Detection of cell-cycle stage by fluorescence in situ hybridization: its application in human interphase cytogenetics.

Distinct cell-cycle-dependent changes in the conformation of centromeric chromatin in a specific human chromosome containing alpha-satellite DNA have been demonstrated by fluorescence in situ hybridization (FISH). This method, based upon specific FISH signal morphology, allows simultaneous analysis of chromosomal aneuploidy and detection of specific cell-cycle stage(s) of human tumor and/or normal cell populations in a single preparation of interphase cells. This interphase cytogenetic procedure might prove useful for both basic and clinical research involving human cells.     

An improved procedure for the cultivation and in situ chromosome preparation of monolayer cell cultures

A method for the cultivation of monolayer cell cultures on microslides in quadruple culture dishes together with a simple procedure for in situ chromosome preparation are described. The cells fixed to the slide can be stained according to standard procedures and analysed microscopically. The method is simple, rapid and reliable and provides many advantages especially for cytogenetic diagnostics with fibroblasts and amniotic fluid cells. It simplifies the performance of cytogenetic mutagenicity testing with primary cultures and permanent cell lines, e.g. the analysis of chromosome aberrations, sister chromatid exchanges (SCEs) and induced aneuploidy, as well as large-scale cytogenetic experiments.     

Recurrent chromosome aberrations in cancer

Cytogenetic investigations of neoplastic cells during the past 25 years have revealed more than 600 acquired, recurrent, balanced chromosome rearrangements, and it has been established that every tumor type, studied in a sufficient number to permit conclusions, may be subdivided on the basis of specific, and even pathognomonic, abnormalities. At the molecular level, the balanced rearrangements exert their action through one of two alternative mechanisms: Deregulation of one gene by relocation to an immunoglobulin or T-cell receptor gene, or the creation of a hybrid gene by the fusion of parts of two genes. At present, nearly 100 genes have been found to be involved in neoplasia-associated chromosomal rearrangements, the great majority in hematological disorders. At the same time, the clinical usefulness of various cytogenetic abnormalities as diagnostic and prognostic aids has been increasingly appreciated. The identification of a recurring chromosome abnormality can assist in the diagnosis and subclassification of a malignant disease and, hence, in the selection of the appropriate treatment. The karyotype is also an independent prognostic factor. In hematological neoplasms, where the knowledge of chromosome abnormalities still is much more complete than is the case with solid tumors, cytogenetic analysis now plays an integral part in the diagnostic work-up of individual patients. Data obtained during recent years strongly suggest that corresponding breakthroughs will be achieved in solid tumors within a not-too-distant future.     

Removal of Stainable Cytoplasmic Substances from Cytogenetic Slide Preparations

A method to remove stainable cytoplasmic substances from cytogenetic preparation using RNase A treatment is reported. The preparations processed with this method are especially useful for the automated analysis of mi-cronuclei of cultured cells with cytochalasin B and of chromosome aberrations induced by radiation.     

Combined RxFISH/G-banding allows refined karyotyping of solid tumors

Chromosome banding analysis of solid tumors often yields incomplete karyotypes because of the complex rearrangements encountered. The addition of fluorescence in situ hybridization (FISH) methods has helped improve the accuracy of solid tumor cytogenetics, but the absence of screening qualities from standard FISH approaches has proved a severe limitation. We describe the cytogenetic analysis of ten solid tumors using G-banding followed by cross-species color banding (RxFISH), a FISH-based screening technique giving a chromosome-specific banding pattern based on the genomic homologies between humans and gibbons. The addition of RxFISH analysis in all cases led to the identification of previously unidentified intra- as well as interchromosomal rearrangements, thus giving a much more certain and detailed karyotype. In two gastric stromal sarcomas, a tumor type for which no cytogenetic data were hitherto available, numerical chromosomal aberrations dominated, but one of the tumors also carried an unbalanced 7;17-translocation with the same breakpoint in chromosome 17 as that seen in endometrial stromal sarcomas. Received: 15 January 1999 / Accepted: 5 March 1999     

The optimization of sample treatment for spectral karyotyping with applications for human tumour cells

