Bacterial SOS Genes mucAB/umuDC Promote Mouse Tumors by Activating Oncogenes Nedd9/Aurkb via a miR-145 Sponge.

The mechanism of cancer induction involves an aberrant expression of oncogenes whose functions can be controlled by RNAi with miRNA. Even foreign bacterial RNA may interfere with the expression of oncogenes. Here we show that bacterial plasmid mucAB and its Escherichia coli genomic homolog umuDC, carrying homologies that match the mouse anti-miR-145, sequestered the miR-145 function in mouse BALB 3T3 cells in a tetracycline (Tet)-inducible manner, activated oncogene Nedd9 and its downstream Aurkb, and further enhanced microcolony formation and cellular transformation as well as the short fragments of the bacterial gene containing the anti-miR-145 sequence. Furthermore, mucAB transgenic mice showed a 1.7-fold elevated tumor incidence compared with wild-type mice after treatments with 3-methylcolanthrene. However, the mutation frequency in intestinal stem cells of the mucAB transgenic mice was unchanged after treatment with X-rays or ethyl-nitrosourea, indicating that the target of mucAB/umuDC is the promotion stage in carcinogenesis. IMPLICATIONS: Foreign bacterial genes can exert oncogenic activity via RNAi, if endogenously expressed. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/9/1271/F1.large.jpg.

Rag-dependent and Rag-independent mechanisms of Notch1 rearrangement in thymic lymphomas of Atm(-/-) and scid mice.

The pathways of thymic lymphomagenesis are classified as Rag-dependent or -independent according to their dependence on recombination-activating gene (Rag1/2) proteins. The role of the two-lymphoma pathways in oncogene rearrangements and the connection between lymphoma pathways and rearrangement mechanisms, however, remain obscure. We compared the incidence and latency of thymic lymphomas, and associated rearrangements of the representative oncogene Notch1 among Rag2(-/-), ataxia telangiectasia mutated (Atm)(-/-), and severe combined immune deficiency (scid) mice combined with Rag2 deficiency. Contrary to expectations, Rag2(-/-) mice were prone to thymic lymphoma development, suggesting the existence of a Rag2-independent lymphoma pathway in Rag2(-/-) mice. The lymphoma incidence in Rag2(-/-)Atm(-/-) mice was lower than that in Atm(-/-) mice, but higher than that in Rag2(-/-) mice, indicating that Atm(-/-) mice develop lymphomas through both pathways. Scid mice developed lymphomas with an incidence and latency similar to Rag2(-/-)scid mice, suggesting that Rag2-mediated V(D)J recombination-driven events are not necessarily required for lymphomagenesis in scid mice. Notch1 rearrangement mechanisms were classified as Rag2-dependent or Rag2-independent based on the presence of recombination signal-like sequences at rearranged sites. In Rag2(-/-) lymphomas, Notch1 must be rearranged independently of Rag2 function, implying that Rag2(-/-) mice are susceptible to lymphomagenesis due to the presence of other rearrangement mechanisms. The results in Atm(-/-) mice suggest that Notch1 was rearranged through both lymphoma pathways. In scid mice, the frequency of Rag2-mediated rearrangements was relatively low compared with that in wild-type mice, suggesting that the Rag2-independent lymphoma pathway prevails in the development of thymic lymphomas in scid mice. Thus, two rearrangement mechanisms underlie the lymphoma pathways and constitute the mechanistic bases for lymphomagenesis, thereby providing the molecular criteria for distinguishing between Rag2-dependent and Rag2-independent lymphoma pathways.

Aggregative organization enhances the DNA end-joining process that is mediated by DNA-dependent protein kinase.

The occurrence of DNA double-strand breaks in the nucleus provokes in its structural organization a large-scale alteration whose molecular basis is still mostly unclear. Here, we show that double-strand breaks trigger preferential assembly of nucleoproteins in human cellular fractions and that they mediate the separation of large protein-DNA aggregates from aqueous solution. The interaction among the aggregative nucleoproteins presents a dynamic condition that allows the effective interaction of nucleoproteins with external molecules like free ATP and facilitates intrinsic DNA end-joining activity. This aggregative organization is functionally coacervate-like. The key component is DNA-dependent protein kinase (DNA-PK), which can be characterized as a DNA-specific aggregation factor as well as a nuclear scaffold/matrix-interactive factor. In the context of aggregation, the kinase activity of DNA-PK is essential for efficient DNA end-joining. The massive and functional concentration of nucleoproteins on DNA in vitro may represent a possible status of nuclear dynamics in vivo, which probably includes the DNA-PK-dependent response to multiple double-strand breaks.

