Regulatory Peptides, 34 (1991) 181-188
181
© 1991 Elsevier Science Publishers B.V. 0167-0115/91/$03.50
REGPEP 01055
Expression of preprodynorphin in human small cell lung carcinoma cell lines Thomas Geijer 1, Jonas Bergh 2 and Lars Terenius 1 1Department of Drug DependenceResearch, Karolinska Institute, Stockholm and 2Department of Oncology, Akademiska Sjukhuset, University of Uppsala, Uppsala (Sweden) (Received 20 December 1990; revised version received and accepted 13 March 1991)
Key words: Opioid; Enkephalin; mRNA; Neuroblastoma; Northern blot; Radioimmunoassay
Summary The expression of preprodynorphin has been studied using the Northern blot technique. Ten human cell lines, six small cell lung carcinoma (SCLC), one large cell carcinoma (LCC), two neuroblastoma and one lymphoblast-like cell line, were screened with a preprodynorphin cRNA-probe. Tryptic digestion followed by radioimmunoassay for Leu-enkephalin-Arg 6 was used to detect possible translation of the preprodynorphin transcript. Of the ten cell lines investigated we found that all expressed preprodynorphin-mRNA to various degrees, and that this transcript is also translated. Two of the cell lines, neuroblastoma SK-N-MC and SCLC H69, also expressed preproenkephalinmRNA. This set of cell lines provides a useful model of human origin in which the regulation of the preprodynorphin gene and the posttranslational processing of its products can be studied and compared.
Introduction The opioid peptides derive from three different prohormones: proopiomelanocortin, proenkephalin and prodynorphin. They are widely expressed in endocrine and neuronal tissues [ 1-7]. It has recently been found that the opioid peptide prohormones are also expressed in various tumors [8,9], particularly of neuroendocrine origin [8]. This has Correspondence: T. Geijer, Dept. of Drug Dependence Research, Karolinska Institute, Box 60500, 104 01 Stockholm, Sweden.
182 raised the interest in opioid peptides as factors potentially influencing tumor growth. Several groups have observed tumor growth inhibition by opioids [ 10-13 ]. Interestingly, a recent paper reported opiate receptors in a series of human lung cancer cell lines and inhibition of tumor growth by the potent opiate etorphine [14]. This inhibition was reversed by low concentrations of nicotine, suggesting that smoking could interfere with a natural tumor growth suppressor system. Clonal tumor cell lines are of great advantage in studying the mechanism by which the opioid prohormone genes are activated. We [15] and others [16] have previously taken advantage of the expression of preproenkephalin in neuroblastoma cell lines. Opioid peptides and their transcripts have also been found in small cell lung carcinoma [17]. SCLC is recruited from cells with neuroendocrine differentiation, while the nonSCLC counterpart is derived from cells with a marked epithelial phenotype [ 18]. Here we show that preprodynorphin is uniformly expressed to different extent in a series of cell lines from human tumors, six small cell lung carcinomas (SCLC), one large cell lung carcinoma (LCC), two neuroblastomas and one leukemia. In contrast, preproenkephalin was only marginally expressed, except in one neuroblastoma and one SCLC. Free, and by trypsin treatment 'cryptic' opioid peptide deriving from preprodynorphin, (Leu-enkephalin-Arg 6) could be demonstrated in all tested (8/lO) cell lines.
Materials and Methods
Cell culture. The cell lines and their origin are shown in Table I. U1285, U1690, U1906, U2020, U2050, H69 and IM9 cells were cultured in humidified 5~o CO2-air in RPMI 1640 medium supplemented with 10~o fetal calf serum and 1 ~o penicillin (100 IU/ml) in 200 ml bottles (Falcon 3024). U1810 and the neuroblastoma cell lines SK-N-MC and SH-SY5Y were cultured as above but in Eagle's medium instead of RPMI 1640 and cell culture dishes (Falcon 3003) instead of bottles. All cell lines used TABLE I Some characteristics of the human cancer cell lines studied Cell line
U 1285 U1690 U1805 U1906 U2020 U2050 H69 SK-N-MC SK-N-SH IM9 a
Subculture Morphology
UI810
SH-SY5Y
SCLC SCLC LCC SCLC SCLC SCLC SCLC neuroblastoma neuroblastoma lymphoblastlike
Marker for neuroendocrinedifferentiation.
