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Loss of heterozygosity for a chromosome 3 sequence presumably at 3p21 in small cell lung cancer
Loss of Heterozygosity for a Chromosome 3 Sequence Presumably at 3p21 in Small Cell Lung Cancer Hans Mooibroek, Jan Osinga, Pieter E. Postmus, Ben Carritt, and Charles H.C.M. Buys
ABSTRACT: A recombinant DNA fragment detecting a chromosome #3 restriction fragment length polymorphism presumably at p21 was hybridized to HindlII-digested DNA isolated from the leukocytes of 12 patients of small cell lung cancer. Four of them appeared to be heterozygous. Analysis of tumor material from these four patients revealed homozygosity for either one or the other restriction fragment in every case. Our findings suggest the presence on the short arm of chromosome #3 of a recessive mutant cancer gene contributing to the development of small cell lung cancer. INTRODUCTION Deletions of a varying length of the short arm of chromosome #3 were found u p o n cytogenetic analysis of a n u m b e r of small cell lung cancer (SCLC) cell lines, shortterm cultures of tumor cells, and a SCLC bone marrow preparation [1]. We confirmed the frequent occurrence of such deletions in a chromosome analysis of three SCLC cell lines and defined p21-p23 as the shortest region of overlap [2, 3]. The h u m a n DNA clone ~,Ch4A-H3, previously m a p p e d to l p by in situ hybridization [4, 5], has recently been shown to originate from chromosome #3. This explains the clustering of grains after in situ hybridization not only over lp36, but also to a significant degree over 3p21 [5]. This clone, and its subclone H3H2, detect restriction fragment length polymorphisms on both chromosomes #1 and # 3 [6,7], that for chromosome # 3 being presumably in the region 3p21. H3H2, therefore, was used as a probe to detect possible deletions of 3p21 directly into DNA from tumors of SCLC patients. MATERIALS AND METHODS Leukocytes were recovered from samples of whole blood after osmotic lysis of erythrocytes and platelets by a m m o n i u m chloride [8]. High molecular weight DNA was obtained by overnight i n c u b a t i o n at 37°C in 10 mM Tris-HC1, pH 8.0, 100 mM From the Departmentsof Human Genetics (H. M., J. O., C. H. C. M. B.) and PulmonaryDiseases (P. E. P.), State Universityof Groningen,The Netherlands, and the MRC Human BiochemicalGenetics Unit (B. C.), UniversityCollege, London,England. Address requests for reprints to Dr. C. H. C. M. Buys, Department of Human Genetics, State University of Groningen, Antonius Deusinglaan 4, NL 9713 A W Groningen, The Netherlands. Received September 9, 1986; accepted November 25, 1986.
361 © 1987 Elsevier SciencePublishingCo., Inc. 52 VanderbiltAve., New York, NY 10017
Cancer Genet Cytogenet27:361-365 (1987) 0165-4608/87/$03.50
362
H. Mooibroek et al. NaC1, 1 mM EDTA, containing 100 ~g proteinase K and 10 mg/ml sodium dodecyl sulfate (SDS). Samples were extracted twice with phenol and twice with a 1:1 (v/ v) phenol-chloroform mixture. DNA was precipitated at room temperature by adding 1/30 volume of a 3 M sodium acetate solution and one volume of isopropanol, pH 5.2, and redissolved in 10 mM Tris-HC1, pH 8.0, 0.1 mM EDTA. HindIII digestion was carried out overnight with a twofold excess of enzyme under standard conditions [9]. Agarose electrophoresis was performed at low voltage using 0.6% gels. Transfer to GeneScreen Plus membrane (New England Nuclear) was performed in 0.4 M NaOH, 0.6 M NaC1 for 18-24 hours. Filters were prehybridized at 65°C in 0.5 M NaHPO4, pH 7.2, containing 7.0% SDS, 1.0 mM EDTA, and 1.0% bovine serum albumin, for 1-2 hours [10]. The internal HindIII fragment of XCh4A-H3 [6] was labeled by nick translation to a specific activity of approximately 108 cpm/~g [11]. For every 10 ml of prehybridization solution, 75 ng of denatured labeled probe DNA was added, and hybridization was carried out overnight at 65°C. Filters were washed at room temperature in 40 mM NaHPO4, pH 7.2, containing 5% SDS, 1.0 mM EDTA, and 0.5% albumin fraction V for 5 minutes, subsequently four to five times in 40 mM NaHPO4 pH 7.2, containing 1.0% SDS, and 1.0 mM EDTA, then three times in 100 mM NaHPO4, pH 7.2, and finally in the same buffer at 65°C for 20 minutes [10]. Filters were covered with plastic wrap and exposed to XAR-5 film (Kodak) backed by a Lightning Plus intensifying screen (Du Pont) at -80°C for 18 hours up to 7 days. Tumor material was obtained from lung resections (patients A, B, and C) and from a metastatic lymph node tumor (patient D). The diagnosis of SCLC was based on a clinical and histologic evaluation. After mincing the tumors into small pieces DNA was isolated as described above.
