Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines
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336 coding region, threenucleotidedifferenceswerefound. Thesedifferences in the secondary structure of the protein, however, were caused by nucleotide substitutions. It was also demonstrated that the level of 67- kDa-lanrinin receptor nrRNA was higher in SBC3 than in IMR90. High frequency of somatically acquired ~53 mutations in small-cell lung cancer cell lines and tumors D’Amico D, Carbone D, Mitsudonri T, Nau M, Fedorko J, Russell E et al. Simmons Cancer Center, University of Tam Southwestern Medical Center, 5323 Harty Hines Boulevard, Dallas, TX 75235.8590. Gncogene 1992;7:339-46. We analysed the p53 open reading frame (ORF) in 16 small-cell lung cancer (SCLC) cell lines by direct sequencing of cDNA/PCR products and in 20 SCLC tumors by chemical cleavage and single-strand conformation polymorphism analyses of genomic DNA/PCR products. Ahnornudities ofp53 were found in 16/16 cell lines (100%) and in 161 20 tumors (80%). In the SCLC cell lines, mutations (59% ntissense, 18 % nonsense and 23 % splicing) changing the coding sequence were dispersed between amino acids 68 and 342. In the tumor samples, while the mutations occurred predominantly in exons 5-8, other mutations were located outside these regions. G to T transversions were common, occurring in 32% of the cases. We found no p53 mutations in the corresponding normal tissue from 19 patients whose tumors had p53 lesions, indicating that the mutations were all somatically acquired. In analysing the clinical data of the patients we found no correlation between tumor response to therapy or survival and the location or type of mutations. We conclude from these. data that: (I) p53 mutations are found in SCLC with high frequency; (2) p53 mutations in a significant fraction of cases generate cDNAs with nonsense or splicing mutations; and (3) to date, these mutations have all been somatically acquired events. (concentration for 50% inhibition of growth, 27-700 ng/ml), all of which had abundant expression of G(M1) ganglioside, the surface receptor for CT. CT-resistant SCLC all had greatly decreased G(MI) expression. In contrast, CT inhibited the growth of only four of 15 NSCLC cell lines. Seven of the I I CT-resistant NSCLC had levels of G(M1) comparable to CT-sensitive NSCLC or SCLC. In a limited panel of cell lines, cyclic AMP (CAMP) agonists including forskolin, 8Br[cAMP], and dibutyryl[cAMP] did not consistently reproduce CT- mediated inhibition of cell growth, nor did these compounds overcome resistance of cells to the growth inhibitory effects of CT. Expression of the R(1) and R(I1) regulatory subunits of CAMP-dependent protein kinase was similar in CT-resistant and CT- sensitive SCLC or NSCLC cell lines. I” the presence of isobutylmethylxanthine, intracellular CAMP levels induced by CT in a CT- resistant, G(Ml)( +) NSCLC cell line were comparable to those achieved in a CT-sensitive NSCLC cell line. We conclude that inhibition of lung carcinoma cell growth by CT in all cases requires expression of G(MI), and in the case of SCLC cell lines the presence of G(M 1) is sufticient. In NSCLC cell lines, expression of G(MI) is not sufficient for growth inhibition by CT. These findings imply refractoriness to growth inhibition by CAMP in G(Ml)( +), CT-resistant NSCLC cell lines and the possibility of “on- CAMP-related mechanisms for growth inhibition in CT-sensitive cell lines. Monoclonal antibody Kl reacts with epithelial mesothelioma hut not with lung adenocarcinoma Chang K, Pai LH, Pass H, Pogrebniak HW, Tsao M-S, Pasta” I et al. Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Building 37, Bethesda, MD 20892. Am J Surg Pathol 1992;16:259-68. Inmnmoperoxidase histochenrical staining of cryostat sections from human tumor tissues revealed that a murine monoclonal antibody (MAb), Kl, can distinguish epithelial mesotheliotnas from lung adenocarcinonras. All of 15 epithelial-type mesothelionras and all four mixed typemesothelionrasamples, butnoneof23 lungadenocarcinonras withdifferent degreeaofhistologicdiffereotiationdemonstratedreactivity with antibody Kl Of the cell populations in each mesothelioma tested, 80 % to 100% showed strong and homogeneous staining with MAb Kl. Inununofluorescence analysis of live cultured cells from a” epithelioid mesothelionta (H-mew) and several lung carcinoma cell lines as well as a pleural &fusion of a patient with mesothelioma also showed selective reactivity of KI with the mesothelioma cells. These data indicate that Kl can be useful as a mesothelial cell marker for thedifferential pathological diagnosis of the epithelial fotmof mesothelionta; Kl may also be useful in the study of the pathogenesis, inununodiagnosis, and inununotherapy of epithelial-type and mixed-type human malignant mesothelionra. Qrowth inhibition by cholera toxin of human lung carcinoma cell lines: Correlation with G(M1) ganglioside expression Kaur G, Viallet 1, Laborda J, Blair 0, Gazdar AF, Minna JD et al. Laboratory of Biological Cbemistty, NCI, NIH, %lW Rockville Pike, Bethesda. MD 20892. Cancer Res 1992;52:3340-6. The effect of cholera toxin (CT) on the growth of 12 small cell lung carcinoma (SCLC) and 15 non-small cell lung carcinoma (NSCLC) cell lines is presented. CT inhibited the growth of nine SCLC cell lines Endogenous receptor-bound umkinase mediates tissue invasion of the human lung carcinoma cell lines A549 and Calu-I Bruckner A, Fildemtan AE, Kirchheimer JC, Binder BR, Remold HG. Rheumatology/immunology Department. Brighnm and Women’s Hospital, LMRC, 221 LongwoodAve., Boston, MA 02115. Cancer Res 1992;52:3043-7. Macrophage colony-stimulating factor (CSF-1) increases the tissue invasive potential of the CSF-I receptor-bearing lung carcinoma cell lines A549 and Calu-1 by increasing thenumberofendogenously bound urokinase-type plasnrinogen activators (u-PA)s on these ceils. CSF-1, at concentrations which optimize invasion of A549 and Calu- I cells into human anmion membranes (250 “g/ml), nraxinrally augments the numberofu-PAreceptorsoccupiedbyendogenouslyproducedumkinase. Preincubatio” of A549 and Calu-I cells with the anti-u-PA monoclonal antibody MPWSUK (25 g/ml) or with a 20- to 40-fold stoichiometric excess of fluid phase type 2 plasnrinogen activator inhibitor abrogates invasiveness, indicating that functionally active cell surface u-PA ‘is essential for tissue invasion. In contrast, fluid phase type I plasnrinogen activator inhibitor (PAI-I, 5-15 units/ml) does not inhibit invasiveness unless preincubated with the anmion membranes. Inhibition of invasion by PAI- 1 isabohshed by presaturating theamnion membranes withanti- vitroneetin monoclonal antibody (IO g/ml) which prevents binding of PAI- to tissue- associated vitronectin. Binding of PAI-I to tissue vitronectin is therefore a prerequistte for its inhibitory action. Thus, endogenously receptor-bound u- PA is the primary protease mediating CSF-l-induced tissue invasiveness of the lung carcinoma cell lines A549 and Calu- 1. Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines Dantstrup L, Rygaard K, Spang-Thomsen M, Paulsen HS. Department of Oncology, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen. Cancer Res 1992;52:308993. Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinitylabeling,andNorthernblotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data demonstrated that the cells bound behveen 3 and 52 fnrollmg protein with a K(D) ranging from 0.5 x lo”’ to 2.7 x IO-” M. EGF binding to the receptor was contirnted by affinity-labeling EGF to the EGF
Transcript of Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines
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coding region, threenucleotidedifferenceswerefound. Thesedifferences in the secondary structure of the protein, however, were caused by nucleotide substitutions. It was also demonstrated that the level of 67- kDa-lanrinin receptor nrRNA was higher in SBC3 than in IMR90.
