Normal Metaphase Research Articles

Genes Proximal and Distal to MYCN Are Highly Expressed in Human Neuroblastoma as Visualized by Comparative Expressed Sequence Hybridization

MYCN amplification is associated with poor prognosis in neuroblastoma disease. To improve our understanding of the influence of the MYCN amplicon and its corresponding expression, we investigated the 2p expression pattern of MYCN amplified (n = 13) and nonamplified (n = 4) cell lines and corresponding primary tumors (n = 3) using the comparative expressed sequence hybridization technique. All but one MYCN amplified cell line displayed overexpression at 2p. Expression peaks were observed frequently at 2pter and less frequently at 2p24 (MYCN locus), 2p23.3-23.2, and/or 2p23.1. Importantly, cell lines and two corresponding primary tumors displayed expression peaks at similar loci. No significant 2p24 expression level was observed for those cell lines displaying a low amplification rate (n = 3) by comparative genomic hybridization. Only the cell lines with an enhanced peak at 2p23.2-23.3 displayed coamplification of the ALK gene (2p23.2), reported to be associated with unfavorable prognosis. Finally, two of four cell lines without MYCN amplification, both derived from patients with poor outcome, also showed an expression peak at 2p23.2. These data indicate that, besides MYCN, other genes proximal and distal to MYCN are highly expressed in neuroblastoma. The prognostic significance of expression peaks at 2p23.2-23.3, independent of MYCN and ALK status, remains to be investigated.

A rule-based computer scheme for centromere identification and polarity assignment of metaphase chromosomes

Automatic centromere identification and polarity assignment are two key factors in the automatic karyotyping of human chromosomes. A multi-stage rule-based computer scheme has been investigated to automatically detect centomeres and determine polarities for both abnormal and normal metaphase chromosomes. The scheme first implements a modified thinning algorithm to identify the medial axis of a chromosome and extracts three feature profiles. Based on a set of pre-optimized classification rules, the scheme adaptively identifies the centromere and then assigns corresponding polarity. An image dataset of 2287 chromosomes acquired from 24 abnormal and 26 normal Giemsa metaphase cells is utilized to optimize and test the scheme. The overall accuracy is 91.4% for centromere identification and 97.4% for polarity assignment. The experimental results demonstrate that our scheme can be successfully applied to diverse chromosomes, which include those severely bent and abnormal chromosomes extracted from cancer cells.

DNA degradation during maturation of erythrocytes – molecular cytogenetic characterization of Howell-Jolly bodies

Howell-Jolly bodies (HJBs) are small DNA-containing inclusions of erythrocytes and are often present after splenectomy. The genetic composition of HJBs is unknown at present. We isolated individual erythrocytes that had inclusion bodies from five splenectomized patients and performed DNA amplification using degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) with subsequent reverse painting on normal male metaphase spreads. We also measured the sizes of HJBs in erythrocytes from a splenectomized patient using an inverted microscope. Two-dimensional positions of HJBs were projected onto a virtual erythrocyte. The average size of HJBs was 0.73 ± 0.17 µm (range 0.4–1.1 µm). Inside the erythrocyte the HJBs were found to be equally distributed. Small HJBs contained DNA from one or two centromeres and larger HJBs contained DNA from up to eight different centromeres. Centromeric DNA from chromosomes 1/5, 7, 8, and 18 was most frequently observed. Signals from the centromeric regions of chromosomes 3, 4, 9, and 10 were not observed. Signals from euchromatic regions were detected in a few cases. We hypothesize that in addition to enucleation and nucleus fragmentation DNA degradation during maturation of erythrocytes preferentially eliminates euchromatic DNA. Similarities between these processes and those described for embryonic stem cells suggest that most stem cells are able to degrade DNA in a genetically controlled manner.

Establishment and molecular cytogenetic characterization of non–small cell lung cancer cell line KU-T1 by multicolor fluorescence in situ hybridization, comparative genomic hybridization, and chromosome microdissection

A human lung adenocarcinoma cell line, designated KU-T1, was established from a Japanese man in Kochi Medical School. Conventional banding and multicolor fluorescence in situ hybridization (M-FISH) analyses of KU-T1 cells revealed a hyperdiploid chromosomal constitution and complex karyotypes. Comparative genomic hybridization showed several chromosomal copy number changes, and five regions that were highly amplified. Two of the five highly amplified regions, 1q and 3q, were identified from distributions of DNA sequences on a metaphase cell by FISH using chromosome microdissection-generated probes hybridized to 1q32∼q34 and 3q26∼q28, respectively. The 3q probe depicted a homogeneously staining region (hsr) in a derivative chromosome 3 of KU-T1. An hsr probe was regenerated by chromosome microdissection and was hybridized back to KU-T1 and normal metaphases. This hybridization experiment confirmed the probe derived from an hsr and indicated original locations of DNA sequences of hsr on normal chromosome 3. Intense hybridized signals shown at three loci (3p12, 3q26.3, and 3q28) suggests that oncogenes may be involved in the hsr formation. The present study provides a comprehensive analysis of the chromosomal abnormalities, including hsr formation and related oncogenes, in the KU-T1 cell line.

