Molecular Characterization of Spontaneous Mesenchymal = Stem Cell Transformation

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Journal=20 List > PLoS=20 ONE > v.3= (1);=20 2008

PubMed=20 articles by:

Rubio, D.

Garcia, S.

Paz, M.

Bernad, = A.

PLoS ONE. 2008; 3(1): e1398.

Published online 2008 January = 2.=20 doi:=20 10.1371/journal.pone.0001398.

Copyright<= /A>=20 Rubio et al. This is an open-access article distributed under the = terms of=20 the Creative Commons Attribution License, which permits = unrestricted use,=20 distribution, and reproduction in any medium, provided the = original author=20 and source are credited.

Molecular Characterization of Spontaneous = Mesenchymal=20 Stem Cell Transformation

Daniel=20 Rubio,¹^=C2=A4a Silvia = Garcia,¹^=C2=A4b=20 Maria F. Paz,¹^=C2=A4b Teresa De la=20 Cueva,¹^=C2=A4c Luis A. = Lopez-Fernandez,²=20 Alison C. Lloyd,³ Javier=20 Garcia-Castro,^1,⁴ and Antonio=20 Bernad¹^*^=C2=A4b

¹Department of Immunology and = Oncology,=20 Centro Nacional de Biotecnolog=C3=ADa, Consejo Superior de = Investigaciones=20 Cient=C3=ADficas (CSIC), Universidad Aut=C3=B3noma de Madrid, = Madrid, Spain

²Servicio de Farmacia, Hospital = General=20 Universitario Gregorio Mara=C3=B1=C3=B3n, Madrid, Spain

³Laboratory for Molecular Cell = Biology,=20 University College London, London, United Kingdom

⁴Andalusian Stem Cell Bank, = Granada,=20 Spain

Joseph Najbauer, Academic=20 Editor

City of Hope Medical Center, United States of = America

* To whom correspondence should = be=20 addressed. E-mail: abernad/at/cnic.es

Conceived and designed the experiments: = DR AB JG.=20 Performed the experiments: DR SG TD MP. Analyzed the data: DR SG = AL JG LL.=20 Contributed reagents/materials/analysis tools: AL. Wrote the = paper: DR AB=20 AL JG.

^=C2=A4aCurrent = address: Centro de=20 Biolog=C3=ADa Molecular Severo Ochoa, Universidad Aut=C3=B3noma de = Madrid, Madrid,=20 Spain

^=C2=A4bCurrent = address: Department of=20 Regenerative Cardiology, Centro Nacional de Investigaciones=20 Cardiovasculares (CNIC), Madrid, Spain

^=C2=A4cCurrent = address: Animalary=20 facility, Centro Nacional de Servicio de Biotecnolog=C3=ADa, = Consejo Superior=20 de Investigaciones Cient=C3=ADficas (CSIC), Universidad = Aut=C3=B3noma de Madrid,=20 Madrid, Spain

Received November 23, 2006; Accepted = December 7,=20 2007.

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"=20 = src=3D"http://www.pubmedcentral.nih.gov/corehtml/pmc/pmcgifs/square.gif" = border=3D0>Abstract

Introduction

Results

Discussion

Materials and=20 Methods

Supporting=20 Information

References

Abstract

Background

We = previously=20 reported the in vitro spontaneous transformation of human = mesenchymal stem cells (MSC) generating a population with = tumorigenic=20 potential, that we termed transformed mesenchymal cells (TMC).

Methodology/Principal=20 Findings

Here we have characterized the molecular changes = associated=20 with TMC generation. Using microarrays techniques we identified a = set of=20 altered pathways and a greater number of downregulated than = upregulated=20 genes during MSC transformation, in part due to the expression of = many=20 untranslated RNAs in MSC. Microarray results were validated by = qRT-PCR and=20 protein detection.

Conclusions/Significance

In our=20 model, the transformation process takes place through two = sequential=20 steps; first MSC bypass senescence by upregulating c-myc and = repressing=20 p16 levels. The cells then bypass cell crisis with acquisition of=20 telomerase activity, Ink4a/Arf locus deletion and Rb = hyperphosphorylation.=20 Other transformation-associated changes include modulation of=20 mitochondrial metabolism, DNA damage-repair proteins and cell = cycle=20 regulators. In this work we have characterized the molecular = mechanisms=20 implicated in TMC generation and we propose a two-stage model by = which a=20 human MSC becomes a tumor cell.

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Abstract

"=20 = src=3D"http://www.pubmedcentral.nih.gov/corehtml/pmc/pmcgifs/square.gif" = border=3D0>Introduction

Results

Discussion

Materials and=20 Methods

Supporting=20 Information

References

Introduction

The development of a solid tumor is considered a multi-step = process in=20 which several molecular checkpoints must be altered to generate a = tumor=20 from a normal cell [1].=20 The acquired capabilities of tumor cells include their ability to=20 proliferate continuously ignoring apoptosis or growth-inhibitory = signals,=20 generating their own mitogenic signals. In advanced phases of = tumor=20 development, a neoangiogenesis process takes place and finally = tumor cells=20 acquire the capacity of tissue invasion and metastasize to other = organs.=20 Generally, it is admitted that most tumors acquire these = characteristics=20 through genome instability, telomere stabilization and disruption = of=20 regulatory circuits [2].

A recent theory suggests the existence of cancer stem cells = (CSC), a=20 subpopulation of cells with tumorigenic potential that is lacked = in the=20 rest of the cells within this tumor. CSC were reported for some = tumor=20 types including breast and lung cancer, leukemia and glioblastoma = [3],=20 [4].=20 However, there is a great ignorance about how the = =E2=80=9Cacquired capabilities=E2=80=9D=20 of tumor cells would take place; directly on adult stem cells, or = on=20 differentiated cells that suffer a dedifferentiation process. In = this=20 regard, CSC share several features with adult stem cells such as=20 self-renewal ability, asymmetric division, and differentiation = potential=20 [5].

Adult human mesenchymal stem cell spontaneous immortalization = and=20 transformation were recently reported by our group [6],=20 supporting the hypothesis of the stem cell origin of CSC. = Independent=20 laboratories have confirmed these data, reporting similar results = using=20 MSC derived from human or murine bone marrow [7]=E2=80=93[11].=20 In this regard, we have previously characterized the cellular = sequence of=20 steps necessary to transform a human MSC into a tumorigenic cell = [6].=20 Following approximately 20 population doublings in vitro, = mesenchymal stem cell cultures enter a senescence phase, but are = able to=20 bypass it at a high frequency. These cells then continue to divide = until=20 they reach a crisis phase. Only some samples are able to escape = from this=20 crisis phase spontaneously, but those that do have undergone = tumorigenic=20 transformation generating TMC.

