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Characterization of the Submicroscopic Deletion in the Small-Cell Lung Carcinoma (SCLC) Cell Line U2020 H~rry A. Drabkln, Mike J. Mendez, Pamela H. Rabbitts, Terry Varkony, Jonas Bergh, Joseph Schlessinger, P'~ul Erickson, ~nd Robert M. Gernmill DMsion of Medical Ontology, University of Colorado Cancer Center (H,AD. T.V., P J:.) ant Eleanor Roosevelt Institute for Cancer Research (H.AD., M.~M, I~M.G.), Denver, Colorado: MRC Clinical Oncotogyand Radiothera~eut cs, Cambridge, U.E (P.H,P~); Depar~nent of Oncology, University of Uppsala, Akademiska Sjukhuset, Uppsala, Sweden (J.B.); and Depa~me~t of Pharmacology, New York University Medical Center, New York New York 05.) The small-cell lung carcinoma cell line U2020 contains a submicroscopic, homoz),gous deletion that removes a chromosomal segment within 3p 13-p 14, including the locus D3S3. We have sublocalized 49 additional probes to the 3p 13-p 14.2 region and have identified 7 new DNA markers that ar'~e from within the U2020 deletion. The estimated size of the deletion, based on marker density, is approximately 4-5 megabases (Mb). Including D3S3, 7 of the 8 markers have been linked by pulsed-field gel (PFG) etectrophoresis over an area of approximately 2 Mb. Including the one unlinked marker, PFG analysis accounts for about 3 Mb of the region. The U2020 deletion appears confined to the 3p 13-p 14.2 region and does not include the candidate tumor suppressor gene, protein-tyrosine phosphatase gamma (PTPG). Genes Chr0m Cancer 5:67-74 (I992). © 1992 Wiley-Uss, Inc. INTRODUCTION Small-cell lung cardnoma (SCLC) is the fifth most common cause of cancer deaths in the United States (Minna et al., 1989). Despite high initital response rates to chemotherapy, very few patients are cured. Better understanding of the pathogenesis of SCLC should lead to more effective interventions. Toward this goal, a number of molecular events have been elucidated in SCLC, including amplification of mem- bers of the MYC family of oncogenes (Little et al., 1983; Nau et al., 1985), autocrine production of bombe- sin (Cuttitta et al., 1985), loss of the retinoblastoma gene in some cases (Horowitz et al., 1990), and muta- tions in the TP53 gene (Takahashi et al., 1989). Whang-Peng et al. (1982a, b) first described the consistent loss of chromosome 3 material from 3p in SCLC. Molecular analyses with polymorphic DNA probes have confirmed this involvement by demon- stinting a near universal loss of 3p heterozygosity (Brauch et al., 1987; Naylor et al., 1987). Isolation of the putative 3p suppressor gene(s) involved in SCLC by positional cloning has met a major obstacle, the large size of the deletion in most tumors and cell lines (Whang-Peng et al., 1982b). Rabbitts et al. (1990) iden- tiffed a homozygous, submicroscopic deletion in the SCLC cell line U2020 that removes the 3p13-p14.2 locus D3S3 (Dmbkin et al., 1989; Gerber et al., 1986). The U2020 deletion has the potential to facilitate the isolation of a tumor suppressor gene important in SCLC. Cytogenetic analysis reported by Rabbitts et al. (1990) was of insufficient resolution to yield with con- fidence an accurate estimate of the size of the deletion. Two probes isolated from a jumpin~g library using D3S3 were also deleted; together, the three deleted probes set a minimum deletion size of 100 to 200 kb. We have described the isolation and mapping of 432 chromosome 3 probes (Drabkin et al., 1990). By using these probes and additional markers obtained from a radiation reduction hybrid, we have found that the U2020 deletion appears confined to the 3p13-p142 region, and that most of the