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Prognostic value of chromosome 1 and 8 copy number in invasive ductal breast carcinoma among Iranian women: an interphase FISH analysis.

Breast cancer is amongst the leading causes of death in women worldwide and the most common cancer amongst Iranian women. Unfortunately, the current clinical and histological criteria can only help 60 percent of women with breast cancer in diagnosis and long-term treatment. Therefore, genetic markers both at single gene and chromosomal level can play an important role in improving the diagnosis and prognosis of breast cancer patients. The aim of this retrospective study was to investigate the role of chromosome 1 and 8 copy number assessed by interphase fluorescence in situ hybridization (FISH), as prognostic parameters in 50 Iranian women, aged 35 to 64 years, with sporadic invasive ductal breast carcinoma. Chromosome 1 and 8 copy numbers were evaluated in relation to established clinicopathological parameters, the immunohistochemical markers ER, PR, P53 and cathepsin D, DNA index by flow cytometry, age and survival status of the patients. FISH using centromeric probes for chromosomes 1 and 8 was applied to interphase cell suspensions prepared from archived, Carnoyfixed tumor cells and selected paraffin-embedded tumor sections. Aneusomy for chromosomes 1 and 8 was present in all 50 patients to different levels. The total abnormality rate for chromosome 1 was 33.92 percent (4.24 percent monosomy and 29.68 percent polysomy), whereas for chromosome 8 this rate was 28.30 percent (6.48 percent monosomy and 21.82 percent polysomy). Statistically significant association (p<0.05) was demonstrated between monosomy 1 and patients' age below 50 years, and between monosomy 1 and poor survival, respectively. Disomy 8 was significantly associated with P53 expression. A borderline significant correlation was demonstrated between polysomy 8 and diploid DNA content, as well as between disomy 1 and disease-free status of the patients. Chromosome 1 and 8 copy numbers may be considered as useful prognostic markers in invasive ductal carcinoma of the breast.

Aneuploidy↗

Proteins tightly bound to DNA in the regions of DNA attachment to the skeletal structures of interphase nuclei and metaphase chromosomes.

Proteins tightly bound to the DNA of mouse L cells were examined both in the regions of DNA attachment to the protein skeleton and in the DNA loops. The interphase nuclear matrix and the metaphase chromosomal scaffold, containing from 2 to 15% of the original DNA, as well as the material released from the skeleton were isolated by mild nuclease treatment. At least six proteins resistant to sarcosyl-Cs2SO4 treatment are associated exclusively with skeleton-attached DNA both in interphase nuclei and metaphase chromosomes. Two (p52 and p60) can be dissociated from the complex by mercapto-ethanol-sarcosyl treatment. Both skeleton-attached and released DNA fractions contain a group of tightly bound low molecular weight polypeptides consisting of a major p18 band and one or two minor bands. They seem to be randomly distributed throughout the whole DNA loop. Some of the specific proteins that are tightly associated with DNA at the site of its interaction with the skeleton may directly be responsible for DNA attachment to the skeleton.

Animals↗

Trisomy 8 detection in Ph+ CML patients using conventional cytogenetic and interphase fluorescence in situ hybridization techniques.

We examined bone marrow (BM) cells from 6 Philadelphia chromosome positive (Ph+) chronic myeloid leukemia (CML) patients in advanced phase of the disease using conventional cytogenetic techniques and fluorescence in situ hybridization (FISH) for detection of an extra chromosome 8. All patients showed mosaicism for trisomy 8 as a secondary chromosome abnormality. For FISH, we used the D8Z5 probe specific for the centromeric region of chromosome 8 and analyzed 300 interphase nuclei and a variable number of mitoses for each patient. The percentages of metaphases carrying trisomy 8 were similar with both techniques, whereas the percentage of interphase nuclei showing three hybridization spots indicative of trisomy 8 was significantly lower than that in metaphases. This finding suggests that cells with a supernumerary chromosome 8 may have a cell cycle time shorter than that of disomic cells.

Aged↗

High incidence of a second BCR-ABL fusion in chronic myeloid leukemia revealed by interphase cytogenetic analysis on blood and bone marrow smears.

