- Email: [email protected]
Direct Evidence for a Membrane-Associated Receptor for a Bombesin-Like Peptide in Human Small Cell Lung Carcinoma H345 Cells
particles secreted basal levels of fibroblast growth promoting activity. Conditioned media collected at various times tween 30 min and 10 h after phagocytosis of zymosan and insoluble particulates (carbonyl iron and chrysotile asbestos) significantly stimulated further increase of 3H-TdR uptake by RLF (p<. 0(1) (Fig 1). In a complementation test, serum-free eM alone resulted in basal fibroblast 3H-TdR incorporation, whereas CM acted synergistically with progression factors in PPP to stimulate fibroblasts approximately tenfold, suggesting the role of this MDGF as a competence factor: Activity in whole eM is detectable at 1:100dilution, is nondialyzable, is stable to heat (lOOOC, io min, 73% retained activity) and acid (1M acetic, 68., retained activity), and is sensitive to a reducing agent (dithiothreitol, SOmM). Freeze-thawing and Concentration plus lyophilization result in no loss of activity. Approximately 90% of the protein and some associated activity are not retained on a cation exchange column (Mono S) at pH 6.0. A single peak ofbound activity elutes from this column at apprOxiinately 0.15 M NaCI just ahead of a small protein peak, A major peak of activity elutes with anapparent molecular weight of 25 KD on a size exclusion column (Spherogel 'rSIC) with a smaller peak of activity detectable in the high molecular weight range, possibly representing an active precursor molecule, protein-bound 25 KD MDGF or a second growth factor in CM. Preincubation (1 h, 30°C) of serum-free CM with a monoclonal antibody to a sis oneogene-predicted synthetic peptide homologous to residues 1-18of the amino terminus of the B chain ofPDGF4 showed a concentration-dependent inhibition of macrophage-derived (MQGF) activity when assayed on RLF (Fi,g2). growth Preincubation of eM with a polyclonal antibody to synthetic rat TFGa. antigenic determinant has shown no inhibition of activity. Recognition by anti-sis 1-18suggests that the macrophage-derived protein contains a sequence homologous to this highly conserved region of the sis proto-oncogene and that this site is available for recognition in the native macrophage protein. In summary, rat alveolar macrophages secrete a heatstable competence factor with cationic properties and immunologic identity to a sis-predicted synthetic peptide, suggesting at least one factor secreted by rat AMs may be homologous With PDGR s This MDGF is differentially secreted depending on the type of particle phagocytized, with sustained secretion observed following phagocytosis of insoluble particulates. Therefore, in parenchymal lung disease in which macrophages may be attracted to sites of particle deposition ~r lung injury, I local secretion of this PDGF-like molecule may be important in the regulation of altered mesenchymal cell growth.
factor
REFERENCES
1 Leibovich 81 Ross R. The role of the macrophage in wound repair: a study with hydrocortisone and anti-macrophage serum. Am J Patholl975; 78:71-100 2 Warheit DB, George G, Hill LH, Snyderman R, Brody AR. Inhaled asbestos activates a complement dependent chemoattractant fOr macrophages. Lab Invest 1985; 52:505-14 3 Warheit DB, Hill LH, George G, Brody AR. TIme course of chemotactic factor generation and the corresponding macrophage response to asbestos inhalation. Am Rev Respir Dis 1986; 134:128-33 4 Niman HL, Thompson AMH, Yu A, Markman M, Willem JJ,
188
Herwig KR, et aI. Antipeptide antibodies detect oncogenerelated proteins in urine. PNAS 1985; 82:7924-28
5 Shimokado K, Raines EW, MadtesDK, BarrettTB, Benditt E~
Ross R. A Significant part of macrophage-derived growth factor consists of at least 2 forms ofPDGE Cell 1985; 277-86
Direct Evidence for a MembraneAssociated Recept~r f()r a Bombesin-Like Peptide in Human Small cell Lung Carcinoma H345 cells· Madelaine Kane, M.D., Ph.D.
