Chromosomal rearrangements and their role in spontaneous immortalization and transformation of rat embryo cells in vitro

Peculiarities of chromosomal rearrangements were studied in cells of the spontaneously immortalized LRec-1 and LRec-3 lines derived from rat embryo fibroblasts, as well as in LRec-1k clone cells and LRec-1sf line cells with autocrine regulation of proliferation at various cell transformation stages. The lines were obtained from rat embryo fibroblasts by cloning during rapid aging of the cultures. Using the G-banding of chromosomes, it was shown that in the process of transformation, cells of the LRec-1 and LRec-3 lines as well as of LRec-1sf maintained diploidy and specific clonal rearrangements of chromosomes 7 and 19, which were revealed earlier at the immortalization stage. In the LRec-1 cells, new clonal rearrangements of chromosomes 10 and 20 were observed, while rearrangements of chromosomes 1, 2, 11, 15, 18, and 19 were observed in the LRec-1sf cells. In the LRec-3 cells, as well as in cells of the LRec-1k clone, new chromosome rearrangements were absent. Loci involved in chromosomal rearrangements were compared with the genes located in them according to RATMAP data. The role of rearrangements of chromosomes 7 and 19 in the immortalization and malignant transformation of embryo fibroblasts is discussed, as well as the roles of other chromosomes during acquisition of the specific signs of the transformed phenotype by the LRec-1 and LRec-1sf cells.     

Characteristics of the spontaneously transformed human endothelial cell line ECV304. I. Multiple chromosomal rearrangements in endothelial cells ECV304

Karyotype of endothelial line ECV304 cells obtained from human umbilicus vein endothelial cells was studied using G-banding chromosome staining. It has been revealed that the cells have a polyploidy karyotype with 96-112 chromosomes and multiple numerical and structural clonal rearrangements. Almost all the chromosomes of the karyotype are involved in structural rearrangements. There are several double chromosome rearrangements revealed including del(9)(p21) as well as two derivatives of chromosome 3 with the breakpoint in the locus p25 - der(3)t(3;12)(3p25;12q11- 12q24.?1) and der(3)t(3;?)(3p25). The role of these rearrangements in the immortalization of endothelial cells and sighs of transformation are discussed. In connection with the information received about the fact that the cells of ECV304 line are not endothelial cells but T24, urinary bladder cancer cells (which karyotype was studied by Hurst et al., 2000), the comparative analysis of the karyotypes of these two lines was carried out. It has been revealed that these two lines differ by all cytogenetic characteristics. Neither identical structural chromosomal rearrangements nor cell characteristic of urinary bladder cancer cells were detected. Our line ECV304 is not identical to the line T24.     

Characterization of the spontaneously transformed human endothelial cell line ECV304: 1. Multiple chromosomal rearrangements in ECV304 cells

Using differential G-staining of chromosomes, the karyotype of the endothelial cell line ECV304 obtained from endotheliocytes of the human umbilical vein was studied. The cells have been shown to have a polyploid karyotype with a number of chromosomes ranging from 96 to 112, as well as multiple numerical and structural clonal chromosome abnormalities. The structural rearrangements involve almost all chromosomes of the karyotype. Several paired chromosomal rearrangements have been revealed and include del(9)(p21), as well as two derivates of chromosome 3 with a breakpoint at the p25 locus, i.e., der(3)t(3;12)(3p25;12q11～12;12q21～24.?1) and der(3)t(3;?)(3p25). The role of these rearrangements in the immortalization of endotheliocytes and in angiogenesis is discussed. A comparison of the karyotypes of the cell line ECV304 and of the bladder carcinoma T24 cell line has shown that these karyotypes differ in all of the main cytogenetic characteristics. No identical structural chromosomal rearrangements, nor rearrangements characteristic of bladder carcinoma cells have been revealed. The studied endothelial cell line ECV304 is not identical to the T24 cell line.     

