HYPOXIA-INDUCED TETRAPLOIDISATION OF A DIPLOID HUMAN-MELANOMA CELL-LINE IN-VITRO

Many human tumours are hyperdiploid, particularly in advanced stages o f growth. The purpose of the present work was to investigate whether e xposure to hypoxia followed by reoxygenation might induce hyperploidis ation of diploid human tumour cells in vitro. The investigation was pe rformed by using the diploid melanoma cell line BEX-c (median chromoso me number, 46; DNA index, 1.10+/-0.04) as test line and the hyperdiplo id melanoma cell line SAX-c (median chromosome number, 61; DNA index, 1.42+/-0.03) as control line. Cell cultures kept in glass dishes in ai r-tight steel chambers were exposed to hypoxia (O-2 concentrations <10 p.p.m, or <100 p.p.m.) at 37 degrees C for 24 h. DNA content was meas ured by flow cytometry. Metaphase spreads banded with trypsin-Versene- Giemsa were examined to determine the number of chromosomes per cell. An electronic particle counter was used to measure cell volume. The ex pression of p53 and pRb was studied by Western blot analysis. Transien t exposure to hypoxia was found to induce a doubling of the number of chromosomes in BEX-c but not in SAX-c. The fraction of the BEX-c metap hase spreads with 92 chromosomes was approximately 10% at 18 h after r eoxygenation, decreased to approximately 2% at 7 days after reoxygenat ion and then increased gradually with time. The whole cell population became tetraploid within 25 weeks. BEX-c and SAX-c behaved differently during the 24 h hypoxia exposure. Cell volume and fraction of cells i n G(2) + M increased with time in BEX-c but remained essentially uncha nged in SAX-c. On the other hand, the expression of p53 and pRb was si milar for the two lines; hypoxia induced increased expression of p53 a nd hypophosphorylation of pRb.