Downregulation of a Candidate Myeloid Tumor Suppressor Gene Isolated from 7q21 Induces Abnormal Mitosis Due to Insufficient Centrosome Maturation.

To isolate myeloid tumor suppressor gene(s) in 7q, we searched microdeletions in a region spanning 21.7 Mb within 7q21.2–7q31.1 using a microarray-based CGH system. By investigation of 21 childhood myeloid leukemia patients with normal karyotype, we identified a common microdeletion cluster spanning approximately 120 Kb in the 7q21.3 subband. Eight (38%) patients shared this microdeletion, which was not detected in normal individuals. Real-time quantitative PCR revealed that this region is also deleted in 9 (29%) out of 31 adult RAEB and AML patients. Database search revealed that this region contains three hypothetical genes. Among them, we chose one previously uncharacterized gene for further investigation and named Miki (mitotic kinetics regulator) for the function of its gene product, described below. Immunoblot analysis revealed high levels of Miki expression in most lymphoid leukemia cell lines, while half of myeloid leukemia cell lines expressed Miki at reduced levels. In six leukemia lines carrying monosomy 7, expression levels were generally low. Miki co-localized with the Golgi apparatus in the interphase and with centrosomes and spindles in the mitotic phase. To test the function of Miki, we used si-RNAs to downregulate Miki expression in HeLa and K562 cells, both of which show basically normal metaphase and nuclear morphology. Cells expressing Miki at reduced levels showed insufficient maturation and disturbed positioning of centrosomes, resulting in unorganized spindles including loss of spindle tension, curled and fragile spindles, or even completely disturbed spindle formation. Time-lapse observation revealed prometaphase and/or metaphase delay with unaligned or even totally scattered chromosomes in prometaphase in virtually all cells in the mitotic phase. As a result, cells underwent pre-anaphase arrest and exited mitosis in the absence of chromosome segregation or terminated mitosis by cell death. In the interphase, there were many cells with chromatid bridges and/or bi- or tri-nuclear or even multinuclear cells with micronuclei that resembled pathological cells routinely observed in the bone marrow pictures of MDS patients. Interestingly, myeloid cell lines with low Miki expression, including those with monosomy 7, generally showed abnormal mitosis such as scattered chromosomes and abnormal nuclear morphologies (multi-nuclear cells with small nuclei) at higher frequency than cell lines expressing Miki at high levels. Moreover, induction of Miki restores normal mitosis in leukemia cells with monosomy 7. Miki was poly(ADP-ribosyl)ated (PARsylated) in late G2 to M phase by tankyrase-1, one of PAR polymerase (PARP), and tankyrase-1 activity was required for the binding of Miki to spindles and centrosomes. These data suggest that loss of Miki gene contributes to the development and progression of MDS by disturbing mitosis.

Brefeldin A disrupts asymmetric spindle positioning in mouse oocytes

Polar body formation in oocytes is an extreme form of asymmetric cell division, but what regulates the asymmetric spindle positioning and cytokinesis is poorly understood. During mouse oocyte maturation, the metaphase I spindle forms at the center but then moves to the cortex prior to anaphase I and first polar body emission. We show here that treating denuded mouse oocytes with brefeldin A, an inhibitor of Golgi-based membrane fusion, abolished the asymmetric positioning of the metaphase I spindle and resulted in the formation of two half-size metaphase II eggs, instead of a full-sized egg and a polar body. The normal metaphase II spindle is similarly asymmetrically positioned in the mature egg, where the spindle lies with its axis parallel to the cortex but becomes perpendicular before anaphase II and emission of the second polar body. When ovulated eggs were activated with strontium in the presence of brefeldin A, the metaphase II spindle failed to assume perpendicular position, and the chromosomes separated without the extrusion of the second polar body. Remarkably, symmetric cytokinesis began following a 3 h delay, forming two half-size eggs each containing a pronucleus. BFA-sensitive intracellular vesicular transport is therefore required for spindle positioning in both MI and MII.