However, until now genetic alterations implied in spontaneous = MSC=20 transformation are little known. Some groups have studied = molecular=20 pathways involved in the artificial transformation of MSC = transduced with=20 oncogenes [12],=20 [13].=20 In this study, we have characterized the molecular mechanisms = implicated=20 in TMC generation and we propose a two-stage model by which a = human MSC=20 becomes in vitro a tumor cell.

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Abstract

Introduction

"=20 = src=3D"http://www.pubmedcentral.nih.gov/corehtml/pmc/pmcgifs/square.gif" = border=3D0>Results

Discussion

Materials and=20 Methods

Supporting=20 Information

References

Results

Comparative gene expression = analysis of=20 MSC transformation by microarray analysis

To analyze = molecular=20 differences associated with TMC generation, we performed = microarray=20 studies using mRNA from pre- and post-senescence MSC, and from = TMC. From a=20 general perspective, data analysis showed that the greatest = changes were=20 associated with TMC generation, as TMC functions were more = different than=20 post-senescence MSC, compared to pre-senescence MSC (Table=20 1). Although in a minor intensity, post-senescence MSC have = the same=20 altered functions that TMC. In both cases the principal category = affected=20 is =E2=80=9Ccancer=E2=80=9D (Table=20 1). However, the main pathways deregulated in both, = post-senescence=20 MSC and TMC, are related to stress, toxic events and mitochondrial = metabolism (Table=20 2). On the other hand, there was more down- than upregulated = RNA=20 transcripts associated with TMC generation (Figure=20 S1). The main differences in mRNA expression profiles between = pre- and=20 post-senescence MSC are shown in Table=20 3. Table=20 4 shows differences between pre-senescence MSC and TMC.

Table=20 1

Comparative table=20 of functions with a higher significance in selected genes = for=20 post-senescence MSC and TMC, obtained by Ingenuity Pathways = Analysis=20 software.

Table=20 2

Top tox = list=20 obtained by Ingenuity Pathways Analysis for post-senescence = MSC and=20 TMC.

Table=20 3

Main mRNA = differences between pre- and post-senescence=20 MSC.

Table=20 4

Main mRNA = differences between pre-senescence MSC and=20 TMC.

Molecular differences in cell=20 cycle-related proteins

We had reported differences in cell = cycle=20 progression in pre- and post-senescence MSC and TMC, including = more rapid=20 cell division in TMC [6].=20 Here we used microarray techniques to explore the cell = cycle-related=20 molecular differences in post-senescence MSC and TMC compared to=20 pre-senescence MSC. Compared to pre-senescence MSC, = post-senescence MSC=20 showed few differences in cell cycle-related proteins. In = contrast, TMC=20 samples showed significant mRNA modulations; Cdk1 and Cdk4 as well = as=20 cyclins B1 and D2 were upregulated, whereas cyclin D1 was = downregulated=20 (Figure=20 1A).

Figure=20 1

Cell = cycle=20 = regulation.

10.1371/journal.pone.00= 01398.g001

We evaluated microarray experiments analyzed by qRT-PCR the = expression=20 of some of these genes. No expression difference was found between = pre-=20 and post-senescent MSC, while TMC overexpressed Cdk2 and Cdk6 (Figure=20 1B). In qRT-PCR experiments we also studied met-TMC, a cell = line=20 derived of lung metastases generated after s.c. inoculation of TMC = in=20 immunodeficient mice (Rubio et al, unpublished = results).

To determine whether the differences in mRNA levels gave rise = to=20 altered protein expression, we compared these samples by western = blot. In=20 TMC, cyclin B1, Cdk2 and Cdk6 were upregulated, whereas Cdk1 and = Cdk4=20 remained constant. Cyclin D1 was downregulated from pre-senescence = MSC to=20 TMC (Figure=20 1C).

Upregulation of DNA repair = pathways in=20 transformed MSC

As DNA repair mechanisms are responsible for = the=20 bypass of senescence and crisis, as well as for tumor maintenance = and=20 progression [14],=20 we analyzed the major proteins linked to these processes. We = tested=20 proteins involved in pathways including non-homologous end joining = (NHEJ),=20 base excision repair (BER), nucleotide excision repair (NER), = mismatch=20 repair (MMR), and homologous recombination (HR).

Microarray analysis showed no mRNA significant differences = between pre-=20 and post-senescence MSC (Figure=20 2A). However protein analysis showed that DNA-PKcs was = repressed in=20 post-senescence MSC compared to pre-senescence MSC and a = downregulation of=20 Rad51 after senescence bypass, although expression was restored = and=20 upregulated after crisis bypass in TMC (Figure=20 2C).

Figure=20 2

DNA = repair=20 = regulation.

10.1371/journal.pone.00= 01398.g002

In contrast, mRNA levels were modulated differentially between = TMC and=20 pre-senescence MSC for several DNA repair-associated proteins.=20 Specifically, DNA-PKcs and PCNA were upregulated in TMC, and XPA = was=20 downregulated compared to pre-senescence MSC. Although DNA = polymerase =CE=BC=20 and ERCC3 upregulation were statistically significant, differences = in mRNA=20 levels appeared to be minor, based on x-fold change and = Z-score=20 values (Figure=20 2A).

We performed qRT-PCR experiments to validate microarray = results. No=20 modulation of expression of DNA repair-related genes was detected = between=20 pre- and post-senescence MSC. In contrast, several of these genes = were=20 overexpressed in TMC, such as DNA-PKcs, DNA polymerase =CE=BC, = RAD51 or ERCC4=20 (Figure=20 2B).

We evaluated the correlation between RNA and protein levels by = western=20 blot analysis. Proteins were elevated in nearly all DNA repair = pathways in=20 TMC compared to pre-senescence MSC. These effects were protein- = rather=20 than pathway-specific. MMR was the only pathway that showed no = differences=20 between MSC and TMC, although we analyzed only PCNA in this = pathway (Figure=20 2C).

Changes in tumor suppressors = and=20 oncogenes expression related with MSC transformation

To = explore the=20 role of tumor suppressor gene inactivation in MSC senescence and = crisis=20 bypass, we analyzed p16, p21 and p53. In a microarray screening = assay, we=20 found no changes in p21 or p53 levels, whereas MSC expressed less = p16 mRNA=20 after senescence bypass (Figure=20 3A), concurring with reports of a role for p16 in senescence = induction=20 [15].=20 p16 downregulation was more marked when we compared TMC and = pre-senescence=20 MSC mRNA levels, and we found slight but significant p21 = downregulation.=20 We detected no differences in p53 mRNA (Figure=20 3A).

