Structural chromosome analysis by whole chromosome painting for assessment of radiation-induced genetic damage.
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Chromosome Aberrations↗
Biomedical subjects
D Pinkel
Publications and source records attributed to D Pinkel.
Therapy of acute lymphoid leukemia in children.
The cure rate of childhood acute lymphoid leukemia (ALL) has improved in the past 20 years due to improved treatment of T cell ALL, better infection control and availability of blood components, and more experience. Several these are proposed as treatment guidelines.
Antineoplastic Combined Chemotherapy Protocols↗
Remission induction and continuation therapy in children with their first relapse of acute lymphoid leukemia. A Pediatric Oncology Group study.
Between January 1979 and April 1983, 113 children undergoing their first relapse of acute lymphoid leukemia (ALL) at any site were registered in Pediatric Oncology Group study 7834; 98 were eligible and evaluable. In addition to radiotherapy administered to sites of local relapse, induction consisted of vincristine, doxorubicin, and prednisone (VAP) chemotherapy. Continuation therapy consisted of triple-drug intrathecal therapy and regimens of 6-thioguanine and cytarabine alternating with vincristine, prednisone, cyclophosphamide, and cytarabine. Randomization in continuation was between VAP pulses or no pulse, regardless of the site of relapse. This report provides long-term follow-up of these patients. Thirty-two of 39 children with bone marrow involvement achieved a complete response (CR). Only one of these is alive in an unmaintained remission, a child who did not have an initial CR. Thirty-four of 36 evaluable children with central nervous system involvement as the site of relapse achieved CR. Of these ten are alive; eight are in continuing CR. Nineteen of 20 boys with testicular relapse achieved CR. Of these, 14 are still alive and not receiving therapy, although only one half received treatment in compliance with the protocol described. These results illustrate the possibility of cure of patients who have relapsed with ALL when it is (1) confined to a meningeal or gonadal site and (2) treated vigorously with radiotherapy and a new regimen of systemic chemotherapy. The results reconfirm the need to prevent an initial relapse at any site.
Antineoplastic Combined Chemotherapy Protocols↗
Two-color hybridization with high complexity chromosome-specific probes and a degenerate alpha satellite probe DNA allows unambiguous discrimination between symmetrical and asymmetrical translocations.
This report describes a fluorescence in situ hybridization approach to chromosome staining that facilitates detection of structural aberrations and allows discrimination between dicentric chromosomes and symmetrically translocated chromosomes. In this approach, selected whole chromosomes are stained in one color by hybridization with composite probes whose elements have DNA sequence homology along the length of the target chromosomes. In addition, all chromosomes are counterstained with a DNA specific dye so that structural aberrations between target and non-target chromosomes are clearly visible. Discrimination between dicentric chromosomes and symmetrical translocations is accomplished by hybridization with a second probe that is homologous to DNA sequences found in the centromeric region of all chromosomes. The centromeric marker is visualized in a different color, so that the number of centromeres per aberrant chromosome can be rapidly determined in the microscope by changing excitation and fluorescence filters.
Base Sequence↗
The male-derived genome after sperm-egg fusion: spatial distribution of chromosomal DNA and paternal-maternal genomic association.
After fusion with an egg, the haploid genome of a mammalian sperm expands from a volume of a few cubic microns to a volume of several thousand cubic microns in a fully developed zygote pronucleus. Using fluorescent in situ hybridization we studied two aspects of the chromatin organization of the sperm-derived genome during this process in a model system involving fusion of human sperm with hamster eggs. We found that (a) from the beginning of sperm head decondensation to early pronuclear stages, the hybridization signal of a probe targeted to the satellite III heterochromatin of chromosome 1 appeared as an irregularly shaped domain. In fully developed pronuclei, the signal became a fiber as much as 20-fold more extended compared with interphase somatic nuclei. The signal appeared as a string of non-uniformly distributed "beads" with interspersed gaps. A similar spatial distribution and appearance recurred after the first cleavage division. (b) Male- and female-derived genomes assembled non-randomly on the first mitotic spindle, and continued to be sequestered from each other in the nuclei of two-cell stages.
