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PubMed1991

Chromatin motion in neuronal interphase nuclei: changes induced by disruption of intermediate filaments.

Motion of nucleoli within interphase nuclei, known as nuclear rotation, may be used as a measure of motion of chromatin domains within the global confines of the nucleus. Mechanisms by which chromatin domains are transposed remain enigmatic. It has been established that nuclei are anchored by a network of intermediate filaments, structural proteins which share epitopes with nuclear lamins and possibly representing a constraint on nuclear rotation. It is postulated that selective removal of this constraint, by acrylamide, would result in increased chromatin motion. Mean rates of nucleolar displacement were quantified in neurons, in vitro. Nuclear rotation increased from a mean control rate of 0.102 +/- 0.002 micron/min (n = 52) to a maximum mean rate of 0.207 +/- 0.026 micron/min (n = 11), after 23 hr of exposure to 4 mM acrylamide. Despite this significant increase in motion of intranuclear domains, cytoplasmic structures in the immediate juxtanuclear area did not exhibit increases in rates of motion. Immunocytochemistry was used to visualize cytoskeletal structures and to assay selective disruption of neurofilaments by acrylamide. Increased rates of chromatin motion coincided with breakdown of the intermediate filament network. Ultrastructural analyses showed that the increase in chromatin motion induced by acrylamide was also associated with a significant (P less than 0.005) change in the thickness of the nuclear lamina, decreasing from 20.9 +/- 5.10 nm (n = 159) in controls to 18.9 +/- 3.1 nm (n = 148), to 19.5 +/- 3.6 nm (n = 240) and to 16.1 +/- 4.4 nm (n = 103) at 4, 8 and 22 hr exposure, respectively. Moreover, the number of mitochondria per unit area changed significantly (P less than 0.0001) with exposure to acrylamide, increasing from 9.1 +/- 2.2 mitochondrial profiles in controls to 16.5 +/- 5.3 profiles after 22 hr exposure to acrylamide. Distribution of other cytoskeletal components, actin and microtubules, was not altered and does not appear to play a significant role in the observed increase in rates of nuclear rotation. We conclude that the removal of the damping effects on chromatin motion normally imposed by the nuclear lamina and by intermediate filaments results in increased chromatin motion.

Acrylamide↗
PubMed2003

Evaluation of interphase fluorescence in situ hybridization for the t(14;18)(q32;q21) translocation in the diagnosis of follicular lymphoma on fine-needle aspirates: a comparison with flow cytometry immunophenotyping.

BACKGROUND: Diagnosing lymphoproliferative disorders on fine-needle aspiration (FNA) can be challenging due to variable cellularity and lack of architecture. Ancillary studies often are required for diagnosis. Follicular lymphoma (FL) is characterized by a monoclonal B-cell proliferation with coexpression of CD19/CD10 and a t(14;18)(q32;q21) reciprocal translocation, resulting in the immunoglobulin heavy chain/BCL-2 fusion gene. These features also can be found, with much lower frequency, in diffuse large B-cell lymphoma (DLBCL) of follicle center cell origin. The objective of the current study was to compare the accuracy in detecting FL and DLBCL of follicle center cell origin by interphase fluorescence in situ hybridization (I-FISH) versus flow cytometry immunophenotyping (FCM) on FNAs. METHODS: Concurrent testing by FISH for t(14;18)(q32;q21) and FCM was performed on 84 FNAs, including 40 FLs and 44 non-FLs (de novo DLBCLs, mantle cell lymphomas, small lymphocytic lymphomas/chronic lymphocytic leukemias [SLLs/CLLs], small B-cell lymphomas, and reactive lymphoid hyperplasias). The final diagnosis was rendered based on the combined information from cytomorphology, FCM, FISH, immunocytochemical staining for Ki-67, monoclonality for kappa and lambda light chains, and, if available, corresponding tissue biopsy, cytogenetic analysis, and polymerase chain reaction analysis. RESULTS: Among 40 FLs, FISH produced positive results for the t(14;18) translocation in 85.0%, negative results in 7.5%, and insufficient results in 7.5%; whereas, with FCM, 75% of cases exhibited a CD19-positive (CD19+)/CD10+ population (28 monoclonal, 2 nonclonal), 12.5% of cases exhibited a CD19+/CD10-negative population (3 monoclonal, 2 nonclonal), and 12.5% of cases were insufficient. All of nonclonal results from FCM and all of the insufficient results from FCM analysis exhibited unequivocal t(14;18) translocation by FISH. In contrast, the three negative results and the three insufficient results from FISH were monoclonal and CD19+/CD10+ on FCM. The results from FISH and FCM were concordant in 75% cases. Of 44 non-FLs, FISH produced positive results for the t(14;18) translocation in 5 DLBCLs and 2 SLLs/CLLs. The latter showed single fusion signals just above the cutoff level. All cases in the non-FL group that failed to show clonality or had insufficient results from FCM were DLBCLs. Among 17 DLBCLs, FISH detected a t(14;18) translocation in 29.4%, whereas FCM demonstrated a CD19+/CD10+ population in 23.5%. CONCLUSIONS: I-FISH for the t(14;18)(q32;q21) translocation provided high overall accuracy in detecting FLs on FNAs. This test can be used for diagnosing or monitoring FL on FNAs when cellularity is limited or when FCM results are noncontributory. For detecting a follicle center cell origin in DLBCLs, I-FISH for the t(14;18) translocation appeared to be slightly more sensitive than FCM for the CD19+/CD10+ immunophenotype.

