Comparison of tissue disaggregation techniques of transitional cell bladder carcinomas for flow cytometry and chromosomal analysis

DNA index (DI) measurements and chromosomal analysis of 42 transitional cell carcinomas were done after mechanical and enzymatical disaggregation of the tumor specimens. The results obtained with these different disaggregation techniques were compared in the 33 cases (79%) that showed recognizable chromosomes. The enzymatically obtained cell suspensions could not be used for chromosomal analysis after short-term culture of 24 hours. In four cases, the DI after enzymatical treatment could not be estimated. In most cases, the DI obtained from the tumor cells was similar for both aggregation techniques, with the exception of four cases of enzymatically treated cell suspensions in which the DI could not be estimated. The average DI of the aneuploid tumors was 13% higher than the corresponding chromosome count. In 19% of the aneuploid tumors the proportion of aneuploid cells could not be measured after enzymatical treatment. In the remaining suspensions the proportion of diploid cells was higher after enzymatical disaggregation than after mechanical treatment. It is concluded that for flow cytometric and direct chromosomal analysis of bladder tumors, the mechanical disaggregation technique is most suitable.     

The reliability of microspectrophotometric and flow cytometric nuclear DNA measurements in adenocarcinomas of the breast

The reliability of microspectrophotometric (MSP) and flow cytometric (FCM) nuclear DNA measurements has been studied in 50 human breast adenocarcinomas. The tumor material was obtained by means of fine-needle aspiration biopsy, and all samples except one were found to be highly representative. The results confirm earlier observations that a good correlation exists between modal value (MV) determined by MSP and DNA index (DI) determined by FCM. However, when tumors were classified into low and high malignant variants according to FCM/DI, FCM/S-phase percentages, and MSP histogram types, the concordance was less pronounced. This was found to be due mainly to the fact that in near-diploid tumors a discrepancy exists between MSP and FCM ploidy, as well as between MSP distribution pattern and the estimated percentages of cells in the S-phase region. Another source of discrepancy was observed in tumors with stemlines in the normal tetraploid region, including cells with highly scattered aneuploid DNA values. These tumors were judged by MSP as aneuploid/high malignant and by FCM as euploid/low malignant. In view of this discrepancy, we conclude that the simple determination of the stemline position by MSP/MV or FCM/DI is not sufficient for adequate cytochemical malignancy grading of breast carcinomas. We suggest that a combination of ploidy and percentage of cells scattered outside the modal peaks is a more sensitive method for optimal cytochemical malignancy grading in breast carcinomas.     

Flow cytometric and cytogenetic analyses in human spontaneous abortions

Cytogenetic and flow cytometric analyses were performed on 38 human spontaneous abortions in an attempt to obtain information on karyotype abnormalities and to compare the two approaches of analysis. In 19 cases, it was not possible to perform cytogenetic analysis because too long a time had passed between surgical sampling and cell culture, and in vitro culture failed. Of the 19 cases analyzed, 10/19 showed a normal karyotype and 5/19 showed a single trisomy (2/5 trisomies involved chromosome 16, 1/5 trisomy involved chromosome 18, 1/5 trisomy involved chromosome 20, and 1/5 was Klinefelter syndrome). Of the remaining 4/19 cases, 2/19 showed a polyploid condition (1 tetraploidy and 1 triploidy), 1/19 a double trisomy (chromosomes 13 and 21), and 1/19 a pentasomy of the sex chromosomes (49,XXXXY). Flow cytometric analysis was performed on all abortive samples. The samples were subdivided, when possible, into two portions conventionally named amniotic and chorionic, using the amniotic membrane as an anatomical reference. Maternal blood lymphocytes were used as a diploid standard for each sample. In the 19 cases not analyzed by the cytogenetic approach, flow cytometric analysis showed 9 diploid and 10 aneuploid DNA distributions. In the remaining 19 cases, analyzed with both approaches, the comparison of DNA estimations using cytogenetic and flow cytometric analyses showed good agreement. In the cases with karyotype abnormalities, flow cytometric measurement provided evidence of an alteration of DNA content with respect to the diploid standard. Flow cytometric analysis showed a diploid distribution, whereas cytogenetic analysis revealed chromosomal abnormalities in only 4/19 cases. These discordant results could be related to mosaic conditions or maternal cell contamination. Moreover, cytogenetic and flow cytometric analyses were performed on 2 amniotic cell cultures, and concordant results were obtained. The results obtained suggest that a combination of these techniques is beneficial in attempts to obtain information about DNA content alterations, even when cultures fail, and in screening studies of human abortions.     

