Phospho-Ku70 induced by DNA damage interacts with RNA Pol II and promotes the formation of phospho-53BP1 foci to ensure optimal cNHEJ.

Canonical non-homologous end-joining (cNHEJ) is the prominent mammalian DNA double-strand breaks (DSBs) repair pathway operative throughout the cell cycle. Phosphorylation of Ku70 at ser27-ser33 (pKu70) is induced by DNA DSBs and has been shown to regulate cNHEJ activity, but the underlying mechanism remained unknown. Here, we established that following DNA damage induction, Ku70 moves from nucleoli to the sites of damage, and once linked to DNA, it is phosphorylated. Notably, the novel emanating functions of pKu70 are evidenced through the recruitment of RNA Pol II and concomitant formation of phospho-53BP1 foci. Phosphorylation is also a prerequisite for the dynamic release of Ku70 from the repair complex through neddylation-dependent ubiquitylation. Although the non-phosphorylable ala-Ku70 form does not compromise the formation of the NHEJ core complex per se, cells expressing this form displayed constitutive and stress-inducible chromosomal instability. Consistently, upon targeted induction of DSBs by the I-SceI meganuclease into an intrachromosomal reporter substrate, cells expressing pKu70, rather than ala-Ku70, are protected against the joining of distal DNA ends. Collectively, our results underpin the essential role of pKu70 in the orchestration of DNA repair execution in living cells and substantiated the way it paves the maintenance of genome stability.

Allelic loss of chromosomes 8 and 19 in MENX-associated rat pheochromocytoma.

Pheochromocytomas are neoplasias of neural crest origin that arise from the chromaffin cells of the adrenal medulla. Pheochromocytomas arise with complete penetrance in rats homozygous for a germ-line frameshift mutation of Cdkn1b, encoding the cell cycle inhibitor p27KIP1 (MENX syndrome). We performed a genome-wide scan for allelic imbalance comparing 20 rat pheochromocytoma DNAs with normal rat DNA to better understand the pathobiology of the tumors and to correlate the findings with human pheochromocytoma. We identified allelic imbalance (AI) at candidate regions on rat chromosomes 8 and 19. Interestingly, the regions often lost in rat tumors are syntenic to regions involved in human pheochromocytomas. Fluorescence in situ hybridization analysis further validated the AI data. Sdhd and Rassf1a were analyzed in detail as they map to regions of AI on chromosome 8 and their homologues are implicated in human pheochromocytoma: we found no genetic mutations nor decreased expression. We also analyzed additional candidate genes, that is, rat homologues of genes predisposing to human pheochromocytoma and known tumor-suppressor genes, but we found no AI. In contrast, we observed frequent overexpression of Cdkn2a and Cdkn2c, encoding the cell cycle inhibitors p16INK4a and p18INK4c, respectively. The relative small number of allelic changes we found in rat pheochromocytoma might be related to their nonmalignant status and losses at chromosomes 8 and 19 are events that precede malignancy. Because of the high concordance of affected loci between rat and human tumors, studies of the MENX-associated pheochromocytomas should facilitate the identification of novel candidate genes implicated in their human counterpart.

Gene expression profiling of alpha-radiation-induced rat osteosarcomas: identification of dysregulated genes involved in radiation-induced tumorigenesis of bone.

To better understand the molecular basis of radiation-induced osteosarcoma (OS), we performed global gene expression profiling of rat OS tumors induced by the bone-seeking alpha emitter (238)Pu, and the expression profiles were compared with those of normal osteoblasts (OB). The expressions of 72 genes were significantly differentially expressed in the tumors related to OB. These included genes involved in the cell adhesion (e.g., Podxl, Col18a1, Cd93, Emcn and Vcl), differentiation, developmental processes (e.g., Hhex, Gata2, P2ry6, P2rx5, Cited2, Osmr and Igsf10), tumor-suppressor function (e.g., Nme3, Blcap and Rrm1), Src tyrosine kinase signaling (e.g., Hck, Shf, Arhgap29, Cttn and Akap12), and Wnt/beta-catenin signaling (e.g., Fzd6, Lzic, Dkk3 and Ctnna1) pathways. Expression changes of several genes were validated by quantitative real-time RT-PCR analysis. Notably, all of the identified genes involved in the Wnt/beta-catenin signaling pathway were known or proposed to be negative regulators of this pathway and were downregulated in the tumors, suggesting the activation of beta-catenin in radiation-induced OS. By using immunohistochemical and immunoblot analyses, constitutive activation of the Wnt/beta-catenin signaling pathway in the tumors was confirmed by observing nuclear and/or cytoplasmic localization of beta-catenin and a decrease in its inactive (phosphorylated) form. Furthermore, we found a significant reduction in the levels of glycogen synthase kinase 3beta (GSK-3beta) protein in the tumors relative to OB. Taken together, these findings provide new insights into the molecular basis of radiation-induced OS.