S
mall cell lung carcinoma (SCLC) cells appear to possess an autocrine growth system (Cuttitta et ale Nature 1985; 309:823). Bombesin-like peptide (BLP) is presumably synthesized intracellularly by posttranscriptional modification of a larger peptide (Spindel et ale PNAS 1984; 81:S699), packaged in vesicles and released from the cells. The released BLP can then interact with specific presumably membrane-associated receptors. This interaction stimulates DNA synthesis and cellular proliferation by as yet undefined biochemical mechanisms, although protein kinase C activation appears to be involved in the BLP-induced mitogenesis in Swiss 3T3 cells (Zachary et al. J Cell BioI 1986; 102:2211). Theoretically, disruption of the interaction between BLP and its receptor would result in inhibition of the proliferation and, possibly, death of SCLC cells. Various potential receptor-directed inhibitors might be bombesin analogs, bombesin-toxin conjugates, and anti-receptor monoclonal antibodies, either alone or linked to toxins or radioisotopes. Thus, further characterization and purification of the receptor for BLP would facilitate development of these inhibitors. Six mammalian systems in which evidence for a specific receptor for BLP has been reported are summarized in Table 1. The transport and binding constants for these intact cell and particulate membrane preparations are all similar, ranging from 0.5-3 nM, values indicating a high affinity receptor. The receptor density has been reported to vary widely. However, Moody and coworkers reported inhibition of bombesin binding to the rat brain membrane receptor by physiologic concentrations of Nael and other salts. Since all of the intact cell studies have been carried out in high salt incubation medium, the receptor densities reported may be quite inaccurate. The goal of the present studies was to further characterize bombesin binding to SCLC cells in vitro. Specifically these studies undertook (1) to confirm specific uptake of 1J5I-[tyr4 ] bombesin by NCI 8345 SCLC cells; (2) to determine optimum conditions for specific uptake; (3) to determine subcellular distribution of radioligand; and (4) to determine the distribution of radioligand after solubilization by detergent. MATERIALS AND METHODS
The experiments were conducted with the use of NCI H345 SCLC cells grown in suspension of HITES medium supplemented with 2% fetal calf serum at 3TC in a humidified 5% CO2 atmosphere. The HITES medium consists of RPMI 1640 supple*From the Division of Medical Oncology, University of Colorado, and Denver Veterans Administration Medical Center, Denver. 29th Aspen Lung Conference
Bombeain-Like Peptitk Receptors TIssue Preparation
KD* (nM)
Non-Peptide Effectors
Density (sites/cell)
(-) NaCI (-) KCI (-) CaC~
Rat brain membranes
3
2,290
Guinea pig pancreatic acini Rat pituitary tumor G8 4CI cells
2
5,000
1.2
3,600
(+) Chloroquine
Human SCLC cells
0.5
2,000
(0) EGTAt
Swiss mouse 3T3 cells
0.5
100,000
Human pancreatic membranes
0.96
lSO,()()()
Reference Moody et al, PNAS 1978; 75:5372 Jensen et al, PNAS 1978; 75:6139 Westendorf and Schonbrunn, JBC 1983; 258:7527 Moody et al, Ufe Sci 1985; 37:105 Zachary and Bozengurt, PNAS 1985; 82:7616 Scemama et al, Pept 1986; 13:125
*KD = dissociation content +EGTA=ethylene glycolbis-Ijs-aminolthyl ether)-N, N, N1, Nl-tetracetic acid mented with hydrocortisone, insulin, transferrin, 17 B estradiol and sodium selenite. The 8345 cells were harvested 48 h after plating at approximately 3 x lOS cells/ml by centrifugation, washed once or twice with uptake incubation buffer at 4°C, and resuspended in incubation buffer. Cells were dispersed by passage through a 22-gauge needle to prepare single-cell suspensions, quantified manually by hemacytometer, and cell viability was determined by trypan blue exclusion. Incubation medium CODsisted of either HITES containing 0.1% BSA or a nonionic Tris buffer made isotonic with sucrose and glucose, hereafter referred to as Tris-isotonic buffer. Specific uptake of lJ5I-[tyr4]-bombesin was determined at 22°C by adding 1-5 X lOS 8345 cells to incubation medium containing radioligand with or without I,OOO-fold excess cold bombesin. Thus, total and nonspecific cell-associated radioactivity could be determined, and the difference was specific uptake. Uptake was considered to include both binding and transport. Incubation was stopped by rapid centrifugation and radioactivity determined in a gamma counter. The effect of the composition of the incubation medium on specific uptake of 2 nM lJ5I-[tyr4]-bombesin by H345 cells was investigated. Cells