La farmacogenetica dei farmaci antitumorali

La variabilità individuale nella risposta ai farmaci è molto importante in quanto può condizionare la riuscita di un trattamento farmacologico ed è dovuta, in gran parte, al profilo genetico. La farmacogenetica e la farmacogenomica studiano tale variabilità ed hanno lo scopo principale di personalizzare le terapie in base all’assetto genetico individuale. Negli ultimi anni, queste discipline hanno assunto sempre maggiore importanza. Allo stato attuale, in alcuni casi i risultati di studi genetici sono stati utilizzati per introdurre test di routine volti a guidare il trattamento farmacologico, mentre molti altri studi in questo campo non hanno ancora trovato un’applicazione nella pratica clinica, ma hanno fornito importanti informazioni. La farmacogenetica e la farmacogenomica sono fondamentali per il trattamento con chemioterapici, farmaci caratterizzati da un alto potenziale tossico. In questo lavoro tratto tre esempi di polimorfismi connessi, in modi diversi, alla risposta ai chemioterapici: una VNTR nel gene UGT1A1 legata alla tossicità del trattamento con irinotecano, un gruppo di mutazioni attivanti del gene EGFR che rendono efficace il trattamento con gefitinib e diversi gruppi funzionali di mutazioni del gene CYP2D6 da cui dipende l’esito del trattamento con tamoxifene

Effects on human transcriptome of two BRCA1-BRCT mutations: M1775R and A1789T

BRCA1 (breast cancer 1, early onset) mutations confer a high risk of breast and ovarian cancer. Most of BRCA1 cancer-predisposing mutations originate truncated proteins, but missense mutations have been also detected in familial breast and ovarian cancer patients. These variants are rare and their role in cancer predisposition is often difficult to ascertain. In the present work I studied the molecular mechanisms affected in human cells by two BRCA1 missense variants, M1775R and A1789T, both located in the second BRCT domain. These variants have been isolated from familial breast cancer patients and their effect on cell transcriptome has been previously investigated in yeast cells. Here I compared the expression profiles of HeLa cells transfected with one or the other variant and HeLa cells transfected with BRCA1 wild-type. Microarray data analysis was performed by three comparisons: M1775R versus wild-type (M1775RvsWT-contrast), A1789T versus wild-type (A1789TvsWT-contrast) and the mutated BRCT domain versus wild-type (MutvsWT-contrast), obtained by considering the two variants as a whole. I found 201 differentially expressed genes in M1775RvsWT-contrast, 313 in A1789TvsWT-contrast and 173 in MutvsWT-contrast. Most of these genes participate in cell processes that are often deregulated in cancer, such as cell cycle progression and DNA damage response and repair. These results represent the first molecular evidence of the pathogenetic role of M1775R, already known by functional studies, and give support to a similar role for A1789T, first hypothesized based on yeast cell experiments

Genes and Aggressive Behavior: Epigenetic Mechanisms Underlying Individual Susceptibility to Aversive Environments

Over the last two decades, the study of the relationship between nature and nurture in shaping human behavior has encountered a renewed interest. Behavioral genetics showed that distinct polymorphisms of genes that code for proteins that control neurotransmitter metabolic and synaptic function are associated with individual vulnerability to aversive experiences, such as stressful and traumatic life events, and may result in an increased risk of developing psychopathologies associated with violence. On the other hand, recent studies indicate that experiencing aversive events modulates gene expression by introducing stable changes to DNA without modifying its sequence, a mechanism known as “epigenetics”. For example, experiencing adversities during periods of maximal sensitivity to the environment, such as prenatal life, infancy and early adolescence, may introduce lasting epigenetic marks in genes that affect maturational processes in brain, thus favoring the emergence of dysfunctional behaviors, including exaggerate aggression in adulthood. The present review discusses data from recent research, both in humans and animals, concerning the epigenetic regulation of four genes belonging to the neuroendocrine, serotonergic and oxytocinergic pathways—Nuclear receptor subfamily 3-group C-member 1 (NR3C1), oxytocin receptor (OXTR), solute carrier-family 6 member 4 (SLC6A4) and monoamine oxidase A (MAOA)—and their role in modulating vulnerability to proactive and reactive aggressive behavior. Behavioral genetics and epigenetics are shedding a new light on the fine interaction between genes and environment, by providing a novel tool to understand the molecular events that underlie aggression. Overall, the findings from these studies carry important implications not only for neuroscience, but also for social sciences, including ethics, philosophy and law

