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PubMed2000

Hypermetaphase and interphase fluorescence in situ hybridisation for monitoring of remission status in Philadelphia chromosome positive chronic myeloid leukaemia.

Interferon (IFN) alone or in combination with cytostatic drugs, can induce major and durable cytogenetic remissions in chronic myelogenous leukaemia (CML) patients. Hypermetaphase (HMF) and interphase (IPF) fluorescence in situ hybridisation (FISH) have been described to be suitable for remission assessment. In the present study we applied HMF and IPF simultaneously to bone marrow (BM) probes from Ph-positive CML patients. As conventional cytogenetics (CC) is still deemed to be the for remission analysis we studied a group of patients analysed with this method as control. A mean of 50 metaphases was available for HMF analysis, whereas only an average of 18.7 metaphases could be analysed by CC. Remission assessment was frequently impossible by CC or HMF due to lack of metaphases, but always possible by applying IPF. Our results show that HMF should replace CC for routinely monitoring the remission status in Ph-positive CML patients and that in case of lack or insufficient number of metaphases in the majority of cases IPF is suitable for remission assessment.

Adolescent↗
PubMed2006

Nuclear exclusion of separase prevents cohesin cleavage in interphase cells.

During mitosis, equal transmission of the duplicated chromosomes demands a strict regulation of separase, which cleaves cohesin and triggers sister chromatid separation in anaphase. Vertebrate separase is inhibited by securin and the inhibitory phosphorylation of separase. However, knockout experiments indicate that securin is dispensable and the inhibitory phosphorylation was observed only in M phase cells. This begs the question how cohesin cleavage by separase is prevented in the absence these two mechanisms. Here we show that separase is excluded from cohesin by the nuclear envelope, which forms in telophase and disassembles in mitosis. The exclusion is achieved passively by its large physical mass and may be backed up by the CRM1-dependent nuclear export. A functional NES motif is identified in separase. We demonstrated that the nuclear envelope is sufficient to prevent active separase from cleaving nuclear cohesin. We propose that the nuclear exclusion is important to prevent cohesin cleavage during interphase in the absence of securin and the phosphorylation inhibition.

Active Transport, Cell Nucleus↗
PubMed2002

Identification of numerical chromosomal changes detected by interphase fluorescence in situ hybridization in high-grade prostate intraepithelial neoplasia as a predictor of carcinoma.

CONTEXT: High-grade prostate intraepithelial neoplasia (HPIN) is the most likely precursor of prostate cancer. The condition of many patients with a diagnosis of HPIN in prostate needle core biopsy could, if left untreated, progress to invasive cancer. Currently there is no available clinical, immunohistochemical, or morphologic criteria that are predictive of this progression. OBJECTIVE: To determine whether chromosomal instability in these precursor lesions could increase their predictive value for cancer detection. DESIGN: Dual-color interphase fluorescence in situ hybridization analysis was performed on archived prostate needle core biopsies from 54 patients with initial diagnosis of isolated HPIN and follow-up of 3 years or more. We used commercially available centromere probes for chromosomes 4, 7, 8, and 10. We had interpretable results in 44 patients as follows: (1) group A: 24 HPIN patients with persistent HPIN and/or benign lesions in the follow-up biopsies, and (2) group B: 20 HPIN patients with progression to prostate carcinoma. RESULTS: Twenty-five percent of the patients in group B displayed numeric chromosomal aberrations. Only 8.3% of the patients from group A had chromosomal abnormalities (P =.1). The observed overall chromosomal changes in HPIN were higher than those in normal or hyperplastic epithelium, with a statistically significant difference (P <.05). All aberrations were detected in the form of chromosomal gain. Overall, the commonest aberration was gain of chromosome 8, followed by gains of chromosomes 7 and 10. CONCLUSION: These results indicated that although no single numeric chromosomal abnormality could be assigned as a predictor of HPIN progression to carcinoma, the overall level of numeric chromosomal abnormalities shows a trend of elevation in HPIN patients whose condition subsequently progressed to carcinoma.

