TSE. In either case, the addition of gluco-corticoids and GR then results in decreasedtranscription, demonstrating interferencewith the activity of the CRE and CRE-binding protein. The presence of GR bind-ing sites that overlap both CREs supports amodel in which negative control is exertedthrough direct GR binding to DNA se-quences that overlap the CREs, with conse-quent blockage ofCRE activity. This mech-anism of interference between transcriptionfactors may apply not only to negative regu-lation by steroid receptors but also to nega-tive regulation in other systems.

glanz, K. Nasmyth, Cell 41, 41 (1985).21. A. Barberis, G. Superti-Furga, M. Busslinger, ibid.

50, 347 (1987).22. T. Edlund, M. D. Walker, P. J. Barr, W. J. Rutter,

Science 230, 912 (1985).23. We thank A. Delegeane and M. Kirven for contrib-

uting to the early experiments, R. Evans and S.Hollenberg for GR expression plasmids, B. Gross

for GR purification, and D. Dean, C. Thompson, J.Windle, and L. Ferland for critical rcading of themanuscript. This work was supported by a grantfrom NIH to P.L.M. and from the Deutsche Fors-chungsgemeinschaft and the Fond der ChemischenIndustrie to M.B.

16 February 1988; accepted 13 May 1988

Abnormalities in Structure and Expression of theHuman Retinoblastoma Gene in SCLC

J. WILLIAM HARBOUR, SHINN-LIANG LAI, JACQUELINE WHANG-PENG,ADI F. GAZDAR, JOHN D. MINNA, FREDERIC J. KAYE*

REFERENCES AND NOTES

1. J. D. Baxter and J. B. Tyrel, in Endocrinology andMetabolism, P. Felig, J. D. Baxter, A. E. Broadus, L.A. Frohman, Eds. (McGraw-Hill, New York,1987), p. 511.

2. F. R. Weiner et al., J. Biol. Chem. 262, 6955(1987); S. M. Frisch and H. E. Ruley, ibid., p.16300; J. Charron and J. Drouin, Proc. Nat!. Acad.Sci. U.S.A. 83, 8903 (1986); A. Israel and S. N.Cohen, Mol. Cell. Biol. 5, 2443 (1985).

3. D. R. Mann, D. Evans, F. Edoimioya, F. Kamel, G.M. Butterstein, Neuroendocrinology 40, 297 (1985);D. E. Suter and N. B. Schwartz, Endocrinology 117,855 (1985).

4. J. G. Pierce and T. F. Parsons, Annu. Rev. Biochem.50, 465 (1981).

5. W. W. Chin, M. A. Shupnik, D. S. Ross, J. F.Habener, E. C. Ridgway, Endocrinology 116, 873(1985).

6. M. A. Shupnik, W. W. Chin, J. F. Habener, E. C.Ridgway, J. Biol. Chem. 260, 2900 (1985); F. E.Carr, E. C. Ridgway, W. W. Chin, Endocrinology117, 1272 (1985); J. H. Nilson, M. T. Nejedlik, J.B. Virgin, M. E. Crowder, T. M. Nett, J. Biol.Chem. 258, 12087 (1983); M. L. Croyle and R. A.Maurer, DNA 3, 231 (1984); S. S. Papavasiliou etal., Endocrinology 119, 691 (1986).

7. A. M. Delegeane, L. H. Feriand, P. L. Mellon, Mol.Cell. Biol. 7, 3994 (1987).

8. S. M. Holienberg, V. Giguere, P. Segui, R. M.Evans, Cell 49, 39 (1987).

9. P. O. Kohler and W. E. Bridson, J. Clin. Endocrinol.32, 683 (1971).

10. A. C. B. Cato, R. M. Miksicek, G. Schutz, J.Arnemann, M. Beato, EMBOJ. 5, 2237 (1986).

11. N. Hynes et al., Proc. Natl. Acad. Sci. U.S.A. 80,3637 (1983); V. L. Chandler, B. A. Mater, K. R.Yamamoto, Cell 33, 489 (1983); M. Karin et al.,ibid. 36, 371 (1984); R. Renkawitz, G. Schutz, D.von der Ahe, M. Beato, ibid. 37, 503 (1984); R.Miesfeld et al., ibid. 46, 389 (1986).

12. B. J. Silver et al., Proc. Natl. Acad. Sci. U.S.A. 84,2198 (1987); P. J. Deutsch, J. L. Jameson, J. F.Habener,J. Biol. Chem. 262, 12169 (1987).

13. M. R. Montriny and L. M. Bilezikjian, Nature 328,175 (1987).

14. C. Scheidereit, S. Geisse, H. M. Westphal, M.Beato, ibid. 304, 749 (1983); D. von der Ahe, J. M.Renoir, T. Buchou, E. E. Baulieu, M. Beato, Proc.Nat!. Acad. Sci. U.S.A. 83, 2817 (1986).