Effects on human transcriptome of mutated BRCA1 BRCT domain: A microarray study

BACKGROUND: BRCA1 (breast cancer 1, early onset) missense mutations have been detected in familial breast and ovarian cancers, but the role of these variants in cancer predisposition is often difficult to ascertain. In this work, the molecular mechanisms affected in human cells by two BRCA1 missense variants, M1775R and A1789T, both located in the second BRCT (BRCA1 C Terminus) domain, have been investigated. Both these variants were isolated from familial breast cancer patients and the study of their effect on yeast cell transcriptome has previously provided interesting clues to their possible role in the pathogenesis of breast cancer. METHODS: We compared by Human Whole Genome Microarrays the expression profiles of HeLa cells transfected with one or the other variant and HeLa cells transfected with BRCA1 wild-type. Microarray data analysis was performed by three comparisons: M1775R versus wild-type (M1775RvsWT-contrast), A1789T versus wild-type (A1789TvsWT-contrast) and the mutated BRCT domain versus wild-type (MutvsWT-contrast), considering the two variants as a single mutation of BRCT domain. RESULTS: 201 differentially expressed genes were found in M1775RvsWT-contrast, 313 in A1789TvsWT-contrast and 173 in MutvsWT-contrast. Most of these genes mapped in pathways deregulated in cancer, such as cell cycle progression and DNA damage response and repair. CONCLUSIONS: Our results represent the first molecular evidence of the pathogenetic role of M1775R, already proposed by functional studies, and give support to a similar role for A1789T that we first hypothesized based on the yeast cell experiments. This is in line with the very recently suggested role of BRCT domain as the main effector of BRCA1 tumor suppressor activity

Carnosol controls the human glioblastoma stemness features through the epithelial-mesenchymal transition modulation and the induction of cancer stem cell apoptosis

A high cell proliferation rate, invasiveness and resistance to chemotherapy are the main features of glioblastoma (GBM). GBM aggressiveness has been widely associated both with a minor population of cells presenting stem-like properties (cancer stem-like cells, CSCs) and with the ability of tumor cells to acquire a mesenchymal phenotype (epithelial-mesenchymal transition, EMT). Carnosol (CAR), a natural inhibitor of MDM2/p53 complex, has been attracted attention for its anti-cancer effects on several tumor types, including GBM. Herein, the effects of CAR on U87MG-derived CSC viability and stemness features were evaluated. CAR decreased the rate of CSC formation and promoted the CSC apoptotic cell death through p53 functional reactivation. Moreover, CAR was able to control the TNF-α/TGF-β-induced EMT, counteracting the effects of the cytokine on EMT master regulator genes (Slug, Snail, Twist and ZEB1) and modulating the activation of miR-200c, a key player in the EMT process. Finally, CAR was able to increase the temozolomide (TMZ) anti-proliferative effects. These findings demonstrate that CAR affected the different intracellular mechanism of the complex machinery that regulates GBM stemness. For the first time, the diterpene was highlighted as a promising lead for the development of agents able to decrease the stemness features, thus controlling GBM aggressiveness

Characterisation of gene expression profiles of yeast cells expressing BRCA1 missense variants

