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PubMed1997

Detection of hyperdiploidy and breakage affecting the 1cen-1q12 region of cultured interphase human lymphocytes treated with various genotoxic agents.

Chromosomal aberrations are associated with cancer, birth defects, and pregnancy loss. Previous studies using banding techniques have revealed that chromosomal alterations induced in human peripheral lymphocytes by many genotoxic agents occur nonrandomly throughout the genome. One of the regions prone to breakage is the centromeric heterochromatin of chromosome 1. We have developed a fluorescence in situ hybridization (FISH) procedure using tandem DNA probes to distinguish hyperdiploidy from breakage occurring in this region. Interphase nuclei exhibiting breakage or exchanges affecting the 1cen-1q12 region can readily be distinguished from nuclei hyperdiploid for this chromosome by identifying the number and location of the hybridization signals. This hybridization approach was tested using cultured human lymphocytes treated with a series of known aneuploidy-inducing agents (colchicine, diethylstilbestrol, and vincristine sulfate), several potent clastogens (ionizing radiation, mitomycin C, and etoposide), as well as sodium arsenite and hydroquinone, agents that have been reported to have relatively weak aneuploidy-inducing and clastogenic activity. Significant increases in chromosomal alterations were seen with all agents tested and the results were generally consistent with those previously seen using standard cytogenetic techniques. Treatment with colchicine, diethylstilbestrol, and vincristine sulfate resulted in high frequencies of primarily hyperdiploid nuclei, and cells exposed to radiation, mitomycin C, and etoposide exhibited elevated frequencies of breakage affecting the 1cen-1q12 region. Sodium arsenite and hydroquinone induced relatively minor but significant increases in both hyperdiploidy and breakage. These results indicate that this tandem labeling approach can be used to distinguish aneuploidy-inducing agents from those causing breakage in interphase human cells and may be a valuable procedure for monitoring human populations exposed to genotoxic agents.

Aneuploidy↗
PubMedDate not supplied

[Structure formation in interphase adsorption layers of lysozyme at liquid boundaries].

In connection with the modelling of biomembranes regularities of the formation and development of interphase adsorption layers of lysozyme at liquid borders under different conditions and depending on the nature of carbohydrate phase were investigated by the determination of mechanical characteristics of such layers. The investigations carried out showed that the most solid layers appeared under the conditions which assured the formation of the maximum number of intermolecular bonds (which in a common case is performed with maximum disorderlinesss of the macromolecules which get at the interphase).

Adsorption↗
PubMed1998

Chromosomal constitution of human spermatocytic seminomas: comparative genomic hybridization supported by conventional and interphase cytogenetics.

No data on the chromosomal constitution of spermatocytic seminomas are available thus far because of their rarity. Ploidy analysis performed on paraffin-embedded cases showed varying results from (near-) diploid to aneuploid. We applied comparative genomic hybridization on four snap-frozen primary spermatocytic seminomas of three different patients. Conventional cytogenetic analysis was successful in one, and "interphase cytogenetics" with centromeric region-specific probes was applied to another. The results from comparative genomic hybridization showed almost exclusively numerical chromosomal aberrations, in agreement with the data from karyotyping. Despite the limited number of cases studied, a nonrandom pattern of chromosome imbalances was detected: chromosome 9 was gained in all spermatocytic seminomas. This suggests that that this aberration plays a role in the development of this cancer. Interphase cytogenetics shows that the copy number of most chromosomes ranges from two to four, with an average of near trisomic. This constitutes the first report on the chromosomal constitution of spermatocytic seminomas.

Aneuploidy↗
PubMed1999

Use of interphase fluorescence in situ hybridization in third trimester fetuses with anomalies and growth retardation.