Animals↗
Construction and characterization of plasmid libraries enriched in sequences from single human chromosomes.
Plasmid libraries enriched in sequences from single chromosome types have been constructed for all human chromosomes. This was accomplished by transferring inserts from the Charon 21A phage libraries constructed by the National Laboratory Gene Library Project into Bluescribe plasmids. Insert material freed by complete digestion of the phage libraries with HindIII or EcoRI was cloned into the corresponding sites in Bluescribe plasmids. The sizes of the Bluescribe library inserts determined by gel electrophoresis range from near 0 to approximately 6 kb. Fluorescence in situ hybridization (FISH) with the plasmid libraries showed that all hybridize along both arms of the expected (target) chromosome type with varying intensity. However, the plasmid libraries for chromosomes 1, 4, 9, 11, 16, 18, and 20 hybridize weakly or not at all near the centromeres of the target chromosome types. The libraries for chromosomes 13, 14, 15, 21, and 22 cross-hybridize near the centromeres of all members of this group and hybridize weakly to the short arms of the target chromosomes. FISH with each library allows specific staining of the target chromosome type in metaphase spreads. The signals resulting from FISH with libraries for chromosomes 1, 4, 8, 9, 13, 14, 17, 18, 21, and Y are sufficiently intense to permit analysis in interphase nuclei. Examples of the use of these libraries for translocation detection, marker chromosome characterization, and interphase aneuploidy analysis are presented.
Chromosome Aberrations↗
Clinical applications of fluorescence in situ hybridization.
We review here the application of fluorescence in situ hybridization with chromosome-specific probes to chromosome classification and to detection of changes in chromosome number or structure associated with genetic disease. Information is presented on probe types that are available for disease detection. We discuss the application of these probes to detection of numerical aberrations important for prenatal diagnosis and to detection and characterization of numerical and structural aberrations in metaphase spreads and in interphase nuclei to facilitate tumor diagnosis.
Chromosome Aberrations↗
Acute renal failure in a female adolescent with leukemia in remission.
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Acute Kidney Injury↗
Molecular cytometry applied to detection and characterization of disease-linked chromosome aberrations.
Flow karyotyping and FISH with chromosome-specific or disease locus-specific probes are powerful adjuncts to conventional cytogenetic analysis. Flow karyotyping is well suited to quantitative analysis of the DNA content changes that occur during structural rearrangement. FISH with probes for repeated sequences allows ready detection of aneuploidy in interphase cells. FISH with whole-chromosome composite probes to metaphase spreads facilitates detection of subtle structural changes and allows detection of structural aberrations that occur at frequencies as low as 10(-3). FISH with locus-specific probes facilitates diagnosis of specific genetic diseases, may allow phenotype-genotype correlation on a cell by cell basis and will permit sensitive detection of residual disease.
Chromosome Aberrations↗
The use of fluorescence in situ hybridisation combined with premature chromosome condensation for the identification of chromosome damage.
The technique of fusing mitotic cells to interphase cells, thereby producing condensation of the chromosomes of the interphase cell (so-called 'premature chromosome condensation' or PCC), has allowed detection of the initial number of chromosome breaks and their repair following ionising radiation. However, the difficulty and tedium of scoring all the chromosome fragments, as well as the inability to readily detect exchange aberrations, has limited the use of PCC. We describe here the use of the recently developed technique of fluorescence in situ hybridisation with whole chromosome libraries to stain individual human chromosomes (also called 'chromosome painting') with the PCC's and show that this overcomes most of the limitations with the analysis of PCC's. First, by focusing on a single chromosome, scoring of breaks in the target chromosome is easy and rapid and greatly expands the radiation dose range over which the PCC technique can be used. Second, it allows the easy recognition of exchange type aberrations. A number of new applications of this technology, such as predicting the radiosensitivity of human tumours in situ, are feasible.
Cell Line↗
Applications of fluorescence in situ hybridization in biological dosimetry and detection of disease-specific chromosome aberrations.
