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CONSTRUCTION AND USE OF EUCARYOTIC INDICATOR CELL LINES FOR THE ASSESSMENT OF RADIATION INDUCED ALTERATIONS LEADING INTO NEW DEFINED PHENOTYPES

Objective


One way to approach the practical problems of human exposure to radiation, such as radiation protection or radiation therapy, is by extrapolating from molecular studies to the doses and dose rates relevant in human exposure. More data using more advanced biological systems are needed towards this goal. With this rationale we have employed increasing amounts of cadmium chloride (5 to 20 uM) in V-79 cells, and cell survival response was followed for 6 days. The results obtained tend to support the aspect that even in the concentration of 20 uM cadmium chloride, during the 4 days of culture, V-79 cells have a significant percentage of survival ranging from 58 to 60%.

Having located the survival rates of V-79 we focussed our attempts on the transfection and selection as well as preliminary characterisation of stably transformed V-79 cells with pBMT3X plasmid.

The data obtained suggest that the inserted plasmid pBMT3X results in the appearance of numerous surviving colonies which stably express the new phenotype (ie resistance to high concentration of cadmium chloride (40, 60 and 80 uM)).

Controls of V-79 cells, subjected to the concentrations mentioned above did not survive as expected. 14 clones were isolated from the transformed cells, after 8 days of culture in 40, 60 and 80 uM cadmium chloride. These resistant clones were cultured for 35 to 40 days and then 1 part of the clones was analysed cytogenetically and the rest were frozen. In all cases the transformed cells became resistant to cadmium toxicity due to the overproduction of metallothionein messenger ribonucleic acid (mRNA) and polypeptide.

Chromosomal analysis of transformed cells, cultured for 5 weeks after initial establishment, revealed a homogeneous cellular preparation. To characterise the chromosome content of the abundant cell in the population, we compared the karyotypes of V-79 and transformed cells. The analysis revealed that the V-79 cell line contains most frequently a total of 21 chro mosomes and for the transformed cells the same chromosome number was found and, moreover, traces of nonintegrated deoxyribonucleic acid (DNA) which is packaged as chromatin and is referred to as a minichromosome without a centromere. This minichromosome may be considered as the cytogenetic equivalent of the multiple episome presence.

Furthermore, in order to demonstrate that clones resistant to 40 and 60 uM cadmium chloride contain the plasmid pBMT3X, DNAs from resistant clones and from V-79 cells were analysed in agarose gel electrophoresis.

In the present study we demonstrate the stable integration of the plasmid into the cellular DNA of the recipient host cell in several independently derived clones. The experimental approach we have adopted to examine the state of the transfected gene involves restriction endonuclease treatment of cellular DNA followed by hybridisation analysis of the DNA fragments which contain information homologous to the gene.

More specifically, in an attempt to unequivocally prove that the resistant clones do contain the whole pBMT3X plasmid and consequently the gene for human metallothionein and the promotor of the mouse metallothionein gene, cellular DNA from clones resistant to 40 uM and 60 uM cadmium chloride and from V-79 cells were isolated. The DNAs were purified and prepared for restriction endonuclease digestion either with EcoRI or with BamHI and hybridisation analysis was performed using as probe the human gene of metallothionein insert. Briefly, purified pBMT3X plasmid DNA was restricted with BamHI endonuclease that generates 2 fragments approximately 14.5 kb and approximately 2.0 kb. The small fragment corresponds to the intact human metallothionein gene, and with EcoRI endonuclease that generates 3 fragments 8.0, 4.5, and 4.0 kb. The 8.0 kb fragment corresponds to the insert that contain the intact human metallothionein. The fragment 2.0 was labelled with biotin-7-d-adenosine triphosphate (biotin-7-dATP).

The restr iction endonuclease BamHI cleaves the 16.5 kb pBMT3X plasmid at 2 sites generating 2 fragments approximately 14.5 and approximately 2.0 kb. The second fragment corresponds to the part of the plasmid that contains the intact gene for human metallothionein. As expected, the control which includes 30 ug of total V-79 digested DNA with EcoRI or BamHI, did not hybridise with human metallothionein gene. In the transformed cellular DNA resistant to cadmium concentrations 40 and 60 uM, the 2 bands at 16.5 and approximately 2.0 kb have identical pattern and intensity, a fact that is concordant with the presence of pBMT3X plasmid and indicated that rearrangement is not involved during transformation.

It has been reported that some bovine papillomavirus (BPV) have acquired foreign DNA sequences either from the host genome or from the carrier DNA and have suffered rearrangements involving deletions and insertions. The faint band at 16.5 kb in the transformed cellular DNA can possibly be attributed to partial digestion of pBMT3X plasmid. This band corresponds to uncut plasmid.

The restriction endonuclease EcoRI cleaves the 16.5 kb pBMT3X plasmid at 3 sites generating 3 fragments approximately 8.0, 4.5 and 4.0 kb. The first fragment corresponds to the part of the plasmid that contains the gene of human metallothionein, the second fragment contains the ampicillin resistance gene of the plasmid and the third fragment corresponds to the part of the plasmid that contains the promotor of the mouse metallothionein gene. As was expected, only 8.0 kb fragment hybridises with the DNAs of transformed cells. The identical pattern and intensity of both transformed V-79 indicated the lack of rearrangement.

Thus, the fact that plasmid DNA and DNA from transformed cells revealed the same pattern after digestion with BamHI or EcoRI may support the aspect that plasmid DNA is able to replicate in a stable manner as a multicopy episome that allows the continuous replication and expression of the cloned foreign gene independently from chromosomal control. In conclusion, BPV DNA establishes itself in transformed cells as multiple episomes, thenumber of which varies in different cell lines, approximately from 20 to 300 copies. However, in a given cell line, their number remains constant over very long periods of time. As the BPV minichromosome does not contain a centromere, its segregation to the daughter cells is likely to be a random event, and the copy number is therefore an average. The factors, if any, that determine the number of episomes in a given cell, and that maintain a constant average number in a given cell line, are unknown. Nor is it known whether BPV DNA replicates at a particular stage of the cell cycle, or whether its synthesis can occur throughout.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Greek Atomic Energy Commission
Address

15310 Attikis
Greece