In the last few years, attention has been focused on the use of interphase fluorescence in situ hybridization (FISH) for prenatal diagnosis with chromosome-specific DNA probes in the second trimester. This technique is accurate, rapid, and detects the most common aneuploidies. We present a preliminary study using FISH technique on uncultured amniotic cells derived from 30 fetuses with ultrasonographic evidence of intrauterine growth retardation (IUGR) in the third trimester. Fifteen fetuses were males and 15 were females. Seven fetuses (23.3%) had abnormal chromosomal constitution: five (18.6%) had trisomy 21, one (2.35%) had trisomy 18, and one (2.35%) showed a mosaic trisomy 18. No abnormalities were detected in the other 23 fetuses. Amniocentesis combined with FISH appears to be a safe, rapid, and accurate alternative to blood sampling in the third trimester, reducing the clinical and emotional stress of the time required to complete chromosome analysis by routine cytogenetics.

Abnormalities, Multiple↗
PubMed1996

Loss of chromosome 9 in tissue sections of transitional cell carcinomas as detected by interphase cytogenetics. A comparison with RFLP analysis.

Interphase cytogenetics by in situ hybridization (ISH) using a panel of centromere-associated DNA probes for chromosomes 1, 7, 9, 10, 11, 16, 17, and 18 was performed on 5 microns thick frozen tissue sections of transitional cell carcinomas (TCCs) of the urinary bladder. By this approach, chromosome ploidy, numerical chromosome aberrations, imbalance between chromosomes, and heterogeneity of aberrations within individual tumours were determined. In 15 of 24 TCCs, loss or underrepresentation of chromosome 9, compared with the ISH copy numbers of at least five other chromosomes, was demonstrated. Independently, RFLP analysis were performed on the same cases to detect loss of heterozygosity (LOH) of chromosome loci 9q34, 11p15, 16q22-24, 17p13, and 18q21. LOH was found in 9 of 19 informative cases for chromosome locus 9q34. Comparison of the ISH and RFLP results showed no correlation between numerical aberration and LOH for the loci on chromosomes 11, 16, 17, and 18. However, numerical loss of chromosome 9 was found in 89 per cent (eight of nine cases) with LOH for 9q34. Conversely, LOH at 9q34 was observed in only 67 per cent (eight of 12 cases) with underrepresentation of chromosome 9. Moreover, in 60 per cent of the non-informative cases (three of five cases), underrepresentation for chromosome 9 was observed. These results indicate that the heterochromatin probe for chromosome 9 can be reliably used in TCC tissue sections for the detection of chromosomal loss. In aneuploid TCCs, this DNA probe can be used for the detection of chromosomal underrepresentation only in combination with other centromere-associated DNA probes.

Blotting, Southern↗
PubMed1996

F-actin forms transient perinuclear shells at the mitosis-interphase transition.

Intermediate filaments and microtubules are known to be involved in establishing and maintaining nuclear shape. F-actin may also be involved in determining nuclear shape, since we have found it associated with reforming nuclei very briefly after cell division. We stained cells from vertebrate tissue cultures (3T3 and NRK-49F) and epidermal cells from an insect with rhodamine-phalloidin and Hoechst #33342 to localize F-actin in relation to the nucleus. We found that F-actin forms shells only around nuclei during reorganization in late mitosis and early interphase. We suggest that perinuclear F-actin shells may be generally present in eukaryotes, but that they are easily missed because of their delicacy and transience.

3T3 Cells↗
PubMed1997

Detection of 14q32.33 translocation and t(11;14) in interphase nuclei of chronic B-cell leukemia/lymphomas by in situ hybridization.