Silencing of Cited2 and Akap12 genes in radiation-induced rat osteosarcomas.

We have previously studied genomic copy number changes and global gene expression patterns in rat osteosarcomas (OS) induced by the bone-seeking alpha emitter (238)Pu by comparative genomic hybridization (CGH) and oligonucleotide microarray analyses, respectively. Among the previously identified genes that were down-regulated in radiation-induced rat OS tumors, Cited2 (Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2) and Akap12 (a kinase anchoring protein, also known as src-suppressed C-kinase substrate, SSeCKS) genes mapped to the most frequently lost regions on chromosome 1p. In the present study, relative copy number losses of Cited2 and Akap12 genes were observed in 8 of 15 (53%) and 10 of 15 (67%) tumors by quantitative PCR analysis. Loss of Cited2 and Akap12 in the tumors was confirmed at the levels of mRNA and protein expression by quantitative RT-PCR and immunoblot analyses, respectively. These results indicate that Cited2 and Akap12 are silenced in radiation-induced OS, and therefore are novel candidate tumor-suppressor genes of this tumor.

Molecular analysis of the Ink4a/Rb1-Arf/Tp53 pathways in radon-induced rat lung tumors.

Inhalation of radon is closely associated with an increased risk of lung cancers. While the involvement of Ink4a in lung tumor development has been widely described, the tumor suppressor gene has not been studied in radon-induced lung tumors. In this study, loss of heterozygosity (LOH) analysis of the Cdkn2a locus, common to the Ink4a and Arf genes, was performed on 33 radon-induced rat lung tumors and showed a DNA loss in 50% of cases. The analysis of p16(Ink4a) protein expression by immunohistochemistry revealed that 50% of the tumors were negative for this protein. Looking for the origin of this lack of expression, we observed a low frequency of homozygous deletion (6%), a lack of mutation, an absence of correlation between promoter methylation and Ink4a mRNA expression and no correlation between LOH and protein expression. However, a tendency for an inverse correlation between p16(Ink4a) and pRb protein expression was observed. The expressions of p19Arf, Mmd2 and Mdm4 were not deregulated and only 14% of the tumors were mutated for Tp53. These results indicated that Ink4a/Cdk4/Rb1 pathway deregulation, more than Arf/Mdm2/Tp53 pathway, has a major role in the development of these tumors through p16(Ink4a) deregulation. However, all known mechanisms of inactivation of the pathway do not play a recurrent role in these tumors and the actual origin of the lack of p16(Ink4a) protein expression remains to be established.

PARP-1 cooperates with Ptc1 to suppress medulloblastoma and basal cell carcinoma.

The patched (Ptc1) protein is a negative regulator of sonic hedgehog signaling, a genetic pathway whose perturbation causes developmental defects and predisposition to specific malignant tumors. Humans and mice with mutated Ptc1 are prone to medulloblastoma and basal cell carcinoma (BCC), both tumors showing dependence on radiation damage for rapid onset and high penetrance. Poly(ADP-ribose) polymerase (PARP-1) is a nuclear enzyme that plays a multifunctional role in DNA damage signaling and repair. In healthy and fertile PARP-1-null mice, radiation exposure reveals an extreme sensitivity and a high genomic instability. To test for interactions between PARP-1 and sonic hedgehog signaling, PARP-1-null mice were crossed to Ptc1 heterozygous mice. PARP-1 deletion further accelerated medulloblastoma development in irradiated Ptc1(+/-) mice, showing that PARP-1 inactivation sensitizes cerebellar cells to radiation tumorigenic effects. In addition to increased formation and slowed down kinetics of disappearance of gamma-H2AX foci, we observed increased apoptosis in PARP-1-deficient granule cell progenitors after irradiation. Double-mutant mice were also strikingly more susceptible to BCC, with >50% of animals developing multiple, large, infiltrative tumors within 30 weeks of age. The results provide genetic evidence that PARP-1 function suppresses sonic hedgehog pathway-associated tumors arising in response to environmental stress.