Isolation and Characterization of a Novel Human Radiosusceptibility Gene, NP95.

The murine nuclear protein Np95 has been shown to underlie resistance to ionizing radiation and other DNA insults or replication arrests in embryonic stem (ES) cells. Using the databases for expressed sequenced tags and a two-step PCR procedure, we isolated human NP95, the full-length human homologue of the murine Np95 cDNA, which consists of 4,327 bp with a single open reading frame (ORF) encoding a polypeptide of 793 amino acids and 73.3% homology to Np95. The ORF of human NP95 cDNA is identical to the UHRF1 (ubiquitin-like protein containing PHD and RING domain 1). The NP95 gene, assigned to 19p13.3, consists of 18 exons, spanning 60 kb. Several stable transformants from HEK293 and WI-38 cells that had been transfected with the antisense NP95 cDNA were, like the murine Np95-knockout ES cells, more sensitive to X rays, UV light and hydroxyurea than the corresponding parental cells. In HEK293 cells, the lack of NP95 did not affect the activities of topoisomerase IIalpha, whose expression had been demonstrated to be regulated by the inverted CCAAT box binding protein of 90 kDa (ICBP90) that closely resembles NP95 in amino acid sequence and in cDNA but differs greatly in genomic organization. These findings collectively indicate that the human NP95 gene is the functional orthologue of the murine Np95 gene.

Regulation of radiation-induced protein kinase Cdelta activation in radiation-induced apoptosis differs between radiosensitive and radioresistant mouse thymic lymphoma cell lines.

Protein kinase Cdelta (PKCdelta) has an important role in radiation-induced apoptosis. The expression and function of PKCdelta in radiation-induced apoptosis were assessed in a radiation-sensitive mouse thymic lymphoma cell line, 3SBH5, and its radioresistant variant, XR223. Rottlerin, a PKCdelta-specific inhibitor, completely abolished radiation-induced apoptosis in 3SBH5. Radiation-induced PKCdelta activation correlated with the degradation of PKCdelta, indicating that PKCdelta activation through degradation is involved in radiation-induced apoptosis in radiosensitive 3SBH5. In radioresistant XR223, radiation-induced PKCdelta activation was lower than that in radiosensitive 3SBH5. Cytosol PKCdelta levels in 3SBH5 decreased markedly after irradiation, while those in XR223 did not. There was no apparent change after irradiation in the membrane fractions of either cell type. In addition, basal cytosol PKCdelta levels in XR223 were higher than those in 3SBH5. These results suggest that the radioresistance in XR223 to radiation-induced apoptosis is due to a difference in the regulation of radiation-induced PKCdelta activation compared to that of 3SBH5. On the other hand, Atm(-/-) mouse thymic lymphoma cells were more radioresistant to radiation-induced apoptosis than wild-type mouse thymic lymphoma cells. Irradiated wild-type cells, but not Atm(-/-) cells, had decreased PKCdelta levels, indicating that the Atm protein is involved in radiation-induced apoptosis through the induction of PKCdelta degradation. The decreased Atm protein levels induced by treatment with Atm small interfering RNA had no effect on radiation-induced apoptosis in 3SBH5 cells. These results suggest that the regulation of radiation-induced PKCdelta activation, which is distinct from the Atm-mediated cascade, determines radiation sensitivity in radiosensitive 3SBH5 cells.

A sensitive transcriptome analysis method that can detect unknown transcripts.