Establishedf r o m
Neuron-specific enolasea (ng/mg protein)
Ref.
pleural fluid pleural fluid pleural fluid cerebral metastasis pleural fluid pleural fluid pleura1 fluid retro-orbitaltumor bone-marrow bone-marrow
380 2100 17 340 1130 850 817
19,20,21 19,21 19,21 19,21 19,21 19,21 19,22 23 23 24
183 in this investigation were examined for mycoplasma infection and found to be negative. RNA and mRNA-preparation. (1-5). 107 cells were harvested from the medium by centrifugation at 1500 g and 4 ° C for 10 min. Before experiments, cell pellets were stored in - 70 ° C. RNA was prepared from cell pellets according to Chomczynski and Sacchi [25] and mRNA as described by Aviv and Leder [26] with modifications according to Chirgwin et al. [27]. Northern blot hybridization. Electrophoretic separation of RNA samples was carried out according to standard procedures [28]. RNA was transferred onto Amersham Hybond-N-membranes and subsequently hybridized as previously described [29]. However, cRNA-probes were used in the present study. The cRNA-probes were derived from 1.3/~g restriction endonuclease-treated plasmid DNA in a transcription system from Promega (Riboprobe) with 50#Ci [~-32p]-UTP (Amersham, 3000 Ci/mmol) present and immediately used in the hybridization assays. A SacI/HaelII 1227 bp fragment containing most of exon 4 of the human preprodynorphin gene subcloned into plasmid psp 64, was used to generate cRNA identifying preprodynorphin-mRNA. A PstI 1150 bp fragment from a mouse fl-actin DNA-clone subcloned into plasmid pGEM blue was used to generate cRNA identifying actin-mRNA, and an EcoRI/PvulI 792 bp fragment from a human preproenkephalin cDNA-subclone in plasmid psp65 was used to generate cRNA identifying preproenkephalin-mRNA. After hybridization, the filters were exposed to Hyperfilm-MP (Amersham, code RPN-6). Exposure was carried out at - 7 0 °C. Exposure times varied from 2 h to 24 h for preprodynorphin signals and from 30 min to 3 h for actin and preproenkephalin signals. Hybridization signals were measured with an Ultrascan XL laser densitometer (LKBPharmacia). The levels of the preprodynorphin-mRNA and the preproenkephalinmRNA hybridization signals were determined relative to the mouse fl-actin hybridization signal. The sizes of the specific RNAs responsible for hybridization signals were calculated from their migration on gels relative to the positions of the 18S and 28S bands whose migration had been determined earlier in relation to an RNA marker (Boehringer-Mannheim, RNA molecular weight marker I). The migration length was measured with an estimated accuracy of + 1 mm. Radioimmunoassay (RIA). (1-5). 107 cells were harvested from the medium by centrifugation at 1500 g and 4 °C for 10 min and then resuspended in 5 ml phosphatebuffered saline (PBS) and recentrifuged. The resuspension and recentrifugation procedure was repeated once and the cell pellets were finally stored in - 7 0 °C until the experiments. Cells were dissolved in 2 ml 1 M acetic acid and boiled for 5 min followed by centrifugation at 13,000 rpm and 4 °C for 10 min. The supernatant was lyophilized and dissolved in 800 #1 of 0.4 M N-ethylmorpholine acetate buffer (pH 8.2), 200/al of this solution was supplied with 50/~1 trypsin (1 #g/#l) and incubated for 6 h at 37 °C. After the incubation, 2 ml of cold 0.018 M pyridine, 0.1 M formic acid buffer (pH 3.2) was added and free Leu-enkephalin-Arg6 was separated by ion-exchange chromatography and then measured by radioimmunoassay performed as previously described [30]. Determination of the protein concentration was performed in the remaining 600 #1 of N-ethyl-morpholine acetate-extracts with a Bio-Rad protein assay according to the manufacturer's instructions.
184
Results The presence ofpreprodynorphin transcripts was determined in ten human cell-lines, six SCLCs, one LCC, two neuroblastomas and one leukemia. In one of the SCLCs and one of the neuroblastoma cell-lines, preproenkephalin transcripts could be detected as well. Data were obtained from two independent Northern blot analyses. The approximately estimated sizes of the transcripts were 2500 + 200 nt for preprodynorphin and 1500 + 150 nt for preproenkephalin (Fig. 1). Hybridization with the preprodynorphincRNA probe also gave a weaker signal at an approximate size of 3300 + 300 nt (Fig. 2). The hybridization signals for preprodynorphin mRNA were in the range 0.4-6.4 (determined in relation to an actin-signal), and the strongest signal was obtained for mRNA from U-1690, an SCLC cell-line. Expression of preproenkephalin has earlier been shown in neuroblastoma SK-N-MC [ 31 ] and SCLC H-69 [ 32] and was confirmed here with the strongest signal for SK-N-MC (16-times the H-69 signal). A
-28S
i!ii!i!i~ii~i,, iii!ii~
_ pr~pro~y~orph,~ -
18S
~!!i i~ii!i~i!i~ii!~~i,iii~il~~!iii ,!i~,,~!!~i ~:i~ ~li~~/i ....................
1
2
3
4
5
6
7
-28
S
_Actin 18S Preproenkephalin
1
2
3
4
5
6
7
Fig. I(A)Northern blot hybridization showing human preprodynorphin RNA. Lanes from left to right: U1285, U1690, U1810, U1906, U2020, H69 and SK-N-MC. 3 #g poly (A)+-RNA was applied to each lane. Exposure time 2 h. (B)The same filter was hybridized with human preproenkephalin and mouse fl-actin probes. Exposure time 35 min.