Abnormalities of chromosome 14 involving band q32.33 are among the most commonly observed cytogenetic alterations in B-cell malignancies. To assess the incidence and pathogenetic implications of 14q32.33 translocation in chronic B-cell leukemia/lymphomas, we performed fluorescence in situ hybridization (FISH) analysis with variable region (V(H)) and gamma constant region (Cgamma) gene probes in 37 patients with these disorders. Chromosome 14q32.33 translocation was detected in 2 of 18 patients with chronic lymphocytic leukemia (CLL), 1 of 2 with CLL of mixed cell types (CLL/PL), 1 of 2 with pro-lymphocytic leukemia (PLL), 5 of 6 with leukemic mantle-cell lymphoma (MCL), 2 of 7 with splenic B-cell leukemia/lymphoma of possible marginal zone origin (SBLL) and 2 with leukemic follicular lymphoma (FL). To further characterize 14q32.33 translocations in these patients, we developed a new procedure using double-color FISH with PRAD1, BCL2, V(H) and Cgamma gene probes. Chromosome t(11;14) was detected in 1 patient with CLL/PL, 1 with PLL and 5 with MCL. Chromosome t(14;18) was detected in 2 patients with FL. In a PLL patient with t(11;14), the cosmid CPP29 containing the PRAD1 gene and its 5'-flanking region split and co-localized with both Cgamma and V(H) gene probes, thus spanning the breakpoint. In CLL and SBLL patients, donor chromosomes were other than chromosomes 2, 11, 18 and 19, suggesting the involvement of a novel oncogene(s) in the pathogenesis of these diseases. Interphase FISH rapidly detected 14q32.33 translocation, t(11;14) and t(14;18) in B-cell malignancies with low mitotic activity at the single-cell level, facilitating the correlation of the molecular features of these translocations with clinical characteristics.

Adolescent↗
PubMed2000

An assessment of the use of interphase FISH with chromosome specific probes as an alternative to cytogenetics in prenatal diagnosis.

We have assessed the effects that would have been observed if we had changed from standard prenatal diagnosis to interphase fluorescence in situ hybridization (FISH) on our amniocentesis samples. We aimed to estimate the number of cases with aberrations other than chromosomes 13, 18, 21, X and Y, which would not have been detectable by FISH and to assess the potential clinical implications for these cases. In 1687 prenatal diagnoses, 111 cases had abnormal cytogenetic reports (6.5% aneuploidy rate). Out of those 111 cases, 14 had chromosomal abnormalities not detectable by FISH but four of these had major structural abnormalities diagnosed on ultrasound, which would have lead to counselling of a very poor prognosis anyway. In 10 cases without abnormal ultrasound findings, if FISH had been used rather than cytogenetics, it appears that there may have had no detrimental effects on the clinical outcomes of the cases studied. Out of those 10 cases, two pregnancies were terminated because of abnormal cytogenetic results (one was due to maternal age and the second one was due to abnormal biochemical screening) (mosaic 46,XY, /47,XY,+mar and 46,X,del(8)(p21) respectively) and their post-mortem results also did not show any abnormalities. One pregnancy was continued in spite of a de novo chromosomal rearrangement and resulted in an apparently normal live birth. Five cases (including a set of twins) with inherited balanced translocations resulted in four normal live births and one unexplained intrauterine death at 32 weeks' gestation and post-mortem was declined. One case with a paternally derived abnormal chromosome 21, decided to continue the pregnancy and resulted in a normal live birth. The last case in this group resulted in a rhesus related intrauterine death in the second trimester, and although an abnormal chromosome 13 insertion (paternally derived known aberration) there was no abnormality found at post-mortem. Therefore, we suggest that it is reasonable to use FISH as an alternative prenatal diagnosis for indications such as advanced maternal age and abnormal maternal serum biochemical screening when high quality ultrasound scanning is performed, but FISH should only be used as an additional test to conventional cytogenetics for the other indications, especially when abnormalities are found on ultrasound scan.

Adult↗
PubMed1996

Quantitative analysis of three-dimensional distribution of AgNOR proteins during interphase in leukemic cells.

