Thesis
Reference
Genomic instability in incuded stem cells and regulation of P14ARF expression
DERELI, Ayguel
Abstract
Part I. Genomic instability in induced stem cells - The ability to reprogram adult cells into stem cells has raised hopes for novel therapies. Expression of oncogenes leads to DNA replication stress and genomic instability, explaining the high frequency of p53 mutations in human cancers. We wondered whether stem cell reprogramming also leads to genomic instability.
We examined stem cells induced by a variety of protocols. Comparative genomic hybridization analysis of stem cells revealed the presence of genomic deletions and amplifications, whose signature was suggestive of oncogene induced DNA replication stress. Part II. Regulation of p14ARF Expression - We examined the role of p14ARF in cancer. p14ARF is encoded by CDKN2A locus, which also encodes p16INK4A. By analysis of mRNA and protein levels, we found that E2F1 acts at the level of p14ARF mRNA. Identifying how p14ARF levels are regulated could be useful in therapeutic applications particularly in p53-defective cancers.
DERELI, Ayguel. Genomic instability in incuded stem cells and regulation of P14ARF expression. Thèse de doctorat : Univ. Genève, 2011, no. Sc. 4335
URN : urn:nbn:ch:unige-170985
DOI : 10.13097/archive-ouverte/unige:17098
Available at:
http://archive-ouverte.unige.ch/unige:17098
Disclaimer: layout of this document may differ from the published version.
UNIVERSITÉ DE GENEVE FACULTÉ DES SCIENCES Département de Biologie Moléculaire Professeur Thanos Halazonetis
GENOMIC INSTABILITY IN INDUCED STEM CELLS AND
REGULATION OF P14ARF EXPRESSION
THÈSE
présentée a la Faculté des sciences de l`Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie
par
Aygul Dereli de
Istanbul (Turquie)
Thèse N° 4335
GENÈVE Uni Mail
2011
ACKNOWLEDGEMENTS
First, I would like to thank my thesis advisor Thanos Halazonetis for his full guidance, support and motivation. I would also like to thank my thesis committee members: Robbie Loewith and Matthias Lutolf.
This is also a great opportunity to thank all members of Halazonetis lab, especially Simona Negrini and Griet Van Houwe for their contributions to this work.
None of this would have been possible without the strength of my family, special thanks to my husband, Altug Öz for his constant support and patience.
ABSTRACT
Part I. Genomic instability in induced stem cells
The ability to reprogram adult cells into stem cells has raised hopes for novel therapies for many human diseases. Typical stem cell reprogramming protocols involve expression of a small number of genes in differentiated somatic cells with the c-Myc and Klf4 proto-oncogenes typically included in this mix. We have previously
shown that expression of oncogenes leads to DNA replication stress and genomic instability, explaining the high frequency of p53 mutations in human cancers.
Consequently, we wondered whether stem cell reprogramming also leads to genomic instability. To test this hypothesis we examined stem cells induced by a variety of protocols. The first protocol, developed specifically for this study, reprogrammed primary mouse mammary cells into mammary stem cells by expressing c-Myc. Two other previously established protocols reprogrammed mouse embryo fibroblasts into induced pluripotent stem (iPS) cells by expressing either 3 genes, Oct4, Sox2 and Klf4 (OSK), or 4 genes, OSK plus c-Myc (OSKC). Comparative genomic hybridization (cGH) analysis of stem cells derived by these protocols revealed the presence of genomic deletions and amplifications, whose signature was suggestive of oncogene- induced DNA replication stress. The genomic aberrations were to a significant degree dependent on c-Myc expression and their presence could explain why p53 inactivation facilitates stem cell reprogramming.
Part II. Regulation of p14ARF Expression
In this project, we examined the role of p14ARF in cancer. P14ARF is
encoded by the CDKN2A locus, a locus that is mutated in more than half of all human cancers. Interestingly, another protein p16INK4A, is also encoded by the same locus.
The p14ARF and p16INK4A transcripts share 2 exons. In this study, conditional E2F1 expression in p53 deficient Saos2 cells was used to up-regulate p14ARF levels.
By analysis of mRNA and protein levels, we found that E2F1 acts at the level of p14ARF mRNA. Further, we showed that the levels of transcripts corresponding to exon1beta of p14ARF increased in response to E2F1 up-regulation suggesting that E2F1 activates transcription. In contrast to p14ARF, E2F1 induction did not affect p16INK4A mRNA or protein levels. In the future it will be interesting to determine how E2F1 modulates p14ARF transcription. We further ruled out an involvement of DDR pathway in E2F1-dependent activation of p14ARF, as well as several other mechanisms by which E2F1 could have affected p14ARF levels, including an effect of E2F1 on Non-Sense-Mediated-Decay. Overall, identifying how p14ARF levels are regulated could be useful in therapeutic applications particularly in p53-defective cancers.
RÉSUMÉ
Partie I. Instabilité du Génome dans les cellules souches induites
La possibilité de reprogrammer les cellules humaines en cellules souches a soulevé l’espoir de découvrir de nouvelles thérapies pour traiter de nombreuses maladies humaines. Les protocoles types de reprogrammation des cellules souches sont basés sur l’expression d’un petit groupe de gènes dans des cellules somatiques différentiées en incluant généralement le c-Myc et les proto-oncogènes Klf4 dans le mélange. Nous avons déjà montré que l’expression des oncogènes conduit à un stress de la réplication de l’ADN et à une instabilité génomique, ce qui explique la
fréquence élevée de mutations de p53 dans les cancers humains.
En conséquence, nous nous sommes demandé si la reprogrammation des cellules souches induit aussi une instabilité génomique. Pour éprouver cette
hypothèse, nous avons examiné des cellules souches induites par divers protocoles. Le premier protocole, développé spécifiquement pour cette étude, visait à reprogrammer les cellules mammaires primaires d’une souris en cellules mammaires souches en exprimant le c-Myc. Les deux protocoles établis précédemment visaient à
reprogrammer les fibroblastes embryonnaires d’une souris en cellules souches
pluripotentes (iPS) induites en exprimant soit 3 gènes, Oct4, Sox2 et Klf4 (OSK), soit 4 gènes, OSK plus c-Myc (OSKC). L’analyse de l’hybridation génomique
comparative (CGH) des cellules souches dérivées de ces protocoles a révélé la présence de délétions ou d’amplifications génomiques dont la signature suggérait un stress de réplication de l’ADN induit par un oncogène. Les aberrations génomiques dépendaient, dans une large mesure, de l’expression du c-Myc et leur présence
pouvait expliquer pourquoi l’inactivation de p53 facilite la reprogrammation des cellules souches.
Partie II. Régulation de l'expression p14ARF
Dans ce projet, nous avons examiné le rôle de p14ARF dans le cancer.
P14ARF est codée par le locus CDKN2A, un locus qui est muté dans plus de la moitié de tous les cancers humains. Fait intéressant, une autre protéine, p16INK4A, est également codée par ce même locus. Les transcrits p14ARF et p16INK4A partagent deux exons. Dans cette étude, l'expression conditionnée E2F1 dans les cellules Saos2 déficientes p53 a été utilisée pour faire une régulation élevée des niveaux p14ARF.
