PURPOSE: Chromosomal aberrations and the nuclear topography of retinoblastoma tumour cells as well as lymphocytes of patients suffering from the familiar or sporadic form of retinoblastoma were studied. METHODS: Fluorescence in situ hybridisation (FISH) on fresh, paraffin-embedded tumour tissues and on peripheral blood leukocytes was used for cytogenetic analysis. The cell cycle profile and induction of apoptosis was studied by flow cytometry and gene expression changes were detected by RT-PCR. RESULTS: Using the repeated FISH technique, the average distances between the nuclear membrane and the fluorescence gravity centre (FGC) of seven selected chromosomes were determined in the same tumour population and three other cell types. Chromosome order in positioning from the nuclear membrane was similar in all cell populations investigated. Our experimental studies were focused on specific genetic loci relevant for retinoblastoma tumour pathogenesis. We revealed a certain heterogeneity in the copy number of the Rb1, N-myc, and TP53 gene loci in tumour cells. In addition, in lymphocytes isolated from peripheral blood of the patients, a high degree of copy number heterogeneity was also detected. In 60% of analysed retinoblastomas we observed numerical aberration involving the centromeric region of chromosome 6. In these tumours, apoptotic bodies were found irrespective of clinical therapy. Chromosome instability seems to be a typical feature of primary retinoblastomas as well as of the human pseudodiploid cell line Y79. These cells, of a hereditary form of retinoblastoma (Y79), were irradiated by gamma rays and exposed to anti-tumour drugs such as etoposide, vincristine, and cisplatin. These treatments induced apoptosis, changes in the cell cycle profile, and specific modifications in the nuclear topography of selected loci. Treatment with a non-lethal concentration of hydroxyurea was shown to induce the loss of the amplified N-myc gene involved in the homogenously staining region (HSR) that was found to be associated with the nuclear membrane of retinoblastoma Y79 cells. CONCLUSIONS: We assume that not only cytological and cytogenetic parameters but also aberrant chromatin structures and their nuclear topography can be useful tools for optimal tumour marker specification.

Institute of Biophysics Academy of Sciences of the Czech Republic Královopolská 135 612 65 Brno Czech Republic

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Antosz H, Kitlinska J, Kwiatkowska-Drabik B, Koczkodaj D, Wach M, Antosz M, Rudzki S, Wojcierowski J (1997) RB1 gene expression in B-cell lymphocytic leukaemia cases with deletion in the 13q14 region. Cytobios 92:111–121 PubMed

Balmer A, Gaillioud C (1983) Retinoblastoma: diagnosis and treatment. Dev Ophthalmol 7:36–100 PubMed

Bártová E, Španová A, Janáková L, Bobková M, Rittich B (1997) Apoptotic damage of DNA in human leukaemic HL-60 cells treated with C2-ceramide was detected after G1 blockade of the cell cycle. Physiol Res 46:155–160 PubMed

Bártová E, Kozubek S, Jirsová P, Kozubek M, Lukášová E, Skalníková M, Cafourková A, Koutná I, Paseková R (2001) Higher-order chromatin structure of human granulocytes. Chromosoma 110:360–370 PubMed

Cano J, Oliveros O, Yunis E (1994) Phenotype variants, malignancy, and additional copies of 6p in retinoblastoma. Cancer Genet Cytogenet 76:112–115 PubMed

Cavenee WK, Dryja TP, Phillips RA, Benedict WF, Godbout R, Gallie BL, Murphree AL, Strong LC, White RL (1983) Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature 305:779–784 PubMed

Chen D, Gallie B, Squire J (2001) Minimal regions of chromosomal imbalance in retinoblastoma detected by comparative genomic hybridisation. Cancer Genet Cytogenet 129:57–63 PubMed

Cowell JK, Hogg A (1992) Genetics and cytogenetics of retinoblastoma. Cancer Genet Cytogenet 64:1–11 PubMed

Cremer T, Kurz A, Zirbel R, Dietzel S, Rinke B, Schrock E, Speicher MR, Mathieu U, Jauch A, Emmerich P, Scherthan H, Ried T, Cremer C, Lichter P (1993) Role of chromosome territories in the functional compartmentalisation of the cell nucleus. Cold Spring Harbor Symp Quant Biol 58:777–792 PubMed

Croft JA, Bridger JM, Boyle S, Perry P, Teague P, Bickmore WA (1999) Differences in the localization and morphology of chromosomes in the human nucleus. J Cell Biol 145:1119–1131 PubMed PMC

Damjanovich J, Ádány R, Berta A, Beck Z, Balázs M (2000) Mutation of the Rb1 gene caused unilateral retinoblastoma in early age. Cancer Genet Cytogenet 119:1–7 PubMed

De Preter K, Speleman F, Combaret V, Lunec J, Laureys G, Eussen BHJ, Francotte N, Board J, Pearson ADJ, De Paepa A, Van Roy N, Vandesompele J (2002) Quantification of MYCN, DDX1, and NAG gene copy number in neuroblastoma using a real-time quantitative PCR assay. Mod Pathol 15:159–166 PubMed

