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Bats are becoming recognised as new model species to study naturally evolved mammalian extended healthspan and disease tolerance. However, this research is limited by the lack of bat specific cellular resources. Here we describe an optimised protocol to develop both primary and immortalised fibroblast cell-lines from wing biopsy punches from the Egyptian fruit bat, Rousettus aegyptiacus. We show that the immortalised cell lines and primary cells show similar characteristics in their proliferative capacity and response to oxidative stress. They also exhibited a similar response in their NF-κB immune response to TLR agonists including SARS-CoV2. As wing punches can be acquired non-lethally, these methods can be used to develop primary and immortalised cells, from potentially any bat species, including those of conservation concern that cannot be sacrificed. This can expand the scope of bat species that can be studied in the future, and the development of key cellular resources required to functionally validate the regulators of bats' unique longevity. [ABSTRACT FROM AUTHOR]

Abstract Bats are becoming recognised as new model species to study naturally evolved mammalian extended healthspan and disease tolerance. However, this research is limited by the lack of bat specific cellular resources. Here we describe an optimised protocol to develop both primary and immortalised fibroblast cell-lines from wing biopsy punches from the Egyptian fruit bat, Rousettus aegyptiacus. We show that the immortalised cell lines and primary cells show similar characteristics in their proliferative capacity and response to oxidative stress. They also exhibited a similar response in their NF-κB immune response to TLR agonists including SARS-CoV2. As wing punches can be acquired non-lethally, these methods can be used to develop primary and immortalised cells, from potentially any bat species, including those of conservation concern that cannot be sacrificed. This can expand the scope of bat species that can be studied in the future, and the development of key cellular resources required to functionally validate the regulators of bats’ unique longevity.

Aims: Adamantinomatous craniopharyngioma (ACP) is a rare benign intracranial tumour that occurs in the sellar region and likely originates from the embryonic craniopharyngeal epithelium (a remnant of the ectoderm, also known as Rathke's pouch). However, progress in ACP research has been slow due to the lack of ACP cell lines. Therefore, in this study, we established an immortalised ACP cell line. Methods: Immortalised ACP cells were established from cultured primary ACP cells by the ectopic expression of human telomerase reverse transcriptase (hTERT). Primary and immortalised cells were compared and characterised by morphological examination, short tandem repeat (STR) profiling, whole exome sequencing (WES) and transcriptome sequencing. The ability of immortalised cells to form tumours was examined using an intracranial tumour formation assay in mice. Immunofluorescence staining was performed to compare the characteristics of human ACP and intracranial xenografts derived from immortalised cells. Results: A novel immortalised ACP cell line, STAM4, was successfully established in a 4‐year‐old male ACP patient. When STAM4 cells were compared with primary ACP cells in terms of morphological characteristics, genetic variants, STR profiles, biological behaviours and transcriptome differences, STAM4 cells were similar to primary ACP cells. Additionally, the STAM4 cells were able to form intracranial tumours that resemble human ACP. Conclusions: This ACP cell line may provide a cell model for further studies on the molecular mechanisms underlying the occurrence and progression of ACP and for the development of new anticancer drugs for ACP. [ABSTRACT FROM AUTHOR]

Mesenchymal (human amniotic mesenchymal stem cell [HAMSC]) and epithelial cells (human amnion epithelial cell [HAEC]) derived from human amniotic membranes possess characteristics of pluripotency. However, the pluripotency of HAMSC and HAEC are sustained only for a finite period. This in vitro cell growth can be extended by cell immortalisation. Many well‐defined immortalisation systems have been used for artificially overexpressing genes such as human telomerase reverse transcriptase in HAMSC and HAEC leading to controlled and prolonged cell proliferation. In recent years, much progress has been made in our understanding of the cellular machinery that regulates the cell cycle when immortalised. This review summarises the current understanding of molecular mechanisms that contribute to cell immortalisation, the strategies that have been employed to immortalise amnion‐derived cell types, and their likely applications in regenerative medicine. [ABSTRACT FROM AUTHOR]

