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• W2001649025 abstract "Osteosarcoma is the most common primary bone cancer. Mutations of the RB gene represent the most frequent molecular defect in this malignancy. A major consequence of this alteration is that the activity of the key cell cycle regulator E2F1 is unleashed from the inhibitory effects of pRb. Studies in animal models and in human cancers have shown that deregulated E2F1 overexpression possesses either “oncogenic” or “oncosuppressor” properties, depending on the cellular context. To address this issue in osteosarcomas, we examined the status of E2F1 relative to cell proliferation and apoptosis in a clinical setting of human primary osteosarcomas and in E2F1-inducible osteosarcoma cell line models that are wild-type and deficient for p53. Collectively, our data demonstrated that high E2F1 levels exerted a growth-suppressing effect that relied on the integrity of the DNA damage response network. Surprisingly, induction of p73, an established E2F1 target, was also DNA damage response-dependent. Furthermore, a global proteome analysis associated with bioinformatics revealed novel E2F1-regulated genes and potential E2F1-driven signaling networks that could provide useful targets in challenging this aggressive neoplasm by innovative therapies. Osteosarcoma is the most common primary bone cancer. Mutations of the RB gene represent the most frequent molecular defect in this malignancy. A major consequence of this alteration is that the activity of the key cell cycle regulator E2F1 is unleashed from the inhibitory effects of pRb. Studies in animal models and in human cancers have shown that deregulated E2F1 overexpression possesses either “oncogenic” or “oncosuppressor” properties, depending on the cellular context. To address this issue in osteosarcomas, we examined the status of E2F1 relative to cell proliferation and apoptosis in a clinical setting of human primary osteosarcomas and in E2F1-inducible osteosarcoma cell line models that are wild-type and deficient for p53. Collectively, our data demonstrated that high E2F1 levels exerted a growth-suppressing effect that relied on the integrity of the DNA damage response network. Surprisingly, induction of p73, an established E2F1 target, was also DNA damage response-dependent. Furthermore, a global proteome analysis associated with bioinformatics revealed novel E2F1-regulated genes and potential E2F1-driven signaling networks that could provide useful targets in challenging this aggressive neoplasm by innovative therapies. Osteosarcoma is a mesenchymal malignancy in which the cells produce bone matrix. It is the most common primary bone cancer, accounting for approximately 20% of primary bone malignancies. Osteosarcomas occur mainly during adolescence, with a peak incidence during the growth spurt, at 15 to 19 years of age. Men are more commonly affected than women with a ratio of 1.6: 1. Notably, most osteosarcomas develop at sites of highest bone growth, where cell proliferation activity is at its crest. Such areas are the metaphyseal regions of the long bones of the extremities.1Meyers PA Gorlick R Osteosarcoma.Pediatr Clin North Am. 1997; 44: 973-989Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar At the molecular level, one of the most frequent defects found in osteosarcomas is genomic alterations of the RB (retinoblastoma) gene. Patients with hereditary retinoblastoma have up to 103 times greater risk for developing such tumors. Among sporadic osteosarcomas, alterations of RB occur in about 70% of cases.2Wadayama B Toguchida J Shimizu T Ishizaki K Sasaki MS Kotoura Y Yamamuro T Mutation spectrum of the retinoblastoma gene in osteosarcomas.Cancer Res. 1994; 54: 3042-3048PubMed Google Scholar The main biochemical activity of the pocket protein pRb is to control the transcription factor E2F1, a central modulator of cell cycle progression, by forming a complex with it during the G1-phase of the cell cycle. On growth stimulating signals pRb becomes hyperphosphorylated, by cyclin-dependent kinases, and releases E2F1, which then activates its target genes and thereby promotes the G1 to S-phase progression.3Tsantoulis PK Gorgoulis VG Involvement of E2F transcription factor family in cancer.Eur J Cancer. 