deleted markers can be linked by PFG electrophoreais, supporting the rela- tively small size of the involved segment. Recently, it has been suggested that the protein- tyrosine phosphatase gamma, PTPG gene, located on the short arm of chromosome 3, may be a tumor suppressor (LaForgia et al., 1991). This suggestion was based on the selective loss of the PTPG gene in some tumors and on its placement in a linkage group with D3S3. Our present study indicates that PTPG is not included in the U2020 deletion. MATERIALS AND METHODS Molecular Probes The isolation and mapping of 432 chromosome 3 probes have been described (Drabkin et al., 1990). These probes were derived from diverse libraries, in- cluding a flow-sorted library as well as NotI and ran- dora (Mbol) lambda libraries constructed from a so- matic cell hybrid containing chromosome 3 as the only human genetic material. Additional molecular probes have been isolated from a radiation reduction Reeelv~l ,~ptumlmr 17. 1991: accepted January 10, 1992. ~dre~ ~pdnt rcxl~s to Dr. IIa~ A. D~bkin, DK~ion of 4~ ~. ~ A~.. Denver. CO 80262. © 1992 ~/ILEY-IJSS, INC.. 50618389

hybrid, R7K/ACI-6, ~ by irmdiati~m of the chromosome 3 contahdng hybrkl, H34 (DrabkJn et al., 1990), with 7,009 rods and sulmeqtmat fusio~ with a second CHO panmt (H.D., unpublished resulm), A par- RTK/ACI-6 DNA in EMBL3 according to standard procedures (Sambrook et al., 1989). Human recombi- nants were identified by hybridization to total human DNA and localized with a 3p-specifac hybrid mapping panel (see below), Additional probes included pYNZ86.1 (D3S30~ B67 (D3S4), RHOlARH12 (Ma- daule and Axel, 1985; Cannizzaro et ak, 1990), and PTPG (LaForgia et al., 1991). Probe pA136E9L was derived from the left end of a D3S3 containing non- chimeric YAC (Mendez et al., manuscript in prepara- tion), 450 kb in size, isolated from the total human YAC library described by Brownstein et al. (1989). Somat|c Ce|| Hybrids For regional mapping experiments, the 3p13-p142 interval was defined by the somatic cell hybrid RI-1, which has 3pter-3p13 as its only chromosome 3 con- tent, and by TL]UC12-8 and 3;814-1, which contain the der(3) and der(8) chromosomes, respectively, from the hereditary renal cell carcinoma 3;8 tmnslocation, t(3;SXpl4.2;qPA.1) (Drabldn et al., 1985,1990). Hybrids de.fling the remaining intervals, including 3p14.2- p14.3, 3p14.3-p21.1 (proximal), 3p21.1 (distal), 3p21.1- p23, 3p24.2-p25, and 3cen-q21, have been described (Drabkin et al., 1989, 1990). Southern Hybridizat|ons DNA from U2020 and from a control B-lympho- blastoid cell line (AGLCL) with no known chromo- somal alterations was digested with either EcoR1 or HindIII, and Southern blot hybridizations were car- ded out under standard conditions (Sambmok et al., 1989). Pu|sed-F|e~d Ge| I-'[eetrapboresls DNA samples were prepared from six human lym- phoblastoid cell lines carrying either normal or trans. located copies of chromosome 3, including T1.8229 (normal, 46,XY); CL21145, tfX;3) (p13;p13); TL9542, t(3;8) (p14.2;q24.1); TL9944, t(3;8) (p14.2;q24.1); CL15874, t(3;7) (p21.1;p13); and CL22767, t(3,~) (p14.3;p12). The cell lines carrying 3;8, 3;7, and 3;6 translocations have been described in association with hereditary renal cell cardnoma (Cohen et al., 1979; Dmbkin et ~, 1985), Greig syndrome (Tomme- rup and Nielson, 1983), and hereditary hematologic malignancies (Markkanen et al., 1987), respectively. DNA samples in some experiments were prepared frown hyba'id UCTP-2/L3 (3pter-3qter) (Gerber et al., 1 2 B 1 2 Figure I. Analysis of probes FTPG and I'1JI557 in U2020 DNA. A. Hindlll digestion of U2020 (lane I) and control (lane 2) DNA hybri- dized with PT/~, demonst~'adng retention of DNA sequences. Bo Ec0RI digested DNA from U2020 (lane I) and control (la~e 2) hybridized with probe MJlSST, showing homoz),gous deletion. 