Abstract Transferring large or multiple genes into primary human stem/progenitor cells is challenged by restrictions in vector capacity, and this hurdle limits the success of gene therapy. A paradigm is Duchenne muscular dystrophy (DMD), an incurable disorder caused by mutations in the largest human gene: dystrophin. The combination of large‐capacity vectors, such as human artificial chromosomes (HACs), with stem/progenitor cells may overcome this limitation. We previously reported amelioration of the dystrophic phenotype in mice transplanted with murine muscle progenitors containing a HAC with the entire dystrophin locus (DYS‐HAC). However, translation of this strategy to human muscle progenitors requires extension of their proliferative potential to withstand clonal cell expansion after HAC transfer. Here, we show that reversible cell immortalisation mediated by lentivirally delivered excisable hTERT and Bmi1 transgenes extended cell proliferation, enabling transfer of a novel DYS‐HAC into DMD satellite cell‐derived myoblasts and perivascular cell‐derived mesoangioblasts. Genetically corrected cells maintained a stable karyotype, did not undergo tumorigenic transformation and retained their migration ability. Cells remained myogenic in vitro (spontaneously or upon MyoD induction) and engrafted murine skeletal muscle upon transplantation. Finally, we combined the aforementioned functions into a next‐generation HAC capable of delivering reversible immortalisation, complete genetic correction, additional dystrophin expression, inducible differentiation and controllable cell death. This work establishes a novel platform for complex gene transfer into clinically relevant human muscle progenitors for DMD gene therapy.

Abstract: Transferring large or multiple genes into primary human stem/progenitor cells is challenged by restrictions in vector capacity, and this hurdle limits the success of gene therapy. A paradigm is Duchenne muscular dystrophy (DMD), an incurable disorder caused by mutations in the largest human gene: dystrophin. The combination of large‐capacity vectors, such as human artificial chromosomes (HACs), with stem/progenitor cells may overcome this limitation. We previously reported amelioration of the dystrophic phenotype in mice transplanted with murine muscle progenitors containing a HAC with the entire dystrophin locus (DYS‐HAC). However, translation of this strategy to human muscle progenitors requires extension of their proliferative potential to withstand clonal cell expansion after HAC transfer. Here, we show that reversible cell immortalisation mediated by lentivirally delivered excisable hTERT and Bmi1 transgenes extended cell proliferation, enabling transfer of a novel DYS‐HAC into DMD satellite cell‐derived myoblasts and perivascular cell‐derived mesoangioblasts. Genetically corrected cells maintained a stable karyotype, did not undergo tumorigenic transformation and retained their migration ability. Cells remained myogenic in vitro (spontaneously or upon MyoD induction) and engrafted murine skeletal muscle upon transplantation. Finally, we combined the aforementioned functions into a next‐generation HAC capable of delivering reversible immortalisation, complete genetic correction, additional dystrophin expression, inducible differentiation and controllable cell death. This work establishes a novel platform for complex gene transfer into clinically relevant human muscle progenitors for DMD gene therapy. [ABSTRACT FROM AUTHOR]

Résumé Le vieillissement attendu de la population générale pourrait générer à terme une situation de pénurie de produits sanguins érythrocytaires. La recherche d’une alternative au don volontaire est donc un enjeu majeur dans les décennies à venir. La possibilité de reproduire in vitro l’érythropoïèse humaine ouvre des perspectives nouvelles depuis que l’amélioration des conditions de culture a permis d’obtenir à partir de cellules souches des globules rouges fonctionnels. Ces érythrocytes peuvent être obtenus à partir de cellules souches pluripotentes induites (iPSCs), ou de cellules souches hématopoïétiques CD34 + (CSH). Les iPSCs sont capables de s’auto-renouveler in vitro, mais, actuellement, leur différenciation érythroïde dirigée entraîne un faible expansion et un défaut de maturation terminale. La différenciation érythroïde issue de CSH présente une meilleure amplification, produit des érythrocytes matures énucléés capables de synthétiser de l’hémoglobine adulte. Toutefois, leur principal inconvénient réside dans leur incapacité à s’auto-renouveler, et ne garantit donc pas l’indépendance au don. L’amélioration de ce système passe donc par un processus d’immortalisation in vitro, capable d’assurer la pérennité de la source cellulaire. La transgenèse inductible de progéniteurs érythroïdes est la première solution. Les modèles les plus aboutis ont utilisé l’induction d’oncogènes puissants, comme c-Myc, Bcl-xL ou surtout d’oncoprotéines virales d’HPV16 dont la dernière version a été testée in vivo chez la souris. Mais la qualité de la différenciation terminale n’est pas optimale et la stabilité à long terme non garantie du fait de l’instabilité génétique induite. L’utilisation de vecteurs viraux codant pour des oncogènes pose également des problèmes de sécurisation des produits obtenus, l’énucléation n’étant jamais totale dans ces systèmes. On voit donc le challenge immense que représente cette immortalisation réversible, et les nombreuses voies d’amélioration nécessaires avant d’envisager une utilisation sécurisée en thérapie transfusionnelle. Population ageing and increase in cancer incidence may lead to a decreased availability of red blood cell units. Thus, finding an alternative source of red blood cells is a highly relevant challenge. The possibility to reproduce in vitro the human erythropoiesis opens a new era, particularly since the improvement in the culture systems allows to produce erythrocytes from induced-Pluripotent Stem Cells (iPSCs), or CD34 + Hematopoietic Stem Cells (HSCs). iPSCs have the advantage of in vitro self-renewal, but lead to poor amplification and maturation defects (high persistence of nucleated erythroid precursors). Erythroid differentiation from HSC allows a far better amplification and adult-like hemoglobin synthesis. But the inability of these progenitors to self-renew in vitro remains a limit in their use as a source of stem cells. A major improvement would consist in immortalizing these erythroid progenitors so that they could expand indefinitively. Inducible transgenesis is the first way to achieve this goal. To date, the best immortalized-cell models involve strong oncogenes induction, such as c-Myc, Bcl-xL, and mostly E6/E7 HPV16 viral oncoproteins. However, the quality of terminal differentiation of erythroid progenitors generated by these oncogenes is not optimal yet and the long-term stability of such systems is unknown. Moreover, viral transgenesis and inducible expression of oncogenes raise important problems in term of safety, since the enucleation rate is not 100% and no nucleated cells having replicative capacities should be present in the final product. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

