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Alport syndrome with intellectual disability (ATS-ID, AMME complex; OMIM #300194) is an X-linked contiguous gene deletion syndrome associated with an Xq22.3 locus mainly characterized by hematuria, renal failure, hearing loss/deafness, neurodevelopmental disorder (NDD), midface retrusion, and elliptocytosis. It is thought that ATS-ID is caused by the loss of function of COL4A5 ( ATS ) and FACL4 (ACSL4) genes through the interstitial (micro)deletion of chromosomal band Xq22.3. We report detailed phenotypic description and results from genome-wide screening of a Czech family with diagnosis ATS-ID (proband, maternal uncle, and two female carriers). Female carriers showed mild clinical features of microscopic hematuria only, while affected males displayed several novel clinical features associated with ATS-ID. Utilization of whole-exome sequencing discovered the presence of approximately 3 Mb of deletion in the Xq23 area, which affected 19 genes from TSC22D3 to CHRDL1. We compared the clinical phenotype with previously reported three ATS-ID families worldwide and correlated their clinical manifestations with the incidence of genes in both telomeric and centromeric regions of the deleted chromosomal area. In addition to previously described phenotypes associated with aberrations in AMMECR1 and FACL4 , we identified two genes, members of tripartite motif family MID2 and subunit of the proteasome PA700/19S complex ( PSMD10 ), respectively, as prime candidate genes responsible for additional clinical features observed in our patients with ATS-ID. Overall, our findings further improve the knowledge about the clinical impact of Xq23 deletions and bring novel information about phenotype/genotype association of this chromosomal aberration., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Smetana, Vallova, Wayhelova, Hladilkova, Filkova, Horinova, Broz, Mikulasova, Gaillyova and Kuglík.)

Aging is an extremely complex phenomenon that has many manifestations at the molecular, cellular, and whole-body levels, and in some form involves virtually all living beings. It is a process characterized by a general progressive deterioration of the physiological functions of the organism leading to increased susceptibility to diseases. This article summarizes the basic features and molecular hallmarks of aging and describes some of the genetic mechanisms of this phenomenon. It deals with the particular genes and molecular pathways involved in the regulation of aging as well as promising possibilities of interventions affecting this process.

Multiple myeloma is the second most common hematological malignancy characterized by focal lesions of malignant plasma cells in the bone marrow. These lesions contain subclones that directly influence survival of patients. Bone marrow biopsies are single-site biopsies and thus cannot contain all information about the tumor. In contrast, liquid biopsies analyze circulating cells and molecules that are secreted from all sites of the tumor. Long noncoding RNA molecules are one class of these molecules. We performed a two-phase biomarker study investigating lncRNA expression profiles in exosomes of peripheral blood serum of newly diagnosed multiple myeloma (MM) patients, monoclonal gammopathy of undetermined significance (MGUS) patients in comparison with healthy donors (HD). Surprisingly, this analysis revealed dysregulation of only one exosomal lncRNA PRINS in MM vs HD. Overall, MM and MGUS patients were distinguished from HD with sensitivity of 84.9% and specificity of 83.3%. Our study suggests a possible diagnostic role for exosomal lncRNA PRINS in monoclonal gammopathies patients., (© 2018 The Authors Hematological Oncology Published by John Wiley & Sons Ltd.)

