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DNA in dividing cells is prone to mutagenesis, with mutations making key contributions to human disease including cancer. The tumour suppressor gene TP53 is the most frequently mutated gene in human tumours. Here, we present a robust protocol for studying TP53 mutagenesis utilising human TP53 knock-in (Hupki) mouse embryonic fibroblasts (HUFs). In the HUF immortalisation assay (HIMA), primary HUFs are treated with known or suspected carcinogens at 3% oxygen and then transferred to 20% atmospheric oxygen to induce senescence. Cells containing mutations (e.g., in TP53) that allow bypassing of senescence eventually emerge as immortalised clonal cell lines after 2-3 months of serial passaging. As not all immortalised HUF cells contain TP53 mutations, we developed a Nutlin-3a counter-screen to select for TP53-mutated clones prior to sequencing. TP53 mutation spectra generated can be compared with those of human tumours recorded in the International Agency for Research on Cancer TP53 mutation database. Environmental mutagens that have demonstrated and validated the utility of the HIMA include ultraviolet radiation, aristolochic acid, and benzo[a]pyrene. The TP53 mutation patterns induced by these mutagens in the HIMA corresponded to those found in human tumours from patients exposed to these mutagens. The approach presented helps to deepen our understanding of human cancer aetiology.

DNA in dividing cells is prone to mutagenesis, with mutations making key contributions to human disease including cancer. The tumour suppressor gene TP53 is the most frequently mutated gene in human tumours. Here, we present a robust protocol for studying TP53 mutagenesis utilising human TP53 knock-in (Hupki) mouse embryonic fibroblasts (HUFs). In the HUF immortalisation assay (HIMA), primary HUFs are treated with known or suspected carcinogens at 3% oxygen and then transferred to 20% atmospheric oxygen to induce senescence. Cells containing mutations (e.g., in TP53) that allow bypassing of senescence eventually emerge as immortalised clonal cell lines after 2-3 months of serial passaging. As not all immortalised HUF cells contain TP53 mutations, we developed a Nutlin-3a counter-screen to select for TP53-mutated clones prior to sequencing. TP53 mutation spectra generated can be compared with those of human tumours recorded in the International Agency for Research on Cancer TP53 mutation database. Environmental mutagens that have demonstrated and validated the utility of the HIMA include ultraviolet radiation, aristolochic acid, and benzo[a]pyrene. The TP53 mutation patterns induced by these mutagens in the HIMA corresponded to those found in human tumours from patients exposed to these mutagens. The approach presented helps to deepen our understanding of human cancer aetiology.

Bacteria and viruses possess circular DNA, whereas eukaryotes with typically very large DNA molecules have had to evolve into linear chromosomes to circumvent the problem of supercoiling circular DNA of that size. Consequently, such organisms possess telomeres to cap chromosome ends. Telomeres are essentially tandem repeats of any DNA sequence that are present at the ends of chromosomes. Their biology has been an enigmatic one, involving various molecules interacting dynamically in an evolutionarily well-trimmed fashion. Telomeres range from canonical hexameric repeats in most eukaryotes to unimaginably random retrotransposons, which attach to chromosome ends and reverse-transcribe to DNA in some plants and insects. Telomeres invariably associate with specialised protein complexes that envelop it, also regulating access of the ends to legitimate enzymes involved in telomere metabolism. They also transcribe into repetitive RNA which also seems to be playing significant roles in telomere maintenance. Telomeres thus form the intersection of DNA, protein, and RNA molecules acting in concert to maintain chromosome integrity. Telomere biology is emerging to appear ever more complex than previously envisaged, with the continual discovery of more molecules and interplays at the telomeres. This review also includes a section dedicated to the history of telomere biology, and intends to target the scientific audience new to the field by rendering an understanding of the phenomenon of chromosome end protection at large, with more emphasis on the biology of human telomeres. The review provides an update on the field and mentions the questions that need to be addressed.