Résumé: Près de 18 % des cancers humains seraient associés à des infections virales impliquant notamment les virus des hépatites chroniques (VHC, VHB), les papillomavirus à haut risque de malignité, les herpesvirus (EBV, HHV8), HTLV-I et plus récemment le polyomavirus associé au carcinome de Merkel. Les liens de causalité reposent sur l’analyse de données épidémiologiques, l’utilisation de modèles animaux, l’investigation biologique des tumeurs et l’analyse du pouvoir immortalisant et transformant de ces virus. Plusieurs virus modèles ont contribué à définir les bases génétiques du cancer, qu’il soit d’origine virale ou non. Cette revue s’appuie sur des synthèses bibliographiques récentes pour illustrer les mécanismes supposés de l’oncogenèse viro-associée chez l’homme et en souligner la complexité. [Copyright &y& Elsevier]

Cellular immortality happens upon impairment of cell-cycle checkpoint pathways (p53/p16/pRb), reactivation or up-regulation of telomerase enzyme, or upregulation of some oncogenes or oncoproteins leading to a higher rate of cell division.There are also some other factors and mechanisms involved in immortalisation, which need to be discovered. Immortalisation of cells derived from different sources and establishment of immortal cell lines has proven useful in understanding the molecular pathways governing cell developmental cascades in eukaryotic, especially human, cells. After the breakthrough of achieving the immortal cells and understanding their critical importance in the field of molecular biology, intense efforts have been dedicated to establish cell lines useful for elucidating the functions of telomerase, developmental lineage of progenitors, self-renewal potency, cellular transformation, differentiation patterns and some bioprocesses, like odontogenesis. Meanwhile, discovering the exact mechanisms of immortality, a major challenge for science yet, is believed to open new gateways toward understanding and treatment of cancer in the long term. This review summarises the methods involved in establishing immortality, its advantages and the challenges still being faced in this field. [ABSTRACT FROM AUTHOR]

Abstract: Cancer stem cells (CSCs) are a sub-population of cancer cells that possess characteristics associated with normal stem cells but with the peculiarity that they are tumourigenic. This property allows them to persist in the tumour population, causing relapse and metastasis by giving rise to new tumours. Accordingly, if the CSCs were eliminated, then the tumour would simply regress due to differentiation and cell death. By selectively targeting CSCs, it may be possible to treat patients with aggressive, non-resectable tumours and prevent the tumour from metastasising. MicroRNAs are involved in all biological processes, and several studies have demonstrated their function in human tumourigenesis. Importantly, microRNAs have been implicated in the regulation of stem cells and CSCs. The most important concept to emerge with regard to CSC therapy is still controversial because a number of signalling pathways unique to normal stem cells may also be operating in CSCs, and these offer new targets for therapy. This article reviews how the modulation of microRNAs may revert tumour proliferation in vivo and in vitro and how this approach could be transferred to the clinic. Although the delivery of therapeutic microRNAs to target cells is a challenge that still needs to be overcome, microRNAs offer the advantage that they are small molecules that can be easily transported by body fluids, which makes them good candidates for cancer therapy. [Copyright &y& Elsevier]