Background: Catastrophic chromosomal event known as chromothripsis was proven to be a significant hallmark of poor prognosis in several cancer diseases. While this phenomenon is very rare in among multiple myeloma (MM) patients, its presence in karyotype is associated with very poor prognosis., Case Presentation: In our case, we report a 62 year female patient with rapid progression of multiple myeloma (MM) into extramedullary disease and short overall survival (OS = 23 months). I-FISH investigation revealed presence of gain 1q21 and hyperdiploidy (+ 5,+ 9,+ 15) in 82% and 86%, respectively, while IgH rearrangements, del(17)(p13) and del(13)(q14) were evaluated as negative.Whole-genome profiling using array-CGH showed complex genomic changes including hyperdiploidy (+ 3,+ 5,+ 9,+ 11, + 15,+ 19), monosomy X, structural gains (1q21-1q23.1, 1q32-1q44, 16p13.13-16p11.2) and losses (1q23.1-1q32.1; 8p23.3-8p11.21) of genetic material and chromothripsis in chromosome 18 with 6 breakpoint areas. Next-generation sequencing showed a total of 338 variants with 1.8% (6/338) of pathological mutations in NRAS (c.181C > A; p.Gln61Lys) or variants of unknown significance in TP53, CUX1 and POU4F1., Conclusions: Our findings suggest that presence of chromothripsis should be considered as another important genetic hallmark of poor prognosis in MM patients and utilization of genome-wide screening techniques such as array-CGH and NGS improves the clinical diagnostics of the disease., Competing Interests: Present case under submission has been approved by the institutional ethics committee of the University Hospital Brno. This process is in accordance with the Helsinki declaration. An informed consent was obtained from the patient in the accordance with the requirement of the institutional ethics committee.Informed written consent was obtained from parents for publication of their clinical details and/or clinical images. A copy of the written consent is available for review by the editor of this journal.The authors declare that they have no competing interests (financial or non-financial).Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Objectives: Long non-coding RNAs (lncRNAs) are RNA transcripts longer than 200 nucleotides that are not translated into proteins. They are involved in pathogenesis of many diseases including cancer and have a potential to serve as diagnostic and prognostic markers. We aimed to investigate lncRNA expression profiles in bone marrow plasma cells (BMPCs) of newly diagnosed multiple myeloma (MM) patients in comparison to normal BMPCs of healthy donors (HD) in a three-phase biomarker study., Methods: Expression profile of 83 lncRNA was performed by RT2 lncRNA PCR Array (Qiagen), followed by quantitative real-time PCR using specific TaqMan non-coding RNA assays analyzing 84 newly diagnosed MM patients and 25 HD., Results: Our analysis revealed dysregulation of two lncRNAs; NEAT1 (sensitivity of 55.0% and specificity of 79.0%) and UCA1 (sensitivity of 85.0% and specificity of 94.7%). UCA1 levels correlated with albumin and monoclonal immunoglobulin serum levels, cytogenetic aberrations, and survival of MM patients., Conclusion: Our study suggests a possible prognostic impact of UCA1 expression levels on MM patients., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Medulloblastoma, an embryonal neuroectodermal tumor of the cerebellum, is the most common malignant brain tumor in children. There are approximately 15 cases diagnosed in the Czech Republic each year. The recent World Health Organization classification recognizes several histopathological subtypes of medulloblastoma: classical, desmoplastic/ nodular with its extensive-nodularity variant, and anaplastic/ large-cell variant. Further molecular analysis identified four basic subgroups of medulloblastoma: WNT, SHH, Group 3, and Group 4. The subgroup of SHH meduloblastoma is associated with somatic mutations of SHH, PTCH1, SUFU, SMO and TP53, while the most common mutations found in infants up to three years of age were PTCH1 and SUFU. The majority of medulloblastomas are sporadic diseases, whereas only about 5- 10% of all cases occur in connection with hereditary genetic syndromes., Case: We present a case of a 21-months old girl diagnosed with a localized posterior fossa tumor. The histopathological examination revealed a desmoplastic/ nodular medulloblastoma. The treatment comprised a radical exstirpation of the tumor followed by adjuvant chemotherapy. With the use of array-CGH, a partial biallelic deletion of the SUFU gene (locus 10q24.32) was detected in the tumor DNA, whereas a monoallelic deletion was found in the peripheral lymphocyte DNA of the patient. These findings were confirmed by an independent qPCR method. Monoallelic germline deletion of SUFU was also identified in the patients mother, who was a healthy carrier. Pedigree of the family suggested a transition of the germline deletion of SUFU, since another brain tumors (including one case diagnosed before the age of three years) were identified in previous generations., Conclusion: Germline mutations in SUFU gene are believed to predispose to infant desmoplastic/ nodular medulloblastomas, basal cell carcinomas and meningiomas. The susceptibility gene shows autosomal dominant inheritance with an incomplete penetrance. There is no evidence-based surveillance strategy suggested for the carriers of germline SUFU mutations/ deletions so far. Our recommendation is based both on a family history of our patient and similar cases described in the literature. Since the germinal mutations in SUFU are responsible for up to 50% of all desmoplastic medulloblastomas in children under three years of age, genetic testing of SUFU should be encouraged in this population of patients.

