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PubMed1996

Cytophotometric determination of heterochromatin base composition in interphase nuclei of plant cells.

The use of DNA base-specific fluorochromes and static cytophotometry at different thresholds of fluorescence intensity is proposed for studying the base composition of differently condensed chromatin fractions in plant interphase nuclei. Results are reported of such analyses on nuclei from leaflets of Posidonia oceanica and Helianthus annuus, which confirm other findings obtained on the same materials by biochemical and molecular methods.

Base Composition↗
PubMed1997

FISH and chips: automation of fluorescent dot counting in interphase cell nuclei.

Fluorescence in situ hybridization allows the enumeration of chromosomal abnormalities in interphase cell nuclei. This process is called dot counting. To estimate the distribution of chromosomes per cell, a large number of cells have to be analyzed, especially when the frequency of aberrant cells is low. Automation of dot counting is required because manual counting is tedious, fatiguing, and time-consuming. We developed a completely automated fluorescence microscope system that can examine 500 cells in approximately 15 min to determine the number of labeled chromosomes (seen as dots) in each cell nucleus. This system works with two fluorescent dyes, one for the DNA hybridization dots and one for the cell nucleus. After the stage has moved to a new field, the image is automatically focused, acquired by a Photometrics KAF 1400 camera (Photometrics Ltd., Tuscon, AZ, USA), and then analyzed on a Macintosh Quadra 840AV (Apple Computer, Inc., Cupertino, CA, USA) computer. After the required number of cells has been analyzed, the user may interact to correct the computer by working with a gallery of the cell images. The automated dot counter has been tested on a number of normal specimens where 4,'6-diamidino-2-phenylindole (DAPI) was used for the nucleus counterstain and a centromeric 8 probe was used to mark the desired chromosome. The slides contained lymphocytes from cultured blood. We compared the results of the dot counter with manual counting. Manually obtained results, published in the literature, were used as the "ground truth." For a normal specimen, 97.5% of cells will have two dots. Fully automated scanning of 13 slides showed that an average of 89% of all nuclei were counted correctly. In other words, an average of 11% has to be interactively corrected, using a monitor display. The machine accuracies, after interactive correction, are comparable to panels of human experts (manual). The fully automatically obtained results are biased with respect to manual counting. An error analysis is given, and different causes are discussed.

Algorithms↗
PubMedDate not supplied

Sex chromosome determination in extragonadal teratomas by interphase cytogenetics: clues to histogenesis.

Teratomas are neoplasms that are composed of tissues from all three germinal layers. The exact histogenetic origin of teratomas, however, is still controversial. In order to gain more insight into histogenesis of extragonadal teratomas (EGTs), the gonosomal status in 13 congenital EGTs was studied by means of interphase cytogenetics using nonradioactive in situ hybridization (NISH) with centromere-specific DNA probes. By use of this technique a direct correlation of cytogenetic results with morphology was possible. In all EGTs analyzed the gonosomal status in tissues derived from the different germinal layers was identical to that of the nontumorous fetal and placental tissue. This was true irrespective of localization, age, histological type, and classification of the EGT. Our results strongly suggest that EGTs arise from pluripotent diploid precursor cells, for example, either premeiotic germ cells that have not yet undergone the first meiotic division or pluripotent ectopic embryonal or extraembryonal cells. Our data do not support the theory of parthenogenetic EGT development, at least in males.

Cell Differentiation↗
PubMed1997

Stereoscopic analysis of microtubule pattern around the centrosome in interphase PK cells after treatment with taxol and nocodazole.