Figure=20 3

Regulation of=20 oncogenes and tumor suppressor=20 = genes.

10.1371/journal.pone.0001398= .g003

To validate the microarray results, we used qRT-PCR to compare = mRNA=20 levels among the three cell types. p16 mRNA levels were = downregulated in=20 post-senescence MSC, to approximately 60% of the pre-senescence = level. We=20 observed no p16 mRNA amplification in TMC samples (Figure=20 3B). p16 protein is downregulated in post-senescence MSC, and = absent=20 in TMC [6],=20 concurring with these results. We analyzed whether complete p16 = repression=20 in TMC was caused by promoter methylation, as reported for this = tumor=20 suppressor [16],=20 [17]=20 studying the methylation status. Methylation analysis of the = Ink4a/Arf=20 promoter showed no methylation (not shown). We used specific = DNA-PCR to=20 detect homozygous deletion of p14, p15 and p16, and found no = amplification=20 (Figure=20 3C=E2=80=93E), indicating that the Ink4A/Arf locus is deleted = specifically in=20 TMC.

Microarray analysis suggested slight p21 downregulation in TMC = compared=20 to pre-senescence MSC, with no difference between pre- and = post-senescence=20 MSC. We found no changes in p21 levels in western blot analysis of = these=20 populations (Figure=20 3F). Although p53 pathway defects have been associated with = the=20 generation of many tumor types [18],=20 this pathway appeared to be functional in our stem cell = transformation=20 model, as p53 was upregulated after UV irradiation and was = phosphorylated=20 in all samples tested (Figure=20 3G). Basal p53 protein levels were also higher in TMC than in = pre- or=20 post-senescence MSC.

Finally, we assayed Rb levels and phosphorylation in pre- and=20 post-senescent MSC, two TMC samples and a met-TMC sample. Rb = protein=20 levels were upregulated in TMC and met-TMC compared to pre- and=20 post-senescence MSC. Rb phosphorylation increased progressively = through=20 the sequence of steps in TMC generation, and was slightly = downregulated in=20 met-TMC compared to TMC (Figure=20 3H).

We compared mRNA regulation differences in the oncogenes c-myc = and=20 telomerase in post-senescence MSC and TMC with pre-senescence MSC. = We=20 found no differences in oncogene mRNA transcript levels in these=20 populations (Figure=20 3A). Nonetheless, in a previous publication we have shown that = c-myc=20 protein overexpression is linked to senescence bypass and is = maintained in=20 TMC; moreover, TMC also have telomerase activity which is not = found in=20 pre- or post-senescence MSC [6].

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Abstract

Introduction

Results

"=20 = src=3D"http://www.pubmedcentral.nih.gov/corehtml/pmc/pmcgifs/square.gif" = border=3D0>Discussion

Materials and=20 Methods

Supporting=20 Information

References

Discussion

Mesenchymal stem cells can be easily isolated and expanded in = culture=20 to generate large numbers of cells for cellular therapies. Human = MSC in=20 early passage are safe although stressful conditions (as they are = cultured=20 for a long time) can turn them in immortal and occasionally they = became=20 tumorogenic [6].=20 Further research is necessary to understand this process in order = to=20 develop better protocols for culture adult stem cells, as it has = been=20 demanded recently [19].=20 Here, we describe several molecular alterations in our spontaneous = human=20 MSC transformation model that affect cell cycle regulation, = oncogene=20 expression, mitochondrial metabolism, DNA repair mechanisms and=20 inactivation of tumor suppressor genes.

TMC versus pre-senescence MSC array analysis showed that = functions with=20 a higher significance are related, as expected, with = transformation,=20 genetic disorders and cell death (Table=20 1). We described previously that post-senescence MSC are=20 non-tumorigenic and their cellular behaviour in culture was very = similar=20 to pre-senescence MSC [6].=20 Interestingly, post-senescence MSC versus pre-senescence MSC array = analysis also showed the same functions altered than TMC, although = in=20 smaller grade (Table=20 1), suggesting a pre-tumoral state of pre-senescence MSC.

We observed expression of many untranslated RNAs in MSC = concurring with=20 reports which show a large and =E2=80=9Csilent=E2=80=9D mRNA pool = in stem cells [20],=20 this could be the reason why, following MSC transformation, we = identified=20 more downregulated than upregulated genes in arrays experiments = (Figure=20 S1). Comparison of mRNA and protein expression in pre- and=20 post-senescence MSC and in TMC showed variation in RNA and protein = regulation. Cyclin D2, Cdk1 and PCNA mRNA were upregulated in TMC = compared=20 to MSC, although their protein levels did not change; whereas = c-Myc, Cdk2,=20 Cdk6, DNA ligase IV and DNA polymerases mRNA levels remained = stable but=20 their protein levels were upregulated. Translational control could = thus be=20 important for adult stem cells, and retention of large numbers of = silenced=20 transcripts might allow rapid stem cell differentiation to other = lineages=20 in response to appropriate stimuli. These data also indicates the=20 limitations of results based on RNA-exclusive analysis of MSC.

Telomerase activity has been found in almost all human tumors = but not=20 in adjacent normal cells [21]=20 and maintenance of telomere stability is required for the = long-term=20 proliferation of tumor cells [22].=20 The escape from cellular senescence and thus becoming immortal by=20 activating telomerase is required by most tumor cells for their = ongoing=20 proliferation [23].=20 In our model, during TMC generation these cells acquire a = detectable=20 telomerase activity [6].=20 Telomerase promotes MSC immortalization and, in conjunction with=20 additional events, produces cell transformation [12],=20 [13],=20 [24].=20 These additional events usually implied an oncogene = deregulation.

One of the most important oncogenes involved in MSC = transformation is=20 c-myc. In our spontaneous model, senescent and post-senescence = MSC, as=20 well as TMC, overexpress c-myc [6].=20 Consistent with our previous results, data from other groups have = shown=20 that c-myc seems to be essential to spontaneously transform MSC [7],=20 [9],=20 [10].=20 In this regard, Funes et al. used retroviral vectors to introduce = human=20 telomerase (TERT), HVP-16 E6 and E7, H-Ras and SV40 small T = antigen (ST),=20 individually or in combination, in human MSC. The combination of = TERT, E6,=20 E7 and H-Ras did not induce MSC transformation. Only MSC = transduced with=20 ST becomes transformed cells [10].=20 ST inactivates phosphatase 2A, resulting in c-myc stabilization [25],=20 suggesting that c-myc might be necessary to transform MSC.

