Cancer susceptibility genes have been classified into two groups: gatekeepers
and caretakers. Gatekeepers are genes that control cell proliferation
and death, whereas caretakers are DNA repair genes ...whose inactivation leads
to genetic instability. Abrogation of both caretaker and gatekeeper function
markedly increases cancer susceptibility. Although the importance of Ku80
in DNA double-strand break repair is well established, neither Ku80 nor other
components of the non-homologous end-joining pathway are known to have a caretaker
role in maintaining genomic stability. Here we show that mouse cells deficient
for Ku80 display a marked increase in chromosomal aberrations, including
breakage, translocations and aneuploidy. Despite the observed chromosome instabilities,
Ku80-/- mice have only a slightly earlier onset of cancer. Loss of p53 synergizes with Ku80 to promote tumorigenesis such
that all Ku80-/-p53-/- mice succumb
to disseminated pro-B-cell lymphoma before three months of age. Tumours result
from a specific set of chromosomal translocations and gene amplifications
involving IgH and c-Myc, reminiscent of Burkitt's lymphoma. We conclude that
Ku80 is a caretaker gene that maintains the integrity of the genome by
a mechanism involving the suppression of chromosomal rearrangements.
Genetic disorders affecting cellular responses to DNA damage are characterized by high rates of translocations involving antigen receptor loci and increased susceptibility to lymphoid malignancies. ...We report that the Nijmegen breakage syndrome protein (NBS1) and histone γ-H2AX, which associate with irradiation-induced DNA double-strand breaks (DSBs), are also found at sites of V(D)J (variable, diversity, joining) recombination-induced DSBs. In developing thymocytes, NBS1 and γ-H2AX form nuclear foci that colocalize with the T cell receptor α locus in response to recombination activating gene (RAG) protein-mediated V(D)J cleavage. Our results suggest that surveillance of T cell receptor recombination intermediates by NBS1 and γ-H2AX may be important for preventing oncogenic translocations.
A system for tetracycline-regulated inducible gene expression was described recently which relies on constitutive expression of a tetracycline-controlled transactivator (tTA) fusion protein combining ...the tetracycline repressor and the transcriptional activation domain of VP16 Gossen, M. \& Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551. This system yielded only low levels of transactivator protein, probably because tTA is toxic. To avoid this difficulty, we placed the tTA gene under the control of the inducible promoter to which tTA binds, making expression of tTA itself inducible and autoregulatory. When used to drive expression of the recombination activating genes 1 and 2 (RAG-1 and RAG-2), the autoregulatory system yielded both substantially higher levels of variable (diversity) joining V(D)J recombination activity (70-fold on average) and inducible expression in a much larger fraction of transfected cells (autoregulatory, 90%, vs. constitutive, 18%). In addition, this system allowed the creation of transgenic mice in which expression of a luciferase transgene was inducible tens to hundreds of times the basal levels in most tissues examined. Induced levels of expression were highest in thymus and lung and appear to be substantially higher than in previously reported inducible luciferase transgenic mice created with the constitutive system. With the modified system, inducible transactivator mRNA and protein were easily detected in cell lines by RNA and Western blotting, and transactivator mRNA was detected by RNA blotting in some tissues of transgenic mice. This autoregulatory system represents an improved strategy for tetracycline-regulated gene expression both in cultured cells and in transgenic animals.
Human epithelial cancers are defined by a recurrent distribution of specific chromosomal aneuploidies, a trait less typical for murine cancer models induced by an oncogenic stimulus. After prolonged ...culture, mouse epithelial cells spontaneously immortalize, transform and become tumorigenic. We assessed genome and transcriptome alterations in cultures derived from bladder and kidney utilizing spectral karyotyping, array CGH, FISH and gene expression profiling. The results show widespread aneuploidy, yet a recurrent and tissue-specific distribution of genomic imbalances, just as in human cancers. Losses of chromosome 4 and gains of chromosome 15 are common and occur early during the transformation process. Global gene expression profiling revealed early and significant transcriptional deregulation. Chromosomal aneuploidy resulted in expression changes of resident genes and consequently in a massive deregulation of the cellular transcriptome. Pathway interrogation of expression changes during the sequential steps of transformation revealed enrichment of genes associated with DNA repair, centrosome regulation, stem cell characteristics and aneuploidy. Genes that modulate the epithelial to mesenchymal transition and genes that define the chromosomal instability phenotype played a dominant role and were changed in a directionality consistent with loss of cell adhesion, invasiveness and proliferation. Comparison with gene expression changes during human bladder and kidney tumorigenesis revealed remarkable overlap with changes observed in the spontaneously transformed murine cultures. Therefore, our novel mouse models faithfully recapitulate the sequence of genomic and transcriptomic events that define human tumorigenesis, hence validating them for both basic and preclinical research.
