The complete spectrum of yeast chromosome instability genes identifies candidate CIN cancer genes and functional roles for ASTRA complex components

Chromosome instability (CIN) is observed in most solid tumors and is linked to somatic mutations in genome integrity maintenance genes. The spectrum of mutations that cause CIN is only partly known and it is not possible to predict a priori all pathways whose disruption might lead to CIN. To address this issue, we generated a catalogue of CIN genes and pathways by screening ～ 2,000 reduction-of-function alleles for 90% of essential genes in Saccharomyces cerevisiae. Integrating this with published CIN phenotypes for other yeast genes generated a systematic CIN gene dataset comprised of 692 genes. Enriched gene ontology terms defined cellular CIN pathways that, together with sequence orthologs, created a list of human CIN candidate genes, which we cross-referenced to published somatic mutation databases revealing hundreds of mutated CIN candidate genes. Characterization of some poorly characterized CIN genes revealed short telomeres in mutants of the ASTRA/TTT components TTI1 and ASA1. High-throughput phenotypic profiling links ASA1 to TTT (Tel2-Tti1-Tti2) complex function and to TORC1 signaling via Tor1p stability, consistent with the role of TTT in PI3-kinase related kinase biogenesis. The comprehensive CIN gene list presented here in principle comprises all conserved eukaryotic genome integrity pathways. Deriving human CIN candidate genes from the list allows direct cross-referencing with tumor mutational data and thus candidate mutations potentially driving CIN in tumors. Overall, the CIN gene spectrum reveals new chromosome biology and will help us to understand CIN phenotypes in human disease.     

Mutations in the APC tumour suppressor gene cause chromosomal instability

Two forms of genetic instability have been described in colorectal cancer: microsatellite instability and chromosomal instability. Microsatellite instability results from mutations in mismatch repair genes; chromosomal instability is the hallmark of many colorectal cancers, although it is not completely understood at the molecular level. As truncations of the Adenomatous Polyposis Coli (APC) gene are found in most colorectal tumours, we thought that mutations in APC might be responsible for chromosomal instability. To test this hypothesis, we examined mouse embryonic stem (ES) cells homozygous for Min (multiple intestinal neoplasia) or Apc1638T alleles. Here we show that Apc mutant ES cells display extensive chromosome and spindle aberrations, providing genetic evidence for a role of APC in chromosome segregation. Consistent with this, APC accumulates at the kinetochore during mitosis. Apc mutant cells form mitotic spindles with an abundance of microtubules that inefficiently connect with kinetochores. This phenotype is recapitulated by the induced expression of a 253-amino-acid carboxy-terminal fragment of APC in microsatellite unstable colorectal cancer cells. We conclude that loss of APC sequences that lie C-terminal to the beta-catenin regulatory domain contributes to chromosomal instability in colorectal cancer.     

No evidence for constitutional chromosome instability in testicular cancer

Summary Chromosome aberrations in 20 lymphocytes of 20 patients with testicular germ cell tumors (TGCT) treated with surgery alone were compared with those of 20 cells from 20 healthy controls using standard G-banding technique. No increase in structural aberrations was found in the cancer group. An unexpected finding was that of more cells with losses of chromosomes being present in the control group. These losses predominantly affected small chromosomes in the control group, whereas the pattern of chromosome loss was different in the cancer group. The literature claiming increased chromosome instability in TGCT patients is reviewed. Point estimates and 95% confidence intervals to exclude such a hypothesis based on our results were calculated.     

Y chromosome instability in testicular cancer

Approximately 15-25% of male infertility cases carry extensive azoospermic factor (AZF) deletions. Moreover, about 80% of Finnish testicular germ cell tumors (TGCT) and about 23-25% of TGCTs from other geographic regions carry short and interstitial AZF deletions. In infertility cases the AZF deficiency occurs in the germ cells of the proband father giving rise to mosaic sperm populations comprising non-deleted and deleted sperms. Fertilization of an oocyte by a Y deleted sperm will give rise to an AZF-deleted and infertile F1 male. In TGCTs the AZF deletions take place in the initial stages of embryogenesis producing individuals that are a mosaic of Y deleted and non-deleted cell lineages. Carcinoma in situ (CIS) is a premalignant lesion that some believe may develop in gonads of male embryos before the ninth week of age due to transformation of a totipotent primordial germ cell. If the transformed cell carries AZF deletions the resultant CIS will also have Y deletions. CIS will differentiate into seminoma or into embryonal carcinoma and non-seminomas in about 1 x 10(-3) of the young adults carrying premalignant CIS outgrowths; if the CIS lesion has AZF deletions the derived forms of testicular cancer will also exhibit these deletions. AZF deletions play no role in the development of testicular cancers. On the other hand, they are a marker of Y chromosome instability and eventually of a more generalized pattern of genome instability associated with the appearance of TGCT. Genetic factors such as malfunction of metabolizing genes, DNA repairing genes, Y-linked or X-linked genes have been considered as possible causes of AZF deletions in testicular cancer. Yet, the exact identification of the genes involved remains elusive. AZF deletions have also been identified in non-Hodgkin lymphomas and in colorectal cancers, two forms of malignancy that have been found to be associated with TGCTs.     