The t(9;22)(q34;q11) is the single most common chromosomal abnormality in leukemias. Recently, dual-color fluorescence in situ hybridization (FISH) protocols for the detection of the BCR-ABL fusion, which is the molecular counterpart of this translocation, have been described. In the present study, we analyzed blood or bone marrow smears of 46 patients (34 with chronic myeloid leukemia [CML] and 12 with acute lymphoblastic leukemia [ALL]) for the presence of a BCR-ABL fusion. On these clinical routine samples, hybridization was performed with high efficiency and the BCR-ABL fusion was detected reliably. This series includes one case with a Philadelphia chromosome (Ph) on banding analysis and negative reverse transcriptase polymerase chain reaction (RT-PCR) results. Surprisingly, in 13 of the 34 CML patients (4 of 17 patients with chronic phase and 9 of 17 patients with blast crisis), and in 1 of the 12 ALL patients, an additional BCR-ABL fusion was diagnosed in 4% to 72.5% of interphase cells. In 10 of these 14 patients, banding data are available; only in two cases was the additional Ph detected by metaphase analysis. The data from this interphase cytogenetic analysis indicate that an additional Ph occurs more frequently than would be assumed based on banding analysis.

Adult↗

p37 is a p97 adaptor required for Golgi and ER biogenesis in interphase and at the end of mitosis.

We previously reported that p97/p47-assisted membrane fusion is important for the reassembly of organelles at the end of mitosis, but not for their maintenance during interphase. We have now identified a p97 adaptor protein, p37, which forms a complex with p97 in the cytosol and localizes to the Golgi and ER. siRNA experiments revealed that p37 is required for Golgi and ER biogenesis. Injection of anti-p37 antibodies into cells at different cell cycle stages showed that p37 plays an important role in both Golgi and ER maintenance during interphase as well as in their reassembly at the end of mitosis. In an in vitro Golgi reassembly assay, the p97/p37 complex has membrane fusion activity. In contrast to the p97/p47 pathway, this pathway requires p115-GM130 tethering and SNARE GS15, but not syntaxin5. Interestingly, although VCIP135 is also required, its deubiquitinating activity is unnecessary for p97/p37-mediated activities.

Adaptor Proteins, Signal Transducing↗

The influence of substrate charge and molecular structure on interphase transfer in cloud point extraction systems.

The specificity of interphase transfer of organic reagents of different types between water and the nonionic-surfactant-rich phases at the cloud point temperature was investigated. In contrast to organic solvents, the ability of the micellar phases to extract highly charged ions of organic reagents was shown. Further evidence of the specificity of interphase transfer in micellar-extraction systems is independent and cooperative influence of substrate molecular structure and hydrophobicity on their distribution. The appearance of host-guest phenomenon in cloud point extraction systems that is inherent in organized systems with molecules of the receptors was established. The possibility of describing reagent distribution using regressions that consider substrate hydrophobicity and molecular structure in the cloud point extraction systems was shown.

Journal Article↗

The effects of interphase and bonding on the elastic modulus of bone: changes with age-related osteoporosis.

A simple shear lag model is developed to analyze the physics of the stress transfer between the organic and mineral constituents of bone tissue in the presence of an interphase and changes in bonding. The analytical model is developed assuming interactions between overlapped bone mineral platelets. The platelets are assumed to carry the axial stresses while the organic matrix transfers the stresses from one platelet to another by shear. A decrease in the interphase mechanical properties decreases the elastic modulus due to increased shear between the overlapped platelets. A decrease in bonding decreases the elastic modulus due to an increase in the axial stress transferred from the ends of the platelet. The implications of the changes in parameters on the age-related disorders of bone (osteoporois) are discussed. It is suggested that the aspect ratio and volume fraction of the mineral in the remaining bone tissue would increase due to a reduction in the density of the bone. The mechanical properties of the organic are hypothesized to increase due to a reduction in the density of bone leading to an increased tendency for damage within the organic.

Blood Platelets↗

Chromosome damage and aneuploidy detected by interphase multicolour FISH in benzene-exposed shale oil workers.