Acidic proteins of the nucleolar organizer regions, selectively stained by silver (AgNOR-proteins), were investigated during interphase in leukemia cells with a confocal scanning laser microscope (CSLM). Simultaneous confocal fluorescence (for specific labeling of DNA, using propidium iodide) and transmitted light microscopy combined with digital deconvolution (for the location of the AgNOR proteins in nonconfocal mode) were used. The distribution of the AgNOR proteins measured by 3D microscopy was described by their number, the volume occupation of the nucleus by the AgNOR aggregates, the distance between each AgNOR, the distance of each AgNOR to the nucleolar border, and their anisotropy. The results of the 3D analysis were compared to those obtained by conventional 2D analysis, cytogenetical analysis of metaphase nucleolar organiser regions (NORs), and cell duplication rate. The descriptive power of these 3D parameters were assessed for nine leukemic cell lines. The measurements of the 3D spatial distribution of AgNORs was a better discriminant parameter than the morphological parameters (i.e., number and volume). The 3D expression of AgNORs is also a reliable parameter for assessing proliferative activity of leukemic cells and seems to be in relation with the differentiation stage of these leukemic cells.

Cell Division↗
PubMed1996

Comparison of automated and manual analysis of interphase in situ hybridization signals in tissue sections and nuclear suspensions.

In this study we compared visual and automated analyses of interphase in situ hybridization (ISH) signals in five prostatic tumor specimens and one normal prostate sample, both in tissue sections and nuclear suspensions. The advantage of tissue sections is preservation of tissue morphology allowing precise analysis of tumor cells only. The advantage of nuclear suspensions is easier access to automated analysis, due to their disaggregated and dispersed cellular appearance. The samples were hybridized with probes for the (peri)centromeric regions of chromosome 1 and Y. The number of ISH signals per nucleus was counted both manually and automatically by means of a commercially available image analysis system. After image analysis the results were interactively corrected using a gallery display. The automatic and manual counts, before and after interactive correction, were then statistically evaluated. We found no significant differences in overall distributions between the automated and the manual counts, before as well as after correction. This was observed for both tissue sections and cellular suspensions. It is therefore concluded that automated analysis of ISH signals is feasible in both nuclear suspensions and in tissue sections, despite a low percentage of nuclei that could be measured on the latter.

Adenocarcinoma↗
PubMed1996

Comparison of cytogenetics, interphase cytogenetics, and DNA flow cytometry in bone tumors.

Twenty-three samples of benign and malignant bone tumors were studied with cytogenetic analysis, interphase cytogenetics (IC) using in situ hybridization with (peri)centromeric probes for chromosomes 1, 7, and/or 8, and DNA flow cytometry (FCM). Our aim was to compare these methods in the detection of numerical chromosome aberrations (NCA) and aneuploidy. IC detected aneuploidy in 91%, FCM in 73%, and cytogenetics in 27% of the malignant tumors. In benign tumors IC detected aneuploid by in 4(33%), FCM in 2(17%), and cytogenetic analysis in 1. All of the benign tumors aneuploid by IC, two of which were also aneuploid by FCM, were histologically potentially aggressive. The clonal aberrations detected with cytogenetics usually agreed with the IC and FCM findings. All malignant tumors which had a normal karyotype were aneuploid either by IC or FCM or by both. In conclusion, IC was the most sensitive method in the detection of NCA and aneuploidy even though it was usually performed with only two (peri)centromeric probes. Aneuploidy was detected by cytogenetic analysis alone in 4 samples (17%), by cytogenetic analysis and/or FCM in 11 samples (48%), and by cytogenetic analysis, FCM, and/or IC in 16 samples (70%). Thus, the combined use of all three methods increased the sensitivity of aneuploidy detection.

Aneuploidy↗
PubMed1998

Automated FISH spot counting in interphase nuclei: statistical validation and data correction.

The evaluation of an automated system for Fluorescence In Situ Hybridization (FISH) spot counting in interphase nuclei is presented in this paper. Different types of experiments have been performed with samples from known populations. In all of them the goal is to detect mosaicism of chromosome X in leukocytes from mixtures in known proportions of healthy male and female blood. First the initial results from the automatic FISH analysis system were obtained and evaluated. Then the analysis was modified to reduce systematic errors, so that the results are closer to what an experienced human operator would have obtained (system calibration step). Finally, an additional control probe of chromosome Y was used to detect and discard cells where incorrect hybridization or other abnormal situations had occurred. In each step the system sensitivity was determined by the use of two statistical validation tests, so that the improvement brought about by the correction methods could be assessed. The results obtained in the study showed that, using both corrections, the system is able to detect 10% monosomies with a significance level alpha = 0.1%.