Abstract: BMI-1 (B lymphoma Mo-MLV insertion region 1 homolog) has been reported to be over-expressed in cell immortalisation and the epithelial–mesenchymal transition (EMT) of cancer cells. The aim of this study is to study the roles of BMI-1 in the human telomerase reverse transcriptase (hTERT)-induced immortalisation and EMT. In this study, hTERT+-OME cells and hTERT+-HaCaT cells were acquired by viral transduction of hTERT to primary cultured oral keratinocytes and HaCaT cells (skin epidermal cells). siRNA transduction was used for the inhibition of BMI-1 expression. RT-PCR and Western blots were performed to detect the expressions of twist, vimentin, BMI-1, hTERT and p16INK4a in these cell lines. EMT was assessed by immunohistochemistry (expressions of cytokertin & vimentin), Western blots (expressions of Twist, vimentin & E-cadherin) and RT-PCR (expression of Twist). The results indicated that hTERT+-OME cells and hTERT+-HaCaT cells underwent EMT spontaneously with high expression of Twist. p16INK4a was silenced in both hTERT-transduced cells but could be detected in HaCaT cells. Moreover, BMI-1 was highly expressed in hTERT+-OME and hTERT+-HaCaT cells but was negative in HaCaT cells. When the expression of BMI-1 was blocked by siRNA transduction, the proliferations of hTERT+-OME and hTERT+-HaCaT cells were inhibited and the mono-spheroid colony formation of these hTERT-transduced cells was decreased. In addition, the expression of p16INK4a was regained while the expressions of EMT markers (twist and vimentin) were down-regulated in these two BMI-1 blocking cell lines. To conclude, this study suggests BMI-1 expression plays a role in hTERT-induced immortalisation and EMT. [Copyright &y& Elsevier]

Background information Evidence has shown that mesenchymal-epithelial transition (MET) and epithelial-mesenchymal transition (EMT) are linked to stem cell properties. We currently lack a model showing how the occurrence of MET and EMT in immortalised cells influences the maintenance of stem cell properties. Thus, we established a project aiming to investigate the roles of EMT and MET in the acquisition of stem cell properties in immortalised oral epithelial cells. Results In this study, a retroviral transfection vector (pLXSN-hTERT) was used to immortalise oral epithelial cells by insertion of the hTERT gene ( hTERT+-oral mucosal epithelial cell line [OME]). The protein and RNA expression of EMT transcriptional factors (Snail, Slug and Twist), their downstream markers (E-cadherin and N-cadherin) and embryonic stem cell markers (OCT4, Nanog and Sox2) were studied by reverse transcription PCR and Western blots in these cells. Some EMT markers were detected at both mRNA and protein levels. Adipocytes and bone cells were noted in the multi-differentiation assay, showing that the immortal cells underwent EMT. The differentiation assay for hTERT+-OME cells revealed the recovery of epithelial phenotypes, implicating the presence of MET. The stem cell properties were confirmed by the detection of appropriate markers. Altered expression of α-tubulin and γ-tubulin in both two-dimensional-cultured (without serum) and three-dimensional-cultured hTERT+-OME spheroids indicated the re-programming of cytoskeleton proteins which is attributed to MET processes in hTERT+-OME cells. Conclusions EMT and MET are essential for hTERT-immortalised cells to maintain their epithelial stem cell properties. [ABSTRACT FROM AUTHOR]

Abstract: New treatments are needed for malignant pleural mesothelioma (MPM), which currently has a poor prognosis. Cellular immortalisation, one of the hallmarks of cancer, depends on the activity of a telomere length maintenance mechanism (TMM) – either telomerase or alternative lengthening of telomeres (ALT). The TMMs are widely regarded as potential targets for cancer therapies and telomerase inhibitors have entered clinical trials. The aim of this study was to determine what proportion of MPMs use ALT and/or telomerase. Forty-three MPMs from 42 patients were examined for telomerase and ALT activity. Telomerase activity was detected by immunoaffinity purification followed by the telomere repeat amplification protocol (TRAP), and ALT activity was determined by the C-circle assay and by assessing telomere lengths using terminal restriction fragment analyses. We found that 43 of 43 MPMs were telomerase-positive[+] and ALT-negative[−]. Therefore, to investigate whether pleural mesothelial cells are unusually susceptible to activation of telomerase, we examined activation of the TMMs in an in vitro model of cellular immortalisation, in which normal pleural mesothelial cells were transduced with simian virus 40 (SV40) oncogenes. We found that normal mesothelial cells were TMM-negative, and that expression of the SV40 oncogenes did not directly activate telomerase or ALT. Immortalisation, which in this experimental system results from additional genetic changes that have not yet been identified, was accompanied by activation of either TMM. Therefore, pleural mesothelial cells are capable of activating either TMM in vitro, and the observation that 100% of MPMs were telomerase[+] suggests that there are factors in vivo that select for telomerase activity during oncogenesis of this tumour type. We conclude that MPM is a tumour that could be considered for anti-telomerase therapy. [Copyright &y& Elsevier]