incubated in Tris-isotonic buffer accumulated approximately five-fold as much radioligand as cells incubated in HITES. Thus, further studies were performed in Tris-isotonic buffer. The time course of 1II5I-[tyr4]-bombesin uptake by 8345 cells was linear over the first two minutes. Uptakeequilibrium was reached by about 5 min, at which time approximately 1,000 cpm or 100 fmol per million cells of specific uptake occurred. No significant effect on specific uptake was observed in cells washed with ms containing 100 mM NaCI prior to uptake. The subcellular distribution of the accumulated radioligand was investigated. The 8345 cells were harvested and incubated with 125I-[tyr4]-bombesin with or without cold bombesin as before. The radioactively labeled cells were then resuspended in hypotonic ms buffer at 4°C and freeze-thawed in a solidified carbon dioxideacetone bath. The lysed cells were then centrifuged at 12,000 x g for I min at 22°C. API particulate fraction, containing most of the cell membranes, and an SI soluble fraction, containing the cytosolic contents, were obtained. The PI fraction was resuspended in'Iiis, 0.05 M, pH 7.25, containing 0.1% BSA and 1% liiton X-l00 in an attempt to solubilize a particulate protein bound to radioligand. The insoluble residue, P2 , and the mton-solubilized supernatant, S" were obtained by centrifuganon as before. The distribution of radioactivity in these preparations was measured. In intact ~345 cells, almost 4,000 cpm of specific uptake was observed, and this was approximately 90 £molper million cells. After
exposure of the intact cells to hypotonic buffer and freeze-thawing,
all of the specific counts were associated with the PI fraction. After 'Iiiton solubilization of the PI fraction, all of the specmc counts were
released into the S. soluble fraction. The SI and S. fractions were
analyzed by Sephadex G50 gel &Jtrationchromatography. More than 90% of the radioactivity in the SI ~ons eluted with free 1111_[ tytA]-
bombesin. Both the unblocked and the blocked 5, fractions contained a void volume peak, presumably representing insoluble membrane fragments, and a peak that coeluted with free lISl_[tytA]_ bombesin. All the specific counts in the unblocked S. fraction eluted with free lJ51-[tyr·]-bombesin.
In summary, specific uptake of 1I5I-[tyr4]-bombesin by 8345 cells was con6rmed. Speciflc uptake was markedly increased in 'Iris-Isotonic buffer compared with HITES. Speci6c counts were associated with particulate membrane fraction. However, radioactivity dissociated from membrane proteins during 'Iriton solubilization under the conditions used. In conclusion, these results indicate that SCLC NCI H345 cells possess a membrane-associated receptor for 1I5I_[tyr4]_ bom~~. . ACKNOWLEDGMENT: This study was supported by the American Cancer Society, the Cancer League of Colorado, a Biomedical Research Support Institutional Grant, .and a VAResearch Advisory Group Support Grant.
Growth Factors for Me_hellal and
~esothelloma Ce~18* E. w Gabriel8on, M.D.;}. F. Lechner, M.D.; B. I. Gerwin, M.D.; M. A LaVeck, M.D.; and C. C. Hania, M.D.
T
he mesothelial cell not only is the progenitor cell type of mesothelioma, but' also is a selective target for the carcinogenic effects of asbestos 6bers. Primary human mesothelial cells, obtained from pleural fluids from patients without malignant disease, are cultured in MCDB 151 medium supplemented with hydrocortisone (. x lQ- 7M), *From the Laboratory of Human Carcinogenesis, National Cancer Institute, Bethesda, Md. CHEST I 91 I 3 I MARCH. 1887 I SUpplement
178
factor
REFERENCES
1 Leibovich 81 Ross R. The role of the macrophage in wound repair: a study with hydrocortisone and anti-macrophage serum. Am J Patholl975; 78:71-100 2 Warheit DB, George G, Hill LH, Snyderman R, Brody AR. Inhaled asbestos activates a complement dependent chemoattractant fOr macrophages. Lab Invest 1985; 52:505-14 3 Warheit DB, Hill LH, George G, Brody AR. TIme course of chemotactic factor generation and the corresponding macrophage response to asbestos inhalation. Am Rev Respir Dis 1986; 134:128-33 4 Niman HL, Thompson AMH, Yu A, Markman M, Willem JJ,
188
Herwig KR, et aI. Antipeptide antibodies detect oncogenerelated proteins in urine. PNAS 1985; 82:7924-28
5 Shimokado K, Raines EW, MadtesDK, BarrettTB, Benditt E~
Ross R. A Significant part of macrophage-derived growth factor consists of at least 2 forms ofPDGE Cell 1985; 277-86
Direct Evidence for a MembraneAssociated Recept~r f()r a Bombesin-Like Peptide in Human Small cell Lung Carcinoma H345 cells· Madelaine Kane, M.D., Ph.D.