2005; 41: 2403-2414Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 4Iaquinta PJ Lees JA Life and death decisions by the E2F transcription factors.Curr Opin Cell Biol. 2007; 19: 649-657Crossref PubMed Scopus (245) Google Scholar Beyond its pivotal role in G1 to S-phase transition, E2F1 also possesses the ability to induce apoptosis in a p53-dependent or -independent manner, mainly via p73.3Tsantoulis PK Gorgoulis VG Involvement of E2F transcription factor family in cancer.Eur J Cancer. 2005; 41: 2403-2414Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 5Bell LA Ryan KM Life and death decisions by E2F-1.Cell Death Differ. 2004; 11: 137-142Crossref PubMed Scopus (123) Google Scholar, 6Crighton D Ryan KM Splicing DNA-damage responses to tumor cell death.Biochim Biophys Acta. 2004; 1705: 3-15PubMed Google Scholar, 7Ginsberg D E2F1 pathways to apoptosis.FEBS Lett. 2002; 529: 122-125Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 8Urist M Tanaka T Poyurovsky MV Prives C p73 induction after DNA damage is regulated by checkpoint kinases Chk1 and Chk2.Genes Dev. 2004; 18: 3041-3054Crossref PubMed Scopus (192) Google Scholar, 9Irwin M Marin MC Phillips AC Seelan RS Smith DI Liu W Flores ER Tsai KY Jacks T Vousden KH Kaelin Jr, WG Role for the p53 homologue p73 in E2F-1-induced apoptosis.Nature. 2000; 407: 645-648Crossref PubMed Scopus (534) Google Scholar, 10Stiewe T Pützer BM Role of the p53-homologue p73 in E2F1-induced apoptosis.Nat Genet. 2000; 26: 464-469Crossref PubMed Scopus (307) Google Scholar The ability of a cellular factor to trigger both proliferation and apoptosis seems paradoxical. However, the rationale behind the connection of opposing functions by the same factor is that the cell consumes less time and energy to control the switch from the proliferation state to the self-destruction program in case of irreversible cellular damage, especially during DNA synthesis. A predicted biochemical consequence of deficient pRb expression is that E2F1 activity is constantly unleashed from the inhibitory effects of pRb. In such a setting the delicate balance between growth promoting and growth suppressing properties of E2F1 would be disrupted and the final outcome would possibly be dictated by the specific cellular background.3Tsantoulis PK Gorgoulis VG Involvement of E2F transcription factor family in cancer.Eur J Cancer. 2005; 41: 2403-2414Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 11La Thangue NB The yin and yang of E2F-1: balancing life and death.Nat Cell Biol. 2003; 5: 587-589Crossref PubMed Scopus (100) Google Scholar The bimodal impact of E2F1 has been demonstrated in various in vitro cellular systems and in vivo animal models.3Tsantoulis PK Gorgoulis VG Involvement of E2F transcription factor family in cancer.Eur J Cancer. 2005; 41: 2403-2414Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar Characteristically, loss of E2F1 in RB+/− mice increases tumor incidence within certain tissues and decreases it in others.12Yamasaki L Bronson R Williams BO Dyson NJ Harlow E Jacks T Loss of E2F-1 reduces tumorigenesis and extends the lifespan of Rb1(+/−) mice.Nat Genet. 