We explored DNA repair mechanisms to elucidate their role in = MSC=20 transformation. Post-senescent MSC showed downregulation of = DNA-PKcs,=20 ERCC3 and Rad51 proteins, each of which is associated to a = distinct DNA=20 repair pathway. Extremely restricted clonal selection takes place = during=20 cell crisis, and only cells with functional DNA repair mechanisms = would=20 continue to grow. TMC have a higher metabolic rate and divide more = rapidly=20 than pre- or post-senescence MSC, with a consequent increase in = DNA=20 damage. Proteins that participate in DNA repair are upregulated in = TMC=20 compared to MSC; this, together with telomere length maintenance, = could=20 permit cell survival, despite oxidative damage to DNA and be = responsible=20 for TMC karyotype stabilization. Recently it has been published = the=20 dependency on oxidative phosphorylation during MSC transformation = [11].=20 We have not detected statistically significant changes of these = genes in=20 our microarray experiments (Table=20 S1), although potential pathways leading to changes in = post-senescence=20 MSC and TMC revealed change in stress, toxic events and = mitochondrial=20 metabolism pathways (Table=20 2). The definitive role of mitochondrial respiration on = spontaneous=20 MSC transformation remains to be investigated.

A chromosome 5 alteration and a (3;11) translocation are = recurrent,=20 stable features of in vitro cultured TMC [6].=20 The telomerase gene map to human chromosome 5, suggests that it is = activated by internal amplification of this chromosome in TMC. = Chromosome=20 11 alterations are recurrent in tumors [26].=20 Although we did not detect a target gene in the 3;11 translocation = in our=20 model, genes involved in cell transformation are likely to be = located in=20 this region [27]=E2=80=93[29].

As tumor suppressor genes are major targets in neoplastic=20 transformation, we analyzed their expression in these cells. The = tumor=20 suppressor Rb is implicated in several cancer types [18].=20 In our model of MSC transformation, Rb protein levels are = upregulated=20 progressively, and Rb is inactivated by a phosphorylation = mechanism in=20 TMC, as described [30].=20 In addition of Rb, loss of p53 function is common in many tumor = types [18],=20 but this pathway appeared to be functional in our model, as p53 = was=20 upregulated and phosphorylated in UV-irradiated cells. We observed = higher=20 basal p53 levels in TMC than in MSC, even when they had not been = exposed=20 to UV irradiation. In TMC, p16 mRNA and protein were entirely = absent, and=20 the Ink4a/Arf locus had been deleted. The increase in basal p53 = may thus=20 be due to stabilization by the ubiquitin protein ligase MDM2, due = to the=20 lack of p16 [31].=20 Identical results, p16 locus deletion and normal p53 activity, was = detected in telomerase-immortalized human MSC [32].=20 The results suggest that p16 inhibition is essential for TMC = generation,=20 as is the case for human malignancies including glioblastoma, = melanoma,=20 pancreatic adenocarcinoma, non-small cell lung cancer, bladder = carcinoma=20 and oropharyngeal cancer, where this tumor suppressor is = frequently lost=20 [33].

Finally, we propose a two-stage model in which a mesenchymal = stem cell=20 becomes a tumor cell (Figure=20 4). The first step, the senescence bypass or M1 phase, is = associated=20 with c-myc overexpression and p16 repression; many DNA repair = proteins are=20 subsequently downregulated. Telomere shortening provokes the cell = crisis=20 phase, or M2, in which cells undergo stringent selection. TMC then = upregulate many DNA repair proteins, which may be necessary for = crisis=20 bypass. Finally, escape from crisis is associated with telomere=20 stabilization, Rb hyperphosphorylation and p16 deletion that seems = to be=20 essential to promote transformation [33],=20 [34].=20 TMC also upregulate many DNA repair proteins, which may be = necessary for=20 crisis bypass. These levels are maintained in TMC and could permit = cell=20 survival, despite oxidative damage to DNA.

Figure=20 4

Model of=20 spontaneous human MSC=20 = transformation.

10.1371/journal.pon= e.0001398.g004

The essential steps in TMC generation described here are = basically in=20 agreement with results of other authors working in MSC = transformation [7]=E2=80=93[11],=20 [32]=20 and these alterations are very similar to molecular changes = associated=20 with transformation of other cell types. In epithelial cells, = spontaneous=20 immortalization of human keratinocytes exhibited a small number of = chromosomal aberrations, reduced p^16INK4amRNA, = elevated=20 telomerase activity and functionally normal p53 [35].=20 Immortalization of human prostate cells by c-myc stabilizes = telomere=20 length through up-regulation of TERT expression and lack=20 Rb/p16^INK4a checkpoint, being easily transformed [36].=20 In mesodermic cells, fibroblast cell lines immortalized either=20 spontaneously or radio-chemically induced maintaining their = telomerase=20 activity, displayed loss of expression of p16^INK4a and=20 hyperphosphorylation of Rb [37].=20 Telomerase-immortalized human fibroblast revealed overexpression = of the=20 c-myc and Bmi-1 oncogenes, as well as loss of p14^ARF = expression=20 [38],=20 [39],=20 while overexpression of c-myc immortalizes freshly isolated human = foreskin=20 fibroblasts displayed a marked decrease in expression of = p14^ARF=20 [40].

In sum, all these evidences strongly suggest that cells with a=20 mesodermal origin could require a common sequence of oncogenic = events to=20 become a tumor cells. How these processes are coordinated or = associated=20 with the critical cell evolution/selection revealed in the culture = [6]=20 remains to be studied in deep. In addition, the cause/consequence=20 relationship of this molecular signature with the recently = characterized=20 mesenchymal to epithelial transition (Rubio, D. et al, in press) = or other=20 potentially involved mechanisms remains also to be=20 determined.

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Abstract

Introduction

Results

Discussion

"=20 = src=3D"http://www.pubmedcentral.nih.gov/corehtml/pmc/pmcgifs/square.gif" = border=3D0>Materials and=20 Methods

Supporting=20 Information

References

Materials and = Methods

Isolation of MSC and cell=20 culture

MSC were isolated as described [6].=20 Briefly, adipose tissue from non-oncogenic patients were digested = with=20 collagenase P and cultured (37=C2=B0C, 5% CO₂) in MSC = medium (DMEM=20 plus 10% FCS, 2 mM glutamine, 50 =C2=B5g/ml gentamycin) and = passaged when they=20 reached 85% confluence. TMC and met-TMC were cultured under the = same=20 conditions.