185
-28S - 3 . 3 kb _ Preprodynorphin -18S
1
2
3
4
5
6
Fig. 2. Northern blot hybridization shows two different species of preprodynorphin RNA. The lower band is at 2500 nt and the upper band at 3300 nt. Lanes from left to right: U1285, U1690, UI810, U1906, U2020 and U2050. 3/~g poly(A)+-RNA was applied to each lane. Exposure time 24 h.
In a d d i t i o n , v e r y w e a k signals for p r e p r o e n k e p h a l i n c o u l d be o b s e r v e d in m o s t celllines after l o n g e r e x p o s u r e - t i m e (see T a b l e II a n d Fig. 1). In o r d e r to d e t e r m i n e w h e t h e r p r e p r o d y n o r p h i n w a s t r a n s l a t e d , cell e x t r a c t s f r o m U 1285, U 1690, U 1810, U1906, U2020, SK-N-MC,
S H - S Y 5 Y a n d I M 9 w e r e t r e a t e d w i t h trypsin (in o r d e r
to yield L e u - e n k e p h a l i n - A r g 6 f r o m larger p r e c u r s o r f o r m s ) a n d m e a s u r e d in a specific radioimmunoassay.
I m m u n o r e a c t i v i t y w a s in the r a n g e 4 0 - 1 1 5 p m o l / m g protein,
w i t h an a v e r a g e o f 82 p m o l / m g p r o t e i n a n d a m e d i a n v a l u e o f 83 p m o l / m g p r o t e i n . H o w e v e r , n o c o r r e l a t i o n w i t h p r e p r o d y n o r p h i n R N A levels c o u l d b e seen. ( T a b l e II).
TABLE II Expression of preprodynorphin and preproenkephalin mRNA in relation to fl-actin mRNA, and Leuenkephalin-Arg6 immunoreactivity Cell line
RNA levels ( x 10) b preprodynorphin/ actin
U1285 a U 1690 a UI810 a U 1906 a U2020 a U2050 H69 SK-N-MC SH-SY5Y IM9 *
1.9 6.4 0.6 4.8 1.3 1.2 1.2 2.2 1.8 0.4
preproenkephalin/ actin weak weak weak none none weak 1.1 16.9 weak none
Leu-enk-Arg6 immunoreactivity (pmol//~g protein)
85 55 115 90 105 N.D. N.D. 75 40 80
a Data obtained from two independent analyses. 'Weak' = less than 1 • 10-4-times the preproenkephalin-signal for SK-N-MC. 'N.D.' = not determined. b No direct comparison of mRNA levels is allowed for two prohormone probes, since no adjustement has been made for the different exposure times and specific activities for the different probes.
186
Discussion The preprodynorphin-transcript seen in the cell lines in this study migrates as approx. 2500 nt. The human gene for preprodynorphin has been characterized [33], and consists of four exons separated from each other by three introns. The gene is 3900 bp between the capping site and polyadenylation site when introns are excluded. Thus, alternative sites for transcriptional initiation and/or polyadenylation, alternative splicing and variable poly(A)-tail length have to be considered in order to explain an approximate 2500 nt transcript from this gene. There is also a possibility that additional introns might be located in exon 1. A weaker signal migrating as approx. 3300 nt (not always detectable) is present in the Northern blots. Whether this represents a differently transcribed or processed species of preprodynorphin-mRNA, hnRNA, an entirely unrelated gene product with some partial homology or even background, was not determined in the present study. However, alternative transcription and/or different hnRNA processing events in these human cells are not unlikely. It is known from Northern blot analysis of rat RNA that there exist two species of preprodynorphinmRNA. The approximate length of preprodynorphin-mRNA is 2400 nt in hypothalamus and 2200 nt in reproductive tissues [6]. Similarly, there exist two species of preproenkephalin-mRNA, the approximate lengths of these transcripts are 1900 nt in testis and 1400 nt in hypothalamus [7]. Furthermore, in a recent study it was shown that the longer preproenkephalin transcript in mouse spermatogenic cells is due to initiation in the first somatic intron (As) downstream from the somatic promoter [34]. In our study, however, both transcripts are observed together in the same cell. Previous work has demonstrated the expression of both opioid receptors and opioid peptides in tumors and tumor cell lines [8,9,14,17,35,36]. In one of these studies opioid agonists were shown to inhibit cell proliferation in an SCLC line, NCI N414, and a non-SCLC line, NCI-H 157 [ 11 ]. Growth inhibiting effects of opioids have also been demonstrated in earlier studies [10-13], suggesting an autocrine system for tumor suppression in cancer cell lines. Opioid peptides might have this effect in some or all of the cell lines used in this investigation. In our study we have shown that ten cell lines, SCLC, LCC, neuroblastoma and lymphoblast-like, express preprodynorphin-mRNA, and that this transcript is also translated. Two of the cell lines, SK-N-MC and H69, also express preproenkephalinmRNA. This set of cell lines provides a useful model of human origin in which studies of the dynorphin gene expression are possible.