TP53 mutations occur in half of all human tumours. Mutagen‐induced or spontaneous TP53 mutagenesis can be studied in vitro using the human TP53 knock‐in (Hupki) mouse embryo fibroblast (HUF) immortalisation assay (HIMA). TP53 mutations arise in up to 30% of mutagen‐treated, immortalised HUFs; however, mutants are not identified until TP53 sequence analysis following immortalisation (2–5 months) and much effort is expended maintaining TP53‐WT cultures. In order to improve the selectivity of the HIMA for HUFs harbouring TP53 mutations, we explored the use of Nutlin‐3a, an MDM2 inhibitor that leads to stabilisation and activation of wild‐type (WT) p53. First, we treated previously established immortal HUF lines carrying WT or mutated TP53 with Nutlin‐3a to examine the effect on cell growth and p53 activation. Nutlin‐3a induced the p53 pathway in TP53‐WT HUFs and inhibited cell growth, whereas most TP53‐mutated HUFs were resistant to Nutlin‐3a. We then assessed whether Nutlin‐3a treatment could discriminate between TP53‐WT and TP53‐mutated cells during the HIMA (n = 72 cultures). As immortal clones emerged from senescent cultures, each was treated with 10 µM Nutlin‐3a for 5 days and observed for sensitivity or resistance. TP53 was subsequently sequenced from all immortalised clones. We found that all Nutlin‐3a‐resistant clones harboured TP53 mutations, which were diverse in position and functional impact, while all but one of the Nutlin‐3a‐sensitive clones were TP53‐WT. These data suggest that including a Nutlin‐3a counter‐screen significantly improves the specificity and efficiency of the HIMA, whereby TP53‐mutated clones are selected prior to sequencing and TP53‐WT clones can be discarded., What's new? Because half of human tumors carry mutations in the TP53 tumor suppressor gene, a mammalian cell culture system reproducing the diverse mutations seen in patients is necessary to properly study functional impact. Here the authors report a significant improvement to an existing model system using fibroblasts from human TP53 knock‐in mice (HIMA). They show that treating cells with Nutlin‐3a, an inhibitor of the destabilizing cofactor MDM2, efficiently selects for the growth of TP53‐mutated immortalized cells, while selecting against TP53‐wild type cells, thus increasing specificity and efficiency of TP53 mutagenesis.

The aim of this project was to systematically identify new interaction partners of the dystrophin protein within differentiated human skeletal muscle cells in order to uncover new roles in which dystrophin is involved, and to better understand how the global interactome is affected by the absence of dystrophin. A more complete understanding of the interactors and roles of dystrophin is vital for increasing the understanding of pathology in patients of Duchenne and Becker muscular dystrophy and other diseases in which dystrophin or dystrophin associated proteins are affected, and will assist in the development and research of treatments aimed at re-establishing the important interactions and pathways that have been lost or disrupted. Initially, a review of existing literature and bioinformatics resources was carried out and used as the basis of an online interactome of dystrophin’s physical interactors. A SILAC-based proteomics approach was then used to identify new interaction partners that were added to this bioinformatics resource. To obtain high protein quantities to maximize sensitivity we required a large number of cells, only feasible though the use of immortalized cell lines. hTERT/cdk4 immortalized myogenic human cell lines represent an important tool for skeletal muscle research however, disruption of the cell cycle has the potential to affect many other cellular processes to which it also linked. We carried out a transcriptome-wide analysis of healthy and diseased lines comparing immortalized lines with their parent primary populations in both differentiated and undifferentiated states in order to test their myogenic character by comparison with non-myogenic cells. We found that immortalization has no measurable effect on the myogenic cascade or on any other cellular processes, and that it was protective against the senescence process - a process observed at higher division counts of primary cells. Selected lines were not aneuploid and did not present any chromosomal rearrangements. In this context the human muscle cell lines are a good in vitro model to study the dystrophin interactome. Having validated our cell lines, we investigated dystrophin’s interactors using a high-sensitivity proteomics approach incorporating (1) isotopic amino acid labelling (SILAC), (2) knockdown of dystrophin