SMURF2 prevents detrimental changes to chromatin, protecting human dermal fibroblasts from chromosomal instability and tumorigenesis.

E3 ubiquitin ligases (E3s) play essential roles in the maintenance of tissue homeostasis under normal and stress conditions, as well as in disease states, particularly in cancer. However, the role of E3s in the initiation of human tumors is poorly understood. Previously, we reported that genetic ablation of the HECT-type E3 ubiquitin ligase Smurf2 induces carcinogenesis in mice; but whether and how these findings are pertinent to the inception of human cancer remain unknown. Here we show that SMURF2 is essential to protect human dermal fibroblasts (HDFs) from malignant transformation, and its depletion converts HDFs into tumorigenic entity. This phenomenon was associated with the radical changes in chromatin structural and epigenetic landscape, dysregulated gene expression and cell-cycle control, mesenchymal-to-epithelial transition and impaired DNA damage response. Furthermore, we show that SMURF2-mediated tumor suppression is interlinked with SMURF2's ability to regulate the expression of two central chromatin modifiers-an E3 ubiquitin ligase RNF20 and histone methyltransferase EZH2. Silencing these factors significantly reduced the growth and transformation capabilities of SMURF2-depleted cells. Finally, we demonstrate that SMURF2-compromised HDFs are highly tumorigenic in nude mice. These findings suggest the critical role that SMURF2 plays in preventing malignant alterations, chromosomal instability and cancer.

Chromosomal integrity of human preimplantation embryos at different days post fertilization.

INTRODUCTION: In order to investigate the dynamics of genomic alterations that occur at different developmental stages in vitro, we examined the chromosome content of human preimplantation embryos by molecular-cytogenetic techniques at the single-cell level, up to 13 days post fertilization. METHODS: The embryos were genetically analyzed several times during their development in culture; each embryo was first analyzed by FISH at 'Day 3' post fertilization, than during its growth in vitro and the third analysis was performed at development arrest, then the entire blastocyst was analyzed by comparative genomic hybridization (CGH/aCGH). RESULTS: We found that while on 'Day 3' only 31% of the embryos were detected as normal, on 'Day 5-6', 44% of the embryos were classified as normal and on 'Day 7', 57% were normal. On 'Days 8-13', 52% of the embryos were classified as chromosomally normal. One third of the embryos that were chromosomally abnormal on 'Day 3', were found to be normal at development arrest point. DISCUSSION: These dynamic changes that occur at early developmental stages suggest that testing a single blastomere at 'Day 3' post fertilization for PGD might inaccurately reflect the embryo ploidy and increase the risk of false aneuploidy diagnosis. Alternatively, blastocyst stage diagnosis may be more appropriate.

Screening of human pluripotent stem cells using CGH and FISH reveals low-grade mosaic aneuploidy and a recurrent amplification of chromosome 1q.

Pluripotency and proliferative capacity of human embryonic stem cells (hESCs) make them a promising source for basic and applied research as well as in therapeutic medicine. The introduction of human induced pluripotent cells (hiPSCs) holds great promise for patient-tailored regenerative medicine therapies. However, for hESCs and hiPSCs to be applied for therapeutic purposes, long-term genomic stability in culture must be maintained. Until recently, G-banding analysis was considered as the default approach for detecting chromosomal abnormalities in stem cells. Our goal in this study was to apply fluorescence in-situ hybridization (FISH) and comparative genomic hybridization (CGH) for the screening of pluripotent stem cells, which will enable us identifying chromosomal abnormalities in stem cells genome with a better resolution. We studied three hESC lines and two hiPSC lines over long-term culture. Aneuploidy rates were evaluated at different passages, using FISH probes (12,13,16,17,18,21,X,Y). Genomic integrity was shown to be maintained at early passages of hESCs and hiPSCs but, at late passages, we observed low rates mosaiciam in hESCs, which implies a direct correlation between number of passages and increased aneuploidy rate. In addition, CGH analysis revealed a recurrent genomic instability, involving the gain of chromosome 1q. This finding was detected in two unrelated cell lines of different origin and implies that gains of chromosome 1q may endow a clonal advantage in culture. These findings, which could only partially be detected by conventional cytogenetic methods, emphasize the importance of using molecular cytogenetic methods for tracking genomic instability in stem cells.