Preimplantation genetic diagnosis (PGD) is a complex approach for detecting genetic abnormalities in early-stage embryos using genetic or molecular cytogenetic methods. Recently, single cell genomic methods based on DNA microarrays have been used for PGD. In the presented paper, we discuss and demonstrate the possibility to detect copy number variation (CNVs) in trophectoderm cells biopsied from 5-day embryos using 60-mer oligonucleotide-based array-CGH with CytoSure 8 × 15K Aneuploidy Array. Whereas this microarray platform was originally designed for analysis of unamplified DNA derived from many cells, the new methods, developed for single-cell genomics, allow the application of oligo arrays technology in preimplanation genetic diagnosis. Preclinical validation of single cell array-CGH was made by analysis of 30 positive and negative controls. Validation process included whole genome amplification of DNA from 5-10 cells with normal karyotype and from samples with known aneuploidies and structural aberrations. Subsequently, we analyzed the whole genome profiles in 118 embryos; aneuploidies of chromosomes were observed in 26.7%; segmental imbalances were proved in 6.8% of embryos. Our first experience confirmed that this oligonucleotide-based array technique enables high-resolution preimplantation aneuploidy screening of all the 23 chromosome pairs and sensitive preimplantation diagnosis of segmental imbalances such as deletions, duplications and amplifications.

Background: Chromosomal aberrations play an important role as prognostic factors in chronic lymphocytic leukemia (CLL). These aberrations are mostly detected by fluorescent in situ hybridization (FISH), as chromosomal banding analysis has been scarce due to low proliferative activity of malignant B-lymphocytes in vitro. In 2006, a new method using stimulation with IL-2 and CpG oligonucleotide DSP30 for metaphase generation in CLL was published [1]. The objective of our study was to verify the efficacy of stimulation and to evaluate if the method is suitable for routine diagnostics., Patients and Methods: In total, peripheral blood samples of 369 CLL patients were analyzed in parallel by chromosomal banding analysis and by FISH probes for 13q14, 11q22-23, CEP12 and 17p13., Results: Out of 369 patients, 307 (83%) were successfully stimulated for metaphase generation. Chromosomal aberrations were detected in 243 (79%) out of 307 patients evaluated by chromosomal banding analysis. Other aberrations that are not included into standard FISH panel were detected in patients karyotypes, e.g. del(6q), del(14q), t(14;18)(q32;q21), t(11;14)(q13;q32) and t(18;22)(q21;q11). One hundred and three (42%) patients showed complex aberrant karyotype not detected by FISH analysis., Conclusion: Stimulation with IL-2 and oligonucleotide DSP30 is an efficient method how to induce proliferation of malignant B-lymphocytes and allows detection of a substantial number of chromosomal aberrations in addition to those detected by standard FISH panel. Using this method in routine diagnostics is helpful particularly in identification of patients with complex aberrant karyotype.

We report an infant with a unique combination of 22q11 deletion syndrome and 14q terminal deletion syndrome. The proband had clinical symptoms compatible with diagnosis of 22q11 deletion syndrome: microcephaly, micrognathia, high-arched palate, hypertelorism, short palpebral fissures, square nasal root, prominent tubular nose, hypoplastic nasal alae, bulbous nasal tip, dysplastic low-set ears, short philtrum, and heart defect, but no cell-mediated immunodeficiency typical for the syndrome. G-banding and fluorescence in situ hybridization analyses revealed a karyotype 45,XY,der(14)t(14;22)(q32.3;q11.2),-22.ish del(14)(q32.33)(D14S1420-),del(22)(q11.2q11.2)(N25-). Subsequent analyses disclosed a translocation between chromosomes 14 and 22 in the proband's mother with a deleted 14q telomere. Using comparative genome hybridization on oligonucleotide-based microarray (array-CGH), the deletion at 22q11.21 in the size of ∼4.25 Mb was revealed in the proband as well as the deletion of the telomeric area at 14q32.33qter (∼3.24 Mb) in the proband and his mother. However, both the proband and his mother showed mild symptoms (microcephaly, thin lips, carp-shaped mouth) typical for patients with the described terminal 14q deletion syndrome.