Chromosomes, Human, Pair 14↗
PubMed1990

Rapid metaphase and interphase detection of radiation-induced chromosome aberrations in human lymphocytes by chromosomal suppression in situ hybridization.

Chromosomal in situ suppression (CISS)-hybridization of biotinylated phage DNA-library inserts from sorted human chromosomes was used to decorate chromosomes 1 and 7 specifically from pter to qter and to detect structural aberrations of these chromosomes in irradiated human peripheral lymphocytes. In addition, probe pUC1.77 was used to mark the 1q12 subregion in normal and aberrant chromosomes 1. Low LET radiation (60Co-gamma-rays; 1.17 and 1.33 MeV) of lymphocyte cultures was performed with various doses (D = 0, 2, 4, 8 Gy) 5 h after stimulation with phytohaemagglutinin. Irradiated cells were cultivated for an additional 67 h before Colcemid arrested metaphase spreads were obtained. Aberrations of the specifically stained chromosomes, such as deletions, dicentrics, and rings, were readily scored after in situ hybridization with either the 1q12 specific probe or DNA-library inserts. By the latter approach, translocations of the specifically stained chromosomes could also be reliably assessed. A linear increase of the percentage of specifically stained aberrant chromosomes was observed when plotted as a function of the square of the dose D. A particular advantage of this new approach is provided by the possibility to delineate numerical and structural chromosome aberrations directly in interphase nuclei. These results indicate that cytogenetic monitoring of ionizing radiation may be considerably facilitated by CISS-hybridization.

Chromosome Aberrations↗
PubMed1995

Karyotypic heterogeneity and its relation to labeling index in interphase breast tumor cells.

We have used fluorescence in situ hybridization (FISH) with chromosome-specific probes and immunofluorescent detection of in vivo bromodeoxyuridine (BrdUrd) incorporation to evaluate simultaneously numerical chromosome aberrations and proliferative activity of breast cancers. The number of distinct hybridization domains specific for repetitive pericentromeric sequences on chromosomes 1, 7, 11, 15, 17, and X was used as an indicator of copy number of these chromosomes in interphase tumor cells from 23 human breast cancers. Every tumor analyzed showed a heterogeneous distribution of copy number for at least one chromosome type. The copy number distribution for different chromosomes within a tumor frequently showed differing patterns. Major cell populations showing monosomy were relatively rare, occurring only in five cases for chromosome 17, once for chromosome 1, and once for chromosome 15. Flow cytometric analysis of DNA ploidy correlated well with FISH analysis, although flow cytometry failed to detect aneuploidy when only a few chromosomes were affected. To determine whether cell populations with different chromosomal copy numbers have identical proliferation characteristics in vivo, BrdUrd incorporation and centromeric copy number were detected simultaneously. Comparison of the chromosome copy number distribution in BrdUrd-positive cells vs. the distribution of the entire cell population showed different distributions in seven of the 20 cases analyzed. This study demonstrates the common occurrence of chromosome copy number heterogeneity and suggests that a cell phenotype (proliferation) may be associated with genotypic subpopulations.