Spectral karyotyping (SKY) represents an important tool for the investigation of the complex chromosomal rearrangements (CCRs) in many human malignancies which may be difficult to characterize by conventional banding techniques. The main goal of our work was to optimize the most important steps in the preparation of molecular cytogenetic slides for a SKY protocol. This approach consisted of optimization of both the aging procedure and protease pretreatment of the slides, with special regard given to the preservation of chromosome structure and shape, as well as to the intensity of hybridization signals. The best results were obtained with a chemical aging procedure using SSC or ethanol in combination with trypsin pretreatment applied at a higher concentration for a shorter period of pretreatment. A resulting protocol for SKY also applicable to human solid tumour cells was subsequently proposed. The practical potential of the SKY technique was demonstrated on examples of two types of human embryonal tumours--neuroblastoma and Wilms' tumour, in which some kinds of chromosomal aberrations were not detectable by means of classic cytogenetic methods.     

Meningioma: a cytogenetic model of a complex benign human tumor, including data on 394 karyotyped cases

Meningioma is the most frequent tumor of neuroectodermal origin in humans. It is usually benign. Only a minority of cases shows progression to an anaplastic tumor (WHO grade II and III). Meningioma is generally a sporadic tumor. Multiple and familial cases are rare and mostly associated with (hereditary) neurofibromatosis 2 (NF2). Meningiomas show an unexpectedly high recurrence rate. Also, completely removed low-grade tumors can recur. Recurrence and multiplicity are correlated with the formation of a peritumoral edema. On the cytogenetic level, meningioma is the best-studied tumor in humans. Grade I tumors show either uniform monosomy 22 or a diploid karyotype. The majority of high-grade, but only a minority of low-grade, meningiomas show loss of merlin, a cytoskeleton-cytoplasm-linker protein. Merlin is the product of the NF2 gene located on chromosome 22. A second tumor suppressor gene on chromosome 22 has not yet been detected. In contrast to other solid tumors, progression of meningiomas is correlated with increasing hypodiploidy, showing characteristic clonal evolutions that mostly include chromosomes 14, 18, and 19 and, more rarely, 6 and 10. Structural aberrations are infrequent, except for the loss of the short arm of chromosome 1, which appears to be the decisive step for anaplastic growth. Comparative histochemical and molecular cytogenetic studies point to the alkaline phosphatase gene (ALPL, liver-bone-kidney type) located on 1p36.1-->p34 as a candidate tumor suppressor gene. A model is proposed that tries to explain - with a minimum number of essential steps - the origin, progression, infiltration, and recurrence of meningiomas.     

Cytogenetic changes in human lymphoma U-937 induced to apoptosis by tumor necrosis factor

The frequency of cytogenetic abnormalities in cell populations of U-937 line in control group and after their exposure to tumor necrosis factor (TNF) was assayed. It has been found that the maximum effect of TNF is observed after 48-h exposure. It was exhibited as apoptosis induction, accumulation of cells with micronuclei and binuclear cells. An increased number of cells with premature chromosome condensation is an early marker of TNF action. Changes in distribution of the cells with various chromosome numbers may lead to the appearance of sublines with properties different from those of the original cell population.     

Flow karyotyping of human melanoma cell lines

Single-laser flow cytometry has been used to study the feasibility of flow karyotyping of human solid tumors. As a model, seven human melanoma cell lines have been used with varying numerical chromosome composition as verified by FCM DNA content measurements and chromosome countings. For all seven cell lines, flow karyotypes that showed a variety of consistent deviations from the normal diploid flow karyotype could be obtained although the resolution of the flow system and varying debris continuum limited the number of resolvable peaks. The predominant changes observed involved the regions normally representing chromosomes 3-8, 9-12, and 13-16. It is concluded that at present the preparation procedure is the main limiting factor for exploring the full potential of flow karyotyping for cytogenetic analysis of solid-tumor cell lines.     