Acknowledgements We thank Dr. S. Numa for providing us with the preprodynorphin genetic probe, Dr. S. Legon for providing the preproenkephalin probe and Dr. H.-J Monstein for constructing the cRNA-generating vectors for both the genetic probes as well as support in numerous other ways. This work was supported by the Swedish Medical Research Council and the National Institute on Drug Abuse, Rockville, MD, U.S.A.T. Geijer has a fellowship from the Ulf Lundahls Minnesfond.
187
References 1 Jingami, H., Nakanishi, S., Imura, H. and Numa, S., Tissue distribution of messenger RNAs coding for opioid peptide precursors and related RNA, Eur. J. Biochem., 142 (1984) 441-447. 2 Civelli, O., Birnberg, N. and Herbert, E., Detection and quantitation of pro-opiomelanocortin mRNA in pituitary and brain tissues from different species, J. Biol. Chem., 257 (1982) 6783-6787. 3 Kazuva, N., Takaaki, Y., Mitsuaki, S., Makoto, S., Yoshio, I., Kyozo, H. and Hiroo, I., Rimorphin (dynorphin B) exists together with a-neoendorphin and dynorphin (dynorphin A) in human hypothalamus, Biochem. Biophys. Res. Commun., 113 (1983) 30-34. 4 Gramsch, C., H611t, V., Pasi, A., Mehraein, P. and Herz, A., Immunoreactive dynorphin in human brain and pituitary, Brain Res., 233 (1982) 65-74. 5 Przewlocki, R., Gramsch, C., Pasi, A. and Herz, A., Characterization and localization ofimmunoreactive dynorphin, a-neoendorphin, met-enkephalin and substance P in human spinal cord, Brain Res., 280 (1983) 95-103. 6 Douglass, J., Cox, B., Quinn, B., Civelli, O. and Herbert, E., Expression of the prodynorphin gene in male and female mammalian reproductive tissues, Endocrinology, 120 (1987) 707-713. 7 Kilpatrick, D. L., Howells, R. D., Noe, M., Bailey, C.L. and Udenfriend, S., Expression of preproenkephalin-like mRNA and its peptide products in mammalian testis and ovary, Proc. Natl. Acad. Sci. USA, 82 (1985) 7467-7469. 8 Bostwick, D.G., Null, W.E., Holmes, D., Weber, E., Barchas, J.D. and Bensch, K.G., Expression of opioid peptides in tumors, N. Engl. J. Med., 317 (1987) 1439-1443. 9 Zagon, I.S., McLaughlin, P.J., Goodman, S.R. and Rhodes, R.E., Opioid receptors and endogenous opioids in diverse human and animal cancers, J. Natl. Cancer Inst., 79 (1987) 1059-1065. 10 Scholar, E.M., Violi, L. and Hexum, T.D., The antimetastatic activity of enkephalin-like peptides, Cancer Lett., 35 (1987) 133-138. 11 Zagon, I. and McLaughlin, P., Naltrexone modulates tumor response in mice with neuroblastoma, Science, 221 (1983) 671-673. 12 Murgo, A.J., Modulation of murine melanoma growth by naloxone, Cancer Lett., 44 (1989) 137-142. 13 Zagon, I. and McLaughlin, P., Heroin prolongs survival time and retards tumor growth in mice with neuroblastoma, Brain Res. Bull., 7 (1981) 25-32. 14 Maneckje, R. and Minna, J. D., Opioid and nicotine receptors affect growth regulation of human cancer cell-lines, Proc. Natl. Acad. Sci. USA 87 (1990) 3294-3298. 15 Folkesson, R., Monstein, H.-J., Geijer, T. and Terenius, L., Modulation of proenkephalin A gene expression by cyclic AMP, Mol. Brain Res. 5 (1989) 211-217. 16 Yoshikawa, K. and Sabol, S. L. Glucocorticoids and cyclic AMP synergistically regulate the abundance of preproenkephalin messenger RNA in neuroblastoma-glioma hybrid cells, Biochem. Biophys. Res. Commun., 139 (1986) 1-10. 17 White, A., Stewart, M.F., Farrell, W. E., Crosby, S.R., Lavender, P. M., Twentyman, P. R., Rees, L.H. and Clark, A. J. L., Proopiomelanocortin gene expression and peptide secretion in small-cell lung cancer cell lines, J. Mol. Endocrinol., 3 (1989) 65-70. 18 Bergh, J., Nilsson, K., Dahl, D., Andersson, L., Virtanen, I. and Lehto, V.-P., Expression of intermediate filaments in established human lung cancer cell lines. An indicator of differentiation and derivation., Lab. Invest., 51 (1984) 307-316. 19 Bergh, J., Bj6rk, P., Westlin, J.-E. and Nilsson, S., Expression of an estramustine-binding associated protein in human lung cancer cell lines, Cancer Res., 48 (1988) 4615-4619. 20 Bergh, J., Larsson, E., Zech, L. and Nilsson, K., Establishment and characterization of two neoplastic cell lines (U1285 and U1568) derived from small cell carcinoma of the lung, Acta. Pathol. Microbiol. Scand. Immun. Sect. A, 90 (1982) 149-158. 21 Bergh, J., Nilsson, K., Ekman, R. and Giovanella, B., Establishment and characterization of cell lines from small cell and large cell carcinoma of the lung, Acta. Pathol. Microbiol. Scand. Immun. Sect. A, 93 (1985) 133-147. 22 Carney, D.N., Gazdar, A.F., Bepler, G., Guccion, G.J., Marangos, P.J., Moody, T.W., Sweig, M.H. and Minna, J.D., Establishment and identification of small cell cancer cell lines having classic and variant features, Cancer Res., 45 (1985) 2913-2923.