15. P. L. Mellon, V. Parker, Y. Gluzman, T. Maniatis,Cell 27, 279 (1981).

16. M. Beato et al.,J. Steroid Biochem. 27, 9 (1987).17. Though the homology of the -97 to -111 region

extends beyond the -152 to -100 fragment usedfor the experiment (Fig. 2), the sequence substitutedby the TKcat plasmid restores the guanine at posi-tion -97.

18. I. E. Akerblom and P. L. Mellon, unpublished data.19. U. Nir, M. D. Walker, W. J. Rutter, Proc. Natl. Acad.

Sci. U.S.A. 83, 3180 (1986); P. Sassone-Corsi andI. M. Verma, Nature 326, 507 (1987); D. Kuhl etal., Cell 50, 1057 (1987); S. Goodboum, H. Bur-stein, T. Maniatis, ibid. 45, 601 (1986).

20. A. H. Brand, L. Breeden, J. Abraham, R. Stem-

Small cell lung cancer (SCLC) has been associated with loss ofheterozygosity at severaldistinct genetic loci including chromosomes 3p, 13q, and 17p. To determine whetherthe retinoblastoma gene (Rb) localized at 13q14, might be the target of recessivemutations in lung cancer, eight primary SCLC tumors and 50 cell lines representing allmajor histologic types oflung cancer were examined with the Rb complementaryDNAprobe. Structural abnormalities within the Rb gene were observed in 1/8 (13%)primary SCLC tumors, 4/22 (18%) SCLC lines, and 1/4 (25%) pulmonary carcinoidlines (comparable to the 20 to 40% observed in retinoblastoma), but were not detectedin other major types oflung cancer. Rb messenger RNA expression was absent in 60%of the SCLC lines and 75% of pulmonary carcinoid lines, including all samples withDNA abnormalities. In contrast, Rb transcripts were found in 90% ofnon-SCLC lungcancer lines and in normal human lung. The finding of abnormalities ofthe Rb gene inSCLC and pulmonary carcinoids (both neuroendocrine tumors) suggests that thisgene may be involved in the pathogenesis of a common adult malignancy.

IN SEVERAL CHILDHOOD TUMORS, IN-

cluding retinoblastoma and Wilm's tu-mor, there is growing evidence to indi-

cate that the inactivation of both alleles ofcertain genes triggers tumorigenesis (1-3).The genomic locus determining susceptibil-ity to retinoblastoma has been mapped tochromosome 13q14 (4), and several groupshave obtained complementary DNA(cDNA) clones derived from this regionthat detect a DNA segment with propertiesofthe putative retinoblastoma (Rb) gene (5-7). Evidence for this gene being the site ofrecessive mutations leading to tumor forma-tion in retinoblastoma is based on the find-ing of structural changes within the gene,including intemal homozygous deletions ina number of retinoblastomas, and the pres-ence of altered or absent messenger RNA(mRNA) expression in the majority of bothsporadic and familial forms of the tumor.

Specific chromosomal deletions have nowbeen reported in various adult tumors (8),suggesting that "recessive oncogenes" maybe important in the pathogenesis of thesemalignancies. In the case of small cell lungcancer (SCLC), a consensus deletion ofDNA in the region 3pi4-3p21 has beenidentified in virtually all cases (9-11). Sever-al studies have also identified nonrandomchanges involving other chromosomes, in-cluding chromosome 13. For example, one

cytogenetic study of SCLC reported thatchromosome 13 was the most frequentlyunderrepresented chromosome, with 17 outof21 SCLC lines having absent or hypodip-loid numbers of chromosome 13 (12). Intwo recent studies, polymorphic probesfrom the long arm of chromosome 13 thatspanned the region 13ql2-13q33 demon-strated a reduction to homozygosity at oneor more informative loci in primary tumortissue from 18 of 23 patients with SCLC(11, 13). SCLC is an aggressive adult tumorwhich phenotypically resembles retinoblas-toma in that both display properties ofneural or neuroendocrine differentiation(14, 15) and both can have deregulated N-myc expression (16, 17). These observations,coupled with the recent report of structuralabnormalities of the Rb gene in -20% ofosteosarcomas and other mesenchymal tu-mors (18), prompted us to examine the

J. W. Harbour, Howard Hughes Medical Institute andNCI-Navy Medical Oncology Branch, National CancerInstitute, NIH, Bethesda, MD 20814.S.-L. Lai and A. F. Gazdar, NCI-Navy Medical Oncolo-gy Branch, National Cancer Institute, NIH, Bethesda,MD 20814.J. Whang-Peng, Medical Oncology Branch, NationalCancer Institute, NIH, Bethesda, MD 20814.J. D. Minna and F. J. Kaye, NCI-Navy Medical Oncolo-gy Branch, National Cancer Institute, NIH, and Uni-formed Services University of the Health Sciences, Be-thesda, MD 20814.