In the interphase PK cells, more than 85% of microtubules radiating from the centrosome were not longer than 1.5 microns. A half of microtubules had their proximal ends free. After nocodazole treatment (20 microM), the number of microtubules attached to the centrosome decreased by 20% after 10 min of treatment, remained the same after 20 min of treatment, and increased after 60 min of nocodazole treatment slightly above the control level. After 5 and 60 min of treatment, the number of attached microtubules with the length over 0.7 micron increased twice as compared to the control level. During the first 20 min of nocodazole treatment, the immunofluorescent staining of cells with antibodies to gamma-tubulin was the same as in the control cells. The number of free microtubules decreased fourfold during the first 5 min, then it decreased slowly (for 20 min) and remained at the same level after 60 min. After 10 min of taxol (12 microM) treatment, the number of attached and free microtubules increased more than two times, whereas the number of attached microtubules with the length over 0.7 micron increased more than tenfold. After 15 min of treatment, the number of attached microtubules was slightly higher, and the number of free microtubules was half of the control level. After 20-60 min of treatment, the number of microtubules of all types decreased. Thus, upon the nocodazole treatment, the microtubules attached to the centrosome were more resistant to depolymerization: however, these microtubules were the most reactive to taxol treatment. The data obtained suggest that (a) in PK cells, the centrosome-attached microtubules occupy not all of the existing templates; (b) during prolonged treatment with inhibitors, the centrosome performed the compensatory reaction-the inhibition of microtubule assembly results in the decrease in the number of active templates on the centrosome; the inhibition of microtubule depolymerization results in the inactivation of hitherto active reserve templates. The microtubules formed on the centrosome within the first minutes disengage from it and then leave the chromosomal region.

Animals↗
PubMed1997

[Interphase analysis of X-aneuploidy using fluorescent in situ hybridization in various tissues of healthy individuals].

The frequency of spontaneous X-chromosome hypo- and hyperploidy was studied in interphase nuclei of cultured and uncultured blood lymphocytes and oral epithelium of 21 healthy individuals by means of fluorescence in situ hybridization of the centromere-specific DNA probe. In women, the frequencies of haploid epithelial cells and cultured and uncultured lymphocytes were 0.84%, 1.59%, and 0.73%, respectively, while the frequency of hyperploid cells was 0.29%, 0.28%, and 0.12% respectively. In men, the X-chromosome spontaneous hyperploidy of epithelial cells, cultured and uncultured lymphocytes was 0.14%, 0.57%, and 0.11% respectively. The upper frequency limit of the spontaneous aneuploidy of various cell types was determined for the purpose of being used for the diagnosis of X-mono- and trisomy mosaicism in women and the disomy in men. In the latter, the frequency of hypoploidy in the cultured lymphocytes was significantly higher than in uncultured lymphocytes. Under conditions of intense cell proliferation in FHA-stimulated cultures, more severe disturbances in chromosome segregation are proposed to occur than in resting cells.

Adult↗
PubMedDate not supplied

Improved technique for investigations on archival formalin-fixed, paraffin-embedded tumors by interphase in-situ hybridisation.

In-situ hybridisation techniques are powerful tools for the analysis of chromosomes in a variety of specimens. Due to hybridisation problems caused by extensive formalin-fixation in tissue samples mainly isolated nuclei were used for chromosomal analysis. In archival tissue, for sections to retain both an optimum of well preserved morphology and hybridisation efficiency a large-scale adjustment of protease digestion steps for each tissue block was required. We sought to develop an easy, reproducible technique by pretreatment in a 90 degrees C glycerol solution and subsequent denaturation in an autoclave 1 bar for 10 minutes. Our experiments were performed on eight up to ten years old human bladder cancer tissue blocks. Comparison with established pretreatment techniques did not reveal hybridisation differences while the morphology of tissue sections kept intact. This technique facilitates retrospective interphase cytogenetic analyses and is a reliable method for detection of chromosomal anomalies in formalin-fixed archival tumors.

Centromere↗
PubMed1998

Interphase cytogenetic evidence for distinct genetic pathways in the development of squamous neoplasia of the uterine cervix.