Microarray labeling

Total = RNA was=20 isolated from four biological replicates of pre- and = post-senescence MSC=20 and from TMC using TriReagent Solution (Sigma) following = manufacturer's=20 instructions. RNAs were purified with MegaClear (Ambion) and = integrity=20 confirmed using the Agilent 2100 Bioanalyzer (Agilent = Technologies). Total=20 RNA (1.5 =C2=B5g each) was amplified using the Amino Allyl = MessageAmp aRNA kit=20 (Ambion); we obtained 15=E2=80=9360 =C2=B5g of amino-allyl = amplified RNA (aRNA). Mean=20 aRNA size was 1500 nucleotides, as measured using the Agilent 2100 = Bioanalyzer. For each sample, 2.5 =C2=B5g of aRNA was labeled with = one aliquot=20 of Cy3 or Cy5 Mono NHS Ester (CyDye Post-labeling Reactive Dye, = Amersham)=20 and purified with the Amino Allyl MessageAmp aRNA kit. Cy3 and Cy5 = incorporation were measured using 1 =C2=B5l probe in a Nanodrop=20 spectrophotometer (Nanodrop Technologies). For each = hybridizations, 80=E2=80=93100=20 pmol of Cy3 and Cy5 probes were mixed, dried by speed-vacuum, and=20 resuspended in 9 =C2=B5l RNase-free water. Labeled aRNA was = fragmented by=20 adding 1 =C2=B5l of 10=C3=97 fragmentation buffer (Ambion) and = incubating (70=C2=B0C, 15=20 min). The reaction was terminated with 1 =C2=B5l stop solution = (Ambion).

Slide treatment and=20 hybridization

Slides containing 22,102 annotated genes = corresponding=20 to the Human 70-mer oligo library (V2.2) (Qiagen-Operon) were = obtained=20 from the Genomics and Microarray Laboratory (Cincinnati = University).=20 Information on printing and the oligo set can be found at http://www.pubmedcentral.nih.gov/redirect3.cgi?&&= ;auth=3D0sg6dZxmFGZhiZxzRQKbxRiq3ZouE0AGUnSk4p52E&reftype=3Dextlink&a= mp;artid=3D2151133&iid=3D157281&jid=3D440&FROM=3DArticle%7CBo= dy&TO=3DExternal%7CLink%7CURI&article-id=3D2151133&journal-id= =3D440&rendering-type=3Dnormal&&http://microarray.uc.edu<= /SPAN>.=20 Slides were prehybridized (42=C2=B0C, 45=E2=80=9360 min) in = 6=C3=97 SSC, 0.5% SDS and 1%=20 BSA, then rinsed 10 times with distilled water. Fragmented Cy5 and = Cy3=20 aRNA probes were mixed (80=E2=80=93100 pmol of each label) with 10 = =C2=B5g PolyA=20 (Sigma) and 5 =C2=B5g Human Cot-DNA (Invitrogen) and dried in a = speed-vacuum.=20 Each probe mix was resuspended in 60 =C2=B5l of hybridization = buffer (50%=20 formamide, 6=C3=97 SSC, 0.5% SDS, 5=C3=97 Denhardt's solution). = Probes were=20 denatured (95=C2=B0C, 5 min) and applied to the slide using a = LifterSlip (Erie=20 Scientific). Slides were incubated (48=C2=B0C, 16 h) in = hybridization chambers=20 (Array-It; Telechem International) in a water bath. After = incubation,=20 slides were washed twice with 0.5=C3=97 SSC, 0.1% SDS (5 min = each), three times=20 with 0.5=C3=97 SSC (5 min) and finally in 0.05=C3=97 SSC (5 min), = then dried by=20 centrifugation (563 g, 1 min). Images from Cy3 and Cy5 channels = were=20 equilibrated and captured with an Axon 4000B scanner and spots = quantified=20 using GenePix 5.1 software.

Four independent biological replicates were =E2=80=9Cdye = swapped=E2=80=9D and studied=20 (8 hybridizations). Data were analyzed using Almazen software. = Each=20 replicate was lowess-normalized and the log ratios merged with the = corresponding standard deviation and z-score. We obtained adjusted = p-values using limma by FDR [40].=20 Differentially expressed genes were selected by filtering signal = intensity=20 (>64), z-score (>3.5 or <=E2=88=923.5) and p-value = (<0.01).

Pathways analysis

By using = Ingenuity=20 Pathways Analysis (IPA), potential pathways leading to changes in=20 MSC-postsenescence and TMC were created. This web-delivered = application=20 reveals relevant networks by comparing gene expression data with = known=20 pathways and interactions between genes (http://www.pubmedcentral.nih.gov/redirect3.cgi?&&= ;auth=3D0qO_ceB5QXHEzUWcSA81PcyRRgUI68j7SJqLEQkZo&reftype=3Dextlink&a= mp;artid=3D2151133&iid=3D157281&jid=3D440&FROM=3DArticle%7CBo= dy&TO=3DExternal%7CLink%7CURI&article-id=3D2151133&journal-id= =3D440&rendering-type=3Dnormal&&http://www.ingenuity.com<= /SPAN>).=20 The filtered expression data set for MSC-postsenescence and TMC = regulated=20 genes were uploaded as tab-delimited text into IPA for generating=20 biological networks. Each gene identifier was mapped to its = corresponding=20 object in the Ingenuity Pathways Knowledge Base. This software = assigned a=20 score for all networks that were ranked on the probability that a=20 collection of genes equal to or greater than the number in a = network could=20 be achieved by chance alone (a score of 2 represents a 99% = confidence=20 level, and 3 a 99.9%). Biological functions are then calculated an = assigned to each network

Quantitative real-time PCR=20 (qRT-PCR)

cDNA was generated from 100 ng of total RNA using = the High=20 Capacity cDNA Archive Kit (Applied Biosystems) in a 10 =C2=B5l = final reaction=20 volume. Real-time PCR reactions were performed in triplicate using = two=20 dilutions (1/50, 1/500; 3 =C2=B5l/well) of each cDNA, 1=C3=97 = TaqMan Assay-On-Demand=20 (Hs00233365_m1, Cdkn2a; Hs00195591_m1, snail; Hs00161904_m1, slug) = or=20 primers described in Table=20 S2, 1=C3=97 SYBR Green PCR Master Mix or 1=C3=97 TaqMan = Universal PCR Master Mix=20 (Applied Biosystems) in a volume of 8 =C2=B5l in 384-well optical = plates, or=20 using Universal ProbeLibrary (Roche). PCR reactions were run on an = ABI=20 Prism 7900HT (Applied Biosystems) and SDS v2.2 software was used = to=20 analyze the results with the Comparative Ct Method = (=CE=94=CE=94Ct).