Aged↗
PubMed1999

[Structure of chromatin and chromosomes in preparations of interphase nucleus derivatives, prepared by removal of the nucleuar envelopes. II. Structure of chromatin and associations of chromosomes in stretched amembranous nuclei and mitotic figures].

Preparations of surface stretched amembranous nuclei and mitotic figures were used for revealing the high order nuclear and chromosomal structures. The preparations were obtained by dropping amembraneous nuclei and mitotic figures suspension in methanol-glacial acetic acid mixture (3:1) on wetted superclean slides. Amembraneous nuclei and mitotic figures were isolated from intact murine and human cells (lines L1210, SK-UT-1B, PHA-stimulated lymphocytes) by means of their 1-5 min prefixational capillary pipetting with freshly prepared 0.018-0.06% Triton X-100 solution in the conditional cultural medium. Stretched amembraneous nuclei and mitotic figures had no features of induced chromatin dispersion and compaction. Stretched interphase amembraneous nuclei showed spatially separated individual structures (thin chromatin fibres, nucleoli, intranuclear bodies), polymorphous pattern of perinucleolar chromatin aggregation and episodically expressed beaded thick chromatin fibres and a chromocenter. The chromomeric pattern of the spread chromosomes of mitotic figures was quite similar but hardly identical with that of G-banding. The stretched prometaphase mitotic figures in all tested cell types always contained loose "residual" nucleoli looking like typical prophase nucleoli as concerns their shape and number per cell (mitotic figure). The majority of chromosomes of stretched mitotic figures and of prophase amembraneous nuclei were attached to the nucleolar material. All tested cell lines showed almost the same variation in number of nucleolus-attached chromosomes, per both prophase amembraneous nucleus and prometaphase mitotic figure. Some chromosomes of stretched mitotic figures were colocated with "residual" nucleoli and looked shortened and strongly condensed. Other chromosomes, locally associated with "residual" nucleoli, were straight and oriented radially to these. Mutual chromosomal arrangements in mitotic cells on smears and in stretched mitotic figures were analogous. Equatorial plates from PBS-washed SK-UT-1B cells displayed a better stretching capacity than those from untreated cells. In the former case metaphase chromosomes were seen more uniformly stretched and well identified after GTG-banding procedure. The number of interchromosomal (mainly telomere-telomeric and telomere-centromeric) connections per stretched mitotic figure (or per stretched prophase amembraneous nucleus) was minimum in late prometaphase, maximum in prophase and early prometaphase, and intermediate in metaphase. The obtained data are discussed in terms of topology and longitudinal heterogeneity of mitotic chromosomes.

Animals↗
PubMed1999

[Comparative analysis of nucleotide sequences of chromosomal DNA isolated from nuclear envelopes and cores of rosette-like structures of murine interphase chromosomes].

Six DNA fragments of interphase chromosomes isolated from nuclear envelopes of murine hepatocytes were cloned and sequenced. Analysis of their structural-functional organization suggests that these fragments are highly specified protein-nonencoding fractions of a eukaryotic genome. In the evolutionary process, they appear already in archaebacteria and may be "ancestral" for DNA sequences involved in structuring chromosomal domains (rosette-like structures) of tissue-specific genes. In their composition, these fragments have nucleotide sequences homologous to the repeats of the SINE and LINE families and to the satellite DNA of murine centromeres.

Animals↗
PubMed2000

Rapid prenatal diagnosis of chromosome aneuploidies by interphase fluorescence in situ hybridization: a one-year clinical experience with high-risk and urgent fetal and postnatal samples.

