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Biogenic amine metabolites in human lung tumor cells: Histochemical and mass-spectrographic demonstration
Life Sciences, Vol. 30, pp. 1355-1360 Printed in the U.S.A.
Pergamon Pres
BIOGENIC AMINE METABOLITES IN HUMAN LUNG TUMORCELLS: HISTOCHEMICAL AND MASS-SPECTROGRAPHICDEMONSTRATION Olive S. P e t t e n g i l l * ,
Nicholas G. Bacopoulos** and George D. Sorenson*
Departments of Pathology* and Pharmacology**, Dartmouth Medical School, Hanover, N.H. 03755 (Received in final form February l, 1982)
Summary Three continuous cell lines isolated from small cell carcinoma of the lung (SCCL) have been examined for t h e i r a b i l i t y to take up and metabolize the biogenic amine precursors, L-DOPA and 5hydroxytryptophane (5-HTP). In two of the three lines formaldehydeinduced fluorescence (FIF) and mass-spectrographic analysis i n d i cated s p e c i f i c uptake and metabolism of L-DOPA and 5-HTP. Small cell carcinoma of the lung (SCCL) is a highly malignant human tumor which is frequently associated-with a v a r i e t y of paraneoplastic syndromes. The l a t t e r are often related to the production of peptide hormones by these neoplasms (14). Continuous SCCL cell l i n e s , DMS 53, DMS 79 and DMS 153, secrete a variety of peptide hormones as well as estrogens (6,16,17,18). In common with the tumors from which they were isolated, cultured c e l l s e x h i b i t dense core vesicles (DCV) in the cytoplasm and i n t e r c e l l u l a r junctional complexes. The cell of o r i g i n of SCCL is generally considered to be the K u l t s c h i t z k y - l i k e ( K - l i k e ) cell of the bronchial epithelium on the basis of morphological s i m i l a r i t i e s (5) although no d e f i n i t i v e evidence is available to support this hypothesis. The a r g y r o p h i l i c K-like cell is usually found as single c e l l s in the basal layer of bronchial epithelium but also occurs as small clumps of c e l l s , called neuroepithelial bodies (NEB) in bronchial, bronchiolar and alveolar e p i t h e l i a (7,11,13). The NEB is thought to function as a chemoreceptor, responding to local changes in i n t r a - a l v e o l a r oxygen levels, by secretion of serotonin which i t has been shown to contain (12). Serotonin may be localized in dense core vesicles which are observed u l t r a s t r u c t u r a l l y in the cytoplasm of the K-like c e l l . In addit i o n , K-like c e l l s contain high levels of acetylcholine and other esterases. They also e x h i b i t formaldehyde-induced fluorescence (FIF), an expression of the presence of biogenic amines, a f t e r nreincubation with a suitable precursor (7). The studies described here have been carried out to characterize SCCL c e l l s for endogenous levels of biogenic amines and t h e i r a b i l i t y to accumulate and metabolize amine precursors, L-DOPA and 5-HTP, c h a r a c t e r i s t i c s which are associated with K-like c e l l s . Gazdar et al (9,10) have described the occurence of FIF in a l l SCCL cell lines established in that laboratory. On the other hand, FIF was not found in a l l our cell lines isolated from SCCL (15). Materials and Methods Cultured c e l l s were preincubated f o r 1 hour at 37Oc with e i t h e r 2.5 x IO-7M L-DOPA or 2.3 x IO-7M 5-HTP in complete growth medium; i . e . RPMI 1640 with 20% f e t a l c a l f serum (FCS) for DMS 79 and DMS 153 and Waymouth's MB 752/I with 20% 0024-3205/82/161355-06503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
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FCS f o r DMS 53 (15,16). DMS 53 has been propagated in v i t r o continuously f o r 6 years as an attached monolayer (15). DMS 153, a f t e r subculture by t r y D s i n i zation, attaches to the substrate, but as larqe clumps form, these detach and f l o a t in the medium (15). DMS 79 c e l l s , propagated f o r 6 years, have consistently grown as large, loose clumps of c e l l s in suspension with no substrate dependency. For FIE and mass spectrographic analyses, c e l l s were harvested by scraping (DMS 53 & 153) or dentrifugation (DMS 79), washed two times in phosphate buffered saline and drained. Specimens f o r mass spectrographic analysis were stored at -70oc u n t i l analyzed. For FIF (8), washed c e l l s were resuspended in complete medium and collected on microscope slides with Cytocentrifuge (Shandon, Sewickly, Pa.). A i r dried slides were freeze-dried overnight and exposed to paraformaldehyde fumes at 80°C f o r 1 hour. Paraformaldehyde was e q u i l i b r a t e d to 58% humidity for 2 weeks p r i o r to use. Preparations were coverslipped with Entellen (EM l a b o r a t o r i e s , Darmstadt, Germany) and immediately examined for fluorescence, u t i l i z i n g a xenon l i g h t source with a Zeiss photoscope equipped with Zeiss fluorescent f i l t e r combination #487711. Mass spectrographic i d e n t i f i c a t i o n of amines: Washed c e l l s were homogenized in 0.5 N HCl and centrifuged at 15,000 x g f o r 20"minutes. Portions of the aqueous supernatant were evaporated to dryness and reacted with