Chromosome Changes Characterizing in Vitroresponse To

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(CANCER RESEARCH 53, 5542-5547, November 15. 1993] Chromosome Changes Characterizing in Vitro Response to Radiation in Human Squamous Cell Carcinoma Lines1

Janet M. Cowan,2 Michael A. Beckett, and Ralph R. Weichselbaum

Department of Radiation and Cellular Oncology, University of Chicago Medical Center, Chicago, Illinois 60637

ABSTRACT tors including tumor microenvironment and inherent (genetic) radia tion susceptibility of cells within a tumor. Oxygen tension and pH We have analyzed the karyotypes of a series of 16 cell lines derived from have been shown to modulate cellular response to radiation, and human squamous cell carcinomas of the head and neck. When the cell changes in the tumor microenvironment can influence blood flow, lines were grouped according to their in vitro response to radiation, it was observed that some recurrent chromosomal changes occurred with differ which in turn changes these parameters. RR cells have been identified ing frequencies between groups. Radiation resistance was associated with in primary cultures of head and neck tumors (22, 23). As the majority clusters of breakpoints in bands Ip22, 3p21, and Spii.i and deletion of of solid tumors examined by molecular and cytogenetic techniques distal 14q, while relative radiation sensitivity was associated with a high have evidence of genetic alterations, it is likely that radiation resis frequency of breakpoints in Ilql3 and duplication of distal I4q. The tance arises as a result of those alterations. regions identified point to the possible locations of genes involved in the Cellular irradiation produces a number of lesions within DNA, response to radiation and could provide a series of markers with which to including single- and double-strand breaks. It is thought that dsb are predict response to radiation therapy. the major lesion, and many studies of radiation response have fo- cussed on dsb repair (24). A study of human squamous cell carcino INTRODUCTION mas of the head and neck noted differences in the initial amount of Cytogenetic analyses of solid tumors have shown that in some types damage (particularly dsb) induced in DNA between RR and relatively of tumor recurrent chromosome changes can be identified, and these RS lines and differences in their ability to repair dsb (25). RS lines had can be used as an aid in differential diagnosis of the tumor (1). more initial damage (measured by DNA neutral elution) and repaired Karyotype changes also have been correlated with outcome (2). For damage more slowly than RR lines. DNA conformation was found to the majority of solid tumors, however, the karyotype changes ob differ between the lines, suggesting that this may influence the initial served are complicated with many unbalanced translocations but no amount of damage sustained and the ability of the cell to repair the specific tumor translocation. The unbalanced translocations result in a damage (26). In addition to the variation in the amount of damage loss and gain of whole or parts of many chromosomes. The duplica induced and the rate of damage repair, it has also been reported that tions and deletions are not random but rather indicate the chromo RR cells spend longer in G2 than RS cells following irradiation (27, somal position of potential tumor suppressor genes (deletions) or 28), suggesting that the response to radiation may involve alteration of oncogenes (duplications) involved in tumorigenesis. cell cycle control genes. HNSCC3 cell lines and cells from primary cultures have shown that In a previous report, we described recurrent chromosome changes the majority have complex karyotypes with typically more than 50% consistent with a multistage model of tumorigenesis in a series of 10 of chromosomes in any karyotype involved in rearrangement (re cell lines derived from HNSCC (3). We have expanded that work to viewed in Ref.3). Recurrent deletion of