expression, (3) immunoprecipitation and (4) mass spectrometry, allowing for the confident identification of dystrophin interactors, eliminating the multitude of false positive proteins pulled down through unspecific binding to both the beads and the antibody. We identified 18 new physical interactors of dystrophin which displayed a high proportion of vesicle transport related proteins and adhesion proteins, strengthening the link between dystrophin and these roles. The proteins determined through previously published data together with the newly identified interactors were incorporated into a web-based data exploration tool: sys-myo.rhcloud.com /dystrophin-interactome, intended to provide an easily accessible and informative view of dystrophins interactions in skeletal muscle. This tool will benefit future understanding and interpretation of the interaction data, by placing these newly identified physically interacting proteins within the mechanistic context of the wider dystrophin interactome., Ziel dieses Projektes war die systematische Aufdeckung neuer Interaktionspartner des Dystrophin-Proteins in differenzierten Skelettmuskelzellen, um neue Funktionen von Dystrophin zu identifizieren und ein eingehenderes Verständnis darüber zu erlangen, welche Auswirkungen das Fehlen von Dystrophin auf das Interaktom insgesamt hat. Eine umfassendere Kenntnis der Wechselspieler und Funktionen von Dystrophin ist entscheidend für ein besseres Verständnis des Krankheitsgeschehens bei Patienten mit Muskeldystrophie vom Typ Duchenne oder Becker und anderer Krankheiten, bei denen Dystrophin oder Dystrophin-assoziierte Proteine beeinträchtigt sind, und wird zur Entwicklung und Erforschung von Therapien beitragen, die auf eine Wiederherstellung fehlender bzw. gestörter fundamentaler Interaktionen und Signalwege abzielen. Zunächst wurde eine Recherche in der vorhandenen Fachliteratur und in Bioinformatik-Ressourcen durchgeführt und als Grundlage für ein Online-Interaktom der physischen Wechselspieler von Dystrophin verwendet. Anschließend wurden mithilfe der in der Proteomik gängigen SILAC- Technik neue Interaktionspartner identifiziert und dieser Bioinformatik- Ressource hinzugefügt. Um im Sinne einer Maximierung der Empfindlichkeit hohe Proteinmengen zu erhalten, benötigten wir eine große Anzahl von Zellen, was nur durch Verwendung immortalisierter Zelllinien möglich war. Immortalisierte myogene Humanzelllinien vom Typ hTERT/cdk4 sind ein wichtiges Hilfsmittel in der Skelettmuskelforschung, allerdings könnten durch die Unterbrechung des Zellzyklus zahlreiche andere zelluläre Prozesse beeinflusst sein, mit denen der Zellzyklus verknüpft ist. Wir führten eine Transkriptom-weite Analyse gesunder Zelllinien und krankheitsspezifischer Zelllinien durch, indem wir immortalisierte Zelllinien mit ihren Primärzell-Elternpopulationen sowohl im differenzierten als auch im undifferenzierten Zustand verglichen, um ihren myogenen Charakter durch Vergleich mit nicht-myogenen Zellen zu prüfen. Bestimmte Zelllinien wurden außerdem im Hinblick auf Aneuploidie und chromosomale Rearrangements überprüft. Wir stellten fest, dass Immortalisierung keine messbaren Auswirkungen auf die myogene Signalkaskase oder auf andere zelluläre Prozesse hatte und dass sie vor den systemischen Auswirkungen von Seneszenz, die nach einer großen Zahl von Teilungen von Primärzellen zu beobachten sind, schützte. Nachdem wir unsere Zelllinien validiert hatten, untersuchten wir Dystrophin-Wechselspieler im Rahmen eines hochempfindlichen Proteomik-Ansatzes mit Knockdown, Isotopenmarkierung von Aminosäuren (SILAC), Knockdown der Dystrophin-Expression, Immunpräzipitation und Massenspektrometrie. Dies ermöglichte eine zuverlässige Identifizierung von Dystrophin-Wechselspielern unter Eliminierung der Präzipitation der zahlreichen falschpositiven Proteine infolge einer unspezifischen Bindung an die Beads und den Antikörper. Wir identifizierten 18 neue physische Wechselspieler von Dystrophin, darunter anteilsmäßig viele Proteine, die am Vesikeltransport beteiligt sind, sowie Adhäsionsproteine, was die Verknüpfung zwischen Dystrophin und diesen Funktionen untermauert. Die aus publizierten Daten ermittelten Proteine sowie die neu identifizierten Wechselspieler wurden in ein internetbasiertes Datenexplorationstool (sys-myo.rhcloud.com /dystrophin-interactome) eingegeben, um eine übersichtliche und informative Darstellung der Dystrophin-Interaktionen im Skelettmuskel zu erhalten. Dieses Tool wird künftig dem Verständnis und der Interpretation der Interaktionsdaten dienlich sein, indem es diese neu identifizierten physisch interagierenden Proteine in den Kontext des größeren Dystrophin-Interaktoms einbindet.