High frequency of somatically acquired ~53 mutations in small-cell lung cancer cell lines and tumors D’Amico D, Carbone D, Mitsudonri T, Nau M, Fedorko J, Russell E et al. Simmons Cancer Center, University of Tam Southwestern Medical Center, 5323 Harty Hines Boulevard, Dallas, TX 75235.8590. Gncogene 1992;7:339-46.
We analysed the p53 open reading frame (ORF) in 16 small-cell lung cancer (SCLC) cell lines by direct sequencing of cDNA/PCR products and in 20 SCLC tumors by chemical cleavage and single-strand conformation polymorphism analyses of genomic DNA/PCR products. Ahnornudities ofp53 were found in 16/16 cell lines (100%) and in 161 20 tumors (80%). In the SCLC cell lines, mutations (59% ntissense, 18 % nonsense and 23 % splicing) changing the coding sequence were dispersed between amino acids 68 and 342. In the tumor samples, while the mutations occurred predominantly in exons 5-8, other mutations were located outside these regions. G to T transversions were common, occurring in 32% of the cases. We found no p53 mutations in the corresponding normal tissue from 19 patients whose tumors had p53 lesions, indicating that the mutations were all somatically acquired. In analysing the clinical data of the patients we found no correlation between tumor response to therapy or survival and the location or type of mutations. We conclude from these. data that: (I) p53 mutations are found in SCLC with high frequency; (2) p53 mutations in a significant fraction of cases generate cDNAs with nonsense or splicing mutations; and (3) to date, these mutations have all been somatically acquired events.
(concentration for 50% inhibition of growth, 27-700 ng/ml), all of which had abundant expression of G(M1) ganglioside, the surface receptor for CT. CT-resistant SCLC all had greatly decreased G(MI) expression. In contrast, CT inhibited the growth of only four of 15 NSCLC cell lines. Seven of the I I CT-resistant NSCLC had levels of G(M1) comparable to CT-sensitive NSCLC or SCLC. In a limited panel of cell lines, cyclic AMP (CAMP) agonists including forskolin, 8Br[cAMP], and dibutyryl[cAMP] did not consistently reproduce CT- mediated inhibition of cell growth, nor did these compounds overcome resistance of cells to the growth inhibitory effects of CT. Expression of the R(1) and R(I1) regulatory subunits of CAMP-dependent protein kinase was similar in CT-resistant and CT- sensitive SCLC or NSCLC cell lines. I” the presence of isobutylmethylxanthine, intracellular CAMP levels induced by CT in a CT- resistant, G(Ml)( +) NSCLC cell line were comparable to those achieved in a CT-sensitive NSCLC cell line. We conclude that inhibition of lung carcinoma cell growth by CT in all cases requires expression of G(MI), and in the case of SCLC cell lines the presence of G(M 1) is sufticient. In NSCLC cell lines, expression of G(MI) is not sufficient for growth inhibition by CT. These findings imply refractoriness to growth inhibition by CAMP in G(Ml)( +), CT-resistant NSCLC cell lines and the possibility of “on- CAMP-related mechanisms for growth inhibition in CT-sensitive cell lines.
Monoclonal antibody Kl reacts with epithelial mesothelioma hut not with lung adenocarcinoma Chang K, Pai LH, Pass H, Pogrebniak HW, Tsao M-S, Pasta” I et al. Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Building 37, Bethesda, MD 20892. Am J Surg Pathol 1992;16:259-68.