Adult↗
PubMed1994

Comparative study of interphase cytogenetics, flow cytometric analysis, and nuclear grade of fine-needle aspirates of breast carcinoma.

The correlation between DNA ploidy and S-phase fraction (SPF) by flow cytometry (FCM) and the detection of numerical chromosomal abnormalities (NCA) by interphase cytogenetics (IC) involving chromosomes 8 and 12 was studied in 20 human breast lesions (17 breast carcinomas, 2 fibroadenomas, and 1 sclerosing adenosis). Initial diagnosis was performed on fine-needle aspiration biopsy (FNAB) material with subsequent histologic confirmation. FCM was performed on formalin-fixed paraffin-embedded tissue while IC by fluorescence in situ hybridization (FISH) was done on alcohol fixed FNAB materials. Sixteen (80%) cases showed concordance between FCM and IC with respect to the presence or absence of aneuploidy. The remainder of the cases (20%), which were all malignant neoplasms, showed discrepancies between the two methods, all four were DNA-diploid with low SPF by FCM but showed various NCA by IC. Nuclear grades (NG) of all the malignant samples were also evaluated and correlated with both FCM and IC studies. Although a good correlation was observed between NG and FCM, a better correlation was seen between NG and IC. This study shows that although IC by FISH correlated well with FCM analysis, it can detect NCA in DNA-diploid, low SPF tumors. It also correlates well with the NG of the tumor. The increased sensitivity provided by IC in detecting aneuploidy may be of great prognostic significance in low stage, DNA-diploid, low SPF breast carcinomas.

Biopsy, Needle↗
PubMed1995

Cytology, flow cytometry, image analysis, and interphase cytogenetics by fluorescence in situ hybridization in the diagnosis of transitional cell carcinoma in bladder washes: a comparative study.

The diagnosis of transitional cell carcinoma (TCC) in bladder washes is a diagnostic challenge to cytology. This study assessed the role of flow cytometry (FCM), image analysis (IA), and interphase cytogenetics by fluorescence in situ hybridization (FISH) as adjuncts in the cytodiagnosis of TCC in bladder washes. Forty separate samples of bladder washes were prospectively evaluated by conventional cytology (CY), FCM, IA, and FISH, and the results were compared with the subsequent surgical biopsy specimens which revealed 26 TCC (3 GR I, 6 GR II, 17 GR III) and 14 benign lesions. Using histology as the "gold standard" and following the previously published criteria for detection of TCC by CY, FCM, IA, and FISH, the concordance rates between histology and CY, FCM, IA, and FISH were 75, 74, 89, and 83%, respectively. CY, FCM, IA, FISH, and histology were concordant in 54% of the cases. The sensitivity of CY, FCM, IA, and FISH were 61, 72, 91, and 73%, respectively, while the specificity were 100, 80, 83, and 100%, respectively. The combined sensitivity of all the parameters was 96%. Interestingly, the false positive cases by FCM and IA showed cystitis. We conclude that IA has the highest sensitivity in detecting TCC in bladder washes followed by FISH, FCM, and CY, while CY and FISH have the highest specificity. This study indicates that FCM, IA, and FISH are useful adjuncts to cytology in the diagnosis of TCC in bladder washes. The finding of DNA-aneuploidy in cystitis warrants further investigation.

Carcinoma, Transitional Cell↗
PubMed2004

Interphase fluorescence in situ hybridization for detection of 8q24/MYC breakpoints on routine histologic sections: validation in Burkitt lymphomas from three geographic regions.

A chromosomal translocation involving the MYC gene is characteristic of Burkitt lymphoma (BL) and represents a molecular disease marker with diagnostic and clinical implications. The detection of MYC breakpoints is hampered by technical problems, including the distribution of the breakpoints over a very large genomic region of approximately 1,000 kb. In this article, we report on the testing and validation of a segregation fluorescence in situ hybridization (FISH) assay for MYC breakpoints on a large series of BLs. A contig of overlapping genomic clones was generated, and two probe sets flanking the MYC gene were selected. Both probe sets were tested in an interphase FISH segregation assay on 8 B-cell lymphoma cell lines and 32 lymphoma samples with proved 8q24/MYC abnormalities and validated in 47 BLs from The Netherlands, Brazil, and Uganda. MYC translocation breakpoints were identified in 98% of the tumors of the test series and in 89% of the cases of the validation series. In 89% of all positive samples, the breakpoints were located between 190 kb 5' and 50 kb 3' of MYC. Nine cases had more distant breakpoints, and in one patient an insertion of MYC into the IGH region was detected. In two of the three BLs lacking CD10 expression, no breakpoint could be detected, suggesting that CD10 is a discriminative marker of BL. We did not find consistent differences between BL and atypical BL in incidence of an MYC breakpoint.