We have developed an AFLP-based gene expression profiling method called 'high coverage expression profiling' (HiCEP) analysis. By making improvements to the selective PCR technique we have reduced the rate of false positive peaks to approximately 4% and consequently the number of peaks, including overlapping peaks, has been markedly decreased. As a result we can determine the relationship between peaks and original transcripts unequivocally. This will make it practical to prepare a database of all peaks, allowing gene assignment without having to isolate individual peaks. This precise selection also enables us to easily clone peaks of interest and predict the corresponding gene for each peak in some species. The procedure is highly reproducible and sensitive enough to detect even a 1.2-fold difference in gene expression. Most importantly, the low false positive rate enables us to analyze gene expression with wide coverage by means of four instead of six nucleotide recognition site restriction enzymes for fingerprinting mRNAs. Therefore, the method detects 70-80% of all transcripts, including non-coding transcripts, unknown and known genes. Moreover, the method requires no sequence information and so is applicable even to eukaryotes for which there is no genome information available.

The association of cyclin A and cyclin kinase inhibitor p21 in response to gamma-irradiation requires the CDK2 binding region, but not the Cy motif.

The cyclin kinase inhibitor p21 associates with and inhibits cyclin-CDKs to retard the progress of the cell cycle in response to DNA damage. The recognition sites for cyclin binding on the various cell cycle-related molecules have been identified as RXL motifs. In the case of p21, the dependence of the Cy1 (18CRRL) or Cy2 (154KRRL) motifs on cyclin E, but not on cyclin A has been demonstrated by in vitro experiments. In this study, to clarify the mechanism of p21 association with cyclin A, we constructed a p21 expression system in mammalian cells. After transfection with an expression vector containing cDNA of various p21-mutants, cells were irradiated with 10 Gy of gamma-rays to introduce DNA damage, followed by quantification of the p21-cyclin A association. The p21-mutant constructs were single or multiple deletions in Cy1, Cy2, and the CDK2 binding region, and a nonphosphorylatable alanine mutant of the C-terminal phosphorylation site. We demonstrated that the association of p21 and cyclin A in response to gamma-irradiation requires the CDK binding region, 49-71 aa, but not the Cy motifs. We believe the mechanism by which p21 inhibits cyclin-CDKs is distinct in each phase of the cell cycle. Furthermore, the increase in the association of p21 and cyclin A was not correlated with the levels of p21. This suggests that DNA damage triggers a signal to the p21 region between 21 and 96 aa to allow cyclin A association.

Resistance against Friend leukemia virus-induced leukemogenesis in DNA-dependent protein kinase (DNA-PK)-deficient scid mice associated with defective viral integration at the Spi-1 and Fli-1 site.

Retroviral DNA integration is mediated by the viral protein integrase. However, elements of the host DNA repair machinery such as the phosphatidylinositol 3-kinase (PI-3K)-related protein kinase family system would play a role in the integration of viral DNA into the host DNA. Here, we show that a host PI-3K-related protein kinase, DNA-dependent protein kinase (DNA-PK), plays a role in the specific integration of retroviral DNA and induction of retroviral diseases in vivo. DNA-PK-deficient scid mice inoculated with Friend leukemia virus (FLV) exhibited a random integration into their genomic DNA and expressed the viral envelope protein gp70. However, the specific integration of FLV at Spi-1 or Fli-1 sites did not occur in association with the significant resistance of scid mice to FLV-induced leukemogenesis. In contrast, the knockout of another member of the PI-3K-related protein kinase family, encoded by the ataxia telangiectasia mutated (ATM) gene, resulted in mice as sensitive to FLV-induced leukemogenesis as the wild type mice. FLV was specifically integrated into the DNA at Spi-1 and Fli-1 sites with significant expression of these transcription factors. These findings indicated that DNA-PK would be essential for controlling the in vivo integration of FLV at specific sites as well as the susceptibility to FLV-induced leukemogenesis.

DNA-dependent protein kinase enhances DNA damage-induced apoptosis in association with Friend gp70.