Chromosomal variations within aneuploid cancer lines.

Aneuploid cancers exhibit a wide spectrum of clinical aggressiveness, possibly because of varying chromosome compositions. To test this, karyotypes from the diploid CCD-34Lu fibroblast and the aneuploid A549 and SUIT-2 cancer lines underwent fluorescence in situ hybridization (FISH) and DAPI counterstaining. The number of DAPI-stained and FISH-identified chromosomes, 1-22, X,Y, as well as structural abnormalities, were counted and compared using the chi(2), Mann-Whitney rank sum test and the Levene's equality of variance. Virtually all of the evaluable diploid CCD-34Lu karyotypes had 46 chromosomes with two normal-appearing homologues. The aneuploid chromosome numbers per karyotype were highly variable, averaging 62 and 72 for the A549 and SUIT-2 lines, respectively. However, the A549 chromosome numbers were more narrowly distributed than the SUIT-2 karyotype chromosome numbers. Furthermore, 25% of the A549 chromosomes had structural abnormalities compared to only 7% of the SUIT-2 chromosomes. The chromosomal compositions of the aneuploid A549 and SUIT-2 cancer lines are widely divergent, suggesting that diverse genetic alterations, rather than chance, may govern the chromosome makeups of aneuploid cancers.     

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.     

Cytogenetic studies of Haplopappus gracilis in both callus and suspension cell cultures

Summary Investigations have been carried out on karyotype change in both callus and suspension cell cultures of Haplopappus gracilis (2n=4). It has been found that polyploidization arises directly in culture to give up to six times the normal diploid chromosome number in some cultures. In polyploid cultures, both chromosome loss and chromosome rearrangements occur to give rise to aneuploid karyotypes displaying chromosomes which differ in morphology from the diploid set. Whole or partial chromosome loss has been observed in the form of lagging chromosomes and chromosome bridges at anaphase, and micronuclei, ring chromosomes and chromosome fragments at other stages in mitosis. C-banded preparations have confirmed the occurrence of chromosomal rearrangements. Comparative investigations suggest that (i) more polyploidy occurs in callus cultures than in suspension cell cultures, and (ii) the presence of cytokinin (kinetin) in the culture medium may reduce the extent of karyotype change.     

Chromosomal composition of aneuploid clones with different DNA contents in head and neck squamous cell carcinomas as determined by combined flow cytometry and fluorescence in situ hybridization.

Studies with DNA flow cytometry (FCM) have shown that DNA contents of aneuploid tumour clones vary in a wide range. The aim of this study was to analyse whether homologous chromosomal changes exist despite the individual differences that may be of general relevance for the development of gross aneuploidy in squamous cell carcinomas of the head and neck. Fluorescence in situ hybridization (FISH) with 13 centromere-specific DNA probes was applied to 3 diploid and 11 aneuploid tumours with DNA indices ranging between 0.8 and 2.2. Disomic and monosomic cell populations were prevalent findings in DNA-diploid tumours. Polysomies were common in aneuploid tumours. Different degrees of aneusomy for identical chromosomes were recurrent features in aneuploid tumours. FISH signal heterogeneity was identified for all chromosomes. The mean number of aneusomic cell populations identified for DNA-aneuploid tumours ranged between 1.6 for chromosome 17 and 3.1 for chromosome 3. Inconsistencies between FISH and FCM data may indicate that centromere-specific DNA probes identify gains and losses of marker DNA due to complex karyotypic rearrangements rather than absolute changes in chromosome numbers. Overall, there was no evidence of the critical involvement of particular chromosomes in the development of different DNA contents.     

High-resolution flow karyotyping and chromosome sorting in Vicia faba lines with standard and reconstructed karyotypes

Flow cytometric analysis has been performed on chromosomes isolated from formaldehyde-fixed root tips in a Vicia faba (2n = 12) line with a standard (wild-type) karyotype and in six V. faba translocation lines with reconstructed karyotypes. The resolution of individual chromosome types on histograms of chromosome fluorescence intensity (flow karyotypes) depended on the type of fluorochrome used for chromosome staining. The highest degree of resolution was achieved with 4,6-diamidino-2-phenylindole (DAPI). The lower resolution obtained after staining with mithramycin A (MIT) and propidium iodide (PI) was probably due to the sensitivity of these stains to changes in chromatin structure induced by formaldehyde fixation. After the staining with DAPI, only 1 chromosome type could be discriminated in the line with a standard karyotype. In the translocation lines, the number of chromosome types resolved on flow karyotypes ranged from 2 in the G and the ACB lines to all (6) chromosome types in the EFK and EF lines. Refined flow karyotyping permitted the sorting of a total of 15 different chromosome types from five of the translocation lines. It is expected that flow sorting of chromosomes from reconstructed karyotypes will become a powerful tool in the study of nuclear genome organisation in V. faba.     