Human papillomavirus (HPV) infection has been implicated as an etiologic factor in most cervical cancers. However, additional genetic alterations are thought to be required for the development of a carcinogenic genotype. In the present study, interphase cytogenetics utilizing pericentromeric probes specific for chromosomes 1, 3, 11, 17, 18, and X was performed on paraffin-embedded tissue sections from 25 high-grade squamous intraepithelial lesions (SILs) and 25 invasive squamous cell carcinomas (ISCCs) of the cervix. HPV infection was determined by both in situ hybridization and broad-spectrum GP5+/GP6+ PCR. HPV was identified in all high-grade SILs (HPV 16, n = 16; 18, n = 2; 26, n = 1; 31, n = 4; 45, n = 1; 66, n = 1) and 23 (92%) ISCCs (HPV 16, n = 19; 18, n = 2; 31, n = 1; 39, n = 1). Aneusomy was identified in 11 (44%) high-grade SILs and 18 (72%) ISCCs. In 18 (62%) of these, relative under-representation of chromosomes 3, 11, 17, and/or 18 was identified (8 high-grade SILs and 10 ISCCs). Tetrasomy of all six chromosomes was present in two high-grade SILs but no ISCCs. Twelve (48%) high-grade SILs and seven (28%) ISCCs were disomic with all six chromosome probes, and there was no relationship between HPV presence or type and chromosome pattern. The presence of distinct patterns of numerical chromosome abnormality in these lesions suggests that progression to high-grade SIL or invasive carcinoma can occur by more than one genetic pathway. The lack of correlation between chromosome pattern and HPV type indicates that these pathways are not HPV type-specific. Whether these patterns reflect differences in early gene expression, possibly related to viral integration, or differences in the biologic properties of HPV type variants remains to be established.

Carcinoma, Squamous Cell↗
PubMed1998

Analysis of meiotic segregation in human nondecondensed interphase spermatozoa by triple colour rapid direct fluorescent in situ hybridization.

Meiotic segregation of chromosomes X,Y and 1 was analyzed by triple colour rapid fluorescent in situ hybridization (FISH) with directly labelled probes on 4506 non-decondensed and non-cleaned interphase spermatozoa from four healthy male donors to test the possibility of rapid sperm FISH by omitting the conventional sperm decondensation and cleaning steps. Only 0.15 per cent of sperms were without any signals which suggested high hybridization success. An abnormal number of signals was seen in 1.6 per cent of sperms. Chromosome specific as well as donor specific segregation error was seen similar to previous reports. There was a wide variation in the ratio of normal X and Y bearing sperm from donor to donor. This study indicated that for segregation studies sperm FISH can be carried out in three hours with directly labelled probes without the steps for separation of sperm from somatic cells (cleaning), sperm swelling and sperm DNA decondensation.

Humans↗
PubMed1998

Use of nonbreakpoint DNA probes to detect the t(X;18) in interphase cells from synovial sarcoma: implications for detection of diagnostic tumor translocations.

Fluorescence in situ hybridization studies using non-breakpoint DNA probes were performed to detect the X;18 translocation on 4-microm sections of synovial sarcoma from paraffin blocks. This was done by using commercially available, large target unique sequence DNA probes for regions of the X chromosome short-arm and the 18 chromosome long-arm together with centromere probes for the alternate chromosomes. We determined that such probe combinations could detect the presence of the diagnostic X;18 translocation in interphase cells. Spatial association of dual color signals from the X centromere and the 18 unique sequence probe, as well as between an 18 centromere and the X unique sequence probe, was seen in a significantly higher percentage of synovial sarcoma cells (81.1% +/- 7.7%, confidence interval 95%) than in control nonsynovial soft tissue sarcomas (14.7% +/- 8.3%) and control peripheral blood lymphocytes (5.6% +/- 0.6%). The observed spatial association supports the use of this strategy to detect the X;18 translocation in synovial sarcoma and suggests that this technique could be applied in the diagnosis of other types of tumors with characteristic translocations when histopathological findings are inconclusive. This study is the first report describing the use of nonbreakpoint unique sequence probes for detecting translocations in tumors on paraffin-embedded slides.

Chromosomes, Human, Pair 18↗
PubMed1998

[Identification of aneuploids in uncultured amniotic fluid cells by interphase fluorescence in situ hybridization].

We report the diagnosis of aneuploids in uncultured amniotic fluid cells by the use of interphase fluorescence in situ hybridization (FISH). About a dozen of cases was studied to detect chromosome aneuploids (trisomies 13, 18 and 21, and monosomy X), especially those in the perinatal period. Results of FISH experiments in each specimen were compared with those of conventional chromosome analysis, and showed no discrepancy between them in most cases. FISH analysis revealed that four 18-trisomies had two different cells, one of which showed two fluorescence signals and the other three signals, the result indicating a maternal cell contamination. Thus, special attention should be paid when FISH analysis is adopted to the diagnosis in uncultured amniotic fluid cells in order to avoid misdiagnosis that may originate in maternal cell contamination.