Studying chromosome instability in the mouse

Aneuploidy has long been recognized as one of the hallmarks of cancer. It nonetheless remains uncertain whether aneuploidy occurring early in the development of a cancer is a primary cause of oncogenic transformation, or whether it is an epiphenomenon that arises from a general breakdown in cell cycle control late in tumorigenesis. The accuracy of chromosome segregation is ensured both by the intrinsic mechanics of mitosis and by an error-checking spindle assembly checkpoint. Many cancers show altered expression of proteins involved in the spindle checkpoint or in proteins implicated in other mitotic processes. To understand the role of aneuploidy in the initiation and progression of cancer, a number of spindle checkpoint genes have been disrupted in mice, most through conventional gene targeting (to create germ-line knockouts). We describe the consequence of these mutations with respect to embryonic development, tumor progression and an unexpected link to premature aging; readers are referred elsewhere [1] for a discussion of other cell cycle regulators.     

Emerging roles for centromeres in meiosis I chromosome segregation

Centromeres are an essential and conserved feature of eukaryotic chromosomes, yet recent research indicates that we are just beginning to understand the numerous roles that centromeres have in chromosome segregation. During meiosis I, in particular, centromeres seem to function in many processes in addition to their canonical role in assembling kinetochores, the sites of microtubule attachment. Here we summarize recent advances that place centromeres at the centre of meiosis I, and discuss how these studies affect a variety of basic research fields and thus hold promise for increasing our understanding of human reproductive defects and disease states.     

Drosophila APC2 and APC1 play overlapping roles in wingless signaling in the embryo and imaginal discs

The regulation of signal transduction plays a key role in cell fate choices, and its disregulation contributes to oncogenesis. This duality is exemplified by the tumor suppressor APC. Originally identified for its role in colon tumors, APC family members were subsequently shown to negatively regulate Wnt signaling in both development and disease. The analysis of the normal roles of APC proteins is complicated by the presence of two APC family members in flies and mice. Previous work demonstrated that, in some tissues, single mutations in each gene have no effect, raising the question of whether there is functional overlap between the two APCs or whether APC-independent mechanisms of Wnt regulation exist. We addressed this by eliminating the function of both Drosophila APC genes simultaneously. We find that APC1 and APC2 play overlapping roles in regulating Wingless signaling in the embryonic epidermis and the imaginal discs. Surprisingly, APC1 function in embryos occurs at levels of expression nearly too low to detect. Further, the overlapping functions exist despite striking differences in the intracellular localization of the two APC family members.     

Distinct chromosome segregation roles for spindle checkpoint proteins

The spindle checkpoint plays a central role in the fidelity of chromosome transmission by ensuring that anaphase is initiated only after kinetochore-microtubule associations of all sister chromatid pairs are complete. In this study, we find that known spindle checkpoint proteins do not contribute equally to chromosome segregation fidelity in Saccharomyces cerevisiae. Loss of Bub1 or Bub3 protein elicits the largest effect. Analysis of Bub1p reveals the presence of two molecular functions. An N-terminal 608-amino acid (nonkinase) portion of the protein supports robust checkpoint activity, and, as expected, contributes to chromosome segregation. A C-terminal kinase-encoding segment independently contributes to chromosome segregation through an unknown mechanism. Both molecular functions depend on association with Bub3p. A 156-amino acid fragment of Bub1p functions in Bub3p binding and in kinetochore localization by one-hybrid assay. An adjacent segment is required for Mad1p binding, detected by deletion analysis and coimmunoprecipitation. Finally, overexpression of wild-type BUB1 or MAD3 genes leads to chromosome instability. Analysis of this activity indicates that the Bub3p-binding domain of Bub1p contributes to this phenotype through disruption of checkpoint activity as well as through introduction of kinetochore or spindle damage.     