New lines of spontaneously transformed cells obtained from "precrisis" cultures of embryonic rat cells

A new approach to selection of lines of spontaneously transformed cells from the rat embryo "precrisis" cultures is described and their phenotypes at the initial and advanced stages during a long-term cultivation are characterized. The new selective system, referred to as 2T7, differs from the well known 3T3, 2T6 and 3T12 systems (Todaro, Green, 1963; Aaronson, Todaro, 1968). It is based on the maintenance of cultures under maximum cell densities. Such an approach facilitated and accelerated the start of the "crisis" stage (up to 3-8 passages) with the following gradual death of almost the whole normal senescent cell population, the colony formation resulting from the proliferation of single clonogenic cells. The frequency of clonogenic cells was about 6 x 10(-6). Six lines of spontaneously transformed cells from embryos of noninbred white rats (LRec-1--LRec-6) and one line from the Wistar embryos (LRec-7) were established. All the lines are characterized as diploid or near-tetraploid, with 1-4 different marker chromosomes formed from chromosome 7, as was reported elsewhere (Artsybasheva et al., 1988). The values of saturation densities and the time of population doubling for all the 7 lines differed from those for the rat embryo primary cultures cells. LRec-1--LRec-6 cells were unable to form the colonies in soft agar, while LRec-7 cells were able to grow in agar. The lines LRec became oncogenic for 1-2 day old rats after different periods of cultivation in vitro--from 3 to 7 months. The line LRec-7 Wistar appeared to be highly oncogenic from the very beginning after its selection. The histological analysis revealed that the LRec-1 tumors could be classified as polymorphocellular sarcoma. Up to 20 passages the LRec-1 line had numerous clonogenic cells (50-60%) in sparse cultures independently on the serum content in the media. By a 3-step selection of LRec-1 cells, on cultivation in media with lower serum contents (1-0.1-0%), a semisuspension of LRec-1sf subline (serum free) was established. This line was highly oncogenic for 1-2 day old rats, was easily cryopreserved and proliferated in the serum-free media for unlimited time, forming small colonies in agar. Thus, the new approach allows to establish with high effectiveness spontaneous lines of rat embryo cells with differently transformed phenotypes, i.e. preneoplastic and oncogenic ones.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phenomenon of evolution of clonal chromosomal abnormalities in childhood acute myeloid leukemia

An analysis of chromosomal abnormalities in bone-marrow cells was performed in 116 children with diagnoses of acute myeloid leukemia (AML). The frequency of the evolution of clonal chromosomal abnormalities in AML constituted 42.3%. Quantitative abnormalities of chromosomes 8, 9, and 21, as well as the secondary structural abnormalities in the chromosomal regions 12p12, 9p22, 9q22, 9q34, 11q14–23, and 6q2, were the most abundant. Quantitative abnormalities were registered in 26.7% cases. The basic mechanism of evolution of the leukemic clone contained trisomy, deletions, and monosomy. The frequency of evolution was seven times higher in the age group of up to 2 years and twofold higher in the age group of up to 5 years. The high frequency of evolution at t(15;17)(q22;q22) was established, while its absence was revealed at inv(16)(p13q22). Patients with clonal evolution were characterized by the increased frequency of relapses and earlier death before reaching remission, which might be explained by the severe initial state of those patients. The conception of abnormalities in the evolution of the clone was proposed to occur at certain stages as follows: (1) appearance of balanced rearrangements; (2) trisomy occurrence; (3) loss of chromosomal material. The occurrence of an unbalanced genome during evolution possesses advantages in the clonal proliferate activity and may be related to its response to chemotherapy. An identity in abnormal chromosomal structure was revealed as a result of the comparison of karyotypes during diagnostics and during relapse, which could be evidence of the initial induction of some types of evolution of chromosomal abnormalities in leukemic cells in AML children by the chemical agents.     

