BMC Cancer 2006, 6:2 http://www.biomedcentral.com/1471-2407/6/2 Table 3: Retained BAC DNAs in cells from recurrent tumor selected for resistance to 10 µ g/ml BCNU. Cell Line # of signals 221H1 212A2 390B3 150C2 229A8 250D10 397C4 268H5 1109B5 299D3 DIR LXR MER 0 1 2 3 4 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 7 9 25 35 18 7 5 10 25 18 20 24 6 2 0 7 36 48 29 5 3 0 0 9 15 39 31 10 2 5 7 26 11 15 36 5 0 0 15 41 20 24 13 5 0 0 7 19 41 24 8 6 12 33 5 9 31 4 0 0 3 30 46 22 7 17 4 1 10 15 37 29 8 3 4 3 32 11 15 25 9 2 2 11 39 21 26 6 3 1 0 3 41 29 21 5 0 0 0 39 5 16 28 21 2 1 2 25 29 41 5 9 3 1 3 55 26 13 4 0 0 0 26 31 19 15 24 3 2 7 19 34 31 10 7 9 6 3 3 18 40 14 0 0 0 29 9 28 9 18 7 4 9 10 39 37 10 6 5 2 2 4 25 32 19 0 0 0 41 8 35 14 12 3 2 7 8 42 45 17 2 4 3 3 9 19 38 16 0 0 0 32 12 17 27 12 2 5 9 14 47 33 12 8 2 4 6 11 34 46 3 0 0 0 24 11 38 14 15 0 2 4 13 59 39 6 5 2 2 began by confirming previous karyotypic results demon- strating over-representation of part or all of chromosome 22 in cells selected for resistance in vitro or in vivo (recur- rent tumor) using whole chromosome paint (WCP) for chromosome 22. There was an increase in the number of fluorescent signals from chromosome 22 in cells selected for resistance to BCNU in vitro or in vivo when compared to cells from primary, untreated tumor. Fragments of chromosome 22 were particularly evident in cells from recurrent tumor MER (Figure 3). To identify the region of chromosome 22 that is over-rep- resented in BCNU resistant cells, we began with pooled BAC probes (Figure 1). Whereas over-representation of BAC DNA sequences from all four regions could be found in some cells such as MER, over-representation of sequences from BAC pools C and D was particularly evi- dent in cells from all recurrent tumors, with an increase seen when these cells were further selected in vitro for BCNU resistance as shown in Figure 4 for tumors ME/ MER. In this figure, the black bars representing BAC pool D can be seen to shift to higher signal numbers when the cells are selected for BCNU resistance. In fact, when cells from recurrent tumor were selected for resistance to BCNU, the region most commonly over-represented cor- responded to BACs in the C and D groups (Figure 4). Logistic Regression was used to compare the portion of each of the signals in each group (A, B, C, D) of chromo- some 22. Analyses were then done to demonstrate statis- tical significance of the results of group A & B compared with C & D. PROC logistic regression was used to analyze ME mock versus ME drug and MER mock versus MER drug. The shift of signals from BAC clone groups C & D in ME mock versus MER mock was statistically significant (<.0001). These was also a statistically significant differ- ence between BAC groups A & B versus C & D when ME mock is compared to ME drug (.0122). Finally, the shift in signal number between MER mock and MER Drug was also statistically significant (<.0001). To further map the region(s) of chromosome 22 retained in cells from recurrent tumor and in cells from recurrent tumor selected for resistance to BCNU, we used individual BACs as FISH probes (Table 3 and Figure 5). This work demonstrated that the region retained most frequently is between BACs bk212A2 and bk299D3, which corre- sponds to 22q12.3–13.32. BACs outside of this region did not show frequent retention in these cells. Differences in the specific pattern of BAC representation in each cell line (Figure 5) provides evidence that the observed similarities are not due to cross-contamination of the cell lines. Discussion Genetic analyses of primary human malignant gliomas have demonstrated numerous alterations including the over-representation of chromosome 7, and normal or under-representation of chromosome 22 in primary untreated glioblastomas multiforme [18]. Molecular Page 7 of 10 (page number not for citation purposes)