Amniotic Fluid↗
PubMed1998

Differences in chromosomal aberrations between nodular and superficial spreading malignant melanoma detected by interphase cytogenetics.

At present, little information is available on specific chromosomal aberrations in malignant melanomas of different subtypes and different growth behaviors. Therefore, we have applied fluorescence in situ hybridization on isolated interphase cells from paraffin sections of 79 primary tumors of malignant melanomas: 47 nodular melanomas and 32 superficial spreading melanomas in various stages. We used centromeric probes for the chromosomes 1, 4, 6, 7, 9, 10, 11, 12, 15, 17, 18, X, and Y and a midisatellite probe localized in 1p36. The number of chromosomal aberrations and the ploidy of the cells rose with the tumor stage in both subtypes, although in superficial spreading melanomas, fewer chromosomal abnormalities were detectable than in nodular melanomas. A deletion in 1p36 could only be found in nodular melanomas (mostly in higher tumor stages), not in superficial spreading melanomas. Our results show that the different histologic subtypes of malignant melanoma of the skin differ also in their chromosomal aberrations. In addition, it seems that there may be a correlation between the growth characteristics and putative tumor suppressor genes on 1p36.

Adult↗
PubMed1998

Interphase cytogenetic study of preoperative core biopsies for the prediction of early serum prostate specific antigen recurrence after radical prostatectomy of clinically localized prostate carcinoma.

BACKGROUND: Current clinical and pathologic methods are imprecise in predicting the extent of prostate carcinoma progression. This study was conducted to evaluate the role of interphase cytogenetics (ICG) with chromosome enumeration probes relative to the role of conventional pathologic characteristics in the preoperative prediction of postoperative tumor classification and recurrence of elevated serum concentrations of prostate specific antigen (PSA). METHODS: The authors performed ICG with enumeration probes for chromosomes 7, 17, and X on 6-microm sections of core biopsies from 75 patients with clinically localized adenocarcinoma of the prostate. Results were compared with pathologic findings (tumor classification, volume, and status of surgical margins) of the corresponding radical prostatectomy specimens and data on serum PSA measurements taken for 72 patients during a follow-up period of up to 42 months (mean, 19.8 months). In addition to ICG, biopsies were reviewed for primary, secondary, highest, and combined Gleason grades, number and bilaterality of positive biopsies, amount of carcinoma (percentage and core length of infiltration), amount of high grade carcinoma, and perineural invasion. RESULTS: Chromosome categories were unevenly distributed among pathologic tumor classification groups (P = 0.0008) and between groups with positive and negative surgical margins (P = 0.0116). Tumors with aneusomic chromosome numbers had larger volumes than those with eusomy and tetrasomy (P = 0.0022). Kaplan-Meier analysis demonstrated that PSA recurrences (> or = 0.4 ng/mL) were more frequent and observed earlier in patients with detected chromosomal aneusomies than in those with eusomic and tetrasomic chromosome numbers (P < 0.0001). Cox regression analysis indicated that ICG was the most valuable independent factor in predicting PSA recurrences. CONCLUSIONS: The detection of numeric chromosomal aberrations in preoperative core biopsies is an adverse prognostic sign. Grading based on the quantification of genetic changes might prove useful in the prognostic stratification of patients with clinically localized prostate carcinoma.

Adenocarcinoma↗
PubMed1998

Correlations between mitotic and apoptotic indices, number of interphase NORs, and histological grading in squamous cell lung cancer.