Germline mutations in breast cancer susceptibility gene 1 (BRCA1) confer high risk of developing breast and ovarian cancers. Even though most BRCA1 cancer-predisposing mutations produce a non-functional truncated protein, 5-10% of them cause single amino acid substitutions. This second type of mutations represents a useful tool for examining BRCA1 molecular functions. Human BRCA1 inhibits cell proliferation in transformed Saccharomyces cerevisiae cells and this effect is abolished by disease-associated mutations in the BRCT domain. Moreover, BRCA1 mutations located both inside and outside the BRCT domain may induce an increase in the homologous recombination frequency in yeast cells. Here we present a microarray analysis of gene expression induced in yeast cells transformed with five BRCA1 missense variants, in comparison with gene expression induced by wildtype BRCA1. Data analysis was performed by grouping the BRCA1 variants into three sets: Recombination (R)-set (Y179C and S1164I), Recombination and Proliferation (RP)-set(I1766S and M1775R) and Proliferation (P)-set (A1789T), according to their effects on yeast cell phenotype. We found 470, 740 and 1136 differentially expressed genes in R-, P- and RP-set, respectively. Our results point to some molecular mechanisms critical for the control of cell proliferation and of genome integrity providing support to a possible pathogenic role of the analysed mutations. They also confirm that yeast, despite the absence of a BRCA1 homologue, represents a valid model system to examine BRCA1 molecular functions, as the molecular pathways activated by BRCA1 variants are conserved in humans

Genetically-Driven Enhancement of Dopaminergic Transmission Affects Moral Acceptability in Females but Not in Males: A Pilot Study

Moral behavior has been a key topic of debate for philosophy and psychology for a long time. In recent years, thanks to the development of novel methodologies in cognitive sciences, the question of how we make moral choices has expanded to the study of neurobiological correlates that subtend the mental processes involved in moral behavior. For instance, in vivo brain imaging studies have shown that distinct patterns of brain neural activity, associated with emotional response and cognitive processes, are involved in moral judgment. Moreover, while it is well-known that responses to the same moral dilemmas differ across individuals, to what extent this variability may be rooted in genetics still remains to be understood. As dopamine is a key modulator of neural processes underlying executive functions, we questioned whether genetic polymorphisms associated with decision-making and dopaminergic neurotransmission modulation would contribute to the observed variability in moral judgment. To this aim, we genotyped five genetic variants of the dopaminergic pathway [rs1800955 in the dopamine receptor D4 (DRD4) gene, DRD4 48 bp variable number of tandem repeat (VNTR), solute carrier family 6 member 3 (SLC6A3) 40 bp VNTR, rs4680 in the catechol-O-methyl transferase (COMT) gene, and rs1800497 in the ankyrin repeat and kinase domain containing 1 (ANKK1) gene] in 200 subjects, who were requested to answer 56 moral dilemmas. As these variants are all located in genes belonging to the dopaminergic pathway, they were combined in multilocus genetic profiles for the association analysis. While no individual variant showed any significant effects on moral dilemma responses, the multilocus genetic profile analysis revealed a significant gender-specific influence on human moral acceptability. Specifically, those genotype combinations that improve dopaminergic signaling selectively increased moral acceptability in females, by making their responses to moral dilemmas more similar to those provided by males. As females usually give more emotionally-based answers and engage the “emotional brain” more than males, our results, though preliminary and therefore in need of replication in independent samples, suggest that this increase in dopamine availability enhances the cognitive and reduces the emotional components of moral decision-making in females, thus favoring a more rationally-driven decision process

α-Synuclein Aggregates with β-Amyloid or Tau in Human Red Blood Cells: Correlation with Antioxidant Capability and Physical Exercise in Human Healthy Subjects