1998; 18: 360-364Crossref PubMed Scopus (253) Google Scholar A similar multifarious pattern of E2F1 response appears to apply to human cancers, as well. In certain types of carcinomas, such as those derived from the lung, breast, thyroid, and pancreas, E2F1 seems to act as an oncogene13Gorgoulis VG Zacharatos P Mariatos G Kotsinas A Bouda M Kletsas D Asimacopoulos PJ Agnantis N Kittas C Papavassiliou AG Transcription factor E2F-1 acts as a growth-promoting factor and is associated with adverse prognosis in non-small cell lung carcinomas.J Pathol. 2002; 198: 142-156Crossref PubMed Scopus (140) Google Scholar, 14Zacharatos P Kotsinas A Evangelou K Karakaidos P Vassiliou LV Rezaei N Kyroudi A Kittas C Patsouris E Papavassiliou AG Gorgoulis VG Distinct expression patterns of the transcription factor E2F-1 in relation to tumor growth parameters in common human carcinomas.J Pathol. 2004; 203: 744-753Crossref PubMed Scopus (73) Google Scholar, 15Saiz AD Overa M Rezk S Florentine BA McCourty A Brynes RK Immunohistochemical expression of cyclin D1. E2F-1, and Ki-67 in benign and malignant thyroid lesions.J Pathol. 2002; 198: 157-162Crossref PubMed Scopus (89) Google Scholar, 16Yamazaki K Yajima T Nagao T Shinkawa H Kondo F Hanami K Asoh A Sugano I Ishida Y Expression of transcription factor E2F-1 in pancreatic ductal carcinoma: an immunohistochemical study.Pathol Res Pract. 2003; 199: 23-28Crossref PubMed Scopus (37) Google Scholar whereas, in others, such as carcinomas of the colon and prostate, an onco-suppressor role has been proposed.14Zacharatos P Kotsinas A Evangelou K Karakaidos P Vassiliou LV Rezaei N Kyroudi A Kittas C Patsouris E Papavassiliou AG Gorgoulis VG Distinct expression patterns of the transcription factor E2F-1 in relation to tumor growth parameters in common human carcinomas.J Pathol. 2004; 203: 744-753Crossref PubMed Scopus (73) Google Scholar The embryological origin of the organ, as exemplified by esophageal cancer17Evangelou K Kotsinas A Mariolis-Sapsakos T Giannopoulos A Tsantoulis PK Constantinides C Troupis TG Salmas M Kyroudis A Kittas C Gorgoulis VG E2F-1 overexpression correlates with decreased proliferation and better prognosis in adenocarcinomas of Barrett's oesophagus.J Clin Pathol. 2008; 61: 601-605Crossref PubMed Scopus (23) Google Scholar, 18Yamazaki K Hasegawa M Ohoka I Hanami K Asoh A Nagao T Sugano I Ishida Y Increased E2F-1 expression via tumor cell proliferation and decreased apoptosis are correlated with adverse prognosis in patients with squamous cell carcinoma of the oesophagus.J Clin Pathol. 2005; 58: 904-910Crossref PubMed Scopus (27) Google Scholar may also modulate the phenotype of E2F1-driven tumors. In contrast to epithelial tumors, there are no data about the expression status and role of endogenous E2F1 in tumors of mesenchymal origin. This information is important in view of E2F1’s role in sensitizing cells to various chemotherapeutic agents.19Banerjee D Schnieders B Fu JZ Adhikari D Zhao S-C Bertino JR Role of E2F-1 in chemosensitivity.Cancer Res. 1998; 58: 4292-4296PubMed Google Scholar To address these questions we examined the status of E2F1 in relation to tumor kinetics [proliferation index: % of tumor cells proliferating, and apoptotic index: % of apoptotic tumor cells in a tumor, respectively], and nodal cell cycle regulators in a series of primary osteosarcoma specimens. The functional basis of the findings obtained with the clinical samples was then investigated in human osteosarcoma cell line models with inducible E2F1, harboring either wild-type or mutant p53. Finally, a global proteome and bioinformatic analysis was performed in an attempt to obtain a broader picture of the potential pathways that could follow E2F1 induction; thus helping us understand better the multifaceted nature of the E2F1-mediated