Insulin Signaling in Mouse Oocytes1

Continuous exposure of follicles/oocytes to elevated levels of insulin compromises embryonic developmental competence, although the underlying cellular mechanisms are unknown. The objectives of the present study were to determine whether mouse oocytes have insulin receptors and a functional insulin signaling cascade, and whether insulin exposure during oocyte growth or maturation influences meiotic progression and chromatin remodeling. Immunoblot and immunocytochemical analyses of germinal vesicle-intact (GVI) oocytes demonstrated the presence of insulin receptor-beta. Insulin receptor expression in oocytes was increased by gonadotropin stimulation, and remained elevated throughout meiotic maturation. Fully grown GVI oocytes contained 3-phosphoinositide-dependent protein kinase-1 (PDPK1), thymoma viral proto-oncogene 1 (AKT1), and glycogen synthase kinase 3 (GSK3). In vitro maturation of GVI oocytes in 5 microg/ml insulin had no influence on meiotic progression or the incidence of normal metaphase II (MII) chromosome condensation. Treatment of oocytes during maturation had no effect on GSK3A/B protein expression or phosphorylation of S21/9. However, the culturing of preantral follicles for 10 days with 5 microg/ml insulin increased the phosphorylation of oocyte GSK3B, indicating GSK3 inactivation. The rates of development to metaphase I (MI) were similar for oocytes obtained from insulin-treated follicles and controls, whereas the incidence of abnormal MI chromatin condensation was significantly higher in oocytes obtained from follicles cultured with insulin compared to those cultured without insulin. These results demonstrate that oocytes contain a functional insulin signaling pathway, and that insulin exposure during oocyte growth results in chromatin remodeling aberrations. These findings begin to elucidate the mechanisms by which chronic elevated insulin influences oocyte meiosis, chromatin remodeling, and embryonic developmental competence.

Current Treatment Options: Impact of Cytogenetics on the Course of Myelodysplasia

The heterogeneity of myelodysplastic syndromes (MDS) has driven the search for unifying biologic and clinical features that would stratify patients into distinct prognostic and therapeutic subgroups. Cytogenetics has been shown to impact the course of myelodysplasia. Despite the presence of non-random cytogenetic abnormalities in approximately 50% of MDS patients, it is significant that only a proportion of metaphases may contain the abnormality. Clonality studies however show that the karyotypically normal metaphases are still part of the MDS clone. This would suggest that the chromosomal abnormality may not be the initiating lesion in MDS, and that the gross karyotypic changes represent clonal evolution in a genetically unstable population. Yet, as will be described below, specific cytogenetic abnormalities are associated with clinically and biologically distinct forms of the disease, most notable in the response of del(5q) patients to lenalidomide. One possible explanation for the appearance of non-random mutational events could relate to the interaction of MDS cells with their microenvironment. Whatever the initiating lesion in the MDS stem cell, the end result is a clonal expansion where the marrow becomes populated by the monoclonal progeny of this cell. Interaction of these cells with a microenvironment which has been shown to be rich in pro-apoptotic cytokines such as tumor necrosis factor alpha (TNFa), leads to increased genetic instability. Hypoxia mediated decrease in DNA repair enzymes could further accelerate mutational events culminating in accumulation of multiple chromosomal abnormalities. Some of these chromosomal changes are associated with increased sensitivity to specific drugs. Lenalidomide has shown a high degree of efficacy in MDS patients with del(5q), although the target for the drug is unknown since a small but significant subset of MDS patients without del(5q) abnormality also respond to the drug. In contrast, the molecular target for imatinib mesylate is known; mutations in tyrosine kinase receptor family of genes found in patients with t(5;12) and del(4q12) make these individuals sensitive to the drug. Patients with isolated trisomy 8 have an immune component to the disease phenotype which can be targeted by cyclosporine and or anti-thymocyte globulin (ATG), especially in the presence of a PNH (paroxysmal nocturnal hemoglobinurea) clone. In the absence of these specific cytogenetic abnormalities described above, the two FDA approved hypomethylating agents 5 azacytidine and decitabine should be considered as therapeutic alternatives.

Cytology of root tips of <i> Dioscoreophyllum cumminsii </i> (STAPF) diel

Dioscoreophyllum cumminsii is a tropical dioecious rainforest vine. It grows in humid and heavily shaded understorey vegetation of closed forest, from May to October. The fruit, serendipity berry, contains a protein sweetener called monellin that could replace sugar in foods for diabetics and dieters. Other parts of the plant, for example, tubers, stems and leaves have medicinal values. This work was set up to study the mitotic chromosomes of root tips of D. cumminsii and establish the number of chromosomes in the genome. This is necessary since it appears that there is no published work on the chromosome number and structure of this important plant. The study was carried out at the Cytology Laboratory of the Department of Botany, University of Nigeria Nsukka. Root tips for the chromosome work were obtained from three sources: runners originating from main vine; sprouting pieces of tubers and redifferentiated root tips from callus. The results showed that normal metaphase plates of root tips of D. cumminsii contained sixteen chromosomes, 2n = 16. These chromosomes showed variations in size based on centromeric positions giving rise to metacentric and sub-metacentric chromosomes. Metaphase cells with more than 16 chromosomes were encountered from root tips regenerated in vitro from callus cells. This cytological knowledge is useful for future micropropagation or breeding activities involving D. cumminsii . Keywords : Aneuploids, Euploids, Monellin, Protein sweetener, Redifferentiation Agro-Science Vol. 4 (1) 2005: pp. 43-45