pentafluoropropionic anhydride as previously described (3). The f i n a l reaction products were dissolved in hexane and injected into a Finnigan 4023 gas-chromatography mass-spectrometry system (GCMS). In subsequent experiments, washed, frozen tumor c e l l s were thawed and homogenized in I0 volumes 0.I N formic acid containing 0.1% sodium m e t a b i s u l f i t e . The homogenate was centrifuged at 20,000 x g f o r 20 minutes at 4°C and the supernatant was saturated with NaCI and extracted with two volumes of ethyl acetate. The organic e x t r a c t was evaporated under nitrogen and treated with pentafluoropropionic anhydride (3). The f i n a l reaction products were dissolved in hexane and injected into GCMS. A l l deuterated internal standards were obtained from Merck and Co. (St. Louis,
Mo.). Results Both DMS 153 and DMS 53 exhibited FIF a f t e r preincubation with e~ther L-DOPA and 5-HTP (FIG. I ) but DMS 79 c e l l s did not. S i m i l a r l y we i d e n t i f i e d and measured dopamine in the two c e l l lines e x h i b i t i n g FIF but not in DMS 79. Aqueous extracts of DMS 53 and DMS 153 c e l l s preincubated with L-DOPA yielded a strong peak with the retention time (3 minutes) of authentic dopamine. Mass spectral analysis of the peak revealed a spectrum i d e n t i c a l to that of dopamine. This could not be due to the interference from L-DOPA which had a retention time of 1.2 minutes, and a c l e a r l y d i f f e r e n t mass spectrum. None of the c e l l lines contained measurable amounts of dopamine in the absence of preincubation with L-DOPA. Following the i n j e c t i o n into the mass spectrograph of extracts from c e l l s of the DMS 53 and DMS 153 c e l l lines preincubated with L-DOPA, we observed a prominent GC peak e l u t i n g at 0.6 minutes and a smaller peak at 1.4 minutes (Fig. 2). Mass-spectral analysis of these peaks revealed that they contained 3,4-dihydroxphenylacetic acid (DOPAC) and homovanillic acid (HVA) r e s p e c t i v e l y . Extracts of DMS 79 c e l l s incubated with L-DOPA did not contain DOPAC and HVA. None of the c e l l lines contained measurable amounts of DOPAC and HVA in the absence of preincubation with L-DOPA.
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Figure l Formaldehyde induced fluorescence (FIF) of SCCL c e l l , preincubated with precursors: Top row, L-DOPA: Bottom row, 5-HTP. (a) and (d) DMS 153; (b) and (e) DMS 79; and (c) and ( f ) DMS 53. In order to make a qualitative comparison, a l l microscopic fields were photographed and printed with the same exposure time, except (e) which was printed with a shorter exposure time for v i s i b i l i t y . Both DMS 53 and DMS 153 exhibit FIF after incubation with either precursor, but DMS 79 is negative for FIF after incubation with both precursors. Not shown is FIF without preincubation in precursor, which was similar to the negative pictures of DMS 79 (b,e). After incubation with 5-HTP, we observed that cells of the 53 and 153 lines contained the acidic metabolite of serotonin, 5-hydroxyindoleacetic acid (5-HIAA) whereas cells of the DMS 79 line did not. No 5-HIAA was detected in any cell line in the absence of preincubation with 5-HTP (Fin. 2). In a separate series of experiments cells incubated with either L-DOPA or 5HTP were homoqenized as described above, with the addition of deuterated internal standards (3,4 dihydroxvphenyl-d 3 acetic-d2 acid and 3-methoxy-4hydroxyphenyl acetic-d 2 acid or 5-hydroxyindole-3-acetic-2,2-d2 acid or 2(3,4-dihydroxypheny]) e t h y l - l , l-d2-amine ) to the homogenizing medium. By mass-spectrometric analysis we established that the peaks observed in the e a r l i e r experiments (Fig. 2) coe,uted with the deuterated internal standards. In addition to the mass-spectrum of the endogenous undeuterated compound, the peaks contained mass fragments shifted by as many mass units as would be expected from the deuterium contribution. This co-chromatographic elution of endogenous and deuterated compound was observed with two different stationary phases (OVl7, 3% on Gas Chrom Q and SE 52 on Gas Chrom Q). The ratio of selected deuterated to undeuterated mass fragment areas, computed by the multiple ion detection programs of the INCOS data system, was used to quantitate the amount of dopamine, DOPAC, HVA and 5-HIAA present in the cell extracts (3). The mass peaks chosen for quantitation of each compound were 415 (endogenous) and 420 for (deuterated) DOPAC: 460 and 462 for HVA; 438 and 440 for 5-HIAA; and 428 and 430 for doDamine. Extracts of control cells or DMS 79 cells incu-
1358
Biogenic Amine Metabolites
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Vol. 30, No. 16, 1982