response and design more rational E2F1-targeted therapeutic strategies in the future. Formalin-fixed, paraffin-embedded sections from 57 surgically removed osteosarcomas were analyzed, after local ethical committee approval. The samples were taken from patients that had not undergone any chemo- or radiotherapy before surgical resection. The majority of patients were diagnosed and treated in the General Hospital of Asklepeion Voula, Athens, Greece. Clinicopathological features of the patients are presented in Table 1.Table 1Clinicopathological DataAgeRange 7–82 yearsMean 22.8 yearsSexMaleFemale32 (56.1%)25 (43.9%)Histologic variantOsteoblasticChondroblasticCombined42 (73.7%)6 (10.5%)9 (15.8%)GradeLowModerateHigh18 (31.6%)20 (35.1%)19 (33.3%)Survival*5 year follow-up period.YesNo13 (22.8%)44 (77.2%)First symptomPainSwellingPain and swellingPain, swelling, and limp22 (38.6%)4 (7%)21 (36.8%)10 (17.6%)Tumor locationLower third femurUpper third tibiaRest femurOther sites**Distribution: 5 Humerus, 3 Thumb, 1 Fibula.22 (38.6%)15 (26.3%)11 (19.3%)9 (15.8%)Duration***Time intervening between the emergence of the first symptom and the final diagnosis.≤3 Months3–6 Months>6 Months29 (50.8%)22 (38.6%)6 (10.6%)NecrosisRange (%)Mean (%)SD (%)20–9671.225.4Extent90%80%70%–50%≤50%20 (35.1%)8 (14%)17 (29.9%)12 (21%)SD, standard deviation.* 5 year follow-up period.** Distribution: 5 Humerus, 3 Thumb, 1 Fibula.*** Time intervening between the emergence of the first symptom and the final diagnosis. Open table in a new tab SD, standard deviation. The following antibodies were used at 1:100 dilution: anti-E2F1 (KH95, Santa Cruz, Bioanalytica, Athens, Greece), anti-Ki-67 (MIB-1, DAKO, Kalifronas, Athens, Greece), anti-p53 (DO7, DAKO, Kalifronas, Athens, Greece), anti-pRB (IF8, Santa Cruz, Bioanalytica, Athens, Greece), anti-phospho-pRb (Ser795-R, Santa Cruz, Bioanalytica, Athens, Greece), anti-phospho-H2AX (Ser139, Millipore, Lab Supplies, Athens, Greece), and anti-phospho-Chk2 (Thr68, Cell Signaling, Bioline, Athens, Greece). Immunohistochemistry was performed according to the indirect streptavidin–biotin–peroxidase method, as previously described with a modification in the heat-mediated antigen retrieval method.13Gorgoulis VG Zacharatos P Mariatos G Kotsinas A Bouda M Kletsas D Asimacopoulos PJ Agnantis N Kittas C Papavassiliou AG Transcription factor E2F-1 acts as a growth-promoting factor and is associated with adverse prognosis in non-small cell lung carcinomas.J Pathol. 2002; 198: 142-156Crossref PubMed Scopus (140) Google Scholar, 14Zacharatos P Kotsinas A Evangelou K Karakaidos P Vassiliou LV Rezaei N Kyroudi A Kittas C Patsouris E Papavassiliou AG Gorgoulis VG Distinct expression patterns of the transcription factor E2F-1 in relation to tumor growth parameters in common human carcinomas.J Pathol. 2004; 203: 744-753Crossref PubMed Scopus (73) Google Scholar Specifically, unmasking of the related proteins was carried out in a 10 mmol/L citrate solution with pH = 6.0 for 50 minutes at 95°C in a steamer. Evaluation of the E2F1, Ki-67 (proliferation index), p53, γH2AX, and Chk2-pT68 expression was performed as previously reported,13Gorgoulis VG Zacharatos P Mariatos G Kotsinas A Bouda M Kletsas D Asimacopoulos PJ Agnantis N Kittas C Papavassiliou AG Transcription factor E2F-1 acts as a growth-promoting factor and is associated with adverse prognosis in non-small cell lung carcinomas.J Pathol. 2002; 198: 142-156Crossref PubMed Scopus (140) Google Scholar, 14Zacharatos P Kotsinas A Evangelou K Karakaidos P Vassiliou LV Rezaei N Kyroudi A Kittas C Patsouris E Papavassiliou AG Gorgoulis VG Distinct expression patterns of the transcription factor E2F-1 in relation to tumor growth parameters in common human carcinomas.J Pathol. 