*To whom correspondence should be addressed.

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DNA and RNA status of the Rb gene inprimary SCLC tumor tissue and in lungcancer cell lines to investigate the possiblerole of the Rb locus in the pathogenesis ofthese tumors.Using the pO.9R and p3.8R cDNA

probes (5), which represent the 4.7-kb Rbtranscript that spans more than 180 kb ofgenomic DNA (6, 18), we analyzed DNAfrom eight SCLC primary tumor samples.We also studied DNA and RNA from 50lung cancer cell lines (19), including 26SCLC lines, 20 non-SCLC lines (eightadenocarcinomas, five large cell carcinomas,four bronchioloalveolar carcinomas, twoadenosquamous carcinomas, and one squa-mous carcinoma), and four pulmonary carci-noid tumors (Table 1).

Structural rearrangements of the Rb genewere detected in DNA from one of theSCLC primary tumors (Fig. 1B, lane 10).Tumor DNA from this specimen exhibitednovel Hind III fragments at 24, 16, and 11kb. The 16-kb band was amplified several-fold relative to the other bands. In addition,the normal 10-kb band was markedly re-duced in intensity, while the 7.5-, 6.2-, 5.5-,4.5-, and 2.1-kb bands appeared normal. Asthe hybridization signal of the 10-kb frag-ment was much less than 50% ofthe expect-ed intensity, this fragment may have beenhomozygously deleted in the tumor, withthe residual band seen on DNA blot analysisrepresenting contaminating normal cells inthe biopsy sample. Similar abnormalitieswere also noted when the DNA was digest-ed with Sst I (20). To expand on theseobservations and to examine correlationsbetween DNA and RNA abnormalities, westudied the DNA and RNA status of Rb inlung cancer cell lines (Table 1).There were structural abnormalities with-

in the Rb gene in four SCLC cell lines and inone carcinoid line (Fig. 1). These structuralchanges were of two types: (i) the homozy-gous loss of a normal Hind III fragment,associated with the appearance of one ortwo novel-sized bands, and (ii) reducedintensity ofone or more normal bands by atleast 50%, confirmed by densitometry, sug-gesting the presence of hemizygous dele-tions. SCLG line H889 displayed a com-plete loss of the 7.5-kb fragment with theappearance of a new band at 10.6 kb asidentified with the p3.8R probe (Fig. 1B,lane 3), while H187 had a complete loss ofthe 14.0-kb Hind III fragment associatedwith the appearance of two new bands of12.0 and 2.8 kb as determined with thepO.9R probe (Fig. lA, lane 4). Additionally,in the carcinoid line H679, the p3.8R probedetected a doublet at 2.25 and 1.95 kb inplace of the normal 2.1-kb band (Fig. 1B,lane 7). In all three cases, these changes were

present in DNA obtained from the cell linesat different time points up to 28-50 pas-sages in culture, suggesting that they werestable abnormalities. To exclude the possi-bility that the different patterns seen inH187, H679, and H889 represented HindIII restriction fragment length polymor-phisms (RFLP), we digested DNA fromthese cell lines with Eco RI and Sst I, and bycomparing to normal thymus DNA we ob-served rearrangements and homozygous lossof bands in these digests as well (20). Inaddition, 18 samples of normal humanDNA probed with both pO.9R and p3.8Rdemonstrated a uniform pattern of restric-tion fragments with no Hind III polymor-phisms (5), and no polymorphism has beenreported for these restriction fragmentsamong over 80 retinoblastomas and othertumors now examined. The homozygousinternal deletion involving the 7.5-kb HindIII fragment, seen in line H889, has beenobserved in a large number of retinoblasto-mas and has been implicated as the possiblesite of a "mutational hotspot" (7).SCLC lines H345 and H378 (Fig. 1,

lanes 1 and 5, respectively) contained allexpected Hind III bands detected by p3.8R,but on visual inspection both lines appearedto contain several bands of decreased inten-sity. To evaluate the possibility of partial Rbdeletions on one allele in H345 and H378,densitometric analysis was performed to

quantitate the DNA band intensities in bothlines. Reproducible densitometric tracingswere obtained and quantitated on autora-diograph films of DNA blots (hybridizedeither with pO.9R or p3.8R) from H345and H378, 13 other lung cancer cell lineswith normal-appearing DNA patterns, anormal B-lymphoblastoid line, and a normalhuman thymus specimen (fresh unculturedtissue). For DNA blots hybridized withpO.9R, all lung cancer lines, including H345and H378, displayed densitometric tracingsvery similar to those for the normal humanDNA samples. For blots hybridized withp3.8R, the tracings for H345 and H378were markedly abnormal (Fig. 2), while the13 other lung cancer lines showed tracingssimilar to those for normal human DNA. Toquantitate these abnormalities, the areas un-der the peaks corresponding to each of theHind III bands detected by p3.8R werecalculated and normalized to the 10.0-kbpeak, which served as an intemal control.Each ofthe 13 lung cancer lines with normaldensitometric tracings had quantitative esti-mates of band intensity ratios similar tothose for the normal DNA samples, withminimal variability. In H345, however,there appeared to be a decrease in intensityby approximately 50% of the 6.2-kb band,suggesting that an internal hemizygous dele-tion of this genomic fragment may haveoccurred. In H378, the abnormalities were

Table 1. Summary ofDNA and RNA status of the Rb gene in 50 lung cancer lines.

