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Expression of receptor tyrosine kinases in lung tumor cell lines
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016
Detection of bomb&n-like peptide receptor a in human lung cancer cell lines with polymerase chain reaction screening. M. Toi, A. Escobedo, G. Johnson, & M. Kane, Denver VAMC, Univ. Cole. HSC, and National Jewish Center, Denver, CO 80220. Most human small cell lung carcinoma (SCLC) cells express an autocrine growth system utilizing bombesin-like peptides (BLP) and the BLP receptor (BLPR) subtypes, gastrin-releasing peptide receptor (GRPR) and/or neuromedin B receptor (NMBR), although novel BLPRs are likely to exist as well. Our goals were to: 1) screen lung cancer cell lines for BLPR rapidly and sensitively using polymerase chain reaction (PCR), and 2) assess the clinical significance of BLPR. Pairs of primers were designed based on published cDNA sequences for BLPR. Primers I & II were predicted to amplify 600 bp sequences from all known BLPRs. Additional primer pairs were constructed to specifically amplify 500 bp sequences from either NMBR or GRPR. PCR was performed with each of the primer pairs on cDNA reverse transcribed from the mRNA of various lung cancer cell lines. With primers I & II, PCR products were obtained from 1 O/l 0 SCLC and 6/6 non-small cell lung carcinoma INSCLC) cell lines. Four SCLC cell lines expressed GRPR only, 1 cell line NMBR only, and 4, both. Four NSCLC expressed GRPR, and 1 NMBR only. PCR products obtained with primers I & II from the other SCLC and NSCLC lines had a novel DNA sequence, possibly representing a novel BLPR. Correlation of clinical data available with BLPR expression has so far revealed no significant difference in survival, age, or smoking history, but the sample size is small at present. Further screening by PCR is needed to evaluate for clinical significance, and may yield information with prognostic implications in patient samples.
IWllJWHISTOCHENICALSTUDYOF EPIOERNALGRONTHFACTOR RECEPTOR(EGFr) ANDTHE ONCOFWOTEIN c-orb62 IN LUNG CANCER _
017 EXPRESSION OF RECEPTOR TYROSINE KINASES IN LUNG TUMOR CELL LINES. Christine Holm and Andrius Kazlauskas, National Jewish Center for lmmunolo and Res iratory Medicine, Division of Basic Sciences, IYenver CO 802 g 6
commonly expressed in epithelial cell lines, we focused subsequent studies on these two rece tor tyrosine kinases. Western blot analysis demonstrated tR at the BPDGFR protein was expressed in a varie of NSCLC cell lines. Northern blot analysis confirmed that I?DR/flk-1 mRNA is expressed in a least a small oup of NSCLC cell lines. Current studies are designed to f etermme . whether the ligands for these owth ?;a! receptors are also expressed in the various NS 8rLC cell
P. Pfeiffer, P.P. Clausen, K. Andersen, C. Rose. Odense University Hospital, Denmark. From 1965 to 1990 tissue samples were obtained from 151 patients (squamous cell=SQ 80; adenocarcinoma=AD 45; large cell carcinoma=LA 16; SCLC 3; carcinoid tumor= Car 4) who were undergoing potentially curative resections for suspected primary non-SCLC. Tissue specimens were immediately frozen at -8OOC and stored until further examination. The expression of the protooncogene products EGFr and c-erbB2 was analyzed on cryostat sections using the peroxidase Labelled Strept-Avidin-Biotin technique. EGFr was recognized by the monoclonal antibody EGFRl (Amersham) and c-erb82 by the polyclonal antibody A465 (DAKO) and the results were correlated with clinical parameters of the patients. The percentage of positively reacting tumorcells within the entire biopsy (presence of definite membrane staining) was evaluated (grade 1: 1-10X; grade 2: ll-49%; grade 3: 50-79%; grade 4: > 80%). 3/3 SCLC and 3/4 carcinoid tumors were negative for EGFr. EGFr staining grade 3 and 4 were more frequent in SO (41% and 44%) than LA (61% and 9%) and AD (47% and 9%). In contrast c-erbB2 staining grade 3 and 4 were more frequent in AD (18% and 51%) EGFr status was not correlated to c-erbB2 status. EGFr and c-erbB2 staining were unrelated to grade, stage, nodal status and size of tumor. Survival was borderline correlated to EGFr status, but unrelated to c-erbB2 status.
