Marja van der Burg

Chromosome Segregation Errors as a Cause of DNA Damage and Structural Chromosome Aberrations

Altering whole-chromosome number can directly increase chromosome structural damage in cancer cells. Various types of chromosomal aberrations, including numerical (aneuploidy) and structural (e.g., translocations, deletions), are commonly found in human tumors and are linked to tumorigenesis. Aneuploidy is a direct consequence of chromosome segregation errors in mitosis, whereas structural aberrations are caused by improperly repaired DNA breaks. Here, we demonstrate that chromosome segregation errors can also result in structural chromosome aberrations. Chromosomes that missegregate are frequently damaged during cytokinesis, triggering a DNA double-strand break response in the respective daughter cells involving ATM, Chk2, and p53. We show that these double-strand breaks can lead to unbalanced translocations in the daughter cells. Our data show that segregation errors can cause translocations and provide insights into the role of whole-chromosome instability in tumorigenesis.

Long–term culture of primary human lymphoblastic leukemia cells in the absence of serum or hematopoietic growth factors

OBJECTIVE B-lineage acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia in lymphatic blastic phase in adults have poor prognoses despite intensive chemotherapy. Novel targeted treatment modalities emerge, but their evaluation requires relevant in vitro models of lymphoblastic leukemia. Presently available cell lines do not fully represent this heterogeneous disease. Available in vitro culturing protocols do not support long-term proliferation of primary cells. We therefore aimed to develop a culture system that allows long-term proliferation of primary human B-lineage lymphoblastic leukemia. MATERIALS AND METHODS Primary lymphoblastic leukemia cells were cultured in a defined serum-free medium, in the absence or presence of human hematopoietic growth factors or serum. RESULTS In the defined serum-free medium, cells from 12 of 34 cases immediately proliferated in vitro. In the absence of hematopoietic growth factors and serum these cases proliferated for more than 1 year without signs of exhaustion. The culturing system supported different subtypes of lymphoblastic leukemia. Two chronic myeloid leukemia in lymphatic blastic phase, four bcr/abl-positive ALL, one etv6/abl-positive ALL, 2 e2a-pbx1-positive ALL, and one t(9;11)-positive ALL could be long-term expanded, as well as two ALL that displayed nontypical cytogenetics. Not all bcr/abl- or e2a-pbx1-positive ALL proliferated in vitro, demonstrating heterogeneity within these subtypes. The proliferating bcr/abl- and etv6/abl-positive cells displayed sensitivity to imatinib, demonstrating that their proliferation depended on the activity of these oncoproteins. CONCLUSION The serum-free culturing system may be a valuable instrument in the study of ALL cell biology, as well as in the evaluation of novel targeted therapeutics.

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.

Optimized amplification and fluorescent labeling of small cell samples for genomic array-CGH

Whole genome amplification (WGA) is usually needed in the genetic analysis of samples containing a low number of cells. In genome‐wide analysis of DNA copy numbers by array comparative genomic hybridization (array‐CGH) it is very important that the genome is evenly represented throughout the amplified product. All currently available WGA techniques are generating some degree of bias.

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.

Rapid detection of genomic imbalances using micro-arrays consisting of pooled BACs covering all human chromosome arms

A strategy is presented to select, pool and spot human BAC clones on an array in such a way that each spot contains five well performing BAC clones, covering one chromosome arm. A mini-array of 240 spots was prepared representing all human chromosome arms in a 5-fold as well as some controls, and used for comparative genomic hybridization (CGH) of 10 cell lines with aneusomies frequently found in clinical cytogenetics and oncology. Spot-to-spot variation within five replicates was below 6% and all expected abnormalities were detected 100% correctly. Sensitivity was such that replacing one BAC clone in a given spot of five by a BAC clone from another chromosome, thus resulting in a change in ratio of 20%, was reproducibly detected. Incubation time of the mini-array was varied and the fluorescently labelled target DNA was diluted. Typically, aneusomies could be detected using 30 ng of non-amplified random primed labelled DNA amounts in a 4 h hybridization reaction. Potential application of these mini-arrays for genomic profiling of disseminated tumour cells or of blastomeres for preimplantation genetic diagnosis, using specially designed DNA amplification methods, are discussed.

Oncogenic functions of hMDMX in in vitro transformation of primary human fibroblasts and embryonic retinoblasts

BackgroundIn around 50% of all human cancers the tumor suppressor p53 is mutated. It is generally assumed that in the remaining tumors the wild-type p53 protein is functionally impaired. The two main inhibitors of p53, hMDM2 (MDM2) and hMDMX (MDMX/MDM4) are frequently overexpressed in wild-type p53 tumors. Whereas the main activity of hMDM2 is to degrade p53 protein, its close homolog hMDMX does not degrade p53, but it represses its transcriptional activity. Here we study the role of hMDMX in the neoplastic transformation of human fibroblasts and embryonic retinoblasts, since a high number of retinoblastomas contain elevated hMDMX levels.MethodsWe made use of an in vitro transformation model using a retroviral system of RNA interference and gene overexpression in primary human fibroblasts and embryonic retinoblasts. Consecutive knockdown of RB and p53, overexpression of SV40-small t, oncogenic HRasV12 and HA-hMDMX resulted in a number of stable cell lines representing different stages of the transformation process, enabling a comparison between loss of p53 and hMDMX overexpression. The cell lines were tested in various assays to assess their oncogenic potential.ResultsBoth p53-knockdown and hMDMX overexpression accelerated proliferation and prevented growth suppression induced by introduction of oncogenic Ras, which was required for anchorage-independent growth and the ability to form tumors in vivo. Furthermore, we found that hMDMX overexpression represses basal p53 activity to some extent. Transformed fibroblasts with very high levels of hMDMX became largely resistant to the p53 reactivating drug Nutlin-3. The Nutlin-3 response of hMDMX transformed retinoblasts was intact and resembled that of retinoblastoma cell lines.ConclusionsOur studies show that hMDMX has the essential properties of an oncogene. Its constitutive expression contributes to the oncogenic phenotype of transformed human cells. Its main function appears to be p53 inactivation. Therefore, developing new drugs targeting hMDMX is a valid approach to obtain new treatments for a subset of human tumors expressing wild-type p53.

Application of multicolor fluorescence in situ hybridization analysis for detection of cross-contamination and in vitro progression in commonly used murine tumor cell lines

Murine tumor models are potent tools for cancer studies, most of which make use of a limited number of murine tumor cell lines that are exchanged by many research groups around the world. Although cross-contamination and in vitro karyotypic progression are well-known risks with respect to the identity of tumor cell lines, these parameters are rarely evaluated. Notably, routine karyotyping of murine cell lines is laborious and technically demanding because mouse chromosomes are morphologically similar. We therefore used a 21-color fluorescence in situ hybridization (FISH) approach (COBRA) for screening two groups of frequently used murine tumor cell lines, each of which shares known immunologic determinants. Multicolor analysis revealed that the sharing of immunologic determinants among three murine lymphoma cell lines (EL-4, MBL-2, and RBL-5) is directly related to their common origin. In several of the cell lines, the chromosomal derivatives had rearranged further, suggesting that the cross-contamination events were not recent. In contrast, karyotypic analysis of three murine colon cancer cell lines (C26, CC36, and C51) showed that these constituted independent tumor clones despite the sharing of immunologic determinants. Our data point out that cross-contamination and in vitro evolution of murine tumor cell lines are a common phenomenon, and that multicolor FISH analysis is an efficient tool for verifying the origin and tracking the evolution of murine cell lines.