1988) and a translocation derivative of UCTP-2A3 designated B-15 (Stein and Glover, 1987). Viable cells were embedded in low-gelling-temperature agarose and prepared for pulsed-field gel analysis as de- scribed previously (Gemmill et al., 1987, 1991). Sam- ples were digested to completion with 8 rare-cutting restriction endonucleases, including BssHII, EagI, NotI, MluI, NruI, SaclI, SalI, and SfiI. The products were separated on pulsed field gels and then trans- ferred to nylon membranes as described (Gemmill et al., 1987, 1991). Filters were hybridized sequentially with the 8 probes found to be deleted in the U2020 cell line. RESULTS Regional Mapping In addition to D3S3, 49 probes have been sublocal- ized to the 3p13-p14,2 region and tested against U2020 DNA. Of these, 7 rmw probes have been identified that are homozygously deleted. An example of one such deleted probe, M11557, is shown in Figure 1B, along with an example of a retained marker, the PTPG gene (Fig. 1A). Ten probes from the adjacent 3p142-p14.3 region, as well as 7_3 probes from other segments of chromosome 3, were also tested against U2020. Each 50618390

DELET/O~ I~/$CI.C CEIL ~ U20;20 69 Deletion Size Estimate M11570, ~t 3 of 60 unique sequence probes isolated from a flow-sorted library that have been mapped to 3p. We have previously shown that the distn'bution of chromosome 3 l~obes isolated from different types of libraries (random MboI partial di- gestz, NotI boundaries, etc.) differs markedly (Dral> kin et al., 1990). The dislribtrtion of the small-insert, unktue sequence probes isolated from the flow-sorted h~rm-y was quire similar to the distnlmti~a o/~ imert lambda phage clones [Xel~'~tt from randomly deaved DNA isolated with use ofan mcrA-B- host. We believe, therefore, tl~t the distn'bufioa of the flow-sorted probes gives a useful estimate, based on probe density, of the size of the region from which these probes were derived. Thus deletion of 3]60 3p probes (5%) would correspond to approximately 5 Mb of the estimated 100 Mb comprising 3p. A similar estimate based on the size of the 3p13-p142 region, containing approximaw.ly 25-30 Mb, yields 3/20 (15%) flow-sorted probes that are deleted. This would TABLE I. DNA Probes Tested, Their Chromosomal Locations, and Presence or Absence in U2020a probe tD num. location U2020 probe I MJ1438 ID3S318 3p13-p14.2 !43 RTKII5 ' !3p]3-p14.2 2 .'VgJ1557 IY3S323 3p13-p14.2 i 44 iR7KII6 ! :3p13 p14.2 ! + 3 MJ1570 D3S3Z4 3p13-p14.2 45 P, TK119 " " :3~i'~:pi4~'- ,1--~ "" 4 NottS7 ID3S332 3p13-p14.2 46 KTK143 [ , ..'_~.p~2 _' ~."~" 5 )A136E9 D3S1226 3p13-p14.2 47 R?K-HW2 [! .,- 3p13-p.I.._4.2_... _.,_+_.. 6 R7KI05 D3S1225 3p13-p14.2 48 R?K-tIW17 7 !RTK142 D3sr224 49 ~ R?K-HW181 3p13-p14.2 + 8 )MS1-37 D3S3 - 50 R7K-HW21 " "~pl 3-p14.2 " " + 10 MS1120 D3S310 + 52 Not94 3ecn-pl3 I ÷ + s~ ~o,~ ~s~.__.~.~_~:pi__3 .... L .+ + 54 M$1221 D3S313 ~3p13-p2L1 i + ss so<~ .... + 56 MJ1498 D3S319 3p14.2~14.3 i + + 61 Notlfi3 .I~'S'~f}I "'3p14.2-p14.3 ! + :" -~--1~'[~I~ :. "3p14.2 p14.3 + 64 R7KI20 i 3p14.2-p14.3 + + 65 R7KI45 i" "3p14.2-p14.3 + 66 Not1?4 i)3S370 "3p1,1.3-21.1 " + + [ 69 Not36 + ,70 re,s7 ! ";~,~.i.~ --...~ + 11 Not251 [ 30 !Not49 3t 32 ;Nml40 33 'Not146 34 'No1187 35 Not192 37 Not215 38 No~220 39 No[232 I ! + ~ 71 Notl9~ t .3p21.1 p21.2 ÷ 3p13-p14.2 3p13-p14.2. 3p13-p14~- !3p13-p14.2 [3p13-p14.2 12 MJI206 !D3S312 3p13-p14.2 13 MJI3$gB D3S314 3p13-p14.2 14 MJl417 D3S315 3p13-p14.2 15 MJ1422 D3S316 3F13-p~4.2 16 MJ1430 ID3S317 3p13-p14~. 