Drosophila APC2 and APC1 have overlapping roles in the larval brain despite their distinct intracellular localizations

The tumor suppressor APC and its homologs, first identified for a role in colon cancer, negatively regulate Wnt signaling in both oncogenesis and normal development, and play Wnt-independent roles in cytoskeletal regulation. Both Drosophila and mammals have two APC family members. We further explored the functions of the Drosophila APCs using the larval brain as a model. We found that both proteins are expressed in the brain. APC2 has a highly dynamic, asymmetric localization through the larval neuroblast cell cycle relative to known mediators of embryonic neuroblast asymmetric divisions. Adherens junction proteins also are asymmetrically localized in neuroblasts. In addition they accumulate with APC2 and APC1 in nerves formed by axons of the progeny of each neuroblast-ganglion mother cell cluster. APC2 and APC1 localize to very different places when expressed in the larval brain: APC2 localizes to the cell cortex and APC1 to centrosomes and microtubules. Despite this, they play redundant roles in the brain; while each single mutant is normal, the zygotic double mutant has severely reduced numbers of larval neuroblasts. Our experiments suggest that this does not result from misregulation of Wg signaling, and thus may involve the cytoskeletal or adhesive roles of APC proteins.     

Modification of chromosome instability in Aspergillus nidulans

Strains of Aspergillus nidulans with a chromosome segment in duplicate show instability at mitosis; their colonies produce faster-growing sectors which arise from nuclei with spontaneous deletions in either duplicate segment. In an attempt to probe the deletion process, the effects of mutations causing sensitivity to UV treatment, and those of manganous ions, have been studied in strains carrying either Dp(I,II) or Dp(III,VIII). For comparison, the effects of Mn2+ on balanced and unbalanced diploids have also been examined. The uvsE allele, which decreases intragenic mitotic crossing over in diploids, increased deletion frequency in strains with either duplication. The uvsB allele, which increases intragenic mitotic crossing over in diploids, increased deletion frequency only in Dp(I,II) strains; in addition, by causing early mitotic crossing over between the homologous segments, it produced some novel deletion products. Mn2+ substantially decreased the deletion frequency in Dp(I,II) strains and decreased mitotic crossing over in diploids; it had no effect on Dp(III,VIII) strains. The results suggest that in haploid duplication strains there are two classes of spontaneous DNA lesions, recombinogenic and non-recombinogenic, both of which, failing repair, lead to deletion.     

Whole chromosome instability and cancer: a complex relationship

Although chromosome mis-segregation is a hallmark of cancer cells, its genetic basis and role in malignant transformation remain poorly understood. In recent years, several mouse models have been generated that harbor gene defects that perturb high-fidelity chromosome segregation. Analysis of these models has revealed that whole chromosome instability (W-CIN) can cause, inhibit or have no effect on tumorigenesis. Here we propose that the effect of W-CIN on tumor development depends on the particular W-CIN gene that is defective, including its other cellular functions, the extent or nature of the gene defect, the affected tissue or cell type and the context of other cancer gene mutations.     

Superoxide dismutase activity and chromosome damage in cultured chromosome instability syndrome cells

The basal levels of superoxide dismutase (SOD) activity and chromosome aberration (CA) and sister-chromatid exchange (SCE) frequencies were examined in cultured fibroblasts or Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs). These cells were derived from patients with chromosome instability syndromes (CISs) including Bloom's syndrome (BS), Fanconi's anemia (FA) and ataxia telangiectasia (AT). Embryonal fibroblasts and LCLs from normal subjects served as controls. Although LCLs tended to exhibit a higher SOD level than fibroblasts due to an elevation of Cu/Zn-SOD activity, BS and FA fibroblasts with increased frequencies of CAs and/or SCEs showed abnormally elevated SOD activity due to the manifold increase of Mn-SOD levels compared with control cells. However, BS and AT LCLs with almost control levels of CA and SCE frequencies showed no, or a slightly elevated, SOD activity, suggesting a possible selection of such cells during EBV transformation. The observed parallelism between the SOD activity and the cytogenetic manifestation may imply an involvement of active oxygen species, especially superoxide radicals, in the increased chromosome damage of CIS cells.     

Origin of diplochromosomal polyploidy in near-senescent fibroblast cultures: heterochromatin, telomeres and chromosomal instability (CIN)

The near-senescence associated phenomena of increases in cells with chromosomal damage (CIN) and in endopolyploid mitotic cells were analyzed for possible inter-relationships by cytogenetics. Gross chromosomal abnormalities in all phases of mitosis were analyzed in situ. Hetrochromatization of telomeres, centromeres and interstitial chromatin regions (i.e., chromocenters/SAHF) were shown to be specific occurrences in the near-senescent phase. Stickiness between such chromatin regions caused breakage/fragmentation by anaphase-pulls on clumped chromosomes. Gluey heterochromatin is therefore, seen as a cause of CIN in near-senescence. Detrimental effects on chromosomes from heterochromatin have been observed for decades, and can be explained from chromatin remodeling in epigenetics. A consequence of genomic damage was re-replication to polyploidy of arrest-escaped cells with G2/M-DNA content. This second synthetic period produced diplochromosomal cells that cycled by bi-polar division into genome reduced cells. This sequence from h-chromatization to CIN and further to cycling endopolyploidy is believed to be a basic mechanism for the production of genetic variability in neoplasia.     

