Joop Wiegant

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 β-catenin regulatory domain contributes to chromosomal instability in colorectal cancer.

Universal features of post-transcriptional gene regulation are critical for Plasmodium zygote development.

A universal feature of metazoan sexual development is the generation of oocyte P granules that withhold certain mRNA species from translation to provide coding potential for proteins during early post-fertilization development. Stabilisation of translationally quiescent mRNA pools in female Plasmodium gametocytes depends on the RNA helicase DOZI, but the molecular machinery involved in the silencing of transcripts in these protozoans is unknown. Using affinity purification coupled with mass-spectrometric analysis we identify a messenger ribonucleoprotein (mRNP) from Plasmodium berghei gametocytes defined by DOZI and the Sm-like factor CITH (homolog of worm CAR-I and fly Trailer Hitch). This mRNP includes 16 major factors, including proteins with homologies to components of metazoan P granules and archaeal proteins. Containing translationally silent transcripts, this mRNP integrates eIF4E and poly(A)-binding protein but excludes P body RNA degradation factors and translation-initiation promoting eIF4G. Gene deletion mutants of 2 core components of this mRNP (DOZI and CITH) are fertilization-competent, but zygotes fail to develop into ookinetes in a female gametocyte-mutant fashion. Through RNA-immunoprecipitation and global expression profiling of CITH-KO mutants we highlight CITH as a crucial repressor of maternally supplied mRNAs. Our data define Plasmodium P granules as an ancient mRNP whose protein core has remained evolutionarily conserved from single-cell organisms to germ cells of multi-cellular animals and stores translationally silent mRNAs that are critical for early post-fertilization development during the initial stages of mosquito infection. Therefore, translational repression may offer avenues as a target for the generation of transmission blocking strategies and contribute to limiting the spread of malaria.

Multiple and sensitive fluorescence in situ hybridization with rhodamine-, fluorescein-, and coumarin-labeled DNAs

We have tested the use of several newly developed red, green, and blue fluorescent dUTPs in direct, multiple, and sensitive fluorescence in situ hybridization procedures. Among the ones tested, the tetramethylrhodamine-dUTP proved to give the best sensitivity; using conventional epifluorescence microscopy, cosmids could be visualized with a hybridization efficiency of 90%. Fluorescein-dUTP permitted visual cosmid detection with 50% efficiency, and, to the human eye, the green signals appeared less bright. With a blue fluorescent coumarin-labeled dUTP, only highly repetitive target sequences could be visualized directly. Imaging with a cooled CCD (charged coupled device) camera for prolonged integration times permitted localization of small targets using probes labeled with red and green fluorochromes. Visualization of a 1.8-kb single copy sequence with a rhodamine-labeled cDNA proved feasible. The strategy of identifying hybridization sites with multiple colors was successfully applied for multiple hybridizations to repetitive and unique targets, using the red and green labels directly and the biotin label indirectly with one layer of avidin-coumarin.

Centrosomal abnormalities, multipolar mitoses, and chromosomal instability in head and neck tumours with dysfunctional telomeres.

Carcinomas of the head and neck typically exhibit complex chromosome aberrations but the underlying mutational mechanisms remain obscure. Evaluation of cell division dynamics in low-passage cell lines from three benign and five malignant head and neck tumours revealed a strong positive correlation between multipolarity of the mitotic spindle and the formation of bridges at anaphase in both benign and malignant tumours. Cells exhibiting a high rate of mitotic abnormalities also showed several chromosome termini lacking TTAGGG repeats and a high frequency of dicentric chromosomes. Multicolour karyotyping demonstrated a preferential involvement in structural rearrangements of chromosomes with deficient telomeres. The majority of malignant, mitotically unstable tumours expressed the reverse transcriptase subunit of telomerase. These data indicate that some of the genomic instability in head and neck tumours is initiated by telomere dysfunction, leading to the formation of dicentric chromosomes. These form chromosome bridges at mitosis that could prevent the normal anaphase-telophase transition. In turn, this may cause an accumulation of centrosomes and mitotic multipolarity. Telomerase expression does not confer total stability to the tumour genome but could be crucial for moderating the rate of chromosomal evolution.

Characterization of chromosome aberrations in salivary gland tumors by FISH, including multicolor COBRA-FISH

Fluorescence in situ hybridization (FISH), including COBRA‐FISH, was used to characterize 11 salivary gland tumors that had been investigated by banding analysis. Five cases were pleomorphic adenoma (PA), three were adenoid cystic carcinoma, and one case each was mucoepidermoid carcinoma, carcinoma ex‐pleomorphic adenoma (CaPA), and adenocarcinoma. All 11 cases were selected on the basis that they had shown rearrangement of 6q or 9p or had unresolved aberrations after karyotyping. The COBRA‐FISH and FISH analyses led to a revised karyotype in all informative cases and made it possible to clarify almost all chromosomal rearrangements occurring in the tumors. Of particular note were the confirmation of the existence of 6q deletions, a common change in salivary gland carcinomas, and the demonstration that a seemingly balanced t(6;9) resulted in del(6q). Other rearrangements that were revealed by FISH included amplification of 12q sequences (MDM2 and CDK4) in one PA. We also investigated the status of the PLAG1 gene in four cases (one PA, one CaPA, one adenoid cystic carcinoma, and one mucoepidermoid carcinoma) with 8q12 rearrangements. Only in the former two cases were the FISH results compatible with intragenic rearrangements. Overall, the results of the study show that, even with good banding quality and in karyotypes of modest complexity, much new information will be gained by supplementing the banding analysis with a multicolor FISH approach, such as COBRA‐FISH.