Role of Water in Chromosome Spreading and Swelling Induced by Acetic Acid Treatment: A Ftir Spectroscopy Study

The so called chromosome preparation is a procedure consisting of three strictly connected stages that enables to obtain chromosomes of quality suitable for cytogenetic analysis. Interestingly, experimental evidence strongly suggested that chromosome spreading and swelling (key processes that allow their counting and detailed structural analysis) are induced in the last fixative-evaporation stage by the interaction, mediated by acetic acid, between water from the environmental humidity, and the cytoplasmic matrix and the chromatin. However, since a considerable variation in the quality of chromosome preparations is observed, strongly depending on the environmental conditions in which the procedure takes place, a better comprehension of the mechanisms underlying chromosome preparation is required. To this aim, here we analysed intact lymphocytes before and at each stage of the chromosome preparation protocol by Fourier transform infrared (FTIR) spectroscopy, a technique widely used for the study not only of isolated biomolecules, but also of complex biological systems, such as whole cells. Interestingly, we found that the chromosome preparation protocol induces significant structural changes of cell proteins and DNA, in particular due to the interaction with acetic acid. Moreover, noteworthy, through the monitoring of changes in the water combination band between 2300 and 1800 cm^–1, we provided evidence at molecular level of the crucial role of the bound water to the cytoplasmic matrix and to the chromatin in determining the chromosome spreading and swelling. Our FTIR results, therefore, underline the need to perform the last fixative-evaporation stage in standardized and optimized temperature and relative humidity conditions, thus providing chromosomes of high quality for the cytogenetic analysis that would lead in this way to more reliable results.Key words: Chromosome preparation, chromosome spreading and swelling, DNA conformational transition, FTIR (micro)spectroscopy, protein, DNA hydration     

A specific chromosomal abnormality in rhabdomyosarcoma

A specific chromosomal abnormality, t(2;13)(q35;q14), was discovered in five cases of advanced rhabdomyosarcoma. It was identified directly in cells that had metastasized from bone marrow in one patient and in xenografts derived from the tumors of four other patients. The translocation was not restricted by histologic subtype, but was found in cases classified as alveolar, undifferentiated, or embryonal. Cytogenetic hallmarks of gene amplification (double minute chromosomes and homogeneously staining regions) were apparent in three cases. Other frequent abnormalities included rearrangements of chromosomes lp and trisomy of chromosome 8. The absence of the t(2;13) in more than 100 cases of other pediatric solid tumors investigated in our laboratory indicates its specificity for rhabdomyosarcoma. These cytogenetic findings suggest directions for further investigation of the molecular events underlying the genesis of this tumor.     

Time-saving in biological dosimetry by using the automatic metaphase finder Metafer2.

The amount of time-saving by using the Metafer2 metaphase finder for routine analysis of radiation-induced chromosome aberrations (biological dosimetry) was determined. Metaphases were prepared by standard methods from cultures of human peripheral blood lymphocytes and stained either with Giemsa or with the FPG method. The metaphase finder was used for detecting metaphases on the microscope slides and for automatically processing the evaluation data. In our laboratory, standardized analysis of 1000 metaphases requires at least 3 working days for cell culturing and slide preparation and 51.5 working hours for cytogenetic analysis. When using the metaphase finder the time required for cytogenetic analysis is reduced to 17.3 working hours (time-saving factor: 51.5/17.3 h = 3.0). In our prolonged method, including more than one scoring of each slide and karyotyping of metaphases with chromosome aberrations, the analysis times for 1000 cells are 132 and 70 working hours, respectively (time saving factor: 132/70 h = 1.9).     

A karyotype study of the Vero cell line cultured long term in a monolayer by static and roller methods

A cytogenetic investigation of Vero cells, before and after adaptation to the medium containing a cattle serum, was carried out by methods of differential chromosome staining. Under these conditions, both the modal number of chromosomes (from 58 to 55) and the karyotype structure, namely the copy number of normal chromosomes and the marker composition were shown to change. The Vero cell karyotype stability was studied in the continued culture by the static (50 passages) and roll-bottle (37 passages) methods. The quantitative changes (the rising percentage of diploid cells, and the change of cell fraction involving the modal number of chromosomes) were shown to occur in spite of the chromosome composition stability, which limits the time of using Vero cells as a substrate for preparation of vaccines.     