13 páginas, 5 figuras, 1 tabla., Cancer is controlled not only by genetic events but also by epigenetic events. The active acquisition of epigenetic changes is a poorly understood but very important process in mammalian development, differentiation, and disease. It is well established that epigenetic events are controlled by a specific subgroup of proteins, such as DNA methyltransferases, histone acetylases histone lysine methyltransferases or histone deacetylases, that influence methylation or acetylation patterns to modulate gene expression. We and others have identified S-adenosylhomocysteine hydrolase in a high-throughput genetic screen focused on discovering novel genes whose inhibition induces immortalisation of primary cells. Herein, we address the importance of genes involved in epigenetic mechanisms during senescence and how their effects might determine senescence bypass and immortalisation. The ways in which genes that regulate epigenetic mechanisms might modulate senescence/immortalisation and how these pathways could influence cancer development are explored. Overall, epigenetic modifications seem to play a major role in cancer, influencing tumour outcome by interfering with key senescence pathways., This study was supported by grants from the Ministerio de Ciencia e Innovacion (SAF2009-08605) and Consejeria de Salud of the Junta de Andalucia (PI-0142) to A.C. and a Marato TV3 project 052130 and AGAUR (Agència de Gestió dÁjuts Universitaris i de Recerca) SGR604 to M.E.LL. M.E.LL is a FIS investigator (CP03/00101).

Cellular senescence, the irreversible proliferative arrest seen in somatic cells after a limited number of divisions, is considered a crucial barrier to cancer, but direct evidence for this in vivo was lacking until recently. The best-known form of human cell senescence is attributed to telomere shortening and a DNA-damage response through p53 and p21. There is also a more rapid form of senescence, dependent on the p16-retinoblastoma pathway. p16 (CDKN2A) is a known melanoma susceptibility gene. Here, we use retrovirally mediated gene transfer to confirm that the normal form of senescence in cultured human melanocytes involves p16, since disruption of the p16/retinoblastoma pathway is required as well as telomerase activation for immortalisation. Expression (immunostaining) patterns of senescence mediators and markers in melanocytic lesions provide strong evidence that cell senescence occurs in benign melanocytic naevi (moles) in vivo and does not involve p53 or p21 upregulation, although p16 is widely expressed. In comparison, dysplastic naevi and early (radial growth-phase, RGP) melanomas show less p16 and some p53 and p21 immunostaining. All RGP melanomas expressed p21, suggesting areas of p53-mediated senescence, while most areas of advanced (vertical growth-phase) melanomas lacked both p16 and p21, implying escape from both forms of senescence (immortalisation). Moreover, nuclear p16 but not p21 expression can be induced in human melanocytes by oncogenic BRAF, as found in around 80% of naevi. We conclude that cell senescence can form a barrier to melanoma development. This also provides a potential explanation of why p16 is a melanoma suppressor gene.