Relationship between chromosomal changes complexity and disease aggressiveness in myeloid and lymphoid disorders

In this paper are presented four cases, with unusual chromosomal abnormalities, identified at the first presentation, among over 100 patients with myeloid and lymphoid acute and chronic leukemias cytogenetically investigated. The complexity and nature of cytogenetic abnormalities was in direct relationship with the disease evolution. The first case, a 22 years old man with acute lymphoblastic leukemia type L3, exhibited many structural changes in bone marrow cells with diploid number of chromosomes: del(3)(q26); del (5)(p13); t(8;14) (q24;q32); del(9)(p11q11);inv(15)(p12qter). The second case, a 62 years old woman, diagnosed as poorly differentiated acute leukemia, refractory to treatment, showed hiperdiploidy (48–54 chromosomes) and 3–4 markers derived from chromosomes 5 and 12. The third case, a young man of 27 years old, diagnosed as acute myeloid leukemia, apart of Philadelphia chromosome, presented trisomy 16, both in diploid and aneuploid cells. None of these three patients did respond to any medical therapy. Their rapid death was a powerful proof of the correlation between the complexity of genome changes and disease aggressiveness. In the fourth case, a constitutional translocation t(3;5)(q26.3;q21) identified in a 72 years old woman with essential thrombocythemia, appeared not to be involved in the etiology of the disease. In this case, the treatment with hydroxyurea was successful and the disease evolution was favourable. In conclusion, we appreciate that in the three cases of myeloid and lymphoid leukemias it was a direct relationship between the complexity of genomic changes and the aggressiveness of the disease.     

(11; 14) translocation in three boys with acute lymphoblastic leukemia of T-cell immunophenotype

Three boys, 12, 15 and 5 years old are presented with acute lymphoblastic leukemia resp. Non-Hodgkin's lymphoma with leukemic transformation. Blast cells could be characterized as being of T-cell origin. Hand mirror variant was the predominant morphologic feature of the blast cells in two patients. Chromosome analysis of the leukemic blast cells revealed a pseudodiploid (modal chromosome number = 46) karyotype in two patients and a pseudotetraploid (modal chromosome number = 92) in one patient. A chromosome translocation (11; 14) with breakpoints at (p 13; q 13) (within the human T-cell receptor alpha chain locus!) was found in the leukemic cells of all three cases plus an additional t (7; 9) (q 22; p 13) in one patient.     

Karyology and karyotype analysis of diploid freshwater planarian populations of the Dugesia gonocephala group (Platyhelminthes,Tricladida) found in Sardinia

Karyology and karyotype analysis were carried out on freshwater planarian populations of the Dugesia gonocephala group. The strains studied were all diploid with chromosomic number 2n = 16; n = 8. They came from 12 sites mainly localized on the west of the island of Sardinia. Three karyotypes indicated with the letters A, B and C were found in which eight homomorphic pairs of chromosomes were easily identified. In karyotype A all chromosomes are metacentric. Ten populations of the twelve examined showed this karyotype which appears to be the most common. In karyotype B the seventh pair of chromosomes is submetacentric. This karyotype is quite common having been previously found in another eight Sardinian localities. Karyotype C differs from the others in having submetacentric third and seventh pairs of the chromosome complement. It was found in only one locality. The differences observed between these three karyotypes could be interpreted either as sign of differentiation at species level, or as an intraspecific variation due to chromosome mutations (pericentric inversions). This revised version was published online in July 2006 with corrections to the Cover Date.     