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Figure 2 Gas-chromatographic and mass spectrometric analysis of e x t r a c t s of human lung tumor c e l l c u l t u r e s . Panels on the l e f t hand side of the f i g u r e show the t o t a l - i o n current chromatogram detected by the mass spectrometer. Chromatographic conditions were as f o l l o w s : a 6 f t . glass column packed with OVI7 (3% on Gas Chrom Q) was operated at 155oc f o r DOPAC and HVA a n a l y s i s and at 180 ~ f o r 5-HIAA. Helium flow was at 30 ml/min. I n j e c t o r temperature was at 225°C, electron energy 55 eV, emission current 0.35 mA, electron m u l t i p l i e r voltage 1500 V. Linear scans between masses 200 and 700 were performed every 2 seconds under computer c o n t r o l . Panels on the r i q h t side show mass spectra corresponding to authentic standards or to peaks in the chromatoqram on the l e f t . The second, smaller peak shown in Panel A contained HVA (mass spectrum not shown). The i n j e c t i o n of c e l l e x t r a c t s which had been homogenized with deuterated i n t e r n a l standards (see t e x t ) resulted in chromatograms i d e n t i c a l to those shown in panels on the l e f t . However, the mass spectra under those conditions were found to contain both endogenous and deuterated mass fragments with the predicted mass s h i f t s . Changing the s t a t i o n a r y phase of the GC column (SE 52 3% on Gas Chrom Q) did not a l t e r the cochromatographic e l u t i o n of deuterated i n t e r n a l standards and the compounds o r i g i n a t i n g from the c e l l e x t r a c t s which gave mass spectra i d e n t i c a l to those of authentic DOPAC, HVA and 5-HIAA. The same r e s u l t s were obtained with DMS 53 c e l l s . Dopamine was i d e n t i f i e d in DMS 53 and DMS 153 c e l l s by s i m i l a r methods.
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Biogenic Amine Metabolites
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bated with L-DOPA or 5-HTP contained only the deuterated i n t e r n a l standards during homogenization, confirming our previous f i n d i n g s t h a t only c e l l s of DMS 53 and DMS 153 c e l l l i n e s metabolized the amino acids (Table I ) . TABLE I Biogenic Amine Metabolism in Cultured SCCL Cells Ngm/gram t i s s u e b Culture Cell Lines
FIF a
DMS 53 Endogenous + L-DOPA + 5-HTP
+++ ++
N.D. 5,500 --
N.D. c 270
DMS 153 Endogenous + L-DOPA + 5-HTP
++++ ++
N.D. 6,400 --
N.D. 500
N.D. 400
N.D.
DMS 79 Endogenous + L- DOPA + 5-HTP
-
N.D.
N.D.
N.D.
N.D.
-
N.D. N.D.
N.D.
N.D.
(a) (b) (c)
D o p a m i n e DOPAC
HVA
5-HIAA
N.D. I00
N.D. 650
N.D.
R e l a t i v e fluorescence was estimated v i s u a l l y by fluorescence microscopv. Concentrations of dopamine, DOPAC, HVA and 5-HIAA were measured by selected ion monitoring as described in t e x t . N.D. = none detectable Discussion
The FIF observed in c e l l l i n e s DMS 53 and DMS 153 a f t e r incubation with L-DOPA is most l i k e l y due to L-DOPA, dopamine or both ( I ) . The i d e n t i f i c a t i o n of 5-HIAA in c e l l s pretreated with 5-HTP s t r o n g l y suggests t h a t t h i s amino acid is also decarboxylated to form 5-hydroxytryptamine (5-HT, or serotonin) which is in turn deaminated to form the a c i d i c metabolite 5-HIAA which we detected by mass-spectrometry. Decarboxylation o f L-DOPA requires the enzyme dopa decarboxylase (DDC) and the presence of HVA in L-DOPA treated c e l l s also demonstrated the occurence of catechol-O-methyl transferase (COMT) in SCCL c e l l s . In another series of 5 continuous c e l l l i n e s , DDC was i d e n t i f i e d as a d i s t i n guishing marker f o r cultured SCCL lung tumor c e l l s , when comoared to l e v e l s o f a c t i v i t y found in continuous c e l l l i n e s of other h i s t o l o g i c types of lung tumors (4). We have p r e v i o u s l y observed t h a t DMS 53 is capable of taking up 3H L-DOPA, but t h a t DMS 79 c e l l s do not (unnublished data). Therefore, DDC may be present in DMS 79 c e l l s , although i t s a c t i v i t v would not be apoarent in these analyses, which depend upon uptake of exoqenous L-DOPA. In the studies described here, we have shown t h a t cultured human small c e l l lung tumor c e l l s (SCCL) take up and metabolize L-DOPA and 5-HTP in v i t r o and t h a t t h i s property is i n t r i n s i c to two c e l l l i n e s and absent from a t h i r d . The above conclusions are supported by two separate experimental apnroaches: Demonstration o f FIF in L-DOPA or 5-HTP incubated c e l l s , and the p o s i t i v e i d e n t i f i c a t i o n o f , t h e metabolites of dopamine and serotonin in these c e l l s . Two c e l l l i n e s , DNS 53 and DMS 153 gave a o o s i t i v e response in terms of the above t e s t systems, w h i l e DMS 79 presented no evidence of e i t h e r L-DOPA or 5-HTP uptake in v i t r o . The s i g n i f i c a n c e of these observations in which n e u r a l - l i k e metabolic patterns
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occur in tumor c e l l s a r i s i n g from the bronchial epithelium is not known and requires f u r t h e r i n v e s t i g a t i o n . However, we have i d e n t i f i e d s i g n i f i c a n t d i f ferences regarding the metabolism of amine precursors between c e l l lines isolated from a s p e c i f i c type of lung tumor and provided evidence that enzymes involved in the biosynthesis and degration of DA and 5-HT are present in 2 of the 3 tumor c e l l l i n e s . References I. 2. 3. 4. 5. 6. 7. 8. 9. I0.