OBJECTIVES: To evaluate the clinical utility of rapid prenatal and postnatal detection of common chromosome aneuploidies by interphase fluorescence in situ hybridization (FISH) analysis with DNA probes. DESIGN: Four hundred and seventy-seven high-risk and/or urgent amniotic fluid, chorionic villus and fetal and postnatal blood samples were prospectively examined by FISH with probes specific for chromosomes 13, 18, 21, X, and Y and results were reported within 48 hours. All FISH results were followed by conventional chromosome analysis, if possible. SETTING: Cytogenetic service laboratory at the tertiary referral center, Rigshospitalet in Copenhagen. MAIN OUTCOME MEASURES: The fraction of clinically significant chromosome aneuploidies that was detected by FISH analysis, and the fraction of terminations that was based on FISH and ultrasound results rather than on conventional cytogenetic results. RESULTS: The FISH assay detected 76% of the clinically significant chromosome abnormalities as determined by subsequent cytogenetic analysis. Seventy-two percent of the terminations of the chromosomally abnormal pregnancies were based on FISH and ultrasound results rather than on conventional cytogenetic results. CONCLUSION: FISH analysis is a clinically useful adjunctive tool to conventional pre- and postnatal cytogenetic analysis. The assay rapidly detects the majority of clinically significant chromosome abnormalities, thus facilitating difficult pre- and postnatal clinical decisions.

Adult↗
PubMed2000

The clinical application of interphase FISH in prenatal diagnosis.

Fluorescence in situ hybridization (FISH) for five chromosomes (13, 18, 21, X and Y) detected 87 of 107 (81%) of the chromosome aberrations identified by conventional chromosome analysis applied to fetal interphase cells obtained by chorionic villus sampling or amniocentesis. The choice of FISH was solely determined by prospective parents after formal genetic counselling concerning the advantages and disadvantages of FISH analysis. Excluding known familial chromosome aberrations, if FISH analysis revealed normal signals, there was an overall residual risk of 1 in 149 for an undetectable chromosome aberration. This risk varied according to the indication for prenatal diagnosis: 1 in 177 for women of advanced maternal age; 1 in 60 for women at increased risk for Down syndrome based on maternal serum screening; and, 1 in 43 for women whose ultrasound examination revealed fetal anomalies. There were 20 cases of discordance between the FISH results and standard karyotype analysis: four were the outcome of a failure to apply the appropriate FISH probe; 16 were not detectable by the available FISH probes. Of these 16, nine were chromosome abnormalities with clinical significance and seven were familial. If FISH is to become a standard part of prenatal genetic diagnosis, genetic counselling that is sensitive to patient health needs must be based on accurate information about the biological and obstetrical implications of the results of FISH analysis.

Adult↗
PubMed2000

[Determination, by in situ hybridization on interphasic nucleus, a cytogenetic DNA index; application to breast cancer; comparison of this DNA index to DNA indexes determined by imaging and flow cytometry].

In oncology, flow cytometry (FCM) and image cytometry (ICM) are commonly used to detect DNA aneuploid cell populations in solid tumors. Agreement between these two approaches is good. The use of both techniques in association minimizes the rate of FCM and ICM false negatives and gives better DNA pattern characterization, particularly for detection of any tumoral component in the FCM DNA diploid peak. Nevertheless, discrepancies exist between the FCM and the ICM DNA index values: the ICM DNA index is often greater than the FCM DNA index. The aim of the present study was to establish a cytogenetic DNA index by determining the chromosomal ploidy using a molecular cytogenetic approach and to compare it to the FCM and ICM DNA indexes. We present here the fluorescence in situ hybridization (FISH) technique we have adapted to the study of breast cancer in order to count the number of copies of the 22 + X human chromosomes in interphasic nuclei. This was achieved using a panel of 21 indirect FITC labeled probes which recognize specific chromosomic DNA sequences. Preliminary results obtained from DNA diploid and DNA aneuploid tumors are discussed.

Breast Neoplasms↗
PubMed2000

[Study on interphase cytogenetic abnormalities in malignant cells in pleural fluids from lung cancer cases].