17 MJ1454 I 3pl 3-p14.2 i !8 [MJ~499 ]D3S320 3p13-p14.2 i 19 [MJI517 [D3S321 3p13-p142 [20 B67iD3S4 3p13-p14 9 [21 PTPG ! 3p13-p14.2 22 [btS125 .D3S304 3p13-p14.2 23 !MSl118 !D3S309 3p13-p14.2 24 ;MS1135 [D3S311 3pt3-p14 o 25 ~Mbog4 ';D3S374 3p~.3.p14.2 26 IMbo157 'D3S378 3pl 3-p14.2 27 Notl3 D3S325 3p13-p149 28 :Not40 'D3S350 3p13-p142 29 Not41 D3S351 3p13-p14~ D3S326 3p13-p14.2 [ + 72 :MJ1437 D3S327 3p13-p14.2 ! -+-..Z' ..7~...]lvff 1__52__6 D3S330 3p13-p14.2 i + i 74 iNot153 i [3p13-p14.2 + ÷ 177 .... !3p_l 3~p_1.4.2 ....... ~i,~-p~,2-'- + + : gZ gTgl12 D3S 277 ..... D3S278 D3S3fil D353¢:2 D3S.R~3 .3p21.2-p2.3. .3p.21.2-p23 .3p21,2-p23 . • p.l.~-p.3 .3p2L2-p23 .3p21.2-p23 .~p~l 2.p23 .3p21 2-p23 .3p21.2-p23 .3p24.2.p25 ~p2-|,2 4.1 IpYNT'.g6.1 .D3S30 .3p13-p14.2 .4+ ; g3 NotlSl D;.~'~'+ 3ccnq21 42 RTKI00 3 13- 14.2 + "__ aDeleted probes ar~ listed as numbers I ~brough 8 aml are indicated by' bold print. 50618391

correspond to 3.75-4.5 Mb d deleted DNA. Likewis~ of the 50 tmal probes in tbe 3p13-p14.2 regioa, 8 (16%) were d~ that ,,voukl correspc~xl to approximately 4.0-4_8 Mb of DNA. Thus a reasomtble size estimate for the U2¢~ delet~ is 4-5 Mh, wh~ is in kee#vg with its submicroscopic nature Examination of the Protein-Tyrosine Phosphata~e Gamma Gene 0~TPG) The PTPG gene b~__s been regionally mapped to 3p14-21 by in sire hybddizatioa (LaForgia et al., 1991). Somatic cell hybrid analysis mapped PTPG proximal to the renal cell carcinoma 3;8 translocat/on breakpoint (LaForgia et al., 1991) located at 3p14.2 (Wang and Perkins, 1984). Because this gene has been implicated as a possible tumor suppressor, we tested it against U2020 DNA. As shown in Figure 1A, PTPG sequences are retained without apparent rearrange- ment in U2020 DNA. Pu|sed.FieM Ge| L|nkage DNA markers deleted in U2020 were tested for physical linkage by hybridization to pulsed-field gel Southern blots. See Figtwe 2 for examples of physical linkage for probes pA136Eg(L), D3S3, M'J1557, and MJ:[438. Figure 2A shows linkage between A136K9(L) and D3S3 in SfiI digests (660 kb fragments); Figure 2B demonstrates linkage between D3S3 and MJ1557 in both NmI (1100 kb) and Sfii (1,400 kb) digests, whereas Figure 2C shows linkage between MJ1557 and MJ1438 in M/uI digests {1,200 kb). Most data obtained from PFG analyses are summarized in Table 2. Frequently, the different cell lines used generated different fragments for a given pmi~ This cell line- specific variation (indicated by italics in Table 2) is presumably due to methyiation differences and was quite useful f~r identification of correct lh~.lm~ges. spec~c fragments that all could not be listed in Table 2. Those listed either demonstrated linkage between markers or yielded particularly strong signals. The data in Table 2 show that most markers were linked to others by multiple restrict/on fragments. For example, A136E9(L) was linked to D3S3 on fragments generated by M/uI (825 kb in two cell lines) and S~cII (510 kb), in addition to the Sill fragments shown in Figure 2A. Marker MJ1557 is linked to MJ1438 on fragments generated by three enzymes, BssHII (1,400, 1,200 kb), E~I (1,400, 750, and 340 kb), and M/uI (1,200, 1,100, and 900 kb). MJ1438 is linked to Not157 on fragments generated by EagI (960, 750, and 340 kb), M/uI {1,200 and 1,100 kb), and NruI (540 and 490 kb). Of note, Not157 detects the same fragments gen- erated by M/uI and EagI as observed with both M'J1557 and MJ1438. As indicated in Table 2, R7K105 and R7K142 can tentatively be linked to the above group (pA136E9(L), D3S3, M~1557, M'J1438, and Not157), although the evidence is less secure. Recent YAC walking