Preimplantation genetic haplotyping a new application for diagnosis of translocation carrier's embryos- preliminary observations of two robertsonian translocation carrier families.

PURPOSE: Preimplantation genetic diagnosis using fluorescence in-situ hybridization (PGD-FISH) is currently the most common reproductive solution for translocation carriers. However, this technique usually does not differentiate between embryos carrying the balanced form of the translocation and those carrying the homologous normal chromosomes. We developed a new application of preimplantation genetic haplotyping (PGH) that can identify and distinguish between all forms of the translocation status in cleavage stage embryos prior to implantation. METHODS: Polymorphic markers were used to identify and differentiate between the alleles that carry the translocation and those that are the normal homologous chromosomes. RESULTS: Embryos from two families of robertsonian translocation carriers were successfully analyzed using polymorphic markers haplotyping. CONCLUSIONS: Our preliminary results indicate that the PGH is capable of distinguishing between normal, balanced and unbalanced translocation carrier embryos. This method will improve PGD and will enable translocation carriers to avoid transmission of the translocation and the associated medical complications to offspring.

Replication timing aberrations and aneuploidy in peripheral blood lymphocytes of breast cancer patients.

BACKGROUND: Peripheral blood lymphocytes of patients with hematological malignancies or solid tumors, such as renal cell carcinoma or prostate cancer, display epigenetic aberrations (loss of synchronous replication of allelic counterparts) and genetic changes (aneuploidy) characteristic of the cancerous phenotype. This study sought to determine whether such alterations could differentiate breast cancer patients from cancer-free subjects. METHODS: The HER2 locus-an oncogene assigned to chromosome 17 whose amplification is associated with breast cancer (BCA)-and the pericentromeric satellite sequence of chromosome 17 (CEN17) were used for replication timing assessments. Aneuploidy was monitored by enumerating the copy numbers of chromosome 17. Replication timing and aneuploidy were detected cytogenetically using fluorescence in situ hybridization technology applied to phytohemagglutinin-stimulated lymphocytes of 20 women with BCA and 10 control subjects. RESULTS: We showed that both the HER2 and CEN17 loci in the stimulated BCA lymphocytes altered their characteristic pattern of synchronous replication and exhibited asynchronicity. In addition, there was an increase in chromosome 17 aneuploidy. The frequency of cells displaying asynchronous replication in the patients' samples was significantly higher (P < 10(-12) for HER2 and P < 10(-6) for CEN17) than the corresponding values in the control samples. Similarly, aneuploidy in patients' cells was significantly higher (P < 10(-9)) than that in the controls. CONCLUSIONS: The HER2 and CEN17 aberrant replication differentiated clearly between BCA patients and control subjects. Thus, monitoring the replication of these genes offers potential blood markers for the detection and monitoring of breast cancer.

True hermaphroditism with ambiguous genitalia due to a complicated mosaic karyotype: clinical features, cytogenetic findings, and literature review.

Abnormal recombination between the X and Y chromosomes during meiosis, occurring outside the pseudoautosomal region, can result in translocation of the SRY gene from the Y to the X chromosome, and consequently in abnormal sexual differentiation, such as the development of 46,XX males or true hermaphroditism. In this report we present clinical, cytogenetic, and molecular-cytogenetic data of a patient with ambiguous genitalia and true hermaphroditism, who had a unique mosaic karyotype, comprising three different cell lines: 46,XX(SRY+), 45,X(SRY+), and 45,X. The mosaic karyotype of our patient probably represents two different events: abnormal recombination between the X and Y chromosomes during paternal meiosis, and postzygotic loss of one of the X chromosomes. Replication studies demonstrated that in 80% of the XX cells, the SRY sequence was located on the active X chromosome. This finding suggests nonrandom X inactivation and, together with the presence of the SRY gene, explains the male phenotype of our patient. On the other hand, the presence of the 45,X cell line may have contributed to genital ambiguity. We conclude that fluorescence in situ hybridization (FISH) analysis with SRY probes is highly recommended and allows accurate diagnosis and optimal management in cases of 46,XX hermaphroditism and ambiguous genitalia.