for LMNA:p.K219T single heterozygotes. Myocardial specimens of doubly heterozygote individuals showed increased nuclear length, sarcomeric disorganization, and myonuclear clustering compared with samples from single heterozygotes. In conclusion, our results show that WES can be used for the identification of causal and modifier variants in families with variable manifestations of DCM. In addition, they not only indicate that LMNA and TTN mutational status may be useful in this family for risk stratification in individuals at risk for DCM but also suggest titin as a modifier for DCM.

Assessment of the 9p21.3 locus in severity of coronary artery disease in the presence and absence of type 2 diabetes.

BACKGROUND: The 9p21.3 locus is strongly associated with the risk of coronary artery disease (CAD) and with type 2 diabetes (T2D). We investigated the association of 9p21.3 variants with severity of CAD (defined by the number of vessel diseased [VD]) in the presence and absence of T2D. METHODS: We tested 11 9p21.3-variants for association in a white Italian study (N = 2,908), and carried out replication in 2 independent white populations, a German study (N = 2,028) and a Canadian Study (N=950). SNP association and permutation analyses were conducted. RESULTS: We identified two 9p21.3-variants, rs4977574 (P < 4×10(-4)) and rs2383207 (P < 1.5×10(-3)) that were associated with severity of CAD in subjects without T2D. Association of rs4977574 with severity of CAD was confirmed in the Canadian Study. Results from subgroup analysis among patients with T2D showed an interaction between rs10738610 and T2D with P = 4.82×10(-2). Further investigation showed that rs10738610 (P < 1.99×10(-2)) was found to be significantly associated with severity of CAD in subjects with T2D. CONCLUSIONS: The 9p21.3 locus is significantly associated with severity of CAD. The number of associations of 9p21.3 variants with severity of CAD is variable to the presence and absence of T2D. In a CAD-susceptible region of 115 kb, there is only one variant associated with the severity of coronary vessel disease in the presence of type 2 diabetes.

Variant on chromosome 9p is associated with left ventricular mass: results from two cohorts of essential hypertensive individuals.

OBJECTIVES: It is well known that among hypertensive patients, an increased left ventricular mass (LVM) is a powerful predictor of cardiovascular morbidity and mortality. However, the mechanisms underlying LVM in hypertension are not completely understood, as the absolute value of blood pressure and other risk factors associated do not predict alone a definite LVM progression. Recently, the 9p21 chromosomal region has been consistently associated with coronary heart disease. METHODS AND RESULTS: We examined the association of 384 single nucleotide polymorphisms (SNPs) in the short arm of chromosome 9 with LVM in 821 hypertensive individuals from northern Italy. We identified a SNP (rs894379) in the intronic region of the centlein, centrosomal protein (CNTLN) gene on chromosome 9p22, whose minor allele G is associated with an increased LVM. We performed a follow-up validation analysis for the top SNP in 1038 hypertensive individuals from southern Italy. We then combined the results and found a nominal association for rs894379 (ß â€Š=  2.46, P  =  0.0026). CONCLUSION: We describe a new variant associated with echocardiography LVM. This result, though it needs to be further investigated, may improve our understanding of the genetic determination of this prognostically relevant trait.

Association study on long-living individuals from Southern Italy identifies rs10491334 in the CAMKIV gene that regulates survival proteins.

Long-living individuals (LLIs) are used to study exceptional longevity. A number of genetic variants have been found associated in LLIs to date, but further identification of variants would improve knowledge on the mechanisms regulating the rate of aging. Therefore, we performed a genome-wide association study on 410 LLIs and 553 young control individuals with a 317K single-nucleotide polymorphism (SNP) chip to identify novel traits associated with aging. Among the top (p < 1 × 10(-4)) SNPs initially identified, we found rs10491334 (CAMKIV) (odds ratio [OR] = 0.55; 95% confidence interval [CI] 0.42-0.73; p = 2.88 × 10(-5)), a variant previously reported associated with diastolic blood pressure, associated also in a replication set of 116 LLIs and 160 controls (OR = 0.54; 95% CI 0.32-0.90; p = 9 × 10(-3)). Furthermore, in vitro analysis established that calcium/calmodulin-dependent protein kinase IV (CAMKIV) activates the survival proteins AKT, SIRT1, and FOXO3A, and we found that homozygous carriers of rs10491334 have a significant reduction in CAMKIV expression. This, together with the observed reduction in minor-allele carriers among centenarians, points to a detrimental role for the SNP. In conclusion, prolongevity genes are activated by CAMKIV, the levels of which are influenced by rs10491334, a SNP associated with human longevity.

Unexpectedly low mutation rates in beta-myosin heavy chain and cardiac myosin binding protein genes in Italian patients with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease. Fourteen sarcomeric and sarcomere-related genes have been implicated in HCM etiology, those encoding ß-myosin heavy chain (MYH7) and cardiac myosin binding protein C (MYBPC3) reported as the most frequently mutated: in fact, these account for around 50% of all cases related to sarcomeric gene mutations, which are collectively responsible for approximately 70% of all HCM cases. Here, we used denaturing high-performance liquid chromatography followed by bidirectional sequencing to screen the coding regions of MYH7 and MYBPC3 in a cohort (n = 125) of Italian patients presenting with HCM. We found 6 MHY7 mutations in 9/125 patients and 18 MYBPC3 mutations in 19/125 patients. Of the three novel MYH7 mutations found, two were missense, and one was a silent mutation; of the eight novel MYBPC3 mutations, one was a substitution, three were stop codons, and four were missense mutations. Thus, our cohort of Italian HCM patients did not harbor the high frequency of mutations usually found in MYH7 and MYBPC3. This finding, coupled to the clinical diversity of our cohort, emphasizes the complexity of HCM and the need for more inclusive investigative approaches in order to fully understand the pathogenesis of this disease.