PIASγ Is Required for Faithful Chromosome Segregation in Human Cells

BackgroundThe precision of the metaphase-anaphase transition ensures stable genetic inheritance. The spindle checkpoint blocks anaphase onset until the last chromosome biorients at metaphase plate, then the bonds between sister chromatids are removed and disjoined chromatids segregate to the spindle poles. But, how sister separation is triggered is not fully understood.Principal FindingsWe identify PIASγ as a human E3 sumo ligase required for timely and efficient sister chromatid separation. In cells lacking PIASγ, normal metaphase plates form, but the spindle checkpoint is activated, leading to a prolonged metaphase block. Sister chromatids remain cohered even if cohesin is removed by depletion of hSgo1, because DNA catenations persist at centromeres. PIASγ-depleted cells cannot properly localize Topoisomerase II at centromeres or in the cores of mitotic chromosomes, providing a functional link between PIASγ and Topoisomerase II.ConclusionsPIASγ directs Topoisomerase II to specific chromosome regions that require efficient removal of DNA catenations prior to anaphase. The lack of this activity activates the spindle checkpoint, protecting cells from non-disjunction. Because DNA catenations persist without PIASγ in the absence of cohesin, removal of catenations and cohesin rings must be regulated in parallel.

A Phase I Trial of Bexarotene, a Retinoid X Receptor Agonist, in non-M3 Acute Myeloid Leukemia: Evidence of Myeloid Differentiation and Clinical Activity.

In vitro, bexarotene has been shown to inhibit the proliferation of non-M3 AML cell lines and induce differentiation of leukemic blasts. This phase I study was designed to evaluate the safety of escalating doses of bexarotene in patients with non-M3 AML. Bexarotene was administered orally daily until disease progression occurred. Prophylactic antihyperlipidemic agents were used in all patients. Six dose levels ranging from 100 to 400mg/m2 are planned. Dose escalation occurred in cohorts of 3–6 patients based on dose-limiting toxicity. Twenty patients have been enrolled in 5 dose cohorts (100–300mg/m2) with enrollment demographics: 13M/7F, median age 69 (range 51–82), 11 prior MDS, 8 primary refractory, median number of induction attempts 2, no prior induction chemotherapy 2, prior autologous stem cell transplant 4, 19 blood transfusion dependent, 13 platelet transfusion dependent, and 16 neutropenic. Consistent with reported toxicity, 2 patients developed hypothyroidism, 7 patients developed grade 2–4 hypertriglyceridemia and 1 patient developed grade 2 pancreatitis. Two patients developed a syndrome reminiscent of retinoic acid syndrome, consisting of dyspnea/hypoxia, pleural/pericardial effusions, weight gain/edema and dry cough in the setting of a rapidly rising neutrophil count. This syndrome resolved within 48 hours of stopping bexarotene and initiating steroids. No CR's were noted, however significant evidence of drug activity were seen. Six patients showed evidence of neutrophil response (pretreatment median ANC 286/μL, range 28–1,037/μL, posttreatment ANC 3,150/μL, range 1,100–27,207/μL). Flow sorted peripheral blood neutrophils were collected from three of these patients and examined by FISH. Between 92–100% of purified neutrophils contained the patient's leukemic cytogenetic abnormality suggesting differentiation of the leukemic blasts. Bone marrow blasts decreased to <6% in 3 patients. Nine patients with platelet counts <100,000/μL had increases in their platelet counts >30,000/μL (peak range 40–292x103/μL). Four of these patients with platelet counts <20,000/μL had improvement in their counts to 40–91,000/μL and became transfusion independent. One patient, with sustained clinical response, had 2 independent leukemic clones (7- and 7q- respectively) and no normal metaphases on bone marrow cytogenetic analysis prior to treatment. After 2 months of treatment, cytogenetic analysis showed 93% normal metaphases and no evidence of the original clones. However, 7% of metaphases consisted of 2 new leukemic clones with multiple cytogenetic abnormalities derived from his original clones. This is suggestive of partial eradication of the original leukemic clones and reestablishment of normal hematopoesis but with clonal evolution of the leukemic clones. Daily oral bexarotene is well tolerated at the dose levels studied to date. Early evidence for clinical activity has been seen as exemplified by improvement in platelet counts, increased neutrophil counts, cytogenetic effects, and decreased bone marrow blast counts. We postulate that bexarotene may work by inducing leukemic blast differentiation in non-M3 AML and may represent a novel non-cytotoxic treatment modality. Patient enrollment is ongoing.