188 23 Biedler, J.L., Helson, L. and Spengler, B.A., Morphology and growth, tumorgenicity and cytogenetics of human neuroblastoma in continuous culture, Cancer Res., 33 (1973) 2643-2652. 24 Fahey, J. L., Buell, D. N. and Sox, H. C., Proliferation and differentiation of lymphoid cells: studies with human lymphoid cell lines and immunoglobulin synthesis, Ann. N.Y. Acad. Sci., 190 (1971) 221-234. 25 Chomczynski, P. and Sacchi, N., Single-step method of RNA isolation by guanidinium thiocyanatephenol-chloroform extraction, Anal. Biochem., 162 (1987) 156-159. 26 Aviv, H. and Leder, P., Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose, Proc. Natl. Acad. Sci. USA, 69 (1972) 1408-1412. 27 Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J., Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease, Biochemistry, 18 (1979) 5294-5299. 28 Maniatis, T., Fritsch, E.F. and Sambrook, J., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1982. 29 Monstein, H.-J., Folkesson, R. and Terenius, L., Proenkephalin A-like mRNA in human leukemia leukocytes and CNS-tissues., Life Sci., 39 (1986) 2237-2241. 30 Bergstrrm, L, Christensson, I, Folkesson, R, Stenstrrm, B. and Terenius, L., An ion-exchange chromatography and radioimmunoassay procedure for measuring opioid peptides and substance P, Life Sci., 33 (1983) 1613-1619. 31 Folkesson, R., Monstein, H.-J., Geijer, T., P~hlman, S., Nilsson, K. and Terenius, L., Expression of the proenkephalin gene in human neuroblastoma cell lines, Mol. Brain Res., 3 (1988) 147-154. 32 Geijer, T., Folkesson, R., Rehfeld, J.F. and Monstein, H.-J., Expression of the cholecystokinin gene in human (small-cell) lung carcinoma cell-line, FEBS Lett., 270 (1990) 30-32. 33 Horikawa, S., Takai, T., Toyosato, M., Takahashi, H., Noda, M., Kakidani, H., Kubo, T., Hirose, T., Tanayama, S., Hayashida, H., Miyata, T. and Numa, S., Isolation and structural organization of the human preproenkephalin B gene, Nature, 306 (1983) 611-614. 34 Kilpatrick, D. L., Zinn, S. A., Fitzgerald, M., Higuchi, H., Sabol, S.L. and Meyerhardt, J., Transcription of the rat and mouse proenkephalin genes is initiated at distinct sites in spermatogenic and somatic cells, Mol. Cell. Biol., 10 (1990) 3717-3726. 35 Roth, K.A. and Barchas, J.D., Small cell carcinoma cell lines contain opioid peptides and receptors, Cancer, 57 (1986) 769-773. 36 McMurray, C.T., Devi, L., Calavetta, L. and Douglass, J.O., Regulated expression of the prodynorphin gene in the R2C Leydig tumor cell line, Endocrinology, 124 (1989) 49-59.
181
© 1991 Elsevier Science Publishers B.V. 0167-0115/91/$03.50
REGPEP 01055
Expression of preprodynorphin in human small cell lung carcinoma cell lines Thomas Geijer 1, Jonas Bergh 2 and Lars Terenius 1 1Department of Drug DependenceResearch, Karolinska Institute, Stockholm and 2Department of Oncology, Akademiska Sjukhuset, University of Uppsala, Uppsala (Sweden) (Received 20 December 1990; revised version received and accepted 13 March 1991)
Key words: Opioid; Enkephalin; mRNA; Neuroblastoma; Northern blot; Radioimmunoassay
Summary The expression of preprodynorphin has been studied using the Northern blot technique. Ten human cell lines, six small cell lung carcinoma (SCLC), one large cell carcinoma (LCC), two neuroblastoma and one lymphoblast-like cell line, were screened with a preprodynorphin cRNA-probe. Tryptic digestion followed by radioimmunoassay for Leu-enkephalin-Arg 6 was used to detect possible translation of the preprodynorphin transcript. Of the ten cell lines investigated we found that all expressed preprodynorphin-mRNA to various degrees, and that this transcript is also translated. Two of the cell lines, neuroblastoma SK-N-MC and SCLC H69, also expressed preproenkephalinmRNA. This set of cell lines provides a useful model of human origin in which the regulation of the preprodynorphin gene and the posttranslational processing of its products can be studied and compared.