2004; 203: 744-753Crossref PubMed Scopus (73) Google Scholar, 20Gorgoulis VG Vassiliou LV Karakaidos P Zacharatos P Kotsinas A Liloglou T Venere M Ditullio Jr, RA Kastrinakis NG Levy B Kletsas D Yoneta A Herlyn M Kittas C Halazonetis TD Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions.Nature. 2005; 434: 907-913Crossref PubMed Scopus (1688) Google Scholar and included counting of an average of 500 cells at ×400 per case, for each antibody, respectively. Only nuclear staining was considered as positive. Three independent observers performed slide examination. Interobserver variability was minimal. MCF-7 breast cancer cells were used as positive control for E2F1 expression. Sections from previously characterized tumors13Gorgoulis VG Zacharatos P Mariatos G Kotsinas A Bouda M Kletsas D Asimacopoulos PJ Agnantis N Kittas C Papavassiliou AG Transcription factor E2F-1 acts as a growth-promoting factor and is associated with adverse prognosis in non-small cell lung carcinomas.J Pathol. 2002; 198: 142-156Crossref PubMed Scopus (140) Google Scholar, 14Zacharatos P Kotsinas A Evangelou K Karakaidos P Vassiliou LV Rezaei N Kyroudi A Kittas C Patsouris E Papavassiliou AG Gorgoulis VG Distinct expression patterns of the transcription factor E2F-1 in relation to tumor growth parameters in common human carcinomas.J Pathol. 2004; 203: 744-753Crossref PubMed Scopus (73) Google Scholar, 20Gorgoulis VG Vassiliou LV Karakaidos P Zacharatos P Kotsinas A Liloglou T Venere M Ditullio Jr, RA Kastrinakis NG Levy B Kletsas D Yoneta A Herlyn M Kittas C Halazonetis TD Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions.Nature. 2005; 434: 907-913Crossref PubMed Scopus (1688) Google Scholar for Ki-67, p53, γH2AX, and Chk2-pT68 staining, were used as positive controls for the corresponding antibodies. Antibodies of the corresponding Ig class, but of unrelated specificity were used as negative controls. Double-stranded DNA breaks were detected by Tdt-mediated dUTP nick end labeling assay, according to the protocol described elsewhere.14Zacharatos P Kotsinas A Evangelou K Karakaidos P Vassiliou LV Rezaei N Kyroudi A Kittas C Patsouris E Papavassiliou AG Gorgoulis VG Distinct expression patterns of the transcription factor E2F-1 in relation to tumor growth parameters in common human carcinomas.J Pathol. 2004; 203: 744-753Crossref PubMed Scopus (73) Google Scholar Cells were considered to undergo apoptosis when nuclear staining from the fragmented DNA, without cytoplasmic background, was observed. The apoptotic index was estimated as the percentage of cancer cells with nuclear staining. Slide examination was performed by three independent observers, with minimal interobserver variability. Sections incubated with DNase I, before treatment with TdT, were used as positive controls, while sections without TdT addition were used as negative controls. The growth index of the samples was estimated by the proliferation to apoptosis index ratio, as previously described.13Gorgoulis VG Zacharatos P Mariatos G Kotsinas A Bouda M Kletsas D Asimacopoulos PJ Agnantis N Kittas C Papavassiliou AG Transcription factor E2F-1 acts as a growth-promoting factor and is associated with adverse prognosis in non-small cell lung carcinomas.J Pathol. 2002; 198: 142-156Crossref PubMed Scopus (140) Google Scholar, 14Zacharatos P Kotsinas A Evangelou K Karakaidos P Vassiliou LV Rezaei N Kyroudi A Kittas C Patsouris E Papavassiliou AG Gorgoulis VG Distinct expression patterns of the transcription factor E2F-1 in relation to tumor growth parameters in common human carcinomas.J Pathol. 