S
mall cell lung carcinoma (SCLC) cells appear to possess an autocrine growth system (Cuttitta et ale Nature 1985; 309:823). Bombesin-like peptide (BLP) is presumably synthesized intracellularly by posttranscriptional modification of a larger peptide (Spindel et ale PNAS 1984; 81:S699), packaged in vesicles and released from the cells. The released BLP can then interact with specific presumably membrane-associated receptors. This interaction stimulates DNA synthesis and cellular proliferation by as yet undefined biochemical mechanisms, although protein kinase C activation appears to be involved in the BLP-induced mitogenesis in Swiss 3T3 cells (Zachary et al. J Cell BioI 1986; 102:2211). Theoretically, disruption of the interaction between BLP and its receptor would result in inhibition of the proliferation and, possibly, death of SCLC cells. Various potential receptor-directed inhibitors might be bombesin analogs, bombesin-toxin conjugates, and anti-receptor monoclonal antibodies, either alone or linked to toxins or radioisotopes. Thus, further characterization and purification of the receptor for BLP would facilitate development of these inhibitors. Six mammalian systems in which evidence for a specific receptor for BLP has been reported are summarized in Table 1. The transport and binding constants for these intact cell and particulate membrane preparations are all similar, ranging from 0.5-3 nM, values indicating a high affinity receptor. The receptor density has been reported to vary widely. However, Moody and coworkers reported inhibition of bombesin binding to the rat brain membrane receptor by physiologic concentrations of Nael and other salts. Since all of the intact cell studies have been carried out in high salt incubation medium, the receptor densities reported may be quite inaccurate. The goal of the present studies was to further characterize bombesin binding to SCLC cells in vitro. Specifically these studies undertook (1) to confirm specific uptake of 1J5I-[tyr4 ] bombesin by NCI 8345 SCLC cells; (2) to determine optimum conditions for specific uptake; (3) to determine subcellular distribution of radioligand; and (4) to determine the distribution of radioligand after solubilization by detergent. MATERIALS AND METHODS
The experiments were conducted with the use of NCI H345 SCLC cells grown in suspension of HITES medium supplemented with 2% fetal calf serum at 3TC in a humidified 5% CO2 atmosphere. The HITES medium consists of RPMI 1640 supple*From the Division of Medical Oncology, University of Colorado, and Denver Veterans Administration Medical Center, Denver. 29th Aspen Lung Conference
Bombeain-Like Peptitk Receptors TIssue Preparation
KD* (nM)
Non-Peptide Effectors
Density (sites/cell)
(-) NaCI (-) KCI (-) CaC~
Rat brain membranes
3
2,290
Guinea pig pancreatic acini Rat pituitary tumor G8 4CI cells
2
5,000
1.2
3,600
(+) Chloroquine
Human SCLC cells
0.5
2,000
(0) EGTAt
Swiss mouse 3T3 cells
0.5
100,000
Human pancreatic membranes
0.96
lSO,()()()
Reference Moody et al, PNAS 1978; 75:5372 Jensen et al, PNAS 1978; 75:6139 Westendorf and Schonbrunn, JBC 1983; 258:7527 Moody et al, Ufe Sci 1985; 37:105 Zachary and Bozengurt, PNAS 1985; 82:7616 Scemama et al, Pept 1986; 13:125
*KD = dissociation content +EGTA=ethylene glycolbis-Ijs-aminolthyl ether)-N, N, N1, Nl-tetracetic acid mented with hydrocortisone, insulin, transferrin, 17 B estradiol and sodium selenite. The 8345 cells were harvested 48 h after plating at approximately 3 x lOS cells/ml by centrifugation, washed once or twice with uptake incubation buffer at 4°C, and resuspended in incubation buffer. Cells were dispersed by passage through a 22-gauge needle to prepare single-cell suspensions, quantified manually by hemacytometer, and cell viability was determined by trypan blue exclusion. Incubation medium CODsisted of either HITES containing 0.1% BSA or a nonionic Tris buffer made isotonic with sucrose and glucose, hereafter referred to as Tris-isotonic buffer. Specific uptake of lJ5I-[tyr4]-bombesin was determined at 22°C by adding 1-5 X lOS 8345 cells