Inmnmoperoxidase histochenrical staining of cryostat sections from human tumor tissues revealed that a murine monoclonal antibody (MAb), Kl, can distinguish epithelial mesotheliotnas from lung adenocarcinonras. All of 15 epithelial-type mesothelionras and all four mixed typemesothelionrasamples, butnoneof23 lungadenocarcinonras withdifferent degreeaofhistologicdiffereotiationdemonstratedreactivity with antibody Kl Of the cell populations in each mesothelioma tested, 80 % to 100% showed strong and homogeneous staining with MAb Kl. Inununofluorescence analysis of live cultured cells from a” epithelioid mesothelionta (H-mew) and several lung carcinoma cell lines as well as a pleural &fusion of a patient with mesothelioma also showed selective reactivity of KI with the mesothelioma cells. These data indicate that Kl can be useful as a mesothelial cell marker for thedifferential pathological diagnosis of the epithelial fotmof mesothelionta; Kl may also be useful in the study of the pathogenesis, inununodiagnosis, and inununotherapy of epithelial-type and mixed-type human malignant mesothelionra.
Qrowth inhibition by cholera toxin of human lung carcinoma cell lines: Correlation with G(M1) ganglioside expression Kaur G, Viallet 1, Laborda J, Blair 0, Gazdar AF, Minna JD et al. Laboratory of Biological Cbemistty, NCI, NIH, %lW Rockville Pike, Bethesda. MD 20892. Cancer Res 1992;52:3340-6.
The effect of cholera toxin (CT) on the growth of 12 small cell lung carcinoma (SCLC) and 15 non-small cell lung carcinoma (NSCLC) cell lines is presented. CT inhibited the growth of nine SCLC cell lines
Endogenous receptor-bound umkinase mediates tissue invasion of the human lung carcinoma cell lines A549 and Calu-I Bruckner A, Fildemtan AE, Kirchheimer JC, Binder BR, Remold HG. Rheumatology/immunology Department. Brighnm and Women’s Hospital, LMRC, 221 LongwoodAve., Boston, MA 02115. Cancer Res 1992;52:3043-7.
Macrophage colony-stimulating factor (CSF-1) increases the tissue invasive potential of the CSF-I receptor-bearing lung carcinoma cell lines A549 and Calu-1 by increasing thenumberofendogenously bound urokinase-type plasnrinogen activators (u-PA)s on these ceils. CSF-1, at concentrations which optimize invasion of A549 and Calu- I cells into human anmion membranes (250 “g/ml), nraxinrally augments the numberofu-PAreceptorsoccupiedbyendogenouslyproducedumkinase. Preincubatio” of A549 and Calu-I cells with the anti-u-PA monoclonal antibody MPWSUK (25 g/ml) or with a 20- to 40-fold stoichiometric excess of fluid phase type 2 plasnrinogen activator inhibitor abrogates invasiveness, indicating that functionally active cell surface u-PA ‘is essential for tissue invasion. In contrast, fluid phase type I plasnrinogen activator inhibitor (PAI-I, 5-15 units/ml) does not inhibit invasiveness unless preincubated with the anmion membranes. Inhibition of invasion by PAI- 1 isabohshed by presaturating theamnion membranes withanti- vitroneetin monoclonal antibody (IO g/ml) which prevents binding of PAI- to tissue- associated vitronectin. Binding of PAI-I to tissue vitronectin is therefore a prerequistte for its inhibitory action. Thus, endogenously receptor-bound u- PA is the primary protease mediating CSF-l-induced tissue invasiveness of the lung carcinoma cell lines A549 and Calu- 1.
Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines Dantstrup L, Rygaard K, Spang-Thomsen M, Paulsen HS. Department of Oncology, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen. Cancer Res 1992;52:308993.
Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinitylabeling,andNorthernblotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data demonstrated that the cells bound behveen 3 and 52 fnrollmg protein with a K(D) ranging from 0.5 x lo”’ to 2.7 x IO-” M. EGF binding to the receptor was contirnted by affinity-labeling EGF to the EGF
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receptor. The cross-linked complex had a M(r) of 17O,OC0-180,ooO. Northern blotting showed the expression of EGF receptor mRNA in all 10 cell lines that were found to be EGF receptor-positive and in one cell line that was found to be EGF receptor-negative in the radioreceptor assay and affinity labeling. Our results provide, for the first time, evidence that a large proportion of a broad panel of small cell lung cancer cell lines express the EGF receptor.