Cell DNA RNA Cell DNA RNAline* changes expres- n chanssiont (histology) g sion

Small cell lung cancer Pulmonary carcinoidsH187 5' rearrangement - H679 (Atypical) 3' rearrangementH345 3' rearrangement - H720 (Atypical) NoneH378 3' rearrangement - H835 (Atypical) NoneH889 3' rearrangement - H727 (Typical) None +H82 NoneN417 None - Non-small cell lung cancerH510 None - H125 (Adenosquamous) None +H524 ND - H226 (Squamous) None +H526 None - H322 (Bronchioloalveolar) None +H735 None - H358 (Bronchioloalveolar) None +H748 ND - H460 (Large cell) None +H774 None - H522 (Adenocarcinoma) None +H1284 None - H661 (Large cell) None +H1304 None - H810 (Large cell) None +H1450 ND - H820 (Bronchioloalveolar) None +H69 None tr H838 (Adenocarcinoma) None +N592 None tr H1373 (Adenocarcinoma) None +H711 ND tr H1385 (Large cell) None +H847 None tr H1404 (Bronchioloalveolar) None +H1622 None tr H1435 (Adenocarcinoma) None +H209 None + H1437 (Adenocarcinoma) None +H841 None + H23 (Adenocarcinoma) None trH1092 None + H1355 (Adenocarcinoma) None trH1105 None + H596 (Adenosquamous) NoneH1184 None + H1155 (Large cell) NoneH1436 None + H1445 (Adenocarcinoma) None ND

*Full designation of the celi lines indudes the prefix "NCI-" t+, easily detectable transcript (comparable tonormal lung expression); tr, trace; -, absent; ND, not determined.

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more complex but included a decrease inintensity of about 50% for the 6.2-kb and2.1-kb bands, consistent with a hemizygousdeletion of the 3'-most end of the gene (seeFig. 1 for genomic structure). The homozy-gous and hemizygous DNA structural ab-normalities described above are similar tothose recently published for retinoblastomaand associated mesenchymal tumors, andthe presence of such changes has been inter-preted as evidence to implicate the Rb locusas the target of recessive mutations in thesetumors (5-7).

Total and poly(A)-selected RNA wereprepared from the lung cancer lines andRNA analysis was performed with thep3.8R probe (Table 1 and Fig. 3). Of 26SCLC lines, 15 expressed no detectable RbmRNA, five expressed trace levels, and sixhad easily detectable levels of the Rb tran-script. In addition, three of four pulmonarycarcinoids expressed no detectable Rb tran-script. Pulmonary carcinoid tumors are simi-lar to SCLC in that both contain neurose-cretory granules and exhibit neuroendocrinedifferentiation. Thus, these two tumors aredistinctive from other types of lung cancerand may represent variants of malignanttransformation in the same neuroendocrineprecursor cell (21). All three tumors lackingthe transcript were characterized as "atypi-cal" carcinoids, with clinical and histologicfeatures intermediate between SCLC andclassic carcinoid (14). In contrast to SCLCand carcinoid, 15 of 19 non-SCLC lines

Akb

14.0

7.0 -6.0 -

1.5 -

1.2 -

1 2 3 4 5 6 7 8

expressed abundant steady-state levels of the4.7-kb transcript, two expressed traceamounts, and two had undetectable levels ofthe Rb mRNA. These data demonstrated acorrelation between the DNA and RNAstatus (Table 1) in that the four SCLC linesand one carcinoid line with gross structuralchanges within the Rb locus expressed nodetectable Rb transcript, while the SCLClines which did express abundant levels ofmRNA, and all of the non-SCLC lines, hadnormal DNA pattems on DNA blots. EightSCLC lines, however, expressed no detect-able mRNA but had normal DNA patternswith the probes used in this study. Aspostulated for retinoblastoma, some or all ofthese cases may have contained small muta-tions in the Rb gene which accounted for theabsent mRNA but which were undetectableby our DNA analysis.

Expression of Rb mRNA was detected intotal RNA from normal adult human lung(Fig. 3A, lane 1) and poly(A)-selected RNAfrom bovine lung (20). Although the cell oforigin of SCLC remains controversial, thefinding of Rb gene expression in lung tissue,as well as in a few SCLC lines and in allother normal tissues examined thus far (6),suggests that Rb may normally be expressedin most cells, including the SCLC precursorcell. Therefore, the lack of expression in theSCLC and carcinoid lines supports the ideathat the Rb gene has been inactivated inthese lines.