Background: The rise of genomic studies in Africa – not least due to projects funded under H3Africa – is associated with the development of a small number of biorepositories across Africa. For the ultimate success of these biorepositories, the creation of cell lines including those from selected H3Africa samples would be beneficial. In this paper, we map ethical challenges in the creation of cell lines. Discussion: The first challenge we identified relates to the moral status of cells living in culture. There is no doubt that cells in culture are alive, and the question is how this characteristic is relevant to ethical decision-making. The second challenge relates to the fact that cells in culture are a source of cell products and mitochondrial DNA. In combination with other technologies, cells in culture could also be used to grow human tissue. Whilst on the one hand, this feature increases the potential utility of the sample and promotes science, on the other it also enables further scientific work that may not have been specifically consented to or approved. The third challenge relates to ownership over samples, particularly in cases where cell lines are created by a biobank, and in a different country than where samples were collected. Relevant questions here concern the export of samples, approval of secondary use and the acceptability of commercialisation. A fourth challenge relates to perceptions of blood and bodily integrity, which may be particularly relevant for African research participants from certain cultures or backgrounds. Finally, we discuss challenges around informed consent and ethical review. Summary: In this paper, we sought to map the myriad of ethical challenges that need to be considered prior to making cell line creation a reality in the H3Africa project. Considering the relative novelty of this practice in Africa, such challenges will need to be considered, discussed and potentially be resolved before cell line creation in Africa becomes financially feasible and sustainable. We suggest that discussions need to be undertaken between stakeholders internationally, considering the international character of the H3Africa project. We also map out avenues for empirical research.

Endometriosis is a common gynaecological disorder associated with dysmenorrhoea, pelvic pain and subfertility and is a leading cause of disability and loss of productivity in women of reproductive age (Olive and Schwartz, 1993). Numerous studies have attempted to dissect the biology of endometriosis. These studies mainly use in vitro culture of stromal cells rather than culture of epithelial cells from endometriotic tissues, because the former cells are more easily and stably cultured for much longer periods than the latter cells (Noble et al, 1997; Gurates et al, 2002). In fact, it is difficult to culture endometriotic epithelial cells in vitro, because these cells lose their proliferative capacity during ongoing cultivation of primary cultures over several days. The inability of endometriotic epithelial cells to survive in vitro is an obstacle to gaining a better understanding of the biology of this disease. In particular, malignant change of endometriosis, especially of ovarian endometrioma, for which epithelial cells are exclusively responsible, has lately attracted considerable clinical attention (Kurman and Craig, 1972; McMeekin et al, 1995; Ness, 2003; Oral et al, 2003). There is therefore an urgent need to establish a stable system for the culture of endometriotic epithelial cells that can be used for research not only into the biology of endometriosis but also into its carcinogenesis. There are two major barriers within epithelial cells that inhibit cell division under usual culture conditions: premature senescence and telomere-dependent senescence (Kiyono et al, 1998). The former is observed during early passage in primary culture and is caused by the activation of Rb that leads to cell cycle arrest, whereas the latter is found at a later stage of culture and is caused by telomere shortening after a considerable number of cell divisions. Inhibition of both Rb function and telomere shortening is therefore required for long-term culture of epithelial cells. In previous studies, we successfully established a stable system for the culture of primary endometrial epithelial cells, in which the human papillomavirus type 16 E6/E7 genes and the human telomerase reverse transcriptase (hTERT) were introduced to inhibit Rb functions and to activate telomerase, respectively (Kyo et al, 2003). These immortal cells were not transformed but retained the original characteristics of endometrial epithelial cells, such as steroid responsiveness. Subsequent studies have demonstrated that overexpression of cyclin D1 or cdk4, instead of HPV E6/E7, effectively inhibited Rb activity and might be an alternative method of overcoming premature senescence in primary epithelial cells of other origins (Ramirez et al, 2004; Sasaki et al, 2009). In the present study, we sought to generate a stable culture of epithelial cells isolated from the ovarian endometriomas by the introduction of various genetic elements. These cells were successfully immortalised without generation of transformed phenotypes and were responsive to progestin and oestrogen. These cells are thus potentially useful as an experimental model for analysis of the mechanisms of steroid hormone functions as well as of carcinogenesis arising from ovarian endometrioma.