RESULTS AND DISCUSSION
Southern analysis of HindIII digested leukocyte DNA from 12 SCLC patients using pH3H2 as a probe revealed homozygosity for the 2.3-Kb fragment in four of them, homozygosity for the 2.0-Kb fragment in four others, and heterozygosity in the remaining four. Thus, in this panel of SCLC patients, each of the alleles of this polymorphism specific for chromosome #3 [6, 7] had a frequency of 0.5, in agreement with our previous finding using a panel of ten unrelated controls [12]. In a recent study [7] on a larger panel (39 individuals) frequencies were found of about 0.4 and 0.6 for the larger and smaller fragment, respectively. These preliminary data suggest that there is no difference in the distribution of alleles among SCLC patients and in the Caucasian population in general. Based on the reported allele frequencies, the frequency of the heterozygous phenotype in the population will be close to the maximum of 50%. This makes the HindlII polymorphism of H3H2 a most suitable one to study a possible loss of the chromosome #3 region involved, presumably 3p21 [5], in tumors where deletions of the short arm of chromosome #3 occur as in SCLC [1-3] or possibly in renal cell carcinomas [13-16]. DNA was isolated from tumor material obtained from the four patients who were heterozygous for the polymorphism in their leukocyte DNA. When these tumor DNA were subjected to the same analysis, all of them showed homozygosity for either the 2.3-Kb fragment (three cases) or the 2.0-Kb fragment (one case). A comparison between the results obtained with constitutional and tumor DNA for the four cases is shown in Figure 1. The additional very faint band in the autoradiogram at 2.0 Kb in the tumor DNA of patient C is most likely explained by the presence of some nontumor cells in the lung resection material. The consistent finding of a loss of heterozygosity for a chromosome #3 polymorphism presumably located at p21 resulting in hemi- or homozygosity in tumor
363
Loss of 3p Heterozygosity in SCLC
L
T A
B
C
D
A
B
C
D
'7
I 8 . 0 k b
-
I
3.
Skb
-
I I
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I
i!/i H
I
I
iJ.......ii
~ ~i ~
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2.3kb
-
2.0kb
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Figure 1. Loss of constitutional heterozygosity in four SCLC patients at the chromosome #3 locus recognized by H3H2. The panel at left represents the patterns obtained with HindIII digested leukocyte DNA (patients A-D). The panel at right represents the tumor DNA samples from the same patients.
DNA means that at least this chromosome # 3 sequence is deleted in the genesis or progression of SCLC. At the moment, we cannot exclude the loss by mitotic nondisjunction of a w h o l e chromosome # 3 h o m o l o g in some cases, but involvement of submicroscopic deletions seems likely in a number of cases in v i e w of failures to find a consistent 3p deletion in SCLC tumor tissue or derived cell lines [17, 18]. Alternatively, loss of a deleted homologue might be compensated by duplication of the morphologically normal one. Our studies are n o w directed to discriminate between these situations and to delimit the possible deletions. A n y h o w , the present findings suggest that some mutant cancer gene at 3p21 or in its vicinity is involved in the development of SCLC. Loss of the normal allele on the other homologue seems to be necessary to allow expression of the mutant gene. Possibly, the normal allele codes for some factor suppressing a proliferation gene or group of genes elsewhere in the genome [12] analogous to m e c h a n i s m s made plausible for childhood cancers [19, 20]. The analogy is even corroborated by the finding of m y c gene amplification in further tumor development both in childhood cancers like retinoblastoma [21] and neuroblastoma [22], as well as in SCLC [12, 23-26]. Suggestive as the analogy might be, present data do not s h o w whether the described loss of heterozygosity of chromosome # 3 is a cause or only a consequence of SCLC development. Supported by grants from the Netherlands Cancer Foundation and the Jan Kornelis de Cock Foundation.