In light of our finding ofDNA and RNA

B 1 2 3 4 s 6 7 8kb

9 10

<-- 24

e 6

10.0 -

7.5 -6.2 -

5.5 -

4.5 -

2.1 -

pO.9R p3.8R

pO.9R p3.8R5' K\\\\' 1 3'

Probe:

14.0 1.2 1.5 6.0 7.0 1.3 5.5 4.5 7.5 5.3 10.0 6.2 2.1El E ElI=IJIJI I IE J 0

Fig. 1. Genomic restriction endonuclease analysis of the Rb in a representative panel of lung cancerlines. DNA (10 ,ug) extracted from seven lung cancer cell lines (H345, H510, H889, H187, H378,H209, and H679; lanes 1 to 7, respectively), normal human thymus (lane 8), and two primary SCLCtumor specimens (lanes 9 to 10, panel B) were digested to completion with Hind III and transferred tonitrocellulose after 0.8% agarose gel electrophoresis. Filters were then hybridized with either the pO.9R(A) or the p3.8R (B) 32P random-primed cDNA probes for 18 hours under standard hybridizationconditions (26). For pO.9R, a 675-bp Eco RI-Hpa I fragment was used as a probe since this removed aGC-rich region that conferred a high background on hybridization. Filters were washed twice at roomtemperature in 2x SSC/0.1% SDS for 30 minutes and then twice in 0.1 x SSC/0.1% SDS at 55°C for 1hour. A diagram at the bottom of the figure depicts the previously described Rb cDNA clone and theHind HI genomic fragments (5, 6) detected by the pO.9R probe (hatched boxes) and the p3.8R probe(open boxes).

15 JULY I988

abnormalities in these cell lines, we analyzedthe karyotypes of 11 of the SCLC lines forwhich adequate metaphase spreads could beobtained, looking for evidence of alterationsspecifically involving chromosome 13. Allthree of the SCLC lines with detectablerearrangements of the Rb gene that wereexamined cytogenetically contained abnor-malities involving the region 13qI4. H889displayed a del(13)(ql4>ter) and had nonormal copy of chromosome 13. H187contained a t(8;13)(q24;ql4) and at(9;13)(ql3;ql4), and H378 contained adel(13)(ql4>ter) and a t(3;13)(p21;ql4).In H5 10, a cell line with no Rb RNAexpression and a normal DNA blot pattern,

Fig. 2. Densitometric analysis. The top panelshows the densitometric tracing observed withnormal human DNA (from fresh thymus tissue)digested to completion with Hind III and hybrid-ized with the p3.8R probe. The five peaks fromleft to right represent, respectively, the Hind IIIfragments at 10, 7.5, 6.2, 5.5, 4.5, and 2.1 kb (seeFig. 1B). The tracings were obtained on a Hoeferscanning densitometer (model GS300). Thirteentumor lines, including H209 shown above, dem-onstrated densitometric patterns similar to thatobtained for normal human DNA. In contrast,cell lines H345 and H378, shown in the lowertwo panels, had anomalous patterns.

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C.) C C)C/) -C.) Cl)

Ui C

C.) 0N C <v C

A 'L I L, i B r-i r1 2 34 5 67 8 9 10 11 12 13 1415 16

fu~ ~ .

C.)-JC.)Ccn

1 2 3 4 5 6 7 8 9 10 11 12

I* I0 Su a:0jw:R fj0

Fig. 3. Rb gene expression in a representative panel of lung cancer lines. (A) Total RNA (15 jig) wasextracted (27) from normal lung tissue obtained from a surgical specimen (lane 1), five non-SCLC lines(H820, H1355, H1373, H1404, and H1437; lanes 2 to 6), three carcinoid lines (H720, H835, andH679; lanes 7 to 9), and seven SCLG lines (H1092, H1622, H748, H1304, H774, H1436, andH1105; lanes 10 to 16). (B) Two micrograms of poly(A)-selected RNA (28) was extracted from twoadditional non-SCLC lines (H125 and H23; lanes 1 and 2) and ten additional SCLC lines (N592,H345, H378, H510, H209, H187, H526, H524, H82, and N417; lanes 3 to 12). The above RNAsamples were transferred to nitrocellulose after formaldehyde-denaturing gel electrophoresis (29) andfilters were hybridized sequentially with the 32P random-primed p3.8R probe which detects the 4.7-kbRb transcript (5-7) or with a 1-actin cDNA probe which detects a 2-kb transcript (30). Autoradiographswere developed on XAR-5 film after 5 days at -70°C for p3.8R and after overnight exposure for -

actin. N, normal lung.

