H y b r i d s M o u s e C h r o m o s o m e N u m b e r H u m a n C h r o m o s o m e N u m b e r Average ±SD Proportion of mouse parental chr.(%) Proportion of Mouse chr. In the hybrid ( % ) AveragelSD Proportion of human parental chr.(%) Proportion of Human chr. In the hybrid (%) F9pBABE 3 9 . 5 1 1 . 5 M G H U l pSV2NEO 8 2 . 8 1 5 . 3 M G F 9cl 3 1 . 7 1 5 . 9 7 9 . 9 1 1 5 . 2 2 9 . 5 1 4 . 4 7 5 . 4 1 6 . 2 8 8 . 7 1 7 . 3 7 0 . 4 1 3 . 7 MGF9c2 3 2 . 1 1 5 . 5 8 2 . 2 1 1 3 . 2 2 9 . 3 1 2 . 5 7 8 . 6 1 6 . 9 9 2 . 4 1 8 . 2 7 1 . 2 1 2 . 9 M GF9c3 3 2 . 7 1 7 . 0 7 7 . 1 1 1 4 . 5 2 9 . 8 1 2 . 6 7 9 . 8 1 1 4 . 5 8 6 . 9 1 1 1 . 1 7 0 . 2 1 2 . 6 M GF9c4 2 9 . 9 1 5 . 5 7 6 . 7 1 1 4 . 1 2 8 . 3 1 4 . 1 7 5 . 3 1 6 . 1 8 7 . 5 1 7 . 1 7 1 . 7 1 4 . 1 M GF9c5 3 1 . 0 1 4 . 6 7 9 . 4 1 1 1 . 9 2 9 . 8 1 1 . 4 7 3 . 5 1 1 3 8 5 . 5 1 1 5 . 1 7 0 . 7 1 1 . 7 M GF9c6 3 4 . 1 1 6 . 9 8 4 . 9 1 1 6 . 3 3 0 . 5 1 2 . 9 7 5 . 1 1 7 . 3 8 7 . 3 1 8 . 4 6 9 . 5 1 2 . 9
Results were derived from minimum o f 10 metaphase cells of each cell line. Chr. Chromosome
FIGURE 6.4
CHROMOSOME ANALYSIS OF THE HYBRIDS AND THEIR PARENTAL CELL LINES
F9pBABE MGHUlpSV2NEO
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MGF9c2 MGF9c5
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FISH was performed on the parental and hybrid metaphase cells using Biotin-labeled human cot-1 DNA as probes. Two representative graphs of the hybrids are shown. The fluorescence-stained chromosomes (green) are human and the red-stained chromosomes are mouse in origin.
6.3 DISCUSSION
In this study, hybrids between a cisplatin-sensitive mouse embryonal carcinoma cell line (F9) and a cisplatin-resistant human bladder cancer cell line (MGHUl) were generated with the aim o f identifying human chromosomes responsible for cisplatin resistance. The resistant parental line MGHUlpSV2NE0 was approximately 11-fold more resistant than the sensitive parental line F9pBABE, providing a large difference in sensitivity to cisplatin.
All the hybrids except MGF9c5 were intermediate in cisplatin sensitivity between F9pBABE and MGHUlpSV2NE0, as observed in previous studies when F9 was fused with another human tumour cell line HT1080 (see Chapter 4.2.1). The four most resistant hybrids (MGF9c3-6) and the two most sensitive hybrids (MGF9cl and 2) were studied for the presence o f human chromosomes using FISH. The results indicated that by passage 10 there was loss of both mouse and human chromosomes in the hybrids. The results are in contrast to previous studies that found little loss of mouse chromosomes in mouse-human hybrids (Illmensee et al, 1978; Cassingena et a l, 1992; Mirakhur et a l, 1996).
The reason why MGF9 hybrids retained the majority of human chromosomes and lost some mouse chromosomes is unknown. All of the six hybrids showed similar results. Interestingly, although different numbers of human and mouse chromosomes were observed in each hybrid, the proportional distribution of the chromosomes in all six hybrids was similar at approximately 70% of human chromosomes and 30% of mouse chromosomes. Technical artifacts could result in some metaphase chromosomes being lost during the preparation. However, a total of 60 metaphase cells from 6 independent hybrid cell lines were examined and consistent data were obtained.
In a previous study (lllemensee et a l, 1978), it was shown that when mouse teratocarcinoma cells were fused with a human fibrosarcoma cell line (HT1080-6TG), the resulting hybrid cells segregated human chromosomes quickly and retained only one to three human chromosomes stably. Similar observations were also reported when the HT1080 cells were fused with established mouse cell lines (Croce, 1976). However, contradictory results were obtained by Miller and co-workers (1976), when these experiments were repeated and a
preferential loss of mouse chromosomes was observed. Another study o f the hybrids between HT1080-6TG and mouse teratocarcinoma cells (Ajiro et a l, 1978) showed that
some hybrids underwent segregation of human chromosomes while others showed loss of mouse chromosomes. However, all these studies showed that there was a higher percentage of loss o f human chromosomes than mouse chromosomes in the hybrids.
In previous studies, the techniques used to identify human chromosomes, such as trypsin/Giemsa staining or C-banding (chromosomes stained by formamide standard saline citrate), might not have been sufficiently sensitive. In present study, human Cot-1 DNA was used as a probe to detect human chromosomes in the hybrids. This probe hybridizes to human specific repeat sequences. In a similar study, however, loss of human chromosomes was observed in hybrids between a mouse fibroblast cell line (A9) and a human ovarian carcinoma cell line (2008/C 13*) (Mirakhur et al, 1996). The human chromosomes were detected by FISH analysis using human total DNA as a probe.
It has also been claimed that it is difficult to generate hybrids between human normal somatic cells and mouse embryonal carcinoma (EC) cells due to their phylogenetic distance and differences in differentiation potential (reviewed by Takagi, 1997). It was first noticed by Featherstone and McBumey (1981) that cell fusion between mouse EC cells and diploid somatic cells from other species failed to generate hybrids. They also failed to obtain any hybrids between mouse EC cells with human cell line such as Hela or a diploid human cell line IMR90 (McBumey and Rogers, 1982). When Takagi (1997) tried to generate hybrids between mouse EC cells with human lymphocytes, it was also failed. Several hybrids are known between mouse EC cells and human cells have been all generated from two cell lines, mouse embryonal carcinoma cell line, OTT6050 and human fibrosarcoma cell line, HT1080-6TG ( Croce, 1976; Meller et a l, 1976, Ajiro et al, 1978; Illmensee et a l, 1978). However, I had no difficulty generating such hybrids in this study and in the fusion performed in chapter 4 between F9 cells and the human fibrosarcoma cell line HT1080.
The aim of this study was to take advantage of the expected loss of human genetic material from the human-mouse hybrid cells in order to observe the effect of the remaining chromosomes on cisplatin sensitivity. Contrary to expectation and previously published
data, the generated hybrids retained a significant proportion of human chromosomes. Consequently, even though changes in sensitivity to cisplatin were observed, the high number of retained human chromosomes precluded the possibility that changes in drug sensitivity could be correlated to any individual human chromosomes in the hybrids.