Cytogenetic comparison of two poorly differentiated human lung squamous cell carcinoma lines Law E, Gilvarry U, Lynch V, Gregory B, Grant G, Clynes M. Nariona2 Cell/Tissue Culture Centre, School of Biological Sciences, Dublin City University, Glasnevin, Dublin9. CancerGenet Cytogenet 1992;59: I1 l- 8.
This paper presents a cytogenetic analysis of two estabhshed but early-passage (passages 5 and 18) cell lines derived from histologically similar, poorly differentiated lymph node metastases of squamous cell carcmoma of the lung. The cell lines showed 3 shared marker chromosomes, del(l)(qll), del(2)(pll. 1) and del(2)(qll. 1). Gneofthe lines (DLKP) had 8 additIona markers including structural rearrangements such as tramlocations and isochromosomes. Five additional markers (including hvo delehons of chromosome 3) were found in DLRP. A notable feature of DLRP was the high incidence of telomeric association evident in the majorityofmetaphaseplates. Over- representationofchromoso~7wasacharactensticfeatureofmetapha~s derived from DLKP. and identltication of i(2lq) in this cell hne was an unusual finchng. Thersultslndicatesigllificilntcytogenetlchderogeneity between these early-passage cell lrnes dewed from two apparently histologically similar tumors.
First establishment of a human monoclonal antibody directed to sulfated glycosphingolipids S(M4s-Gal) and S(M4g), from a patient with lung cancer Miyake M, Taki T, Karma@ R, Hltomi S. Department of i%oraoc Surgery, Tazuke Kofi&i Medical Res. Inst., Kitano Hospital. 13-3, Kamiyamncho, Kitaku, Osaka. Cancar Res 1992;52:2292-7.
Several human monoclonal antibodies directed to hlmor-associated glycolipid antigens have been established, but more than one-half of themreactwithganglios,desandtheothersreaLt withneutralglycolipids. We report here the first establishment of a human IgM monoclonal antibody directed to the sulfated glycolipid. This monoclonal antibody, Ml4-376, did not react with S(M3) and S(Bla) which have a terminal HSO, 3Gall31 R,, but with the simple sulfolipids S(M4s-Gal) and S(M4g) which contam a terminal HSO, 3GalBl 0 CH, 5; however, lyso-S(M4s-Gal)and lyso-S(M4g) d,dnot bindMl4-376. These resuIIs suggest that terminal HSO, 3Gal and part of the hydrophobic region of the glycolipld are recognized by M 14- 376. Dmxtly biotmylated Ml4- 376 was used for immunohistochemical staining of 140 formalin-fixed, paraffin-embedded lung cancer twue sections lo study the distribution of the antigen. A high ,ncidence of positwe staining was found in adenocarcinoma (39.5%, 17 of 43), followed by large cell carcinoma (20.0%, 5 of 25), while thrs anhgen was rarely detected in small cell carcinoma (4.7%, 1 of 21) and squamous cell carcinoma (3.9%, 2 of 51). Thmlayerchromatography ,mmunostamingofglycolip,drextracted from lung cancer tissues showed the presence of only S(M4s-Gal) in adenocarcinoma, but S(M4g) was not found in any subtype of lung cancer. Immunohistochemical stammg revealed that this ant,gan was expressed in normal krdney, testis, and bram, but erythrocytes, granulocytes, and lymphocytes were negative ,n cytofluorometric a”alys,s.