A multicolour tandem-labelling fluorescence in situ hybridization (FISH) procedure was used to detect chromosome alterations in peripheral blood cells of a group of Estonian petrochemistry workers. Twelve workers employed in benzene production and five cokery workers, together with eight unexposed rural controls, were enrolled in the study. The methodology employed, based on the in situ hybridization of adjacent centromeric and pericentromeric regions, allowed the simultaneous detection of both chromosome breakage, involving damage-prone pericentromeric regions, and hyperploidy in interphase cells. Blood smears from all subjects were hybridized with chromosome 1 specific probes, in order to detect genotoxic damage in circulating lymphocytes and granulocytes. Moreover, lymphocyte cultures were established, harvested 48 h following mitogen stimulation and hybridized with the tandem chromosomes 1 and 9 probes. No significant difference in the incidence of breakage was detected in the nucleated cells of blood smears of exposed vs. control subjects. In contrast, modest but significantly increased frequencies of breakage affecting both chromosomes 1 and 9 were observed in the cultured lymphocytes of the benzene-exposed workers compared to the unexposed controls, suggesting an expression of premutagenic lesions during the S-phase in vitro. Across the entire study group, the frequencies of breakage affecting chromosomes 1 and 9 in the stimulated lymphocytes were highly intercorrelated (p < 0.001). No significant difference was found in the incidence of hyperploidy among the study groups, although a tendency to higher values was observed in benzene-exposed workers. Although the relatively small size of the study groups does not allow firm conclusions on the role of occupational exposure, the observed patterns are suggestive of effects in the benzene-exposed workers. This work also shows that tandem labelling FISH can be usefully applied in human biomonitoring, allowing the simultaneous detection of both hyperploidy and chromosome breakage at interphase in different cell types.

Adult↗

Understanding Interphase Boundary Dynamics by In Situ High-Resolution and Energy-Filtering Transmission Electron Microscopy and Real-Time Image Simulation.

: This study discusses the use of in situ high-resolution transmission electron microscropy (HRTEM) techniques to determine the structure, composition, and interphase boundary dynamics during phase transformations at the atomic level. Three main in situ HRTEM techniques are described: (1) in situ HRTEM dynamic studies that are performed on the same precipitate plates from different viewing directions to determine the three-dimensional structure of the interfaces; (2) in situ compositional mapping of precipitate interfaces obtained by energy-filtering TEM experiments at temperature in a HRTEM, and (3) real-time HRTEM image simulations that are being created for comparison with and interpretation of experimental in situ HRTEM dynamic observations. The results from these studies demonstrate that it is possible to understand the mechanisms and kinetics of interphase boundary motion at the atomic level.

Journal Article↗

Effect of high-viscosity interphases on drainage between hydrophilic surfaces.

Drainage of water from the region between an advancing probe tip and a flat sample is reconsidered under the assumption that the tip and sample surfaces are both coated by a thin water "interphase" (of width approximately a few nanometers) whose viscosity is much higher than that of the bulk liquid. A formula derived by solving the Navier-Stokes equations allows one to extract an interphase viscosity of approximately 59 kPa x s (or approximately 6.6 x 10(7) times the viscosity of bulk water at 25 degrees C) from interfacial force microscope measurements with both tip and sample functionalized hydrophilic by OH-terminated tri(ethylene glycol) undecylthiol, self-assambled monolayers.

Journal Article↗

Frequent RET rearrangements in thyroid papillary microcarcinoma detected by interphase fluorescence in situ hybridization.

Papillary thyroid microcarcinomas (measuring 1 cm or less in diameter) are very common thyroid tumors, which are present in 10% to 35% of post-mortem histopathological examinations of individuals whose death was due to a cause other than thyroid cancer. The molecular basis of this tumor is still poorly understood. Somatic mutations are better characterized in clinically evident papillary thyroid carcinomas (PTCs), the most common involving the proto-oncogene RET, which maps to 10q11.2. Molecular alterations of RET always lead to intra- or interchromosomal rearrangements. In this study we have investigated the status of RET in 21 microcarcinomas, by means of interphase fluorescence in situ hybridization (FISH). RET was rearranged in 52% of microcarcinomas, a statistically significant higher frequency than that found previously in clinically evident PTCs using the same technique. Moreover, interphase FISH allowed us to detect a putative novel type of rearrangement in a microcarcinoma, and we observed trisomies of chromosome 10 and other chromosomes in two adenomas surrounding two of the microcarcinomas. The strikingly high frequency of RET rearrangements in microcarcinomas strongly suggests that RET plays a role in the initiation of thyroid tumorigenesis but does not seem to be necessary for the further progression of the tumor.