The authors report a case of a 64-year-old man with chronic lymphocytic leukaemia (CLL) diagnosed 5 years ago. Recently, the patient was admitted with a tumour of the skin in the left lumbar region. Histological and immunohistochemical examinations established the diagnosis of Merkel cell carcinoma (MCC). Electron-microscopic examination revealed the formation of spherical aggregates of intermediate-sized filaments in the perinuclear region. The coincidence of MCC and CLl is rather rare and in published cases, no cytogenetic examinations were performed. We examined the RB1 gene using the interphase FISH method. A biallelic deletion in CLL tumour cells was detected; in MCC tumour cells, biallelic deletion was found in 33% of the cells and monoallelic deletion in 57% of the cells. In addition, chromosome 6 trisomy and 1p36 deletion were detected. Examination of non-neoplastic cells of the patient's skin showed a biallelic presence of the RB1 gene. According to the relevant literature, examination of the RB1 gene in CLL has informational value as a prognostic factor. The relationship between deletion of the RB1 gene and prognosis in MCC has not yet been determined and needs more research.

Submicroscopic structural chromosomal aberrations (microduplications and microdeletions) are believed to be common causes of mental retardation. These so-called copy number variations can now be routinely detected using various platforms for array-based comparative genomic hybridization (array-CGH), which allow genome-wide identification of pathogenic genomic imbalances. In this study, oligonucleotide-based array-CGH was used to investigate a panel of 23 patients with mental retardation and developmental delay, dysmorphic features or congenital anomalies. Array-CGH confirmed or revealed 16 chromosomal aberrations in a total of 12 patients. Analysis of parental samples showed that five aberrations had occurred de novo: del(1)(p36.33p36.23), del(4)(p16.3p16.2) joined with dup(8)(p23.3p23.1), del(6)(q14.1q15), del(11)(q13.1q13.4). Three aberrations appeared to be inherited from an unaffected parent: dup(3)(q29), del(6)(q12), dup(16)(p13.11). Six aberrations appeared to be inherited from a parental carrier: del(1)(p36.33) joined with dup(12)(q24.32), del(21)(q22.2q22.3) joined with dup(11)(q24.2q25), del(X)(q22.3) and del(1)(q21.1). In two cases, parents were not available for testing: del(17)(q11.2q12) and del(2)(q24.3q31.1). Our results show that the use of oligonucleotide-based array- CGH in a clinical diagnostic laboratory increases the detection rate of pathogenic submicroscopic chromosomal aberrations in patients with mental retardation and congenital abnormalities, but it also presents challenges for clinical interpretation of the results (i.e., distinguishing between pathogenic and benign variants). Difficulties with analysis notwithstanding, the array-CGH is shown to be a sensitive, fast and reliable method for genome-wide screening of chromosomal aberrations in patients with mental retardation and congenital abnormalities.

The presence of multiple centrosomes in tumor cells is associated with the formation of multipolar mitotic spindles and results in aneuploidy of both daughter cells. Centrosome amplification is a feature of all cancer cells. We have previously described centrosome amplification in abnormal B cells. Further studies of centrosome amplification in different stages of B lineage development could provide important information about multiple myeloma pathogenesis.

Multiple myeloma is a malignant disease of terminal developmental stages of B-lymphocytes, i.e. plasma cells. During the progress of the disease genetic changes are cumulated in these cells which are the cause of their fully malignant phenotype. The affection of plasma cells is characterized by the presence of various numerical and structural chromosomal aberrations. A frequent cytogenetic finding is the presence of reciprocal translocations originating within the process of the so-called illegitimate switch recombination, affecting the gene for heavy chains of immunoglobulins (IgH) in the 14q32 chromosome area. As a result of this recombination the oncogenes on derived chromosomes get under control of the molecular enhancers of the IgH locus. This paper summarizes the current knowledge concerning the formation mechanism of chromosomal translocations in multiple myeloma and the areas of their formation; it also mentions the way of their detection. The paper presents the most frequent types of chromosomal translocations affecting the area of chromosome 14q32 in the karyotype of people diseased with multiple myeloma, including the frequency of their detection and the prognostic significance for the patients.