Adolescent↗
PubMed2005

Delineation of distinct subgroups of multiple myeloma and a model for clonal evolution based on interphase cytogenetics.

To delineate multiple myeloma (MM) subgroups and their clonal evolution, we analyzed 81 newly diagnosed patients by interphase fluorescence in situ hybridization using a comprehensive probe set for 10 chromosomes and two IGH rearrangements. A median of 5 probes per patient displayed aberrant signal numbers (range, 1-10). Additional copies most frequently found were for 15q22, 19q13, 9q34, 11q23, and 1q21. Losses commonly observed were of 13q14.3, 17p13, and 22q11. Predominance of gain or loss was quantified by a copy number score (CS) for each patient. Two peaks (CS = +3 and CS = 0) were found by plotting patient copy number scores over CS values corresponding to hyperdiploid and nonhyperdiploid MM. Cluster analysis revealed four major branches: (i) gain of 9q, 15q, 19q, and/or 11q; (ii) deletion of 13q and t(4;14); (iii) t(11;14); and (iv) gain of 1q. Statistical modeling of an oncogenetic tree indicated that early independent events were gain of 15q/9q and/or 11q, t(11;14); deletion of 13q followed by t(4;14); and gain of 1q. Aberrations of 17p13, 22q11, 8p12, and 6q21 were found as subsequent events. MM with gain of 1q was delineated as a subentity with significantly higher beta-2-microglobulin and lower hemoglobin levels, indicating a poor prognosis. From our results, we propose a model of MM for clonal evolution.

Evolution, Molecular↗
PubMed1992

Analysis of chromosome 12 aneuploidy in interphase cells from human male germ cell tumors by fluorescence in situ hybridization.

The i(12p) marker chromosome has been found to be a highly nonrandom chromosome abnormality associated with germ cell tumors (GCTs). We have previously shown that a chromosome 12 centromere specific alpha-satellite DNA probe detects the i(12p) by virtue of differences in the size of the signal originating from the i(12p) and normal chromosome 12 centromeres after fluorescence in situ hybridization (FISH) in metaphase and interphase cells of cultured GCT cell lines. We have now extended this analysis to 72 fresh GCT tumor biopsy specimens. Banded cytogenetic analysis was attempted on each of these tumors, 45 of which were found to be clonally abnormal. Data on i(12p) and chromosome 12 copy number obtained by FISH agreed well with those obtained by cytogenetic analysis. In addition, the FISH method made possible the detection and determination of i(12p) and the chromosome 12 copy number in cases in which conventional cytogenetic analysis was unsuccessful. We found the incidence of i(12p) in seminomas to be low (7%) compared to that in nonseminomas (75%) when tumor biopsy specimens were studied by FISH. Our results show that the FISH technique can be used reliably for detection of the diagnostically and prognostically useful i(12p) marker in GCT tumor biopsy specimens.

Aneuploidy↗
PubMed1993

Complementarity of interphase and metaphase chromosome analysis in human renal tumors.

Fluorescence in situ hybridization (FISH) was used as a complement to earlier cytogenetic studies of human renal tumors. Chromosome-specific para-centromeric probes were applied to cells disaggregated from tissue blocks of tumors, fresh samples from which had yielded cytogenetic results after short-term culture. Cells were dissociated from thick sections of paraffin-embedded, formalin-fixed tissues. Biotin-labeled probes specific for chromosomes 1, 3, 7, 11, 12, 15, 17, and the Y chromosome were applied in individual cases and were detected by fluorescence. Probes for chromosomes 1, 7, and 17 yielded clean signals with disomic control frequencies near 90%, and 97% of controls showed a single bright spot for the Y chromosome. The 9 cases selected all provided ample numbers of dissociated cells which were hybridized successfully, although some chromosomal signals were poor in individual cases. Abnormal copy numbers of chromosomes 1 and/or 17, not identified in culture, were observed in 7 cases. Trisomy 7 observed in culture was substantiated by FISH on the original tissues, as was loss of the Y, in each of 4 cases, respectively. Our results include limited validation of culture cytogenetics, evidence of selection in culture of tumor subpopulations, and demonstration that interphase cytogenetics by FISH is applicable to archival tissue blocks after prolonged periods of storage. Conditions of culture before harvest and inherent heterogeneity within tumors permit selection for nonrepresentative subgroups within tumors and emphasize the need for multiple approaches to evaluation.