Fatty acid percentage in erythrocyte membranes of atrial flutter/fibrillation patients and controls.

PURPOSE: Several epidemiological published data support the protective role of omega-3 consumption in coronary artery disease, sudden cardiac death and ventricular arrhythmias, but interestingly, this is not the case for atrial arrhythmias. The purpose of this study is to evaluate different fatty acid profile between AF/AFL subjects and healthy controls. METHODS: Gas chromatography was employed to determine fatty acid percentage of erythrocyte membranes from 40 idiopathic AFL/AF patients and 53 healthy control subjects. RESULTS: AFL/AF erythrocyte membranes had significantly lower percentage of saturated fatty acid (43.1 +/- SD2.2 versus 47.8 +/- SD9.6, p < 0.001), monounsaturated fatty acid (18.2 +/- SD2.5 versus 22.6 +/- SD5.2, p < 0.001) and total trans fatty acid (0.2 +/- SD0.1 vs 1.3 +/- SD1.1, p < 0.001) than controls. Furthermore, fatty acid (FA) profiles of arrhythmic individuals showed an increased percent of total polyunsaturated fatty acid (PUFA) (36.7 +/- SD2.4 versus 26.4 +/- SD10.4, p < 0.001), PUFA n-3 (5.3 +/- SD1.1 versus 2.8 +/- SD1.8, p < 0.001) and n-6 (31.4 +/- SD2.2 versus 23.5 +/- SD9.9, p < 0.001). CONCLUSION: This study shows that the erythrocyte membranes FA composition of AF/AFL subjects differs from that of healthy controls.

Association of the FOXO3A locus with extreme longevity in a southern Italian centenarian study.

A number of potential candidate genes in a variety of biological pathways have been associated with longevity in model organisms. Many of these genes have human homologs and thus have the potential to provide insights into human longevity. Recently, several studies suggested that FOXO3A functions as a key bridge for various signaling pathways that influence aging and longevity. Interestingly, Willcox and colleagues identified several variants that displayed significant genotype-gender interaction in male human longevity. In particular, a nested case-control study was performed in an ethnic Japanese population in Hawaii, and five candidate longevity genes were chosen based on links to the insulin-insulin-like growth factor-1 (IGF-1) signaling pathway. In the Willcox study, the investigated genetic variations (rs2802292, rs2764264, and rs13217795) within the FOXO3A gene were significantly associated with longevity in male centenarians. We validated the association of FOXO3A polymorphisms with extreme longevity in males from the Southern Italian Centenarian Study. Particularly, rs2802288, a proxy of rs2802292, showed the best allelic association--minor allele frequency (MAF) = 0.49; p = 0.003; odds ratio (OR) = 1.51; 95% confidence interval (CI), 1.15-1.98). Furthermore, we undertook a meta-analysis to explore the significance of rs2802292 association with longevity by combining the association results of the current study and the findings coming from the Willcox et al. investigation. Our data point to a key role of FOXO3A in human longevity and confirm the feasibility of the identification of such genes with centenarian-controls studies. Moreover, we hypothesize the susceptibility to the longevity phenotype may well be the result of complex interactions involving genes and environmental factors but also gender.

Lack of replication of genetic associations with human longevity.

The exceptional longevity of centenarians is due in part to inherited genetic factors, as deduced from data that show that first degree relatives of centenarians live longer and have reduced overall mortality. In recent years, a number of groups have performed genetic association studies on long-living individuals (LLI) and young controls to identify alleles that are either positively or negatively selected in the centenarian population as consequence of a demographic pressure. Many of the reported studies have shown genetic loci associated with longevity. Of these, with the exception of APOE, none have been convincingly reproduced. We validated our populations by typing the APOE locus. In addition, we used 749 American Caucasian LLI, organized in two independent tiers and 355 American Caucasian controls in the attempt to replicate previously published findings. We tested Klotho (KL)-VS variant (rs952706), Cholesteryl Ester Transfer Protein (CETP) I405V (rs5882), Paraoxonase 1 (PON1) Q192R (rs662), Apolipoprotein C-III (APOC3) -641C/A (rs2542052), Microsomal Transfer Protein (MTP) -493G/T (rs2866164) and apolipoprotein E (APOE) epsilon2 and epsilon4 isoforms, (rs7412 and rs429358) haplotypes respectively. Our results show that, at present, except for APOE, none of the selected genes show association with longevity if carefully tested in a large cohort of LLI and their controls, pointing to the need of larger populations for case-control studies in extreme longevity.

Neuronal apoptosis is accompanied by amyloid beta-protein accumulation in the endoplasmic reticulum.

A series of evidences suggests that enhanced susceptibility to programmed cell death (PCD) is a major pathogenetic factor in Alzheimer's disease (AD). We investigated this issue, analyzing amyloid beta-protein (A beta) production in a model of neuronal PCD, induced by staurosporine in a murine neuroblastoma cell line. When PCD was induced, a 280-290% secreted A beta occurred, in spite of a 20% metabolism and an unchanged A betaPP expression. The increased intracellular A beta reactivity largely colocalized with a marker of ER. Inhibition of caspases blocked the cleavage at the C-terminus of beta PP, but only partially rescued A beta overproduction caused by staurosporine treatment. Our findings suggest that PCD fosters the physiological pathways of A beta production characteristic of neuronal cells, and they confirm the theory that unbalance of PCD is a central event in AD pathogenesis. Moreover, our data indicate that still unidentified cellular mechanisms, other than caspases activation, are responsible of the specific alteration of A betaPP processing during PCD.