Reducing MCM levels in human primary T cells during the G(0)-->G(1) transition causes genomic instability during the first cell cycle.

DNA replication is tightly regulated, but paradoxically there is reported to be an excess of MCM DNA replication proteins over the number of replication origins. Here, we show that MCM levels in primary human T cells are induced during the G(0)-->G(1) transition and are not in excess in proliferating cells. The level of induction is critical as we show that a 50% reduction leads to increased centromere separation, premature chromatid separation (PCS) and gross chromosomal abnormalities typical of genomic instability syndromes. We investigated the mechanisms involved and show that a reduction in MCM levels causes dose-dependent DNA damage involving activation of ATR & ATM and Chk1 & Chk2. There is increased DNA mis-repair by non-homologous end joining (NHEJ) and both NHEJ and homologous recombination are necessary for Mcm7-depleted cells to progress to metaphase. Therefore, a simple reduction in MCM loading onto DNA, which occurs in cancers as a result of aberrant cell cycle control, is sufficient to cause PCS and gross genomic instability within one cell cycle.

Chromosomal aberrations in the peripheral blood of patients with metal-on-metal hip bearings.

BACKGROUND: Approximately one-third of patients undergoing joint replacement are under sixty years of age. Many of these patients may be exposed to wear debris from the orthopaedic implant for several decades. Clinical follow-up of this group of patients has been short compared with the lifetimes of the patients, and the long-term effects of this chronic exposure are unknown. METHODS: By using cytogenetic biomarkers (twenty-four-color fluorescent in situ hybridization [FISH]), we analyzed the peripheral blood leukocytes for chromosomal aberrations in three groups of subjects: (1) six age and sex-matched control subjects who had no implant and did not smoke (control group), (2) five subjects in whom an implant with a metal-on-metal articulation had been in situ for an average of thirty-five years (metal-on-metal group), and (3) four subjects in whom a metal-on-metal implant had been revised to a metal-on-polyethylene articulation at an average of twenty-two years (revised group). RESULTS: The number of chromosomal aberrations in the metal-on-metal group was greater than that in the control group. Specifically, the percentage of aneuploidy gain was three times greater (p = 0.01) in the metal-on-metal group. Structural aberrations were not seen in the control group, and this difference was highly significant (p = 0.003). Also, the number of chromosomal aberrations in the metal-on-metal group was greater than that in the revised group. Specifically, the percentage of structural aberrations was thirty-one-fold higher (p = 0.013). The percentage of aneuploidy gain in the metal-on-metal group was about twice that in the revised group, although this difference was not significant (p = 0.37). The percentage of aneuploidy gain in the revised group was about double that in the control group, although this difference was also not significant (p = 0.41). Translocations were seen only in subjects with a metal-on-metal articulation. CONCLUSIONS: The clinical consequences of the chromosomal changes seen in this study are unknown, and it is unknown if the changes are present in other cells in the body. The results emphasize the need for additional investigations into the effect of chronic exposure to elevated levels of metal ions produced by orthopaedic implants.

The role of the surface chemistry of CoCr alloy particles in the phagocytosis and DNA damage of fibroblast cells.

Surface chemistry of CoCr particles is demonstrated to be fundamental to the process of phagocytosis by fibroblast cells in vitro. Particles preincubated in serum for 5 days and washed in water before addition to cell cultures were phagocytosed less readily than were particles preincubated in minimal essential medium (MEM) for 1 h and washed in water. This was explained by the coating of calcium phosphate and protein on the serum-immersed particles investigated by time-of-flight secondary ion mass spectroscopy. The cells incubated with the serum-immersed particles had a reduced mitotic index when compared with the MEM-immersed particles, indicating that the phagocytosed particles were causing cell cycle arrest. The release of soluble ions measured by electrothermal atomic absorption spectroscopy within the first hour of particle immersion in MEM was identified as the most likely cause for the DNA damage measured by single cell gel electrophoresis ("Comet" assay). Cryofocused ion beam SEM with a spatial resolution of 8 nm was used to cross section cells, to investigate the location of the phagocytosed particles, some of which were found within the nuclear membrane. This paper demonstrated that consideration of the surface chemistry is essential to understand the processes of the effects of orthopedic wear debris.

Effects of hTERT on metal ion-induced genomic instability.