parts or all of chromosome include an additional 6 lines and have identified karyotype changes numbers 1, 3, 4, 7, 8, 9, 10, 11, 13, and 18 have been reported (3-12). that correlate with an in vitro response to radiation. Clusters of translocation breakpoints have been reported in chromo some 1 band Ip22 and chromosome 11 band ql3 (3, 13). The karyotype changes seen in HNSCC show similarity to those MATERIALS AND METHODS seen in esophageal and lung tumors with deletion of 3p being a change Cell Lines. Cell lines were established according to methods published common to all three tumor types (14-16). As tobacco use has been previously (29, 30). Table 1 summarizes details of the site of the tumor, Du, implicated in the etiology of all of these tumors, the observations SF2, modal chromosome number, and ploidy of each line used in this analysis. suggest that chromosome 3p may be the site of a gene preferentially Cell lines are listed according to their Dn, from most sensitive to most resistant. affected by a component of tobacco smoke. Additionally, amplifica Karyotypes of 10 lines used in this analysis have been reported previously (3). tion of a group of putative oncogenes on chromosome Ilql3, HST1, X-ray survival curves and cytogenetic harvest were undertaken on cells of the BCL1, INT2, and PRAD1, has been reported in HNSCC and esopha same passage whenever possible. If different passages were used, there was no geal cancer (17-20). more than 3 passages difference. Despite emphasis on improving early diagnosis, survival rates for Cytogenetic Harvest and Analysis. Cells in an exponential growth phase patients with head and neck tumors have not improved dramatically in were harvested according to methods published previously (3). Following G-banding using trypsin-Giemsa, 15 metaphase spreads were photographed recent years. One major problem in designing treatment protocols is and analyzed fully for each line. Metaphase spreads were selected for analysis the spontaneous occurrence of radiation resistance, resulting in the based on technical quality. failure to achieve local control of disease. Investigators report that, In order to identify regions of chromosomal loss or gain in HNSCC lines, overall, in vitro radiation resistance is associated with poor prognosis each line was assigned a ploidy number when the karyotype was completed. (21). Tumor response to radiation therapy involves a number of fac- Ploidy was primarily based on the number of chromosomes in the modal cell population and was rounded to the nearest multiple of 23. However, the num Received 8/28/92; accepted 9/14/93. ber of copies of each chromosome was also taken into account. For instance, The costs of publication of this article were defrayed in part by the payment of page a cell line with a count of 57 and 2 copies of each of 13 or more of the 22 charges. This article must therefore be hereby marked advertisement in accordance with autosome centromeres would be diploid. However, if there were three copies 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported in part by The Center for Radiation Therapy, The Chicago of most the chromosomes and one copy of a few chromosomes, the cell line Tumor Institute, and N1H Grant CA 42596. would be triploid. This method of deriving the number of copies of a chro 2 To whom requests for reprints should be addressed, at Cytogenetics Laboratory, New mosome expected was used because it seemed more likely that aneuploidy England Medical Center, 75ÃœWashington Street, Box 188, Boston. MA 02111. 3 The abbreviations used are: HNSCC, head and neck squamous cell carcinoma; dsb. arises as a result of aberrant mitotic separation, such as tripolar mitoses, than double-strand breaks; RR. radiorcsistant; RS. radiosensitive. by simultaneous duplication of the majority of chromosomes within a cell by 5542 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1993 American Association for Cancer Research. KARYÃœTYPE CHANGES CORRELATED WITH RADIOSENSITIVITY