a portion ofchromosome 13 was attached atband q14 to an unidentified chromosomalfragment, and there was no normal copy of13. Two other cell lines with trace or absentRb expression (H69 and H526) had lost onecopy of chromosome 13 on the basis ofexpected modal number. In two SCLC lines

with apparently intact structure and expres-

sion of Rb (H209 and H841) and in threelines with absent or trace expression (H82,N417, and H847) no abnormalities ofnum-ber or structure of chromosome 13 were

detected.The presence of deletions within the Rb

locus and lack of expression of the gene inSCLC is analogous to observations recentlymade in retinoblastoma and associated mes-enchymal tumors where the gene is believedto contribute to tumorigenesis by a recessive"two-hit" mechanism (1). However, in con-

trast to retinoblastoma, which occurs in a

familial form in 40% of cases (22), SCLC isnot known to have a clear hereditary pat-tem. Although it is possible that an inherit-ed abnormality ofthe Rb gene might predis-pose to SCLC (23), there is no establishedclinical association between retinoblastomaand SCLC, either in the same patient or

within families. This would suggest thatmutations at both alleles of the Rb gene inSCLC occur as somatic events. SCLC isclosely associated with heavy cigarette smok-ing (24), and carcinogen exposure fromtobacco may generate an increased mutationrate in the bronchial epithelium. Extremelylarge genes, such as Rb, may be especiallysusceptible to mutation and, thus, may com-

356

monly become inactivated in the bronchialepithelium of smokers.From this study, it appears that abnormal-

ities in structure and RNA expression of theRb gene occur commonly in SCLC andpulmonary carcinoid but infrequently inother types of lung cancer. Further, thepresence of DNA abnormalities of Rb inprimary SCLC tumor tissue argues thatthese changes occur in vivo and are not an

artifact of cell culture. Among the SCLCspecimens examined, one primary tumorand 18% of cell lines demonstrated evidencefor gross structural abnormalities of the Rbgene, and 60% expressed no detectablemRNA. Another 20% of SCLC lines ex-

pressed only trace amounts of the mRNA,suggesting that alterations in Rb gene

expression may have occurred in these linesas well. Similar abnormalities of structureand expression were observed in our "atypi-cal" pulmonary carcinoid lines. These per-

centages for DNA and RNA abnormalitieswere comparable to those that have beenreported for retinoblastoma and associatedmesenchymal tumors (5-7, 18). In compari-son, no alterations of the Rb gene were

detected in any of our non-SCLC lines, and90% of these lines expressed Rb mRNA.Recent reports of chromosomal deletions at3p in many non-SCLC tumors, as well as inSCLC (13, 25), suggest that the chromo-some 3 deletion may be a common pathoge-netic step to most or all major types of lungcancer. In contrast, a loss of heterozygosityof chromosome 13q markers appeared tooccur mostly in SCLC when primary tumor

tissue was examined (11, 13), and our datasupport this observation in that abnormali-ties of the Rb gene were more specific toSCLC and "atypical" pulmonary carcinoid.The finding of abnormalities of the Rb genein a large percentage of both of these typesof pulmonary neuroendocrine tumors sug-gests that inactivation of this gene may be animportant event in the pathogenesis of lungtumors of neuroendocrine differentiation.These findings indicate that the Rb gene maybe involved in the pathogenesis of a com-mon non-ocular adult malignancy, suggest-ing that it may play an important physiolog-ical role and may contribute to malignanttransformation in various tissues besides theretina. Ultimately, functional studies will berequired to prove a definitive role for thisgene in the genesis of human tumors.

REFERENCES AND NOTES

1. A. G. Knudson, Jr., Proc. Natl. Acad. Sci. U.S.A. 68,820 (1971).

2. W. F. Benedict et al., Science 219, 973 (1983); W.K. Cavenee et al., Nature 305, 779 (1983); M. F.Hansen et al., Proc. Natl. Acad. Sci. U.S.A. 82, 6216(1985).

3. S. H. Friend, T. P. Dryia, R. A. Weinberg, N. Engl.J. Med. 318, 618 (1988).

4. A. G. Knudson, Jr., A. T. Meadows, W. W. Nichols,R. Hill, ibid. 295, 1120 (1976); J. J. Yunis and N.Ramsay, Am.J. Dis. Child. 132, 161 (1978); R. S.Sparkes et al., Science 219, 971 (1983).

5. S. H. Friend et al., Nature 323, 643 (1986).6. W.H. Lee et al., Science 235, 1394 (1987).7. Y.-K. T. Fung et al., ibid. 236, 1657 (1987).8. G. Klein, ibid. 238, 1539 (1987); B. R. Seizinger et

al., ibid. 236, 317 (1987); I. U. Ali, R. Lidereau, C.Theillet, R. Callahan, ibid. 238, 185 (1987); E.Solomon et al., Nature 328, 616 (1987).

