BMC Cancer 2006, 6:2 http://www.biomedcentral.com/1471-2407/6/2 FCihgruormeo1some 22 ideogram Chromosome 22 ideogram. Chromosome 22 ideogram showing the size and location of the individual BACs and the 4 grouped BAC pools used in this study. a bp position obtained from The Sanger Center; b set denotes BAC pools. Within a year all three required surgery for recurrent tumor (Table 1). Primary tumors were given a random 2-letter code desig- nation, and the recurrent tumor from the same patient received the same code with the addition of "R" (ME/ MER, LX/LXR, DI/DIR). Cell lines were derived as described [10,11] and grown in Waymouth MAB 87/3 medium (MAB) with 20% fetal calf serum (FCS). The pas- sage number of the cells used for all experiments varied (LX/LXR 15–32, DI 5–25, DIR 15–40, ME 18–41; MER 52–120); however, the results of repeats done at different passage numbers were consistent. Cells were selected for resistance to 10 µ g/ml BCNU as described [10,12], and mock treatment of the cells was done in parallel. Briefly, cells were washed with MAB without serum 3 times; they were then mock-treated using MAB without serum or drug, or treated with BCNU in MAB without serum for 1 hour at 37°C with 5% CO2. Cells were washed and fed with MAB containing 20% serum. The cells were treated with the same concentration of BCNU for three consecu- tive days, after which the cells were allowed to grow. This step was repeated several times until the resulting cell population was resistant, as evidenced by the absence of cytopathic effect and cell debris in the media which would indicate cell death after treatment when compared to the mock-treated controls. When cells appeared resistant to a FKiagruyoretyp2es Karyotypes. (A) Representative karyotype from the pri- mary LX tumor cell passage 2. Karyotypic deviation: 57XX, +2, i(6)(p10), +7, +7, +8, +9, der(9)t(9;12)(p13;q11)ins(9;?)(p13;?), +11, +11, +12, +14, +15, +16, -17, del(17)(p11.2), +21, -22, + mar. (B) Repre- sentative karyotype from the recurrent LXR tumor cell pas- sage 3. Karyotypic deviation: 49XX, del(1)(q12), +der(2)t(2;9)(p13;q13), der(3)ins(3;?)(p23;?), add(3)(q21), der(4)t(4;8)(q13.2;q11.2), -5, add(5)(q11.1), -6, der(6)t(1;6)(p11;q11), +add(7)(q21.2), -9, add(9)(q32), -10, add(11)(p11.2), -12, add(12)(q24.3), -13, add(15)(q21.2), -16, del(17)(p11.2) ×2, +add(20)(q11.1), -21, +22, +22, +5 mar. particular dose of BCNU, subsequent treatment was done at a higher dose (2.5, 5.0, 7.5, 10.0 µ g/ml) until cells were resistant to 10 µ g/ml. The time required to select for a resistant cell population varied for the different cell lines. For example, DI cells required approximately 3 months to get a cell line fully resistant to 10 µ g/ml BCNU. Tumor cell lines LX and ME required 4–6 months. Selection for resist- ant cells from recurrent tumors (DIR, LXR, MER) was complete in 2–3 months for all cell lines, reflecting the higher level of intrinsic resistance in cells from the recur- rent tumors compared to cells from the primary tumor. Page 3 of 10 (page number not for citation purposes)