Analogues of Insulin-like Growth Factor 1 Inhibition of Growth of Prostatic Cancer Cell Lines by Peptide Updated

We have investigated three prostatic cancer cell lines, PC-3, DU-145, and LNCa.FGC, and found that all three cell lines can grow in serum-free medium without the addition of exogenous growth factors. All three cell lines produce substantial amounts of insulin-like growth factor 1 (IGF-1) that is secreted in the medium and they all display constitutively autophosphorylated IGF-1 receptors; two of the cell lines overexpress IGF-1 receptor RNA. The growth of all three cell lines is inhibited by an antisense oligodeoxynucleotide to IGF-1 receptor RNA or by peptide analogues of IGF-1 that compete with IGF-1 for binding to its receptor. Our results indicate that these three cell lines grow by an autocrine loop in which the overproduced IGF-1 activates its receptor. Interference with the activation of the receptor leads to cessation of growth. INTRODUCTION The activation of the IGF-11 receptor by its ligands (IGF-1, IGF-2, or insulin at supraphysiological concentrations) seems to play a piv otal role in the proliferation of a variety of cell types, such as fibroblasts, smooth muscle cells, chondrocytes, hemopoietic cells, and others (for a review, see Ref. 1). Recently, we have shown that 3T3 cells constitutively overexpressing the human IGF-1 receptor grow in serum-free medium supplemented solely with IGF-1, to the total ex clusion of the receptors for the other 2 growth factors that are required by 3T3 cells, platelet-derived growth factor and EOF (2). The impor tance of the IGF-l/IGF-1 receptor interaction in cell proliferation was confirmed by other findings from our laboratory: (a) overexpression of c-mvb abrogates the requirement for IGF-1 in 3T3 cells, by induc ing an increase in the expression of IGF-1 itself (3); (b) EGF markedly increases the expression of IGF-1 and IGF-1 mRNA, and growth stimulation by EGF indirectly requires a functional IGF-1 receptor (4); and (c) the SV40 large T-antigen also markedly increases the expression of IGF-1 and IGF-1 mRNA, leading to reduced growth factor requirements for SV40-transformed cells (5). In a recent review, Macaulay (6) has summarized the evidence indicating the role played by IGF-1, IGF-2, and their receptors in the growth of a variety of human tumors. Because the IGF-l/IGF-1 receptor autocrine loop oc curs frequently in different types of tumors, we thought it desirable to determine whether IGF-1 was required for the growth of prostatic cancer cell lines in culture. Our results show that the 3 prostatic cancer cell lines examined grow in serum-free medium, produce abundant amounts of IGF-1, and require an activated IGF-1 receptor for growth. MATERIALS AND METHODS Cell Lines. The following cell lines were obtained from the American Type Culture Collection: PC-3 (7) originating from a human adenocarcinoma of the prostate; LNCa.FGC (LNC; see Ref. 8) from a human metastatic adenocarciReceived 9/17/92; accepted 12/17/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by N1H Grants GM 33694 and CA 56309. 1 To whom requests for reprints should be addressed. 'The abbreviations used are: IGF-1. insulin-like growth factor 1; EGF, epidermal growth factor; RT-PRC, reverse-transcriptase polymerase chain reaction. noma of the prostate; and DU-145 (9) from a human carcinoma of the prostate metastatic to the brain. The cells were passaged as recommended by the American Type Culture Collection; they are now grown in the following serum-free medium: DU-145 in minimum essential medium supplemented with 1 UNIferrous sulfate; 1 HIMsodium pyruvale and 0.1% bovine serum albumin. The same supplements were added to Dulbecco's modified Eagle's medium:RPMl 1640 (1:1) to grow PC-3 cells and to RPMI 1640 for LNC cells. Growth in Serum-free Medium. For these experiments, the cells were plated first in \07c calf serum; the growth medium was removed after 24 h and replaced with serum-free medium, with the sole additions of bovine serum albumin (0.5 mg/ml) and 1.0 UMferrous sulfate (2). The number of cells was determined by standard methods at the times indicated in each individual experiment. RT-PCR. We followed the method of Rappolee et al. (IO), with the slight modifications used in our laboratory (II). RNA was extracted from cells using the method of Chomczynski and Sacchi (12). Amplimers and probe for the IGF-1 receptor RNA were chosen on the basis of the published complementary DNA sequence of the human IGF-1 receptor (13): 5' amplimer, 5' ACC ATT GAT TCT GTT ACT TC 3'; 3' amplimer, 5' ATA CTC TGT GAC ATT CTT AA 3'; probe 5' CTG CTC CTC TCC TAG GAT GA 3'. Labeling of probes and hybridization were carried out by standard methods (14, 15). The various controls we use in our RT-PCR assays (elimination of DNA, rejection of samples that give signals without reverse transcriptase, and multiple amplifi cation cycles) have been repeatedly described in articles from our laboratory (2, 4, 16). RNA amounts were monitored with amplimers and probe for the pHE 7 complementary DNA, the cognate RNA of which is expressed con stantly under different conditions of growth (16). Antisense Experiments. The antisense and sense oligodeoxynucleotides corresponded to codons 21-29 of the signal sequence of the human IGF-1 receptor (13). The actual sequences are given in the article by Pietrzkowski et al. (2) and the antisense oligodeoxynucleotide has already been shown to inhibit cell growth and to decrease the number of IGF-1 binding sites (2, 4). The oligodeoxynucleotides were synthesized on an Applied Biosystem Model 391 EP DNA synthesizer as already described (2, 4, 16). The oligodeoxynu cleotides were added to the medium after 48 h in serum-free medium (40 ug/ml) and the treatment was repeated the next day (20 ng/ml). The cells were counted 48 h after the second addition. Autophosphorylation of the IGF-1 Receptor. This was carried out essen tially by the method of Lammers et al. ( 17), using the monoclonal antibody to the IGF-1 receptor from Oncogene Sciences (Uniondale, NY), an anti-phosphotyrosine antibody from UBI (Saranac Lake, NY), and the enhanced chemiluminescence detection system from Amersham (Arlington Heights, IL). IGF-1 Radioimmunoassay. This has been described in detail in a previous paper (4), following the instructions of the manufacturer of the kit (Amersham, Arlington Heights. IL). IGF-1 Peptide Analogues. The IGF-1 peptide analogues used in these experiments have been described in a paper by Pietrzkowski et al. ( 18). They inhibit autophosphorylation of the IGF-1 receptor by IGF-1 and inhibit the growth of cells that require IGF-1 for growth.