Three major cytogenetic subgroups can be identified among chromosomally abnormal solitary lipomas

Summary We have investigated cytogenetically a total of 35 solitary lipomas, 10 of which have been reported previously. Of the 25 tumours presented herein for the first time, clonal chromosome aberrations were detected in 17. The remaining eight had normal karyotypes, although two of them had nonclonal aberrations in about one quarter of the cells. Based on the cytogenetic findings in all 35 lipomas, four major subgroups can be distinguished. These are characterized by: (I) hyperdiploid karyotypes including one or more supernumerary ring chromosomes (5 cases); (II) diploid karyotypes with mostly balanced rearrangements involving 12q13-14 (13 cases), including the rearrangement t(3;12) (q27-28;q13-14) in 4 cases; (III) hypodiploid or diploid karyotypes with other aberrations than ring chromosomes or rearrangements of 12q13-14 (8 cases); and (IV) normal karyotypes (9 cases).     

Cytogenetic and molecular cytogenetic investigations in relapse of B-cell chronic lymphocytic leukemia

Сhromosomal abnormalities have been analyzed in bone marrow cells of 61 patients with relapse of B-cell chronic lymphocytic leukemia. The cytogenetic results have allowed the structural stratification of the obtained karyotypes into ten groups of clones: normal, normal/near tetraploid, abnormal/normal, abnormal/ near tetraploid/normal, evolution of clonal chromosome abnormalities; evolution of clonal chromosome abnormalities/normal, evolution of clonal chromosome abnormalities/near tetraploid/normal, independent clones, independent/normal clones; and independent/near tetraploid/normal clones. The identified structural rearrangements included translocations, deletions, insertions, and duplications; however, deletions with the involvement of bands 17p12, 13q12–q14, 11q14, and 11q23 dominated (63.8%). The application of i-FISH helped to show the presence of one to four abnormalities per karyotype. The identified cytogenetic and molecular cytogenetic rearrangements may signify a multilevel nature of the process underlying the development of resistant karyotypes. The results obtained under both methods have revealed the presence of a heterogenic cell population with possibly different levels of chemotherapy resistance.     

Double partial trisomy of 6p23-pter and 9pter-q21.2 in a neonate resulting from 4:2 meiotic segregation of a maternal complex t(6;7;9)(p23;p15;q21.2) translocation

We report, a newborn presenting multiple congenital abnormalities with karyotype; 47,XY,der(7)t(6;7)(pter-p23::p15-->qter),+der(9)t(7;9)(pter-->p15::q21.2--> pter)t(6;7;9)(p23;p15;q21.2)mat[20]. The mother and her phenotypically normal daughter were carriers of a complex chromosomal rearrangement with karyotypes; 46,XX,t(6;7;9)(p23;p15;q21.2)[20]. Paternal chromosomes were normal. In our case the extra derivative chromosome was the result of a 4:2 segregation of the chromosomes involved in translocation during oogenesis. Double partial trisomy in newborns resulting from 4:2 segregation is a rare event, and double partial trisomies of the 6p23-pter and trisomy 9pter-q22 regions have not reported to date.     

Clonal evolution through loss of chromosomes and subsequent polyploidization in chondrosarcoma

Near-haploid chromosome numbers have been found in less than 1% of cytogenetically reported tumors, but seem to be more common in certain neoplasms including the malignant cartilage-producing tumor chondrosarcoma. By a literature survey of published karyotypes from chondrosarcomas we could confirm that loss of chromosomes resulting in hyperhaploid-hypodiploid cells is common and that these cells may polyploidize. Sixteen chondrosarcomas were investigated by single nucleotide polymorphism (SNP) array and the majority displayed SNP patterns indicative of a hyperhaploid-hypodiploid origin, with or without subsequent polyploidization. Except for chromosomes 5, 7, 19, 20 and 21, autosomal loss of heterozygosity was commonly found, resulting from chromosome loss and subsequent duplication of monosomic chromosomes giving rise to uniparental disomy. Additional gains, losses and rearrangements of genetic material, and even repeated rounds of polyploidization, may affect chondrosarcoma cells resulting in highly complex karyotypes. Loss of chromosomes and subsequent polyploidization was not restricted to a particular chondrosarcoma subtype and, although commonly found in chondrosarcoma, binucleated cells did not seem to be involved in these events.     