Neurodegenerative disorders (NDs) are characterized by abnormal accumulation/misfolding of specific proteins, primarily α-synuclein (α-syn), β-amyloid1–42 (Aβ), and tau, in both brain and peripheral tissue. In addition to homo-oligomers, the role of α-syn interactions with Aβ or tau has gradually emerged. The altered protein accumulation has been related to both oxidative stress and physical activity; nevertheless, no correlation among the presence of peripheral α-syn hetero-aggregates, antioxidant capacity, and physical exercise has been discovered as of yet. Herein, the content of α-syn, Aβ, tau, and of their heterocomplexes was determined in red blood cells (RBCs) of healthy subjects (sedentary and athletes). Such parameters were related to the extent of the antioxidant capability (AOC), a key marker of oxidative stress in aging-related pathologies, and to physical exercise, which is known to play an important preventive role in NDs and to modulate oxidative stress. Tau content and plasma AOC toward hydroxyl radicals were both reduced in older or sedentary subjects; in contrast, α-syn and Aβ accumulated in elderly subjects and showed an inverse correlation with both hydroxyl AOC and the level of physical activity. For the first time, α-syn heterocomplexes with Aβ or tau were quantified and demonstrated to be inversely related to hydroxyl AOC. Furthermore, α-syn/Aβ aggregates were significantly reduced in athletes and inversely correlated with physical activity level, independent of age. The positive correlation between antioxidant capability/physical activity and reduced protein accumulation was confirmed by these data and suggested that peripheral α-syn heterocomplexes may represent new indicators of ND-related protein misfolding

Analisi del ruolo del gene Xbsx nello sviluppo dell'organo pineale di Xenopus Laevis

L’organo pineale nei vertebrati è un importante sito di produzione della melatonina, un ormone coinvolto nella regolazione di ritmi vitali come quelli circadiani e della riproduzione. In tutti i vertebrati la produzione di questo ormone è sottoposta al controllo di un orologio endogeno il quale, a sua volta, è regolato in parte dalle condizioni ambientali, essenzialmente dalla luce. Nei vertebrati superiori, in particolare nei mammiferi, la struttura che costituisce l’orologio endogeno principale e il luogo di produzione della melatonina sono separati; infatti l’orologio endogeno è situato nel nucleo suprachiasmatico e la melatonina è prodotta nell’organo pineale. Nei vertebrati inferiori, come ad esempio l’anfibio anuro Xenopus laevis, invece troviamo che l’organo pineale è la sede di entrambi: svolge direttamente la funzione di orologio endogeno e rappresenta il sito principale di produzione dell’ormone melatonina. Esistono geni la cui trascrizione ha un andamento che corrisponde nettamente ad un ritmo giorno-notte. In particolare, dalla letteratura conosciamo geni espressi nell’organo pineale che codificano per enzimi della via di biosintesi della melatonina, come Aanat (Serotonina-N-acetil-transferasi) e Tph (Triptofano idrossilasi), la cui trascrizione ha un andamento ciclico con un’ espressione alta di notte e bassa di giorno. Inoltre sono stati individuati alcuni geni considerati fondamentali per l’orologio circadiano, tra cui i fattori di trascrizione Clock e B-mal; alcuni di questi geni hanno a loro volta un’espressione ritmica. Nel mio lavoro di tesi ho studiato Brain Specific homeobox (Xbsx), un gene espresso specificamente nel territorio dell’organo pineale durante lo sviluppo di Xenopus laevis. Attualmente la funzione di questo gene non è nota, ma data la presenza della sequenza homeobox si può ipotizzare che codifichi per un fattore di trascrizione. Informazioni cruciali per la determinazione del ruolo svolto da Xbsx possono essere ottenute da un’analisi dettagliata dell’espressione di questo gene. In una prima serie di esperimenti ho voluto valutare se Xbsx fosse espresso in cellule in proliferazione o in differenziamento. Utilizzando l’analogo di base bromodeossiuridina (che viene incorporato dalle cellule in divisione) e confrontando la sua incorporazione con l’espressione di Xbsx a vari stadi (24, 32, 37 e 40) ho trovato che a stadio 24 Xbsx è espresso soprattutto in cellule differenziate con una piccola sovrapposizione con la zona in proliferazione, mentre a stadio 32 e negli stadi successivi è nettamente espresso soltanto in cellule differenziate. Visto ciò, ho cercato di capire in quali tipi di cellule differenziate Xbsx fosse espresso. Nell’organo pineale ci sono essenzialmente due tipi di cellule: i fotorecettori e le cellule gangliari. Ho confrontato, mediante ibridazione in situ su sezione, l’espressione di Xbsx con quella di XOtx5, un gene espresso specificamente nei fotorecettori e con quella di Hermes, un marcatore di cellule gangliari. I dati ottenuti hanno dimostrato che Xbsx è espresso soltanto nei fotorecettori, avendo un’espressione completamente sovrapponibile a quella di XOtx5 e complementare ad Hermes. Essendo un gene espresso specificamente nei fotorecettori, nei quali è prodotta la melatonina, ho poi cercato un possibile coinvolgimento di Xbsx nella regolazione dei ritmi circadiani. In particolare ho voluto accertare se la sua espressione fosse di tipo ritmico. A questo scopo ho confrontato la sua espressione con quelle di geni noti avere un’espressione circadiana in Xenopus laevis, come Aanat-2 e Tph. Ho quindi utilizzato un sistema di crescita degli embrioni in dodici ore di luce e dodici di buio alternate fino allo stadio 46. A questo stadio ho fissato gli embrioni ad orari prestabiliti (6:30, 12:30, 18:30, 00:30) e ne ho prelevato i cervelli. Sui cervelli ho effettuato ibridazioni in situ whole-mount, confrontando l’espressione di Xbsx con quelle di Aanat-2 e Tph. Questi esperimenti hanno dimostrato che i livelli di espressione di Xbsx sono analoghi a quelli di questi due geni, cioè di tipo ritmico, alti di notte e bassi di giorno. Avendo studiato questi aspetti descrittivi, in futuro potrà essere utile procedere ad analisi di tipo funzionale, esaminando gli effetti della perdita e del guadagno di funzione di Xbsx. Uno dei metodi più efficaci per generare la perdita di funzione in Xenopus consiste nella microiniezione di oligonucleotidi antisenso. In particolare ho intenzione di utilizzare un morpholino di cui per ora ho saggiato la specificità. Il saggio di specificità consiste nell’utilizzare due costrutti recanti una parte della regione codificante per la proteina Myc riconosciuta da anticorpi commerciali. Un costrutto contiene, oltre a questa regione, la sequenza bersaglio esatta del morpholino mentre l’altro presenta una mutazione in questa sequenza. Ho quindi microiniettato questi costrutti in embrioni a stadio di 2 cellule ed ho effettuato un’analisi di western blotting per verificare la presenza delle proteine di fusione negli embrioni a stadio 10. Così ho potuto verificare che il nostro morpholino è effettivamente specifico: soltanto negli embrioni microiniettati con la sequenza bersaglio esatta è stata inibita la traduzione della proteina di fusione. Esperimenti futuri saranno volti a determinare se la perdita di funzione di Xbsx sia in grado di influenzare in qualche modo l’espressione di geni chiave dell’orologio circadiano come Clock, Bmal e Period e anche quella degli stessi Aanat-2 e Tph