Adenocarcinoma↗
PubMed1995

Interphase cytogenetics of prostatic adenocarcinoma and precursor lesions: analysis of 25 radical prostatectomies and 17 adjacent prostatic intraepithelial neoplasias.

Twenty-five radical prostatectomy specimens were screened for the presence of numerical chromosome changes within the adenocarcinoma as well as 17 adjacent prostatic intraepithelial neoplasias (PIN) by means of interphase in situ hybridization (ISH) to routinely processed tissue sections. To this end a defined alfoid repetitive DNA probe set was used, specific for the centromeres of chromosomes 1, 7, 8, 10, 15, and Y. The cytogenetic information was correlated with histopathological and clinical features as well as with DNA ploidy. Numerical aberrations of at least one chromosome were shown in 13 of 25 cases (52%). Alterations of chromosome 8 and loss of the Y chromosome were the most frequent findings (both 20%), followed by loss of chromosomes 15 (16%) and 10 (12%). Gain of chromosome 7 was seen in 8% of cases. No aberrations of chromosomes 7, 8, 10, and 15 were found in the adjacent PIN lesions, whereas loss of the Y chromosome in both PIN and tumor occurred in two cases. Also, (low level) aneuploidy was observed in 76% of these PIN lesions. Ploidy of the carcinomas as assessed by ISH correlated well with ploidy measured by DNA flow cytometry (FCM; P < 0.02). Due to the more specific correspondence between ISH and tumor pathology, pathologic grade correlated with ISH aneuploidy (P < 0.05), whereas FCM ploidy did not. Furthermore, genetic heterogeneity within a tumor was seen, as judged by the focal appearance of chromosomal aberrations. Chromosomal alterations occurred in all grades and stages, although loss of chromosome 10, gain of chromosome 7, and aberrations of chromosome 8 tended to predominate in more advanced cancers.

Adenocarcinoma↗
PubMed1996

Interphase cytogenetic studies of human hepatocellular carcinomas by fluorescent in situ hybridization.

Although numerous allelic chromosome losses have been reported in hepatocellular carcinomas (HCC), chromosome analysis by cytogenetic methods has rarely been performed in these tumors, unlike other solid malignant tumors. The purpose of the current study was to analyze primary liver tumors by conventional cytogenetic methods and by a new molecular cytogenetic technique, called fluorescent in situ hybridization (FISH), a technique that has been recently proposed to count the number of chromosome copies in interphase nuclei with chromosome centromeric probes. Primary cultures of tumoral cells were prepared to obtain metaphases. Specific chromosomes probes 7, 17, and 20 were used to perform in situ hybridization on isolated intact tumoral cells. Seven cases of primary liver tumors (six cases of HCC and one case of benign focal hepatic nodular hyperplasia) were investigated. A few metaphases were obtained in five of the seven tumors, and in most cases numerical abnormalities were difficult to interpret. In contrast with in situ hybridization, all cases of HCC showed losses and/or gains of chromosomes. Loss of one to three chromosomes occurred in five tumors. A gain of two chromosomes was observed in two of these five tumors. In only one case, a gain of only three chromosomes occurred. In addition, a loss of chromosome 17 was recorded for the benign tumor. These results demonstrate that FISH with specific probes can provide information on chromosome number in the tumoral cells of primary liver tumors even in the absence of analyzable metaphases. This technique opens new possibilities for the investigation of chromosome abnormalities in HCC.

Adult↗
PubMed2002

Interphase FISH assays for the detection of translocations with breakpoints in immunoglobulin light chain loci.