steps have confirmed the linkage of R7K105 to D3S3 and placed this marker between D3S3 and MJ1557 (data not shown). We have been unable to detect any evidence of linkage between MJ1570 and any of the above probes. We used the data in Table 2 to develop a probable probes: A136EgILI D3S3 D3S3 M J1557 D3S3 MJ1557 MJ1557 MJ1438 Sill Nrul Sfil Mlu I Figure Z ~ Linkage be~veen probes A136Eg(L) and D353 on an S~-generamd ~-agrnent obs~ved in two cell I~nes, CL21145 It(X;3)] and CL 15874 [t~3;7)]. The digested DNA sm'nples were resolved on a pulsed-field gel at t pulse time of 60 s ~cl an electric field of 5 VJcm for 48 h. B: Unka~e between probes D353 and HJ 1557 on Nrul and Sfiggenecated fragments. The I, 100 kb Nml fragment is specific to the CL21145 cell line. The 1,400 kb Sill fragment is apparently a ~artia~-cligest product. Although both the 1.400 and 1,250 kb fragments were observed in many other cell lines, the slgrmls were strongest in the 13- IS cell fine shown here. The DhlA samples were resolved at a pulse dine o~ 4 m and an electric field o~'4 VJcm foe 413 h. C: Unkage between probes HJI557 ar*d ~4JI438 on M#Jl-~nerated fi-agrn~nts~ Separation was addeved under the same conc~iom as in B. 50618392

~t~ler fi~- these nmrkers, as slmwn in the map pre- sented in Figure ~ This map is designed only to show ~ ~le ~ ~ ~ ~te d ~e d~ ~~ ~t m~ ~ ~ ~ ~ ~ ~t ~n~ ~ ~ ~le ~fi~s ha~ ~t ~ ~~ F~o~ m~ o~ (~., ~7, ~1~, md Not157 on ~I md S~ ~). ~ in~s~d~ ~d ~ ~ed by doubl~di~fi~ ~e~en~ At ~e pr~ ~t time, ~ ~ve no ~ ~~ the ~m~ ~lo~e ofimmfi~ of ~ ~k~. ~be ~1570 li~ ou~ide ~s ~ ~oup and ~ ~ pla~ at ei~ en& Fi~ly, we do nor ~ow wh~er ~e ~- merits obse~,ed on PFG gels extend outside the U20~ deletio~ DISCUSSION The identification of a homozyg~m, submicro- scopic deletion in the U20~0 ~ life suggests that this marks the location of a tumor suppressor gene critical to the pathogenesis of SCLC. Our current studies have identified 7 new probes in the deleted interval, all of which (including D3S3)map to 3plOp14.2. The size of the deletion, as estimated by probe density, is approx- imately 4-5 Mb. Pulsed-field gel studies have provided evidence linking 7 of the 8 probes and have established the TABLE 2. Large DNA Fr-a~menr~ Detected by Pulsed-Field Gel Analysis With Probes From Within the U2020 Deletion= ENZYMES: PROBES: ND aFragmenr sizes are listed in kiloba~es. Fra~nenrs decect~ I~ more than o~e probe are boxed: fragments listed in italics are call line-specific; and CZ in&cam d~e probe hFbrldized m the ~ or~in or the compresskm zone. respectively. The probes and restriction endonudeases 50618393

3 cen tlX;31(pl $:p15114.1~]) ~($;$)(p14.2;¢]24,1 t 3pter iota ) SCLC deletion I000=~o Nrul MIuI BaIHII Sail 8tll Sagll Eagl A1361 0 zoo zoo 700 D~S3 1000 I I $00 j 300 400 SO0 I I ZOO t I 1100 4~10 ioo Nrul Mlul zoo' BssHll I ~-~ 8all Sill Saoll j o~o Elgl Rgure 3. Pulsed-field gel linkages within the U2020 deletion. We used the data In Table 2.to generate a probable order and approxirr~ate distsnce between marker=, as shown here. The two breakpoints chat define the 3pi3 to pi4.2 region are shown at the rap. The approximate region of 3p commonly deleted in SCLC is shown underneath the 3p ideogram.The U2020 deletion, represented bya black box, lies within the Intarval defined by the eransloca- tions, At the boctem of the figure those PFG fragments, drawn te scale, are shown that were uzeful for detecting linkages between probes. The positions of'these fragments are not known with accuracy. Circles indicate that dlca have not yet been obtained to confirm hybridization to the approprlace fragment. Scale bar is marked In kllobase=.