Abnormal Mitosis and Genetic Instability Induced by Downregulation of Candidate Myeloid Tumor Suppressor Genes Isolated from the Long Arm of Chromosome 7.

To isolate myeloid tumor suppressor genes from 7q, we tried to detect microdeletions (< 100 Kb) that might be present in MDS/AML cells carrying apparently normal chromosome 7. For this purpose, we developed our original microarray-based CGH technology. In this system, instead of BAC clones generally used as probes, we applied short (3–5 Kb) genomic DNA fragments containing strictly no repetitive sequences. We made 235 probes in a region spanning 21.7 Mb within 7q21.3–7q31.1. Although we selected MDS/AML patients whose marrow did not show cytogenetically visible 7q deletions, gross copy number changes frequently observed in adult patients prevented us from identification of common microdeletions. By investigation of 21 childhood myeloid leukemia patients with normal karyotype, we successfully identified a common microdeletion spanning approximately 120 Kb. Eight (38%) patients shared this microdeletion, which was not detected in normal individuals. Database search revealed that this region contains three hypothetical genes. Only vertebrates have these genes that likely evolved from one common ancestral gene of fish. Real-time quantitative PCR revealed that 9 (29%) out of 31 adult MDS/AML harbors microdeletions in at least one of these three genes. None of these genes had been well characterized nor has known motifs that would suggest function of the gene products. We named them Miki, Titan and Kasumi. Immunoblot analysis revealed expression of all three genes at high levels in most lymphoid leukemia cell lines, while half of myeloid cell lines lacked at least one of their expression. In leukemia cells carrying monosomy 7, expression levels were generally low. Miki, a heavily glycosylated protein, co-localized with centrosomes and spindles in the mitotic phase. To test the function of Miki, we used si-RNAs to downregulate Miki expression in HeLa and K562 cells, both of which show basically normal metaphase and nuclear morphology. Cells expressing Miki at reduced levels showed small and fragmented centrosomes, loss of spindle tension, tripolar mitosis or even completely disturbed spindle formation. As a result, anaphase lagging, colchicine-mitosis (C-mitosis), premature chromosome decondensation and chromatid bridges were observed in virtually all cells in the mitotic phase. In the interphase, bi- or tri-nuclear or even multinuclear cells with micronuclei, all of which are characteristic to MDS, were frequently observed. On the other hand, proteomic analysis revealed that Titan and Kasumi bind to the DNA-PK complex, which plays critical roles in the non-homologous end joining (NHEJ) of double stranded DNA (dsDNA) breaks. Indeed, these proteins were translocated from cytoplasm to nucleus by ionizing radiation (IR) or by treatment of drugs that yield dsDNA breaks. Cells expressing Kasumi at reduced levels by si-RNA showed increased radiosensitivity, sister chromatid exchange, and number of background-level phosphorylated histone H2AX foci (i.e., foci formed without IR), which are co-localized with dsDNA breaks. These results indicate that the genes we isolated are promising candidates for anti-leukemic genes located in 7q, because downregulation of these gene products by 7q deletions would cause the abnormal morphology of MDS and genetic instability.

High Density SNP Arrays Reveal That Distinct Clonal Lesions Including Uniparental Disomy Can Be Detected in a Proportion of Patients with Aplastic Anemia with Normal Metaphase Cytogenetics.