Many B-cell malignancies bear chromosomal translocations juxtaposing immunoglobulin (IG) genes with oncogenes, resulting in deregulated expression of the latter. Translocations affecting the IG heavy chain (IGH) locus in chromosomal region 14q32 are most prevalent. However, variant translocations involving the IG kappa (IGK) locus in 2p12 or the IG lambda (IGL) locus in 22q11 occur recurrently in B-cell neoplasias. No routine methods for the detection of all breakpoints involving IG light chain loci independently of the translocation partner have been described. For this reason, we have designed 2 novel interphase fluorescence in situ hybridization (FISH) assays using differentially labeled probes flanking the IGK and IGL locus, respectively. Based on extensive control studies, the diagnostic thresholds for the detection of breakpoints were set at 0.3% for IGK and 1.4% for IGL. Fifteen cases of B-cell malignancies with cytogenetically detectable chromosomal abnormalities in 2p11-14 were investigated with the FISH assay for IGK. Breakpoints affecting the IGK locus were detected in 7 cases including all 4 variant Burkitt's translocations t(2;8)(p12;q24) and a variant BCL2-associated translocation t(2;18)(p12;q21). Other translocation partners were chromosome bands 7q21 and 16q24. Ten cases with abnormalities in 22q11-12 were investigated with the FISH assay for IGL. Breakpoints in the IGL locus were diagnosed in 7 cases including both variant Burkitt's translocations t(8;22)(q24;q11) and a t(3;22)(q27;q11) involving the BCL6 locus. Other translocation partners were 2p13-14, 4q13 and 16p12. Our results show that these FISH assays provide flexible, simple and reliable tools in the diagnosis and characterization of genetic changes in B-cell malignancies.

Adolescent↗
PubMed1995

Sensitive detection of numerical and structural aberrations of chromosome 1 in neuroblastoma by interphase fluorescence in situ hybridization. Comparison with restriction fragment length polymorphism and conventional cytogenetic analyses.

Chromosome I abnormalities are indicators of prognosis in neuroblastoma (NB) but are not yet routinely exploited because conventional methods are technically demanding. We evaluated the pertinence of interphase cytogenetics fluorescence in situ hybridization (FISH) for the analysis of chromosome I in NB, compared with conventional methods. Deletion of Ip was detected in 8 of 9 cell lines analyzed by both FISH and restriction fragment length polymorphism (RFLP), but was evidenced in only 2 cases by conventional cytogenetics, painting analysis being required to reveal the other cases. The chromosome I number evaluated by FISH reflected the total chromosome modal number obtained by cytogenetics. Twenty-eight specimens obtained from ultrasound-guided punctures, surgical biopsies of the primary tumor and bone-marrow aspirates were studied by FISH on frozen cytocentrifuged smears; 12 had a chromosome I trisomy and 16 a disomy. Requirements for a reliable control analysis of Ip deletion by RFLP were met in only 23 cases. The retention of 2 alleles was observed in 15 cases and Ip deletion in 7, by both techniques. In one case, an interstitial deletion of Ip was evidenced only by RFLP, and one of 5 cases analyzed only by FISH had a Ip deletion. Although FISH might be improved by using additional probes, it presents major advantages for routine exploitation. Determining Ip deletion in individual cells makes it possible to analyze small and heterogeneous tumoral specimens; the technique requires only a few hours and can easily be standardized in non-specialized laboratories. The number of chromosome I homologues per cell might serve as a rapid screening for ploidy.

Biopsy↗
PubMed1986

Conjugation of [125I]ubiquitin to cellular proteins in permeabilized mammalian cells: comparison of mitotic and interphase cells.

[125I]Ubiquitin introduced into permeabilized hepatoma tissue culture (HTC) cells rapidly forms conjugates with endogenous proteins. A characteristic pattern of low mol. wt conjugates is obtained which includes the ubiquitinated histone, uH2A, and unknown molecular species with MrS of 14, 23, 26 (two bands) and 29 kd. A broad spectrum of higher mol. wt conjugates is also produced. The formation of all conjugates is absolutely dependent on ATP, and upon depletion of ATP they are rapidly broken down. The 14, 23 and 29 kd species are found in all subcellular fractions examined. uH2A is located exclusively in the nuclear fraction. The pair of 26 kd bands is specifically associated with the ribosome fraction. A considerable percentage of the higher mol. wt conjugates sediments with the small particle (100,000 g) fraction in the ultracentrifuge but is solubilized with deoxycholate, indicating that there are many membrane-associated conjugates. The pattern of ubiquitin conjugation in interphase and metaphase cells was compared. The incorporation of ubiquitin into uH2A was markedly reduced in metaphase cells whereas its incorporation into other low mol. wt conjugates and into high mol. wt conjugates was affected slightly, if at all. This shows that the known decrease of uH2A levels in metaphase is due to a specific effect on histone ubiquitination and not to a general decrease in ubiquitination activity or increase of isopeptidase activity. Changes in the levels of uH2A during mitosis measured by immunoblotting were similar to those estimated in permeabilized cells. These experiments indicate that permeabilized cells provide a useful approach to the study of rapidly turning over ubiquitin conjugates in mammalian cells.