364
H. M o o i b r o e k et al.
REFERENCES 1. Whang-Peng J, Bunn Jr PA, Kao-Shan CS, Lee EC, Carney DN, Gazdar A, Minna JD (1982): A n o n r a n d o m chromosomal abnormality, del 3p(14-23), in h u m a n small cell lung cancer (SCLC). Cancer Genet Cytogenet 6:119-134. 2. Buys CHCM, van der Veen AY, de Leij L (1984): Chromosome analysis of three cell lines established from small cell carcinoma of the lung. Chromosomes Today 8:301. 3. De Leij L, Postmus PE, Buys CHCM, Elema JD, Ramaekers F, Poppema S, Brouwer M, van der Veen AY, Mesander G, The Th (1985): Characterization of three new variant type cell lines derived from small cell carcinoma of the lung. Cancer Res 45:6024-6033. 4. Harper ME, Saunders GF (1981): Localization of single copy DNA sequences on G-banded h u m a n chromosomes by in situ hybridization. Chromosoma 83:431--439. 5. Donlon TA, Magenis RE (1984): Localization of the cloned segment Ch4A-H3 to chromosome 1 band p36: A confirmation of locus D1S1. (HGM7) Cytogenet Cell Genet 37:454. 6. Carritt B, Welch HM, Parry-Jones NJ (1986): Sequences homologous to the h u m a n D1S1 locus present on h u m a n chromosome 3. Am J Hum Genet 38:428-436. 7. Goode ME, van Tuinen P, Ledbetter DH, Daiger SP (1986): The anonymous polymorphic DNA clone DIS1, previously mapped to h u m a n chromosome lp36 by in situ hybridization, is from chromosome 3 and is duplicated on chromosome 1. Am J Hum Genet 38:437-446. 8. Dioguardi N, Agostani A, Fiorelli G, Lomanto B (1963): Characterization of lactic dehydrogenase of normal h u m a n granulocytes. J Lab Clin Med 61:713-723. 9. Maniatis T, Fritsch EF, Sambrook J (1982): Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 104-105. 10. Church GM, Gilbert W (1984): Genomic sequencing. Proc Natl Acad Sci USA 81:19911995, 11. Rigby PW, Dieckman M, Rhodes C, Berg P (1977): Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Molec Biol 113:237251. 12. Buys CHCM, Osinga J, van der Veen AY, Mooibroek H, van der Hout AH, de Leij L, Postmus PE, Carritt B (1986): Genome analysis of small cell lung cancer (SCLC) and clinical significance. Eur J Resp Dis (in press) 13. Cohen AJ, Li FP, Berg S, Marchetto DJ, Tsai S, Jacobs SC, Brown RS (1979): Hereditary renal cell carcinoma associated with a chromosomal translocation. N Engl J Med 301:592595. 14. Pathak S, Strong LC, Ferrell RE, Trinidade A (1982): Familial renal cell carcinoma with a 3;11 chromosome translocation limited to tumor cells. Science 217:939-940. 15. Wang N, Perkins KL (1984): Involvement of band 3p14 in t(3;8) hereditary renal carcinoma. Cancer Genet Cytogenet 11:479-481. 16. Yoshida MA, Ohyashiki K, Ochi H, Gibas Z, Pontes JE, Prout Jr GR, Huben R, Sandberg AA (1986): Cytogenetic studies of tumor tissue from patients with nonfamilial renal cell carcinoma. Cancer Res 46:2139-2147. 17. Wurster-Hill, Cannizzaro LA, Pettengill OS, Sorenson GD, Cate CC, Maurer LH (1984): Cytogenetics of small cell carcinoma of the lung. Cancer Genet Cytogenet 13:303-330. 18. Zech L, Bergh J, Nilsson K (1985): Karyotypic characterization of established cell lines and short time cultures of h u m a n lung cancers. Cancer Genet Cytogenet 15:335-347. 19. Knudson Jr AG (1985): Hereditary cancer, oncogenes, and anti-oncogenes. Cancer Res 45:1437-1443. 20. Cavenee WK, Koufos A, Hansen MF (1986): Recessive mutant genes predisposing to human cancer. Mutation Res 168:3-14. 21. Lee W-H, Murphree AL, Benedict WF (1984): Expresson and amplification of the N-myc gene in primary retinoblastoma. Nature 309:458-460. 22. Brodeur GM, Seeger RC, Schwab M, Varmus HE, Bishop JM (1984): Amplification of Nmyc in untreated h u m a n neuroblastomas correlates with advanced disease stage. Science 224:1121-1124. 23. Little CD, Nau MM, Carney DN, Gazdar AF, Minna JD (1983): Amplification and expression of the c-myc oncogene in h u m a n lung cancer cell lines. Nature 306:194-196.