9. J. Whang-Peng et al., Cancer Genet. Cytogenet. 6, 119(1982); J. Whang-Peng et al., Science 215, 181(1982).

10. S. L. Naylor, B. E. Johnson, J. D. Minna, A. Y.Sakaguchi, Nature 329, 451 (1987); H. Brauch etal., N. Engl.J. Med. 317, 1109 (1987).

11. B. E. Johnson et al., J. Clin. Invest., in press.12. D. H. Wurster-Hill et al., Cancer Genet. Cytogenet.

13, 303 (1984).13. J. Yokota, M. Wada, Y. Shimosato, M. Terada, T.

Sugimura, Proc. Natl. Acad. Sci. U.S.A. 84, 9252(1987).

14. K. L. Becker and A. F. Gazdar, Eds; The EndocrineLung in Health and Disease (Saunders, Philadelphia,1984).

15. A. P. Kyritsis, M. Tsokos, T. J. Triche, G. J. Chader,Nature 307, 471 (1984).

16. W.-H. Lee, A. L. Murphree, W. F. Benedict, ibid.309, 458 (1984).

17. M. M. Nau et al., Proc. Natl. Acad. Sci. U.S.A. 83,1092 (1986).

18. S. H. Friend et al., ibid. 84, 9059 (1987).19. D. N. Carney et al., Cancer Res. 45, 2913 (1985); A.

F. Gazdar et al., ibid., p. 2924; A. F. Gazdar,unpublished data.

20. J. W. Harbour et al., data not shown.21. K. G. Bensch, B. Corrin, R. Pariente, H. Spencer,

Cancer 22, 1163 (1968).22. F. Vogel, Hum. Genet. 52, 1 (1979).23. E. P. Messmer, H.-J. Richter, W. Hopping, W.

Havers, W. Alberti, Klin. Mbl. Augenheik. 191, 299(1987).

24. W. Weiss et al., J. Am. Med. Assoc. 222, 799 (1972).25. K. Kok et al., Nature 330, 578 (1987).26. L. G. Davis, M. D. Dibner, J. F. Battey, Basic

Methods in Molecular Biology (Elsevier/North-Hol-land, Amsterdam, 1986).

27. J. Chirgwin, A. Przybyla, R. Macdonald, W. Rutter,Biochemistry 18, 5294 (1979).

SCIENCE, VOL. 24I

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probes; B. E. Johnson for the primary tumor materi-al; W. Nash for some of the cytogenetic data; and J.F. Battey, E. A. Sausville, I. R. Kirsch, M. J. Birrer,M. Zajac-Kaye, and M. M. Nau for critical review ofthe manuscript. J.W.H. is a Howard Hughes Medi-cal Institute-NIH Research Scholar.

24 March 1988; accepted 7 June 1988

Type-Restricted Neutralization of Molecular Clonesof Human Immunodeficiency Virus

DAVID J. LOONEY,* AMANDA G. FISHER, Scorr D. PUTNEY,JAMES R. RUSCHE, ROBERT R. REDFIELD, DONALD S. BuRKE,ROBERT C. GALLO, F. WONG-STAAL

In a study of the immunologic significance of the genetic diversity present withinsingle isolates ofhuman immunodeficiency virus type 1 (HIV-1), the neutralization ofviruses derived from molecular clones of the HIV-1 strain HTLV-IIIB by an extensivepanel of sera was compared. Sera from HIV-1-infected patients and from goatsimmunized with polyacrylamide gel-purified HIV-1 envelope glycoprotein (gpl20),native gp120, or gp120-derived recombinant peptides, showed marked heterogeneityin neutralizing activity against these dosely related viruses. The change of a singleamino acid residue in gpl20 may account for such "donal restriction" of neutralizingactivity.

NE STRATEGY FOR IMMUNOPRO-

phylaxis against acquired immuno-deficiency syndrome is the admin-

istration of an immunogen intended to elicitprotective virus neutralizing antibodies.Neutralizing activity (NA) against HIV-1has been documented by many investigatorsusing a variety oftechniques (1-5). Domainswithin the HIV-1 envelope protein (gpl20)(6-8) and the transmembrane glycoprotein(gp4l) (9) have been shown to elicit or

absorb NA. Sera from infected patients gen-erally neutralize a broad range of isolates. Incontrast, antisera produced in experimentalanimals by immunization with gpl20 or

recombinant or synthetic peptides displayrestricted capacity to neutralize isolates oth-er than that from which the immunogen was

derived (10-12). Recent observations indi-cate that individual isolates of HIV-1 are

composed of populations of genetically andbiologically distinct variants (13). To deter-mine the implications of such heterogeneityfor vaccine development and evaluation, wecompared the susceptibility of three virusesmolecularly cloned from HTLV-IIIB (HIV-1/NIH/USA/1983/ HTLV-IIIB) to the neu-

D. J. Looney, R. R. Redfield, D. S. Burke, Departmentof Viral Diseases, Walter Reed Army Institute of Re-search, Washington, DC 20307.A. G. Fisher, R. C. Galo, F. Wong-Staal, Laboratory ofTumor Cell Biology, National Cancer Institute, Bethes-da, MD 20892.S. D. Putney and J. R. Rusche, Repligen Corporation,One Kendall Square, Building 700, Cambridge, MA02139.