Epithelial cell lines were established from the transition and peripheral zones of human prostate by transduction with cdk4 and hTERT. The properties of these lines were investigated using immunocytochemical markers, ability to generate anchorage-independent colonies and by spectral karyotyping (SKY). Cells were exposed to fractionated doses of gamma irradiation to investigate their ability to transform. Cell lines were established from the transition and peripheral zones of human prostate. The expression of CD133, CK5, CK14, CK18, p16, PSCA, p63 and c-myc varied between the lines from the two regions. The line derived from the peripheral zone exhibited properties of a tumour line. A similar pattern was observed in two separate transductions. It was thus unlikely to be an in vitro transformation event, which is very rarely observed with human cells in vitro, and thus more likely to be derived from the immortalisation of a quiescent tumour clone. Fractionated irradiation of the transition zone cell line resulted in forming of transformed colonies. The transformed and tumour line had marked chromosomal rearrangements as demonstrated by SKY analysis. Cell lines have been derived from different zones of human prostate for studies on radiation carcinogenesis. The unirradiated cell line derived from the peripheral zone exhibited chromosomal rearrangements similar to those observed in prostate carcinoma. The cell line derived from the transitional zone exhibited a near diploid karyotype and could be transformed following exposure to fractionated doses of gamma irradiation.

We established an immortal human foreskin fibroblast line to use as feeder cells for human embryonic stem cells (hESC), as well as induced pluripotent stem (iPS) cells that were generated by genetic engineering from the same parental fibroblasts. The fibroblasts were conditionally immortalised by lentiviral vector (LV) mediated transduction of Bmi-1 and telomerase (hTERT) genes and further modified by LV-mediated transduction of a secreted form of the basic fibroblast growth factor (bFGF) gene product. Four hESC lines derived in three laboratories were used to test the established feeder cells. Immortalised fibroblast cells secreting stable amounts of bFGF supported growth of all hESC lines, which remained pluripotent and had a normal karyotype for at least ten passages. The feeders were tested up to passage 50 and they were still supportive. We then generated human iPS cells from parental fibroblasts by LV-mediated transduction with Oct 4, Sox2, Nanog, and Lin 28 genes, using their own immortalised bFGF-transduced descendants as feeders. At passage seven these iPS cells expressed pluripotency markers, and had hESC morphology. In vitro differentiation within embryoid bodies resulted in tissue components of the three embryonic germ layers. These immortalised bFGF-producing foreskin fibroblasts can thus be used as a genetically-defined feeder cell line for reproducible and cost-effective culture of both hESC and iPS cells.

Previously, we reported successful immortalisation following hTERT introduction in primary human fibroblasts, strain VH25. Since one subclone in that study developed some abnormalities, we decided to study eight additional independent immortalised clones to get an indication of the frequency and type of abnormalities that develop after hTERT-mediated immortalisation. We show that although some cell lines can maintain a normal phenotype for 500 population doublings (PDs), in four clones after 150-300PDs changes developed in basal and radiation-induced p53 and p21(WAF-1,CIP-1) levels. Our experiments demonstrate that, after prolonged culture, cells with abnormalities in cell cycle control parameters can take over the population. This calls for caution when working with hTERT-immortalised cells in vitro as well as in vivo.

Epithelial ovarian carcinoma is thought to derive from ovarian surface epithelium (OSE). The black box of the early molecular changes in ovarian carcinogenesis is being interpreted by the development of experimental systems employing immortalised human OSE cells. However, the existing cell lines of the OSE cells have limited utility due to chromosomal instability. Our goal was to establish new immortalised human OSE cells that retain the original characteristics of the primary cells without chromosomal alterations. Using primary human OSE cells obtained from a postmenopausal patient with endometrial cancer, five cell lines ('HOSE1' lines) were newly established by infection with retroviral expression vectors containing type 16 human papillomavirus (HPV-16) E6, E7, a variant E6 (E6delta151), and Bmi1 polycomb gene, in combination with telomerase reverse transcriptase (hTERT). Consequently, five HOSE1s cell lines, HOSE1s-E6/hTERT, -E7/hTERT, -E6/E7/hTERT, -E6delta151/E7/hTERT, and -E6delta151/Bmi1/hTERT, grew beyond the population doubling number of 200. These cell lines, except for HOSE1-E6/hTERT, essentially showed the original features of the primary human OSE cells. Of them, HOSE1-E7/hTERT preserved diploidy in a kariotype analysis, and did not show transformed phenotypes in anchorage-independent growth and tumour formation. Thus, HOSE1-E7/hTERT may provide a novel model system with which to investigate the mechanisms of early molecular changes.