2004; 203: 744-753Crossref PubMed Scopus (73) Google Scholar DNA extraction was performed according to previous protocol.13Gorgoulis VG Zacharatos P Mariatos G Kotsinas A Bouda M Kletsas D Asimacopoulos PJ Agnantis N Kittas C Papavassiliou AG Transcription factor E2F-1 acts as a growth-promoting factor and is associated with adverse prognosis in non-small cell lung carcinomas.J Pathol. 2002; 198: 142-156Crossref PubMed Scopus (140) Google Scholar Genomic analysis of exons 4 to 10 of p53 and exons 2 to 14 of p73 for mutation presence, respectively, was accomplished as formerly mentioned13Gorgoulis VG Zacharatos P Mariatos G Kotsinas A Bouda M Kletsas D Asimacopoulos PJ Agnantis N Kittas C Papavassiliou AG Transcription factor E2F-1 acts as a growth-promoting factor and is associated with adverse prognosis in non-small cell lung carcinomas.J Pathol. 2002; 198: 142-156Crossref PubMed Scopus (140) Google Scholar, 14Zacharatos P Kotsinas A Evangelou K Karakaidos P Vassiliou LV Rezaei N Kyroudi A Kittas C Patsouris E Papavassiliou AG Gorgoulis VG Distinct expression patterns of the transcription factor E2F-1 in relation to tumor growth parameters in common human carcinomas.J Pathol. 2004; 203: 744-753Crossref PubMed Scopus (73) Google Scholar, 21Yoshikawa H Nagashima M Khan MA McMenamin MG Hagiwara K Harris CC Mutational analysis of p73 and p53 in human cancer cell lines.Oncogene. 1999; 18: 3415-3421Crossref PubMed Scopus (56) Google Scholar on an ABI-PRISM 377 automatic sequencer (Applied Biosystems, Biosolutions, Athens, Greece). To assess allelic imbalance presence at the TP53 locus, the D17S179E polymorphic pentanucleotide marker, located within the first TP53 intron, was analyzed as previously described,14Zacharatos P Kotsinas A Evangelou K Karakaidos P Vassiliou LV Rezaei N Kyroudi A Kittas C Patsouris E Papavassiliou AG Gorgoulis VG Distinct expression patterns of the transcription factor E2F-1 in relation to tumor growth parameters in common human carcinomas.J Pathol. 2004; 203: 744-753Crossref PubMed Scopus (73) Google Scholar on an ABI-PRISM 377 automatic sequencer (Applied Biosystems, Biosolutions, Athens, Greece). Gene amplification was assessed with differential-PCR as described elsewhere.22Liontos M Koutsami M Sideridou M Evangelou K Kletsas D Levy B Kotsinas A Nahum O Zoumpourlis V Kouloukoussa M Lygerou Z Taraviras S Kittas C Bartkova J Papavassiliou AG Bartek J Halazonetis TD Gorgoulis VG Deregulated overexpression of hCdt1 and hCdc6 promotes malignant behavior.Cancer Res. 2007; 67: 10899-10909Crossref PubMed Scopus (159) Google Scholar Primers and annealing temperature used for MDM2 gene amplification were: 5′-TACCATGATCTACAGGAACTT-3′ and 5′-CTCAGTATGTGGTTTTAGTTC-3′ at 60°C. The U2OS E2F1-ER and Saos2 E2F1-ER cells20Gorgoulis VG Vassiliou LV Karakaidos P Zacharatos P Kotsinas A Liloglou T Venere M Ditullio Jr, RA Kastrinakis NG Levy B Kletsas D Yoneta A Herlyn M Kittas C Halazonetis TD Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions.Nature. 2005; 434: 907-913Crossref PubMed Scopus (1688) Google Scholar, 23Bartkova J Horejsí Z Koed K Krämer A Tort F Zieger K Guldberg P Sehested M Nesland JM Lukas C Ørntoft T Lukas J Bartek J DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis.Nature. 2005; 434: 864-870Crossref PubMed Scopus (2224) Google Scholar, 24Hershko T Chaussepied M Oren M Ginsberg D Novel link between E2F and p53: proapoptotic cofactors of p53 are transcriptionally upregulated by E2F.Cell Death Differ. 