Western blot

Cell extracts = were=20 fractionated in 6%=E2=80=9315% SDS-PAGE, followed by transfer to = PVDF membranes.=20 We used antibodies to cyclins A clone E23 (1/200), D1 DCS-11 = (1/1000), and=20 D2 DCS-3.1 (1/1000) from Labvision; cyclin B1 sc-595 (1/200), = cyclin D3=20 sc-182 (1/200), cdc2 sc-747 (1/200), cdk2 sc-163 (1/200), cdk4 = sc-260=20 (1/200) and cdk6 sc-177 (1/200) were from Santa Cruz = Biotechnology.=20 DNA-PKcs Ab-1 (1/200) was from Calbiochem, and Ku-70 sc-9033 = (1/200),=20 XRCC4 sc-8285 (1/200), DNA ligase IV sc-11748 (1/200) were from = Santa Cruz=20 Biotechnology. We also used anti-DNA polymerase =CE=BC [25],=20 -PCNA Ab-1 (Calbiochem, 1/100), and -DNA polymerase =CE=B2 Ab-1 = (1/500), -ERCC1=20 Ab-2 (1/200), -XPA Ab-1 (1/200), -XPF Ab-1 (1/200), and -XPG Ab-1 = (1/200)=20 from Labvision, -Rad-51 (Pharmingen, 1/5000), -p21 sc-397 (Santa = Cruz=20 Biotechnology 1/1000), -p53 DO-1 (Merck, 1/1000), and -Rb (1/2000, = overnight, 4=C2=B0C), -phospho Ser 780-Rb (1/1000, overnight, = 4=C2=B0C), -phospho=20 Ser 795-Rb (1/1000, overnight, 4=C2=B0C), -phospho Ser 807/811-Rb = (1/1000,=20 overnight, 4=C2=B0C) from Cell Signalling. We used anti-tubulin = 9026 (Sigma,=20 1/5000). Incubation was 1 h at room temperature unless otherwise=20 specified, followed by peroxidase-labelled goat anti-mouse, goat=20 anti-rabbit or rabbit anti-goat antibody (Dako, 1/2000, 1 h, RT). = Blots=20 were developed using ECL (Amersham).

p53 activation assay

We = induced p53=20 upregulation and activation in UV-irradiated pre- and = post-senescence MSC,=20 TMC, and met-TMC (15 JU/m²). Extracts were collected 18 = h after=20 irradiation and used in western blot with anti-p53 or -phospho = Ser15-p53=20 antibodies. =CE=B1-tubulin was used as control.

Analysis of p16ink4a, p15ink4b = and p14ARF=20 CpG island methylation status

We determined DNA methylation = patterns=20 in the CpG islands of p16ink4a, p15ink4b and p14ARF tumor = suppressor genes=20 by chemical conversion of unmethylated, but not methylated, = cytosine to=20 uracil, followed by methyl-specific PCR (MSP) amplification using = primers=20 specific for methylated or modified unmethylated DNA [41],=20 [42].=20 Placental DNA treated in vitro with Sss I = methyltransferase was=20 used as positive control, and DNA from normal lymphocytes as = negative=20 control for methylated alleles. Each PCR sample (12 =C2=B5l) was = separated in=20 non-denaturing 6% polyacrylamide gels, ethidium bromide-stained, = and=20 visualized with UV illumination. Promoter methylation status of = these=20 genes was verified by bisulfite genomic sequencing of CpG islands. = Both=20 strands were sequenced. Primers for bisulfite genomic sequencing = and=20 methylation-specific PCR were designed according to genomic = sequences=20 around presumed transcription start sites of the genes studied. = Primer=20 sequences and PCR conditions for methylation analysis are = available on=20 request.

Bisulfite = treatment

Genomic DNA was=20 EcoRI-digested to shear DNA and achieve complete chemical = conversion after=20 bisulfite treatment. Sodium bisulfite conversion of genomic DNA (1 = =C2=B5g) was=20 performed as described [41],=20 [42],=20 with modifications. Briefly, NaOH was added to denature DNA (0.3 M = final=20 concentration) and incubated (15 min, 37=C2=B0C). Fresh bisulfite = solution (2.5=20 M sodium metabisulfite and 125 mM hydroquinone, pH 5.0) was added = to each=20 sample, and incubation continued (16 h, 50=C2=B0C, in the dark). = Modified DNA=20 was purified using Wizard DNA purification resin (Promega) and = eluted in=20 water at 60=C2=B0C. After desulfonation with NaOH (0.3 M final = concentration;=20 10 min, 37=C2=B0C), isolation was continued with 0.3 volume of = 10.5 M ammonium=20 acetate, followed by incubation (5 min, RT). Modified DNA was = precipitated=20 using 2.5 volumes of 100% ethanol and glycogen (5 mg/ml) as a = carrier. The=20 pellet was washed with 70% ethanol, dried, and eluted in distilled = water.

Homozygous deletion = analysis

We=20 analyzed fragments of the p16INK4a-E1=CE=B1, E2, p14ARF-E1=CE=B2, = and p15INK4b genes=20 as described [16]=20 to detect homozygous deletion in TMC. Comparative multiplex PCR = was=20 performed as described [43]=20 to analyze each gene locus, using the =CE=B2-actin fragment as = internal=20 control. Normal lymphocytes (NL) were used as negative control of = tumor=20 suppressor gene methylation, HCT116 (colorectal cancer line) as = positive=20 control of Ink4a/Arf locus methylation and MDA-MB231 (mammary=20 adenocarcinoma) as control of Ink4a/Arf locus deletion.

Top

Abstract

Introduction

Results

Discussion

Materials and=20 Methods

"=20 = src=3D"http://www.pubmedcentral.nih.gov/corehtml/pmc/pmcgifs/square.gif" = border=3D0>Supporting=20 Information

References

Supporting Information

Figure S1

Comparison of mRNA differences between pre- and post-senescence = MSC and=20 in TMC. Microarray analysis pattern of overall mRNA differences = between=20 pre- and post-senescence MSC (A), and pre-senescence MSC and TMC = (B). MA=20 plots are shown, being A: log-ratio of two expression intensities = vs. M:=20 the mean log-expression of the two.

(4.09 MB TIF)

Click=20 here for additional data file.^{=20
(3.9M)}

Table S1

Main mRNA differences between pre-senescence MSC and TMC = focused in=20 genes implicated in bioenergetic pathways.