OBJECTIVE: To study the interphase cytogenetic abnormalities in malignant cells in pleural fluids from lung cancer cases by fluorescence in situ hybridization(FISH) and compare the result of FISH with that of conventional cytology. METHODS: Twenty-six pleural fluids from the lung cancer cases were detected by dual-fluorescence in situ hybridization centromere DNA probes of chromosomes 7, 11, 17, and X. RESULTS: In 19 positive pleural fluids the rates of hyperdiploid in chromosomes 7, X, 17 and 11 were 16(84.2%), 14(73. 7%), 12(63.2%) and 8(42.1%), respectively. In 2 suspicious malignant pleural fluids, both chromosomes 7 and X showed hyperdiploid. Two cases of hyperplasia from 5 negative specimens also showed gain of chromosome X. Another 3 negative pleural fluids had a normal number of chromosome. CONCLUSION: One of the important abnormalities occurred in tumor cells derived from pleural fluid of lung cancer is hyperdiploid, which can be detected by FISH. Therefore, hyperdiploid in pleural fluid cells can be an implication for the malignancy that has not been detected or confirmed by cytological examination.

Chromosome Aberrations↗
PubMed2000

[The interphase fluorescence in situ hybridization (I-FISH) technique in patients with chronic lymphatic leukemia (CLL)].

BACKGROUND: Trisomy 12 was found to be the most frequent chromosomal aberration identified by conventional cytogenetic studies of bone marrow cells and peripheral lymphocytes of patients with CLL. Molecular-cytogenetic techniques which enable examination of dividing and/or non-diving interphase nuclei (I-FISH), proved existence of other chromosomal abnormalities, mainly deletions, which could have in CLL patients relation to the origin, course and prognosis of the disease. METHODS AND RESULTS: During the last two years bone marrow chromosomes of all patients with CLL were examined by G-banding and by I-FISH. The numerical changes of chromosome 12 were followed by centromeric DNA probe in dividing and non-dividing cells. The small deletions were ascertained by locus specific probes for 13q14 (Rb gene), 17p13 (p53 protein) and 11q23 (MLL gene). These genes are responsible for cell division and their function is probably in connection with neoplastic process. It is of interest whether numerical and structural chromosomal rearrangements are primary or secondary changes and what is their impact on etiology of CLL. 93 patients were examined by DNA prove CEP12 and trisomy 12 was found in 24 of them (25.8%), the range of the clone was 2.5-75.5% of the screened cells. Deletion del(13)(q14) was examined by probe D13S319 in 73 patients and proved in 24 of them (32.8%), pathological clone ranged 2.5-80.0% of the cells. Deletion del(17)(p13) was found in 14 patients out of 61 examined by probe LSI p53 (22.9%). The extent of the clone was 2.5-34.0% of examined cells. Deletion 11q23 was not ascertained in any of 11 patients by means of probe LSI 11q23 (MLL). All probes used for FISH were manufactured by VYSIS. CONCLUSIONS: FISH is very sensitive method, suitable for molecular-cytogenetic examination of leukemic patients. With I-FISH the deletion of 13q14 was ascertained as the most frequent chromosomal aberration in series of 73 patients with CLL. We continue to increase the number of patients screened by I-FISH with all eligible DNA probes and start the prospective study on patients with chromosomal pathology. We will correlate the immunophenotype, morphology, clinical course and prognosis with karyotypic findings.

Adult↗
PubMed2000

Interphase microtubules in cultured cells: long or short?

Presently, the question about the length of microtubules in the interphase cell became actual, since the parameters of dynamic instability of the plus end measured in vivo do not allow one to explain the rapid turnover of the long microtubule system. The problem may be solved if one of the following suppositions is assumed: either microtubules undergo rapid depolymerization from the minus end or they are on the average much shorter than it is usually considered. To check the last hypothesis, we have reconstructed microtubules using stereophotography of electron microscopic sections. Microtubules around the cell center in cultures of epithelial cells (kidney of pig embryo (PK) and bovine trachea (FBT)) and fibroblasts (MEF, primary mouse embryo fibroblasts, and L cells), as well as at the periphery of PK cells were studied. All in all, no less than 200 microtubules were found near the centrosome in each cell culture. From 2.5 to 8% microtubules were beyond the studied volume (4.0 x 5.5 x 1.5 microm). Most of microtubules in all studied cell lines were up to 1 microm and about 1/3 of them were 0.2-0.4 microm long. The mean length of microtubules surrounding the centrosome in different cell lines differed insignificantly and equalled 0.4-0.8 microm. In this case, the microtubules attached to the centrosome were on the average slightly shorter than the free ones. Thus, almost all microtubules around the centrosome are short, and the majority of those attached to it do not reach the cell periphery. A similar reconstruction of a part of the PK cell cytoplasm (10 x 35 microm) has shown that at the periphery, the mean length of microtubules is about 1.6 microm and most of them are 0.5 to 1.5 microm long. Thus, our data confirm the recent hypothesis of Vorobjev et al. (I. A. Vorobjev, T. M. Svitkina, and G. G. Borisy, J. Cell Sci. 110:2635-2645 (1997)) that most of microtubules in the cells are not connected with the centrosomes.