Doz F, Peter M, Schleiermacher G, Vielh P, Validire P, Putterman M, Blanquet V, Desjardins L, Dufier JL, Zucker JM, Mosseri V, Thomas G, Magdelénat H, Delattre O (1996) N-myc amplification, loss of heterozygosity on the short arm of chromosome 1 and DNA ploidy in retinoblastoma. Eur J Cancer 32A:645–649 PubMed

Francastel C, Walters MC, Groudine M, Martin DIK (1999) A functional enhancer suppresses silencing of a transgene and prevents its localisation close to centromeric heterochromatin. Cell 99:259–269 PubMed

Herzog S, Lohmann DR, Buiting K, Schüler A, Horsthemke B, Rehder H, Rieder H (2001) Marked differences in unilateral isolated retinoblastomas from young and older children studied by comparative genomic hybridisation. Human Genet 108:98–104 PubMed

Hogg A, Bia B, Onadim Z, Cowell JK (1993) Molecular mechanism of oncogenic mutations in tumors from patients with bilateral and unilateral retinoblastoma. Proc Natl Acad Sci USA 90:7351–7355 PubMed PMC

Horsthemke B (1992) Genetics and cytogenetics of retinoblastoma. Cancer Genet Cytogenet 63:1–7 PubMed

Jirsová P, Kozubek S, Bártová E, Kozubek M, Lukášová E, Cafourková A, Koutná I, Skalníková M (2001) Spatial distribution of selected genetic loci in nuclei of human leukemia cells after irradiation. Radiat Res 155:311–319 PubMed

Jones PA (1999) The DNA methylation paradox. Trends Genet 15:34–37 PubMed

Kallioniemi A, Kallioniemi OP, Sudar D, Rutovitz SD, Gray JW, Waldman F, Pinkel D (1992) Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 258:818–821 PubMed

Knudson AG (1971) Mutation and cancer: statistic study of retinoblastoma. Proc Natl Acad Sci USA 68:820–823 PubMed PMC

Knudson AG (1993) Antioncogenes and human cancer. Proc Natl Acad Sci USA 90:10914–10921 PubMed PMC

Kozubek M, Kozubek S, Lukášová E, Marečková A, Bártová E, SkalnŢková M, Jergová A (1999) High-resolution cytometry of FISH dots in interphase cell nuclei. Cytometry 36:279–293 PubMed

Kozubek S, Lukášová E, Rýznar L, Kozubek M, Lišková A, Govorun RD, Krasavin EA, Horneck G (1997) Distribution of ABL and BCR genes in cell nuclei of normal and irradiated lymphocytes. Blood 89:4537–4545 PubMed

Kusnetsova LE, Prigogina EL, Pogosianz HE, Belkina BM (1982) Similar chromosomal abnormalities in several retinoblastomas. Human Genet 61:201–204 PubMed

Lee WH, Murphee AL, Benedict WF (1984) Expression and amplification of the N-myc gene in primary retinoblastoma. Nature 309:458–460 PubMed

Lichter P, Cremer C, Borden J, Mauelidis L, Ward DC (1988) Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridisation using recombinant DNA libraries. Human Genet 80:224–234 PubMed

Oliveros O, Yunis E (1995) Chromosome evolution in retinoblastoma. Cancer Genet Cytogenet 82:155–160 PubMed

Pinkel D, Landegent J, Collins C, Fuscoe J, Segraves R, Lucas J, Gray J (1988) Fluorescence in situ hybridisation with human chromosome-specific libraries: detection of trisomy 21 and translocation of chromosome 4. Proc Natl Acad Sci USA 85:9138–9142 PubMed PMC

Potluri VR, Helson L, Ellsworth R, Reid T, Gilbert F (1986) Chromosomal abnormalities in human retinoblastoma. Cancer 58:663–671 PubMed

Ramzi C, Vinay K, Tucker-Robbins C (1999) Pathologic basis of disease. Saunders, Philadelphia, USA

Sadoni N, Langer S, Fauth C, Bernardi G, Cremer T, Turner BM, Zink D (1999) Nuclear organisation of mammalian genomes: polar chromosome territories build up functionally distinct higher order compartments. J Cell Biol 146:1211–1226 PubMed PMC

Sakai T, Toguchida J, Ohtani N, Yandell DW, Rapaport JM, Dryja TP (1991) Allele-specific hypermethylation of the retinoblastoma tumour-suppressor gene. Am J Hum Genet 48:880–888 PubMed PMC

Savelyeva L, Schwab M (2001) Amplification of oncogenes revisited: from expression profiling to clinical application. Cancer Lett 167:115–123 PubMed

Schwab M, Ellison J, Busch M, Rosenau W, Varmus HE, Bishop M (1984) Enhanced expression of human gene N-myc consequent to amplification of DNA may contribute to malignant progression of neuroblastoma. Proc Natl Acad Sci USA 81:4940–4944 PubMed PMC

Snapka RM, Varshavsky A (1983) Loss of unstably amplified dihydrofolate reductase genes from mouse cells is greatly accelerated by hydroxyurea. Proc Natl Acad Sci USA 80:7533–7537 PubMed PMC

Solovei I, Kienle D, Little G, Eils R, Savelyeva L, Schwab M, Jager W, Cremer C, Cremer T (2000) Topology of double minutes (dmins) and homogeneously staining region (HSRs) in nuclei of human neuroblastoma cell lines. Genes Chrom Cancer 29:297–308 PubMed