Abstract: Background: The development of urothelial malignancy is not solely a consequence of loss of proliferation constraints but also involves loss of cellular differentiation, defined histopathologically as grade. Although tumour grade is an independent prognostic marker for urothelial carcinoma (UC), the molecular events underpinning the loss of urothelial differentiation are poorly understood. Objective: To examine the effect of gene alterations implicated in UC development on the ability of human urothelial cells to undergo molecular differentiation and form a functional urothelial barrier. Design, setting, and participants: Laboratory study. Intervention: Normal human urothelial (NHU) cell cultures were transduced with recombinant retroviruses to produce stable sublines overexpressing wild-type or oncogenic mutated fibroblast growth factor receptor 3 or human telomerase reverse transcriptase (hTERT). Previously generated NHU sublines carrying dominant-negative CDK4 and p53 mutant genes or immortalised with the human papillomavirus 16 E6 oncoprotein were included. Measurements: The activity of introduced transgenes was demonstrated by comparing phenotypes of transgene-expressing and isogenic control NHU cells. Modified and control sublines were compared for changes in generational potential (life span) and capacity to respond to differentiation-inducing signals by transcript expression of uroplakins 2 and 3. The ability to form a barrier epithelium was assessed by measuring the transepithelial electrical resistance. Results and limitations: By contrast to tumour suppressor loss of function or oncogene overactivation, hTERT overexpression alone led to life span extension and immortalisation. The hTERT immortalised cells carried no gross genomic alterations but became progressively insensitive to differentiation signals and lost the ability to form an epithelial barrier. Further characterisation of hTERT cells revealed a downregulation of p16 cyclin-dependent kinase inhibitor expression and loss of responsiveness to peroxisome proliferator-activated receptor γ, providing mechanistic explanations for the subjugation of senescence constraints and the abrogation of differentiation capability, respectively. Although immortalised urothelial cell lines without karyotypic aberrations may be generated, such cell lines are compromised in terms of differentiation and functional capacity. Conclusions: Overexpression of hTERT promotes development of an immortalised differentiation-insensitive urothelial cell phenotype. Although such cells offer a useful insight into the grade/stage paradigm of UC, they have limited value for investigating normal urothelial cell/tissue biology and physiology. [Copyright &y& Elsevier]

Abstract: The specialised DNA polymerase μ (pol μ) affects a sub-class of immunoglobulin genes rearrangements and haematopoietic development in vivo. These effects appear linked to double-strand breaks (DSBs) repair, but it is still unclear how and to what extent pol μ intervenes in this process. Using high-resolution quantitative imaging of DNA damage in irradiated wild-type and pol μ−/− mouse embryonic fibroblasts (MEFs) we show that lack of pol μ results in delayed DSB repair kinetics and in persistent DNA damage. DNA damage triggers cellular senescence, and this response is thought to suppress cancer. Independent investigations either report or not a proliferative decline for MEFs lacking pol μ. Here we show pronounced senescence in pol μ−/− MEFs, associated with high levels of the tumor-suppressor p16INK4A and the DNA damage response kinase CHK2. Importantly, cellular senescence is induced by culture stress and exacerbated by low doses of irradiation in pol μ−/− MEFs. We also found that low doses of irradiation provoke delayed immortalisation in MEFs lacking pol μ. Pol μ−/− MEFs thus exhibit a robust anti-proliferative defence in response to irreparable DNA damage. These findings indicate that sub-optimal DSB repair, due to the absence of an auxiliary DNA damage repair factor, can impact on cell fitness and thereby on cell fate. [ABSTRACT FROM AUTHOR]