018 EXPRESSION OF THE (;RP AUTOCRINE NCI-Aj4b
SCLC CELLS ilEQuLREs
GROWTH SYSTEM llrr CELGCELL INTERACTIONS
VIA THE H-A-V SEOUENCE OF THE CADHERIN ‘S FIRST EXTRACELLULAR DOMAIN. S.M. Aguayo, CL. Sundell, T.D. McDermott, and K.D. Schuyler. Div. of Pulmonary and Critical Care Medicine, Atlantn VA Medical Center, Emory University School of Medicine, Atlanta, GA. NCI-H345 small cell lung cancer (SCLC) cells grow in suspension in serwn-free, HITES-RPMI 1640 medium as cell clumps that exp=ss gastrin releasing peptide (GRP) in a constitutive fashion. However, GRP mRNA and peptide can he transiently upregulated by mechanically disaggregating cell clumps and plating cells af lower densities. This transient peak of GRP expression is typically followed by a rapid growth phase that is inhibited by anti-GRP monoclonal antibodies. Chunped cells plated at similar densities do not exhibit GRP upregulation or a rapid growth phase. Of note. disaggregated cells do not clump, express GRP, or even remain viable if plated in freshly made calcium-free medium or medium pretreated with EJJTA. Because GRF’upregulation and cell growth appear related to this calcium-dependent re-aggngation of cells, we hypothesized a role for cell adhesion molecules of-&cadherin family. Tb-test this hypothesis, we cultured cells with an inhibitor of cadherlns-mediated adhesion. a decapeptide containing tbe histldine-alanine-valine (HAV) cell adhesion recognition sequence that is common to the first extracellular domain of all known cadherins. Although clumped cells were not significantly affected, disaggregated cells plated with HAV-decapeptlde (1 mg/ml) did not reaggregate or upqulate their GRP expression, and began to die within 24. hours. In contrast, different control peptides did not affect aggregation, GRP expression, cell viability, or rate of cell growth. Next, we examined the relationship between cadherin-mediated call-cell contact and GRP mRNA expression using in situ mRNA hybridization with a human GRP cRNA probe. In siru hybridization demonstrated GRP mRNA expression within the re-aggregated, clumped cells, but not within the single. disaggregated cells that were cultured with the HAV-decapeptide. Our findings suggest that calcium-dependent cell-cell interactions during the re-aggregation 01 cells are mediated by cadherins, and that such cadherin-mediated cell adhesion regulates the expression of the GRP auto&w growth system in SCLC NCI-H345 cells. Therefore, while downregularion of cadherins expression may be advantageous for the metastatic potential of tumors. cadherins expression may indeed still be necessary for tamor viability
016
Detection of bomb&n-like peptide receptor a in human lung cancer cell lines with polymerase chain reaction screening. M. Toi, A. Escobedo, G. Johnson, & M. Kane, Denver VAMC, Univ. Cole. HSC, and National Jewish Center, Denver, CO 80220. Most human small cell lung carcinoma (SCLC) cells express an autocrine growth system utilizing bombesin-like peptides (BLP) and the BLP receptor (BLPR) subtypes, gastrin-releasing peptide receptor (GRPR) and/or neuromedin B receptor (NMBR), although novel BLPRs are likely to exist as well. Our goals were to: 1) screen lung cancer cell lines for BLPR rapidly and sensitively using polymerase chain reaction (PCR), and 2) assess the clinical significance of BLPR. Pairs of primers were designed based on published cDNA sequences for BLPR. Primers I & II were predicted to amplify 600 bp sequences from all known BLPRs. Additional primer pairs were constructed to specifically amplify 500 bp sequences from either NMBR or GRPR. PCR was performed with each of the primer pairs on cDNA reverse transcribed from the mRNA of various lung cancer cell lines. With primers I & II, PCR products were obtained from 1 O/l 0 SCLC and 6/6 non-small cell lung carcinoma INSCLC) cell lines. Four SCLC cell lines expressed GRPR only, 1 cell line NMBR only, and 4, both. Four NSCLC expressed GRPR, and 1 NMBR only. PCR products obtained with primers I & II from the other SCLC and NSCLC lines had a novel DNA sequence, possibly representing a novel BLPR. Correlation of clinical data available with BLPR expression has so far revealed no significant difference in survival, age, or smoking history, but the sample size is small at present. Further screening by PCR is needed to evaluate for clinical significance, and may yield information with prognostic implications in patient samples.
