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Single-Cell Analysis of Ploidy and Centrosomes Underscores the Peculiarity of Normal Hepatocytes

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Single-Cell Analysis of Ploidy and Centrosomes Underscores the Peculiarity of Normal Hepatocytes Università degli Studi di Milano Scuola di Dottorato in Medicina Molecolare Curriculum di Genomics, Proteomics e tecnologie correlate Ciclo XXV Anno Accademico 2011/2012 Settore disciplinare BIO10 Dottorando: Francesca FAGGIOLI Single-cell analysis of ploidy and centrosomes underscores the peculiarity of normal hepatocytes Direttore della Scuola: Ch.mo Prof. Mario CLERICI Tutore: Prof. Cristina BATTAGLIA Correlatore: Prof. Paolo Maria VEZZONI Albert Einstein College of Medicine, New York Istituto clinico Humanitas, Rozzano, Milano Alla mia famiglia “Only the ideas that we really live have any value” Hermann Hesse ABSTRACT STATE OF ART: The literature reports that, contrary to most other cell types, adult hepatocytes are polyploid cells with a DNA content of 4, 8 or even 16 haploid genomes. In fetal and early neonatal life, hepatocytes are mononucleated diploid cells that, quite abruptly, become binucleated and polyploid soon after weaning. The generation of tetraploid intermediates is not an uncommon event in the liver. These cells have the potential to generate aneuploid progeny in the subsequent cell division, because of the presence of four centrosomes. Normally in diploid cells, at the beginning of mitosis, a single centrosome duplicates and the mother and daughter organelles migrate to opposite cell poles, directing the formation of the spindle, to guarantee a balanced chromosomal segregation. Four centrosomes can cluster together, mimicking a bipolar spindle, or, as reported for tumoral cells, act as single entities that generate multipolar spindle. The result of a multipolar division is a progeny with an unbalanced DNA content, differing in 1 or a few chromosomes. The formation of aneuploid progeny in hepatocytes has never been supported by experimental evidence for two reasons: 1) the classical approaches chosen to assess the hepatocyte ploidy lack the sensitivity to detect the small differences in DNA content that result from unbalanced chromosomal segregation; in addition, a quantitative and behavioural analysis of centrosomes in normal liver cells has not been thoroughly investigated; thus the presence of extranumerary centrosomes (i.e.,more than four) has never been postulated 2) The detrimental effects commonly attributed to aneuploidy made difficult to only hypothesize that a highly regenerative tissue such as the liver can contain aneuploid cells. It is thus of interest to note that some tissues are naturally aneuploid, like the embryonic brain, and in these tissues chromosome instability confers advantages properties to the cells. AIM: The main goal of my present work is to determine whether aneuploidy is a common feature of hepatocytes in physiological conditions. To address this goal I performed a quantitative analysis of the DNA content in normal hepatocytes during liver development and adulthood (at 18.5 post coitum, at 15 days, 1.5 months, 4 months) combined with a quantitative and behavioural analysis of centrosomes. MATERIALS AND METHODS: I applied a novel approach employing a 2-color FISH on interphase cells that provides highly quantitative and reproducible polyploidy data for individual chromosomes by assessing ploidy of a cell based on a comparison between an autosome (17 or 18) and a sexual chromosome (Y). I used a double staining for centrosome associated proteins to assess the number of centrosomes at different time point and I combined this approach to the interphase FISH to determine a correspondence between the DNA content and the number of centrosomes. RESULTS: I have demonstrated that aneuploidy and unbalanced DNA content in binucleated hepatocytes are common features of the normal adult liver. Despite the common belief that hepatocytes contain 1, 2 or no more than 4 centrosomes, our double staining for centrosome associated proteins reveals extranumerary centrosomes in a high percentage of cells as early as 15 days of age. I showed that in mice the period between 15 days and 1.5 months marks the transition from a liver with a prevalence of mononucleated cells to a liver with up to 75% of binucleated cells. My data demonstrate that this timing correlates with a switch of I specific centrosomes numbers. At 15 days, in addition to cells that show the expected number of centrosomes (1 or 2), we also found several hepatocytes with 3 centrosomes; at 1.5 months the percentage of cells with 3 centrosomes decreased concomitantly with the increase of cells with more than 4 centrosomes. My analysis shows that the number of extranumerary centrosomes develops in concomitance with the process of binucleation and polyploidization. In addition, supernumerary centrosomes