II. 12. 13. 14. 15. 16. 17. 18. 19.
E.T. ANGELAKOS, and M. KING. Fluorescence histochemistrv of the biogenic amines. In: Psychopharmacology: A Review of Proqress 1957-1967 (D.H. Efron, ed.) pn. 27-36, U.S.P.H.S. Publication No. 1836, 1968. N.G. BACOPOULOS, D.E. REDMONDand R.H. ROTH, J. Neurochem. 32:1215-1218, 1979. N.G. BACOPOULOS, S.E. HATTOX and R.H. ROTH, Euronean J. Pharmacol. 56:225236, 1979. S.B. BAYLIN, M.D. ABELOFF, G. GOODWIN, D.N. CAREY and A.F. GAZDAR, Cancer Res. 40:19q0-1994, 1980. K.G. B~NSCH, B. CORRIN, R. PARIENTE and H. SPENCER, Cancer 22:1163-I172, 1968. T. BRINCK-JOHNSEN, O.S. PETTENGILL, K. BRINCK-JOHNSEN, G.D. SORENSON and L.H. MAURER, J. Steroid Biochem. 10:339-340, 1979. E. CUTZ, W. CHAN, V. WONGand P.E. CONEN, Lab Invest. 30:458-464, 1974. B. FALCK and OWMAN, C., Acta Univ. Lund., Sect. I I ( 7 ) 7 - 2 3 , 1965. A.F. GAZDAR, D.N. CARNEY, E.K. RUSSEL, H.L. SIMS, S.B. BAYLIN, P.A. BUNN, JR., J.G. GUCCION and J.D. MINNA, Cancer Res. 40:3502-3507, 1980. A.F. GAZDAR, D.N. CARNEY, J.G. GUCCION and S.B. BAYLIN. Small c e l l carcinoma of the lung: C e l l u l a r o r i g i n & r e l a t i o n s h i p to other pulmonary tumors. In: Small Cell Lung Cancer (F.A. Greco, R.K. Oldham and P.A. Bunn, J r . , Eds.) pp.145-176, GrUne and Stratton, 1981. E. HAGE, Acta Path. Microbiol. Scand. Sect. A, 80:225-234, 1972. J.M. LAUWERYNS, M. COKELAERE, T. LERUT and P. THEUNYNCK, Cell Tiss. Res., 193, 373-386, 1978. J.M. LAUWERYNS and M. COKELAERE, Experientia 29:1384-1386, 1973. W.D. ODELL and A. WOLFSEN. Ectopic Hormone Secretion by Tumors. In: Cancer, a Comprehensive Treatise (F.F. Becker, Ed.) Dp. 81-97, Plenum Press, New York, 1975. O.S. PETTENGILL and G.D. SORENSON. Tissue Culture and in V i t r o characteristics. In: Small Cell Lunn Cancer (F.A. Greco, R.K. hldham and P.A. Bunn, J r . , Eds.) pp. 79-94, Grune and Stratton, 1981. O.S. PETTENGILL, G.D. SORENSON, D.H. WURSTER-HILL, T.J. CURPHEY, W.W. NOLL, C.C. CATE and L.H. MAURER, Cancer 45:906-918, 198fl. G.D. SORENSON and T. BRINCK-JOHNSEN, Proc. Amer. Assoc. Cancer Res., 18: 248, 1977. G.D. SORENSON, O.S. PETTENGILL, T. BRINCK-JOHNSEN, C.C. CATE and L.H. MAURER, Cancer 47:1289-1296, 1981. These studies we--re supported in part by CA 2~845, CA 23108 and BRSG RR05392. We thank Andrea Goggin f o r technical expertise in carrying out FIF studies. I
Pergamon Pres
BIOGENIC AMINE METABOLITES IN HUMAN LUNG TUMORCELLS: HISTOCHEMICAL AND MASS-SPECTROGRAPHICDEMONSTRATION Olive S. P e t t e n g i l l * ,
Nicholas G. Bacopoulos** and George D. Sorenson*
Departments of Pathology* and Pharmacology**, Dartmouth Medical School, Hanover, N.H. 03755 (Received in final form February l, 1982)
Summary Three continuous cell lines isolated from small cell carcinoma of the lung (SCCL) have been examined for t h e i r a b i l i t y to take up and metabolize the biogenic amine precursors, L-DOPA and 5hydroxytryptophane (5-HTP). In two of the three lines formaldehydeinduced fluorescence (FIF) and mass-spectrographic analysis i n d i cated s p e c i f i c uptake and metabolism of L-DOPA and 5-HTP. Small cell carcinoma of the lung (SCCL) is a highly malignant human tumor which is frequently associated-with a v a r i e t y of paraneoplastic syndromes. The l a t t e r are often related to the production of peptide hormones by these neoplasms (14). Continuous SCCL cell l i n e s , DMS 53, DMS 79 and DMS 153, secrete a variety of peptide hormones as well as estrogens (6,16,17,18). In common with the tumors from which they were isolated, cultured c e l l s e x h i b i t dense core vesicles (DCV) in the cytoplasm and i n t e r c e l l u l a r junctional complexes. The cell of o r i g i n of SCCL is generally considered to be the K u l t s c h i t z k y - l i k e ( K - l i k e ) cell of the bronchial epithelium on the basis of morphological s i m i l a r i t i e s (5) although no d e f i n i t i v e evidence is available to support this hypothesis. The a r g y r o p h i l i c K-like cell is usually found as single c e l l s in the basal layer of bronchial epithelium but also occurs as small clumps of c e l l s , called neuroepithelial bodies (NEB) in bronchial, bronchiolar and alveolar e p i t h e l i a (7,11,13). The NEB is thought to function as a chemoreceptor, responding to local changes in i n t r a - a l v e o l a r oxygen levels, by secretion of serotonin which i t has been