2005; 12: 377-383Crossref PubMed Scopus (91) Google Scholar were grown in Dulbeco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and incubated at 37°C and 5% CO2. The phoenix amphotropic helper-free retrovirus producer line was used to construct retroviruses containing the pRETROSUPER-short-hairpin(sh)p53, pRETROSUPER-shp14alternate reading frame(ARF) and the corresponding pRETROSUPERshLaz control.22Liontos M Koutsami M Sideridou M Evangelou K Kletsas D Levy B Kotsinas A Nahum O Zoumpourlis V Kouloukoussa M Lygerou Z Taraviras S Kittas C Bartkova J Papavassiliou AG Bartek J Halazonetis TD Gorgoulis VG Deregulated overexpression of hCdt1 and hCdc6 promotes malignant behavior.Cancer Res. 2007; 67: 10899-10909Crossref PubMed Scopus (159) Google Scholar Infection of the U2OS E2F1-ER or Saos2 E2F1-ER cells was performed at 12 hours intervals in 60-mm dishes, as previously described.22Liontos M Koutsami M Sideridou M Evangelou K Kletsas D Levy B Kotsinas A Nahum O Zoumpourlis V Kouloukoussa M Lygerou Z Taraviras S Kittas C Bartkova J Papavassiliou AG Bartek J Halazonetis TD Gorgoulis VG Deregulated overexpression of hCdt1 and hCdc6 promotes malignant behavior.Cancer Res. 2007; 67: 10899-10909Crossref PubMed Scopus (159) Google Scholar For ATM or p73 silencing, 3 × 105 cells were plated in 60-mm dishes and next day transfected using Lipofectamine 2000 (Invitrogen, Anti-Sel, Athens, Greece) with the appropriate Stealth Select RNAi pool or the corresponding RNAi negative control (Invitrogen, Anti-Sel, Athens, Greece), respectively, according to manufacturer’s instructions. To study the effects of p53, ATM, p73, and p14ARF on E2F1 behavior we activated the U2OS E2F1-ER or Saos2 E2F1-ER cellular systems 24 hours after siRNA interference.25Brummelkamp TR Bernards R Agami R A system for stable expression of short interfering RNAs in mammalian cells.Science. 2002; 296: 550-553Crossref PubMed Scopus (3949) Google Scholar To observe effects later than 72 hours, the cells of interest were retransfected. Indirect immunofluorescence analysis was performed according to a previously described protocol.22Liontos M Koutsami M Sideridou M Evangelou K Kletsas D Levy B Kotsinas A Nahum O Zoumpourlis V Kouloukoussa M Lygerou Z Taraviras S Kittas C Bartkova J Papavassiliou AG Bartek J Halazonetis TD Gorgoulis VG Deregulated overexpression of hCdt1 and hCdc6 promotes malignant behavior.Cancer Res. 2007; 67: 10899-10909Crossref PubMed Scopus (159) Google Scholar In brief, cells grown on coverslips were fixed with methanol and subsequently incubated with the primary antibody overnight at 4°C. Subsequently, cells were incubated with the Oregon Green-conjugated secondary antibody (O-6380, Invitrogen, Anti-Sel, Athens, Greece), counterstained with 4,6-diamidino-2-phenylindole (Sigma) and mounted with Fluoromount G (#0100–01, SouthernBiotech, AlterChem, Athens, Greece). The antibodies used were: anti-E2F1 (1:100)(KH-95, Santa Cruz, Bioanalytica, Athens, Greece), anti-Cyclin B1 (1:100)(H-20, Santa Cruz, Bioanalytica, Athens, Greece), and anti-γH2AX-pS139 (1:100)(JBW301, Millipore, Lab-Supplies, Athens, Greece). Total protein extraction from cells was performed according to protocols described previously.20Gorgoulis VG Vassiliou LV Karakaidos P Zacharatos P Kotsinas A Liloglou T Venere M Ditullio Jr, RA Kastrinakis NG Levy B Kletsas D Yoneta A Herlyn M Kittas C Halazonetis TD Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions.Nature. 2005; 434: 907-913Crossref PubMed Scopus (1688) Google Scholar, 22Liontos M Koutsami M Sideridou M Evangelou K Kletsas D Levy B Kotsinas A Nahum O Zoumpourlis V Kouloukoussa M Lygerou Z Taraviras S Kittas C Bartkova J Papavassiliou AG Bartek J Halazonetis TD Gorgoulis VG Deregulated overexpression of hCdt1 and hCdc6 promotes malignant behavior.Cancer Res. 2007; 67: 10899-10909Crossref PubMed Scopus (159) Google Scholar Thirty μg of protein from total extracts from each sample were adjusted with NuPAGE LDS Sample Buffer (Invitrogen, Anti-Sel, Athens, Greece) and loaded on 4% to 12% gradient NuPAGE precast gels (Invitrogen, Anti-Sel, Athens, Greece), according to manufacturer’s instructions. Gel electrophoresis, transfer to polyvinylidene difluoride membrane (Millipore, Lab Supplies, Athens, Greece), blotting, immunodetection, and signal development were performed as previously described.20Gorgoulis VG Vassiliou LV Karakaidos P Zacharatos P Kotsinas A Liloglou T Venere M Ditullio Jr, RA Kastrinakis NG Levy B Kletsas D Yoneta A Herlyn M Kittas C Halazonetis TD Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions.Nature. 