Characterization of preneoplastic and neoplastic rat mesothelial cell lines: the involvement of TETs, DNMTs, and 5-hydroxymethylcytosine.

Malignant mesothelioma (MM) is one of the worst cancers in terms of clinical outcome, urging the need to establish and characterize new preclinical tools for investigation of the tumorigenic process, improvement of early diagnosis and evaluation of new therapeutic strategies. For these purposes, we characterized a collection of 27 cell lines established from F344 rats, after 136 to 415 days of induction with crocidolite asbestos administered intraperitoneally. Four mesotheliomas were distinguished from 23 preneoplastic mesothelial cell lines (PN) according to their propensity to generate tumors after orthotopic transplantation into syngeneic rats, their growth pattern, and the expression profile of three genes. PN cell lines were further discriminated into groups / subgroups according to morphology in culture and the expression profiles of 14 additional genes. This approach was completed by analysis of positive and negative immunohistochemical MM markers in the four tumors, of karyotype alterations in the most aggressive MM cell line in comparison with a PN epithelioid cell line, and of human normal mesothelial and mesothelioma cells and a tissue array. Our results showed that both the rat and human MM cell lines shared in common a dramatic decrease in the relative expression of Cdkn2a and of epigenetic regulators, in comparison with PN and normal human mesothelial cells, respectively. In particular, we identified the involvement of the relative expression of the Ten-Eleven Translocation (TET) family of dioxygenases and Dnmt3a in relation to the 5-hydroxymethylcytosine level in malignant transformation and the acquisition of metastatic potential.

Quantitative FISH analysis on interphase nuclei may improve diagnosis of DNA diploid breast cancers.

The detection of DNA aneuploid cells using flow cytometry is an indication for the presence of tumor cells, but when DNA diploid cells are found in 25-33% of the cases, the diagnostic and prognostic significance of DNA ploidy is more limited. We analyzed interphase nuclei after in situ hybridization and using image cytometry on 50 breast tumors with diploid DNA content to investigate whether early chromosome rearrangements were detectable and if their occurrence was clinically significant. Imbalances between the two arms of chromosome 1 were found in 55% of the cases and values ranged from 1.5-3.0. Comparison with histological data showed that Grade I tumors mainly have imbalances (67%) and that Grade III tumors were mainly without the imbalance (67%), whereas Grade II tumors were intermediate (50% imbalance). These data suggest that the diagnosis of DNA diploid cases may be improved by using interphase FISH. In addition, the data also indicates that early breast tumors may have different genetic origins, which is important in the comprehension of tumor malignancy in early stages, especially for preinvasive lesions.

Cytogenetic and molecular characterization of plutonium-induced rat osteosarcomas.

The association between ionizing radiation and the subsequent development of osteosarcoma has been well described, but little is known about the cytogenetic and molecular events, which could be involved in the formation of radiation-induced osteosarcomas. Here, we performed comparative genomic hybridization (CGH) to detect chromosomal copy number changes in a series of 16 rat osteosarcomas induced by injection of plutonium-238. Recurrent gains/amplifications were observed at chromosomal regions 3p12-q12, 3q41-qter, 4q41-qter, 6q12-q16, 7q22-q34, 8q11-q23, 9q11-q22, 10q32.1-qter, and 12q, whereas recurrent losses were observed at 1p, 1q, 3q23-q35, 5q21-q33, 8q24-q31, 10q22-q25, 15p, 15q, and 18q. The gained region at 7q22-q34 was homologous to human chromosome bands 12q13-q15/8q24/22q11-q13, including the loci of Mdm2, Cdk4, c-Myc and Pdgf-b genes. The lost regions at 5q21-q33, 10q22-q25 and 15q contained tumor suppressor genes such as p16INK4a/p19ARF, Tp53 and Rb1. To identify potential target gene(s) for the chromosomal aberrations, we compared the expression levels of several candidate genes, located within the regions of frequent chromosomal aberrations, between the tumors and normal osteoblasts by using quantitative RT-PCR analysis. The Cdk4, c-Myc, Pdgf-b and p57KIP2 genes were thought to be possible target genes for the frequent chromosomal gain at 7q22-34 and loss at 1q in the tumors, respectively. In addition, mutations of the Tp53 gene were found in 27% (4 of 15) osteosarcomas. Our data may contribute to further understanding of the molecular mechanisms underlying osteosarcomas induced by ionizing radiation in human.

