EXPERIMENTAL

CELL

RESEARCH

192,

414-417 (1991)

Insulin-like Growth Factor Binding Protein Expression in Human Small Cell Lung Cancer Cell Lines P. KIEFER,

G. JAQUES,’

J. SCH~NEBERGER,

G. HEINRICH,*

AND K. HAVEMANN

Philipps-University, Medical Center, Division of Hematology/Oncology, D-3.550 Marburg, Federal Republic of Germany; and *Department of Biotechnology, Preclinical Research, Sandoz Ltd., CH-4002 Basel, Switzerland

Insulin-like growth factor binding proteins (IGF-BP) are secreted by several human small cell lung cancer cell lines (SCLC). In order to identify the IGF-BPS from SCLC cell lines the RNA from 10 different SCLC cell lines was analyzed by Northern blot analysis with the probes for three different IGF-BPS, IGFBP-1, IGFBP2, and IGFBP-3. No hybridization signal could be detected with the probes encoding for IGFBP-1 and IGFBP-3. The hybridization with different IGFBPa-specific oligodeoxynucleotide probes and with the corresponding full-length cDNA showed that all SCLC cell lines which secreted IGF-BPS express IGFBP-2. (L 1991

Academic

Press,

Inc.

been shown to modulate growth-promoting effects in vitro [6-lo]. At present cloning of three different IGF-BPS has been reported: IGFBP-1 from human amniotic fluid [ 111, IGFBP-2, the human equivalent of the rat BRL-3A IGF-BP [ 121, and the plasma-derived growth hormonedependent IGFBP-3 [13]. Recently we could demonstrate that SCLC ceil lines secrete IGF-BPS into serumfree culture medium in the molecular mass range of 24 to 32 kDa. These studies now were undertaken to characterize the IGF-BPS which are expressed by SCLC cell lines. For this reason, we have analyzed the RNA from 10 different SCLC cell lines with IGFBP-l-, IGFBP-2-, and IGFBP-3-specific probes. MATERIALS

INTRODUCTION

Insulin-like growth factor (IGF)‘-1 and IGF-2 are potent mitogens for many normal and malignant cells in. uiuo and in vitro [l]. In malignant cells it has been suggested that IGFs are part of an autocrine system in which they interact with specific receptors to stimulate growth of the cells which secreted them. Previously we and others have shown that IGF-1 and IGF-2 may be involved in an autocrine growth regulation mechanism in small cell lung cancer (SCLC) cell lines [2-41. Nakanishi et al. [3] reported that the mitogenie effect of insulin, IGF-1, and IGF-2 was mediated by the IGF-1 receptor, since a monoclonal antibody that specifically binds to the IGF-1 receptor, aIR3 [5], inhibited the insulin-, IGF-l-, and IGF-2-mediated proliferation in SCLC. Unlike insulin both IGFs circulate in plasma bound to specific IGF-binding proteins (IGF-BP). These proteins have a high affinity for both IGF-1 and IGF-2 and have

i To whom correspondence and reprint requests should be addressed. * Abbreviations used: Insulin-like growth factor 1 (2), IGF-1 (2); small cell lung cancer, SCLC; nonsmall cell lung cancer, NSCLC; IGF-binding protein, IGF-BP; sodium dodecyl sulfate, SDS; polyacrylamide gel electrophoresis, PAGE.

$3.00

Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

METHODS

Cell lines. The permanent human SCLC cell lines SCLC21H, SCLC22H, and SCLC86Ml and the NSCLC cell line MSTO-211H were established and characterized in our laboratory [15, 161. The SCLC cell lines NCI-H69, NCI-H82, NCI-H146, NCI-N417, NCIH526, and NCI-N592 and the NSCLC cell line A-549 were obtained from Drs. A. F. Gazdar and J. Minna, National Cancer Institute (Bethesda, MD) [17,18], the SCLC cell line DMS-79 was from Dr. G. D. Sorenson, Dartmouth Hitchcock Medical Center (Hanover, NH) (191, the SCLC cell line FRE was from Dr. J. Reeve, MRC Centre (Cambridge, UK) [20], and the NSCLC cell lines U-1752 and U-1810 were from Dr. J. Bergh (University of Uppsala, Sweden) [21]. Cell lines SCLC22H, SCLC86M1, NCI-H69, NCI-H146, NCI-N592, and DMS-79 were classified as classic SCLC cell lines; all other cell lines of SCLC origin were of the variant SCLC subtype [17]. The human breast carcinoma cell line MCF-7 and the rat hepatoma cell line BRL3A were obtained from American Type Culture Collection, Rockville, MD. Cell lines were maintained in RPM1 1640 (GIBCO Europe, Paisley, UK) supplemented with 10% fetal calf serum (GIBCO). Crosslinking analysis. Serum-free conditioned media were obtained after 4 days and crosslinking analysis was performed as previously described (141. In brief, 50 ~1 of conditioned media was incubated with [‘251]IGF-I alone and with unlabeled IGF-I for 24 h; crosslinking was accomplished by adding disuccinimidyl suberate in dimethyl sulfoxide to obtain a final concentration of 0.5 mM. The proteins were then separated by 15% SDS-PAGE under nonreducing conditions and visualized by autoradiography [14]. Northern blot analysis. Total RNA was prepared from cells in logarithmic growth phase using the guanidinium thiocyanate method [22] with fractionation of RNA through cesium chloride gradient; poly(A)+RNA was selected using oligo(dT)-cellulose [23]. Human adult liver poly(A)+RNA was obtained from Clontech (Cat. No. 6516. 414