Abstract: Maintenance of telomere length has been reported to be an absolute requirement for unlimited growth of human tumour cells and in about 85% of cases, this is achieved by reactivation of telomerase, the enzyme that elongates telomeres. Only in rare cases, like in human medullary thyroid carcinomas (MTC), telomerase activity (TA) is low or undetectable; however, this does not limit tumours to become clinically significant. Here, we report that very low TA (below 5% of HEK293) observed in MTC cell strains derived from different patients, although not sufficient for immortalising the cells, is necessary for prolonging their replicative life span. Telomere erosion led to induction of a crisis period after long-term in vitro cultivation, which was reached earlier when treating the cells with MST-312, a telomerase inhibitor at non-toxic concentrations. Crisis was bypassed either by ectopic hTERT introduction or by infrequent spontaneous immortalisation, the latter of which was always associated with telomerase reactivation and changes of the cellular phenotype. While confirming the high importance of telomerase for tumour development, these data draw attention to the relevance of low TA: although insufficient for telomere stabilisation, it allows MTC cells to reach more population doublings, increasing both cell numbers as well as the risk of accumulating mutations and thus might support the development of clinically significant MTC. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

The CDKN2A locus is frequently inactivated in urothelial cell carcinoma (UCC), yet how this alteration contributes to bladder tumorigenesis is not known. Although most UCC express telomerase, inactivation of the p16/Rb pathway is generally required for in vitro immortalisation. This and the involvement of p16 in senescence of normal human urothelial cells (NHUC) suggest that CDKN2A deletion may aid bypass of senescence and allow immortalisation. CDKN2A encodes p16 and p14ARF and therefore inactivation of this locus can disrupt both the Rb and p53 tumour suppressor pathways. Retrovirus-mediated transduction was used to specifically modulate the p16/Rb and/or p53 tumour suppressor pathways in NHUC and to express human telomerase reverse transcriptase (hTERT). Expression of hTERT bypassed Rb and p53 pathway-dependent barriers to proliferation and immortalised NHUC. TERT-NHUC had normal karyotypes, were non-tumorigenic and unexpectedly retained CDKN2A. Thus, the phenotypic significance of inactivation of CDKN2A in UCC may not be solely related to bypass of senescence. Phenotypic assays in human urothelial cells have relied on cell strains derived from invasive tumours or NHUC immortalised by expression of SV40-large T. The production of genetically normal but immortal NHUC lines now provides a valuable platform for experiments to examine the timing and combination of events necessary for UCC tumorigenesis.Oncogene (2006) 25, 5037–5045. doi:10.1038/sj.onc.1209513; published online 17 April 2006 [ABSTRACT FROM AUTHOR]

Abstract: Transplantation of genetically modified or unmodified hepatocytes appears to be a less invasive alternative to liver transplantation. However, clinical trials performed for the treatment of metabolic deficiencies resulted in a partial and transitory correction due to an insufficient number of engrafted and functional hepatocytes. In vitro, adult hepatocytes do not proliferate and the lack of organ donors limits their availability. Concomitantly, numerous works on hepatocyte transplantation in rodents have shown that cell engraftment was inefficient in normal livers. It is therefore necessary to explore the therapeutic potential of new cell sources such as stem cells and to develop pre-clinical models of transplantation. Foetal liver progenitor cells (hepatoblasts) are bipotent and express markers of both foetal hepatocytes and cholangiocytes. We have immortalized one clone of primate hepatoblasts using a retroviral vector expressing SV40 Large T and have characterized the cells at different population doublings (PDs). After 500 days in culture, immortalized cells remained bipotent and kept contact inhibition, in spite of numerous chromosomal rearrangements. After transplantation into athymic mice, the cells expressed hepatocyte functions but did not proliferate. We isolated, phenotypically characterized, transduced and cryopreserved early human hepatoblasts. These cells repopulate up to 7% of recipient immunodeficient mouse livers. This indicates that early progenitor cells display molecular characteristics related to proliferation and migration that allow these cells to engraft within hepatic parenchyma more efficiently than adult hepatocytes. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Summary: Odontoblasts and osteoblasts differ functionally and histologically. Because of their close relationship, mesenchymal cells derived from teeth and bone are difficult to distinguish ex vivo. Indeed, the main non-collagenous components of the odontoblastic extracellular matrix, dentin sialoprotein (DSP) or dentin matrix protein 1 (DMP1), have also been detected in osteoblasts. The need to develop cellular models of odontoblast differentiation and to identify markers specific for the odontoblast lineage, has led us to establish clonal cell lines from tooth germs of day 18 mouse embryos transgenic for an adenovirus-SV40 recombinant plasmid. In this study, we analyzed the phenotypes of three independent clones by RT-PCR and Western blot. These clones synthesised DSP, DMP1 and other extracellular matrix proteins typical of the odontoblast and are therefore likely to be derived from the pulp. Transcripts encoding a set of homeobox proteins involved in craniofacial development, such as Pax9, Msx1, Cbfa1, Dlx2 and 5 were also expressed albeit at a different level. These features of the pulpal clones are shared by the C1 mesodermal cells that are capable of differentiating along osteogenic, chondrogenic or adipogenic lineages In contrast, transcripts for two LIM-domain homeobox family genes (Lhx6 and Lhx7) were only detected in the dental clones. Since these genes are preferentially expressed in the mesenchyme of the developing tooth, this suggests that our transgenic-derived cell lines retain intrinsic properties of odontoblastic cells. They may help to characterise genes specifying the odontoblast phenotype and the signalling pathways underlying odontoblast differentiation. [Copyright &y& Elsevier]