En analysant l’ARN messager et les niveaux de protéine, nous avons trouvé que le E2F1 agit au niveau de l’ARN messager p14ARF. Par ailleurs, nous avons mis en évidence que les niveaux des transcrits correspondant à l’exon1beta de la p14ARF augmentaient en réponse à la régulation élevée E2F1, suggérant que E2F1 active la transcription. Contrairement à p14ARF, l'induction de E2F1 n'a pas affecté l’ARN messager p16INK4A ni les niveaux de protéine. À l'avenir, il sera intéressant de déterminer la façon dont E2F1 module la transcription de p14ARF. De plus, nous avons exclu l’implication d’un des chemins qui répondent à un dommage sur l’ADN lors de l'activation de p14ARF dépendante de E2F1, tout comme d'autres mécanismes par lesquels E2F1 aurait pu affecter les niveaux de p14ARF, dont un effet de E2F1 sur la dégradation des ARNm non-sens. Dans l’ensemble, l'identification de la façon dont les niveaux de p14ARF sont régulés pourraient servir à des applications
thérapeutiques, en particulier dans les cancers défectueux en p53.
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
ABSTRACT iii
RÉSUMÉ v
TABLE OF CONTENTS vii
CHAPTER 1: INTRODUCTION 1
PART I. GENOMIC INSTABILITY IN INDUCED STEM CELLS 1
1.1 The Discovery of iPSCs 1
1.2 Nuclear Transfer 2
1.3 Fusion Hybrids and ESCs 3
1.4 Transcription factor transduction 6
1.5 Induced Pluripotent StemCells (iPSCs) 7
1.5.1 Sox2 and Oct4 8
1.5.2 Klf4 10
1.5.3 c-Myc 13
1.6 P53 14
1.6.1 Role of p53 in Reprogramming 15
1.6.2 Role of p53 in Oncogene induced DNA Replication Stress 16
1.7 Chromosome Fragile Sites 18
1.7.1 Rare fragile sites 18
1.7.2 Common fragile sites (CFS) 19
1.7.2.1 Evolutionary conservation of common fragile sites (CFS) 19
1.7.2.2 Common fragile instability in tumors 20 1.7.2.3 Mechanisms that regulate CFS instability 22
PART II. ROLE OF P14ARF EXPRESSION 24
1.8 CDKN2A locus 24
1.8.1 P16INK4A pathway 25
1.8.2 p14ARF pathway 26
1.9 p53 pathways 28
1.10 Non-sense mediated decay (NMD) Mechanism 30 1.10.1 Cis- and trans-acting NMD determinants 30
1.11 Thesis summary 32
CHAPTER 2: GENOMIC INSTABILITY IN INDUCED STEM CELLS 38
2.1 MATERIALS AND METHODS 39
2.1.1 Induced mammary stem cells 39
2.1.2 Lentiviral infections 41
2.1.3 Transplantation experiments 41
2.1.4 iPS cells induced by OSK 42
2.1.5 iPS cells induced by OSKC 43
2.1.6 Aphidicolin-induced DNA replication stress 44
2.1.7 cGH analysis 44
2.2 RESULTS 45
2.2.1 c-Myc induced mammary stem cells 45
2.2.2 Genomic instability in iPS cells 48
2.2.3 Genomic lesions induced by DNA replication stress 49
CHAPTER 3: REGULATION OF P14ARF EXPRESSION 61
3.1 MATERIALS AND METHODS 62
3.1.1 Cell lines 62
3.1.2 Preparation of the cell extracts 62
3.1.3 Immunoblotting and antibodies 63
3.1.4 Treatment protocols 63
3.1.5 mRNA Extraction and qRT PCR settings 64 3.1.6 Small interfering RNA (siRNA) Transfection 65
3.2 RESULTS 67
3.2.1 Wortmannin suppresses NMD and cause accumulation of p53 67 3.2.2 p14ARF is not a specific target for NMD mechanism 68 3.2.3 E2F1 induction increases p14ARF protein levels 69
3.2.4 Autophagy does not regulate p14ARF 70
3.2.5 Transcriptional regulation of p14ARF 71
CHAPTER 4: DISCUSSION 88
PART I. GENOMIC INSTABILITY IN INDUCED STEM CELLS 88 PART II. ROLE OF p14ARF EXPRESSION 96
REFERENCES 102
CHAPTER 1
INTRODUCTION
Part I. Genomic instability in induced Stem Cells
1.1 The Discovery of iPSCs
Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somatic cell, by inducing a forced expression of specific genes. These cells meet the defining criteria for pluripotent stem cells with the potential to differentiate into any of the three germ layers: endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), or ectoderm (epidermal tissues and nervous system) Pluripotent stem cells can give rise to any fetal or adult cell type. However, alone they cannot develop into a fetal or adult animal because they lack the potential to contribute to extraembryonic tissue, such as the placenta.
The discovery of” induced pluripotency” includes the principles and technologies that have been developed over the last six decades. First, with somatic cell nuclear transfer (SCNT) experiments it was demonstrated that differentiated cells retain the same genetic information as early embryonic cells. The nuclear transfer from intestinal oocytes was sufficient to reprogram the nucleus and produce frog clones. Two decades later, fusion experiments provided a second way to reprogram cells. Both techniques indicated that trans-acting factors had to be responsible for the reprogramming of a differentiated nucleus. Additionally many techniques were
developed that allowed researchers to derive, culture and study pluripotent cell lines.
Furthermore, the fascinating finding that enforced expression of transcription factors can switch the mature cell type into another, contributed to the generation of induced pluripotent stem cells (iPSCs) (Stadtfeld and Hochedlinger 2010).
1.2 Nuclear Transfer
During mammalian development, cells gradually lose pluripotency potential and become progressively differentiated to perform the specialized functions of somatic tissues. When a nucleus from a differentiated somatic cell, such as an intestinal cell, is transplanted into an enucleated oocyte, nuclear reprogramming is initiated, allowing the generation of an entire individual, a genetically identical clone of the original somatic cell. Nuclear transfer experiments have demonstrated that all the genes required to create an entire organism are present in the nucleus of the specialized cell and can be activated upon exposure to reprogramming factors present in the oocytes.
During 1950s, Briggs and King established the “cloning“ technique of SCNT to probe the developmental potential of nuclei isolated from late-stage embryos and tadpoles by transplanting them into enucleated oocytes. However, they failed to reproduce this finding with cells from more specialized tissues and they suggested that this could be due to loss of plasticity as cells differentiate (Briggs and King 1952). Later in 1962 John Gurdon introduced the nucleus of fully differentiated adult tadpole intestinal cells into ultraviolet-light-irradiated oocytes, obtaining not only tadpoles but also normal adult frogs. He interpreted his findings as evidence that the process of cell specialization did not require irreversible nuclear changes (Gurdon 1962).
Next, the cloning of Dolly the sheep and other mammals from adult cells including terminally differentiated cells showed that the genome of fully specialized cells remains genetically totipotent with the ability to support the development of an entire organism (Wilmut, Schnieke et al. 1997; Hochedlinger and Jaenisch 2002;
Eggan, Baldwin et al. 2004; Inoue, Wakao et al. 2005). A wide range of species have now been successfully cloned using SCNT, ranging from domestic animals such as dogs and goats, and their hybrids such as mules, to wild animals such as African wildcats and wolves (Thuan, Kishigami et al. 2010). Also, nuclei from frozen tissues have been transplanted into enucleated oocytes a decade after tissue freezing (Wakayama, Ohta et al. 2008). A low efficiency of nuclear cloning (1-2%) is typical of mice which are the most widely used experimental animal system (Thuan, Kishigami et al. 2010). However, it should be mentioned that most of the cloned animals showed phenotypic and gene expression abnormalities. These are; aberrant gene expression in embryos, telomere elongation, obesity in adults, impaired immune systems and often increased cancer susceptibility and premature death (Thuan, Kishigami et al. 2010). The developmental defects in cloned animals have been suggested to occur due to a failure to erase epigenetic memory completely, so that a better understanding of epigenetic memory is required to improve nuclear transfer experiments (Stadtfeld and Hochedlinger 2010).