Evidence for chromosome instability in vivo in bloom syndrome: Increased numbers of micronuclei in exfoliated cells

Summary The incidence of exfoliated epithelial cells containing micronuclei was determined in two small human populations, one homozygous and the other heterozygous for the Bloom syndrome gene (bl). The objectives of the study were two: (1) to learn whether the chromosome instability featured so prominently by Bloom syndrome (BS) cells proliferating in vitro also occurs in vivo, and (2) as part of a broad survey of various cancer-prone populations, to determine whether estimating micronucleus frequencies in exfoliated cell samples might be useful for identifying individuals with genetically determined chromosome instability. Eight individuals homozygous (bl/bl) for the BS gene, i.e., persons with the clinical syndrome, were examined, along with 11 obligate heterozygotes (bl/+), parents of affected persons. Exfoliated cells were obtained from two sites, the oral cavity and the urinary tract. Striking and statistically highly significant elevations in the frequencies of cells with micronuclei were observed in cells from both sites in bl/bl individuals compared to that in bl/+ (P<0.001) and in a control population, indicating that chromosome instability occurs in vivo in BS. In contrast, micronucleus frequencies at either site did not differ significantly between bl/+ individuals and the control population. This survey, in combination with similar earlier ones of populations predisposed to cancer not on a genetic basis but because of exposure to some environmental carcinogen, suggests that the exfoliated cell micronucleus test identifies individuals whose somatic genetic material has, for either genetic or environmental reasons, been damaged in a way that produces chromosome breakage and rearrangement.     

Chromosome synapsis and genetic recombination: their roles in meiotic chromosome segregation

Chromosome synapsis and genetic recombination ensure the faithful segregation of chromosomes at meiosis I by establishing physical connections between homologs. Recent observations suggest that recombination may also play a role in the homology search process that precedes synapsis.     

Novel roles for APC family members and Wingless/Wnt signaling during Drosophila brain development

Construction of the brain is one of the most complex developmental challenges. Wnt signals shape all tissues, including the brain, and the tumor suppressor adenomatous polyposis coli (APC) is a key negative regulator of Wnt/Wingless (Wg) signaling. We carried out the first assessment of the role of APC proteins in brain development, simultaneously inactivating both APC1 and APC2 in clones of cells in the Drosophila larval optic lobe. We focused on the medulla, where epithelial neural progenitors shift from symmetric to asymmetric divisions across the lateral-medial axis. Loss of both APCs triggers dramatic defects in optic lobe development. Double mutant cells segregate from wild-type neighbors, while double mutant neurons form tangled axonal knots, suggesting changes in cell adhesion. Strikingly, phenotypes are graded along the anterior-posterior axis. Activation of Wg signaling downstream of APC mimics these phenotypes, a dominant-negative TCF blocks them, and a known Wg target, decapentaplegic, is activated in double mutant clones, strongly suggesting that the phenotypes result from activated Wg signaling. We also explored the roles of classic cadherins in differential adhesion. Finally, we propose a model suggesting that Wg signaling regulates fine scale cell fates along the anterior-posterior axis, in part by creating an adhesion gradient and consider possible alternate explanations for our observations.     

Karyotypic determinants of chromosome instability in aneuploid budding yeast

Recent studies in cancer cells and budding yeast demonstrated that aneuploidy, the state of having abnormal chromosome numbers, correlates with elevated chromosome instability (CIN), i.e. the propensity of gaining and losing chromosomes at a high frequency. Here we have investigated ploidy- and chromosome-specific determinants underlying aneuploidy-induced CIN by observing karyotype dynamics in fully isogenic aneuploid yeast strains with ploidies between 1N and 2N obtained through a random meiotic process. The aneuploid strains exhibited various levels of whole-chromosome instability (i.e. chromosome gains and losses). CIN correlates with cellular ploidy in an unexpected way: cells with a chromosomal content close to the haploid state are significantly more stable than cells displaying an apparent ploidy between 1.5 and 2N. We propose that the capacity for accurate chromosome segregation by the mitotic system does not scale continuously with an increasing number of chromosomes, but may occur via discrete steps each time a full set of chromosomes is added to the genome. On top of such general ploidy-related effect, CIN is also associated with the presence of specific aneuploid chromosomes as well as dosage imbalance between specific chromosome pairs. Our findings potentially help reconcile the divide between gene-centric versus genome-centric theories in cancer evolution.