to incubation medium containing radioligand with or without I,OOO-fold excess cold bombesin. Thus, total and nonspecific cell-associated radioactivity could be determined, and the difference was specific uptake. Uptake was considered to include both binding and transport. Incubation was stopped by rapid centrifugation and radioactivity determined in a gamma counter. The effect of the composition of the incubation medium on specific uptake of 2 nM lJ5I-[tyr4]-bombesin by H345 cells was investigated. Cells incubated in Tris-isotonic buffer accumulated approximately five-fold as much radioligand as cells incubated in HITES. Thus, further studies were performed in Tris-isotonic buffer. The time course of 1II5I-[tyr4]-bombesin uptake by 8345 cells was linear over the first two minutes. Uptakeequilibrium was reached by about 5 min, at which time approximately 1,000 cpm or 100 fmol per million cells of specific uptake occurred. No significant effect on specific uptake was observed in cells washed with ms containing 100 mM NaCI prior to uptake. The subcellular distribution of the accumulated radioligand was investigated. The 8345 cells were harvested and incubated with 125I-[tyr4]-bombesin with or without cold bombesin as before. The radioactively labeled cells were then resuspended in hypotonic ms buffer at 4°C and freeze-thawed in a solidified carbon dioxideacetone bath. The lysed cells were then centrifuged at 12,000 x g for I min at 22°C. API particulate fraction, containing most of the cell membranes, and an SI soluble fraction, containing the cytosolic contents, were obtained. The PI fraction was resuspended in'Iiis, 0.05 M, pH 7.25, containing 0.1% BSA and 1% liiton X-l00 in an attempt to solubilize a particulate protein bound to radioligand. The insoluble residue, P2 , and the mton-solubilized supernatant, S" were obtained by centrifuganon as before. The distribution of radioactivity in these preparations was measured. In intact ~345 cells, almost 4,000 cpm of specific uptake was observed, and this was approximately 90 £molper million cells. After
exposure of the intact cells to hypotonic buffer and freeze-thawing,
all of the specific counts were associated with the PI fraction. After 'Iiiton solubilization of the PI fraction, all of the specmc counts were
released into the S. soluble fraction. The SI and S. fractions were
analyzed by Sephadex G50 gel &Jtrationchromatography. More than 90% of the radioactivity in the SI ~ons eluted with free 1111_[ tytA]-
bombesin. Both the unblocked and the blocked 5, fractions contained a void volume peak, presumably representing insoluble membrane fragments, and a peak that coeluted with free lISl_[tytA]_ bombesin. All the specific counts in the unblocked S. fraction eluted with free lJ51-[tyr·]-bombesin.
In summary, specific uptake of 1I5I-[tyr4]-bombesin by 8345 cells was con6rmed. Speciflc uptake was markedly increased in 'Iris-Isotonic buffer compared with HITES. Speci6c counts were associated with particulate membrane fraction. However, radioactivity dissociated from membrane proteins during 'Iriton solubilization under the conditions used. In conclusion, these results indicate that SCLC NCI H345 cells possess a membrane-associated receptor for 1I5I_[tyr4]_ bom~~. . ACKNOWLEDGMENT: This study was supported by the American Cancer Society, the Cancer League of Colorado, a Biomedical Research Support Institutional Grant, .and a VAResearch Advisory Group Support Grant.
Growth Factors for Me_hellal and
~esothelloma Ce~18* E. w Gabriel8on, M.D.;}. F. Lechner, M.D.; B. I. Gerwin, M.D.; M. A LaVeck, M.D.; and C. C. Hania, M.D.
T
he mesothelial cell not only is the progenitor cell type of mesothelioma, but' also is a selective target for the carcinogenic effects of asbestos 6bers. Primary human mesothelial cells, obtained from pleural fluids from patients without malignant disease, are cultured in MCDB 151 medium supplemented with hydrocortisone (. x lQ- 7M), *From the Laboratory of Human Carcinogenesis, National Cancer Institute, Bethesda, Md. CHEST I 91 I 3 I MARCH. 1887 I SUpplement
178