Allelotype of non-small cell lung carcinoma - Comparison between loss of heterozygosity in squamous cell carcinoma and adenocarcinoma Tsuchiya E, Nakamura Y, Weng S-Y, Nakagawa K, Tsuchiya S, Sugano H et al. Department of Pathology, Cancer Institute, l-37-1, Kami-lkebukuro, Toshima-ku. Tokyo 170. Cancer Res 1992;52:2478- 81.
We examined loss of heterozygosity (LOH) on all autosomal chromosomes in 53 non-small cell lung carcinomas. Frequent LOH was observedon thelongarmsofchromosomes 1(37%), 2(31%), 5 (30%), 8 (3 1%). and I3 (32%), and theshortarmsofchromosomes 3 (54%) and 17 (62%). LOH on chromosomes 3p and 17p was observed ,n all informativecasesofsquamouscell carcinoma, hutwas significantly less frequent m adenocarcinomas (P = 0.003 and 0.001, respecbvely). Similarly, LOH on chromosome 13q was observed frequently ,n squamouscell carcmomas(5 of9 informativecases, or56%), but inonly 5 of26, or 19 %, ofadenocarc,nomas. In contrast, LOH onchromosome 2qwasobservedonlyinadenocarcinomas. Inadditron, thischromosornal arm was lost more frequently in poorly differentiated, compared to well differentiated adenocarcinomas. Furthermore. a correlation between fractional allelic loss and pathohistologlcal grade was identified. These results implicate the presence of several tumor suppressor genes associated wth development andiorprogression of non-small cell lung carcinomas.
Polymorphic sits within the MCC and APC loci reveal very frequent loss of heterozygosity in human small cell lung cancer D’Amico D, Carbonc DP, Johnson BE, Mel&r SJ, Minna JD. Simmons Cancer Center, Chiversify of Texas, Southwestern Medical Center, 5323 Harry Hitws Boulevard, Dallas, TX 75235-8593. Cancer Res 1992;52:1996-9.
Using single-strand conformation polymorplusm we have found two polymorphic Sites, AAC to AAT at codon 511 (exon 12) and GCT to GCG at codon 708 (exon 15), ,n the MCC gene. These wes and an RsaI polymorphrc site ,n APC allowed us to study 23 human small cell lung cancer (SCLC) and 7 non- small cell lung cancer samples for allele loss. Of the 23 SCLC samples, 21 (91%) were rnformative for one or more of these markers, and we found allele loss in more than 80 % (17 of 2 I). In non-small cell lung cancer samples, 5 of 7 (71%) were informatlbe, andreduction orlossofoneallelewas found ,n 2of 5 (40%). Sevencases were ,nformative for both genes, loss of heterozygoslty occurred tar both genes in fwe, one retained heterozygosrty for both, and one SCL.C had loss ofhcterozygouty for APC but not for MCC. We conclude that loss of heterozygowty occurs frequently for MCC and APC ,n lung cancer of all histolog,cal types and IS very frequent ,n SCLC. Tlus suggests the presence of tumor suppressor gene(s) ,n rhe MCCiAPC regmn of 5q21 ,nvolvcd in human lung cancer.
Applicationofmoleculargenetics to the early diagnosis and screening of lung cancer Birrer MJ, Brown PH. Lintformed Svcs. Hlth. Sciences Univ., Naval Hospital Bethesda, Bethesda. MD 20814. Cancer Res 1992;52:Suppl. 2658s-64s.
Recent studies of the molecular biology of lung cancer have identified multiple abnormalities. Despite ttus vast cataloging of genetic lesions, the chronology of these events and those which occur early remains essentially unknown. This review summaw.es thegenetic abnormalrtirs ,n lung cancer cells, including mutation, amplification, and overexpression of dominant protooncogenes as well as deletion and mutation of recessive oncogenes. In addition, possible candidate genes eX,st which may participate ,n the early actwation events of lung cancer, and evidence for thw role rn the early development of cancer IS discussed. These lesions may be helpful in developmg strateeles to screen for lung cancer