1.3 Fusion Hybrids and ESCs
By definition, cell fusion techniques involve fusing two or more cell types to form a single entity. This allows the impact of one genome on another to be investigated,
and in this way the existence of trans- acting repressors and tumor suppressor proteins were discovered in the late 1960s.
Interestingly, fusion experiments have revealed that when mouse fibroblasts were fused with a hamster melanocyte or rat hepatocyte, melanin and tyrosine aminotransferase, respectively, ceased to be synthesized (Davidson, Ephrussi et al.
1966) (Weiss and Chaplain 1971). According to these pioneer studies, gene expression was not only regulated by cis- acting elements but also by trans-acting repressors. Subsequent experiments with normal and tumor cell hybrids demonstrated the existence of trans-acting tumor suppressor proteins, that lead to dominant normal state overcoming the transformed state. Moreover, other experiments have shown that previously silent genes could be activated in mammals by producing heterokaryons (Blau, Chiu et al. 1983), which are short-lived, non-dividing multinucleate fusion products of two distinct cell types. Activation of silent genes was initially demonstrated in heterokaryons of muscle cells and amniotic cells (Blau, Chiu et al.
1983). Later, heterokaryons from diverse cell types, including human fibroblasts, hepatocytes and keratinocytes indicated that previously silent muscle genes could be activated in cells representative of all three embryonic lineages (Blau, Chiu et al.
1983; Blau, Pavlath et al. 1985). These experiments converged on the conclusion that expression of previously silent genes typical of diverse differentiated mammalian cell types, could be induced in other differentiated cell types, even in vivo (Johansson, Youssef et al. 2008) (Weimann, Charlton et al. 2003).
Therefore, the differentiated state was highlighted as not fixed, but as a continuously regulated state characterized by plasticity.
Further work allowed nuclear reprogramming of somatic cells in proliferative hybrids and involved fusing female embryonic germ cells, which are pluripotent stem
cells derived from primordial germ cells, with thymocytes from adult mice. Later observations showed that fused tetraploid cells were pluripotent: the cells could contribute to the three germ layers in chimaeric embryos (Tada, Tada et al. 1997).
Additionally, Tada and colleagues revealed that somatic cells can acquire a pluripotent state after being fused with ES cells (Tada, Takahama et al. 2001). Later findings pointed out that human somatic cells could be reprogrammed by human ES fusion in tetraploid hybrids in a 1:1 ratio (Cowan, Atienza et al. 2005). Recent studies have concluded that the rapid rate of reprogramming in heterokaryons (in contrast to hybrids), makes them beneficial for novel studies that focus on reprogramming initiation to a pluripotent state.
Earlier, embryonic stem cells (ESCs) were isolated from the inner cell mass of mouse blastocysts (Evans and Kaufman 1981) and from human embryos (Thomson, Itskovitz-Eldor et al. 1998) and were shown to be karyotypically normal. These cells efficiently contribute to all adult tissues, including the germline. Transplantation of these cells into nude mice results in the formation of teratomas consisting of various tissues from all three germ layers, confirming the pluripotency of these cells. Beside ES cells, other pluripotent cells have been derived upon explantation. They are epiblast-derived stem cells( EpiSCs) that have been isolated from post-implantation embryos, embryonic germ cells (EGCs) that have been isolated from primordial germ cells of mid-gestation embryo, and multipotent germline stem cells (mGSCs) that have been isolated from neonatal and adult mouse testicular cells. All these cells originate from as either early embryos or germ lineage cells that are possibly the only cells that harbor the epigenetic state that is permissive for a pluripotent state conversion. Also, endogenous Oct4 expression is a molecular marker that is common to pluripotent cells lines.
Among pluripotent cells, it is suggested that ESCs carry balanced parental imprints and that is why they are the only cells that pass the most stringent tetraploid complementation assay. However, all the pluripotent cell lines tested so far (ESCs, ECCs, EGCs) can to induce pluripotency in somatic cells after cellular fusion, indicating their capacity for reprogramming (Stadtfeld and Hochedlinger 2010).
1.4 Transcription factor transduction
Transcription factors help to establish and maintain cellular identity during development by driving expression of cell type specific genes and by suppressing lineage inappropriate genes. It was discovered that transcription factors can change the cell fate when ectopically expressed in certain heterologous cells. The pioneer work in the field was done in D.melanogaster larvae, ectopically expressing the homeotic gene Antennapedia under the control of a heat-shock gene promoter. This experiment led to interesting outcome: an additional set of legs was formed instead of antennae (Schneuwly, Klemenz et al. 1987). Moreover, ectopic expression of eyeless in the same model system led to the development of functional eyes on legs, wings and antennae.
Furthermore, Davis and his co-workers showed that fibroblast cell lines transduced with retroviral vectors expressing the skeletal muscle factor MyoD can form myofibers. Moreover, B and T cells upon overexpression of the myeloid transcription factor C/EBPα could be converted into functional macrophages (Xie, Ye et al. 2004). Fibroblasts could also be converted into neurons by activation of neural factors such as Ascl1, Brn2 and Myt1l. These studies suggest that “master genes” can drive cell reprogramming. It also became evident that lineage conversions are not
restricted to cell types within the same lineage or germ layer because fibroblasts are mesodermal in origin and neurons are derived from ectoderm (Stadtfeld and Hochedlinger 2010).
1.5 Induced Pluripotent StemCells (iPSCs)
The aim to identify transcriptional regulators that can reprogram cells brought Takahashi and Yamanaka in 2006 to perform a revolutionary work demonstrating that somatic cells can be reprogrammed back to a pluripotent state highly similar to that of ES cells. iPSCs express ES cell markers such as E–Ras, Cripto, Dax1, Zfp296 and Fgf4, exhibit ES cell morphology and growth properties, differentiate into tissues
from the three germ layers, and contribute to embryonic development. The authors used retroviral vectors to introduce into mouse embryonic and adult fibroblasts a mini-complementary-DNA library of 24 genes expressed by ES cells, and these genes were then tested for their collective ability to induce pluripotency. Pluripotency was assayed by examining for activation of a reporter gene construct containing the promoter of a gene previously identified as being specific to ES cells. Clones in which the Fbx15 promoter was activated produced a reporter protein that rendered them resistant to the drug neomycin, and these drug-resistant clones had similar morphology, growth properties and gene expression characteristics as ES cells (Takahashi and Yamanaka 2006).
The first generation iPSCs did not generate viable chimeras when injected into blastocysts and endogenous Oct4 and Nanog promoters were still methylated (Takahashi and Yamanaka 2006). Subsequently, fully reprogrammed iPSCs could be generated by reinforced expression of Yamanaka factors followed by selection for
endogenous expression of Oct4 and Nanog. Fully reprogrammed iPSCs were afterwards demonstrated to generate viable chimeras, contribute to the germline and to be unmethylated at their Oct4 and Nanog promoters (Okita, Ichisaka et al. 2007;
Wernig, Meissner et al. 2007).
iPSCs overcome the ethical issues related to ES cell derivation from human embryos since they are generated from adult cells. Furthermore because they are patient specific cells, they would not be rejected by the immune system of the hostas would be the case for ES cells.
Accordingly, Takahashi and Yamanaka revealed four key factors (which are also named as Yamanaka factors) that suffice to induce pluripotency in fibroblasts:
Sox2, Oct4, Klf4 and c-Myc.
1.5.1 Sox2 and Oct4
Sox2, which is required for iPS generation, belongs to the Sox family (sex determining region Y, SRY- related box proteins) that function during all stages of mammalian development and share highly conserved DNA-binding domains of 80 a.a. that are referred to high mobility group (HMG) box domains. The second Yamanaka factor, Oct4 is a member of the octamer protein binding family, first identified by virtue of its binding abilities to an eight-base-pair DNA motif found within regulatory region of many genes. Each of octamer- protein family member contains a bipartite POU domain which is responsible for DNA binding. Oct4 has become the most widely studied octamer binding protein, because it is exclusively found in stem cells during development and in a growing list of tumors (Scholer, Ruppert et al. 1990; Hu, Liu et al. 2008).