Loss of 3p H e t e r o z y g o s i t y in SCLC
365
24. Nau MM, Carney DN, Battey J, Johnson B, Little C, Gazdar A, Minna JD (1984): Amplification, expression and rearrangement of c-myc and N-myc oncogenes in human lung cancer. Current Topics Microbiol Immunol 113:172-177. 25. Saksela K, Bergh J, Lehto V-P, Nilsson K, Alitalo K (1985): Amplification of the c-myc oncogene in a subpopulation of human small cell lung cancer. Cancer Res 45:1823-1827. 26. Nau MM, Brooks BJ, Battey J, Sausville E, Gazdar AF, Kitsch IR, McBride OW, Bertness V, Hollis G, Minna JD (1985): L-myc, a new myc-related gene amplified and expressed in human small cell lung cancer. Nature 318:69-73.
ABSTRACT: A recombinant DNA fragment detecting a chromosome #3 restriction fragment length polymorphism presumably at p21 was hybridized to HindlII-digested DNA isolated from the leukocytes of 12 patients of small cell lung cancer. Four of them appeared to be heterozygous. Analysis of tumor material from these four patients revealed homozygosity for either one or the other restriction fragment in every case. Our findings suggest the presence on the short arm of chromosome #3 of a recessive mutant cancer gene contributing to the development of small cell lung cancer. INTRODUCTION Deletions of a varying length of the short arm of chromosome #3 were found u p o n cytogenetic analysis of a n u m b e r of small cell lung cancer (SCLC) cell lines, shortterm cultures of tumor cells, and a SCLC bone marrow preparation [1]. We confirmed the frequent occurrence of such deletions in a chromosome analysis of three SCLC cell lines and defined p21-p23 as the shortest region of overlap [2, 3]. The h u m a n DNA clone ~,Ch4A-H3, previously m a p p e d to l p by in situ hybridization [4, 5], has recently been shown to originate from chromosome #3. This explains the clustering of grains after in situ hybridization not only over lp36, but also to a significant degree over 3p21 [5]. This clone, and its subclone H3H2, detect restriction fragment length polymorphisms on both chromosomes #1 and # 3 [6,7], that for chromosome # 3 being presumably in the region 3p21. H3H2, therefore, was used as a probe to detect possible deletions of 3p21 directly into DNA from tumors of SCLC patients. MATERIALS AND METHODS Leukocytes were recovered from samples of whole blood after osmotic lysis of erythrocytes and platelets by a m m o n i u m chloride [8]. High molecular weight DNA was obtained by overnight i n c u b a t i o n at 37°C in 10 mM Tris-HC1, pH 8.0, 100 mM From the Departmentsof Human Genetics (H. M., J. O., C. H. C. M. B.) and PulmonaryDiseases (P. E. P.), State Universityof Groningen,The Netherlands, and the MRC Human BiochemicalGenetics Unit (B. C.), UniversityCollege, London,England. Address requests for reprints to Dr. C. H. C. M. Buys, Department of Human Genetics, State University of Groningen, Antonius Deusinglaan 4, NL 9713 A W Groningen, The Netherlands. Received September 9, 1986; accepted November 25, 1986.