There is currently a great interest in delayed chromosomal and other damaging effects of low-dose exposure to a variety of pollutants which appear collectively to act through induction of stress-response pathways related to oxidative stress and ageing. These have been studied mostly in the radiation field but evidence is accumulating that the mechanisms can also be triggered by chemicals, especially heavy metals. Humans are exposed to metals, including chromium (Cr) (VI) and vanadium (V) (V), from the environment, industry and surgical implants. Thus, the impact of low-dose stress responses may be larger than expected from individual toxicity projections. In this study, a short (24 h) exposure of human fibroblasts to low doses of Cr (VI) and V (V) caused both acute chromosome damage and genomic instability in the progeny of exposed cells for at least 30 days after exposure. Acutely, Cr (VI) caused chromatid breaks without aneuploidy while V (V) caused aneuploidy without chromatid breaks. The longer-term genomic instability was similar but depended on hTERT positivity. In telomerase-negative hTERT- cells, Cr (VI) and V (V) caused a long lasting and transmissible induction of dicentric chromosomes, nucleoplasmic bridges, micronuclei and aneuploidy. There was also a long term and transmissible reduction of clonogenic survival, with an increased beta-galactosidase staining and apoptosis. This instability was not present in telomerase-positive hTERT+ cells. In contrast, in hTERT+ cells the metals caused a persistent induction of tetraploidy, which was not noted in hTERT- cells. The growth and survival of both metal-exposed hTERT+ and hTERT- cells differed if they were cultured at subconfluent levels or plated out as colonies. Genomic instability is considered to be a driving force towards cancer. This study suggests that the type of genomic instability in human cells may depend critically on whether they are telomerase-positive or -negative and that their sensitivities to metals could depend on whether they are clustered or diffuse.

Donor lymphocyte infusion followed by interferon-alpha plus low dose cyclosporine A for modulation of donor CD3 cells activity with monitoring of minimal residual disease and cellular chimerism in a patient with first hematologic relapse of chronic myelogenous leukemia after allogeneic bone marrow transplantation.

A 15-year-old girl with Ph-positive chronic myelogenous leukemia in first chronic phase received bone marrow from her human leukocyte antigen matched brother. Twenty three months after bone marrow transplantation hematological relapse occured which was treated with two infusions of donor lymphocytes (DLI) (0.5x10(8) CD3/kg b.w./infusion). To enforce the graft-versus-leukemia effect (GvL), the first DLI was followed by administration of interferon-alpha (INF-alpha) 6x10(6) U/day for 30 days, whereas, after the second infusion INF-alpha was given at the same dose until hematological remission was achieved (80 doses). Minimal residual disease (MRD) was detected by conventional cytogenetics (Ph chromosome), fluorescence in situ hybridization (FISH) cytogenetics (BCR/ABL translocation) and reverse transcriptase-polymerase chain reaction (RT-PCR) Ecotropic virus integration site-1 (EVI-1 gene expression), whereas cellular chimerism was monitored by assessment of microsatellite markers PCR and Y-chromosomal DNA content FISH. When hematological remission was achieved the pancytopenia was observed and the cytogenetic and molecular investigations revealed only partial remission and mixed chimerism, however, with predominance of donor origin hematopoiesis. To diminish the myelosupressive effect of donor CD3 cells without switching-off the GvL effect, a low dose of cyclosporine A was given. Further observation revealed significant improvement of hematopoiesis with parallel gradual decline of MRD and increase of donor hematopoiesis up to complete chimerism. Graft-versus-host disease was not observed at any stage of the treatment.

Molecular follow up of donor lymphocyte infusion in CML children after allogeneic bone marrow transplantation.

In this study we present the applicability of fluorescence in situ hybridisation (FISH) and RT-PCR to detect the minimal residual disease (MRD) in relapsed Ph+ children after donor lymphocyte infusion (DLI) post bone marrow transplantation (BMT). In both patients BCR/ABL fusion was detected and its transcript at the moment of relapse. After the DLI treatment in short time intervals a decreasing number of cells with BCR/ABL fusion were noticed and the expression of the hybrid gene disappeared. These results demonstrate that all the methods presented in this study provide a feasible, rapid and accurate way for the detecting of the minimal residual disease after DLI in Ph positive CML patients.

Chromosome identification and assignment of DNA clones in the dog using a red fox and dog comparative map.

We have developed a novel method for identifying dog chromosomes and unambiguously mapping specific clones onto canine chromosomes. This method uses a previously established red fox/dog comparative chromosome map to guide the FISH mapping of cloned canine DNA. Mixing metaphase preparations of the red fox and dog enabled a single hybridization to be performed on both species. We used this approach to map the chromosomal locations of twenty-six canine cosmids. Each cosmid contains highly polymorphic microsatellite markers currently used by the DogMap project to compile the canine linkage map. All but two cosmids were successfully assigned to subchromosomal regions on red fox and dog chromosomes. For eight cosmids previously mapped on dog chromosomes, we confirmed and refined the canine chromosomal assignments of seven cosmids and corrected an erroneous assignment regarding cosmid CanBern1. These results demonstrate that the red fox and dog comparative chromosome map can greatly improve the accuracy and efficiency of chromosomal assignments of canine genetic markers by FISH.

Chromosomal assignment of two putative canine keratin gene clusters.

Mutations in keratin genes account for a number of inherited keratodermas in humans. The groups of basic and acidic keratin genes are clustered on human chromosomes 12 and 17, respectively. The present authors have assigned the two putative keratin gene clusters to canine chromosomes using canine cosmid clones. Successful fluorescence in situ hybridization mapped the putative cluster of canine acidic genes to dog chromosome 20 and the putative cluster of basic keratin genes to a small autosome not yet included in the partial canine standard karyotype.