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Background: Epithelial cells of endometriotic tissues are difficult to propagate in vitro as experimental material is scarce owing to their limited life span. However, there is an increasing concern regarding their malignant transformation in ovaries. The present study sought to generate their stable culture system.Methods and Results :Purified epithelial cells isolated from ovarian endometriomas using microscopic manipulation were successfully immortalised by combinatorial transfection of human cyclinD1, cdk4 and human telomerase reverse transcriptase (hTERT) genes, whereas the introduction of hTERT alone, or together with cdk4, was insufficient for immortalisation, leading to cellular senescence. We confirmed stable cytokeratin expression in the immortalised cells, proving their epithelial origin. These cells expressed progesterone receptor B and showed significant growth inhibition by various progestins. Oestrogen receptor (ER) expression was detected in these cells, albeit at low levels. Additional overexpression of ERα generated stable cells with oestrogen-dependent growth activation. Soft-agar colony formation assay and nude mice xenograft experiments demonstrated that these cells, even those with additional inactivation of p53, did not have transformed phenotypes. Conclusion: We for the first time generated immortalised epithelial cells from ovarian endometrioma that retained sex steroid responsiveness. These cells are invaluable tools not only for the consistent in vitro work but also for the study of molecular pathogenesis or carcinogenesis of endometriosis. © 2012 Cancer Research UK All rights reserved., [総説 / Reviews][博士課程優秀論文] 第11回 高安賞優秀論文賞受賞論文, British Journal of Cancer 106(6), pp.1205-1213(2012年2月掲載), Bono, Y., Kyo, S., Takakura, M., Maida, Y., Mizumoto, Y., Nakamura, M., Nomura, K., Kiyono, T., Inoue, M. "Creation of immortalised epithelial cells from ovarian endometrioma.", http://dx.doi.org/10.1038/bjc.2012.26

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Telomeres are specialised structures, consisting of TTAGGG DNA repeats and binding proteins, that cap the ends of human chromosomes and maintain chromosome integrity. It has been shown that telomeres shorten with each round of cell division in most normal human somatic cells. It has become generally accepted that this shortening is due, in part, to the inability of DNA polymerases to replicate the extreme ends of chromosomes which is a phenomenon known as the 'end replication problem'. An intriguing hypothesis that has emerged from these observations is that critically shortened telomeres trigger growth arrest and senescence. This is regarded as a key determining factor in the limited lifespan of normal cells in culture and is commonly known as the 'Telomere Hypothesis of Senescence'. In support of this hypothesis it has been demonstrated that immortalised human cells, that have an unlimited lifespan in culture, maintain stable telomere lengths and do not undergo progressive telomere shortening. In most cases this is due to the ribonucleoprotein enzyme telomerase, the activation of which is as a key step in the immortalisation process. Telomerase compensates for sequential telomere shortening by utilising an RNA template to catalyse the addition of repeat sequences by reverse transcription. It is absent from most normal tissue but is present in the germline and is presumably downregulated during development. Significantly, analysis of human tumour cells has shown that a majority also have active telomerase, which supports the importance of immortalisation in tumourigenesis. Previous work in this laboratory has shown that, although the majority of in vitro immortalised cells and tumour cells that have been studied maintain telomeres by reactivation of telomerase, a proportion do not have detectable telomerase activity. These telomerase-negative cells still maintain telomeres, however, and this is via a mechanism(s) yet to be fully elucidated known as Alternative Lengthening of Telomeres (ALT). ALT is characterised, in addition to lack of telomerase activity, by extreme telomere length heterogeneity with telomere lengths ranging from over 50 kilobases (kb) of DNA to almost undetectable. This phenotype is evident, by Southern analysis and fluorescent in situ hybridisation (FISH), in all ALT cells. Alternative mechanisms of telomere maintenance, via retrotransposition and recombination, had already been characterised in lower eukaryotes. It has been shown in this laboratory that ALT cell lines and tumours contain a novel type of PML body, referred to as ALT-associated PML bodies (APBs). APBs have been found in all of the ALT cell lines so far tested and also in archival tumour sections, and contain a number of factors which co-localise. These include PML, TTAGGG repeats, TRF 1 & TRF 2 telomere binding proteins and proteins involved in homologous