We described a rare malignant fibrous histiocytoma of the parotid gland (MFH) in a 63-year-old woman. During six months the tumour size became 10 cm in diameter with skin ulceration. The tumour was examined morphologically, by immunohistochemistry and molecular biology methods - FASAY and CGH. The histology revealed a storiform-pleomorphic type of MFH with high mitotic rate. The FASAY method identified a non-mutated p53 gene. The chromosomal changes were identified by the CGH method and 6 cytogenetic changes were found in the tumour cells (deletions at 8p12-p22, 13q32-qter, 14q24-qter, and gains of chromosomal material at 5p, 8q12-q23, and Xq25-qter). The patient died shortly after the beginning of chemotherapy. Autopsy revealed brain and cerebellar haemorrhage. No other tumour foci were proved. In view of short course of disease we lack the data about the influence of the non-mutated p53 gene on the prognosis and therapy.

Comparative genomic hybridization (CGH) is a molecular cytogenetic technique that allows the genome-wide analysis of DNA sequence copy number differences. We applied conventional CGH and the recently developed high-resolution CGH (HR-CGH) to tumour samples from 18 patients with glioblastoma multiforme (GBM) in order to compare the sensitivity of CGH and HR-CGH in the screening of chromosomal abnormalities. The abnormalities were studied in topologically different central and peripheral tumour parts. A total of 78 different changes were observed using CGH (0-16 per tumour, median 3.5) and 154 using HR-CGH (0-21 per tumour, median 6). Using HR-CGH, losses were more frequent than gains. The representation of the most prominent changes revealed by both methods was similar and was comprised of the amplification of 7q12 and 12q13-q15, the gain of 7, 3q and 19, and the loss of 10, 9p, and 13q. However, HR-CGH detected certain other abnormalities (the loss of 6, 14q, 15q and 18q, and the gain of 19), which were rarely revealed by CGH. Using HR-CGH, the numbers and types of chromosomal changes detected in the central and peripheral parts of GBM were almost the same. The loss of chromosomes 10 and 9p and the gain of chromosomes 7 and 19 were the most frequent chromosomal alterations in both tumour parts. Our results from the GBM analysis show that HR-CGH technology can reveal new, recurrent genetic alterations involving the genes known to participate in tumorigenesis and in the progression of several human malignancies, thus allowing for a more accurate genetic characterization of these tumours.

Glioblastoma multiforme (GBM) is the most common as well as the most aggressive type of primary brain tumor of astrocytic origin in adults. GBM is characterized by a high degree of intratumoral heterogeneity both in histomorphology and genetic changes. Trisomy/polysomy of chromosome 7, monosomy of chromosome 10, EGFR gene amplification and p53 deletion have been described as the typical genetic markers for tumor classification and prediction of possible response to therapy. Our work was based on detection of these four main genetic changes both in central and peripheral parts of the tumors to evaluate possible differences in the topological incidence of these genetic markers. Chromosomal abnormalities in tumor samples from a group of 21 patients surgically treated for GBM were characterized by means of the interphase-fluorescence in situ hybridization (I-FISH) technique using sets of centromere and locus-specific DNA probes. In addition, we performed a detailed analysis of one selected tumor sample using a genomic microarray system (GenoSensor Array 300) to characterize copy number changes of specific sequences and refine results obtained by I-FISH. However, the data show no significant differences in occurrence of the described genetic markers in either part of the tumor.

Multiple myeloma is a malignant disease caused by malign transformation of B-cells, their clonal proliferation and the accumulation of myeloma cells in the bone marrow. These cells are set apart by a pronounced genetic instability. Chromosomal abnormalities are probably the most important prognostic factors in myeloma which influence the division of the patients into individual sub-groups each with a different developmental process of the disease and thus a different approach during treatment. The study summarizes the methodological approaches and current options in classical and molecular cytogenetics during the examination of cytogenetic changes in multiple myeloma. It presents the most common types of numerical and structural chromosomal changes found in the karyotype of multiple myeloma patients, and their prognostic importance.

