gms | German Medical Science

102. Jahrestagung der DOG

Deutsche Ophthalmologische Gesellschaft e. V.

23. bis 26.09.2004, Berlin

Genetic and cytological characterization of retinoblastoma

Meeting Abstract

  • E. Bártová - Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno/CZ
  • H. Gajová - Department of Ophthalmology, Children's Hospital, Faculty of Medicine, Masaryk University Brno, Brno/CZ
  • corresponding author R. Autrata - Department of Ophthalmology, Children's Hospital, Faculty of Medicine, Masaryk University Brno, Brno/CZ
  • S. Kozubek - Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno/CZ

Evidenzbasierte Medizin - Anspruch und Wirklichkeit. 102. Jahrestagung der Deutschen Ophthalmologischen Gesellschaft. Berlin, 23.-26.09.2004. Düsseldorf, Köln: German Medical Science; 2004. Doc04dogP 187

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Veröffentlicht: 22. September 2004

© 2004 Bártová et al.
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Gliederung

Text

Objective

Retinoblastoma, tumour of childhood age, occurs either as a sporadic or familiar form. The hereditary tumours develop at an earlier age than the sporadic and usually appear as a bilateral or multicentric cancer or both. Deletion of the long arm of chromosome 13 has been associated with this disease. Two mutations in retinoblastoma gene locus Rb1 (13q14) have to be presented for the manifestation of the hereditary form (1). Various techniques were proposed for the screening of specific loci alteration in tumour cells. Comparative genomic hybridisation (CGH) revealed certain imbalances in following loci: +1q, +2p, +6p, -16q. High-level of amplification were observed in 2p23-25 and 1q21 regions (2). Standard karyotyping, which can be applied only on mitotic cells, enables the identification of deletions, but has limited resolution. Fluorescence in situ hybridisation (FISH) is a well-established technique for identifying gene modification occurring in low percentage of interphase nuclei as well as metaphase chromosomes. The recent work was focused on the study of genetic loci such as Rb1, N-myc, p53 and chromosomes 6, 13, X and others in human retinoblastoma tumours. Fresh tissue or paraffin-embedded sections were used for chromatin analyses.

Methods

Using classical FISH techniques and FISH on paraffin embedded sections RT-PCR, immunohistochemical studies, flow cytometric analysis of the cell cycle and apoptosis we studied structural as well numerical aberrations and higher order chromatin structure modifications in retinoblastoma tumour cells, in patient's lymphocytes and in human cell line Y79, originally established from bilateral retinoblastoma. Treatment of anticancer agents such as vincristine, etoposide, cisplatin and gamma-radiation was used in order to find modification in the chromatin arrangements, cell cycle profile and induction of apoptosis in Y79 cells.

Results

Heterogeneity in the copy number of genetic elements has been found in tumour cells and lymphocytes of the patients with diagnosed unilateral or bilateral retinoblastoma. Numerical aberrations lead to a general assertion that retinoblastoma tumours are characterised by heterogeneous nuclear structure that we also revealed in retinoblastoma cell line Y79. On the other hand, centre-to-locus distances of studied genetic structures were similar for aneuploid cells as well as for diploid population. Topographic analyses of the N-myc gene and its organisation into homogenously staining region (HSR) revealed the peripheral location of interphase HSR within the nuclei of Y79 cells. Induction of cell death apoptotosis we observed not only in in vitro experiments, but also in tumour tissue of patients suffering from retinoblastoma.

Conclusions

Numerical aberrations lead to a general assertion that retinoblastoma tumours are characterised by heterogeneous nuclear structure that we also revealed in retinoblastoma cell line Y79. On the other hand, centre-to-locus distances of studied genetic structures were similar for aneuploid cells as well as for diploid population. Reduced Rb1 gene activity was observed by RT-PCR analysis. Translocated region including chromosome X was found to be heavy methylated. Our observations support an assertion that heterochromatin-mediated gene silencing and methylation plays an important role in development of unilateral retinoblastoma. Induction of cell death apoptotosis we observed not only in in vitro experiments, but also in tumour tissue of patients suffering from retinoblastoma.