Friend leukemia virus (FLV) infection strongly enhances gamma-irradiation-induced apoptosis of hematopoietic cells of C3H hosts leading to a lethal anemia. Experiments using p53 knockout mice with the C3H background have clarified that the apoptosis is p53-dependent and would not be associated with changes of cell populations caused by the infection with FLV. In bone marrow cells of FLV + total body irradiation (TBI)-treated C3H mice, the p53 protein was prominently activated to overexpress p21 and bax suggesting that apoptosis-enhancing mechanisms lay upstream of p53 protein in the signaling pathway. Neither of DNA-dependent protein kinase (DNA-PK)-deficient SCID mice nor ataxia telangiectasia mutated (ATM) gene knockout mice with the C3H background exhibited a remarkable enhancement of apoptosis or p53 activation on FLV + TBI-treatment indicating that DNA-PK and ATM were both essential. ATM appeared necessary for introducing DNA damage-induced apoptosis, while DNA-PK enhanced p53-dependent apoptosis under FLV-infection. Surprisingly, viral envelope protein, gp70, was co-precipitated with DNA-PK but not with ATM in FLV + TBI-treated C3H mice. These results indicated that FLV-infection enhances DNA damage-induced apoptosis via p53 activation and that DNA-PK, in association with gp70, might play critical roles in modulating the signaling pathway.

Involvement of DNA-dependent protein kinase in down-regulation of cell cycle progression.

The catalytic polypeptide of DNA-dependent protein kinase (p470) is encoded by the gene responsible for murine severe combined immunodeficiency (SCID) devoid of DNA double-strand break repair and V(D)J recombination. Here, we have characterized the role of p470 in cell proliferation using SCID mice and the cell lines. In accord with DNA histogram patterns, SCID cell lines (SD/SD-eA and SC3VA2) expressing extremely low level of DNA-PK activity grew faster than a normal mouse cell line (CB/CB-eB) and SC3VA2 complemented with human p470 gene (RD13B2). In regenerating liver after partial hepatectomy, de novo DNA synthesis determined by [(3)H]thymidine incorporation started at 30h in C.B-17/Icr-SCID (SCID) mice and at around 36h in C.B-17/Icr (C.B-17) mice. Compared with normal cells, SCID cells contained slightly higher levels of transcripts of cyclin A, cyclin E, B-Myb and dihydrofolate reductase, which are regulated by E2F-1. E2F-1 playing a key role in G1- to S-phase progression was phosphorylated in vitro by DNA-PK. Importantly, the E2F-1 promoter transcriptional activity in SCID cell lines (SD/SD-eA and SC3VA2) was 4-5-fold higher than that in CB/CB-eB and RD13B2. These results suggest that p470 is involved in down-regulation of cell cycle progression through E2F-1-responsible genes.

Effect of Atm disruption on spontaneously arising and radiation-induced deletion mutations in mouse liver.

Deletion mutations were efficiently recovered in mouse liver after total-body irradiation with X rays by using a transgenic mouse "gpt-delta" system that harbored a lambda EG10 shuttle vector with the red and gam genes for Spi- (sensitive to P2 lysogen interference) selection. We incorporated this system into homozygous Atm-knockout mice as a model of the radiosensitive hereditary disease ataxia telangiectasia (AT). Lambda phages recovered from the livers of X-irradiated mice with the Atm+/+ genotype showed a dose-dependent increase in the Spi- mutant frequency up to sixfold at 50 Gy over the unirradiated control of 2.8x10(-6). The livers from Atm-/- mice yielded a virtually identical dose-response curve for X rays with a background fraction of 2.4x10(-6). Structural analyses revealed no significant difference in the proportion of -1 frameshifts and larger deletions between Atm+/+ and Atm-/- mice, although larger deletions prevailed in X-ray-induced Spi- mutants irrespective of Atm status. While a possible defect in DNA repair after irradiation has been strongly indicated in the literature for nondividing cultured cells in vitro from AT patients, the Atm disruption does not significantly affect radiation mutagenesis in the stationary mouse liver in vivo.

Sequence analysis of 193.4 and 83.9 kbp of mouse and chicken genomic DNAs containing the entire Prkdc (DNA-PKcs) gene.

The catalytic subunit of DNA-dependent protein kinase plays critical roles in nonhomologous end joining in repair of DNA double-strand breaks and V(D)J recombination. In addition to the SCID phenotype, it has been suggested that the molecule contributes to the polymorphic variations in radiosensitivity and susceptibility to cancer in mouse strains. Here we show the nucleotide sequence of approximately 193-kbp and 84-kbp genomic regions encoding the entire Prkdc gene (also known as DNA-PKcs) in the mouse and chicken, respectively. A large retroposon was found in intron 51 in the mouse but not in the human or chicken. Comparative analyses of the genome strongly suggested that the region contains only two genes for Prkdc and Mcm4; however, several conserved sequences and cis elements were also predicted.