Background: Cytogenetic abnormalities of chromosome 13 are emerging as important prognostic factors in multiple myeloma and have been associated with poor prognosis., Methods and Results: The occurrence of 13q14 deletion and other standard laboratory parameters were determined in 40 patients with multiple myeloma. We found that interphase fluorescence in situ hybridization using a locus specific probe for RB1 gene on immunomagnetically selected myeloma cells was more sensitive than non selected cells. The 13q14 deletion was found in 10 of 40 (25.0%) of bone marrow samples without cell selection and in 25 of 40 (62.5%) of samples with CD138+ enriched myeloma cells. Negative correlation was found between albumin and the 13q14 deletion in separated (p = 0.003) as well as in cells without selection (p = 0.010). No significant correlation was found in overall survival of separated and unseparated cells (p = 0.830; p = 0.260) and a similar result was obtained for treatment response after transplantation of separated cells (p = 0.520) or non-separated cells (0.190)., Conclusions: Our results confirm that immunomagnetic selection of CD138+ cells increases the probability of detection of the 13q14 deletion in bone marrow samples. The correlation was found between albumin and the 13q14 deletion in both of type of cells.

We present two patients with Ph-negative chronic myeloid leukemia (CML) and fusion signal BCR/ABL on both chromosomes 9, located in region 9q34. The first case was a 27 years old man with CML. Molecular studies (RT-PCR) revealed the rearrangement in the major-BCR region and expression of chimeric BCR/ABL mRNA of b3a2 configuration. By classical cytogenetic studies (G-banding) karyotype 46,XY was found in short-term cultivated bone marrow cells and phytohemagglutinin (PHA) stimulated peripheral lymphocytes. FISH studies revealed the BCR/ABL fusion signals on both chromosomes 9 and green BCR signals on both chromosomes 22 in all mitoses studied. Detection of the alleles of ABL1 intragenic STR locus by fluorescence PCR followed by fragmentation analysis in the patient and his parents provided no information about transmission of the ABL gene. Quantitative assessment of BCR/ABL transcript level by RT-PCR showed 60 and 70% BCR/ABL positivity in two peripheral blood samples at 6,5 and 10,5 months after diagnosis, respectively, which does not correspond to the expression from two identical BCR/ABL hybrid genes. Therefore, the possible mechanism of the origin of two BCR/ABL fusion signals present on both chromosomes 9 could not be resolved and remains speculative. The second case was a 53 years old male with diagnosis of chronic phase of CML, with first sign of acceleration one month after diagnosis and death because of sepsis in blastic phase within four months. The cytogenetic findings were identical to those in case No. 1., i.e. karyotype 46, XY by G-banding, two BCR/ABL fusion signals on both chromosomes 9 and RT-PCR molecular studies proved b3a2 breakpoints. It is generally accepted that prognosis of the patients with fused BCR/ABL gene located on chromosome 9 is poor. The presence of two fused genes could be anticipated as two Ph chromosomes in accelerated and blastic phases of the disease. However, in our study, quantitative findings of BCR/ABL transcripts did not corresponded to the expression of two BCR/ABL genes originating from duplication. If this assumption is correct then the expression of both fused genes BCR/ABL was in case No. 1 equally suppressed and total expression reached about the level of one BCR/ABL gene.

Accurate prognostic evaluation of patients with multiple myeloma (MM) is required for their stratification for more adequate therapy. Chromosomal G-banding and interphase fluorescence in situ hybridization (FISH) on cell-nonspecific samples and on myeloma cells selected by magnetic-activated cell separation (MACS) were used to study 13 samples from 12 multiple myeloma (MM) patients. Bone marrow (BM) samples were analysed using three approaches. Standard mitotic samples were prepared and analysed after G-banding. Interphase FISH was performed to detect the 13q14 deletion in unselected BM cells. In parallel, myeloma cells were selected from the BM using the CD138-specific antibody. The high-purity myeloma cell suspension was then analysed by interphase FISH for the 13q14 deletion. Magnetic separation yielded enriched myeloma cell suspensions with the mean viability of 98.0% (range: 97.0%-99.0%), and the purity of 97.6% (range: 87.2%-99.2%) as detected morphologically, and 85.2% (range: 44.8%-98.4%) as detected by immunophenotyping for CD138+ cells. Interphase FISH revealed the 13q14.3 deletion in 5 of 13 (38.5%) of cell-nonspecific samples and in 9 of 13 (69.2%) of enriched myeloma cell suspensions. In conclusion, interphase FISH on immunomagnetically selected MM cells increases the detection of the 13q14 deletion in BM samples from the patients with MM.