(0.11 MB DOC)

Click=20 here for additional data file.^{=20
(104K)}

Table S2

Primers used for q-RT-PCR analysis with Universal ProbeLibrary=20 protocol.

(0.04 MB DOC)

Click=20 here for additional data file.^{=20
(35K)}

Acknowledgments

We thank L. Almonacid for qRT-PCR analysis, C. Mark and C. = Pantoll for=20 editorial support.

Footnotes

Competing Interests: The = authors=20 have declared that no competing interests exist.

Funding: DR and SG received = predoctoral=20 fellowships from the Spanish Ministry of Education and Science, = JG-C and L=20 L-F received postdoctoral fellowships from the Ministry of Science = and=20 Technology and the Ministerio de Sanidad y Consumo (FIS; CP03/0031 = and=20 CP06/0267). This work was partially supported by Spanish Ministry = of=20 Science and Technology (CICYT) grants SAF2001-2262, SAF2005-0864 = and=20 GEN2001-4856-C13-02 to AB. The Department of Immunology and = Oncology was=20 founded and is supported by the Spanish National Research Council = (CSIC)=20 and by Pfizer.

Top

Abstract

Introduction

Results

Discussion

Materials and=20 Methods

Supporting=20 Information

"=20 = src=3D"http://www.pubmedcentral.nih.gov/corehtml/pmc/pmcgifs/square.gif" = border=3D0>References

<= /TD>

References

Hahn WC, Weinberg RA. Rules for making human = tumor=20 cells. N Engl J=20 Med. 2002;347:1593=E2=80=931603. [PubMed]

Hahn WC, Weinberg RA. Modelling the molecular = circuitry=20 of cancer. Nat=20 Rev Cancer. 2002;2:331=E2=80=93341. [PubMed]

Reya T, Morrison SJ, Clarke MF, Weissman IL. = Stem=20 cells, cancer, and cancer stem cells. Nature.=20 2001;414:105=E2=80=93111. [PubMed]

Dalerba P, Cho RW, Clarke MF. Cancer stem = cells: models=20 and concepts. Annu Rev Med. 2007;58:267=E2=80=93284. [PubMed]

Pardal R, Clarke MF, Morrison SJ. Applying = the=20 principles of stem-cell biology to cancer. Nat Rev = Cancer.=20 2003;3:895=E2=80=93902. = [PubMed]

Rubio D, Garcia-Castro J, Martin MC, de la = Fuente R,=20 Cigudosa JC, et al. Spontaneous human adult stem cell = transformation.=20 Cancer Res.=20 2005;65:3035=E2=80=933039. [PubMed]

Miura M, Miura Y, Padilla-Nash HM, Molinolo = AA, Fu B,=20 et al. Accumulated Chromosomal Instability in Murine Bone Marrow=20 Mesenchymal Stem Cells Leads to Malignant Transformation. Stem = Cells.=20 2006;24:1095=E2=80=931103. [PubMed]

Tolar J, Nauta AJ, Osborn MJ, Panoskaltsis = Mortari A,=20 McElmurry RT, et al. Sarcoma Derived from Cultured Mesenchymal = Stem Cells.=20 Stem = Cells.=20 2006;25:371=E2=80=93379. [PubMed]

Wang Y, Huso DL, Harrington J, Kellner J, = Jeong DK, et=20 al. Outgrowth of a transformed cell population derived from normal = human=20 BM mesenchymal stem cell culture. Cytotherapy. 2005;7:509=E2=80=93519. [PubMed]

10.

Zhou YZ, Bosch-Marce M, Okuyama H, = Krishnamachary B,=20 Kimura H, et al. Spontaneous Transformation of Cultured Mouse Bone = Marrow=E2=80=93Derived Stromal Cells. 2006;66:10849=E2=80=9310854.

11.

Li H, Fan X, Kovi RC, Jo Y, Moquin B, et al.=20 Spontaneous Expression of Embryonic Factors and p53 Point = Mutations in=20 Aged Mesenchymal Stem Cells: A Model of Age-Related Tumorigenesis = In Mice.=20 Cancer Res.=20 2007;67:10889=E2=80=9310898. [PubMed]

12.

Funes JM, Quintero M, Henderson S, Martinez = D, Qureshi=20 U, et al. Transformation of human mesenchymal stem cells increases = their=20 dependency on oxidative phosphorylation for energy production. = Proc Natl = Acad Sci U S=20 A. 2007;104:6223=E2=80=936228. [PubMed]

13.

Serakinci N, Guldberg P, Burns JS, Abdallah = B,=20 Schrodder H, et al. Adult human mesenchymal stem cell as a target = for=20 neoplastic transformation. Oncogene. 2004;23:5095=E2=80=935098. [PubMed]

14.

Kastan MB, Bartek J. Cell-cycle checkpoints = and cancer.=20 Nature.=20 2004;432:316=E2=80=93323. [PubMed]

15.

Jacobs JJ, Kieboom K, Marino S, DePinho RA, = van=20 Lohuizen M. The oncogene and Polycomb-group gene bmi-1 regulates = cell=20 proliferation and senescence through the ink4a locus. Nature.=20 1999;397:164=E2=80=93168. [PubMed]

16.

Mund C, Beier V, Bewerunge P, Dahms M, Lyko = F, et al.=20 Array-based analysis of genomic DNA methylation patterns of the = tumour=20 suppressor gene p16INK4A promoter in colon carcinoma cell lines. = Nucleic = Acids Res.=20 2005;33:e73. [PubMed]

17.

Xing EP, Nie Y, Wang LD, Yang GY, Yang CS. = Aberrant=20 methylation of p16INK4a and deletion of p15INK4b are frequent = events in=20 human esophageal cancer in Linxian, China. Carcinogenesis.=20 1999;20:77=E2=80=9384. = [PubMed]

18.

Sherr CJ, McCormick F. The RB and p53 = pathways in=20 cancer. Cancer=20 Cell. 2002;2:103=E2=80=93112. [PubMed]

19.

Prockop DJ, Olson SD. Clinical trials with = adult=20 stem/progenitor cells for tissue repair: let's not overlook some = essential=20 precautions. Blood. 2007;109:3147=E2=80=933151. [PubMed]

20.

Tremain N, Korkko J, Ibberson D, Kopen GC, = DiGirolamo=20 C, et al. MicroSAGE analysis of 2,353 expressed genes in a single=20 cell-derived colony of undifferentiated human mesenchymal stem = cells=20 reveals mRNAs of multiple cell lineages. Stem = Cells.=20 2001;19:408=E2=80=93418. [PubMed]

21.