Dual color FISH with whole chromosome and pan-centromere probes facilitates rapid detection of stable structural aberrations such as translocations. This approach should allow analysis of translocations for assessment of genetic damage at long times after exposure or as a result of chronic exposure during a long period of time. Multi-color FISH with locus specific probes allows assessment of the frequency of cells carrying specific aberrations known to be associated with tumorigenesis, analysis of the series of genetic changes that occur during tumor evolution and correlation between genotype and phenotype. The power of FISH for analysis of random and tumor related events will increase steadily as informative probes are developed during the course of the International Human Genome Project.
Chromosome Aberrations↗
Detection of aneuploidy involving chromosomes 13, 18, or 21, by fluorescence in situ hybridization (FISH) to interphase and metaphase amniocytes.
Fluorescence in situ hybridization (FISH) with chromosome-specific probes has been applied to detection of numerical aberrations involving chromosomes 13, 18, and 21 in metaphase and interphase amniocytes. High-complexity, composite probes for chromosomes 13, 18, and 21 were used as hybridization probes for this study. These probes were constructed as chromosome-specific libraries in Bluescribe plasmids and are designated pBS-13, pBS-18, and pBS-21. Elements of these probes bind at numerous sites along the target chromosome and, when detected fluorescently, stain essentially the entire long arm of the target chromosome. The target chromosome number (i.e., the number of chromosomes of the type for which the probe was specific) was correctly determined in 20 of 20 samples in which metaphase spreads were analyzed and in 43 of 43 samples in which interphase nuclei were analyzed; all of these studies were conducted in blind fashion. These results suggest the utility of FISH with composite probes for rapid detection of numerical aberrations in metaphase and interphase amniotic cells.
Aneuploidy↗
Detection of bcr-abl fusion in chronic myelogeneous leukemia by in situ hybridization.
Chronic myelogeneous leukemia (CML) is genetically characterized by fusion of the bcr and abl genes on chromosomes 22 and 9, respectively. In most cases, the fusion involves a reciprocal translocation t(9;22)(q34;q11), which produces the cytogenetically distinctive Philadelphia chromosome (Ph1). Fusion can be detected by Southern (DNA) analysis or by in vitro amplification of the messenger RNA from the fusion gene with polymerase chain reaction (PCR). These techniques are sensitive but cannot be applied to single cells. Two-color fluorescence in situ hybridization (FISH) was used with probes from portions of the bcr and abl genes to detect the bcr-abl fusion in individual blood and bone marrow cells from six patients. The fusion event was detected in all samples analyzed, of which three were cytogenetically Ph1-negative. One of the Ph1-negative samples was also PCR-negative. This approach is fast and sensitive, and provides potential for determining the frequency of the abnormality in different cell lineages.
Chromosomes, Human, Pair 22↗
Clinically isolated mandibular relapse in childhood acute leukemia.
With improved methods for preventing extramedullary relapse in the leptomeninges and gonads, the problem of clinically isolated relapse at other sites has become more significant. The authors report here two children with acute leukemia who developed mandibular relapse while in complete hematologic remission. One had been off chemotherapy for acute lymphoid leukemia for 2.5 years. The other child is apparently the first patient with promyeloid morphologic features to experience relapse at this site. Both children are in second complete remission and off treatment after local radiation therapy and second courses of chemotherapy. Review of these two and five previously reported isolated mandibular relapses in childhood leukemia indicate that they are usually delayed until after cessation of therapy. Treatment with radiation and combination chemotherapy can result in long remission and possibly cure.
Child, Preschool↗
Fluorescence in situ hybridization to Y chromosomes in decondensed human sperm nuclei.
Human sperm nuclei were isolated with mixed alkyltrimethylammonium bromide and dithiothreitol (MATAB/DTT) and decondensed by treatments with lithium diiodosalicylate (LIS), sodium chloride, or Tris salts. Concentrations as low as 1 mM LIS induced measurable nuclear swelling compared to 600 mM required for the other two salts. As measured by image analyses, the projected nuclear area increased linearly up to approximately fivefold with LIS concentrations up to 10 mM. Swollen nuclei also maintained the elliptical shapes characteristic of the human sperm head. Expanded sperm nuclei of three men were hybridized with a fluorescently labeled 3.4 kb Y chromosome-specific repetitive DNA probe; 50.1% of the nuclei of each semen sample showed fluorescent labeling over a part of the nucleus indicating presence of the Y chromosome. In comparison, unswollen sperm did not yield reliable hybridization signals. This procedure is suitable for determining the proportion of human sperm with Y chromosomes and can be used to evaluate sperm separation techniques. The availability of probes specific for most human chromosomes suggests that this procedure may find general application in studies of sperm chromosomal constitution.