Simultaneous molecular karyotyping and mapping of viral DNA integration sites by 25‐color COBRA‐FISH

Combined binary ratio labeling (COBRA) fluorescence in situ hybridization (FISH) allows 24‐color FISH karyotyping of human metaphase chromosomes utilizing only four fluorochromes, instead of the five required for combinatorial labeling procedures. Here we show that by introduction of a fifth fluorochrome, COBRA‐FISH permits molecular cytogenetic mapping of viral integration sites in complex karyotypes in the context of a 24‐color hybridization. We were able to detect a single copy of the human papillomavirus 16 in the SiHa cell line and to confirm the site of integration at 13q21–31. We also demonstrate the gene mapping possibility of 25‐color hybridization by detecting a MYC cosmid on normal metaphase chromosomes. The possibility of mapping single‐copy probes in the background of 24‐color hybridization expands the tools for cytogenetic mapping of DNA sequences and will contribute to the understanding of the role of viral integration and chromosome rearrangement in virus‐mediated carcinogenesis. Genes Chromosomes Cancer 28:92–97, 2000.

Simultaneous mapping of human papillomavirus integration sites and molecular karyotyping in short-term cultures of cervical carcinomas by using 49-color combined binary ratio labeling fluorescence in situ hybridization

Infection with high-risk type human papillomavirus (HPV) is a necessary causal factor in the pathogenesis of cervical carcinoma. In most invasive cervical cancers, HPV is integrated in the host cell genome, and additional genetic aberrations are observed among which are chromosomal aberrations. To analyze in detail such often complex chromosomal changes and simultaneously map HPV integration sites, we extended the multiplicity of the combined binary ratio labeling fluorescence in situ hybridization (COBRA-FISH) technique to 49 by inclusion of a large Stokes shift fluorochrome as the third binary label. The technique allows mapping of the integrated HPV genome in the context of p- and q-arm COBRA-FISH, with a sensitivity of one copy of the HPV genome as tested for HPV 16 in SiHa cells. We investigated the molecular karyotypes and integration patterns of HPV types 16 and 18 in metaphase spreads from short-term cultures of primary cervical carcinomas (n=5). Of the tested cervical carcinomas, two contained integrated HPV at 8q24, one of which in addition harbored the integrated virus near a translocation breakpoint. Two carcinomas had integrated HPV at 17q21 through 23 in a morphologically normal chromosome 17. One carcinoma contained HPV at 1q42 in a morphologically normal chromosome 1. Our data illustrate the efficacy of 49-color COBRA-FISH to resolve complex karyotypes and simultaneously map specific sequences in metaphases obtained from short-term solid tumor cultures.

Individuals with abnormal phenotype and normal G-banding karyotype: improvement and limitations in the diagnosis by the use of 24-colour FISH

Abstract. The simultaneous identification, by fluorescence in situ hybridisation (FISH), of each chromosome in a distinct colour became feasible a few years ago. The key question in the application of this and many other developments in molecular cytogenetics to clinical situations is whether the results add significant further information that is relevant to the diagnosis. So far, limited data exist regarding how much improvement the technique brings to the diagnosis of phenotypically abnormal individuals in whom no abnormalities have been detected by conventional G-banding analysis. Because of the lack of a conclusive diagnosis, genetic counselling, estimation of recurrence risk and prenatal diagnosis of these individuals and their relatives is problematic. We report a study with 24-colour whole-chromosome painting of 10 familial and 11 isolated cases with abnormal phenotypes and normal G-banding karyotypes. Previously undetected unbalanced translocations were revealed in two cases. The value and current cost-effectiveness of multicolour FISH for cytogenetic diagnosis is discussed.

Telomeric DNA Mediates De Novo PML Body Formation

The cell nucleus harbors a variety of different bodies that vary in number, composition, and size. Although these bodies coordinate important nuclear processes, little is known about how they are formed. Among the most intensively studied bodies in recent years is the PML body. These bodies have been implicated in gene regulation and other cellular processes and are disrupted in cells from patients suffering from acute promyelocytic leukemia. Using live cell imaging microscopy and immunofluorescence, we show in several cell types that PML bodies are formed at telomeric DNA during interphase. Recent studies revealed that both SUMO modification sites and SUMO interaction motifs in the promyelocytic leukemia (PML) protein are required for PML body formation. We show that SMC5, a component of the SUMO ligase MMS21-containing SMC5/6 complex, localizes temporarily at telomeric DNA during PML body formation, suggesting a possible role for SUMO in the formation of PML bodies at telomeric DNA. Our data identify a novel role of telomeric DNA during PML body formation.

Array comparative genomic hybridization with cyanin cis-platinum-labeled DNAs.

Fluorescent cis-platinum compounds that react with the N7 atom of guanine are useful for labeling nucleic acids influorescence hybridization applications. Here we report that cyanin (CyN) cis-platinum labeling of DNA samples for array comparative genomic hybridizations (arrayCGH) can be achieved reproducibly and reliably. We demonstrate that degrees of labeling of approximately 1% of all nucleotides in test and reference DNA samples with CyN3- and CyN5-cis-platinum produces arrayCGH signal-to-background ratios ranging from 30 to 40. The arrayCGH results achieved during analyses of mouse and human tumor samples were comparable to those achieved using enzymatic labeling. Thus, we conclude that Cy-cis-platinum labeling is an alternative to enzymatic labeling for arrayCGH.