shown to contain (12). Serotonin may be localized in dense core vesicles which are observed u l t r a s t r u c t u r a l l y in the cytoplasm of the K-like c e l l . In addit i o n , K-like c e l l s contain high levels of acetylcholine and other esterases. They also e x h i b i t formaldehyde-induced fluorescence (FIF), an expression of the presence of biogenic amines, a f t e r nreincubation with a suitable precursor (7). The studies described here have been carried out to characterize SCCL c e l l s for endogenous levels of biogenic amines and t h e i r a b i l i t y to accumulate and metabolize amine precursors, L-DOPA and 5-HTP, c h a r a c t e r i s t i c s which are associated with K-like c e l l s . Gazdar et al (9,10) have described the occurence of FIF in a l l SCCL cell lines established in that laboratory. On the other hand, FIF was not found in a l l our cell lines isolated from SCCL (15). Materials and Methods Cultured c e l l s were preincubated f o r 1 hour at 37Oc with e i t h e r 2.5 x IO-7M L-DOPA or 2.3 x IO-7M 5-HTP in complete growth medium; i . e . RPMI 1640 with 20% f e t a l c a l f serum (FCS) for DMS 79 and DMS 153 and Waymouth's MB 752/I with 20% 0024-3205/82/161355-06503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
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FCS f o r DMS 53 (15,16). DMS 53 has been propagated in v i t r o continuously f o r 6 years as an attached monolayer (15). DMS 153, a f t e r subculture by t r y D s i n i zation, attaches to the substrate, but as larqe clumps form, these detach and f l o a t in the medium (15). DMS 79 c e l l s , propagated f o r 6 years, have consistently grown as large, loose clumps of c e l l s in suspension with no substrate dependency. For FIE and mass spectrographic analyses, c e l l s were harvested by scraping (DMS 53 & 153) or dentrifugation (DMS 79), washed two times in phosphate buffered saline and drained. Specimens f o r mass spectrographic analysis were stored at -70oc u n t i l analyzed. For FIF (8), washed c e l l s were resuspended in complete medium and collected on microscope slides with Cytocentrifuge (Shandon, Sewickly, Pa.). A i r dried slides were freeze-dried overnight and exposed to paraformaldehyde fumes at 80°C f o r 1 hour. Paraformaldehyde was e q u i l i b r a t e d to 58% humidity for 2 weeks p r i o r to use. Preparations were coverslipped with Entellen (EM l a b o r a t o r i e s , Darmstadt, Germany) and immediately examined for fluorescence, u t i l i z i n g a xenon l i g h t source with a Zeiss photoscope equipped with Zeiss fluorescent f i l t e r combination #487711. Mass spectrographic i d e n t i f i c a t i o n of amines: Washed c e l l s were homogenized in 0.5 N HCl and centrifuged at 15,000 x g f o r 20"minutes. Portions of the aqueous supernatant were evaporated to dryness and reacted with pentafluoropropionic anhydride as previously described (3). The f i n a l reaction products were dissolved in hexane and injected into a Finnigan 4023 gas-chromatography mass-spectrometry system (GCMS). In subsequent experiments, washed, frozen tumor c e l l s were thawed and homogenized in I0 volumes 0.I N formic acid containing 0.1% sodium m e t a b i s u l f i t e . The homogenate was centrifuged at 20,000 x g f o r 20 minutes at 4°C and the supernatant was saturated with NaCI and extracted with two volumes of ethyl acetate. The organic e x t r a c t was evaporated under nitrogen and treated with pentafluoropropionic anhydride (3). The f i n a l reaction products were dissolved in hexane and injected into GCMS. A l l deuterated internal standards were obtained from Merck and Co. (St. Louis,
Mo.). Results Both DMS 153 and DMS 53 exhibited FIF a f t e r preincubation with e~ther L-DOPA and 5-HTP (FIG. I ) but DMS 79 c e l l s did not. S i m i l a r l y we i d e n t i f i e d and measured dopamine in the two c e l l lines e x h i b i t i n g FIF but not in DMS 79. Aqueous extracts of DMS 53 and DMS 153 c e l l s preincubated with L-DOPA yielded a strong peak with the retention time (3 minutes) of authentic dopamine. Mass spectral analysis of the peak revealed a spectrum i d e n t i c a l to that of dopamine. This could not be due to the interference from L-DOPA which had a retention time of 1.2 minutes, and a c l e a r l y d i f f e r e n t mass spectrum. None of the c e l l lines