maintain the ability to nucleate α-tubulin, one of the main components of the cytoskeleton and of the mitotic spindle. This observation is intriguing based on the knowledge that adult hepatocytes are commonly considered to reside in G0 phase. Finally, by integrating interphase FISH and immunofluorescent approaches, we detected an imbalance between centrosome number and DNA content in liver cells that deviates from the equilibrium expected in normal cells. CONCLUSIONS: We demonstrated that half of the mature hepatocytes in mice are aneuploid. This discovery could have a different impact to several fields. On one hand it provides new insights on the role of aneuploidy in adult somatic tissues. Thus, the low tumorigenicity of liver suggests that this unique feature is relevant to the peculiar biological function of hepatic cells, which are continuously challenged by metabolic stress and other insults rather than a cause of tumor formation. We can speculate that the liver, with its high level of aneuploidy detected consistently overall the ages analyzed can be pictured as a store of well tolerated genetic heterogeneity. In response to toxic stresses and diseases the liver may select the more beneficial chromosomal pattern to promote cellular fitness against cell deterioration. On the other hand, not less important is the contribution of this discovery to the field of liver cell therapies with mature hepatocytes and the consequences of aneuploidy after transplantation. II SOMMARIO SITUAZIONE INIZIALE: La letteratura riporta, che contrariamente alla maggior parte dei tipi cellulari, gli epatociti adulti sono cellule poliploidi con un contenuto di DNA pari a 4, 8 o anche 16 genomi aploidi. Durante lo sviluppo fetale e nella vita neonatale, gli epatociti si presentano come cellule mononucleate diploidi, che abbastanza bruscamente, diventano binucleate e poliploidi subito dopo il periodo di svezzamento. Una delle peculiarità del fegato è la presenza di intermedi tetraploidi. Queste cellule hanno il potenziale di generare progenie aneuploide nelle successive divisioni cellulari, a causa della presenza di quattro centrosomi. Normalmente in cellule diploidi, all’inizio della mitosi, un singolo centrosoma si duplica, a ciò segue la migrazione dei due organelli, madre e figlia, ai lati opposti della cellula, dove andranno ad organizzare la formazione del fuso mitotico e garantiranno una corretta segregazione dei cromosomi. Quattro centrosomi possono formare dei cluster, mimando una divisione bipolare, oppure, come è già stato descritto per le cellule tumorali, agire come singole entità e generare un fuso mitotico multipolare. Il risultato di questa divisione è una progenie con una quantità di DNA non bilanciata, che differisce di uno o più cromosomi dall’atteso contenuto diploide. La formazione di progenie aneuploide nel fegato non è mai stata documentata sperimentalmente per due motivi: 1) gli approcci classici utilizzati nel determinare la ploidia degli epatociti mancano della sensibilità necessaria per rilevare piccole differenze di contenuto di DNA, come i singoli cromosomi, che derivano da una segregazione non bilanciata; inoltre un’analisi quantitativa e funzionale dei centrosomi e quindi dell’eventuale presenza di extra-centrosomi non è mai stata realizzata nel fegato in condizioni fisiologiche, tanto da supporre eventi di segregazione non corretti; 2) Gli effetti negativi comunemente legati al concetto di aneuploidia hanno reso difficile anche solo ipotizzare che un tessuto altamente rigenerativo come il fegato possa contenere delle cellule aneuploidi. Ma è certamente importante sottolineare che non è strano trovare cellule aneuploidi in condizioni fisiologiche; alcuni tessuti sono naturalmente aneuploidi, come il cervello durante lo sviluppo embrionale, e in quei tessuti l’instabilità dei cromosomi addirittura conferisce dei vantaggi alle cellule, invece che creare un danno. SCOPO: Il principale obiettivo del mio presente lavoro è quello di determinare se l’aneuploidia è una caratteristica tipica degli epatociti in condizioni fisiologiche. Per rispondere a questo quesito io ho realizzato un’analisi quantitativa del contenuto di DNA in epatociti in condizioni fisiologiche durante lo sviluppo del fegato e durante l’età adulta considerando diversi time points, quali i 18.5 giorni post coitum, 15 giorni, 1.5 mesi e 4 mesi. Gli stessi time points sono stati utilizzati per un’analisi quantitativa e funzionale dei centrosomi. MATERIALI E METODI: Il mio approccio nel determinare la ploidia degli epatociti consiste nell’utilizzo di una FISH a due colori applicata a cellule in interfase, perché basata sul confronto tra i segnali derivanti da un autosoma (17 o 18) e un cromosoma sessuale (Y) applicati all’interno della stessa cellula. Per stabilire il numero di centrosomi nei diversi time points ho utilizzato un doppio staining, costituito dall’uso
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