Growth of large chromosomally abnormal T cell clones in ataxia telangiectasia patients is associated with translocation at 14q11

Summary Human T cell malignancies often show chromosome breaks at 14q11, within the chain locus of the human T cell antigen receptor, with translocation of the distal portion of 14 to one of several sites. In patients with ataxia telangiectasia (A-T) the majority of T cell chromosome translocations associated with this disorder appear to occur at the sites of the T cell antigen receptor genes 7p14, 7q35, and 14q11 and may result in clone formation. In three large proliferating A-T T cell clones we have observed (including one which became malignant) and in most T cell tumours reported, the clonal chromosome exchange involves one breakpoint at 14q11 with the second breakpoint occurring in a gene not involved in the immunoglobulin supergene family. Our observations on A-T patients confirm the suggestion that chromosome exchanges involving either t(7;14)(p14;q11), t(7;14)(q35;q11), inv(7) (p14q35), or t(7;7)(p14;q35) confer only a small proliferative advantage on T cells in vivo without the capacity for malignant transformation and that the potential for malignant change is not a feature of all these rearrangements, but is restricted to cells or clones with other chromosome exchanges.     

New recurrent chromosome alterations in patients with multiple myeloma and plasma cell leukemia

Chromosome abnormalities detected in metaphases from multiple myeloma (MM) cells have a clear impact on prognosis and response to therapy. Thirteen out of 50 (26%) patients with plasma cell disorders and abnormal karyotypes (11 with MM and 2 with plasma cell leukemia (PCL)) were selected for inclusion in the present report based on the presence of karyotypes with new and/or infrequent structural aberrations. Thirty-three new rearrangements, including a novel recurrent aberration: psu dic(5;1)(q35;q10), were detected. Chromosome 1 was the most frequently involved. Gains of genetic material (57%) were noted more frequently than losses (43%). Three rearrangements that were observed only once in the literature appear to be recurrent from our data: del(16)(q13), del(5)(p13) and i(3)(q10), the latter being a single structural aberration in the karyotype. Clinical parameters from our series were compared with 2 control groups: 20 MM cases with recurrent aberrations in MM/PCL with a similar distribution of abnormalities associated with poor prognosis (group 1), and 40 with normal karyotypes and fluorescence in situ hybridization analysis (group 2). Significantly increased serum calcium levels (p = 0.022) in patients with new and/or infrequent chromosome changes with respect to both control groups, and a higher percentage of bone marrow plasma cell infiltration (p = 0.005), β(2) microglobulin, and lactate dehydrogenase levels (p < 0.0001) compared to group 2 were observed. Our results suggest that some of these novel rearrangements may be capable to deregulate genetic mechanisms related to the development and/or progression of the disease. The finding of new recurrent aberrations supports this hypothesis.     

Discrepancies in cytogenetic results between different tissues in two fetuses with Wolf- Hirschhorn syndrome

Discrepant unbalanced structural chromosome aberrations between placental and fetal tissue, both involving the short arm of chromosome 4, were found in two human fetuses affected with Wolf-Hirschhorn syndrome. In the first instance, placental chromosome examination revealed a del(4) (p14), whereas fetal fibroblast chromosomes showed an unbalanced der(4)t(4;13)(p14;q11) translocation. In the second instance, placental karyotyping revealed a 4p+ chromosome, while amniocytes showed a submicroscopic deletion at 4p16.3. Since confirmation of structural aberrations from placental tissue is mostly not sought if the phenotype of the fetus is abnor- mal, discrepancies between karyotypes obtained from placental tissue and amniocytes or fetal tissues might be more frequent than the rare reports published so far would suggest. It is unclear whether the simple deletion or the more complex rearrangement is the primary aberration from which the other derived. Structural chromosome aberrations often have a much more complex mechanism of formation than the end product would suggest, and secondary rearrangements of a given aberration in the zygote are more frequent than expected.     