recombination: RAD51 & RAD52. More recently, it has been shown that the RAD50/Mre11/Nbs1 complex, which is involved in cell cycle checkpoint control and repair of DNA damage, is also present in APBs. The presence of these RAD proteins in APBs is of great interest as a recombination between telomeres has been proposed as the central mechanism by which ALT lengthens telomeres. Studies in yeast have identified such a mechanism and it was proposed that a similar process occurred in human immortal cells that utilise ALT. It has now been shown by this laboratory that a recombination mechanism is indeed evident at the telomeres of ALT cells. To date all in vitro immortalised cell lines and most tumour cell types that have been studied have a telomere maintenance mechanism either via telomerase or ALT. Targeting telomerase has become a major focus of anti-cancer research as inhibitors have the potential to treat a wide variety of different tumour types. An understanding of ALT and its regulation is likely to be important in such therapeutic strategies, as selective pressure due to telomerase inhibition may result in ALT revertants within the tumour mass. Development of inhibitors of both telomerase and ALT may therefore be required when targeting telomere maintenance. The main focus of this thesis is the understanding of ALT repression in the SV40 immortalised skin fibroblast cell line GM847, as a means to further understanding the mechanism of ALT. The data presented provide new insights into the repression of ALT and also the relationship between telomerase and ALT which is important for our understanding of telomere maintenance in human cancer. Hybrids formed by fusion of normal cells and ALT cells underwent rapid telomere loss followed by senescence, indicating that normal cells contain factors that repress ALT. This strongly suggests that ALT is recessive and is activated in part by loss or mutation of repressors. Similar experiments were performed with ALT cells and telomerasepositive cells, and the resulting hybrids were all telomerase-positive and ALT repressed. It is possible that the same negative regulators are involved as additional data show that telomerase does not act as an ALT inhibitor. Exogenous expression of telomerase in ALT cells did not repress ALT, but both mechanisms co-existed in these transfected cells. This result provides a further argument for targeting both ALT and telomerase in any future treatments of tumours as it demonstrates in principle that these mechanisms are not mutually exclusive. A serendipitous finding was that a dominant-negative telomerase catalytic subunit caused telomere shortening in ALT cells, had not been reported elsewhere, and indeed was in contrast to previous findings. This provided further evidence for a link between telomerase and ALT as it suggested that there were essential components that were common to both pathways. As a further means to understanding ALT repression, a series of experiments was performed to determine the chromosomal localisation of ALT repressor(s) by microcell mediated chromosome transfer. This was done to facilitate the eventual isolation of repressors. A repressor of ALT in the chemically immortalised fibroblast cell line SUSM-1, had been reported to be localised to chromosome 7. This result could not be repeated in the GM847 cell line, but ALT repression was evident in GM847 cells upon transfer of chromosome 6. Another important question regarding the nature of ALT is the structure and sequence of the long heterogeneous telomeres generated by ALT specific recombination, which is the focus of the final series of data that is presented. ALT telomere length heterogeneity was detected under denaturing conditions, ruling out the possibility that it was an artefact of electrophoresis conditions due to novel secondary structure. Although the hybridisation signal intensity of TTAGGG increases at the onset of immortalisation in ALT cells, it had been demonstrated by restriction digests that degenerate repeats did exist at the telomeres of some ALT cell lines. Sequences containing telomere repeats were cloned from the ALT cell line WI38 VA13/2RA (SV40 immortalised fibroblasts) and these were found to be interspersed with a number of other sequence fragments. The significance of these sequences in relation to the mechanism of ALT is discussed.

Immortal lymphoblastoid cell lines of B and T lineages have been derived from blood cell cultures of Duroc, Miniature (histocompatible) or unknown porcine breeds. The lymphoblast immortalization has been putatively attributed to an oncogenic virus belonging either to the Herpesviridae or to the Retroviridae groups. But a definitive proof of a viral etiology is still lacking, impeding some therapeutic use of tissue culture products secreted by these cell lines. Nevertheless, immortal cell lines have many advantages over finite ones, owing to their ease of maintenance in simple media and to the acquisition of an infinite lifespan. The different cell lines are mainly used to develop the hybridoma technology in this species for cytogenetics and xenogenic research.