Algorithms↗
PubMed1999

High-resolution cytometry of FISH dots in interphase cell nuclei.

BACKGROUND: Flow cytometry (FCM) and laser scanning cytometry (LSCM) provide indispensable tools for measuring large number of cells with low resolution. Confocal microscopy, on the other hand, is used for measuring small number of cells with high resolution. In this paper, we present a reasonable compromise between the two extremes. METHODS: We have developed a completely automated, high-resolution system (high-resolution cytometer, HRCM) capable of analyzing microscope slides with FISH-stained interphase nuclei in two dimensions as well as in three dimensions using a fully motorized epi-fluorescence microscope and a cooled digital CCD camera fully controlled by a high-performance computer which performs both acquisition and related on-line image analysis. The images of different dyes are acquired sequentially using highly specific filters and superimposed in computer memory. For each nucleus and each hybridization dot, user-selected attributes (such as position, size, intensity, etc.) are computed off-line using another processor or computer connected with a network. RESULTS: Using HRCM, it is possible to analyze multi-color preparations including UV-excited dyes as well as repeatedly hybridized preparations reacquiring individual nuclei. The speed of the acquisition and analysis is about 50 nuclei per minute in two dimensions and 1 nucleus per minute in three dimensions, but depends on the density of nuclei on the slide; the precision of the lateral and axial measurements is approximately 100 nm. CONCLUSIONS: Thus, using overnight acquisition, quantities comparable to those of FCM or LSCM measurements can be analyzed with an accuracy comparable to confocal microscopy. HRCM is suitable for a number of clinical and scientific tasks: routine diagnostics, follow-up of therapy, studies of chromatin structure, and many other different aspects of cell research.

Cell Nucleus↗
PubMed1997

Ploidy analysis by in situ hybridization of interphase cell nuclei in fine-needle aspirates from breast carcinomas: correlation with cytologic grading.

Fine-needle aspirates from 54 breast cancer patients were investigated for numeric aberrations in chromosomes 6, 7, 12, and 17 by in situ hybridization (ISH) of interphase cell nuclei. Ploidy findings were compared with cytologic grading of tumors. Aneuploidy was found in 73% of cases. Chromosomes 6 and 7 showed numeric abnormalities in 63% and 62% of cases, respectively, whereas chromosome 17 retained a disome pattern in 2/3 of the tumors. Thirteen cancers (28% of 47 with four analyzed probes) had a normal signal number in all four chromosomes. In 17 (36%), all four had signal gain. Another 17 showed a mixed disome/aneusome pattern. They presented a continuum of increasing numeric abnormalities, 82% disomy for chromosome 17, and 13 of them were grade 2, indicating intermediate biologic properties. Correlation between grading and ploidy was good, with 10 of 11 grade 1 carcinomas showing diploidy, whereas 33 of 36 grade 2 and 3 tumors had numeric aberrations.

Breast Neoplasms↗
PubMed1996

Detection of numerical chromosome anomalies in interphase cells of ovarian carcinomas using fluorescence in situ hybridization.

Fluorescence in situ hybridization was used in interphase cells of 30 ovarian carcinomas to detect numerical changes in copy number of 13 different centromeres (1, 2, 3, 4, 6, 7, 8, 10, 11, 12, 17, 18, and X). Thirty-seven percent of samples (11/30) were near diploid and demonstrated only minor changes in centromere copy number, involving gain and/or loss of one or a few centromeres. The most common changes included loss of centromeres 4,6, 17, and 18 and gain of centromere 1. The remaining 63% of samples were hyperdiploid and demonstrated a general increase in copy number of most or all centromeres examined. Among these samples, the centromere of chromosome I was most often found to be at higher copy number. Centromeres that were less often at increased copy or deleted within the hyperdiploid samples include centromeres 4, 17, 18, and X. These results suggest that tumor-suppressor genes that are located on chromosomes 4, 6, 17, and 18 may be involved in the development and progression of ovarian cancer.