In vivo cytogenetics: mammalian germ cells

This chapter summarizes the most relevant methodologies available for evaluation of cytogenetic damage induced in vivo in mammalian germ cells. Protocols are provided for the following endpoints: numerical and structural chromosome aberrations in secondary oocytes or first-cleavage zygotes, reciprocal translocations in primary spermatocytes, chromosome counting in secondary spermatocytes, numerical and structural chromosome aberrations, and sister chromatid exchanges (SCE) in spermatogonia, micronuclei in early spermatids, aneuploidy in mature sperm. The significance of each methodology is discussed. The contribution of novel molecular cytogenetic approaches to the detection of chromosome damage in rodent germ cells is also considered.     

Short-term culture of spleen lymphocytes for chromosome analysis of small rodents

In order to improve the methods for the cytogenetic analysis of small rodents with regard to metaphase number, quality of chromosome resolution and duration of cultivation, a short-term culture technique from spleen cells, based on the capacity of Concanavalin A supernatant to stimulate the proliferation of normal T lymphocytes, was developed. Protocols of culture and chromosome preparation are described in detail. This technique not only makes it possible to obtain a high number of cells in mitosis from the spleen of very small rodents in a short time, but also insures metaphases of good quality, due to optimal control of cell cycle and processing thanks to cultivation in small aliquots. Although the method applies mainly to rodents because of species specificity of interleukin 2, it can probably be easily adapted to species from other mammalian orders.     

Protocols for cytogenetic studies of human embryonic stem cells

All cultured cells develop chromosome changes over time, including cultures of human embryonic stem cells (hESC), but only those cells with adaptive chromosomes changes survive. The most frequent chromosome changes in hESC cultures are trisomy 12 and trisomy 17. Cells with these trisomies are indistinguishable from normal cells by appearance and also demonstrate typical markers of pluripotency, making them difficult to identify without cytogenetic analysis. Early detection of these cells is essential since cells with trisomy 12 and 17 can replace the normal cell population in 5-10 passages. Cytogenetic analysis using G-banding is considered to be the gold standard for detecting chromosome abnormalities and, when used in combination with interphase FISH, provides a sensitive method for early detection of cytogenetic aberrations, such as full and partial trisomies of chromosomes 12 and 17. The following discussion describes the cytogenetic methods used in our laboratory to study cultured hESCs, along with recommendations for integrating these methods into a plan for routine cell line quality control.     

宽体沙鳅的染色体核型与DNA含量分析

为了解宽体沙鳅(Sinibotia reevesae)的种质特征,以野生宽体沙鳅为材料,采用腹腔注射植物血球凝集素(PHA)和秋水仙素,肾组织细胞短期培养、常规空气干燥法制备染色体标本,并对其核型进行分析。以鸡(Gallus gallus)血细胞DNA含量(2.50 pg/2c,2c指二倍体)为标准,用流式细胞仪测定宽体沙鳅外周血细胞的DNA含量。结果表明:(1)宽体沙鳅的染色体数目为2n=96,核型组成公式为2n=36m+14sm+20st+26t,染色体总臂数NF=146;未发现与性别相关的异型染色体。(2)宽体沙鳅的DNA含量为(2.60±0.36)pg/2c。通过与其他26种鳅科鱼类核型进行比较,发现宽体沙鳅属于鳅科鱼类中的特化类群,其染色体核型经历了罗伯逊易位和染色体多倍化等过程。本研究结果可为宽体沙鳅种质资源保护和细胞遗传学研究提供基础资料。     

Detection and chromosomal assignment of SV40-DNA integration in Chinese hamster cell lines by chromosome sorting and dot blot hybridization

A combination of cytometric (chromosome sorting), molecular (dot blot hybridization using radio-active and/or biotinylated DNA probes) and cytogenetic (G-banding) evaluation is described which allows the rapid identification of single copy and repetitive viral integrates and their assignment to chromosome groups or even individual chromosomes. In the case of Chinese hamster cell line CO 631 it could be demonstrated that SV40 DNA was solely integrated into a submetacentric marker chromosome. Such a cytometric/molecular/cytogenetic "identogram" may prove to be a useful tool in many areas of cell and tumor biology. Furthermore, amounts of chromosomes sufficient for analysis as well as subsequent cloning experiments can be accumulated.