Table 1 Details of head and neck cell lines studied, including site of primary, in vitro Dlf, SF,?, modal chromosomal number, passage, and assigned ploidy of line

LineSCC ofprimaryTongueTongueTonsilLarynxParotidParapharyngealTonsilPyriform(Gy)1.071.421.461.541.541.561.811.852.172.172.262.272.272.392.562.633.33SF2Â«0.2680.3590.3500.3910.5370.5790.6500.4690.5320.5320.5090.5720.5630.5690.4000.6740.727Modal'no.(range)46 oflinec2N4N2N3N3N2N3N3N4N4N4N3N4N3N3N2N3N

61dSCC (45^*6)72 25"*SO (59-78)52 90HNSCC (48-58)69(64-71)73 28HNSCC 152HNSCC (62-74)49 255HNSCC (47-50)70 167aSCC (68-73)65 35rfHNSCC sinusTongueTonguePyriform (63-69)82 rfHNSCC 151 (75-85)82 rfSOSlÂ«*HNSCC151 (75-85)85 sinusHard (84-92)77 I35dHNSCC palateTongueLarynxTongueNasal (70-80)80 294rfSQ (77-81)68 20BrfHNSCC (64-69)55 58JSQS''HNSCC (53-58)57 vestibuleOropharynxDo (56-62)70 143Site (67-74)ChromosomePassage*MidLateLateLateMidLateEarlyMidEarlyLateEarlyEarlyLateLateLateMidPloidy " Fraction of cells which survive 2 Gy. ''Early, ^7 passages; mid. 7-15 passages; late. ^:1 passages. '' 2N, diploid; 3N. triploid; 4N, tetraploid. '' Previously reported (3). some other mechanism. Mitotic nondisjunction and tripolar mitoses have no chromosomal rearrangements were seen in common among the been described as characteristics of tumor cells (31). lines. Intermetaphase variation was observed in some cell lines with Survival Curves. The D0 for each line was determined with X-ray survival additional marker chromosomes and/or losses or gains of one or two curves as published previously (22). Briefly, exponential phase cells were irradiated with a GE Maximar X-ray generator at 250 kV peak and 26 mA, whole chromosomes in some but not all metaphases. There was no yielding a dose rate of 107 rads/min (1 rad = 1 cGy). The cells were grown for clear cosegregation of additional or missing chromosomes and/or an additional 18-24 days after which they were fixed and stained with crystal marker chromosomes in any of the lines. The intraline variation was violet. Colonies of >50 cells were scored as survivors. Dn. the inverse of the always less than the interline variation. The consensus karyotype of slope of the radiation survival curve, was calculated from the results of 2-4 each line included all markers seen in at least three cells. As it was not experiments. Also, cell survival was assessed by a second measure, SF2, the possible to identify the components of all marker chromosomes in all fraction of cells surviving irradiation with 2 Gy. lines with G-banding, there is underscoring of some breakpoints and some regions of deletion/duplication. There was no correlation be RESULTS tween karyotype homogeneity in the cell lines and Dâ€ž. As reported previously and illustrated in Figs. 1-4 , the karyotypes Eight lines were found to have D0 of <1.85 Gy (relatively RS) and of the cell lines were extremely complex (3). However, consistent 8 lines had Dâ€žof>2.17 Gy (RR). Relative RR was defined as a Â£>â€ž rearrangements could be identified in all cells of each line, although of greater than the upper limit of the range of D(, for normal fibro-

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6 10 11 12 1 N! 'i 13 15 16 17i u18 21 22 19 X? Fig. 1. Representative karyotype of cell line SQ9G. Modal chromosome number, 53 (diploid). This karyotype was interpreted as: 56, X, ?X, der(l)t(l;7Xp34;pl3), del(2)(q23). der(2)l(2;3)(q31;q21), + der(2)t(2;12Xpl3;pll.2), + i(2p), i(3q), der(4)t(4;?9;ll)(4pter-*q21::9ql3^q34::llql3^qter), 5q + , der(5)t(5;?llXqll.2;ql3), + del(5)(qll.2), del(6)- (q23), + del(6)(q21), der(7)t(7;ll)(q22;ql3), + del(7)(qll.l), + psu dic(7) t(7;ll;?13)(llqter^llql3::i(7p)::llql3^11q23::13ql2â€”qter), + der(8) t(8;?22Xpll.2;qll.2), dup(9)- (qll.2q21.2), der( 10)1(10;l())(pl 1.2;q22), psu dic(lI)t(tl;19)(q21;pl3.1), psu dic(ll)t(7;ll;22) (Ilpter^llq23::7q22--7q32::22pll.2 ->22qter), der(l2)t(12;?14)(q24.1;q24), Â¡(13q), der(14)t(14;21)(P11.2;qll.2), 7del(15)(q22q24), + i(15q), -16, + der(17) t(?6;17)(q21;pl3), der(18)t(18;?)(ql2.2;?), 19p+, + der(19) I(19;?)(pl3.1;?), -I- 20, -22, der(22)t(14;19)- (qll.2;pl3.1) + ring. In this cell, arrowheads indicate telomeric association between 3 and 19; 11 and 13; 12 and 21; and 14 and der(19). This cell also has a ring chromosome (r) not seen in other cells. 5543