Adult↗

Comparison of chromosome 1 aneusomy detected by interphase cytogenetics and DNA ploidy in carcinoma of the breast.

Aneuploidy is an important prognostic factor in many cancers. Chromosome 1 abnormalities are present in most breast carcinomas. These may be part of a non-specific increase in DNA (aneuploid status) or represent a restricted chromosomal abnormality. In 16 breast carcinomas we compared chromosome 1 aneusomy with ploidy status. Patients were selected from a mammographically screened population and interphase tumour nuclei were studied by in situ hybridization using a chromosome 1 pericentromeric probe. Ploidy status was assessed by image cytometry on disaggregated cells from paraffin blocks. Of eight cases showing chromosome 1 aneusomy, six (75%) were aneuploid and two diploid. Six (75%) of the eight eusomic cases were aneuploid. This study demonstrates that chromosome 1 aneusomy does not always reflect a gross aneuploid status but, in some tumours, is part of a more restricted chromosomal abnormality. Interphase cytogenetics, possibly using a small panel of pericentromeric probes, may be more sensitive than DNA cytometry for detecting abnormal nuclear DNA content.

Aneuploidy↗

Analysis of interphase chromosome damage by means of premature chromosome condensation after X- and ultraviolet-irradiation.

Sendai virus-mediated fusion between mitotic and interphase mammalian cells causes the rapid condensation of the interphase chromosomes into distinct structures, a process termed premature chromosome condensation. This phenomenon has been used to assess the immediate action of x-rays and ultraviolet light on the chromosomes of HeLa cells irradiated in the G1 phase of the life cycle. X-irradiation produces fragmented chromosomes; but even the most finely chopped fragments retain the condensed morphology characteristic of the premature chromosome condensation of unirradiated G1 cells. For doses up to about 1800 rads, the increase in the number of fragments is linearly related to the dose. One mean lethal dose (about 100 rads) yields a net increase of 10-15 fragments per G1 cell, which is considerably larger than previous estimates based on scoring of mitotic chromosomes. Incubation of irradiated cells produces a rapid (within 2 hr) reduction in the number of fragments, indicative of a rejoining process. The decrease in the number of pieces is not accompanied by unscheduled DNA synthesis detectable by radioautography. G1 chromosomes of cells irradiated with UV light in G1 phase are not appreciably fragmented but are elongated and attenuated so that they resemble the premature-chromosome-condensation chromosomes of unirradiated S-phase cells. Both the degree of "S-like" character attained by the G1 chromosomes in a cell and the percentage of the cell population displaying the G1 --> S transition increase with the dose and incubation time after irradiation. Thus, in contrast to the immediate manifestation of damage from x-rays, the maximum induction of the "S-like" state does not occur until about 2 hr after irradiation. The "S-like" chromosomes are capable of unscheduled DNA synthesis. We suggest that the difference in chromosome morphology found after UV- and x-irradiation underlies the reason why the former, but not the latter, induces unscheduled DNA synthesis in G1 cells.

Cell Fusion↗

Interphases of chain molecules: Monolayers and lipid bilayer membranes.

Using the lattice model for a liquid, we treat the packing of short-chain molecules in interphases such as bilayer membranes. The constant density in the interphase imposes intermolecular constraints on the configurations of the flexible chains. The statistical theory here presented predicts a diffuse distribution of chain ends near the bilayer midplane; no adjustable parameters are required. Inasmuch as some of the chains terminate relatively near the polar interface, the number of chains reaching deeper planar layers is diminished. Consequently, configurational freedom increases with depth. This is the source of the well-known disorder gradient.

Journal Article↗

Hidden aberrations diagnosed by interphase fluorescence in situ hybridisation and spectral karyotyping in childhood acute lymphoblastic leukaemia.