Cell Nucleus↗
Biochemical interactions between methotrexate and 1-beta-D-arabinofuranosylcytosine in hematopoietic cells of children: a Pediatric Oncology Group study.
Children with acute lymphocytic leukemia (ALL) in remission were treated with overlapping sequential infusions of methotrexate (MTX) and 1-beta-D-arabinofuranosylcytosine (araC) as part of continuation therapy. The doses and the sequence were chosen to mimic conditions that produced greater than additive antineoplastic activity with these two drugs in preclinical studies. To assess the potential for the drug combination to exhibit greater than additive effect in vivo, we investigated several biochemical parameters that had been associated with synergism in vitro. Because the patients were in remission, the intracellular parameters could only be measured in cytologically normal hematopoietic cells. We observed that (1) the mean plasma concentrations of MTX and araC were above those required to obtain a greater than additive cytotoxicity with the two drugs in tissue culture; (2) MTX did not have a significant antipurine effect in bone marrow mononuclear cells; (3) the mean intracellular concentration of deoxycytidine triphosphate (dCTP) was significantly lower after treatment with the drug combination than after therapy with araC alone; and (4) the ratio of araC triphosphate (araCTP) to dCTP was 2.6 times higher after treatment with the combination than after araC alone. These results indicate that it is possible to achieve in patients the biochemical conditions associated with the greater than additive antineoplastic activity of MTX and araC in vitro.
Antineoplastic Combined Chemotherapy Protocols↗
Detection of aneuploidy and aneuploidy-inducing agents in human lymphocytes using fluorescence in situ hybridization with chromosome-specific DNA probes.
The feasibility of utilizing fluorescence in situ hybridization with chromosome-specific DNA probes as the basis of an assay to detect aneuploidy and aneuploidy-inducing agents in interphase human lymphocytes has been investigated. The assay involves counting the number of hybridization regions in interphase cells to determine the number of copies of a specific chromosome of interest, 22,000 interphase nuclei from untreated 72-h lymphocyte cultures were examined following hybridization with probes for chromosomes 1, 7, 9, 17, X or Y. The combined frequencies of nuclei containing 0, 1, 2, 3 and 4 hybridization regions for the various autosomal chromosomes were 0.004, 0.084, 0.909, 0.003 and 0.001, respectively. Based on these frequencies, scoring 1000-2000 cells should allow detection of aneuploid cells with a 0.012 frequency of hyperdiploidy or a 0.11 frequency of hypodiploidy for a specific chromosome of interest (alpha = 0.05, beta = 0.80). This difference in test sensitivity is related to the higher frequency of cells with one apparent spot. A comparison of the ratio of hybridization region to nuclear area in the two-dimensional images used for this analysis indicates that an overlap of the two regions probably accounts for the high frequency of apparent monosomy observed in normal cells. Treatment with the aneuploidy-inducing chemicals, colchicine, vincristine sulfate and diethylstilbestrol resulted in significant dose-related increases in the number of nuclei containing 3 or more hybridization regions. Treatment with the clastogen sodium arsenite produced only a minor increase in apparently hyperdiploid cells whereas treatment with ionizing radiation, another potent clastogen, resulted in a significant increase in nuclei containing multiple hybridization regions. These results suggest that ionizing radiation is an aneuploidy-inducing agent under these conditions although chromosomal breakage within the hybridization region may account for a portion of the increased frequency of nuclei with multiple hybridization regions. These results indicate that the use of fluorescence in situ hybridization with DNA probes is capable of detecting aneuploid cells occurring at relatively low frequencies within a population of cells. Assays based on these techniques should facilitate a more rapid identification of aneuploidy-inducing environmental and therapeutic agents.
Aneuploidy↗
Fluorescence in situ hybridization with DNA probes.
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2-Acetylaminofluorene↗