Animals↗
PubMed2001

[Study of the structural and structure-functional subdomains in the interphase nucleus by photostabilization].

The role of the nuclear matrix components in the organization of structural and functional domains of interphase nuclei was studied using irradiation with blue light in the presence of a photosensibilized agent (Ethidium bromide). Nuclear domain resistance to extractive solution (2 M NaCl) treatment served as a criterion of irradiation-induced stabilization of different nuclear domains. The following results have been obtained: 1) the structural organization of the complexes of chromatin and clusters of replication does not depend on the state of the nuclear matrix in isolated nuclei; 2) chemical stabilization of the nuclear matrix by Cu(2+)-ions is not sufficient for the organization of chromatin domains; 3) irradiation in the presence of Ethidium bromide stabilizes domains of the nuclei, but does not lead to stabilization of the nuclear matrix internal network. Hence, the irradiation prevented extraction from the nuclear domains of nonhistone proteins which were not standard matrix proteins. Based on the results obtained, a hypothesis was proposed about a coexistence of two groups of nonhistone proteins in the cell nucleus. The first group includes proteins of the nuclear matrix involved in immobilization of scafford attachment regions and active genes. The second group includes some hypothetical structural proteins participating only in compaction of DNA of condensed chromatin.

Animals↗
PubMedDate not supplied

[Rejoining pathways underlying intrachange formation depend on interphase chromosome structure].

Fluorescence in situ hybridization technique with dual-arm painting of the same chromosome allows to study radiation induced intrachromosomal exchange aberrations (intrachanges). The formal method for analysis of FISH data developed by Savage and Simpson includes an arrangement of chromosomal reactive breaks on chromosomes and chromosome arms (CAB system). That method predicts types and frequencies of aberrations assuming random restitution/rejoining of breaks free ends. However experimental data suggest that many aberrations are formed non-uniformly along the chromosomes and chromosome arms. In the present paper the system for classifying rejoining pathways underlying intrachange formation is proposed which does not require random restitution/rejoining suggestion. It takes into account the parameters of interphase chromosome structure. Types and frequencies of intrachanges are considered by exploring different hypotheses about exchange initiating chromosomal lesions.

Chromosome Aberrations↗
PubMed2002

Biological and prognostic significance of interphase fluorescence in situ hybridization detection of chromosome 13 abnormalities (delta13) in multiple myeloma: an eastern cooperative oncology group study.

Chromosome 13 abnormalities (Delta13) have been associated with an unfavorable prognosis in patients with multiple myeloma (MM). The significance of this has been unresolved because of diverse methods of detection and heterogeneous groups of patients. We conducted a study of Delta13 in patients entered into the Eastern Cooperative Oncology Group trial E9486/E9487. Patients with newly diagnosed MM (median follow-up of survivors >100 months) were studied for Delta13, using bone marrow samples obtained at study enrollment. We used interphase fluorescence in situ hybridization with the probes LSI13 (Rb)/D13S319 with simultaneous immunofluorescence detection of bone marrow plasma cells (PCs). We detected Delta13 in 176 of 325 (54%) evaluable patients. Patients with Delta13 were more likely to have a serum monoclonal protein at a concentration < or =1 g/dl (22 versus 13%; P = 0.04), light-chain-only MM (19.3 versus 10.8%; P = 0.04), gamma light chain (42 versus 28%; P = 0.027), stage III (56 versus 42%; P = 0.014), and be female (60 versus 50%; P = 0.087). The PC labeling index and Delta13 correlated (P = 0.03). Patients with Delta13 were less likely to respond to treatment (74 versus 63%; P = 0.041) and had a significantly shorter median overall survival (34.9 versus 51 months; P = 0.021). The association of Delta13 and survival remained an independent prognostic variable in a regression model. Among patients with Delta13, those receiving IFN had a worse overall survival that those not receiving the medication (P = 0.03). The presence of Delta13 is an important and independent adverse prognostic factor in newly diagnosed MM and is associated with specific biological features.