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16 17 18 13 15 Â». Â« I JKÂ» m 19 20 21 22 Fig. 2. Representative karyotype of cell line HNSCC 28. Modal chromosome number, 69 (triploid). Karyotype interpreted as: 69, X, -sex, -sex, del(lXpll), 2 X der(2)t(2;15)- (pl3;q21.2). der(3)t(3:5)(pll;pl2), del(4Xq31.3), psu dic(4)t(4;9Xq21;q34) 4ps+, + der(4)t(l;4Xq23;q21) 4ps + , i(5p), + i(5p), + der(6)t(6;12Xql3;pll.2), + ider(6)t(2:6)- (2pter-*pll.2::6ql5 ->6cen::6cen->6ql5::2pll.2 >2pter), i(7p), +der(7)t(7;?Xqll.l:?), i(8q). der(8Xpter^q24.1::?HSR), der(l())l(10:14Xpll.l;q24), der(ll)t(9;ll;13)(llpter Â»ql3. 3::9qll -*9q22::13ql2 -Bqter), -13, der(13)t(13;13)(pl2;ql3), der(13)t(13;?)(pl2;?), -14, i(14q), -15, -15, der(15)t(l;15)(p22;pll.2), +16, -18, -22, 2 X i(22q), + 2 small markers.

blasts, which is around 1.80 Gy. When the second measure of cell resents one breakpoint occurring in one cell line, and multiple copies survival, 5F2- was used, the majority of lines remained within the of the same rearrangement within a line were scored once. same general grouping. The SF2 of a normal keratinocyte line grown It can be seen that the breakpoints were not distributed at random under the same conditions was found to be 0.424 (32). Five of 16 lines throughout the genome but instead tended to cluster, particularly in the had SF2 of <0.424, and 11 of 16 lines were relatively RR (SF2 > pericentric regions of heterochromatin. Breaks were considered to normal keratinocytes). cluster in a chromosome band if a20% of the breaks on a particular In order to identify chromosome bands that may be the sites of chromosome were in one band. For some chromosomes, however, the known or unknown oncogenes and which may be activated in HNSCC total number of breaks on the chromosome was sufficiently low that as a result of chromosomal breakage and translocation, all identified even one or two breaks constituted a cluster (e.g., chromosome 16). breakpoints were plotted on an ideogram. Fig. 5 illustrates the results The distribution of breakpoints differed between RR and RS lines when the lines were grouped according to D0. Each arrowhead rep (grouped according to DH), although the total number of breaks was

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Fig. 3. Representative karyotypc of cell line HNSCC 58. Modal chromosome number, 56 (triploid). The karyotype was interpreted as: 56, XY, -sex, der(l)- (lpter^pl3::HSR:lpl3 ^qter), deI(lXq25), + ?der(lXlp22-Â»lq21::HSR::?), der(2)t(2;8)(pl3;ql3), i(2p), der(3)t(3;3Xpl3;q25), -4, -4, 2 X Â¡(5p),+ del(5)(ql3). der(6)t(6;7)- (6pter Â«6cen::7cen >7pler), Â¡(6p).der(7)t(l;7)(7pter .7plO::lqlO Â»Iqter), -8, i(8q), -9, i(9p), -10, der(10)t(5;10Xqll.l;pll.2). del(ll)(ql4q21), + der(ll)t(6;ll)(ql5;ql4.2). der(12)t(?5;12)(qll.l;pll.l), -13. der(13)t(13;15)(pll.l;qll.l), der(13)t(?9:13)(ql3;q21.2), der(14)t(14;?)(q32;?), -15, der(15)t(?8;15)(pll.2;pll.2), der(15)t(5;15)(ql3;pll.2), -16, -17, -18, -18, der(18)l(?4;18)(q21;pter), +der(19)t(19;22)(ql2;qll.2), 20p + , -21, -21, -21, -22, -22, der(22)t(9;22)(9pter >9cen::22cen Â«22qter).