Report on the Eleventh International Workshop on the Identification of Transcribed Sequences 2001. November 9-11, 2001. Washington, DC, USA

Glioblastoma multiforme (GBM) is characterized by intratumoral heterogeneity as to both histomorphology and genetic changes, displaying a wide variety of numerical chromosome aberrations the most common of which are monosomy 10 and trisomy 7. Moreover, GBM in vitro are known to have variable karyotypes within a given tumor cell culture leading to rapid karyotype evolution through a high incidence of secondary numerical chromosome aberrations. The aim of our study was to investigate to what extent this mitotic instability of glioblastoma cells is also present in vivo. We assessed the spatial distribution patterns of numerical chromosome aberrations in vivo in a series of 24 GBM using two-color in situ hybridization for chromosomes 7/10, 8/17, and 12/18 on consecutive 6-microm paraffin-embedded tissue slides. The chromosome aberration patterns were compared with the histomorphology of the investigated tumor assessed from a consecutive HE-stained section, and with the in vitro karyotype of cell cultures established from the tumors. All investigated chromosomes showed mitotic instability, i.e., numerical aberrations within significant amounts of tumor cells in a scattered distribution through the tumor tissue. As to chromosomes 10 and 17, only monosomy occurred, as to chromosome 7 only trisomy/polysomy, apparently as a result of selection in favor of the respective aberration. Conversely, chromosomes 8, 12, and 18 displayed scattered patterns of monosomy as well as trisomy within a given tumor reflecting a high mitotic error rate without selective effects. The karyotypes of the tumor cell cultures showed less variability of numerical aberrations apparently due to clonal adaptation to in vitro conditions. We conclude that glioblastoma cells in vivo are characterized by an extensive tendency to mitotic errors. The resulting clonal diversity of chromosomally aberrant cells may be an important biological constituent of the well-known ability of glioblastomas to preserve viable tumor cell clones under adaptive stress in vivo, in clinical terms to rapidly recur after antitumoral therapy including radio- or chemotherapy.     

Complete or partial trisomy for the long arm of chromosome 1 in patients with various hematologic malignancies

This study contains data obtained from a cytogenetic investigation of six patients with acute and chronic leukaemia. The karyotypes of bone marrow or blood cells of these patients showed a partial or complete trisomy for the long arm of chromosome 1. Three observations revealed a pronounced resistance of cell clones with 1q+ towards cytostatic therapy, and a comparatively short life span of patients after detection of 1q+. The importance of these changes for the role of some chromosomes and chromosome loci in leukaemogenesis is discussed.     

Karyotypic rearrangements in 20 uterine leiomyomas

Short-term cultures from 106 uterine leiomyomas have been cytogenetically investigated. In 29 cases the number of metaphases was insufficient for analysis. A normal female karyotype was found in 57 tumors and clonal chromosome rearrangements in 20. A reciprocal translocation, t(12;14) (q14----q15;q23----q24), was observed in 10 tumors and probably represents a primary change of tumorigenic importance. In four of the tumors containing this specific anomaly, secondary chromosome changes were also present. The 10 karyotypically abnormal leiomyomas without a t(12;14) had various structural and numerical aberrations involving chromosomes 1, 2, 3, 4, 6, 8, 9, 10, 11, 12, 13, and 19. Different structural changes of chromosome 1 were the second most frequent abnormalities, being found in five tumors. Ring chromosomes were observed in three cases, but never as the sole change.     

Pericentric inversions of chromosome 12 in two families

Summary Two cases of pericentric inversion of chromosome 12 are presented, one 46,XX,inv(12)(p13;q11) and the other 47,XX,+21,inv(12)(p13;q13). In both cases one of the parents was also a heterozygotic carrier of the inversion. These inversions were detected among 4035 cytogenetic analyses carried out in patients with psychosomatic retardation and/or malformations (357 with a Down phenotype) and in patients with histories of miscarriages, sterility, or growth failure.In cases studied from a review of the literature together with our own we found that among 3235 cases of Down syndrome there were 7 patients with trisomy 21 and inherited balanced reciprocal translocation involving chromosomes other than pair 21. The frequent participation of some chromosomes in these balanced reciprocal translocations, above all those of group A (1–3), suggests that these and probably other rearrangements could make the segregation of chromosome 21 easier.     