contained measurable amounts of dopamine in the absence of preincubation with L-DOPA. Following the i n j e c t i o n into the mass spectrograph of extracts from c e l l s of the DMS 53 and DMS 153 c e l l lines preincubated with L-DOPA, we observed a prominent GC peak e l u t i n g at 0.6 minutes and a smaller peak at 1.4 minutes (Fig. 2). Mass-spectral analysis of these peaks revealed that they contained 3,4-dihydroxphenylacetic acid (DOPAC) and homovanillic acid (HVA) r e s p e c t i v e l y . Extracts of DMS 79 c e l l s incubated with L-DOPA did not contain DOPAC and HVA. None of the c e l l lines contained measurable amounts of DOPAC and HVA in the absence of preincubation with L-DOPA.
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Biogenic AmCne Metabolltes in SCCL
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Figure l Formaldehyde induced fluorescence (FIF) of SCCL c e l l , preincubated with precursors: Top row, L-DOPA: Bottom row, 5-HTP. (a) and (d) DMS 153; (b) and (e) DMS 79; and (c) and ( f ) DMS 53. In order to make a qualitative comparison, a l l microscopic fields were photographed and printed with the same exposure time, except (e) which was printed with a shorter exposure time for v i s i b i l i t y . Both DMS 53 and DMS 153 exhibit FIF after incubation with either precursor, but DMS 79 is negative for FIF after incubation with both precursors. Not shown is FIF without preincubation in precursor, which was similar to the negative pictures of DMS 79 (b,e). After incubation with 5-HTP, we observed that cells of the 53 and 153 lines contained the acidic metabolite of serotonin, 5-hydroxyindoleacetic acid (5-HIAA) whereas cells of the DMS 79 line did not. No 5-HIAA was detected in any cell line in the absence of preincubation with 5-HTP (Fin. 2). In a separate series of experiments cells incubated with either L-DOPA or 5HTP were homoqenized as described above, with the addition of deuterated internal standards (3,4 dihydroxvphenyl-d 3 acetic-d2 acid and 3-methoxy-4hydroxyphenyl acetic-d 2 acid or 5-hydroxyindole-3-acetic-2,2-d2 acid or 2(3,4-dihydroxypheny]) e t h y l - l , l-d2-amine ) to the homogenizing medium. By mass-spectrometric analysis we established that the peaks observed in the e a r l i e r experiments (Fig. 2) coe,uted with the deuterated internal standards. In addition to the mass-spectrum of the endogenous undeuterated compound, the peaks contained mass fragments shifted by as many mass units as would be expected from the deuterium contribution. This co-chromatographic elution of endogenous and deuterated compound was observed with two different stationary phases (OVl7, 3% on Gas Chrom Q and SE 52 on Gas Chrom Q). The ratio of selected deuterated to undeuterated mass fragment areas, computed by the multiple ion detection programs of the INCOS data system, was used to quantitate the amount of dopamine, DOPAC, HVA and 5-HIAA present in the cell extracts (3). The mass peaks chosen for quantitation of each compound were 415 (endogenous) and 420 for (deuterated) DOPAC: 460 and 462 for HVA; 438 and 440 for 5-HIAA; and 428 and 430 for doDamine. Extracts of control cells or DMS 79 cells incu-
1358
Biogenic Amine Metabolites
in SCCL
A
Vol. 30, No. 16, 1982
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Figure 2 Gas-chromatographic and mass spectrometric analysis of e x t r a c t s of human lung tumor c e l l c u l t u r e s . Panels on the l e f t hand side of the f i g u r e show the t o t a l - i o n current chromatogram detected by the mass spectrometer. Chromatographic conditions were as f o l l o w s : a 6 f t . glass column packed with OVI7 (3% on Gas Chrom Q) was operated at 155oc f o r DOPAC and HVA a n a l y s i s and at 180 ~ f o r 5-HIAA. Helium flow was at 30 ml/min. I n j e c t o r temperature was at 225°C, electron energy 55 eV, emission current 0.35 mA, electron m u l t i p l i e r voltage 1500 V. Linear scans between masses 200 and 700 were performed every 2 seconds under computer c o n t r o l . Panels on the r i q h t side show mass spectra corresponding to authentic standards or to peaks in the chromatoqram on the l e f t . The second, smaller peak shown in Panel A contained HVA (mass spectrum not shown). The i n j e c t i o n of c e l l e x t r a c t s which had been homogenized with deuterated i n t e r n a l standards (see t e x t ) resulted in