Aplastic anemia (AA) and myelodysplastic syndrome (MDS) show pathophysiologic and clinical overlap; e.g., MDS can present with hypocellular marrow and a substantial proportion of AA patients develop MDS. A majority of AA patients respond to immunosuppression (IS), also effective in some patients with MDS. Patients with MDS show expansion of single hematopoietic clone frequently characterized by chromosomal aberrations while AA is oligoclonal, likely due to a decrease in available stem cells. Owing to the low resolution of metaphase cytogenetics and its dependence on cell growth in vitro, the ability to detect clonal chromosomal defects is limited in AA and the test is often negative or non-informative in MDS. High-density SNP arrays (SNP-A) can be used for precise identification of unbalanced genomic lesions. We hypothesize that cryptic clonal genomic aberrations do exist in some patients with AA. Their detection may have important clinical implications; it could help to distinguish patients unresponsive to IS, indicate propensity to MDS evolution and point towards pathogenetic genetic lesions. We have applied 50K and 250K SNP-A to analyze bone marrow (N=17), blood (N=49) or both (N=3) of patients with AA (N=26), PNH (N=33) and hypoplastic MDS (N=10). The results were analyzed using CNAG and Exemplar software. Defects identified in marrow of healthy controls (N=36) were used to define the minimal pathologic lesion (only 1 lesion was identified). In AA and PNH, clonal chromosomal abnormalities were found in 5/10 marrow and 15/52 blood samples, respectively, and in 4/10 bone marrows from hypocellular MDS. Of these lesions, known chromosomal defects (following progression to MDS) were confirmed in 3 AA patients and most significantly we also found previously cryptic abnormalities in 17 patients with AA (4 marrow, 13 blood) and normal metaphase cytogenetics. The new lesions involved segmental uniparental disomy (UPD) involving portions of chromosomes 1,2,6,7,8, 9,10,13 and 14. For example, we found LOH due to UPD at 7q (q21.11, q31.33, N=3). The results of 50K SNP-A were validated by 250K scan. LOH due to deletions (monoploid) or UDP (diploid) were confirmed by microsatellite and Taqman PCR copy number analysis, respectively. The lesions were somatic and not present in nonclonal lymphocytes. Overlapping UDP at 2p21.1–p22.1 (2AA, 1PNH) and 2p16.1 (2AA, 1hypoMDS) was detected. Examples of genes linked to SNP in deleted lesions include FANCL, LOC151445 and VRK2. Moreover, LOH due to UDP resulted in homozygosity for very minor alleles of SNPs linked to KIA1607 or NHMT (control population frequency 20%, 19%) and NRX1 or SMEK3 (14%, 27%) in 2q21 and 2p16, respectively. Hematologic response to IS was seen in 7/12 (58%) AA patients in whom lesions were found. However, only 5 of these patients were sampled prior to IS, 2 subsequently responded and 3 remained refractory. In contrast, 16/22 AA patients with normal chromosomes by SNP-A treated with IS showed a hematologic response (72%). Our study, representing the first application of high-density SNP-A for karyotyping of patients with AA, demonstrates that some patients with AA may harbor cryptic clonal defects possibly consistent with the diagnosis of MDS. It is likely that these lesions have clinical and prognostic significance.

Additional Cytogenetic Abnormalities and/or Philadelphia Chromosome Metaphase Mosaicism Might Adversely Influence Survival and Imatinib Response in Chronic Myeloid Leukemia.

Background: Chronic myeloid leukemia (CML) is invariably associated with the reciprocal translocation of BCR and ABL to form the Philadelphia chromosome (Ph), t(9;22)(q34;q11). At diagnosis, a small proportion of patients display additional cytogenetic abnormalities and/or a variable proportion of cytogenetically normal metaphases that coexist with Ph-positive metaphases. The objective of the current study was to examine the prognostic relevance of these two scenarios.Methods: The study population consisted of 65 (47.7% female) consecutive, newly diagnosed CML patients seen at the Mayo Clinic with a median age of 59 years. 44.5 percent were initially treated with interferon alpha (IFN) resulting in complete cytogenetic remission in 10.7 percent for a median length of 5.83 years, partial cytogenetic remission in 3.57 percent for a median length of 2 years. 55.5 percent were treated with imatinib (Gleevec) resulting in complete cytogenetic remission in 54.1 percent for a median length of 3.08 years, partial cytogenetic remission in 13.5% for a median length of 1.08 years.Results: Survival at five years through Kaplan-Meier analysis was approximately 92 percent in patients demonstrating only the Ph chromosome (n = 53), versus 60 percent in patients with additional chromosomal abnormalities (n = 12; p < 0.0001 by both Logrank and Breslow-Gehan-Wilcox analysis). Furthermore, patients with additional chromosomal abnormalities demonstrated lower rates of either complete or partial cytogenetic remission with imatinib therapy. Similarly, five year survival of patients with less than 90% Ph-positive metaphases at diagnosis (n = 4) was approximately 60% compared to 92% in patients with greater than 90% Ph-positive metaphases (n = 61; p = 0.01 by Logrank and p = 0.001 by Breslow-Gehan-Wilcox analysis). Additionally, the former group of patients was significantly less responsive to imatinib. Finally, there was significant correlation between Ph-positive metaphase mosaicism and the presence of additional chromosomal abnormalities (p = 0.05).Conclusion: Although it is tempting to speculate the possibility that Ph-chromosome mosaicism and/or additional cytogenetic abnormalities at presentation of CML is a surrogate for the presence of Ph-negative imatinib-resistant clones, the preliminary results from the current study require validation from a larger study. [Display omitted] [Display omitted]

The specificity of interphase FISH translocation probes in formalin fixed paraffin embedded tissue sections is readily assessed using automated staining and scoring of tissue microarrays constructed from murine xenografts