tentative orcl~ ~/n Figure 3. The PFG map of this region myers ai~tely 2 Mb of DNA f~ the linked set cff la-Oi~ ancl another I Mh of DNA sur- ~l~ rapid i~k~ ~ ~ o~i~ ~ d~ h~ by ~ ~ ~ su~ ~e ~- d~ly ~1 s~ of ~e ~le~ ~t. As sh~ in F~e 3, ~s d~e~ se~t ~ ~- ~ ~ ~ ~ ~ ~a~uci~ of~e cl~ si~.~s m~ ~r~ ~likely to ~ ~ ove~ (G~in~ et al., ~). ~e pm~ Not157 d~ not mn~n a dete~ble Noll site ~d may Mve ~n d~ved by ~le~t~ate li~tio~ ~ has ~n £ound in ~me No~ lib~ ~all~ et ~., 1989). Probm ~m ~ re, on ~ve ~so ~ ~und to ~ ~a~ ~th i~o~ of ~h AT ~nt~t ~ ~- d~, ~monal ~i~tion). ~ g~al, such ~ ~ons ~e a~oda~ ~th Giem~ dark ~ds ~er- ~di, 1989). ~us a l~dy lo~fion of ~e U20~ d~efion is ~th~ band 3p14.1. T~s is in ~eement ~ fluor~nce ~ sire hybfi~fion s~di~ (~bc~- ~n et ak, 1989) plad~ D~ at ~e ~d~ of the Gi~ light band 3p13 and ~e Giemsa d~k band 3p14. Thee obse~ations on ~e na~e of the DNA ~ the U2020 deletion should not be ~t~re~ as ~t- ing ~t no gen~ ~e contained in ~is int~, but mth~ ~t ~e ~ numb~ of gen~ ~ ~is ~ent may be considerably lower than that in Cp~rich ar~s. This is p~haps fo~uitous, bemuse flae ~timated s~ of ~e deletion is still considerable. YAC ~ng~ ~e und~ development for this re~ as a pr~equisi~ to ~e idenfifi~fion of an involved gene or g~. Finely, the ~ gene did not show evidence of loss or r~ngement in U~20, although ~e ~ pro~ is not a ~ll-le~h cDNA ~aFor~a ~ al., 1~1). It would be unlikely, ~o~h not impo~le, for one of the U2020 br~k~ints to d~ly involve ~is gene. YA~ and subsequ~t prob~ that d~ne the ends of • e delet~ se~ent should ~cilitate ~e evaluation of gen~ t~t may be ~ pro~miW m ~e deletion, and ~eby potbelly aff~ted due to c~nges in ~gh~ order chro~fin ~ation. ACKNOWLEDGMENTS This work was supported by NIH grants HG 00353 and HG 00358 to RaM.G. and H.A.D., respectively, and by HD23826. R.M.G. was additionally suppca'ted by the Lucile P. Markey Charitable Trust. REFERENCES Albert~n DG, Sherrkngton PD, Rabbitts PH (19~9) Localization of poly- morpMc DNA pmb~ frequemly deleted ~n hmg cardn~ Hum Gemt 83:1gg-13~. ~ S. Sc~e~;on GD, P~e= BJ, ~ J, ~ M, 7~ B 317:11~1113. ~ D. $:1~1~1. ~ M, Li FP. I~ S, M~ DJ, Tmi g Ja~ ~, Brown RS ~tfi~ K ~ey 1~, Mul~ine J, Mhna ~ 0 9~ ~mb~in-like ~d~ ~ ~ncfi~ as aut~ne ~wth ha~s h~ human ~11~1 I~ ~n~. Harm 316~ ~b~n H~ Bradley C. ~tiun of c-myc in the her~i~ ~iatM with a ~3:8) ~fl Amd ~i L%A ~:~. ~abkin HA, Smith D. Jon~s C, Jonah M, ~ M, ~ld S. Glover