chromatograms i d e n t i c a l to those shown in panels on the l e f t . However, the mass spectra under those conditions were found to contain both endogenous and deuterated mass fragments with the predicted mass s h i f t s . Changing the s t a t i o n a r y phase of the GC column (SE 52 3% on Gas Chrom Q) did not a l t e r the cochromatographic e l u t i o n of deuterated i n t e r n a l standards and the compounds o r i g i n a t i n g from the c e l l e x t r a c t s which gave mass spectra i d e n t i c a l to those of authentic DOPAC, HVA and 5-HIAA. The same r e s u l t s were obtained with DMS 53 c e l l s . Dopamine was i d e n t i f i e d in DMS 53 and DMS 153 c e l l s by s i m i l a r methods.
Vol. 30, No. 16, 1982
Biogenic Amine Metabolites
in SCCL
1359
bated with L-DOPA or 5-HTP contained only the deuterated i n t e r n a l standards during homogenization, confirming our previous f i n d i n g s t h a t only c e l l s of DMS 53 and DMS 153 c e l l l i n e s metabolized the amino acids (Table I ) . TABLE I Biogenic Amine Metabolism in Cultured SCCL Cells Ngm/gram t i s s u e b Culture Cell Lines
FIF a
DMS 53 Endogenous + L-DOPA + 5-HTP
+++ ++
N.D. 5,500 --
N.D. c 270
DMS 153 Endogenous + L-DOPA + 5-HTP
++++ ++
N.D. 6,400 --
N.D. 500
N.D. 400
N.D.
DMS 79 Endogenous + L- DOPA + 5-HTP
-
N.D.
N.D.
N.D.
N.D.
-
N.D. N.D.
N.D.
N.D.
(a) (b) (c)
D o p a m i n e DOPAC
HVA
5-HIAA
N.D. I00
N.D. 650
N.D.
R e l a t i v e fluorescence was estimated v i s u a l l y by fluorescence microscopv. Concentrations of dopamine, DOPAC, HVA and 5-HIAA were measured by selected ion monitoring as described in t e x t . N.D. = none detectable Discussion
The FIF observed in c e l l l i n e s DMS 53 and DMS 153 a f t e r incubation with L-DOPA is most l i k e l y due to L-DOPA, dopamine or both ( I ) . The i d e n t i f i c a t i o n of 5-HIAA in c e l l s pretreated with 5-HTP s t r o n g l y suggests t h a t t h i s amino acid is also decarboxylated to form 5-hydroxytryptamine (5-HT, or serotonin) which is in turn deaminated to form the a c i d i c metabolite 5-HIAA which we detected by mass-spectrometry. Decarboxylation o f L-DOPA requires the enzyme dopa decarboxylase (DDC) and the presence of HVA in L-DOPA treated c e l l s also demonstrated the occurence of catechol-O-methyl transferase (COMT) in SCCL c e l l s . In another series of 5 continuous c e l l l i n e s , DDC was i d e n t i f i e d as a d i s t i n guishing marker f o r cultured SCCL lung tumor c e l l s , when comoared to l e v e l s o f a c t i v i t y found in continuous c e l l l i n e s of other h i s t o l o g i c types of lung tumors (4). We have p r e v i o u s l y observed t h a t DMS 53 is capable of taking up 3H L-DOPA, but t h a t DMS 79 c e l l s do not (unnublished data). Therefore, DDC may be present in DMS 79 c e l l s , although i t s a c t i v i t v would not be apoarent in these analyses, which depend upon uptake of exoqenous L-DOPA. In the studies described here, we have shown t h a t cultured human small c e l l lung tumor c e l l s (SCCL) take up and metabolize L-DOPA and 5-HTP in v i t r o and t h a t t h i s property is i n t r i n s i c to two c e l l l i n e s and absent from a t h i r d . The above conclusions are supported by two separate experimental apnroaches: Demonstration o f FIF in L-DOPA or 5-HTP incubated c e l l s , and the p o s i t i v e i d e n t i f i c a t i o n o f , t h e metabolites of dopamine and serotonin in these c e l l s . Two c e l l l i n e s , DNS 53 and DMS 153 gave a o o s i t i v e response in terms of the above t e s t systems, w h i l e DMS 79 presented no evidence of e i t h e r L-DOPA or 5-HTP uptake in v i t r o . The s i g n i f i c a n c e of these observations in which n e u r a l - l i k e metabolic patterns
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Vol. 30, No. 16, 1982
occur in tumor c e l l s a r i s i n g from the bronchial epithelium is not known and requires f u r t h e r i n v e s t i g a t i o n . However, we have i d e n t i f i e d s i g n i f i c a n t d i f ferences regarding the metabolism of amine precursors between c e l l lines isolated from a s p e c i f i c type of lung tumor and provided evidence that enzymes involved in the biosynthesis and degration of DA and 5-HT are present in 2 of the 3 tumor c e l l l i n e s . References I. 2. 3. 4. 5. 6. 7. 8. 9. I0.