0014.4827/91

AND

IGFBP-2

Mr110-~ 69

k-

L6 k30 k-

FIG. 1. Electrophoretic analysis and autoradiography of [““I]IGF-l-binding complexes in conditioned media of SCLC cell lines under nonreducing conditions. Molecular weight markers M, (X10m3) are shown on the left side.

1). For Northern blot analysis 20 pg total RNA or 2 pg poly(A)+RNA was subjected to electrophoresis in 1.4% agarose gels after denaturation with glyoxal and DMSO, and blotted onto Hybond-N membranes (Amersham, U.K.) as described [24]. The resulting blots were hybridized with an IGFBP-1 cDNA clone (~85) [ll], with the complete 1.43.kb IGFBP-2 cDNA and with two different IGFBP-2-specific oligonucleotides [12] and with a IGFBP-3-specific oligonucleotide probe [13]. The sequences of the IGFBP-2-specific oligonucleotide probes were: 5’.GAGCTCGGTGCTGGTCTCTTTCCAAATATAAATAC-3’ (oligonucleotide 3’) and 5’-TCGCAGCGCACCACGGGACCC-3’ (oligonucleotide 5’). The sequence of the IGFBP-S-specific oligonucleotide probe was deduced from the published BP-53 cDNA and was 5’-TCGCACGGCTCGCAGCGCACCACGGGACCC-3’ [Nt 10221311 [13]. The random primer extension method was used to label the cDNA probes with [n-32P]dCTP [25], the oligonucleotide probes were labeled with [y-“‘P]ATP (Amersham, UK). RESULTS

AND

DISCUSSION

As previously shown incubation of radiolabeled IGF-1 and IGF-2 with serum-free conditioned media of SCLC cell lines followed by gel filtration revealed the presence of IGF-BPS with molecular masses in the range 24-32 kDa [14]. Figure 1 shows the result of a SDS-PAGE under nonreducing conditions of [‘“51]IGF-I crosslinked to conditioned media of six different SCLC cell lines. The conditioned media of NCI-H82, NCI-H69, NCIN417, NCI-N592, and DMS-79 showed specific binding for [1251]IGF-I, which is totally abolished by the addition of unlabeled IGF-I. Under these conditions no IGF-BPS were detected in the conditioned media of SCLC-22H. Since the IGF-binding activity released by SCLC cell lines corresponds in molecular size to three different, already cloned IGF-BPS, we analyzed 10 SCLC cell lines for transcription of the IGFBP-1, the IGFBP-2, and the IGFBP-3 genes. An IGFBP-1 cDNA [ll], IGFBP-2-spe-

IN SCLC

415

citic oligonucleotides from the 3’ and 5’ end and the corresponding cDNA [12] and an IGFBP-3-specific oligonucleotide [13] were used as hybridization probes in Northern blot analysis. Total RNA and/or poly(A)+RNA of different lung tumor cell lines, the breast cancer cell line MCF-7, the rat hepatoma cell line BRL-3A, and adult liver were used for hybridization experiments. While the Northern blot analysis performed with the probes specific for IGFBP-1 and IGFBP-3 showed no positive hybridization in all tested SCLC cell lines, positive signals were found after hybridization with the different probes specific for IGFBP-2. Figure 2 shows data gained from the hybridization with the 35merit-3’-oligonucleotide probe. This probe has a 33135 nucleotide identity with the IGF-BP gene from BRL-3A and detects both human IGFBP-2 and its rat homologue. A single transcript of approximately 1.6 kb was identified in the RNA from 7/8 SCLC cell lines, comparable in size to poly(A)+RNA from adult liver or the RNA from the rat hepatoma cell line BRL-3A. The same result was obtained with the corresponding IGFBP-2-specific cDNA (data not shown). In contrast to the SCLC cell lines SCLC-BlH, NCI-H69, DMS-79, NCI-H82, NCI-N592, and FRE, no hybridization signals were detected with the RNA prepared from the SCLC cell line NCI-H526, the NSCLC cell lines MSTO211H, A-549, U-1752, U-1810, and the breast cancer cell line MCF-7. In addition, a larger band that comigrated with 28 S ribosomal RNA was detected in all total RNA probes. Since this transcript was only found in hybridization experiments with total RNA and not with poly(A)+RNA, we conclude that this transcript represents a cross-hybridization between the 28 S ribosomal RNA and the 3’-oligonucleotide probe. When hybridized against the 3’-oligonucleotide probe, in addition to the expected 1.6 kb (NCI-H69), transcripts of approximately 3.0, 1.0, and 0.7 kb with the RNA from SCLC22H, and 1.3 to 1.9 kb and 0.5 kb with the RNA from NCI-N417 were detected (Fig. 3). In order to explain these transcripts in the RNA from SCLC-22H and from NCI-N417 we additionally performed Northern blot analysis with an oligonucleotide probe from the 5’ nontranslated region of the IGFBP-2 cDNA. Since cell specifically regulated genes often differ in their 5’ noncoding sequences as a consequence of alternative splicing or alternative promotors, we chose this oligonucleotide probe. The analysis of poly(A)+RNA of SCLC-22H with the 5’ located oligonucleotide probe showed no hybridization signals in comparison to the RNA from NCI-N417 and NCI-H69 (Fig. 3). These data suggest that the IGFBP-2 related transcripts in SCLC-22H might be different from the other SCLC cell lines in their 5’ part. Consistent with this RNA data, no IGF-binding protein was detected by crosslinking analysis in the conditioned medium of SCLC-22H. In comparison to SCLC-22H a