5544 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1993 American Association for Cancer Research. KARYOTYPE CHANGES CORRELATED WITH RADIOSENSITIVITY IK) )l\ 4 5 Fig. 4. Representative karyotype of cell line HNSCC 151. Miniai chromosome number, 82 (tcl- raploid). The karyolype was interpreted as: 82, XXXX, + der(X)l(X;?2Xp22.1;q31), dcr(l)t(I;7)- Â«*ufi w Â«l (Icen;pl3), -2, 2 X del(3)(ql2q25), -5, del(6)- (q23), der(7)l(7;l3)(pl5;ql4), i(8q), der(10)t- (10;13)(pl1.l;qll), der(10)1(10:22)(p11.1;ql1.2), der(ll)(llptcr >llql3::HSR). -12. psu dic(12)t- 10 11 12 (12;19)(pll.l;pl3.3), -13, -13, -15, -16, -18, -19, +20, -21, -21, -22. In this cell, der(1) is with the 7's. U Ã¨tti Iff ,.f i:;l 13 14 15 16 18 19

â€¢¿IÂ« 999 Ulf* â€¢¿â€¢¿ 19 20 21 22 similar in both groups (228 and 225 in RR and RS lines, respectively). group. Several apparent clusters became associated with the opposite Additionally, the frequency of isochromosome formation (indicated phenotype, e.g., Ipl3 cluster in D0 RR lines became a cluster in SF2 by the boxes with arrowheads) in RS lines was double that in RR lines RS lines, suggesting no phenotype specificity. Several clusters re (from 24 to 12). The major breakpoint clusters observed in RR lines mained associated with the same phenotype regardless of whether D(i were in Ip22, Ipl3, and the centromeric regions of chromosomes 7, or SF2 was used for sorting (Ip22, 3p21, and 8pll.2 with RR). The 13, and 14. For the RS lines, the major peak was in Ilql3 and the cluster observed in 1Iql3 was lessened, but 5 breaks were observed in centromeric regions of chromosomes 5, 13, and 14. 5 lines, compared with 6 breaks in the 11 RR lines (data not shown). The distribution of breakpoints differed slightly when lines were In order to determine the sites of possible tumor suppressor genes grouped according to SF2, with the 5 lines with the lowest SF2 which are most likely to be in regions of frequent deletion, a com becoming the RS group (SF2 <0.424) and the remainder the RR parison was made of the number of copies of a region present in a line with the number expected from the ploidy designation, and any de viations were scored as deletions or duplications. For each line, loss or SS SS ER ES gain of a chromosomal region was recorded once, although in many cases multiple copies of the region were lost or gained. Overall, deletions appeared to be more frequent than duplications. Fig. 6 shows RR RS the occurrence of duplication and deletion in all the cell lines ana lyzed, listed according to D0 and SF2, from low to high in each case. I: HRr"1 It can be seen that some deletions occurred in a majority of lines (a 10 of 16): deletion of 3p; 4p; 4q; 8p; lOp; 18p; 18q; and the short arm regions of the acrocentric chromosomes (13, 14, 15, 21, and 22). Two regions, 3q and 7p, were duplicated in the majority of lines. When the lines were sorted according to Â£>,,,itappeared that several chromo some changes were complementary between the RR and RS lines: 12p was duplicated in 3 of 8 RS lines and deleted in 5 of 8 RR lines; lOq was duplicated in 4 of 8 RS lines and deleted in 5 of 8 RR lines; distal ES SS RE SS 14q was duplicated in 3 of 8 RS lines and deleted in 5 of 8 lines. When "Â»Â¡S the lines were arranged according to SF2, changes in distal 14q con Â»HÂ« * tinued to segregate with phenotype, while those of lOq and 12p did not. Interestingly, 5 of 5 SF2 RS lines had 5q deletions, suggesting that this may be an additional region for investigation.