Partial trisomy of the distal part of 10q: a report of two Egyptian cases

Partial trisomy of the distal third of the long arm of chromosome 10 is a well defined but rare syndrome. Most cases result from an unbalanced translocation. Growth retardation, developmental delay and characteristic dysmorphic features are well described in the syndrome. This report includes 2 Egyptian cases with partial 10q trisomy involving different breakpoints. Cases were subjected to full clinical examination and detailed cytogenetic analysis using conventional and FISH studies. Results showed that the karyotype of case 1 was 46,XX,der(7)t(7;10)(p22;q23).ish(wcp7+;wcpl0+) and the karyotype of case 2 was 46,XX,der(7)t(7;10)(p22;q25).ish(wcp7+;wcp 10+). The chromosomal abnormalities in case 1 resulted from a paternal balanced translocation while case 2 resulted from a maternal balanced translocation involving chromosomes 10 and 7 in both cases. The probands' phenotypes were correlated to the breakpoints and compared to previously reported cases with partial trisomy 10q. Both cases had the well characterized phenotype of the distal trisomy of 10q in the form of mental retardation, microcephaly, characteristic dysmorphic facies and limb anomalies as trisomy in both cases involved the 10q25-->qter region. However, case 1 with 10q23-->qter duplication showed more severe clinical manifestations than case 2 with less extensive 10q25-->qter trisomy. These included severe failure to thrive, cardiac involvement and death from respiratory and heart failure. This study confirmed that unbalanced chromosome regions of the long arm of chromosome 10 play an important role in developmental malformations and that a more severe form is associated with involvement of 10q23. It also emphasizes the importance of increasing public awareness regarding these chromosomal rearrangements and the importance of genetic counseling and prenatal diagnosis to avoid recurrences and associated family stress. This was clearly demonstrated in the second family in this study as the couple refused any follow up or further investigations due to religious beliefs despite their social and educational level.     

Additional chromosome in a child as a result of a balanced reciprocal translocation t(12;18)(p13;q12) in his mother's karyotype

In this case report we present a child with an additional chromosome in the karyotype. The karyotypes of the boy and his parents were analyzed by use of a conventional banding technique (GTG) and fluorescence in situ hybridization (FISH). Probes painting whole chromosomes 12 and 18 were used in FISH. Cytogenetic examination of the parents revealed that his mother was carrying balanced reciprocal translocation between chromosomes 12 and 18. Her karyotype was described as 46,XX,t(12;18)(p13;q12). Father's karyotype was normal, described as 46,XY. The boy's karyotype was defined as 47,XY,+der(18)t(12;18)(p13;q12). The additional chromosome appeared probably due to 3:1 meiotic disjunction of the maternal balanced translocation, known as tertiary trisomy. The mother displayed a normal phenotype and delivered earlier a healthy child. However, the boy with the unbalanced karyotype shows multiple congenital abnormalities.     

Cytogenetic study of spontaneous lympholeukemia in AKR mice in the process of its transplantation to an isogeneic line of animals]

Spontaneous leucosis was cytogenetically studied in subsequent generations (from 1 to 150) of AKR mice. By means of the differential staining technique of chromosomes, large variations in chromosome numbers were found in the karyotypes of leucotic cells of different generations, and the formation of cell clones containing different marker chromosomes as well as the dominance of a hyperdiploid clone with 41-42 chromosomes was revealed. Chromosome analysis of such hyperdiploid cells of the 150th generation has indicated that the supernumerary chromosomes (in 88.0% of cases examined) belong to the smallest chromosomes of the mouse karyogramm (to the 18-19th chromosome pairs or to chromosomes smaller than those of 19th pair). Similar trisomy was also observed in hypodiploid and pseudodiploid leucosis cells. It is suggested that the cell clone with trisomy for the smallest chromosomes is specific to the spontaneous lympholeucosis in AKR mice as well as to the leucosis transplanted to isogenic mice for a number of subsequent generations. Increased rate of hyperdiploid cells was associated with a generalization of leucosis. It was concluded that the development rate and the severity of transplanted lympholeucosis in AKR mice was determined by the domination of the cell clone with trisomy for the 18-19th chromosome pairs in the population of leucotic cells.