CELL LINES IC50 ± SD (ng ml * COLONY FORMING ABILITY
CISPLATIN) (% ± S D ) PARENTAL LINES 2 2 . 8 ± 2 . 8 13.5 ± 7.3 17.4 ± 2.7 3.3 ± 0.9 17.4 ± 7.3 6.2 ± 0.5 18.2 ± 5.0 6 . 0 ± 0 . 6 8^ 14.5 ± 2.8 5.2 ± 2.4 8^ 16.3 ± 1.5 9.3 ± 4.4 11.8 ± 4.0 7.2 ± 0.9 17.6 ± 5.8 16.1 ± 4.0 SELF-CROSSES G V 16.7 ± 0.5 3.7 ± 0.4 s " s f 58.4 ± 5.2 9.1 ± 0.6
SV-1
13.2 ± 3.7 5.0 ± 2.1 8^8^ 10.3 ± 2.1 6.3 ± 0.9 c V 27.3 ± 5.8 7.4 ± 1.7 HYBRIDS G V 10.6 ± 3.2 9.8 ± 3.8 G^C^ 20.3 ± 2.2 7.7 ± 0.7 G^S^ 16.1 ± 1.9 7.4 ± 0.1 G^S^ 18.4 ± 4.4 1 . 8 ± 1 . 2 G V 37.2 ± 5.9 9.8 ± 5.3 G^8^-l 21.8 ± 5.6 4.9 ± 1.0 G V 19.2 ± 5.9 5.1 ± 2.8 s V 43.5 ± 7.7 12.5 ± 4.6 s V - 1 34.9 ± 10.9 10.3 ± 0.9 S^8^ 12.9 ± 1.9 4.6 ± 3.6 8 V 14.4 ± 3.0 1.7 ± 0.4 8^C^ 11.4 ± 0.6 7.0 ± 2.0 C^S^ 1 2 . 6 ± 2 . 6 10.6 ± 3.0 C^S^ 23.1 ± 7.7 6.9 ± 2.93.3 DISCUSSION
In this study, cell fusions were performed between four human testicular tumour cell lines and 16 hybrids were generated. The origin of the hybrids was confirmed by karyotyping and PCR analysis of VNTRs. Karyotyping analysis showed that the hybrids contained chromosome numbers similar to the sum of the numbers of chromosomes in the parental lines, indicating that there was little or no loss of genetic material as a result of cell fusion. PCR analysis suggested that in a small proportion of the hybrids one of the VNTR repeats was lost, as previously observed (Wang et ah, 1996). However, in every case the bands obtained from the hybrid cells matched those of the parental cells, and as two independent loci were examined it was possible to confirm the origin the hybrids.
Had complementation occurred, we would have expected the hybrids to be more resistant than the parental cells, as shown in our previous study when testicular cells were fused with a cisplatin sensitive cell line D98°^ (Wang et a l, 1996). In these previous studies, the ratios of the IC50S of the hybrid to the parental testicular lines ranged from 2.1 to 6 . 8 fold. Cisplatin IC5 0S ranged from 43.7 to 146 ng/ml for the hybrids, compared to 20.6 to 21.4 ng/ml for the
3 testicular tumour cell lines (SuSa, 833K, GH). These earlier data (Wang et al, 1996) provide a positive control for the present study, indicating how complementation can occur when the mechanisms controlling sensitivity to cisplatin differ.
The fusions in this study were made between cell lines containing a dominant selectable marker. The transfected sublines retained their hypersensitivity to cisplatin, indicating that neither transfection nor the presence of the dominant selectable marker influenced drug sensitivity (data not shown). Self-crosses were used to control for the influence of gene dosage on drug sensitivity. In one self-cross (SfS^) the resulting hybrids were resistant to cisplatin, both in the uncloned parent population and in clones isolated from this population (data not shown). The anomalous result was therefore not due to clonal variation. However, on repeating the fusion, the cloned hybrid cells (S^S^-1) were similar to the parental cells in
their sensitivity to cisplatin. It is possible that the cisplatin resistance in the first fusion was due to a mutation or genetic loss in the hybrid cells, but further analysis would be necessary to identify the cause of the resistance. All the other three self-crosses gave hybrids with cisplatin sensitivities similar to the parent cells.
All four testicular tumour cell lines were hybridized with one another in all possible combinations. Consequently, for each cross, two pairs of cell lines were generated containing the opposite selection markers. If the genetic basis of sensitivity had been different in one of the cell lines, this could have been confirmed in two ways. Firstly, the matched hybrid with the reverse selection markers should also have been resistant. Secondly, all hybrids generated between this cell line and all other lines with a different mechanism controlling cisplatin sensitivity should have been relatively resistant. Neither of these patterns were observed, and with two exceptions all the hybrids were similar in cisplatin sensitivity to the parental cells, indicating that complementation had not occurred. The implication of these data is that a single or common genes control the sensitivity of all 4 testicular tumour cell lines to cisplatin.
Although consistent evidence of complementation was not observed, two clones were generated that were relatively resistant. Consequently these fusions were repeated and independent clones generated. In one case (G^8^), the second clone was sensitive to cisplatin. The first clone (G**8^) may have contained a mutation or genetic loss as previously suggested for S^S^, but further work would be necessary to determine the mechanism of resistance. In the second case (S^8^), the second independent clone was also more resistant to cisplatin, although the reverse cross (S^8**) was sensitive. It seems unlikely that this anomalous result was generated by chance on two separate occasions, and it is possible that one of the parental lines contained a mutation which could result, under appropriate circumstances, in cisplatin resistance. Both the hybrids showing the anomalous resistance contained 8^, and it is conceivable that the neomycin gene had been incorporated in the host DNA at a site that could result in aberrant gene expression.
The data in this study showed that the hybrids between four testicular cancer cell lines had similar sensitivity to cisplatin as their parents. This indicates that there was no complementation between the four testicular cancer cell lines studied. It is therefore possible that the sensitivity of these cell lines to cisplatin could be accounted for by a single mechanism.