Both Sox2 and Oct4 are essential for self renewal and pluripotency of ES cells. Also Oct3/4 and Sox2 expression has been reported of an expanding number of cancers. It has been found that inactivation of the Sox2 gene or Oct3/4 results in embryonic lethality during the peri-implantation stage of development. Further experiments have revealed that knock-down of either Sox2 or Oct3/4 in ES cells promotes their differentiation into trophectoderm-like cells (Rizzino 2009).Therefore, Oct4 and Sox2 are both esssential for the self renewal and pluripotency of ES cells.
CHIP-chip experiments performed by Boyer et. al determined that Sox2 and Oct3/4 co-occupy several hundred genes, a large percentage of which are expressed in ES cells (Boyer, Lee et al. 2005). The vast majority of the genes co-occupied by Sox2 and Oct3/4 and expressed in ES cells are turned off when ES cells differentiate, probably because Sox2 and Oct3/4 expression is extinguished. Sequential experiments determined that these two transcription factors are bound simultaneously to at least six different genes in mouse ES cells (Loh, Wu et al. 2006). Another interesting finding is that Sox2 and Oct3/4 co-occupy two classes of genes, one that is expressed in ES cells and a second class of genes that is expressed only after ES cells undergo differentiation. A possible explanation for this is that Sox2 and Oct4 may contribute to silencing of the second class of genes in ES cells, by helping to recruit transcriptional repressive machinery, but this possibility needs to be investigated.
Further studies also highlighted the importance of master regulation to occur to maintain the self-renewal of ES cells. It has been observed that small changes in the levels of Oct3/4 promote the differentiation of ES cells and that the cell types formed were heavily influenced by the expression of Oct3/4. In particular, reductions in the level of Oct3/4 promote the differentiation of ES cells into trophoectoderm-like cells,
whereas a small increase in Oct3/4 levels promotes their differentiation into cells that exhibit markers for endoderm and mesoderm (Rizzino 2009).
Overall, it is clear that more progress needs to be made to understand the mechanisms by which these transcription factors act. Understanding how Sox2 and Oct4 function will be useful in ES biology and in improving new protocols for iPSCs generation, including their development without the aid of viruses.
1.5.2. Klf4
Kruppel-like-factor 4 (Klf4) is a transcription factor - that belongs to the relatively large family of Sp1-like transcription factors with over 20 members- that can both activate and repress genes involved in cell-cycle regulation and differentiation. The 438 a.a mouse KLF4 protein contains 3 Kruppel-type zinc fingers in its immediate carboxyl terminus, which is preceded by a 20 a.a segment rich in basic residues that serves as nuclear localization signal. KLF4 is found in various tissues such as epithelial, lung, testis, thymus, cornea, lymphocytes, vascular endothelial cells and cardiac myocytes. These studies point out that KLF4 is usually expressed in adult tissues that have a high rate of cell turnover (Nandan and Yang 2009).
KLF4 regulates the expression of a set of genes to co-ordinately inhibit cellular proliferation. Over-expression of KLF4 has been observed as inhibitor for cell proliferation in culture with a possible checkpoint activity G1/S and G2/M.
Moreover, KLF4 expression has been found to increase following DNA damage, cell- cycle arrest in response to serum withdrawal and contact inhibition (Shields, Christy et al. 1996)((Zhang, Geiman et al. 2000). Surprisingly, elevated Klf4 levels have also
been linked to cancer; its mRNA and protein are over-expressed in up to 70% of mammary carcinomas. Experiments have also shown that KLF4 was frequently over- expressed in squamous-cell carcinomas of the oropharynx (Foster, Ren et al. 1999).
Recently, ectopic Klf4 expression in mice has been shown to induce squamous epithelial dysplasia (Foster, Liu et al. 2005). Klf4 has also been shown to help to bypass Ras V12-dependent senescence by inhibiting p53-expression. Klf4`s ability to transform cells was reported in adenovirus E1A-immortalized rat kidney epithelial cells. A recent study supported these findings by demonstrating an anti-apoptotic effect of KLF4 following DNA damage due to its ability to inhibit p53-mediated activation of the pro-apoptotic gene BAX (Ghaleb, Katz et al. 2007). The mechanism by which KLF4 regulates ES cell renewal may regulate to the fact that its expression is upregulated by LIF signalling in ES cells. Further, ES cells over-expressing KLF4 had a great propensity for self renewal based on secondary embryoid body formation (EB). It was concluded that KLF4-transduced EBs expressed higher levels of Oct4, consistent with the notion that KLF4 regulates ES cell renewal. Global analysis of promoter occupancy by the four somatic cell reprogramming factors (Klf4, Oct4, Sox2 and c-Myc) pointed out a transcriptional hierarchy within these factors, with auto-regulatory and feed-forward regulation. This study additionally suggested that KLF4 is an upstream regulator of a feed-forward loop that contains Oct4, Sox2 and c- Myc, as well as other downstream targets such as Nanog. Consequently, KLF4 plays an important role in somatic cell reprogramming and maintenance of ES cell self- renewal.
The ability of KLF4 in maintaining immortality of iPS cells is closely associated with c-Myc. Possibly, KLF4 and c-Myc act together, similar to how KLF4 and Ras which cooperatively induce cell transformation (Rowland and Peeper 2006)
in order to affect iPS cell self-renewal. It could be that suggested that KLF4 suppresses apoptosis induced by c-Myc, while c-Myc neutralizes the cytostatic effect of KLF4 by suprressing p21. Thus, the balance between KLF4 and c-Myc might play a critical role in the establishment of an immortalized state of iPS cells (Yamanaka 2007).
Subsequent experiments addressed whether iPS clones could be generated by substitution of homologs: Klf2 or Klf5 for Klf4; Sox1 for Sox2; L-Myc or N-Myc for c-Myc. In all cases, iPC clones were generated but with lower efficiency (Nakagawa, Koyanagi et al. 2008). Depletion of the Kruppel-like factors showed that, single depletion of Klf2, Klf4 or Klf4 in mouse ES cells failed to influence ES self-renewal, when three factors were depleted the ES cells underwent differentiation. Chromatin immunoprecipitation experiments suggested that, the three Klfs share Nanog as a common target. So, when one Klf factor was depleted, the other two still bind to their targets. These results converged to the conclusion that the three Klf factors form a circuit that regulates self-renewal of ES cells (Jiang, Chan et al. 2008). Another report has revealed that Klf5 is critical for derivation and self-renewal of mouse ES cells.
The authors reported that the Klf5-knockout embryos failed to develop past E6.5 due to failure of implantation resulting from trophoectoderm defects. The Klf5-KO ES cells were pluripotent, but differentiated upon prolonged culture in vitro, displaying decreased G1 cell cycle progression and increased p21 expression. Moreover, Klf5 over-expression in ES cells led to elevated Tcl1 expression, Akt phosphorylation and increased cell proliferation. Thus, Klf5 over-expression could maintain ES cell self- renewal through stimulation of the Akt pathway. In subsequent experiments, Klf4 over-expression was shown to supress differentiation of Klf5-KO ES cells, but with decreased cell proliferation, suggesting that Klf4 and Klf5 function similarly to
suppress differentiation, but have opposing effects on cellular proliferation (Ema, Mori et al. 2008).
1.5.3 c-Myc
The basic helix-loop-helix protein Myc is a transcription factor controlling various aspects of cell physiology that allow efficient proliferation of somatic cells.