Kim NW, Piatyszek MA, Prowse KR, Harley CB, = West MD, et=20 al. Specific association of human telomerase activity with = immortal cells=20 and cancer. Science. 1994;266:2011=E2=80=932015. [PubMed]

22.

Shay JW, Wright WE. Telomerase activity in = human=20 cancer. Curr.=20 Opin. Oncol. 1996;8:66=E2=80=9371. [PubMed]

23.

Shay JW, Zou Y, Hiyama E, Wright WE. = Telomerase and=20 cancer. Hum Mol=20 Genet. 2001;10:677=E2=80=93685. [PubMed]

24.

Serakinci N, Hoare SF, Kassem M, Atkinson SP, = Keith WN.=20 Telomerase promoter reprogramming and interaction with general=20 transcription factors in the human mesenchymal stem cell. Regen Med. = 2006;1:125=E2=80=93131. = [PubMed]

25.

Yeh E, Cunningham M, Arnold H, Chasse D, = Monteith T, et=20 al. Signalling pathway controlling c-Myc degradation that impacts=20 oncogenic transformation of human cells. Nat Cell = Biol.=20 2004;6:308=E2=80=93318. = [PubMed]

26.

Daser A, Rabbitts TH. Extending the = repertoire of the=20 mixed-lineage leukemia gene MLL in leukemogenesis. Genes Dev. = 2004;18:965=E2=80=93974. [PubMed]

27.

Daibata M, Nemoto Y, Komatsu N, Machida H, = Miyoshi I,=20 et al. Constitutional t(3;11)(p21;q23) in a family, including one = member=20 with lymphoma: establishment of novel cell lines with this = translocation.=20 Cancer Genet=20 Cytogenet. 2000;117:28=E2=80=9331. [PubMed]

28.

Pegram LD, Megonigal MD, Lange BJ, Nowell PC, = Rowley=20 JD, et al. t(3;11) translocation in treatment-related acute = myeloid=20 leukemia fuses MLL with the GMPS (guanosine 5=E2=80=B2 = monophosphate synthetase)=20 gene. Blood.=20 2000;96:4360=E2=80=934362. [PubMed]

29.

Kourlas PJ, Strout MP, Becknell B, Veronese = ML, Croce=20 CM, et al. Identification of a gene at 11q23 encoding a guanine = nucleotide=20 exchange factor: evidence for its fusion with MLL in acute myeloid = leukemia. Proc=20 Natl Acad Sci U S A. 2000;97:2145=E2=80=932150. [PubMed]

30.

Chau BN, Wang JY. Coordinated regulation of = life and=20 death by RB. Nat=20 Rev Cancer. 2003;3:130=E2=80=93138. [PubMed]

31.

Gorgoulis VG, Zacharatos P, Kotsinas A, = Liloglou T,=20 Kyroudi A, et al. Alterations of the p16-pRb pathway and the = chromosome=20 locus 9p21-22 in non-small-cell lung carcinomas: relationship with = p53 and=20 MDM2 protein expression. Am J Pathol. 1998;153:1749=E2=80=931765. [PubMed]

32.

Burns JS, Abdallah BM, Guldberg P, Rygaard J, = Schroder=20 HD, et al. Tumorigenic heterogeneity in cancer stem cells evolved = from=20 long-term cultures of telomerase-immortalized human mesenchymal = stem=20 cells. Cancer=20 Res. 2005;65:3126=E2=80=933135. [PubMed]

33.

Sharpless NE. INK4a/ARF: a multifunctional = tumor=20 suppressor locus. Mutat Res. 2005;576:22=E2=80=9338. [PubMed]

34.

Matheu A, Pantoja C, Efeyan A, Criado LM,=20 Martin-Caballero J, et al. Increased gene dosage of Ink4a/Arf = results in=20 cancer resistance and normal aging. Genes Dev. = 2004;18:2736=E2=80=932746. [PubMed]

35.

Rea MA, Zhou L, Qin Q, Barrandon Y, Easley = KW, et al.=20 Spontaneous immortalization of human epidermal cells with = naturally=20 elevated telomerase. J Invest Dermatol. 2006;126:2507=E2=80=932515. [PubMed]

36.

Gil J, Kerai P, Lleonart M, Bernard D, = Cigudosa JC, et=20 al. Immortalization of primary human prostate epithelial cells by = c-Myc.=20 Cancer Res.=20 2005;65:2179=E2=80=932185. [PubMed]

37.

Tsutsui T, Kumakura S, Yamamoto A, Kanai H, = Tamura, et=20 al. Association of p16(INK4a) and pRb inactivation with = immortalization of=20 human cells Carcinogenesis. 2002;23:2111=E2=80=932117.

38.

Milyavsky M, Shats I, Erez N, Tang X, = Senderovich S, et=20 al. Prolonged culture of telomerase-immortalized human fibroblasts = leads=20 to a premalignant phenotype. Cancer Res. 2003;63:7147=E2=80=937157. [PubMed]

39.

Zongaro S, de Stanchina E, Colombo T, = D'Incalci M,=20 Giulotto E, et al. Stepwise neoplastic transformation of a = telomerase=20 immortalized fibroblast cell line. Cancer Res. 2005;65:11411=E2=80=9311418. [PubMed]

40.

Benanti JA, Wang ML, Myers HE, Robinson KL, = Grandori C,=20 et al. Epigenetic Down-Regulation of ARF Expression Is a Selection = Step in=20 Immortalization of Human Fibroblasts by c-Myc. Mol Cancer = Res.=20 2007;5:OF1=E2=80=93OF9.

41.

Wettenhall JM, Smyth GK. LimmaGUI: a = graphical user=20 interface for linear modeling of microarray data. Bioinformatics.=20 2004;20:3705=E2=80=933706. [PubMed]

42.

Ruiz JF, Lucas D, Garcia-Palomero E, Saez AI, = Gonzalez=20 MA, et al. Overexpression of human DNA polymerase mu (Pol mu) in a = Burkitt's lymphoma cell line affects the somatic hypermutation = rate. Nucleic = Acids Res.=20 2004;32:5861=E2=80=935873. [PubMed]

43.

Herman JG, Graff JR, Myohanen S, Nelkin BD, = Baylin SB.=20 Methylation-specific PCR: a novel PCR assay for methylation status = of CpG=20 islands. Proc=20 Natl Acad Sci U S A. 1996;93:9821=E2=80=939826. [PubMed]

Articles=20 from PLoS ONE = are provided=20 here courtesy of
Public Library of Science

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