Adult↗
PubMed2001

[Dynamic monitoring of Ph positive cells during early post-transplant period in chronic granulocytic leukemia by interphase FISH].

OBJECTIVE: To assess the effectiveness of transplantation and observe the kinetics of residual leukemic cells during early post-transplantation period in chronic granulocytic leukemia (CGL). METHODS: Ten normal controls, seven untreated CGL patients and eleven CGL patients after allogeneic bone marrow transplantation (allo-BMT) or peripheral blood stem cell transplantation (PBSCT) were studied by interphase fluorescence in situ hybridization (I-FISH) with the FITC fluorescein labeled BCR probe. Conventional cytogenetic analysis (CCA) and reverse transcriptase-polymerase chain reaction (RT-PCR) were also employed. RESULTS: The normal cutoff was defined as less than 6.68% (x +/- 3s) from the analysis of normal controls. The reproduciblity of FISH method was confirmed by serial dilutions by mixing Ph positive cells and normal cells. Forty specimens from 11 CGL patients at 1 week to 6 years post-BMT were investigated after PBSCT and the results showed that (1) FISH and RT-PCR could accurately evaluate Ph (+) cells during early transplantation period (within one month), but RT-PCR was more sensitive in six months after BMT. (2) Residual Ph (+) cells tended to be linear decreasing with the time after transplantation and fell in normal range in most samples three months later. The mean time of normaligation was 57 days (20 to 170 days). (3) One case relapsed and received a second transplantation. FISH presented more precise data for predicting relapse as compared to CCA and RT-PCR. CONCLUSIONS: I-FISH as a useful tool can not only offer more precise assessment of residual leukemic cells compared with CCA and RT-PCR, but also provide dynamic monitoring during early post-BMT period to evaluate the transplantation outcome and predict relapse.

Adult↗
PubMedDate not supplied

Interphase chromosome locus displacement induced by low-doses of radiation.

The most important stage in the making of mutations is a reparation of different DNA damage, including the more deleterious double-strand DNA breaks (DSB). The first stage of adaptive response--fundamental antimutagenic cell reaction, purposeful to reparation for induced DSB repair--is investigated in present work. Non-radioactive in situ hybridization of biotin-labeled DNA probe was used to mark chromosome 1 pericentromeric regions (PR) in G0 human lymphocytes. It was shown that under 3-10 cGy (X-radiation, 160 kV) PR become displaced from a nucleus periphery to inner territory of a nucleus. The moving process realizes during several hours after an irradiation. As far as some non-specific gene repressors are co-localized with chromosome centromeric regions it is possible hypothesizes that the displacement cause changing expression of some genes. It is possible to propose that an absence of radiation induced chromosome locus displacement may be one of causes DSB repair disturbance. This hypothesis was tested by the model. It is assumed that one consequence of the underlying defect may be inappropriate involvement of cell's recombination machinery in the repair of DSB. We studied lymphocytes of patients with hereditary BRCA2 mutation. It is thought that this gene takes part in DSB repair. The significant differences of the PR moving between control samples and the cases were revealed under 10 cGy. Similar results were observed on lymphocytes of patients with Fanconi syndrome. Thus, abnormal moving of interphase nucleus chromosomes conditioned by low-dose irradiation may suggest on imperfect machinery of DSB repair, i.e. genetic risk. We realize that further investigations are needed for definitive conclusion.

Chromosomes, Human, Pair 1↗