The Myc family is composed of three evolutionary conserved bHLHZip transcription factors; c-Myc, N-Myc and L-Myc that regulate cell growth, cell cycle progression, biosynthetic metabolism, apoptosis, angiogenesis, invasion, stromal remodelling and inflammation (Eisenman 2001). The targets of Myc proteins comprise a very large repertoire: RNA Pol II transcribed genes, RNA Pol I and RNA III RNAs involved in translation and growth, as well as miRNAs that have key roles in cell proliferation, cancer and stem cell maintenance (Arabi, Wu et al. 2005; Grandori, Gomez-Roman et al. 2005) (Gomez-Roman, Grandori et al. 2003) (Lotterman, Kent et al. 2008).
Additionally it seems that there are very few genes for which Myc is the sole transcriptional regulator. Most likely, Myc generates global alterations in chromatin structure, which in turn modulate transcription (Grandori, Cowley et al. 2000). Germ line deletions of either c-Myc or N-Myc lead to embryonic death at E11 because of failures in organ and tissue growth. Also it was found that fibroblasts lacking c-Myc proliferate very slowly and inefficiently. Reports have indicated that in normal cells, Myc function is regulated by developmental or mitogenic signals. Myc mRNAs and proteins are very short lived and, in the absence of such proactive signals, Myc expression is decreased and Myc protein levels rapidly fall, tirggering growth arrest.
Myc activity is deregulated in tumor cells, sometimes through mutations within the
Myc genes, but more often, through gene amplification or induction by upstream oncogenic signals in pathways such as Ras, Wnt-b-catenin or Notch (Soucek and Evan 2010).
Since c-Myc is one of the Yamanaka factors, the concern was raised that iPS cells might have genomic instability. In this context, iPS-derived animals developed tumours because of the reactivation of the c-Myc virus (Okita, Ichisaka et al. 2007) whereas chimeric mice derived from c-Myc free iPS cells showed substantially less tumour formation (Nakagawa, Koyanagi et al. 2008). However, another study revealed that the use of a c-Myc retrovirus did not enhance the teratoma forming propensity of iPS cells (Miura, Okada et al. 2009). Thus, the mechanisms underlying different teratoma forming propensities of iPS cells remain elusive, and the risk of tumorigenesis needs to be defined for the iPS cells to be used in the clinic.
1.6 P53
Direct mutations or indirect inactivation of the p53 tumour-suppressor-protein network occur in most human cancers, underlying the role of p53 in tumour prevention.
In brief, p53 is a master transcription factor that under normal conditions is functionally inactive due to its rapid degradation by the ubiquitin ligase MDM2.
However, upon almost any cellular stress, but particularly in response to DNA damage, MDM2-driven degradation is halted, and p53 accumulates and gains full competence in transcriptional activation. p53 coordinates a wide range of cellular responses: activation of programmed cell death (apoptosis), activation of cell cycle
arrest or promotion of senescence via transcriptional dependent and independent mechanisms.
1.6.1 Role of p53 in Reprogramming
Beside its tumour suppressive role, p53 has roles during development and differentiation. p53-null mice have birth defects, such as defects in neural tube closure, defects in bone development and polydactyly. It has been shown that p53 has many functions during differentiation, depending on the cell type and lineage. p53 is reported to regulate fertility in mice and humans via its direct interaction with the LIF gene promoter, thus regulating LIF expression during pregnancy. As a consequence, low levels of p53 affect human and mice fertility (Menendez, Camus et al. 2010). LIF is an important factor maintaining mouse ES in the pluripotent state. Moreover, knock-out of the p53 gene increases the spontaneous generation of embryonic stem (ES) and embryonic germ (EG) cells from primordial germ cells, suggesting the contribution of p53 pathway in the process. The first demonstration that suggested a direct link between p53 and somatic cell reprogramming was established by showing that the SV-40 oncogenic protein (the SV40 antigenthat targets p53) increases reprogramming efficiency (Mali, Ye et al. 2008).
More recently, five groups in the same issue of Nature (2009) have demonstrated that inhibition of cell cycle checkpoints regulated by p53 and the INK4A-ARF locus limit reprogramming and that disruption of these pathways causes a significant increase in reprogramming efficiency (Hong, Takahashi et al. 2009;
Kawamura, Suzuki et al. 2009; Li, Collado et al. 2009; Marion, Strati et al. 2009;
Utikal, Polo et al. 2009). Marion, Strati et al. (2009) showed that p53-deficient iPS
cells are genomically unstable and not fit enough to efficiently produce mice. This observation was confirmed by the work of Hong and his co-workers who demonstrated that mice derived from p53-deficient iPS cells eventually develop tumours. They presented also evidence for contribution of the p53-p21 pathway as a barrier for iPS cell generation. Furthermore Kawamura et al. reported that inactivating p53 or its target gene p21 or inhibiting apoptosis in mouse fibroblasts increases reprogramming efficiency. This group was also able to generate IPS cells with only 2 factors, Oct4 and Sox2 in a p53-deficient background. Utikal et al. demonstrated that acute genetic ablation of p53 in cells that normally fail to reprogram rescues their ability to produce iPS cells. In this study the Arf-p53 pathway in mice has been observed as a barrier to iPS cell formation.
Other observations pointed out that the Ink4a/Arf locus (which encodes Ink4a - p16(cell-cycle inhibitor) and Arf -p14 (indirect p53 activator) is silenced during iPS reprogramming.
1.6.2 Role of p53 in Oncogene induced DNA Replication Stress
The cellular machinery, named DNA Damage Response (DDR), that responds to damaged DNA is formed by a dynamic network of hierarchically ordered proteins and multi-protein complexes capable of detecting DNA lesions and signaling their presence to activate pathways that delay cell cycle progression (the so-called checkpoints), repair the DNA lesions, or eliminate the genetically unstable cells by inducing cell death. A current model that has been proposed by us and others, highlights the critical role of the DDR machinery as an inducible barrier against progression of tumors beyond their early stages. This model, that was initially
proposed to explain the high frequency of p53 mutations in human cancers, was based on the ability of oncogenes` to generate genomic instability. According to this model, activated oncogenes induce DNA replication stress and DNA damage, which in turn activate DDR. DDR is a cascade of Ser/Thr kinases that includes ATM, ATR, Chk1 and Chk2, which phosphorylate p53 leading to cell cycle arrest, apoptosis and/or senescence. Characterization of mice genetically manipulated with a knocked-in p53 that cannot be phosphorylated at two of the main residues targeted by DDR kinases, namely Ser 18 and Ser23 (Ser 15 and Ser 20 in human p53), indicates an important role of these phosphorylation sites in DNA damage induced p53-dependent responses (Chao, Herr et al. 2006). Importantly, in precancerous lesions, wild type p53 function and p53-dependent apoptosis and/or senescence, are maintained, thus limiting the growth of the lesion. When p53 function is lost, the cells can escape the apoptotic and senescence effects of p53, and the precancerous lesion can become cancerous (Bartkova, Horejsi et al. 2005; Gorgoulis, Vassiliou et al. 2005; Halazonetis, Gorgoulis et al. 2008). It is know that the common fragile sites (CFS) are the genomic regions that are most unstable in the presence of DNA replication stress. Thus, the initial evidence that activated oncogenes induce DNA replication stress was provided by the analysis of allelic imbalances (LOH) in a small number of precancerous lesions focusing on CFS. This analysis showed preferential targeting of common fragile sites, consistent with the DNA damage in precancerous lesions being due to DNA replication stress (Bartkova, Horejsi et al. 2005; Gorgoulis, Vassiliou et al. 2005).