361 © 1987 Elsevier SciencePublishingCo., Inc. 52 VanderbiltAve., New York, NY 10017
Cancer Genet Cytogenet27:361-365 (1987) 0165-4608/87/$03.50
362
H. Mooibroek et al. NaC1, 1 mM EDTA, containing 100 ~g proteinase K and 10 mg/ml sodium dodecyl sulfate (SDS). Samples were extracted twice with phenol and twice with a 1:1 (v/ v) phenol-chloroform mixture. DNA was precipitated at room temperature by adding 1/30 volume of a 3 M sodium acetate solution and one volume of isopropanol, pH 5.2, and redissolved in 10 mM Tris-HC1, pH 8.0, 0.1 mM EDTA. HindIII digestion was carried out overnight with a twofold excess of enzyme under standard conditions [9]. Agarose electrophoresis was performed at low voltage using 0.6% gels. Transfer to GeneScreen Plus membrane (New England Nuclear) was performed in 0.4 M NaOH, 0.6 M NaC1 for 18-24 hours. Filters were prehybridized at 65°C in 0.5 M NaHPO4, pH 7.2, containing 7.0% SDS, 1.0 mM EDTA, and 1.0% bovine serum albumin, for 1-2 hours [10]. The internal HindIII fragment of XCh4A-H3 [6] was labeled by nick translation to a specific activity of approximately 108 cpm/~g [11]. For every 10 ml of prehybridization solution, 75 ng of denatured labeled probe DNA was added, and hybridization was carried out overnight at 65°C. Filters were washed at room temperature in 40 mM NaHPO4, pH 7.2, containing 5% SDS, 1.0 mM EDTA, and 0.5% albumin fraction V for 5 minutes, subsequently four to five times in 40 mM NaHPO4 pH 7.2, containing 1.0% SDS, and 1.0 mM EDTA, then three times in 100 mM NaHPO4, pH 7.2, and finally in the same buffer at 65°C for 20 minutes [10]. Filters were covered with plastic wrap and exposed to XAR-5 film (Kodak) backed by a Lightning Plus intensifying screen (Du Pont) at -80°C for 18 hours up to 7 days. Tumor material was obtained from lung resections (patients A, B, and C) and from a metastatic lymph node tumor (patient D). The diagnosis of SCLC was based on a clinical and histologic evaluation. After mincing the tumors into small pieces DNA was isolated as described above.
RESULTS AND DISCUSSION
Southern analysis of HindIII digested leukocyte DNA from 12 SCLC patients using pH3H2 as a probe revealed homozygosity for the 2.3-Kb fragment in four of them, homozygosity for the 2.0-Kb fragment in four others, and heterozygosity in the remaining four. Thus, in this panel of SCLC patients, each of the alleles of this polymorphism specific for chromosome #3 [6, 7] had a frequency of 0.5, in agreement with our previous finding using a panel of ten unrelated controls [12]. In a recent study [7] on a larger panel (39 individuals) frequencies were found of about 0.4 and 0.6 for the larger and smaller fragment, respectively. These preliminary data suggest that there is no difference in the distribution of alleles among SCLC patients and in the Caucasian population in general. Based on the reported allele frequencies, the frequency of the heterozygous phenotype in the population will be close to the maximum of 50%. This makes the HindlII polymorphism of H3H2 a most suitable one to study a possible loss of the chromosome #3 region involved, presumably 3p21 [5], in tumors where deletions of the short arm of chromosome #3 occur as in SCLC [1-3] or possibly in renal cell carcinomas [13-16]. DNA was isolated from tumor material obtained from the four patients who were heterozygous for the polymorphism in their leukocyte DNA. When these tumor DNA were subjected to the same analysis, all of them showed homozygosity for either the 2.3-Kb fragment (three cases) or the 2.0-Kb fragment (one case). A comparison between the results obtained with constitutional and tumor DNA for the four cases is shown in Figure 1. The additional very faint band in the autoradiogram at 2.0 Kb in the tumor DNA of patient C is most likely explained by the presence of some nontumor cells in the lung resection material. The consistent finding of a loss of heterozygosity for a chromosome #3 polymorphism presumably located at p21 resulting in hemi- or homozygosity in tumor
363
Loss of 3p Heterozygosity in SCLC
L
T A
B
C
D
A
B
C
D
'7
I 8 . 0 k b
-
I
3.