Introduction The opioid peptides derive from three different prohormones: proopiomelanocortin, proenkephalin and prodynorphin. They are widely expressed in endocrine and neuronal tissues [ 1-7]. It has recently been found that the opioid peptide prohormones are also expressed in various tumors [8,9], particularly of neuroendocrine origin [8]. This has Correspondence: T. Geijer, Dept. of Drug Dependence Research, Karolinska Institute, Box 60500, 104 01 Stockholm, Sweden.
182 raised the interest in opioid peptides as factors potentially influencing tumor growth. Several groups have observed tumor growth inhibition by opioids [ 10-13 ]. Interestingly, a recent paper reported opiate receptors in a series of human lung cancer cell lines and inhibition of tumor growth by the potent opiate etorphine [14]. This inhibition was reversed by low concentrations of nicotine, suggesting that smoking could interfere with a natural tumor growth suppressor system. Clonal tumor cell lines are of great advantage in studying the mechanism by which the opioid prohormone genes are activated. We [15] and others [16] have previously taken advantage of the expression of preproenkephalin in neuroblastoma cell lines. Opioid peptides and their transcripts have also been found in small cell lung carcinoma [17]. SCLC is recruited from cells with neuroendocrine differentiation, while the nonSCLC counterpart is derived from cells with a marked epithelial phenotype [ 18]. Here we show that preprodynorphin is uniformly expressed to different extent in a series of cell lines from human tumors, six small cell lung carcinomas (SCLC), one large cell lung carcinoma (LCC), two neuroblastomas and one leukemia. In contrast, preproenkephalin was only marginally expressed, except in one neuroblastoma and one SCLC. Free, and by trypsin treatment 'cryptic' opioid peptide deriving from preprodynorphin, (Leu-enkephalin-Arg 6) could be demonstrated in all tested (8/lO) cell lines.
Materials and Methods
Cell culture. The cell lines and their origin are shown in Table I. U1285, U1690, U1906, U2020, U2050, H69 and IM9 cells were cultured in humidified 5~o CO2-air in RPMI 1640 medium supplemented with 10~o fetal calf serum and 1 ~o penicillin (100 IU/ml) in 200 ml bottles (Falcon 3024). U1810 and the neuroblastoma cell lines SK-N-MC and SH-SY5Y were cultured as above but in Eagle's medium instead of RPMI 1640 and cell culture dishes (Falcon 3003) instead of bottles. All cell lines used TABLE I Some characteristics of the human cancer cell lines studied Cell line
U 1285 U1690 U1805 U1906 U2020 U2050 H69 SK-N-MC SK-N-SH IM9 a
Subculture Morphology
UI810
SH-SY5Y
SCLC SCLC LCC SCLC SCLC SCLC SCLC neuroblastoma neuroblastoma lymphoblastlike
Marker for neuroendocrinedifferentiation.
Establishedf r o m
Neuron-specific enolasea (ng/mg protein)
Ref.