II. 12. 13. 14. 15. 16. 17. 18. 19.
E.T. ANGELAKOS, and M. KING. Fluorescence histochemistrv of the biogenic amines. In: Psychopharmacology: A Review of Proqress 1957-1967 (D.H. Efron, ed.) pn. 27-36, U.S.P.H.S. Publication No. 1836, 1968. N.G. BACOPOULOS, D.E. REDMONDand R.H. ROTH, J. Neurochem. 32:1215-1218, 1979. N.G. BACOPOULOS, S.E. HATTOX and R.H. ROTH, Euronean J. Pharmacol. 56:225236, 1979. S.B. BAYLIN, M.D. ABELOFF, G. GOODWIN, D.N. CAREY and A.F. GAZDAR, Cancer Res. 40:19q0-1994, 1980. K.G. B~NSCH, B. CORRIN, R. PARIENTE and H. SPENCER, Cancer 22:1163-I172, 1968. T. BRINCK-JOHNSEN, O.S. PETTENGILL, K. BRINCK-JOHNSEN, G.D. SORENSON and L.H. MAURER, J. Steroid Biochem. 10:339-340, 1979. E. CUTZ, W. CHAN, V. WONGand P.E. CONEN, Lab Invest. 30:458-464, 1974. B. FALCK and OWMAN, C., Acta Univ. Lund., Sect. I I ( 7 ) 7 - 2 3 , 1965. A.F. GAZDAR, D.N. CARNEY, E.K. RUSSEL, H.L. SIMS, S.B. BAYLIN, P.A. BUNN, JR., J.G. GUCCION and J.D. MINNA, Cancer Res. 40:3502-3507, 1980. A.F. GAZDAR, D.N. CARNEY, J.G. GUCCION and S.B. BAYLIN. Small c e l l carcinoma of the lung: C e l l u l a r o r i g i n & r e l a t i o n s h i p to other pulmonary tumors. In: Small Cell Lung Cancer (F.A. Greco, R.K. Oldham and P.A. Bunn, J r . , Eds.) pp.145-176, GrUne and Stratton, 1981. E. HAGE, Acta Path. Microbiol. Scand. Sect. A, 80:225-234, 1972. J.M. LAUWERYNS, M. COKELAERE, T. LERUT and P. THEUNYNCK, Cell Tiss. Res., 193, 373-386, 1978. J.M. LAUWERYNS and M. COKELAERE, Experientia 29:1384-1386, 1973. W.D. ODELL and A. WOLFSEN. Ectopic Hormone Secretion by Tumors. In: Cancer, a Comprehensive Treatise (F.F. Becker, Ed.) Dp. 81-97, Plenum Press, New York, 1975. O.S. PETTENGILL and G.D. SORENSON. Tissue Culture and in V i t r o characteristics. In: Small Cell Lunn Cancer (F.A. Greco, R.K. hldham and P.A. Bunn, J r . , Eds.) pp. 79-94, Grune and Stratton, 1981. O.S. PETTENGILL, G.D. SORENSON, D.H. WURSTER-HILL, T.J. CURPHEY, W.W. NOLL, C.C. CATE and L.H. MAURER, Cancer 45:906-918, 198fl. G.D. SORENSON and T. BRINCK-JOHNSEN, Proc. Amer. Assoc. Cancer Res., 18: 248, 1977. G.D. SORENSON, O.S. PETTENGILL, T. BRINCK-JOHNSEN, C.C. CATE and L.H. MAURER, Cancer 47:1289-1296, 1981. These studies we--re supported in part by CA 2~845, CA 23108 and BRSG RR05392. We thank Andrea Goggin f o r technical expertise in carrying out FIF studies. I