Tumor cells can escape DNA-damaging cisplatin through DNA endoreduplication and reversible polyploidy.

Cancer chemotherapy can induce tumor regression followed, in many cases, by relapse in the long-term. Thus this study was performed to assess the determinants of such phenomenon using an in vivo cancer model and in vitro approaches. When animals bearing an established tumor are treated by cisplatin, the tumor initially undergoes a dramatic shrinkage and is characterized by giant tumor cells that do not proliferate but maintain DNA synthesis. After several weeks of latency, the tumor resumes its progression and consists of small proliferating cells. Similarly, when tumor cells are exposed in vitro to pharmacological concentrations of cisplatin, mitotic activity stops initially but cells maintain DNA duplication. This DNA endoreduplication generates giant polyploid cells that then initiate abortive mitoses and can die through mitotic catastrophe. However, many polyploid cells survive for weeks as non-proliferating mono- or multi-nucleated giant cells which acquire a senescence phenotype. Prolonged observation of these cells sheds light on the delayed emergence of a limited number of extensive colonies which originate from polyploid cells, as demonstrated by cell sorting analysis. Theses colonies are made of small diploid cells which differ from parental cells by stereotyped chromosomal aberrations and an increased resistance to cytotoxic drugs. These data suggest that a multistep pathway, including DNA endoreduplication, polyploidy, then depolyploidization and generation of clonogenic escape cells can account for tumor relapse after initial efficient chemotherapy.

Rapid prenatal diagnosis of Down syndrome using quantitative fluorescence in situ hybridization on interphase nuclei.

OBJECTIVES: Presently, conventional cytogenetic analysis of metaphase chromosomes remains the reference approach in prenatal diagnosis. However, this method is labor-intensive and time-consuming. The first step toward the rapid identification of aneuploidies is achieved by interphase fluorescence in situ hybridization (FISH) with centromeric or locus-specific probes. Spot counting using this type of probes is a reliable approach, but is very time-consuming with some technical and biological limitations. In this study, we present a new FISH method using image cytometry for the detection of trisomy 21 within interphase nuclei. METHODS: The method is based on a comparative quantitation of the fluorescence signals emitted by whole chromosome 21 and 22 painting probes cohybridized on interphase nuclei. The chromosomal imbalance was determined with an automated image cytometer by detecting an abnormal ratio of both fluorescence emissions when compared with the ratio obtained in normal cells. RESULTS: Ten blood samples and twenty amniotic fluids were analyzed. Results from FISH and standard cytogenetics were compared and 100% correlation was achieved. CONCLUSIONS: This method, which enables an easy detection of chromosomal imbalances without a need for metaphase preparations, can be applied to the diagnosis of trisomy 21 and extended to other disorders with chromosomal imbalances. Compared to other interphase FISH techniques, it avoids spot-scoring difficulties.

Quantitative fish determination of chromosome 3 arm imbalances in lung tumors by automated image cytometry.

BACKGROUND: Clonal heterogeneity is a major difficulty in the analysis of chromosome rearrangements within tumor tissue. Using in situ hybridization, a cell-to-cell analysis can be performed and should allow a better understanding of the genetic process. In addition, detection of pre-neoplastic lesions with only a few cells involved may improve the diagnosis of such lesions and their precocious treatment. MATERIAL/METHODS: Automated analysis was performed on tissue sections with our previously described two-color fluorescence in situ hybridization-based method for quantitative determination of chromosome arm imbalance. The imbalance between the long and short arms of chromosome 3 was determined in 24 cases of non-small-cell and small-cell lung cancers in which only small snap-frozen sections were used, allowing other simultaneous molecular analyses, such as TP53 gene mutation detection. RESULTS: Specifically developed software allowed localization of each nucleus within the section with regard to its chromosome imbalance and to reconstitute a multi-clonal panel within an apparently homogeneous sample. In some cases, discrepancies in the imbalance values were observed between the biopsy and the tumor obtained after surgery from the same patient. CONCLUSIONS: The discrepancies observed between biopsies and tumors, likely linked to the samples' heterogeneity, demonstrate the necessity to analyze tissue sections collected at various locations. The fully automated approach developed in this study rendered such investigations possible.