Further studies have supported this finding by showing that activated oncogenes induce collapse of DNA replication forks and replication dependent induction of DNA DSBs (Bartkova, Rezaei et al. 2006; Di Micco, Fumagalli et al. 2006).
1.7. Chromosome Fragile Sites
Chromosome Fragile Sites are generally defined as chromosomal loci that are particularly sensitive to stresses, forming cytogenetically visible gaps and breaks on metaphase chromosomes. These sites are generally classified into two main categories, rare and common, based on their population frequency, pattern of inheritance and method of induction (Durkin and Glover 2007).
1.7.1 Rare fragile sites
Rare fragile sites are observed only in a small proportion (less that 5%) of a population and are inherited in Mendelian manner. Increased breakage at these sites is most often caused by the expansion of nucleotide repeats. The major group of rare fragile sites is the folate-sensitive group, harboring CGG-repeat expansion. This group includes the sites: FRAXA, FRAXE, FRAXF, FRA16A and FRA11B. Apart from their fragility, the CGG CCG repeat expansions in both FRAXA and FRAXE cause X-linked mental retardation by interfering with gene expression. Another group of rare fragile sites is characterized by expanded AT-rich minisatellite repeats, fragility at these sites is induced by bromodeoxyuridine (BrdU) or distamycin. A.
These fragile sites are FRA10B and FRA16B. A common feature of the sequences that form rare fragile sites is that they are able to form secondary structures. CTG, CAG, CGG, and AT repeats can form stable hairpin and even multiple hairpins (Durkin and Glover 2007).
1.7.2 Common fragile sites (CFS)
Common fragile sites were first discovered in a Fragile X patient having gaps and breaks on chromosomal locations other than the fragile X locus. Follow up work published in 1984 showed that when cells were cultured in the presence of the DNA polymerase inhibitor aphidicolin (APH), a number of sites in the genome were specifically and reproducibly induced to form gaps and breaks on metaphase chromosomes (Glover, Berger et al. 1984). CFSs occur in all individuals in specific regions of many chromosomes. In tissue culture they can be observed in metaphase spreads prepared from cells treated with aphidicolin. Following the administration of aphidicolin, CFS can be they are involved in translocations, sister-chromatid exchanges and interchromosomal rearrangements. Like rare fragile sites, CFSs are late-replicating regions and this was first demonstrated for FRA3B, APH treatment further delayed replication, leaving the FRA3B sites unreplicated in the G2 phase of the cell cycle in about 16.5% of the APH-treated cells (Le Beau, Rassool et al. 1998).
Additional studies showed that other CFSs, such as FRA16D and FRA7H, may also experience difficulty in replication fork progression (Palakodeti, Han et al. 2004) (Hellman, Rahat et al. 2000). These studies suggest a model in which CFS regions initiate replication properly but are slow to complete the process, thus resulting in regions of unreplicated DNA, which manifest as chromosome breaks.
1.7.2.1 Evolutionary conservation of common fragile sites (CFS)
CFSs are highly conserved during mammalian evolution. Orthologs of human fragile sites have been found in other mammals such as cat, dog, pig, horse, cow,
Indian mole rat, deer mouse and laboratory mouse. Mice experiments indicated at least 8 CFSs: Fra14A2 (FRA3B), Fra8E1 (FRA16D), Fra6C1 (FRA4F), Fra12C1 (FRA7K), Fra2D (FRA2G), Fra6A3.1 (FRA7G), Fra6C1 (FRA7H) and Fra4C2 (FRA9E) that have human CFS orthologs. It was also proposed that in lower eukaryotes like Saccharomyces cerevisiae replication slow zones (RSZ) are analogous to CFS in metazoans. This idea came up after the observations that in yeast mec1 (ATR ortholog) mutants, double strand breaks formed in specific regions characterized by slowly moving replication forks (Cha and Kleckner 2002). The fact that CFSs persist in the genomes of organisms from yeast to human suggests that they serve a conserved function in the cell. Other experiments have examined the roles that yeast replicative polymerases play in deletions and translocations. Several studies have determined that yeast preferentially form DSBs and translocations at tandem inverted repeats, and that the frequency of these events increases dramatically by mutations in either DNA polymerase alpha or delta (Lemoine, Degtyareva et al. 2005) (Ruskin and Fink 1993; Freudenreich, Kantrow et al. 1998).
1.7.2.2 Common fragile instability in tumors
Many groups have concluded that CFSs are sites of frequent chromosome breakage and rearrangements in cancer cells. The most frequently expressed and the best characterized CFS are FRA3B and FRA16D, which lie within large genes, FHIT and WWOX espectively. FHIT (fragile histidine triad gene) catalyzes hydrolysis of diadenosine polyphosphates, produced by action of the aminoacyl t-RNA synthetases.
High frequency of allelic loss or homozygous deletions within FRA3B under FHIT have been described in esophegeal adenocarcinomas, gastric cancer, head and neck
squamous cell carcinoma, lung cancers and B-cell lymphomas (Durkin and Glover 2007). FRA16D maps within regions of frequent loss of hetorozygousity (LOH) in breast and prostate cancers and is associated with homozygous deletions in various adenocarcinomas and with chromosomal translocations in multiple myeloma.
Similarly to FRA3B, these deletions include deletions of ten to hundreds of kilobase pairs directly within FRA16D that inactivate the WWOX gene (Ried, Finnis et al.
2000) (Bednarek, Keck-Waggoner et al. 2001) (Paige, Taylor et al. 2001).
Deletions in cancer cells have been detected within other CFSs and their associated genes, including FRA16D, FRA6E, FRA9E and FRA7G.
While deletion breakpoints within fragile sites in cancer cells are common, translocations involving common fragile sites are reported. Translocations involving FRA3B have been found in small number of tumor cell lines, including hepatocellar, esophageal and breast carcinoma (Fang, Arlt et al. 2001) (Keck, Zimonjic et al. 1999) (Popovici, Basset et al. 2002). Other translocations also have been revealed in other fragile sites, including like FRA16D and FRA8C (Bednarek, Laflin et al. 2000) (Ferber, Eilers et al. 2004). However it should be noted that these translocations do not necessarily form a fusion protein, but rather inactivate the involved genes.
In addition to deletion and translocation events, CFSs have been associated with gene amplification events in tumor cells. Several studies have shown that breakage at fragile sites can initiate breakage-fusion-bridge cycles, a mechanism responsible for accumulation of intrachromosomal amplicons (Kuo, Vyas et al. 1994;
Coquelle, Pipiras et al. 1997). Indeed, one study identified FRA7I and FRA7G at boundaries of amplicons found in tumor-derived cell lines (Hellman, Zlotorynski et al. 2002). Boundaries of amplicons involving the MET oncogene and FRA7G have
also been identified in six primary esophageal adenocarcinomas (Miller, Lin et al.
2006) (Coquelle, Pipiras et al. 1997).
1.7.2.3 Mechanisms that regulate CFS instability
Unlike rare fragile sites, which are caused by expansions of di-, or tri- nucleotide repeats, such repeats have not been identified within CFS. The only common feature of CFS is a relatively high amount of AT-rich sequences. FlexStab, a computer-program designed by Mishmar, measures (Mishmar, Rahat et al. 1998) local variation in twist angle between bases within a sequence, revealing areas of high flexibility, termed “flexibility peaks” within CFS. FRA2G, FRA3B, FRAXB, FRA7E, FRA7H, FRA8C and FRA16D have been analyzed this way and contain a high number of flexibility peaks relative to non-fragile regions (Arlt, Durkin et al.
2006). It has been speculated that these sequences may lead to increased potential to form secondary structures and thus perturb replication at these sites. On the other hand, other findings indicate that loss of large sequences within FRA3B in cells with various deletions in FRA3B, do not affect fragility (Corbin, Neilly et al. 2002).