T. ~bmvi¢ A. BrMley WE~, G~ill R (1~) R~onal ~d phyMml ~ppi~ smdim involvi~ ~ngem~ of human c~omo~me 3. ~nmr ~lls ~bkin H. Wright M. Jonsen Morn ~ Mend= M. Efick~n P cell hybrid mapping imnd and ~l~lar pro~ for human ch~ some 3. ~nomi~ 8:43~6. ~rdin~ K, Horis~rger M. Kmus J, Tan~vahi U, KomnMrg J, Rao V, ~dy S, ~tterson D 0~) Analysis of human chrom~me 21: ~lation of phys:cal and ~'togemfic ma~ g~e and C~ isl~d dis~fions. E~O J ~2~, Gmmill RM, Coyle-Mut ris jF. McP~k FD Jr; Wa~Uri~ LF, Hecht F ~ ~ion of long-range ~icfion ~ in human DNA ~ing pul~ field gel el~oph~is. Gene A~ T~n ~11~131. C~mill ~, V~ella-Gamia M, Smi~ DI, Efi~n P, Golembieski W, ~ll~ ~, ~yleMoMs J. Tomm~p N,~bkin ~ (1~1) A ~Mb phys~l map within 3p21.1 s~ns ~e br~int a~tM ~th GrOg ~phalopolys~mdac~,ly s~m~ ~nomim 1]:~--10Z ~ ~, Miller YE, Dmbkin HA, ~#n CH merit of tM ~bm~rphi¢ pro~ D~ m 3p14 by ~l~ul~ hybrid- i~fion. Cytogeuet ~11 Genet 4~72-74. Ger~ MJ, Dmbkln HA. FimhaMr C, Miller YE, ~og#n CH, Smith DI (19~) Re#~mai localization men~ by using a hybrid ~11 delefi~ ~ppi~ ~nel. Am J llum ~net ~:~2.-~1. Horowi=~, ~ark SH, Bogmmmnn E, C~ JC, Yan~ll DW, ~ye FJ, Mi~a JD, ~'ja TP. WelnMrg ~ (1~) F~u~t i~ctivati~ of the ~tinoblastoma ami.oncogene is ~aM to a su~t oi human ~or ceK¢ IY~k" Natl At~td ~For~ S, Mo~ B. LeW J, Barn= G, ~niz~ ~ghosian-~ll 1., Glick J, W~ton & Ha~s ~, ~bkin H, PaNer- ~n D, Cr~ C~ L Schl~iuger J, Hmbner K (1~1) R~ptor pmtein- ~ine pho~phatase 8 is a ~d~te ~r ~uppr~or ~ne at human chromtx~m~e re#on 3p21. ~le ~, Nau MM, Carney DE, GaMar ~', Minna JD (l~) Amplifi~- ~n and exp~inn of the c-myc onm~e in hu~n lung ~ncer ceil lin~. Nature 30~:19-I Madaule P, Axel R ~1 ~) A novd m~mla~ g~e family. ~1141:31~. ~ & Ruum T, ~8i V, Fr~ila ~ ~uufiM ~ de la ~a~e A (I~ ~nstitutio~l ~1~ ~6) ~14~11) ~ml~c ~li~a~i~. ~ G=a Cy~net ~-~ ~n~ ~, ~ II. Glatst~n E, lhde D (1~) ~ of the lung. In ~Vi~ VC Jr. ~ t~n & R~n~g ~ (Ms): ~ ~pl~ and ~ of ~colo~y. ~ M. Phi~ddphh: Lipplnm~. Nau ~ By,ks BJ. l~ey J. ~e E. G~ ~, K~h IR. ~e OW, ~ V, Holl~ GF, myc-r~tM ge~ amplifiM aM ~ ~ h~ s~ll ~I1 lu~ ~. Na~m 318:~ Nayl~ ~ J~n BF~ Mi~ ~, ~ AY (1~ ~ of Na~e ~451 ~ P, B~gh J, ~ a ~11 c~41 lu~ ~ ~ ~ ~ ~1-~ 50618395

Wal~:e I'~ Fommin JW'. Brmam AM. ~ F fl~ D~-~ct con- V~mng-Peug J, B,ann PA Jr, Kao-Shan CS, L~ EC, C~mey DN, Gazdar A, Minm.}D (l~eb) A nmrand~xa chromosomal almonmlity, • :3p04-23), in htanan smal] c~l lung cancer k~CL~ Cancer Genct 50618396