Acute lymphoblastic leukaemia (ALL) is the most common oncologic disease in childhood, accounting for approximately 25% of all paediatric malignancies. Based on clinical risk criteria and modern laboratory investigations including immunophenotyping, cytogenetics and molecular genetics, patients can be divided into prognostic groups and assigned to risk-adjusted treatment protocols. The karyotype is an independent prognostic indicator and has for some aberrations that are associated with a poor outcome a direct impact on the choice of treatment. Cytogenetic analysis in ALL is often hampered by poor chromosome morphology, few malignant metaphases, undetectable chromosomal rearrangements due to regions of a similar size and banding pattern and sometimes only normal metaphases derived from normal cells are found after cell culture. Structural as well as numerical aberrations may therefore remain undetected using conventional G-banding. The application of modern molecular cytogenetic techniques including a broad set of fluorescence in situ hybridisation (FISH) methods and recent developments in comparative genomic hybridisation to DNA microarrays, together with molecular methods such as Southern blotting and RT-PCR has greatly improved the detection rate of genetic changes in ALL. This review emphasises the value of increasing the resolving power of the cytogenetic investigation by spectral karyotyping (SKY) and interphase FISH in identifying prognostically important and novel chromosomal rearrangements as a complement to conventional banding analysis. The results of investigations performed on cases with ALL have shown that interphase FISH is valuable and in many cases even mandatory for the detection of prognostically important genetic abnormalities and should therefore consistently be employed in the routine cytogenetic investigations in ALL. Likewise, SKY is a valuable tool for the cytogenetic analysis. Thus, the results of several different investigations described in this review revealed that SKY yielded additional information in 97/157 (62%) cases with chromosomal aberrations detected by G-banding, and in 10/66 (15%) cases with normal G-banding.

Blotting, Southern↗

Okadaic acid induces interphase to mitotic-like microtubule dynamic instability by inactivating rescue.

We used high-resolution video microscopy to visualize microtubule dynamic instability in extracts of interphase sea urchin eggs and to analyze the changes that occur upon addition of 0.8-2.5 microM okadaic acid, an inhibitor of phosphatase 1 and 2A (PP1, PP2a) (Bialojan, D., and A. Takai. 1988. Biochem. J. 256:283-290). Microtubule plus-ends in these extracts oscillated between the elongation and shortening phases of dynamic instability at frequencies typical for interphase cells. Switching from elongation to shortening (catastrophe) was frequent, but microtubules persisted and grew long because of frequent switching back to elongation (rescue). Addition of okadaic acid to the extract induced rapid (< 5 min) conversion to short, dynamic microtubules typical of mitosis. The frequency of catastrophe doubled and the velocities of elongation and shortening increased slightly; however, the major change was an elimination of rescue. Thus, modulation of the rescue frequency by phosphorylation-dependent mechanisms may be a major regulatory pathway for selectively controlling microtubule dynamics without dramatically changing velocities of microtubule elongation and shortening.

Animals↗

Nucleocytoplasmic exchanges during early interphase.

Colloidal gold was injected into the cytoplasm of amebae (Amoeba proteus) approximately 5 min, 1 hr, and 2 hr after cytokinesis. Later, interphase cells were similarly treated. All of the amebae were fixed about 50 min after injection and were examined with the electron microscope in order to determine the distribution of the gold. It was found that for a period of 2 hr after division the uptake of gold by the nuclei was significantly greater than that during late interphase. Correlation of the gold distribution with the morphology of the nuclear envelope indicated that an inverse relationship exists between the rate of incorporation of colloid into the nucleoplasm and the degree of reconstitution of the fibrous lamina ("honeycomb" structure). These data support the view that the fibrous lamina functions in regulating nucleocytoplasmic exchanges.

Amoeba↗

Fine structural alterations of interphase nuclei of lymphocytes stimulated to grwoth activity in vitro.

This report describes fine structural changes of interphase nuclei of human peripheral blood lymphocytes stimulated to growth by short-term culture with phytohemagglutinin. Chromatin is found highly labile, its changes accompanying the sequential increases of RNA and DNA synthesis which are known to occur in lymphocyte cultures. In "resting" lymphocytes, abundant condensed chromatin appears as a network of large and small aggregates. Early in the response to phytohemagglutinin, small aggregates disappear during increase of diffuse chromatin regions. Small aggregates soon reappear, probably resulting from disaggregation of large masses of condensed chromatin. Loosened and highly dispersed forms then appear prior to the formation of prophase chromosomes. The loosened state is found by radioautography to be most active in DNA synthesis. Small nucleoli of resting lymphocytes have concentric agranular, fibrillar, and granular zones with small amounts of intranucleolar chromatin. Enlarging interphase nucleoli change chiefly (1) by increase in amount of intranucleolar chromatin and alteration of its state of aggregation and (2) by increase in granular components in close association with fibrillar components.

Autoradiography↗