Adenosine Triphosphate↗
PubMed1994

A fission yeast RCC1-related protein is required for the mitosis to interphase transition.

The isolation and characterization of the mutant dcdts (defect in chromatin decondensation) has led to the identification of two conserved proteins required for the re-establishment of the interphase state following the completion of mitosis. The gene that rescues the dcdts mutant encodes a protein similar to the human chromatin binding protein, RCC1. A suppressor of dcdts encodes a protein nearly identical to the human GTP-binding protein, RAN, encoded by the TC4 gene. These results indicate that completion of mitosis is regulated at least in part by a GTPase molecular switch. The gene and suppressor of dcdts are identical to the previously described Schizosaccharomyces pombe genes pim1 (premature initiation of mitosis) and spi1 (suppressor of pim), but the dcdts mutant does not enter mitosis prematurely, a phenotype that has been reported for the pim1-46ts mutant. Based on our studies we propose that the pim1 gene product is required for regulating chromatin condensation with a primary role at the end of mitosis and pleiotropic effects on other aspects of cell behavior.

Alleles↗
PubMed2006

The significance of telomeric aggregates in the interphase nuclei of tumor cells.

Telomeres are TTAGGG repetitive motifs found at the ends of vertebrate chromosomes. In humans, telomeres are protected by shelterin, a complex of six proteins (de Lange [2005] Genes Dev. 19: 2100-2110). Since (Müller [1938] Collecting Net. 13: 181-198; McClintock [1941] Genetics 26: 234-282), their function in maintaining chromosome stability has been intensively studied. This interest, especially in cancer biology, stems from the fact that telomere dysfunction is linked to genomic instability and tumorigenesis (Gisselsson et al. [2001] Proc. Natl. Acad. Sci. USA 98: 12683-12688; Deng et al. [2003] Genes Chromosomes Cancer 37: 92-97; DePinho and Polyak [2004] Nat. Genetics 36: 932-934; Meeker et al. [2004] Clin. Cancer Res. 10: 3317-3326). In the present overview, we will discuss the role of telomeres in genome stability, recent findings on three-dimensional (3D) changes of telomeres in tumor interphase nuclei, and outline future avenues of research.

Animals↗
PubMed2002

P2P-R protein localizes to the nucleolus of interphase cells and the periphery of chromosomes in mitotic cells which show maximum P2P-R immunoreactivity.

P2P-R is a nuclear protein that can bind both p53 and Rb1. Its functions include roles in the control of RNA metabolism, apoptosis, and p53-dependent transcription. The expression of P2P-R also is repressed in G1 arrested terminally differentiated cells. The current studies therefore evaluated if P2P-R undergoes cell cycle-associated changes in its abundance and/or localization. Western blots show that relative to G0 quiescent cells, P2P-R protein levels are higher in populations of G2/M cells prepared by the physiological parasynchronization technique of serum deprivation followed by serum stimulation. More striking is the > 10-fold enrichment of P2P-R protein in specimens of highly purified mitotic cells prepared by the mitotic shake-select technique, or by synchrony with the mitotic spindle disruption agents nocodazole or vinblastine. These changes in P2P-R protein occur without a concomitant change in P2P-R mRNA expression suggesting that P2P-R immunoreactivity increases during mitosis. Confocal microscopy next established the localization of P2P-R to nucleoli in interphase cells and at the periphery of chromosomes in mitotic cells that lack nucleoli. The high levels of P2P-R localized to the periphery of chromosomes in mitotic cells suggest that P2P-R shares characteristics with other nucleolar proteins that associate with the periphery of chromosomes during mitosis. These include: nucleolin, B23, Ki67, and fibrillarin.

3T3 Cells↗