Epithelial ovarian cancer is the most common form of gynaecological malignancy. This lethal disease is thought to arise in ovarian surface epithelial (OSE) cells. The biology of these cells is not well understood, due to the limited amount of tissue that can be obtained from a single biopsy and their limited life span in culture. To overcome these problems, we have conditionally immortalised OSE cells with the catalytic subunit of telomerase (hTERT) and a temperature-sensitive form of SV40 Large T antigen (tsT). We have maintained these cells (designated OSE-C2) in culture for more than 100 population doublings after introduction of the immortalising genes. Early passage OSE-C2 cells have a near-tetraploid karyotype and exhibit a dual mesenchymal–epithelial phenotype, with consistent expression of vimentin and variable expression of cytokeratins and type III collagen, and absence of E cadherin expression. OSE-C2 cells proliferate steadily at the permissive temperature of 33°C, but fail to increase in number at the nonpermissive temperature of 39°C. Serum-deprived OSE-C2 cells are stimulated to grow at 33°C by EGF, whereas they are growth inhibited at 33°C by TGFβ in the presence or the absence of serum. When temperature shifted to the nonpermissive temperature, OSE-C2 cells modulate to a more mesenchymal phenotype, and a proportion of the cells undergo senescence and/or apoptosis. Moreover, at the nonpermissive temperature, the levels of p53 and SV40 Large T antigen diminish, whilst the level of p21 increases, whereas the level of p16 and telomerase activity is unchanged. This experimental system shows that expression of telomerase alone only allows limited proliferative potential of OSE cells; expression of tsT is necessary to maintain these cells in culture for longer periods, perhaps by its ability to inactivate components of the p53/Rb pathway. OSE-C2 cells may be useful in studying the physiology and differentiation of human OSE cells and provide insight into the poorly understood earliest stages of epithelial ovarian cancer. [Copyright &y& Elsevier]

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Oncogenic human papillomavirus (HPV) plays a possible aetiological role in a subset of head and neck cancers, particularly in tonsillar carcinomas. For establishing a model to study mechanisms involved in HPV-associated tonsillar carcinogenesis, normal human tonsillar epithelial (HTE) cells were transfected with full-length HPV-16 DNA. The transfections produced four immortalised cell lines, designated HTE-114/K1, HTE-114/K2, HTE-114/K3 and HTE-114/B. All transfected HTE cell lines were cytogenetically abnormal. They exhibited altered morphology and impaired expression of cytokeratins in organotypic cultures. They failed to form colonies in soft agarose and formed no tumours in nude mice within 6 months. Each of them contained integrated viral DNA in a distinctive pattern as shown by Southern blot hybridisation. Early viral transcripts containing the E7 gene were detected by northern blot hybridisation. In conclusion, primary HTE cells can be immortalised following transfection with full-length HPV-16 DNA; the immortalised cell lines had partially retained epithelial characteristics in their morphology and function. They seem to represent early stages of premalignant epithelial cells and thus provide a useful model for studying further the multistep molecular events of HPV-16-associated tonsillar carcinogenesis. [Copyright &y& Elsevier]