Recent studies have demonstrated that DNA cleavage during V(D)J recombination is mediated by the RAG1 and RAG2 proteins. These proteins must therefore bind to the recombination signals, but the ...specific binding interaction has been difficult to study in vitro. Here, we use an in vivo one-hybrid DNA binding assay to demonstrate that RAG1, in the absence of RAG2, can mediate signal recognition via the nonamer, with the heptamer acting to enhance its binding. A region of RAG1 with sequence similarity to bacterial invertases is essential for DNA binding. Localization of RAG2 to the signal is dependent upon the presence of RAG1 and is substantially more efficient with a 12 bp spacer signal than with a 23 bp spacer signal.
Histone H2AX becomes phosphorylated in chromatin domains flanking sites of DNA double-strand breakage associated with γ-irradiation, meiotic recombination, DNA replication, and antigen receptor ...rearrangements. Here, we show that loss of a single
H2AX allele compromises genomic integrity and enhances the susceptibility to cancer in the absence of p53. In comparison with heterozygotes, tumors arise earlier in the H2AX homozygous null background, and
H2AX
−/−
p53
−/− lymphomas harbor an increased frequency of clonal nonreciprocal translocations and amplifications. These include complex rearrangements that juxtapose the c-
myc oncogene to antigen receptor loci. Restoration of the
H2AX null allele with wild-type
H2AX restores genomic stability and radiation resistance, but this effect is abolished by substitution of the conserved serine phosphorylation sites in H2AX with alanine or glutamic acid residues. Our results establish
H2AX as genomic caretaker that requires the function of both gene alleles for optimal protection against tumorigenesis.
There is a wide spectrum of tumor responsiveness of rectal adenocarcinomas to preoperative chemoradiotherapy ranging from complete response to complete resistance. This study aimed to investigate ...whether parallel gene expression profiling of the primary tumor can contribute to stratification of patients into groups of responders or nonresponders.
Pretherapeutic biopsies from 30 locally advanced rectal carcinomas were analyzed for gene expression signatures using microarrays. All patients were participants of a phase III clinical trial (CAO/ARO/AIO-94, German Rectal Cancer Trial) and were randomized to receive a preoperative combined-modality therapy including fluorouracil and radiation. Class comparison was used to identify a set of genes that were differentially expressed between responders and nonresponders as measured by T level downsizing and histopathologic tumor regression grading.
In an initial set of 23 patients, responders and nonresponders showed significantly different expression levels for 54 genes (P < .001). The ability to predict response to therapy using gene expression profiles was rigorously evaluated using leave-one-out cross-validation. Tumor behavior was correctly predicted in 83% of patients (P = .02). Sensitivity (correct prediction of response) was 78%, and specificity (correct prediction of nonresponse) was 86%, with a positive and negative predictive value of 78% and 86%, respectively.
Our results suggest that pretherapeutic gene expression profiling may assist in response prediction of rectal adenocarcinomas to preoperative chemoradiotherapy. The implementation of gene expression profiles for treatment stratification and clinical management of cancer patients requires validation in large, independent studies, which are now warranted.
Many carcinomas have recurrent chromosomal aneuploidies specific to the tissue of tumor origin. The reason for this specificity is not completely understood.
In this study, we looked at the frequency ...of chromosomal arm gains and losses in different cancer types from the The Cancer Genome Atlas (TCGA) and compared them to the mean gene expression of each chromosome arm in corresponding normal tissues of origin from the Genotype-Tissue Expression (GTEx) database, in addition to the distribution of tissue-specific oncogenes and tumor suppressors on different chromosome arms.
This analysis revealed a complex picture of factors driving tumor karyotype evolution in which some recurrent chromosomal copy number reflect the chromosome arm-wide gene expression levels of the their normal tissue of tumor origin.
We conclude that the cancer type-specific distribution of chromosomal arm gains and losses is potentially "hardwiring" gene expression levels characteristic of the normal tissue of tumor origin, in addition to broadly modulating the expression of tissue-specific tumor driver genes.
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