pleural fluid pleural fluid pleural fluid cerebral metastasis pleural fluid pleural fluid pleura1 fluid retro-orbitaltumor bone-marrow bone-marrow
380 2100 17 340 1130 850 817
19,20,21 19,21 19,21 19,21 19,21 19,21 19,22 23 23 24
183 in this investigation were examined for mycoplasma infection and found to be negative. RNA and mRNA-preparation. (1-5). 107 cells were harvested from the medium by centrifugation at 1500 g and 4 ° C for 10 min. Before experiments, cell pellets were stored in - 70 ° C. RNA was prepared from cell pellets according to Chomczynski and Sacchi [25] and mRNA as described by Aviv and Leder [26] with modifications according to Chirgwin et al. [27]. Northern blot hybridization. Electrophoretic separation of RNA samples was carried out according to standard procedures [28]. RNA was transferred onto Amersham Hybond-N-membranes and subsequently hybridized as previously described [29]. However, cRNA-probes were used in the present study. The cRNA-probes were derived from 1.3/~g restriction endonuclease-treated plasmid DNA in a transcription system from Promega (Riboprobe) with 50#Ci [~-32p]-UTP (Amersham, 3000 Ci/mmol) present and immediately used in the hybridization assays. A SacI/HaelII 1227 bp fragment containing most of exon 4 of the human preprodynorphin gene subcloned into plasmid psp 64, was used to generate cRNA identifying preprodynorphin-mRNA. A PstI 1150 bp fragment from a mouse fl-actin DNA-clone subcloned into plasmid pGEM blue was used to generate cRNA identifying actin-mRNA, and an EcoRI/PvulI 792 bp fragment from a human preproenkephalin cDNA-subclone in plasmid psp65 was used to generate cRNA identifying preproenkephalin-mRNA. After hybridization, the filters were exposed to Hyperfilm-MP (Amersham, code RPN-6). Exposure was carried out at - 7 0 °C. Exposure times varied from 2 h to 24 h for preprodynorphin signals and from 30 min to 3 h for actin and preproenkephalin signals. Hybridization signals were measured with an Ultrascan XL laser densitometer (LKBPharmacia). The levels of the preprodynorphin-mRNA and the preproenkephalinmRNA hybridization signals were determined relative to the mouse fl-actin hybridization signal. The sizes of the specific RNAs responsible for hybridization signals were calculated from their migration on gels relative to the positions of the 18S and 28S bands whose migration had been determined earlier in relation to an RNA marker (Boehringer-Mannheim, RNA molecular weight marker I). The migration length was measured with an estimated accuracy of + 1 mm. Radioimmunoassay (RIA). (1-5). 107 cells were harvested from the medium by centrifugation at 1500 g and 4 °C for 10 min and then resuspended in 5 ml phosphatebuffered saline (PBS) and recentrifuged. The resuspension and recentrifugation procedure was repeated once and the cell pellets were finally stored in - 7 0 °C until the experiments. Cells were dissolved in 2 ml 1 M acetic acid and boiled for 5 min followed by centrifugation at 13,000 rpm and 4 °C for 10 min. The supernatant was lyophilized and dissolved in 800 #1 of 0.4 M N-ethylmorpholine acetate buffer (pH 8.2), 200/al of this solution was supplied with 50/~1 trypsin (1 #g/#l) and incubated for 6 h at 37 °C. After the incubation, 2 ml of cold 0.018 M pyridine, 0.1 M formic acid buffer (pH 3.2) was added and free Leu-enkephalin-Arg6 was separated by ion-exchange chromatography and then measured by radioimmunoassay performed as previously described [30]. Determination of the protein concentration was performed in the remaining 600 #1 of N-ethyl-morpholine acetate-extracts with a Bio-Rad protein assay according to the manufacturer's instructions.
184
Results The presence ofpreprodynorphin transcripts was determined in ten human cell-lines, six SCLCs, one LCC, two neuroblastomas and one leukemia. In one of the SCLCs and one of the neuroblastoma cell-lines, preproenkephalin transcripts could be detected as well. Data were obtained from two independent Northern blot analyses. The approximately estimated sizes of the transcripts were 2500 + 200 nt for preprodynorphin and 1500 + 150 nt for preproenkephalin (Fig. 1). Hybridization with the preprodynorphincRNA probe also gave a weaker signal at an approximate size of 3300 + 300 nt (Fig. 2). The hybridization signals for preprodynorphin mRNA were in the range 0.4-6.4 (determined in relation to an actin-signal), and the strongest signal was obtained for mRNA from U-1690, an SCLC cell-line. Expression of preproenkephalin has earlier been shown in neuroblastoma SK-N-MC [ 31 ] and SCLC H-69 [ 32] and was confirmed here with the strongest signal for SK-N-MC (16-times the H-69 signal). A
-28S
i!ii!i!i~ii~i,, iii!ii~
_ pr~pro~y~orph,~ -
18S
~!!i i~ii!i~i!i~ii!~~i,iii~il~~!iii ,!i~,,~!!~i ~:i~ ~li~~/i ....................
1
2
3
4
5
6
7
-28
S
_Actin 18S Preproenkephalin
1
2
3
4
5
6
7
Fig. I(A)Northern blot hybridization showing human preprodynorphin RNA. Lanes from left to right: U1285, U1690, U1810, U1906, U2020, H69 and SK-N-MC. 3 #g poly (A)+-RNA was applied to each lane. Exposure time 2 h. (B)The same filter was hybridized with human preproenkephalin and mouse fl-actin probes. Exposure time 35 min.
185
-28S - 3 . 3 kb _ Preprodynorphin -18S
1
2
3
4
5
6
Fig. 2. Northern blot hybridization shows two different species of preprodynorphin RNA. The lower band is at 2500 nt and the upper band at 3300 nt. Lanes from left to right: U1285, U1690, UI810, U1906, U2020 and U2050. 3/~g poly(A)+-RNA was applied to each lane. Exposure time 24 h.