Nature of nontargeted radiation effects observed during fractionated irradiation-induced thymic lymphomagenesis in mice.

Changes in the thymic microenvironment lead to radiation-induced thymic lymphomagenesis, but the phenomena are not fully understood. Here we show that radiation-induced chromosomal instability and bystander effects occur in thymocytes and are involved in lymphomagenesis in C57BL/6 mice that have been irradiated four times with 1.8-Gy γ-rays. Reactive oxygen species (ROS) were generated in descendants of irradiated thymocytes during recovery from radiation-induced thymic atrophy. Concomitantly, descendants of irradiated thymocytes manifested DNA lesions as revealed by γ-H2AX foci, chromosomal instability, aneuploidy with trisomy 15 and bystander effects on chromosomal aberration induction in co-cultured ROS-sensitive mutant cells, suggesting that the delayed generation of ROS is a primary cause of these phenomena. Abolishing the bystander effect of post-irradiation thymocytes by superoxide dismutase and catalase supports ROS involvement. Chromosomal instability in thymocytes resulted in the generation of abnormal cell clones bearing trisomy 15 and aberrant karyotypes in the thymus. The emergence of thymic lymphomas from the thymocyte population containing abnormal cell clones indicated that clones with trisomy 15 and altered karyotypes were prelymphoma cells with the potential to develop into thymic lymphomas. The oncogene Notch1 was rearranged after the prelymphoma cells were established. Thus, delayed nontargeted radiation effects drive thymic lymphomagenesis through the induction of characteristic changes in intrathymic immature T cells and the generation of prelymphoma cells.

Targeted disruption of Np95 gene renders murine embryonic stem cells hypersensitive to DNA damaging agents and DNA replication blocks.

NP95, which contains a ubiquitin-like domain, a cyclin A/E-Cdk2 phosphorylation site, a retinoblastoma (Rb) binding motif, and a ring finger domain, has been shown to be colocalized as foci with proliferating cell nuclear antigen in early and mid-S phase nuclei. We established Np95 nulligous embryonic stem cells by replacing the exons 2-7 of the Np95 gene with a neo cassette and by selecting out a spontaneously occurring homologous chromosome crossing over with a higher concentration of neomycin. Np95-null cells were more sensitive to x-rays, UV light, N-methyl-N"-nitro-N-nitrosoguanidine (MNNG), and hydroxyurea than embryonic stem wild type (Np95(+/+)) or heterozygously inactivated (Np95(+/-)) cells. Expression of transfected Np95 cDNA in Np95-null cells restored the resistance to x-rays, UV, MNNG, or hydroxyurea concurrently to a level similar to that of Np95(+/-) cells, although slightly below that of wild type (Np95(+/+)) cells. These findings suggest that NP95 plays a role in the repair of DNA damage incurred by these agents. The frequency of spontaneous sister chromatid exchange was significantly higher for Np95-null cells than for Np95(+/+) cells or Np95(+/-) cells (p < 0.001). We conclude that NP95 functions as a common component in the multiple response pathways against DNA damage and replication arrest and thereby contributes to genomic stability.

Identification of the regulatory region required for ubiquitination of the cyclin kinase inhibitor, p21.

The expression of cyclin kinase inhibitor p21 is regulated by the ubiquitin-proteasome protein degradation system, as well as by transcriptional regulation. Generally, ubiquitination is regulated by the phosphorylation of the substrate. In this study, we identified the region of p21 responsible for the regulation of ubiquitination. Since the phosphorylation sites of p21 are distributed in the C-terminal region, we constructed sequential C-terminal truncated fragments and examined their ubiquitination in eukaryotic cells. The ubiquitination was observed in the 1-164 (full length) and 1-157 fragments with the same efficiency, but not in the 1-147 fragment. The lack of ubiquitination in the 1-147 fragment was unlikely due to the removal of a Lys residue at position 154, since the p21 K154R mutant was ubiquitinated as efficiently as the full-length p21. Furthermore, the 148-157 deleted form of p21 was not ubiquitinated, just like the 1-147 fragment. Thus, the C-terminal 148-157 region, not a ubiquitination site by itself, should contain an essential regulatory region for the efficient ubiquitination of p21.