*To whom all correspondence should be addressed.

IS JULY I988

tralizing effects of a number of antisera.The molecularly cloned viruses designated

HX10, HXB2, and HXB3, were obtainedfrom a single cell line that was infected withHIV-1 (14, 15). They differ by less than 1%in nucleotide sequence, at 19 sites (24 ami-no acids) in the envelope region. ClonesHXB2 and HXB3 were constructed by in-serting fragments containing fiull-lengthproviral sequences into the expression vectorpSP62, transfecting DH-1 bacteria, and re-

covering virus by protoplast fusion (16-18).BX10 was derived from the XBH10 phageproviral molecular clone (14), which lacks a

190-bp segment in the 5'-LTR, by insertingthe 8.1-kb Cla I-Xho I fragment ofXBH10

(which contains sequences coding for resi-due 14 ofgag through residue 443 of the nefregion) into pHXB2gpt2, and virus was

recovered by protoplast fusion (17). Thecloned viruses and the parent isolate repli-cate well in neoplastic T cell lines and pos-

sess similar infectivity and cytopathogenicityfor CD4+ cells (17).We initially examined the NA of a panel

of 38 human sera from 25 seropositivepatients against HTLV-IIIB, HX10, HXB2,and HXB3. The NA ofsome sera against thedifferent viruses differed (Table 1). Someneutralized one or two clones in high(.1:256) titer although they showed low(< 1: 256) NA against other clones (seeTables 1, HS-01 and HXB3, HS-12 andHX1O, and HS-23 and HXB2); others neu-

tralized all viruses to some degree (HS-06)or lacked appreciable NA against any virus

100 15 20

Time (months)

Fig. 1. Variation mn neutralization of HTLV-IIIB

and its clones by sequential patient sera. Sequen-

tial serum samples obtained from a single patient

at the specified intervals were used to investigate

the time course of NA against HTLV-IIB (0)

and its clones. Neutralization assays were per-

formed with an inoculum of 20x TCID50 of the

respective viruses, and 50% reduction of virus

expression was the end point used. The titers

shown represent the inverse geometric mean titers

(GMT) of duplicate determinations. 0, HXLO;

*, HXB2; O, HXB3.

Table 1. Restricted neutralization of viruses derived from HTLV-IIIB by selected patient sera. Inversegeometric mean titers (GMT) and standard errors of paired duplicate determinations on different viruspreparations with inocula of four times the 50% infectious dose (4x TCID50) are shown. Completeinhibition of viral expression at 2 weeks was used as the end point. Sera samples in parentheses sharesimilar patterns of NA. All viruses were grown in H9 lymphoblastoid cells, and supematants wereconcentrated 1000-fold by ultracentrifugation. Titrations and neutralization assays were done with animmunofluoresence technique to detect HIV-1 p24 expression (1). A 14- to 16-day incubation timewas used for both neutralization assays and calculation of TCID50 from eight parallel dilutions.

Virus neutralization (inverse GMT) for virusSera

HTLV-IIIB HX10 HXB2 HXB3

HS-06 (HS-15, -20) 360 ± 240 430 ± 290 510 ± 320 4000 ± 0

HS-12 (HS-07, -26) 180 ± 150 1600 ± 730 64 ± 0 64 ± 60

HS-23 (HS-22) 23 ± 16 130 ± 100 1000 ± 600 510 ± 390

HS-01 (HS-10, -13, -16) 1 ± 2 11 ± 8 16 ± 0 2000 ± 800

HS-08 (HS-04, -05, 1 0 2 ± 1 4 0 2 ± 1-18, -21)

REPORTS 357

28. H. Aviv and P. Leder, Proc. Natl. Acad. Sci. U.S.A.69, 1408 (1972).

29. H. Lehrach, D. Diamond, J. M. Wozney, H.Boedtker, Biochemistry 16, 4743 (1977).

30. P. Ponte, S. Y. Ng, J. Engel, P. Gunning, L. Kedes,Nucleic Acids Res. 12, 1687 (1984).

31. We thank S. H. Friend, R. A. Weinberg, and T. P.Dryja for the generous gift of the pO.9R and p3.8R

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Abnormalities in structure and expression of the human retinoblastoma gene in SCLCJW Harbour, SL Lai, J Whang-Peng, AF Gazdar, JD Minna and FJ Kaye

DOI: 10.1126/science.2838909 (4863), 353-357.241Science 

ARTICLE TOOLS http://science.sciencemag.org/content/241/4863/353

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