In a d d i t i o n , v e r y w e a k signals for p r e p r o e n k e p h a l i n c o u l d be o b s e r v e d in m o s t celllines after l o n g e r e x p o s u r e - t i m e (see T a b l e II a n d Fig. 1). In o r d e r to d e t e r m i n e w h e t h e r p r e p r o d y n o r p h i n w a s t r a n s l a t e d , cell e x t r a c t s f r o m U 1285, U 1690, U 1810, U1906, U2020, SK-N-MC,
S H - S Y 5 Y a n d I M 9 w e r e t r e a t e d w i t h trypsin (in o r d e r
to yield L e u - e n k e p h a l i n - A r g 6 f r o m larger p r e c u r s o r f o r m s ) a n d m e a s u r e d in a specific radioimmunoassay.
I m m u n o r e a c t i v i t y w a s in the r a n g e 4 0 - 1 1 5 p m o l / m g protein,
w i t h an a v e r a g e o f 82 p m o l / m g p r o t e i n a n d a m e d i a n v a l u e o f 83 p m o l / m g p r o t e i n . H o w e v e r , n o c o r r e l a t i o n w i t h p r e p r o d y n o r p h i n R N A levels c o u l d b e seen. ( T a b l e II).
TABLE II Expression of preprodynorphin and preproenkephalin mRNA in relation to fl-actin mRNA, and Leuenkephalin-Arg6 immunoreactivity Cell line
RNA levels ( x 10) b preprodynorphin/ actin
U1285 a U 1690 a UI810 a U 1906 a U2020 a U2050 H69 SK-N-MC SH-SY5Y IM9 *
1.9 6.4 0.6 4.8 1.3 1.2 1.2 2.2 1.8 0.4
preproenkephalin/ actin weak weak weak none none weak 1.1 16.9 weak none
Leu-enk-Arg6 immunoreactivity (pmol//~g protein)
85 55 115 90 105 N.D. N.D. 75 40 80
a Data obtained from two independent analyses. 'Weak' = less than 1 • 10-4-times the preproenkephalin-signal for SK-N-MC. 'N.D.' = not determined. b No direct comparison of mRNA levels is allowed for two prohormone probes, since no adjustement has been made for the different exposure times and specific activities for the different probes.
186
Discussion The preprodynorphin-transcript seen in the cell lines in this study migrates as approx. 2500 nt. The human gene for preprodynorphin has been characterized [33], and consists of four exons separated from each other by three introns. The gene is 3900 bp between the capping site and polyadenylation site when introns are excluded. Thus, alternative sites for transcriptional initiation and/or polyadenylation, alternative splicing and variable poly(A)-tail length have to be considered in order to explain an approximate 2500 nt transcript from this gene. There is also a possibility that additional introns might be located in exon 1. A weaker signal migrating as approx. 3300 nt (not always detectable) is present in the Northern blots. Whether this represents a differently transcribed or processed species of preprodynorphin-mRNA, hnRNA, an entirely unrelated gene product with some partial homology or even background, was not determined in the present study. However, alternative transcription and/or different hnRNA processing events in these human cells are not unlikely. It is known from Northern blot analysis of rat RNA that there exist two species of preprodynorphinmRNA. The approximate length of preprodynorphin-mRNA is 2400 nt in hypothalamus and 2200 nt in reproductive tissues [6]. Similarly, there exist two species of preproenkephalin-mRNA, the approximate lengths of these transcripts are 1900 nt in testis and 1400 nt in hypothalamus [7]. Furthermore, in a recent study it was shown that the longer preproenkephalin transcript in mouse spermatogenic cells is due to initiation in the first somatic intron (As) downstream from the somatic promoter [34]. In our study, however, both transcripts are observed together in the same cell. Previous work has demonstrated the expression of both opioid receptors and opioid peptides in tumors and tumor cell lines [8,9,14,17,35,36]. In one of these studies opioid agonists were shown to inhibit cell proliferation in an SCLC line, NCI N414, and a non-SCLC line, NCI-H 157 [ 11 ]. Growth inhibiting effects of opioids have also been demonstrated in earlier studies [10-13], suggesting an autocrine system for tumor suppression in cancer cell lines. Opioid peptides might have this effect in some or all of the cell lines used in this investigation. In our study we have shown that ten cell lines, SCLC, LCC, neuroblastoma and lymphoblast-like, express preprodynorphin-mRNA, and that this transcript is also translated. Two of the cell lines, SK-N-MC and H69, also express preproenkephalinmRNA. This set of cell lines provides a useful model of human origin in which studies of the dynorphin gene expression are possible.
Acknowledgements We thank Dr. S. Numa for providing us with the preprodynorphin genetic probe, Dr. S. Legon for providing the preproenkephalin probe and Dr. H.-J Monstein for constructing the cRNA-generating vectors for both the genetic probes as well as support in numerous other ways. This work was supported by the Swedish Medical Research Council and the National Institute on Drug Abuse, Rockville, MD, U.S.A.T. Geijer has a fellowship from the Ulf Lundahls Minnesfond.
187
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