Further mapping of the regions of frequent breakpoints in cancer cells showed no correlation between flexibility peaks and breakpoints in FRA3B (Corbin, Neilly et al.
2002).
The late replication observed in common fragile sites is possibly due to formation of secondary structures that inhibit replication fork progression, or other factors affecting replication dynamics in these regions. FRA3B and FRA16D contain polymorphic AT- repeats and it is speculated that AT-repeat sequences have the potential of forming hairpin and cruciform structures that could lead to replication
fork collapse. It is known that aphidicolin uncouples replicative polymerases from the helicase/topoisomerase complex. Thus, after polymerase stalling (or slowing down) at CFSs, helicase/topoI complex continues to unwind DNA giving rise to long stretches of ssDNA with the potential to form secondary structures in the AT- rich sequence.
These structures could then destabilize replication. The majority of replication perturbations should afterwards be detected by the DNA damage checkpoint and DNA repair machinery. The lesions that escape repair would then manifest as breaks on metaphase chromosomes. This idea is supported by the observation that low doses a campthotecin -topoisomerase I inhibitor – can prevent breaks at CFSs induced by APH.
According to the oncogene induced replication stress model, CFSs are indicators of replication stress during early stages of tumorigenesis. Furthermore, when ATR (the kinase that responds to DNA replication stress) is depleted, common fragile site expression increases significantly after low doses of aphidicolin treatment.
Moreover, ATR deficiency alone, without addition of replication inhibitors, induces gaps and breaks at fragile sites. These findings demonstrate that some level of replication stalling occurs normally at fragile sites that is under regulation of ATR (Casper, Nghiem et al. 2002). Another study found that cells from individuals with Seckel syndrome, which contain a hypormorphic mutation in ATR, show dose- responsive increased breakage at fragile sites. This form of Seckel syndrome is the first human genetic disorder with increased susceptibility to fragile site instability.
Another study, supported these findings by demonstrating that Chk1 deficiency results in significant breakage rate at common fragile sites, but no effect was observed when CHK2 was inhibited (Durkin, Arlt et al. 2006). These results converge to a common conclusion that points out ATR, but not ATM regulates CFS expression.
Based on the appearance of deletions and chromosome rearrangements in cultured and tumor cells, it is clear that DSBs can occur at common fragile sites, either directly or as a result of repair defects at stalled replication forks. Homologous recombination plays a major role in responding to DSBs and stalled or collapsed replication forks during S and G2 when sister chromatids are present. Sister chromatid exchanges are a consequence of HR repair. Consistent with this, in cells treated with aphidicolin most gaps and breaks at FRA3B involve a sister chromatid exchange at that site, indicating the repair role of HR at these sites (Glover and Stein 1987).
Replication stress induced by aphidicolin additionally increases the number of RAD51 foci and phosphorylated DNA-PKcs, key components of HR and NHEJ, DSB repair pathways respectively (Schwartz, Zlotorynski et al. 2005). Furthermore, replication stress has been shown to result in DSB markers, gamma H2AX, MDC1, which colocalize with RAD51 and phospho DNA PKcs. Especially, γ-H2AX and phospho-DNA-PKcs were localized at expressed CFSs on metaphase chromosomes (Schwartz, Zlotorynski et al. 2005). These findings reveal some information about how CFS are repaired, but more studies will be needed to determine the exact nature of the lesions at fragile sites and their repair.
Part II. ROLE OF P14ARF EXPRESSION
1.8 CDKN2A locus
In humans, deletion of the CDKN2A locus (situated on chromosome 9p21) is a common genetic lesion detected in ~ 50 % of tumours such as glioblastoma, melanoma, pancreatic adeno-carcinoma, non-small cell lung cancer, bladder
carcinoma and oropharyngeal cancer. CDKN2A encodes two distinct proteins, p16INK4A and p14ARF and this locus confers tumour suppressor activity via two distinct pathways: the “Rb pathway” involving p16INK4A and the “p53 pathway”
involving p14ARF (Sharpless 2005). In some cancers, the CDKN2A locus suffers point mutations that most often target p16INK4A and not p14ARF. In most cancers, however, the entire CDKN2A locus is deleted, resulting in loss of both p14ARF and p16INK4A expression (Negrini, Gorgoulis et al. 2010).
1.8.1 p16INK4A pathway
In late 1993 and early 1994 research converged to identify a new tumor suppressor gene located on human chromosome 9p21. The first report was of the existence of 16-kDa protein that associated with cyclin dependent kinases when human diploid fibroblasts were transformed by SV40 virus (Xiong, Zhang et al.
1993). The interaction with CDK4 was exploited to isolate the corresponding cDNA in a yeast two-hybrid screen, and it was found that p16INK4A acted as a CDK4 inhibitor (Serrano, Hannon et al. 1993).
In mammals, the INK4-class of cell cycle inhibitors consists of P15INK4B, P16INK4A, P18INK4C, and P19INK4D. These proteins consist of four or more ankyrin repeats that are highly conserved. Evolutionary P15INK4B and P16INK4A share a common ancestor, as do P18INK4C and P19INK4D. In mammals, P18INK4C and P19INK4D are highly expressed during development, while P15INK4B and P16INK4A are associated with tumour suppression (Kamijo, Zindy et al. 1997). All four INK4 members are well conserved at the amino acid level, especially in the second and third ankyrin binding domains. It seems that the functional differences
among INK4 members relate to different patterns of expression in response to different genetic and environmental stimuli.
The INK4 proteins bind to CDK4 and CDK6 and inhibit their kinase activity for Rb and other targets. P16INK4A was characterized as a CDK4-associated protein capable of inhibiting CDK4/6-mediated phosphorylation of the retinoblastoma tumour suppressor protein (pRb) (Serrano, Hannon et al. 1993). Hypophosphorylated pRb binds to and represses E2F transcriptional activity and thus preventing G1/S progression. Therefore, P16INK4A expression results in cell cycle arrest. Studies have shown that in response to diverse stimuli such as: passage in cell culture, growth at high density, DNA damage, oncogene activation and advancing age, P16INK4A levels increase (Alcorta, Xiong et al. 1996; Reznikoff, Yeager et al. 1996; Serrano, Lin et al. 1997; Robles and Adami 1998; Shapiro, Edwards et al. 1998; Zindy, Eischen et al. 1998) (Pavey, Conroy et al. 1999; Wieser, Faust et al. 1999; Piepkorn 2000). It has been also found that mice lacking P16INK4A are phenotypically normal except prone to tumours and sensitive to carcinogens suggesting a role of P16INK4A in limiting aberrant cell proliferation (Sharpless 2005).
1.8.2 p14ARF pathway
The second transcript of the CDKN2A gene originates from an exon 1β (13 kb upstream of the exon 1α of P16INK4A). This transcript is spliced to a common second exon that is common with the transcript originating from exon1α. The β transcript is translated into a protein called p14ARF. P16INK4A and p14ARF are not isoforms and share no amino-acid homology, because their transcripts utilize different reading frames. While alternate reading frames are common in viruses and bacteria,
such a structure is practically unique in the mammalian genome. Evidence that p14ARF is a tumour suppressor came from the observation that the protein was a potent cell cycle inhibitor, and that mice lacking exon 1β of p14ARF were highly prone to spontaneous and carcinogen-induced tumours, similar to exons 2 and 3 deficient mice, that lack both P16INK4A and p14ARF (Quelle, Zindy et al. 1995) (Serrano, Lee et al. 1996; Kamijo, Zindy et al. 1997). ARF expression can induce cell cycle arrest even in cells with enforced cyclin D expression, suggesting that its function is distinct from that of p16INK4A (Quelle, Zindy et al. 1995). p14ARF was tested in the same assays that had been applied to INK4A proteins. Importantly, the protein did not bind to CDKs or did not directly interfere with their activity (Arap, Knudsen et al. 1997). Additionally it was reported that p19ARF loss obviated the need for p53 inactivation to immortalize murine embryonic fibroblasts, suggesting that ARF and p53 belong to a common genetic pathway (Kamijo, Zindy et al. 1997).