12 13 DISCUSSION

We and others have reported recurrent karyotype changes in cell RS 85 SB RS lines and primary tumors derived from HNSCC. The most frequent changes were deletions of 18q, lOp, 3p, 8p, duplication of 7p, and clustering of breakpoints in Ip and 1Iq (3-12, 33). Molecular studies on HNSCC cell lines and primary tumors have confirmed some of these cytogenetic observations: loss of sequences from the short arm 15 16 17 IB 19 20 2l 22 of chromosome 3, between bands 3pl4 and 3p25 (34); overexpression Fig. 5. Diagrammatic representation of the location of all identified breakpoints from 16 HNSCC cell lines. Breakpoints identified in RR lines are on the left of the chromo of the epidermal growth factor receptor, located on chromosome 7p some, and those in RS lines are on the right. Each arrowhead represents one breakpoint. (35^0); and amplification of a group of putative oncogenes on chro The boxes with arrows represent the formation of an isochromosome, with the arrow mosome Ilql3, HST1, BCL1, INT2, and PRADl (17-20). Other mo indicating which arm is duplicated. Multiple copies of the same marker chromosome in a line were scored once. lecular alterations identified in HNSCC, including overexpression of 5545 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1993 American Association for Cancer Research. KARYOTYPE CHANGES CORRELATED WITH RADIOSENSITIVITY

