BMC Cancer 2006, 6:2 http://www.biomedcentral.com/1471-2407/6/2 Table 2: Karyotype analysis of primary/recurrent tumor cells Clonal Designation Karyotypic deviation DI DIR ME MER LX LXR 45,X,-X,+7,-10[9]/45,idem,-9,der(9)t(9;11)(p22;q21),-11[13]/45,idem,-9,der(9)t(9;11)(p22;q21),-11,-14,-18,del(11) (q21),der(14)t(14;18)(P11;q12),der(22)t(22:?)(p11;?)[44]/90,idem ×2[5]/88,idem ×2,+6,+11,-11,+15,15,+19 [cp12]/85- 86,idem,+X,-3,+5,-8,+11,-11,+12,+12,-13,+15,-15,i(15q),-19,+20,-20,+21[31] non-clonal cells [1] 46,XX[13] 45,X,-X,+7,-9,-10,-11,-14,-18,der(9)t(9;11)(p22;q21),del(11)(q21),der(14)t(14;18)(P11;q12),der(22)t(22:?) (p11;?)[159] non-clonal cells[7] 45,XX,del(9)(p13),add(9)(p21),+9-13,-17[9]/47,idem,+mar1,+mar2,1-3dmin[6]/48,idem,+7[8]/49,idem,+mar3[13]/ 90,idem×2[5]/80-85,idem×2,-1,+8,-8,-10,+11,-11,+12,-12,-19,-21,-21-22,+mar3,3-7dmin[cp38] non-clonal cells [9] 46,XX[6] 55,X,der(X)t(X:?)(q25;?),der(1)t(1;?)((p32;?),×2,+1,inv(2)(p13q21),der(2)t(2;14)(q23;q13),add(2)(p25.1),+2,add(3) (q29) ×2,+3,-4,del(5)(q13),-5,iso(7)(p11.1),der(9)t(9;1)(p22;p13),del(10)(p11.2), der(10)t(10;?)(q22;?),inv(11)(q13q23)×2,+11,- 12,-13,-15, -17,add(18)(q23),add(19)(q13.3), add(20)(q13.3),-22,+mar1,+mar2,+mar3,+mar4,+mar5[16]/56,idem, +7,[36]/ 57,+7,+mar6 [5] non-clonal cells [23] 56-57,XX,+1,-1,+2,+2,+6,-6,i(6)(p10),+7,+7,+8,+9,der(9)t(9;12)(p13;q11),ins(9;?)(p13;?),+11,+14,+15,+16,- 17,del(17)(p11.2),+21,-22,2-3mar[cp11]69,XX,+1,+3, add(3) (q29),x2,+3,+3,+4,del(5)(p13.3p15.1)del(6)q22)×2,+6,+7,+7,+7,del(9)(p13) ×2,+11,+11,+12,+15, +15,-17,-17,+18, +18,+20,+20,+21,+21,+mar3,+mar4,+mar5,[21]/68,idem,-10,-mar3,+mar5[19]/67,idem,-3, -10,+17,-mar3,+mar6[10] non-clonal cells [6] 46-49,XX, +1, -1, del(1)(p13), del(1)(q12), del(1)(q25), der(?)t(1;?)(q21;?),-2, -2, der(2)t(2;9)(p13;q13),der(2)ins(2;4)(q11.2;12q34), add(2)(q31), -3, add(3)(q21), del(3)(p22;p23),-4, der(4)t(4;8)(q13.2;q11.2), -5, del(5)(p15.1;p15.2), add(5)(q11.1), -6, der(6)t(1;6)(p11;q11), del(6)(q21), add(7)(q21.2), +der(7)t(7;?)(q11.2;?), -9, add(9)(q32), -10, +11, 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, 5, -6, mar[cp41] non-clonal (16) Cells were re-treated with 10 µ g/ml BCNU every 8–10 pas- sages to maintain the resistant phenotype. Karyotype analysis Karyotype analysis was done on low passage cells as described previously [13]. Briefly, mitotic cells from mock-treated or BCNU-resistant cultures were harvested by banging the sides of the flasks to release dividing cells into the medium. Cells were pelleted, swelled using 0.075 M KCl at 37°C for 17 min and 22 sec, and fixed with methanol/acetic acid (3:1). Cells were then dropped onto slides, rinsed with methanol/acetic acid, dried, and stored until use [14]. The nomenclature follows ISCN recom- mendations [15,16]. Chromosomal abnormalities were classified as clonal if two or more metaphases had an identical structural abnormality, or five or more met- aphases had gained or lost a specific chromosome. Non- clonal cells represent cells in which unique structural arrangements, telomeric fusions, somatic exchanges and/ or different states of chromosome contraction occur. Probes for FISH analyses Whole chromosome paint for chromosome 22 (Vysis, Inc., Downers Grove, IL) was used following conditions specified by the manufacturer. Bacterial artificial chromo- somes (BACs) spanning the q arm of chromosome 22 at 2-megabase intervals (2 Mb BAC Set; a kind gift from The Sanger Institute, Cambridge, UK) were used to identify the regions of chromosome 22 retained in BCNU resistant cells (Figure 1). The BACs were grown and purified according to standard protocols [17]. To verify that there was no cross hybridization to other chromosomes, BACs were tested against CGH Metaphase Target Slides which contain metaphases derived from karyotypically normal male cultured lymphocytes (Vysis Inc.). Labeling with SpectrumGreen™ or SpectrumOrange™ was done with a Nick Translation kit (Vysis) under conditions specified by the manufacturer. For identifying the region of chromo- some 22 retained in BCNU-resistant cells, BAC DNAs were individually labeled and tested prior to being sepa- rated into one of 4 groups (Figure 1). Pooled probes con- tained 0.1 µ g of each BAC DNA, 5 µ g salmon sperm DNA, and 6 µ g Cot-I DNA. Individual BAC probes contained 0.1 µ g of BAC DNA, 5 µ g salmon sperm DNA, and 1 µ g Cot-I DNA. To verify the hybridization efficiency of our probes the BACs were separated into 2 groups (Pools A+B and C+D) and tested using CGH Metaphase Target Slides (Vysis). Ninety five percent (102/107 cells counted) of the met- aphase spreads were diploid when pool A+B was used as probe and 94% (113/120) were diploid using C+D. Cell preparation and FISH analysis Mitotic cells from mock-treated or BCNU-resistant cul- tures were harvested by banging the sides of the flasks to Page 4 of 10 (page number not for citation purposes)