2005; 434: 907-913Crossref PubMed Scopus (1688) Google Scholar, 22Liontos M Koutsami M Sideridou M Evangelou K Kletsas D Levy B Kotsinas A Nahum O Zoumpourlis V Kouloukoussa M Lygerou Z Taraviras S Kittas C Bartkova J Papavassiliou AG Bartek J Halazonetis TD Gorgoulis VG Deregulated overexpression of hCdt1 and hCdc6 promotes malignant behavior.Cancer Res. 2007; 67: 10899-10909Crossref PubMed Scopus (159) Google Scholar The antibodies used were: anti-γH2AX-pS139 (1:500) (Millipore, Lab-Supplies, Athens, Greece), anti-p53-pS15 (1:500) (Cell Signaling, Bioline, Athens, Greece), anti-p53 (1:500) (DAKO, Kalifronas, Athens, Greece), anti-pChk2 T68 (1:500) (Cell Signaling, Bioline, Athens, Greece), anti-Chk2 (hybridoma supernatant),20Gorgoulis VG Vassiliou LV Karakaidos P Zacharatos P Kotsinas A Liloglou T Venere M Ditullio Jr, RA Kastrinakis NG Levy B Kletsas D Yoneta A Herlyn M Kittas C Halazonetis TD Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions.Nature. 2005; 434: 907-913Crossref PubMed Scopus (1688) Google Scholar anti-Cyclin B1 (1:500) (SantaCruz, Bioanalytica, Athens, Greece), anti-p73 (1:300) (NeoMarkers, Bioanalytica, Athens, Greece), anti-β-actin (1:1000) (Abcam, Anti-Sel, Athens, Greece), anti-Csn8 (1:500) (SantaCruz, Bioanalytica, Athens, Greece), anti-ATM (1:500) (Abcam, Anti-Sel, Athens, Greece), anti-p14ARF (1:500) (Neomarkers, Bioanalytica, Athens, Greece), anti-BUB3 (1:500) (Abcam, Anti-Sel, Athens, Greece), anti-COP9 subunit8 (1:500) (Abcam, Anti-Sel, Athens, Greece), anti-Cdt1 (1:500) (SantaCruz, Bioanalytica, Athens, Greece), anti AuroraA (1:500) (Abcam, Anti-Sel, Athens, Greece), and anti-HNRPU (a kind gift from Dr Guialis A.). The growth of cells was monitored as mentioned elsewhere.22Liontos M Koutsami M Sideridou M Evangelou K Kletsas D Levy B Kotsinas A Nahum O Zoumpourlis V Kouloukoussa M Lygerou Z Taraviras S Kittas C Bartkova J Papavassiliou AG Bartek J Halazonetis TD Gorgoulis VG Deregulated overexpression of hCdt1 and hCdc6 promotes malignant behavior.Cancer Res. 2007; 67: 10899-10909Crossref PubMed Scopus (159) Google Scholar Cells were seeded into 12-well plates at a density of 25 × 103 cells/ml in a total volume of 1 ml/well. One day after seeding, 4-OH Tamoxifen (Sigma Hellas, Athens, Greece) was added to the medium of half of the wells at a final concentration of 300 nmol/L. Cells were harvested at days 0, 1, 2, 3, 4, and 5 after 4-OH Tamoxifen administration. For each time point the number of cells from four wells was counted: two wells with 4-OH Tamoxifen treatment and the corresponding untreated ones. Cells were trypsinized, centrifuged, and resuspended with 1 ml of medium and counted with a Newbauer Hematocytometer. The whole procedure was repeated in triplicate and counting was performed by two independent investigators. Approximately 5 × 105 cells were seeded per 100-mm dish and cultured according to t" @default.
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• W2001649025 title "Modulation of the E2F1-Driven Cancer Cell Fate by the DNA Damage Response Machinery and Potential Novel E2F1 Targets in Osteosarcomas" @default.
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• W2001649025 doi "https://doi.org/10.2353/ajpath.2009.081160" @default.
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