Skb
-
I I
I I
I
i!/i H
I
I
iJ.......ii
~ ~i ~
~ ~!
2.3kb
-
2.0kb
il
~ii~ ¸
Figure 1. Loss of constitutional heterozygosity in four SCLC patients at the chromosome #3 locus recognized by H3H2. The panel at left represents the patterns obtained with HindIII digested leukocyte DNA (patients A-D). The panel at right represents the tumor DNA samples from the same patients.
DNA means that at least this chromosome # 3 sequence is deleted in the genesis or progression of SCLC. At the moment, we cannot exclude the loss by mitotic nondisjunction of a w h o l e chromosome # 3 h o m o l o g in some cases, but involvement of submicroscopic deletions seems likely in a number of cases in v i e w of failures to find a consistent 3p deletion in SCLC tumor tissue or derived cell lines [17, 18]. Alternatively, loss of a deleted homologue might be compensated by duplication of the morphologically normal one. Our studies are n o w directed to discriminate between these situations and to delimit the possible deletions. A n y h o w , the present findings suggest that some mutant cancer gene at 3p21 or in its vicinity is involved in the development of SCLC. Loss of the normal allele on the other homologue seems to be necessary to allow expression of the mutant gene. Possibly, the normal allele codes for some factor suppressing a proliferation gene or group of genes elsewhere in the genome [12] analogous to m e c h a n i s m s made plausible for childhood cancers [19, 20]. The analogy is even corroborated by the finding of m y c gene amplification in further tumor development both in childhood cancers like retinoblastoma [21] and neuroblastoma [22], as well as in SCLC [12, 23-26]. Suggestive as the analogy might be, present data do not s h o w whether the described loss of heterozygosity of chromosome # 3 is a cause or only a consequence of SCLC development. Supported by grants from the Netherlands Cancer Foundation and the Jan Kornelis de Cock Foundation.
364
H. M o o i b r o e k et al.
REFERENCES 1. Whang-Peng J, Bunn Jr PA, Kao-Shan CS, Lee EC, Carney DN, Gazdar A, Minna JD (1982): A n o n r a n d o m chromosomal abnormality, del 3p(14-23), in h u m a n small cell lung cancer (SCLC). Cancer Genet Cytogenet 6:119-134. 2. Buys CHCM, van der Veen AY, de Leij L (1984): Chromosome analysis of three cell lines established from small cell carcinoma of the lung. Chromosomes Today 8:301. 3. De Leij L, Postmus PE, Buys CHCM, Elema JD, Ramaekers F, Poppema S, Brouwer M, van der Veen AY, Mesander G, The Th (1985): Characterization of three new variant type cell lines derived from small cell carcinoma of the lung. Cancer Res 45:6024-6033. 4. Harper ME, Saunders GF (1981): Localization of single copy DNA sequences on G-banded h u m a n chromosomes by in situ hybridization. Chromosoma 83:431--439. 5. Donlon TA, Magenis RE (1984): Localization of the cloned segment Ch4A-H3 to chromosome 1 band p36: A confirmation of locus D1S1. (HGM7) Cytogenet Cell Genet 37:454. 6. Carritt B, Welch HM, Parry-Jones NJ (1986): Sequences homologous to the h u m a n D1S1 locus present on h u m a n chromosome 3. Am J Hum Genet 38:428-436. 