BackgroundAdhesion of haematopoietic progenitor cells (HPC) to human bone marrow endothelial cells (HBMEC) plays a key role in homing of HPC to bone marrow. Here we describe four new HBMEC cell lines that can be used to study the (specific) adhesion of HPC to HBMEC. DesignHBMEC were immortalised with a retroviral construct containing the human papilloma virus 16 E6/E7 genes. Four cell lines were characterised. ResultsThe cell lines showed their endothelial nature by the expression of von Willebrand Factor and VE-cadherin (CD144). Electron microscopic analysis revealed normal endothelial-cell characteristics, including the presence of Weibel-Palade bodies and intercellular junction structures. An extensive phenotypic analysis of the cell-lines was performed, they were found to resemble primary HBMEC. The only difference found was the absence of expression of E-selectin (CD62e) and VCAM-1 (CD106) on resting HBMEC cell lines. Upon stimulation with IL-1β the expression of E-selectin, VCAM-1 and ICAM-1 (CD54) was upregulated. All resting cell lines bound CD34+ HPC. Adhesion was increased by addition of the phorbol ester PMA. Two cell lines showed increased binding upon IL-1β prestimulation. Highest adhesion was observed after the combination of IL-1β prestimulation of the endothelial cells and addition of PMA. Binding of CD34+ HPC to HBMEC was compared with the binding to human umbilical vein endothelial cell lines and to a human dermal microvascular endothelial cell line (HMEC-1). So far, we have only found relatively less binding of HPC to IL-1β prestimulated HMEC-1 cells, which could be explained by a reduced induction of E-selectin and VCAM-1 upon IL-1β stimulation of these cells. ConclusionThe immortalised HBMEC cell lines have maintained their normal phenotype for the majority of characteristics examined. The expression of E-selectin and VCAM-1, which are not constitutively expressed on the cell lines, can be... [ABSTRACT FROM AUTHOR]

DNA-like sequences of the p53 and pRB-inactivating simian virus 40 large T-antigen (SV40 LTag) have recently been found in mesotheliomas in the United States and several European countries. Nuclear expression of SV40 LTag, possibly in concert with detectable telomerase activity, could be responsible for immortalisation of (pre)malignant clones, as suggested by the mesothelioma-specific latency period. Depending on the antibody used, different results have been observed regarding the subcellular expression of SV40 LTag in mesotheliomas with SV40 LTag-like DNA sequences. In this study, we screened 28 Belgian mesothelioma tumour samples for the presence of SV40 LTag-like DNA and its gene product by polymerase chain reaction amplification, using the SV.for3/SV.rev primer set, and by tyramine-amplified immunohistochemistry, using the pAb419 and the pAb101 SV40 LTag antibodies. Amplicons were found in 13 of the 28 (46%) mesotheliomas. Cytoplasmic, but no nuclear, staining was found in 10 of these 13 cases. Although our study confirms the presence of SV40 LTag-like DNA sequences in Belgian mesotheliomas, we did not detect nuclear expression of the viral oncoprotein, which makes a pathogenic role of SV40 LTag in mesothelioma carcinogenesis questionable. [ABSTRACT FROM AUTHOR]

The current lack of continuous prawn cell lines suitable for the isolation and growth of prawn viruses is a major setback for diagnosis of viral diseases of prawns; isolation and identification of the causative agents are severely hindered and the development of other diagnostic procedures is slowed. To date, there are few, if any, examples of continuous prawn cell lines suitable for virus isolation and growth. Recent studies indicate that clastogenic agents such as ionising radiation are more effective than powerful point mutagens as immortalising agents for mammalian cells. Such studies with mammalian cell cultures indicate that certain mutagenic agents and/or prolonged treatment with a combination of mutagens may prove more useful in the production of immortal prawn cell lines in vitro rather than other, more limited and specific procedures. This report provides a brief review of the use of mutagens to induce immortalisation -- the acquisition of unlimited growth potential -- in mammalian cells in culture, i.e. the production of continuous cell lines from primary cultures. Aspects of cell transformation vs. immortalisation, the control of cell growth in vitro, and the application of mutagenic agents to induce immortalisation in primary cultures of prawn tissues will be discussed. [ABSTRACT FROM AUTHOR]

A cell line derived from the urothelium lining the ureter of a 12-year-old girl was immortalised using a temperature-sensitive SV40 large T-antigen gene construct, and designated UROtsa. Following immortalisation, UROtsa cells expressed SV40 large T-antigen, but did not acquire characteristics of neoplastic transformation, including growth in soft agar or the development of tumours in nude mice. Metaphase spreads had a normal chromosomal appearance and number. UROtsa cells remained permissive for cell growth at 39°C, indicating that they did not retain temperature sensitivity. UROtsa provides an in vitro model of 'normal' urothelium. [ABSTRACT FROM AUTHOR]

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., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Where authors are identified as personnel of the International Agency for Research on Cancer/World Health Organization, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer/World Health Organization.