Adult↗
PubMed2000

A DNA probe combination for improved detection of MLL/11q23 breakpoints by double-color interphase-FISH in acute leukemias.

Reciprocal translocations involving the MLL gene on chromosome band 11q23 have been observed in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). In AML, identification of MLL breakpoints is an important prognostic factor. Breakpoints are clustered in an 8 kb DNA fragment (bcr) and can be detected by Southern blotting or fluorescence in situ hybridization (FISH) analysis. Our objective in this study was to design a DNA probe set that enables optimal detection of MLL rearrangements using interphase FISH. Two PAC clones, 217A21 and 167K13, spanning the MLL gene with a minimal overlap in the bcr were isolated and labeled. Twenty-seven AML/ALL patients with cytogenetic 11q23 abnormalities, seven AML/ALL patients without 11q23 abnormalities but MLL rearrangement by Southern blotting, and eight healthy donors were analyzed by FISH. We compared this double-color FISH analysis with FISH using a YAC clone (yB22B2) and with Southern blotting. The PAC probe combination detects an MLL breakpoint in all cases with MLL rearrangement detected by Southern blotting except for cases with a partial tandem duplication detected by reverse transcriptase-polymerase chain reaction (RT-PCR). FISH using the PAC probes also detected MLL breakpoints in four cases with MLL deletions telomeric to the breakpoint that could not be detected by the single probe yB22B2. This new probe set provides a reliable and rapid assay for the diagnosis of AML and ALL patients with MLL/11q23 breakpoints.

Acute Disease↗
PubMed2005

Interphase FISH screening for the LCR-mediated common rearrangement of isochromosome 17q in primary myelofibrosis.

Non-allelic homologous recombination (NAHR) between low-copy repeats (LCRs) has been implicated recently in somatic rearrangements including isochromosome i(17q), which is associated with hematologic malignancies as well as solid tumors. In hematological malignancies, the most common i(17q) breakpoint results from LCR-mediated NAHR, which creates a dicentric chromosome, idic(17)(p11.2). We report an elderly patient who presented with primary myelofibrosis (MF) with myeloid metaplasia (MMM), associated with idic(17)(p11.2) as the sole chromosomal abnormality, making this the first idic(17)(p11.2) myeloproliferative case reported in which the breakpoints are mapped to the breakpoint cluster region in proximal 17p. The rearrangement breakpoint maps to the previously defined LCR cluster, further suggesting that the genomic architecture of proximal 17p may be responsible for the formation of the majority of i(17q) cases. We describe our development of a rapid screening test using interphase FISH to detect idic(17)(p11.2), discuss the potential prognostic value of this molecular diagnostic test, and examine the relevance of LCR-mediated NAHR to common rearrangements in neoplasms.

Aged↗
PubMed2002

Arrest of cell cycle progression during first interphase in murine zygotes microinjected with anti-PCM-1 antibodies.

To investigate the function of the centrosome protein PCM-1, antibodies against PCM-1 were microinjected into either germinal vesicle stage meiotic oocytes or fertilized mouse eggs, and cell cycle progression events (i.e., microtubule assembly, chromosome and centrosome organization, meiotic maturation) were assayed. These studies determined that microinjected PCM-1 antibodies arrested cell cycle progression, with anti-PCM-1 arresting fertilized eggs at the pronucleate stage when injected during G1. Analysis of the injected eggs determined that centrosome disruption and microtubule cytaster disorganization accompanied the cell cycle arrest. Anti-PCM-1 blocked neither pronuclear centration, completion of mitosis when microinjected into zygotes at G2, nor meiotic maturation when microinjected into immature oocytes. These results identify a novel role for PCM- 1 in cell cycle regulation, and indicate that PCM-1 must fulfill an essential function for cells to complete interphase.

Animals↗