Human Neural Stem Cell Aging Is Counteracted by α-Glycerylphosphorylethanolamine

Neural stem cells (NSCs) represent a subpopulation of cells, located in specific regions of the adult mammalian brain, with the ability of self-renewing and generating neurons and glia. In aged NSCs, modifications in the amount and composition of membrane proteins/lipids, which lead to a reduction in membrane fluidity and cholinergic activities, have been reported. In this respect, molecules that are effective at normalizing the membrane composition and cholinergic signaling could counteract stem cell aging. α-Glycerylphosphorylethanolamine (GPE), a nootropic drug, plays a role in phospholipid biosynthesis and acetylcholine release. Herein, GPE was assayed on human NSC cultures and on hydroxyurea-aged cells. Using cell counting, colorimetric, and fluorimetric analyses, immunoenzymatic assays, and real time PCR experiments, NSC culture proliferation, senescence, reactive oxygen species, and ADP/ATP levels were assessed. Aged NSCs exhibited cellular senescence, decreased proliferation, and an impairment in mitochondrial metabolism. These changes included a substantial induction in the nuclear factor NF-κB, a key inflammatory mediator. GPE cell treatment significantly protected the redox state and functional integrity of mitochondria, and counteracted senescence and NF-κB activation. In conclusion, our data show the beneficial properties of GPE in this model of stem cell aging