Treatment of HL-60 cells with micromolar concentrations of N(6)-benzyladenosine (N(6)-benzylaminopurine riboside [BAPR]) led to the occurrence of apoptosis in a concentration-dependent manner. Incubation period as short as 2 h in the presence of BAPR was sufficient for triggering irreversible changes leading to apoptosis even after the transfer of cells to the standard medium (without BAPR). Cell death induced by BAPR proceeded rapidly and in a very synchronous fashion. Detailed study of temporal changes in a chromatin structure and DNA integrity showed that the movement of chromatin toward the nuclear periphery is the fundamental event within dying cells. We demonstrated that this rearrangement of chromatin is irreversible and it takes place without apparent DNA degradation. The extensive DNA cleavage seems to be a rather late event, as it was observed in cells that exhibited a typical apoptotic morphology (apoptotic bodies). On the basis of temporal changes in the ATP level within dying cells, it is concluded that ATP is essential for the movement of chromatin toward the nuclear envelope but not for the subsequent chromatin condensation leading to the formation of apoptotic bodies. DNA fragmentation also seems to be ATP independent. BAPR interfered with neither pyrimidine nor purine biosynthesis, as none of the tested bases and the corresponding nucleosides prevented or reduced apoptosis in BAPR-treated cells. Adenosine was the only exception that substantially reduced the effect of BAPR. Since transport of exogenous adenosine into cells was essential to manifest its protective effect, we assume that adenosine is a competitive inhibitor of adenosine kinase and thus reduces intracellular phosphorylation of BAPR. Indeed, 4-amino-3-iodo-1(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine, a potent inhibitor of adenosine kinase, fully prevented BAPR-induced apoptosis. These results suggest that cytotoxic effect of BAPR is related to its activation by phosphorylation within cells, rather than to its interaction with extracellular adenosine receptors., (Copyright 2000 Wiley-Liss, Inc.)

The Somatic Mutation and Recombination Test (SMART) in wing cells of Drosophila melanogaster, the Vicia faba cytogenetic tests-Sister Chromatid Exchange (SCE) and Micronucleus Test (MN), and the Müller test for gametic mutations in Arabidopsis thaliana were used for genotoxicity testing of environmental samples of pollutants from the surroundings of LACHEMA chemical factory (Brno, Czech Republic) and DEZA factory in Valasské Mezirící (Moravia, Czech Republic). Tested soil and air samples were taken from the near vicinity of both factories. The surroundings of both sites are heavy loaded by exhalation of chemicals from the factories. Chemical analyses of the 16 Polycyclic Aromatic Hydrocarbons (PAHs) according to the United States Environmental Protection Agency (US EPA) list of priority pollutants and heavy metals were performed in both soil and air samples. The Drosophila wing spot test was positive in 70.6% of the tested samples, the Vicia sister chromatid exchange test in 62.5%, and the Arabidopsis Müller test in 58.9%. The micronucleus Vicia faba test was quite insensitive in tested environmental samples. The concordance between SMART and SCE was 62.5%, between SMART and Müller test 76.5%, and between Müller test and SCE 100%. Total concordance of these three tests was 79.7%. Müller test for gametic mutation in Arabidopsis thaliana and cytogenetic SCE test in Vicia faba seem to be quite sensitive and convenient plant bioassays for assessing the mutagenic potential of environmental agents, when compared to the SMART test in Drosophila melanogaster.

In a population of plant meristematic cells of Vicia faba the frequency of sister chromatid exchanges (SCEs), incidence of chromosomal aberrations and mitotic activity of cells was evaluated after short-term treatment (1 h) with antitumour active platinum complexes cis-DDP, CHIP IV, CBDCA, oxo-Pt and antitumour inactive trans-DDP. It was found that the action of platinum compounds in equimolar concentration 3.33 microM increases the level of SCEs 1.3 to 6.4-fold. The maximum effect in terms of SCE formation was observed after cis-DDP. Comparison of the incidence of SCEs and chromosomal aberrations in plant cells demonstrated that the tested drugs had a greater effect on SCE formation than on chromosomal aberration induction. Inhibition of mitotic activity correlated with the total cytogenetic damage to chromosomes of Vicia faba cells.