Ectopic expression of ARF leads to activation of p53 with consequent up-regulation of MDM2, a gene known to be transcriptional target of p53. Further studies have shown that ARF associates directly with Mdm2 to block its ability to interact productively with p53, both by localizing Mdm2 within the nucleolus, and by inhibiting Mdm2`s E3 Ubiquitin ligase activity (Weber, Taylor et al. 1999).
The ARF protein in mouse is highly basic and is composed of 169 amino acids (thus named P19ARF) without homology to other known proteins. The human ARF is predicted to be 132 amino acids long (thus named P14ARF). Interestingly, the predicted sequences of ARF proteins of man, mouse and oposum show only modest homology between these species. The portion of the protein encoded by exon 1β exhibits only 44% homology (53% similarity) between mouse and human although there is significant conservation of the first 25 amino acids. In contrast there is 54 %
homology (63% similarity) for the carboxyl terminus, but it is the amino-terminal portion of the protein that has a role to stabilize p53 and induce cell-cycle arrest when over-expressed in ARF null cells (Sharpless and DePinho 1999). In addition, it has been found that the chicken exon 1β splices to a different reading frame from that of mammals and there is no conservation outside exon 1β between chicken and mammals in ARF (Kim, Mitchell et al. 2003).
Many mitogenic stimuli such as E1A (de Stanchina, McCurrach et al. 1998), myc (Zindy, Eischen et al. 1998), oncogenic ras (Palmero, Pantoja et al. 1998), V-abl
(Radfar, Unnikrishnan et al. 1998) and E2F-1 (Bates, Phillips et al. 1998) have been shown to upregulate ARF leading to p53 stabilization. A reasonable hypothesis is that oncogenes induce selective pressure for perturbation of the ARF-p53-MDM2 pathway and that such perturbation occurs and needs to be suppressed during immortalization of rodent cells (Sharpless and DePinho 1999).
1.9 p53 pathways
Among the various cellular stresses present during malignant transformation, two have been particularly well-studied in relation to p53 due to their universal occurrence in cancer, namely DNA damage and oncogenic signalling. As previously explained, DDR activates p53 via the ATM/ATR/Chk1/Chk2 kinases and probably through other kinases, such as p38, JNK/SAPK and c-Abl (Milne, Campbell et al.
1995) (Hu, Qiu et al. 1997) (Bulavin, Saito et al. 1999) (Buschmann, Potapova et al.
2001). Furthermore, oncogenic signalling is known to activate p53 through ARF, which interacts with MDM2 inhibiting its p53-ubiquitin ligase activity. It has been established that under normal conditions, p53 levels are very low because of MDM2
dependent proteasomal degradation. Exposure of cells to harmful stimuli like oncogenic stress results in number of modifications in p53 (phosphorylation and acetylation), which suppress binding of p53 to MDM2 and which lead to accumulation and increased transcriptional activity of p53. In parallel, ARF- dependent stabilization of p53 results in a dramatic increase in p53 activity.
Interestingly, mice lacking p14ARF have a remarkable tumor-prone phenotype, although not as severe as p53-deficient mice and there is a good evidence in mice supporting the relevance of ARF/MDM2/p53 axis in tumor suppression (Donehower, Harvey et al. 1992).
Recent high-throughput sequencing studies in human sporadic cancers have been performed by several consortia and their results have been summarized in recent reviews (Negrini, Gorgoulis et al. 2010). Thousands of mutations have been sequenced in primary cancers and early passage cancer cell lines or xenographs.
Interestingly, TP53 mutations and amplification of MDM2 and MDM4 were mutually exclusive, consistent with the well documented role of MDM2 and MDM4 in targeting p53 for ubiquitin-dependent degradation. On the other hand, CDKN2A and TP53 mutations were not mutually exclusive, whereas CDKN2A mutations were mutually exclusive with CDK4, CDK6 and RB1 mutations. Since the CDKN2A locus encodes both P16INK4A and P14ARF, these results suggest that the inactivation of p16INK4A may be the driving force for the CDKN2A deletions in human cancers, as p16INK4A functions in the same pathway as CDK4, CDK6 and RB1.
1.10 Non-sense mediated decay (NMD) Mechanism
Non sense mediated decay is a mechanism that allows eukaryotic cells to degrade abnormal mRNAs that prematurely terminate translation. Such transcripts can be formed due to genomic frameshift or nonsense mutations, many of which cause diseases because of failure to produce functional proteins. NMD targets can also arise as a result of error in cellular processes: such as splicing. Targets of NMD include inefficiently spliced pre-mRNAs as well as transcripts from T-cell receptor genes, immunoglobulin genes and other antigen receptor genes that have undergone error-prone somatic-cell rearrangements and hyper-mutations during lymphocyte development. The importance of NMD becomes evident from the observation that there are numerous recessively inherited diseases that acquire a dominant negative phenotype when NMD fails to target premature transcripts (Isken and Maquat 2008).
1.10.1 Cis- and trans-acting NMD determinants
NMD is usually triggered when translation terminates prematurely, i.e. at a premature termination codon (PTC). The RNA features and protein factors that are generally necessary for a nonsense codon to trigger NMD can vary depending on the organism. In S. cerevisiae, an abnormally long distance between a termination event and the 3` poly(A) tail, as defined by the presence of poly (A)-binding protein 1 seems to be required to initiate NMD. In mammals, premature termination codons (PTCs) when located more than 50-54 nucleotides upstream of the last exon-exon junction, are able to target a mRNA for NMD. Presently it is known that during pre- mRNA splicing, protein complexes called exon junction complexes (EJCs) assemble
20-24 nucleotides upstream of each exon- exon juction. The EJC is a dynamic structure with a heterologous protein composition that evolves throughout the mRNA life cycle. Core EJC components consist of elF4III, RNA-binding-motif protein 14, mago nashi homologue (MAGOH) and Barentsz (BTZ). There is supporting evidence that EJCs are essential for PTC definition and NMD activation (Isken and Maquat 2008). According to the current model, EJCs are defined as “the marks” used to discriminate premature from normal termination. Thus, EJCs assemble during splicing next to each exon-exon junction and serve as anchoring point for the NMD factors UPF2 and UPF3.
Regardless of the species, the core set of NDM factors are UPF1, UPF2, UPF3 which were first identified in Saccharomyces cerevisiae and later found to be essential for NMD in all organisms studied. UPF3 consists of two isoforms in mammals that are encoded by distinct genes. In humans UPF3 is encoded by an autosomal gene, whereas UPF3X is encoded by an X-linked gene. UPF3 seems to partially substitute UPF3X when the latter is absent. hUPF1 is both ATP-dependent helicase and an RNA-dependent ATPase, whose activation by phosphorylation is essential for NMD activation. hUPF2 is thought to function as an adaptor for UPF1 and UPF3 binding since it includes two domains mediating interactions with UPF1 and UPF3 (Silva and Romao 2009).
NMD activation has been shown to be impaired by the expression of a dominant negative hUPF1 mutant, which contains the point mutation R844C in its RNA helicase domain. Additionally, the depletion of hUPF1, hUPF2, hUPF3 by RNAi inhibits NMD.
Recent studies have shown that there are four proteins that play an important role in phosphorylation and dephosphorylation of UPF1: SMG-1, SMG-5, SMG-6