Proliferative activity of tumors is strongly associated with prognosis and response to therapy. The reason for faster and uncontrolled growth rate of tumors compared with normal tissue may be caused by the greater proliferation of cells, the smaller rate of cell death, or both. Cell production vs. cell loss rates, and their correlation with a grade of tumor cell differentiation (G) was estimated in 45 cases of squamous cell lung cancers (SCLC) by the use of mitotic indices (MI), number of interphase NORs, and apoptotic indices (AI) as parameters. The mitotic figures as well as apoptotic cells were observed on paraffin sections (4-microm thick) stained with haematoxylin and eosin, and with Feulgen reaction with Schiff-type reagent containing 0.5% Toluidine Blue. According to our results, all three parameters distinguish significantly (P < 0.05) between well and moderately or poorly differentiated groups, but not between the first two groups, and clearly discriminate between low- and high-grade malignancy. These results suggest classification of squamous cell lung cancers into two groups, a group of low and a group of high proliferative activity, despite their morphological appearance. Regression analysis revealed a significant (P < 0.0005) correlation between MI and AgNOR counts per cell nucleus as proliferative markers and AI as a marker of cell loss. The number of mitoses and apoptoses, especially when they are expressed as a percentage of the total number of tumor cells, are markers of tumor proliferation rate. They both can be used in biofunctional staging, based on cell kinetics, to provide more prognostic information about lung cancers than clinicopathological staging.

Adult↗
PubMed1997

Interphase karyotypic analysis of chromosomes 11, 17 and X in invasive squamous-cell carcinoma of the cervix: morphological correlation with HPV infection.

Human papillomavirus (HPV) infection has been widely implicated in cervical carcinogenesis, but it appears to be an early event, with other genetic abnormalities being required for biological transformation. In this study, interphase cytogenetic analysis of numerical abnormalities of chromosomes 11, 17 and X was performed on paraffin-embedded tissue sections from 25 invasive squamous-cell carcinomas of the cervix and compared with both histopathological features and the morphological distribution of HPV sequences as determined by in situ hybridisation. Numerical differences between chromosomes were identified in 76% of cases, with underrepresentation of chromosomes 11 and/or 17 relative to X in 64% of the total; 22 of 25 cases were HPV-positive, containing either HPV 16, 18 or 31. There was no relationship between the distribution of viral sequences and chromosomal pattern, suggesting that HPV infection precedes karyotypic changes. Our findings suggest that relative reduction in number of chromosomes 11 and 17 is important in the development of invasive cervical neoplasia and are consistent with the putative presence of relevant tumour-suppressor genes on these chromosomes.

Carcinoma, Squamous Cell↗
PubMed1997

Modified interphase cytogenetics technique as an adjunct in the analysis of atypical cells in body fluids.

Our objective was to determine the value of a modified interphase cytogenetics technique (MICT) by fluorescence in situ hybridization (FISH) in the study of atypical cells in body fluids in previously stained slides, allowing a direct morphologic-cytogenetic correlation. Thirty-five cases (29 bladder washes, four pleural fluids, two ascitic fluids) initially diagnosed as "atypical" with subsequent histologic confirmation were included. Histologically, there were 25 malignant four dysplastic, and six benign lesions. Previously Papanicolaou or Diff-Quick-stained slides were marked to determine the location of the cells of interest prior to FISH analysis. A pretreatment modification using pepsin digestion was utilized. Chromosome-specific probe 8 (Vysis) was used to detect numerical chromosomal abnormalities (NCA) involving chromosome 8. Various NCA (aneuploid) were detected in the atypical cells of histologically proven malignant cases but not in the benign cases. Using histology as a "gold standard," FISH has a sensitivity of 83% and a specificity of 100%. In conclusion this study shows that a MICT by FISH on previously stained slides can serve as an adjunct in the study of atypical cells in body fluids. This technique allows a direct morphologic-cytogenetic correlation which in the future may aid in the better understanding of carcinogenesis.

Body Fluids↗
PubMed1988

Modulation of nuclear rotation in neuronal interphase nuclei by nerve growth factor, by gamma-aminobutyric acid, and by changes in intracellular calcium.