Implementation of interphase fluorescence in situ hybridization (FISH) assays in the clinical laboratory requires validation against established methods. Validation tools in common use include exchange of consecutive sections with another institution that has already established the FISH assay, comparison with conventional banded metaphase cytogenetics, confirmation of specificity using probed normal metaphases, consecutive paraffin sections of a validation set tested by a reference laboratory, and specificity assessment against well characterized cell lines. We have investigated the feasibility of using tissue microarrays (TMA) constructed from murine xenografts as a preliminary specificity-screening tool for validation of interphase FISH assays. Cell lines currently in use for FISH controls are used to generate xenografts in SCID mice which are fixed in formalin and paraffin embedded. A TMA is constructed using duplicate donor cores from the xenograft blocks. Xenografts used represent a wide range of translocations used routinely for formalin fixed paraffin embedded sections evaluated by FISH. Probe cocktails (Abbott-Vysis), for several non-random translocations associated with hematologic neoplasms and soft tissue sarcomas have been used in this manner. On-line deparaffinization, cell conditioning, and prehybridization steps are automated using a staining workstation (Ventana Discovery XT); hybridization and stringency washes are performed manually offline. FISH-probed TMAs are tracked using a Metasystems image scanner and analyzed using classifiers specifically developed for each molecular abnormality. FISH results for each xenograft in the TMA correspond exactly to the genotype previously established for the parent cell line from which the xenograft was prepared. Moderate complexity tissue microarrays constructed from murine xenografts are excellent validation tools for initial assessment of interphase FISH probe specificity.

Butyrolactone I reversibly alters nuclear configuration, periooplasmic microtubules and development of porcine oocytes

In the present study, we investigated the effects of specific cdc2 kinase inhibitor, butyrolactone I (BL I) on the prevention of germinal vesicle breakdown, changes of microtubular structures, and development of porcine oocytes after removal of the drug. In Experiment 1, cumulus–oocyte complexes (COCs) were cultured (44 h) in NCSU-23 medium containing different concentrations of BL I. The percentages of oocytes remaining at GV stage were 0, 0, 32, 80, and 84% ( P < 0.05), and the maturation rates were 86, 63, 30, 0, and 0% ( P < 0.05) for oocytes treated with 0, 10, 20, 40, and 80 μM of BL I, respectively. When oocytes were released from BL I incubation (Experiment 2) and cultured for an additional 44 h, 79, 84, and 83% of oocytes resumed meiosis, but only 52, 38 and 17% of oocytes reached normal metaphase II (MII) stage in the groups treated with 20, 40 and 80 μM BL I, respectively. In Experiments 3–5, reversibility and development of oocytes and embryos were evaluated after removal of the inhibitor. A reduced duration of BL I incubation (22 h) at 20 μM increased the percentage of oocytes remaining at the GV stage compared to the control group (85% versus 9%, P < 0.05). Blastocyst rates were lower in treatment groups than in the control (44 h) group (0–14% versus 24%; P < 0.05). However, all developing blastocysts possessed similar cell numbers, regardless of the drug-treated or non-treated controls. Taken together, treatment with 20–80 μM of BL I effectively prevented the resumption of meiosis and polymerization of periooplasmic microtubules. Furthermore, reversibility of the oocytes after reduced duration of BL I treatment was satisfactory.

Mammalian CLASPs are required for mitotic spindle organization and kinetochore alignment

CLASP1 and CLASP2 are homologous mammalian proteins, which associate with the ends of growing microtubules, as well as the cell cortex and the kinetochores of mitotic chromosomes. Previous studies have shown that in interphase cells CLASPs can attach microtubule plus ends to the cortex and stabilize them by repeatedly rescuing them from depolymerization. Here we show that CLASP1 and 2 play similar and redundant roles in organizing the mitotic apparatus in HeLa cells. Simultaneous depletion of both CLASPs causes mitotic spindle defects and a significant metaphase delay, which often results in abnormal exit from mitosis. Metaphase delay is associated with decreased kinetochore tension, increased kinetochore oscillations and more rapid microtubule growth. We show that the association of CLASP2 with the kinetochores relies on its C-terminal domain, but is independent of microtubules or association with CLIP-170. We propose that CLASPs exhibit at the kinetochores an activity similar to that at the cortex, providing apparent stabilization of microtubules by locally reducing the amplitude of growth/shortening episodes at the microtubule ends. This local stabilization of microtubules is essential for the formation of normal metaphase spindle, completion of anaphase and cytokinesis.

Control of the AtMAP65-1 interaction with microtubules through the cell cycle

Cell division depends on the fine control of both microtubule dynamics and microtubule organisation. The microtubule bundling protein MAP65 is a ;midzone MAP' essential for the integrity of the anaphase spindle and cell division. Arabidopsis thaliana MAP65-1 (AtMAP65-1) binds and bundles microtubules by forming 25 nm cross-bridges. Moreover, as AtMAP65-1 bundles microtubules in interphase, anaphase and telophase but does not bind microtubules in prophase or metaphase, its activity through the cell cycle must be under tight control. Here we show that AtMAP65-1 is hyperphosphorylated during prometaphase and metaphase and that CDK and MAPK are involved in this phosphorylation. This phosphorylation inhibits AtMAP65-1 activity. Expression of non-phosphorylatable AtMAP65-1 has a negative effect on mitotic progression resulting in excessive accumulation of microtubules in the metaphase spindle midzone causing a delay in mitosis. We conclude that normal metaphase spindle organisation and the transition to anaphase is dependent on inactivation of AtMAP65-1.

Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization

Chromogenic (CISH) and fluorescent (FISH) in situ hybridization have emerged as reliable techniques to identify amplifications and chromosomal translocations. CISH provides a spatial distribution of gene copy number changes in tumour tissue and allows a direct correlation between copy number changes and the morphological features of neoplastic cells. However, the limited number of commercially available gene probes has hindered the use of this technique. We have devised a protocol to generate probes for CISH that can be applied to formalin-fixed, paraffin-embedded tissue sections (FFPETS). Bacterial artificial chromosomes (BACs) containing fragments of human DNA which map to specific genomic regions of interest are amplified with φ29 polymerase and random primer labelled with biotin. The genomic location of these can be readily confirmed by BAC end pair sequencing and FISH mapping on normal lymphocyte metaphase spreads. To demonstrate the reliability of the probes generated with this protocol, four strategies were employed: (i) probes mapping to cyclin D1 (CCND1) were generated and their performance was compared with that of a commercially available probe for the same gene in a series of 10 FFPETS of breast cancer samples of which five harboured CCND1 amplification; (ii) probes targeting cyclin-dependent kinase 4 were used to validate an amplification identified by microarray-based comparative genomic hybridization (aCGH) in a pleomorphic adenoma; (iii) probes targeting fibroblast growth factor receptor 1 and CCND1 were used to validate amplifications mapping to these regions, as defined by aCGH, in an invasive lobular breast carcinoma with FISH and CISH; and (iv) gene-specific probes for ETV6 and NTRK3 were used to demonstrate the presence of t(12;15)(p12;q25) translocation in a case of breast secretory carcinoma with dual colour FISH. In summary, this protocol enables the generation of probes mapping to any gene of interest that can be applied to FFPETS, allowing correlation of morphological features with gene copy number.

Submicroscopic Interstitial Deletions in 13q14 Are Detectable in Metaphase Cells by Fluorescence In Situ Hybridization (FISH) with D13S319 in Chronic Lymphocytic Leukemia (B-CLL).

Background: Results of conventional cytogenetic studies on specimens processed without B-cell mitogens are usually normal in B-CLL: this observation is often attributed to lack of mitotic B-CLL cells. Using metaphase FISH with D13S319, we now have evidence that these apparently normal mitotic B-CLL cells often exhibit submicroscopic interstitial deletions in 13q14. This observation indicates B-CLL cells do divide in unstimulated cultures, but the abnormal metaphases are not detected because the abnormalities are submicroscopic and represent a low percentage of proliferating cells.Methods: We studied 12 patients with B-CLL who had normal metaphases by conventional cytogenetics, but abnormal interphase nuclei by FISH with D13S319. This cohort consisted of 7 females and 5 males who ranged in age from 50 to 87 years. Clinical information was available for 11 patients: Rai stages were 0, I, II, III, or IV for 6, 1, 1, 2, and 1 patients, respectively; 8 patients received treatment and 3 have died. Conventional cytogenetic studies and metaphase FISH were done on bone marrow specimens that were processed by standard 24 and 48-hour unstimulated cultures with Chang Medium BMC. Interphase FISH studies were done on blood or bone marrow (collected within 3 days of specimens for conventional cytogenetic studies) using probes to detect anomalies of chromosomes 6, 11, 12, 13, 14 and 17. For each patient, metaphase FISH was done with commercial probes from Vysis Inc., Downers Grove, IL for D13S319/13q34 using leftover fixed cells from conventional cytogenetics studies.Results: Metaphase FISH results were normal for 3 bone marrow specimens: the remaining 9 specimens had 1.9 to 13.8% metaphases with interstitial deletions of 13q14. One patient had a cytogenetically visible chromosome 13 deletion in 2.2% of metaphases. In the remaining 8 specimens, the size of the abnormal 13 was similar to the normal 13 homolog and other acrocentric chromosomes. By comparison, interphase FISH revealed 13 to 93% nuclei with 13q-x1 or 13q-x2. Seven patients had additonal anomalies in their interphase nuclei; 3 had 11q-, 2 had 17p-, 1 had +12 and 1 had +IGH.Conclusions: This investigation indicates that metaphase FISH detects submicroscopic deletions in chromosome 13 as well as infrequent mitotic B-CLL cells in patients who have normal conventional cytogenetic results. Therefore, metaphase FISH is a novel and useful technique to detect abnormal mitotic cells in B-CLL in cultures without special B-cell mitogens. The prognostic significance of abnormal metaphase FISH results is still under investigation. We speculate that this method will allow for identification of more aggressive proliferating disease even in so-called “favorable” prognostic groups based on interphase FISH results.