Establishment of the AU-bek cell line and comparison with two other bovine cell lines

Summary Establishment of a new bovine cell line, AU-BEK, is reported. The cell line developed in a culture initiated from bovine embryonic kidneys by spontaneous cultural alteration to epithelioid cells that are indefinitely propagable. Epithelioid cells gradually increased to become the predominant cell. Whereas normal bovine cells have a diploid number of 60 chromosomes, of which only the two sex chromosomes are biarmed, AU-BEK cells at the 80th passage had a modal chromosome number of 84 and an average of 30 biarmed chromosomes per cell. AU-BEK cells are now in their 220th passage. Of the AU-BEK, MDBK, and CKT-1 bovine cell lines, the CKT-1 cell line had a karyotype closest to that of normal bovine cells. Their modal chromosome number was 57, and only three biarmed chromosomes were usually present. The bovine character of AU-BEK and CKT-1 cells was established by cytotoxic and viral susceptibility tests. Supported by the Alabama Agricultural Experiment Station. Publication No. 1115, School of Veterinary Medicine, Auburn University.     

The effects of growth in vitro on the chromosome complement of Daucus carota (L.) suspension cultures

The chromosome number distributions and modal karyotypes of several suspension culture lines of Daucus carota L. have been analysed at various times after initiation. All lines had stable modal chromosome numbers and karyotypes, with small but significant variation about the modes. Some lines showed a predominance of diploid cells with a karyotype similar to the plant. Polyploid multiples of the modal chromosome number were present in all lines at low frequency. Variation of the 2,4-D concentration in the culture medium produced little alteration of the chromosome number distributions, but omission of 2,4-D produced a significant drop in the frequency of multipolar mitoses in those culture lines in which this treatment induced differentiation. There was no evidence of any direct effect of 2,4-D on general mitotic dynamics. Alteration of the frequency with which cultures were transferred to fresh medium showed that stationary phase was critical in the maintenance of the low frequency of tetraploids present in a predominantly diploid culture line. The results are explicable in terms of a competitive selection for cells with the dominant modal chromosome number in the presence of various mechanisms continuously producing polyploid, aneuploid and structurally altered karyotypes.     

Characterizations of the murine mesenchymal cell line expressing GFP

We established and characterized a murine mesenchymal stem cell line from the bone marrow of a transgenic C57BL mouse that ubiquitously expressed green fluorescent protein (GFP). Immunostaining revealed the presence of several markers common for mesenchymal stem cells (MSCs). The cells expressed specific fibroblast proteins, such as smooth muscle actin, which is localized in stress fibrils, and vimentin, a major protein of intermediate filaments in connective tissue cells. These proteins are responsible for the ability to differentiate into adipocytes or osteoblasts under appropriate conditions. The MSC karyotype was unstable. At the 6th passage cells, were aneuploid and genetically heterogeneous. The number of chromosomes ranged from near 2n to 8n. 80% of cells had chromosome numbers between 50 and 85 without a well-defined modal class. Differential G-staining of metaphase spreads showed variability in the copy numbers of individual chromosomes and presence of random chromosome rearrangements, such as ectopic associations of nonhomologous chromosomes. All cells analyzed contained a single dicentric marker chromosome. Some cells also had mini-chromosomes regarded as indicators of gene amplification. We suppose that the karyotypic instability of MSCs that express GFP is provoked by the insertion of foreign GFP transgenes into the murine genome. These cells could be useful for the study of genomic alterations during the spontaneous oncogenic transformation of stem cells.     

Karyotypic determinants of chromosome instability in aneuploid budding yeast

Recent studies in cancer cells and budding yeast demonstrated that aneuploidy, the state of having abnormal chromosome numbers, correlates with elevated chromosome instability (CIN), i.e. the propensity of gaining and losing chromosomes at a high frequency. Here we have investigated ploidy- and chromosome-specific determinants underlying aneuploidy-induced CIN by observing karyotype dynamics in fully isogenic aneuploid yeast strains with ploidies between 1N and 2N obtained through a random meiotic process. The aneuploid strains exhibited various levels of whole-chromosome instability (i.e. chromosome gains and losses). CIN correlates with cellular ploidy in an unexpected way: cells with a chromosomal content close to the haploid state are significantly more stable than cells displaying an apparent ploidy between 1.5 and 2N. We propose that the capacity for accurate chromosome segregation by the mitotic system does not scale continuously with an increasing number of chromosomes, but may occur via discrete steps each time a full set of chromosomes is added to the genome. On top of such general ploidy-related effect, CIN is also associated with the presence of specific aneuploid chromosomes as well as dosage imbalance between specific chromosome pairs. Our findings potentially help reconcile the divide between gene-centric versus genome-centric theories in cancer evolution.     

