MARCO CONCEPTUAL

OF THE CISPLATIN RESISTANT PRIMARY

a

Metaphase cells w^re^gg^ared from three cisplatin resistant primary transfectants F9cpc53, c87 and c l 3 3 / pcONAI/NEG plasmid was labelled with biotin and used as a probe for detecting the presence of the pcDNAI/NEO plasmid in these cell lines. Images a and b

were captured using a MRC 600 (BioRad) laser confocal microscope under an oil-merged lOOx objective lens. Image c was captured from a CCD camera using a Zeiss fluorescence microscope and a software from VISYS.

FIGURE 4.5d

ENLARGED IMAGE OF F9cpc53

The image was captured from a CCD camera using a Zeiss fluorescence microscope and software from VISYS under a lOOx oil-merged objective.

4.3 DISCUSSION

The aim of this study was to clone genes conferring cisplatin resistance to F9 cells. Firstly, the conditions for fusion of F9 and HT1080 cells were established and the cisplatin sensitivity of the resulting hybrids was measured. The results indicated that the hybrids were more resistant to cisplatin than F9 cells. The preliminary experiments indicated that HT1080 cells contain genetic material that confers greater resistance to cisplatin than F9 cells.

The fact that the control transfection resulted in 42 colonies suggested that the selection procedures applied in this study allowed a low background of parental cell survival. This provided an opportunity for selection of cisplatin-resistant primary transfectants after the transfection o f the cDNA library. However, this selection resulted in the total isolation of 270 primary transfectant colonies indicating that the cisplatin selection could be harsher so that the amount o f secondary selection work would be reduced. If there were genes conferring cisplatin resistance to F9 cells presented in the HT1080 cDNA library, they would have been expected to be included amongst the 270 colonies isolated.

Following the secondary selection, five primary G418 resistant transfectants, which showed increased resistance to cisplatin compared to the untransfected parental cell line F9, were isolated. FISH analysis showed that a maximum of 3 different integration sites of the pcDNAI/NEO plasmid were detected in the 3 primary transfectants. Despite the fact that a large number (88) o f the secondary transfectants were tested, none o f them showed cisplatin resistance when compared to F9. These results suggest that the cisplatin resistance in the primary transfectants was not due to the effect of the transfected DNA from the library.

It is possible that the inserted DNA was damaged during the generation of genomic DNA from the primary transfectant and that this resulted in its inability to induce cisplatin

resistance in the secondary transfectants. However, the maximum size o f the cloning plasmid pcDNAI/NEO plus the inserted DNA in the library was 12kb, and a high percentage DNA isolated from the primary transfectants was sheared into fragments of 25- 50Kb (see Figure 2.3). This meant that the G418 and cisplatin resistance genes remained associated in a large proportion of the fragments. It is also therefore likely that the cDNA

postulated to confer increased cisplatin resistance in the primary transfectants had also been transferred to the secondary transfectants. Even if events such as transfection-induced mutations, unexpected recombination of the transfected plasmid or instability of the transfectants had occurred during the secondary transfection, as reported in previous studies (Drinkwater et al, 1986; Roth et a l, 1985; Wigler et a l, 1979), the number o f secondary transfectants tested (88 clones) should have been enough to exclude these possibilities.

There are several possible explanations for generating o f cisplatin-resistant primary transfectants that are not due to the effect of the transfected insert DNA.

It has been shown that spontaneous mutations occur in tumour cells in vitro which could result in drug resistant variants in the tumour cell population (Goldie and Goldman, 1979; Cifone and Fidler, 1981; Sager et a l, 1985; Otto et a l, 1989). It is possible that the cisplatin resistant primary transfectants isolated in this study might be cells carrying mutations in the transfected cell population. These pre-existing mutant cells were then selected by cisplatin. Thus we were unable to transfer cisplatin resistance to the secondary transfectants.

Acquired cisplatin resistance can also be generated by continuous exposure o f cisplatin to the sensitive cells over a period of a few months (Kelland et al, 1992). It is also possible that the cisplatin resistant primary transfectants were generated by the cisplatin selection procedures used in this study. However, the transfected F9 cells were only exposed to two doses of cisplatin over a 4 day period so it is unlikely to induce acquired resistance. This is supported by a study on doxorubicin induced resistant cells (Chen et al, 1994). Human sarcoma cells were treated with a single dose of doxorubicin and the resistant colonies were selected. Using Luria-Delbruck fluctuation analysis, it found that these resistant cells were spontaneous mutant cells selected by doxorubicin and not caused by the treatment of doxorubicin. This suggests that a single dose doxorubicin might not be able to cause either mutations or acquired resistance.

Another possible reason for the generation of cisplatin resistance in F9 cells might include the random integration o f the transfected plasmid. It has been shovm that transfection of pSV2NE0 and pCMVNEO markers into rat embryonal cells generated radioresistance in some of the transfectants (Pardo et a l, 1991). This group showed that the neo transfection

and the clonal selection process could modulate the radiation sensitivity of both mouse and human cells. Similar results have been observed in other studies (Arlett et a l, 1988; Green

et al, 1985; Mumane et al, 1985). Such effects could depend on a variety of factors such as plasmid integration into a regulatory element. In the present study, it is possible that the pcDNAI/NEO vector integrated randomly into the genome. Consequently a gene involved in the acquisition o f cisplatin resistance may have been mutated or activated by insertion of the plasmid DNA within the gene or adjacent to it, resulting in cisplatin resistance in the primary transfectants. When the secondary transfection was performed, the plasmid integrated into a different site in the genome, thus failing to confer cisplatin resistance.

It has been shown that when a plasmid containing 2 selective markers was transfected into human cells and the transfectants were selected for only one marker, only a small percentage of the transfectants retained the unselected marker in the cellular DNA after 4-8 weeks of culture (Mayne et a l, 1988). This suggests that while transfectants are selected in the selective medium, only the selective marker gene remains intact. It is possible that other genes linked to the marker might be altered, as they are not selected for. In the present study, the primary transfectants were selected for the neo marker for up to 8 weeks before isolation of DNA for secondary transfections. It is possible that some genetic changes had occurred during that period on the inserted DNA. However, all the primary transfectants retained their resistance to cisplatin even 5 months after they were isolated. This suggests that neither the non-selective conditions for the insert DNA nor long term culture had any effect on their resistance to cisplatin.

It is also possible that gene(s) which complement the sensitivity of F9 cells to cisplatin were either not present in the HT1080 cDNA library or that human genes might not function well in mouse cells resulting in the failure of this study. This was observed in the HF hybrids that showed less resistance than the parental line HT1080 (see Figure 4.3). However, the fact that hybrids of F9 and HT1080 cells showed increased resistance to cisplatin compared to F9 allowed the selection of the resistant transfectants from the F9 cell background. This raises another possibility that there might be more than one gene involved in the sensitivity of F9 cells to cisplatin. It may explain the fact that while cell hybrids o f F9 and HT1080 showed some complementation of the sensitivity of F9 to cisplatin, we failed to achieve it when individual cDNAs from HT 1080 library were transfected into F9 cells.

Future studies should also include using the same approach with a different cDNA library derived from a human cell line that is more resistant to cisplatin than HT1080 cells. This may allow more efficient selection of the transfectants. Or a mouse library instead of human library should be used to allow the full complementation of cisplatin sensitivity in F9 cells. Alternatively, different approaches can be used for identifying genes responsible for complementing the sensitivity of testicular tumour cells. The following chapters will outline several alternative strategies.

Chapter 5