Nuclear rotation (NR) is typically measured as motion of nucleoli within nuclei of cells in vitro. This occurs in cycling cells. However, its observation in neurons arrested in interphase indicates that mechanisms related to mitosis are not a prerequisite. We have recently shown that NR occurs in three dimensions within the nuclear space, that it occurs within the space delineated by the outer nuclear membrane and that it includes chromatin domains in addition to nucleoli and have postulated that this motion of chromatin domains is related to changes in gene expression. We now show that exposure of dorsal root, sensory neurons in vitro to nerve growth factor (NGF) or to gamma-aminobutyric acid (GABA), agents which alter gene expression, and to agents causing redistribution of calcium, such as EGTA and the calcium ionophore A23187, significantly alters NR. The NGF increased the mean rate of NR and did so at a time after exposure when activity of RNA polymerases have been shown to rise. Exposure to GABA resulted, within minutes, in shifts of the nucleolus within the three-dimensional space of the nucleus, associated in some neurons with significant, sigmoidal increases in the rate of NR. The calcium ionophore A23187 as well as chelation of extracellular calcium with EGTA similarly increased rates. Importantly, excess calcium, with EGTA remaining present, returned NR of all nucleoli to rates not different from controls. This indicates that the increase in NR seen with EGTA is specific to the chelation of calcium and not an nonspecific response to EGTA. It is difficult to link the action of agents which alter gene expression or transmembrane ion balance with changes in NR. Nevertheless, in support of our hypothesis, the results presented here show that agents known to alter gene expression, alter NR in a temporally coincident manner and that they do so, possibly, by calcium-dependent mechanisms.

Animals↗
PubMed1995

A 62-kDa mitotic apparatus protein required for mitotic progression is sequestered to the interphase nucleus by associating with the chromosomes during anaphase.

A protein component of 62-kDa (p62) in the mitotic apparatus of the sea urchin embryo has been shown to be important for the proper progression of mitosis [Dinsmore and Sloboda, 1989: Cell 57:127-134]. To study the subcellular distribution of p62 during the cell cycle of sea urchin embryos, indirect immunofluorescence microscopy was used coupled to a modified detergent extraction procedure. The improved fluorescent images obtained by this procedure provide new information concerning the subcellular localization of p62 during the cell cycle that could not be obtained with previous conventional staining procedures [Johnston and Sloboda, 1992: J. Cell Biol. 119:843-854]. Using affinity purified antibodies to p62, we observed a cell cycle-dependent localization of p62 to the chromosomes/chromatin. Prior to nuclear envelope breakdown of the first or second cell cycle, p62 localizes to chromatin in the nucleus. During mitosis, p62 associates with the region of the spindle occupied by the microtubules of the mitotic apparatus. As anaphase proceeds, but before the nuclear envelope reforms, p62 becomes progressively associated with the chromosomes. Thus, p62 is incorporated into the forming interphase nucleus due to its association with chromosomes during late anaphase, rather than by active translocation into the newly formed daughter nuclei through the nuclear pores. The protein is not unique to marine embryos, as demonstrated by immunofluorescence of Y-1 cells, a mouse adrenal tumor cell line. In these cells, the localization of p62 is similar to the localization of the protein in echinoderm embryos, suggesting its possible function in mitotic progression in mammalian somatic cells as well.

Anaphase↗
PubMed2005

Smart 3D-FISH: automation of distance analysis in nuclei of interphase cells by image processing.

BACKGROUND: Detection of fluorescent probes by fluorescence in situ hybridization in cells with preserved three-dimensional nuclear structures (3D-FISH) is useful for studying the organization of chromatin and localization of genes in interphase nuclei. Fast and reliable measurements of the relative positioning of fluorescent spots specific to subchromosomal regions and genes would improve understanding of cell structure and function. METHODS: 3D-FISH protocol, confocal microscopy, and digital image analysis were used. RESULTS: New software (Smart 3D-FISH) has been developed to automate the process of spot segmentation and distance measurements in images from 3D-FISH experiments. It can handle any number of fluorescent spots and incorporate images of 4',6-diamino-2-phenylindole counterstained nuclei to measure the relative positioning of spot loci in the nucleus and inter-spot distance. Results from a pilot experiment using Smart 3D-FISH on ENL, MLL, and AF4 genes in two lymphoblastic cell lines were satisfactory and consistent with data published in the literature. CONCLUSION: Smart 3D-FISH should greatly facilitate image processing and analysis of 3D-FISH images by providing a useful tool to overcome the laborious task of image segmentation based on user-defined parameters and decrease subjectivity in data analysis. It is available as a set of plugins for ImageJ software.

Algorithms↗