同源四倍体青花菜的核型分析

以四倍体青花菜为材料,采用常规压片法进行核型分析和有丝分裂观察.结果表明:四倍体青花菜核型公式为2n=4x=36=16m+20sm(4 SAT),其中第3、4、7、8对为中着丝粒染色体,第1、2、5、6、9对为近中着丝粒染色体,第6对染色体具随体;核型类型属于2A型,为基本对称型;染色体相对长度组成为2n=36=16 M_2+20 M_1,表明该四倍体青花菜是二倍体加倍得到,为同源四倍体.在部分四倍体根尖中发现非整倍体细胞,其染色体数目变异较大;与二倍体相比,四倍体有丝分裂过程存在双核仁、体细胞配对、染色体桥等异常现象.     

Indirect mitotic nondisjunction in Vicia faba and Chinese hamster cells

The hypothesis of indirect mitotic nondisjunction was tested in plant and mammalian cells. This hypothesis states that micronuclei derived from lagging chromosomes or chromatids are able to perform DNA synthesis and undergo mitotic condensation synchronously with main nuclei. Hence, as chromosomes, they can be moved to spindle poles together with the chromosomes of the main nuclei during mitosis. In that way chromosomes lost as micro-nuclei can be reincorporated in the main nuclei. In order to test this, both Vicia faba meristematic cells and cells of a Chinese hamster line (Cl-1) were treated with low doses of colchicine. Mitotic anomalies, micronuclei and cells with a polyploid or aneuploid karyotype were scored at different fixation times. A detailed analysis was performed on single chromosome misdistributions, as well as on micronuclei and cells with aneuploid karyotypes derived from single chromosome misdistributions. Indirect mitotic nondisjunction was shown to play a primary role in the origin of aneuploid karyotypes in Vicia faba, but not in Cl-1 cells.     

广东四个墨兰品种的核型研究

研究了广东四个墨兰品种金嘴墨兰、银边墨兰、企剑黑墨和企剑白墨的染色体数目和核型。结果表明,企剑黑墨和企剑白墨的染色体数目为2n=40,为二倍体,核型公式分别为：2n=2x=40=30m＋10sm和2n=2x=40=2M＋36m＋2st;金嘴墨兰和银边睾兰的染色体数目为2n=41,为非整倍体。4个墨兰品种的染色体结构主要由中部着丝粒染色体组成,除银边墨兰为1B型外,其它均为2B型。     

Cytogenetic characterization of the Zebra Mussel Dreissena polymorpha (Pallas) from Miedwie Lake, Poland

Cytogenetic characterization of D. polymorpha was carried out using banding techniques such as C-banding, fluorochrome CMA3 and silver nitrate treatment. The diploid chromosome number of both investigated D. polymorpha forms (typical and albinotic) was the same 2n = 32 (NF = 56). The karyotype consisted of 5 pairs of metacentric, 7 pairs of submetacentric and four pairs of subtelo-acrocentric chromosomes. Ag-NORs were located in the telomeric position on the largest subtelo-acrocentric chromosome pair. C banding patterns indicate many sites of constitutive heterochromatin mainly located in the telomeric regions and interstitially in some chromosomes. CMA3-sites were observed in almost all chromosomes; apart from the Ag-NORs sites, they were located terminally on the chromosome arms and interstitially on three chromosome pairs. Sixteen chromosomes could be counted at the diakinesis stage of meiosis. No differences in banding chromosome patterns were found neither between both analyzed forms of D. polymorpha nor between males and females.     

The cytogenetics of acute leukemia

Methodological prerequisites for the culture of leukaemic blasts and the fluorescence banding of spread metaphases are represented in detail. By using an improved method the percentage of aneuploid karyotype findings increased in AML from 49% to 68% and ALL from 65% to 81% of all evaluable patients. Specific aberrations known up to the present day are discussed. They could be diagnosed in 46% of aneuploid AML-clones and in 59% of ALL-clones. The fluorescence banding according to the three-colour technique is demonstrated by three examples of "non-specific" aberrations: a) Tandem translocation of 1q and 7 q (64a, male, FAB:M5B), b) Karyotype instability with variability of chromosome numbers arising from trisomy, tetrasomy and polyploidisation of single normal as well as marker chromosomes (57a, female, FAB:M6), c) Identification of a marker chromosome as a derivative chromosome 16 (33a, female, FAB:L2, c-ALL).