have been observed in yolk sac tumors (47) and the large cell diffuse 89101112 13 14 1516 17 18 19 20 21 22 lymphoma subgroup of non-Hodgkin's lymphomas (48). The RS SCC61 \AAAA breakpoint hotspot in Ilql3 is the site of an amplicon that includes SCC25 HST1, BCL1, INT2, and PRAD1, which has been demonstrated to SQ9G HNSCC28 have altered expression in HNSCC. The role, if any, of other genes HNSCC152 remains to be determined. HNSCC255 Differences in response to radiation between cell lines are likely to HNSCC167 be the result of alterations of many cellular systems, including DNA SCC35 HNSCC151 repair systems and possibly cell cycle control changes. The absolute SCB1 number of genes involved in each of the many mammalian DNA' HNSCC135 repair pathways is presently unknown, but complementation experi HNSCC294 ments between different RS mutant cell lines suggest there are 8-9 SQ20B HNSCC58 complementation groups (49). To date, only one X-ray response gene [SCO has been cloned, XRCC1, and it has been localized to human chro HNSCC143 mosome 19ql3.2 (50). The gene corrects a defect in single strand B. SF2 from graph intercept break joining in a hamster mutant cell line. Three other X-ray response 12345678 910111213141516171819202122 genes have been mapped by complementation studies: XRCC2 to chromosome 7; XRCC4 to chromosome 5; and XRCC5 to chromo some 2 (51-53). It has been suggested that chromatin structure differences may contribute to radiation response (26). Analysis of nucleoids in four of the lines studied showed differences between RR lines (JSQ-3; SQ20B) and RS lines (SCC25; SQ9G) consistent with alterations in supercoiling (26). Such changes may result in less efficient repair of damage in RS lines following irradiation as a result of altered or more relaxed DNA conformation. Additionally, alterations in chromatin structure may lead to centromeric replication errors and result in isochromosomes, which are thought to be the result of misdivision of the centromere (54). The more frequent isochromosome formation observed in RS lines (24 compared with 12 in RR lines) may be a result of such modifications. KEY: Subtle changes in cycle control could account for differences in response to radiation with sensitivity associated with a shortened deletion duplication I duplication and deletion in same arm period for repair. RS lines had frequent breaks in Ilql3, the band to Fig. 6. Regions of duplication and deletion within cell lines. Each region is scored which PRAD1 has been assigned (18). PRAD1 is a G, cyclin first once, although in many cases multiple copies of a region were gained or lost. In some cases, duplication and deletion were observed within the same chromosome arm. SCC61: identified in a subset of benign parathyroid tumors, which is also dup(20)(pl0^pl3), del(20)(pl3 .pier); SQ9G: dup(2)(ql0^q31) del(2)(q31^qter), amplified in mammary tumors (18, 20). Overexpression of this gene dup(6)(qlO-*q21) del(6)(q23^qter); /W5CC2Ã„: dup(2)(plO-Â»pll.2) del(2)(pl3^pter), del(13)(ql0^ql2) dup(13)(ql4^qter); HNSCCI52: dup(3)(pl3^p21) del(3)- may result in the failure of arrest in G, following irradiation with (p23-Â»pter); HNSCC167: dup(ll)(plO-^pl3) del(ll)(pl3^pter), del(13)(qlO-*q21) concomitant failure of completion of DNA repair in RS lines. dup(13)(q21^qter), dup(18)(ql() .q21) del(18)(q21^qter), dup(20)qlO-*ql3.1) del- (20)ql3.1^qter); SCC35: dup(10)(plO-->pl3) del(10)(pl5), dup(ll)(pl0^pll.2) del- In summary, we have shown that in addition to the recurring chro (ll)(pll.2^pler), dup(ll)(ql3) del(ll)(q21^q23); SQ3I: dup(5)(p!0-^pl5.1) dei(5)- mosome changes described for HNSCC tumors, there are additional (pl5.1-*pter), dup(14)(qlO-^q32) del(14)(q32â€”qter); HNSCC135: del(4XplO-Â»pl2) recurring changes that correlate with in vitro response to X-rays. The dup(4)(p!2^pter), dup(7)(qlO-q22) del(7)(q22-Â»qter), dup(1l)(ql0^q23) del(ll)- changes included deletion of 14q in one-third of RR cell lines and (q23->qler); JSQ3: del(l)(ql0^q21) dup(l)(q21^qter); HNSCC143: dup(8)(qll. 2->q21.2) del(8)(q21.2^qter). duplication of the same region in one-third of RS cell lines. Also, as RS cell lines frequently had breaks in chromosome 11 at band ql3, a region frequently amplified in HNSCC, sensitivity may result from myc and ras oncogenes, have been reported (for review, see Ref. 41), as well as loss and/or mutation of the p53 gene (42-44). Overexpression ofPRADl, a G, cyclin, resulting in passage to mitosis before full repair of DNA damage with persistence of lethal DNA Inherently RR cells have been identified in HNSCC and it has been lesions. These observations suggest that radiation sensitivity would be suggested that these may account for therapy failure (22). Identifica the dominant phenotype in fusion experiments. We suggest that rela tion of any changes that mark such cells could aid in prediction of tive radiation sensitivity also may be associated with altered chroma- treatment outcome. When we compared the karyotypes of HNSCC tin structure, which in turn may lead to an increased frequency of cell lines that were RR with those that were RS in vitro, we found several karyotype changes which segregated with cell survival fol centromere replication errors. lowing irradiation. These included a cluster of breakpoints at Ip22 and deletion of distal 14q in RR lines; RS lines were characterized by ACKNOWLEDGMENTS more frequent breakpoints in Ilql3 and duplications of distal 14q. The regions of interest identified above contain several genes which We thank Terri Williams for technical assistance and Dr. Arthur Brothman might show altered expression in HNSCC. 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5547 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1993 American Association for Cancer Research. Chromosome Changes Characterizing in Vitro Response to Radiation in Human Squamous Cell Carcinoma Lines

Janet M. Cowan, Michael A. Beckett and Ralph R. Weichselbaum

Cancer Res 1993;53:5542-5547.

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