7. Goode ME, van Tuinen P, Ledbetter DH, Daiger SP (1986): The anonymous polymorphic DNA clone DIS1, previously mapped to h u m a n chromosome lp36 by in situ hybridization, is from chromosome 3 and is duplicated on chromosome 1. Am J Hum Genet 38:437-446. 8. Dioguardi N, Agostani A, Fiorelli G, Lomanto B (1963): Characterization of lactic dehydrogenase of normal h u m a n granulocytes. J Lab Clin Med 61:713-723. 9. Maniatis T, Fritsch EF, Sambrook J (1982): Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 104-105. 10. Church GM, Gilbert W (1984): Genomic sequencing. Proc Natl Acad Sci USA 81:19911995, 11. Rigby PW, Dieckman M, Rhodes C, Berg P (1977): Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Molec Biol 113:237251. 12. Buys CHCM, Osinga J, van der Veen AY, Mooibroek H, van der Hout AH, de Leij L, Postmus PE, Carritt B (1986): Genome analysis of small cell lung cancer (SCLC) and clinical significance. Eur J Resp Dis (in press) 13. Cohen AJ, Li FP, Berg S, Marchetto DJ, Tsai S, Jacobs SC, Brown RS (1979): Hereditary renal cell carcinoma associated with a chromosomal translocation. N Engl J Med 301:592595. 14. Pathak S, Strong LC, Ferrell RE, Trinidade A (1982): Familial renal cell carcinoma with a 3;11 chromosome translocation limited to tumor cells. Science 217:939-940. 15. Wang N, Perkins KL (1984): Involvement of band 3p14 in t(3;8) hereditary renal carcinoma. Cancer Genet Cytogenet 11:479-481. 16. Yoshida MA, Ohyashiki K, Ochi H, Gibas Z, Pontes JE, Prout Jr GR, Huben R, Sandberg AA (1986): Cytogenetic studies of tumor tissue from patients with nonfamilial renal cell carcinoma. Cancer Res 46:2139-2147. 17. Wurster-Hill, Cannizzaro LA, Pettengill OS, Sorenson GD, Cate CC, Maurer LH (1984): Cytogenetics of small cell carcinoma of the lung. Cancer Genet Cytogenet 13:303-330. 18. Zech L, Bergh J, Nilsson K (1985): Karyotypic characterization of established cell lines and short time cultures of h u m a n lung cancers. Cancer Genet Cytogenet 15:335-347. 19. Knudson Jr AG (1985): Hereditary cancer, oncogenes, and anti-oncogenes. Cancer Res 45:1437-1443. 20. Cavenee WK, Koufos A, Hansen MF (1986): Recessive mutant genes predisposing to human cancer. Mutation Res 168:3-14. 21. Lee W-H, Murphree AL, Benedict WF (1984): Expresson and amplification of the N-myc gene in primary retinoblastoma. Nature 309:458-460. 22. Brodeur GM, Seeger RC, Schwab M, Varmus HE, Bishop JM (1984): Amplification of Nmyc in untreated h u m a n neuroblastomas correlates with advanced disease stage. Science 224:1121-1124. 23. Little CD, Nau MM, Carney DN, Gazdar AF, Minna JD (1983): Amplification and expression of the c-myc oncogene in h u m a n lung cancer cell lines. Nature 306:194-196.
Loss of 3p H e t e r o z y g o s i t y in SCLC
365
24. Nau MM, Carney DN, Battey J, Johnson B, Little C, Gazdar A, Minna JD (1984): Amplification, expression and rearrangement of c-myc and N-myc oncogenes in human lung cancer. Current Topics Microbiol Immunol 113:172-177. 25. Saksela K, Bergh J, Lehto V-P, Nilsson K, Alitalo K (1985): Amplification of the c-myc oncogene in a subpopulation of human small cell lung cancer. Cancer Res 45:1823-1827. 26. Nau MM, Brooks BJ, Battey J, Sausville E, Gazdar AF, Kitsch IR, McBride OW, Bertness V, Hollis G, Minna JD (1985): L-myc, a new myc-related gene amplified and expressed in human small cell lung cancer. Nature 318:69-73.