IWllJWHISTOCHENICALSTUDYOF EPIOERNALGRONTHFACTOR RECEPTOR(EGFr) ANDTHE ONCOFWOTEIN c-orb62 IN LUNG CANCER _
017 EXPRESSION OF RECEPTOR TYROSINE KINASES IN LUNG TUMOR CELL LINES. Christine Holm and Andrius Kazlauskas, National Jewish Center for lmmunolo and Res iratory Medicine, Division of Basic Sciences, IYenver CO 802 g 6
commonly expressed in epithelial cell lines, we focused subsequent studies on these two rece tor tyrosine kinases. Western blot analysis demonstrated tR at the BPDGFR protein was expressed in a varie of NSCLC cell lines. Northern blot analysis confirmed that I?DR/flk-1 mRNA is expressed in a least a small oup of NSCLC cell lines. Current studies are designed to f etermme . whether the ligands for these owth ?;a! receptors are also expressed in the various NS 8rLC cell
P. Pfeiffer, P.P. Clausen, K. Andersen, C. Rose. Odense University Hospital, Denmark. From 1965 to 1990 tissue samples were obtained from 151 patients (squamous cell=SQ 80; adenocarcinoma=AD 45; large cell carcinoma=LA 16; SCLC 3; carcinoid tumor= Car 4) who were undergoing potentially curative resections for suspected primary non-SCLC. Tissue specimens were immediately frozen at -8OOC and stored until further examination. The expression of the protooncogene products EGFr and c-erbB2 was analyzed on cryostat sections using the peroxidase Labelled Strept-Avidin-Biotin technique. EGFr was recognized by the monoclonal antibody EGFRl (Amersham) and c-erb82 by the polyclonal antibody A465 (DAKO) and the results were correlated with clinical parameters of the patients. The percentage of positively reacting tumorcells within the entire biopsy (presence of definite membrane staining) was evaluated (grade 1: 1-10X; grade 2: ll-49%; grade 3: 50-79%; grade 4: > 80%). 3/3 SCLC and 3/4 carcinoid tumors were negative for EGFr. EGFr staining grade 3 and 4 were more frequent in SO (41% and 44%) than LA (61% and 9%) and AD (47% and 9%). In contrast c-erbB2 staining grade 3 and 4 were more frequent in AD (18% and 51%) EGFr status was not correlated to c-erbB2 status. EGFr and c-erbB2 staining were unrelated to grade, stage, nodal status and size of tumor. Survival was borderline correlated to EGFr status, but unrelated to c-erbB2 status.
018 EXPRESSION OF THE (;RP AUTOCRINE NCI-Aj4b
SCLC CELLS ilEQuLREs
GROWTH SYSTEM llrr CELGCELL INTERACTIONS
VIA THE H-A-V SEOUENCE OF THE CADHERIN ‘S FIRST EXTRACELLULAR DOMAIN. S.M. Aguayo, CL. Sundell, T.D. McDermott, and K.D. Schuyler. Div. of Pulmonary and Critical Care Medicine, Atlantn VA Medical Center, Emory University School of Medicine, Atlanta, GA. NCI-H345 small cell lung cancer (SCLC) cells grow in suspension in serwn-free, HITES-RPMI 1640 medium as cell clumps that exp=ss gastrin releasing peptide (GRP) in a constitutive fashion. However, GRP mRNA and peptide can he transiently upregulated by mechanically disaggregating cell clumps and plating cells af lower densities. This transient peak of GRP expression is typically followed by a rapid growth phase that is inhibited by anti-GRP monoclonal antibodies. Chunped cells plated at similar densities do not exhibit GRP upregulation or a rapid growth phase. Of note. disaggregated cells do not clump, express GRP, or even remain viable if plated in freshly made calcium-free medium or medium pretreated with EJJTA. Because GRF’upregulation and cell growth appear related to this calcium-dependent re-aggngation of cells, we hypothesized a role for cell adhesion molecules of-&cadherin family. Tb-test this hypothesis, we cultured cells with an inhibitor of cadherlns-mediated adhesion. a decapeptide containing tbe histldine-alanine-valine (HAV) cell adhesion recognition sequence that is common to the first extracellular domain of all known cadherins. Although clumped cells were not significantly affected, disaggregated cells plated with HAV-decapeptlde (1 mg/ml) did not reaggregate or upqulate their GRP expression, and began to die within 24. hours. In contrast, different control peptides did not affect aggregation, GRP expression, cell viability, or rate of cell growth. Next, we examined the relationship between cadherin-mediated call-cell contact and GRP mRNA expression using in situ mRNA hybridization with a human GRP cRNA probe. In siru hybridization demonstrated GRP mRNA expression within the re-aggregated, clumped cells, but not within the single. disaggregated cells that were cultured with the HAV-decapeptide. Our findings suggest that calcium-dependent cell-cell interactions during the re-aggregation 01 cells are mediated by cadherins, and that such cadherin-mediated cell adhesion regulates the expression of the GRP auto&w growth system in SCLC NCI-H345 cells. Therefore, while downregularion of cadherins expression may be advantageous for the metastatic potential of tumors. cadherins expression may indeed still be necessary for tamor viability