416

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ET AL.

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NSCLC

- 6.6 Lb - 4.4 kb

- 2.3 Lb 16FrlP-2 (1.6 Lb)

-

- 1.4 Lb - 0.6 Lb

oligonucleotide

probe:

3‘

FIG. 2. Northern blot analysis of SCLC cell lines using the IGFBP-2-specific oligonucleotide IGFBP-2-specific transcript of 1.6 kb are noted. Transcript sizes are indicated in kb.

different hybridization pattern was found in the RNA and poly(A)+RNA prepared from NCI-H69 and NCIN417 (Fig. 3): Both oligonucleotide probes detected the expected transcripts (NCI-H69) of approximately 1.6 kb and additional bands in the range of 1.4 kb-1.9 kb. The possibility that other closely related IGF-BP RNA species are transcribed by NCI-N417 cannot be excluded. The corresponding data gained from the crosslinking analysis showed the presence of IGF-BP in the supernatant of NCI-N417. This study demonstrates that all examined SCLC cell lines which release IGF-BPS in the conditioned media express IGFBP-2-specific transcripts. No hybridization signal was detected with the IGFBP-2-specific probes with the RNA of four NSCLC cell lines and the breast

- 6.6 kb 28

S-

- 4.4 Lb

- 2.3 Lb IGFBP-2 (I.6 Lb)

-

ill

of the 28 S and the size of the

cancer cell line MCF-7. We further found no expression with probes encoding for IGFBP-1 and IGFBP-3 in all examined SCLC cell lines. The RNA levels were different in the individual cell lines and did not constantly reflect the protein levels found in the culture medium. The different molecular masses and expression levels in the SCLC cell lines might be a result of post-translational modification of IGFBP-2. IGFBP-1 and IGFBP-2 are proteins which contain an -Arg-Gly-Asp sequence (RGD motif) which could be involved in cell-surface binding [26]. This observation strengthens the possibility that the small IGF-BPS secreted by SCLC cell lines might be modulators of IGF action at the cell surface. IGFBP-1 can potentiate the mitogenic effect of IGF-1 in human fibroblasts and muscle cells [6, 271, an effect which seems to be mediated by the association of the IGF-BPS to the cell surface [28]. Insulin, IGF-1, and IGF-2 are potent mitogens for SCLC cell lines; the interaction between IGFBP-2 and the IGF-1 receptor could modulate the action of the IGFs at the cell surface in SCLC. The authors thank Dr. A. Brinkman for the gift of the IGFBP-1 cDNA and Dr. Abel for preparation of the IGFBP-3specific oligonucleotides (Behringwerke AG, Marburg, FRG). We thank P. Halboth, E. Achenbach, and M. Kopcke for expert technical assistance. The excellent secretarial assistance of C. Born and the help in correcting the manuscript by Dr. A. Rodden are greatly appreciated. This work was supported by the SFB 215 of the German Research Society.

- 1.4 kb 1

REFERENCES

I”

- 0.6 kb *

probe:

3’. Migration

oligonucleotide

3‘

oligonucleotide

5‘

FIG. 3. Northern blot analysis of total and poly(A)+RNA from three different SCLC cell lines using the 3’- and 5’specific oligonucleotides. Migration of the 28 S ribosomal RNA and the size of the IGFBP-Z-specific transcript of 1.6 kb are noted. Transcript sizes are indicated in kb.

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Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18, 5294-5299. Aviv, H., and Leder, P. (1972) Proc. Natl. Acad. Sci. USA 77, 1408-1412. Thomas, P. S. (1980) Proc. Natl. Acad. Sci. LISA 77,5201-5202. Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13. Ruoslathi, E., and Pirschbacher, M. D. (1986) Cell 44, 517-518.

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