(CANCER RESEARCH 51.712-717, January 15. 1991] Inhibition of Cell Growth by Lovastatin Is Independent of ras Function Jeffrey E. DeClue, William C. Vass, Alex G. Papageorge, Douglas R. Lowy, and Berthe M. Willumsen Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, Maryland 20892 ¡J.E. D., W. C. V.,A. G. P., D. R. L.J, and University Microbiology Institute, Osterfarimagsgade 2A, D-1353 Copenhagen, Denmark ¡B.M. W.] ABSTRACT We have investigated the inhibition of cell growth by lovastatin (pre viously known as mevinolin), an antagonist of hydroxymethylglutaryl coenzyme A reducÃ-ase which blocks the processing and membrane local ization of ras proteins via inhibition of polyisoprenylation. A series of NIH 3T3 cells transformed by oncogenes with activities that are depend ent or independent of isoprenylated ras were studied, including cells transformed by myristylated ras protein that is isoprenylation independ ent. Treatment with lovastatin at concentrations ranging from 5 to IS MM for up to 96 h resulted in a time- and dose-dependent inhibition of cell growth in all lines tested. The inhibition ranged from 25 to 50% when cells were treated with 5 nM lovastatin for 48 h, to 72-90% for cells treated with 15 MMlovastatin for 96 h. Cells transformed by c-ras, \-ras, \-src, \-raf, and the myristylated ras genes displayed similar sensitivities; the parental NIH 3T3 line was the most resistant of the lines tested. Metabolic labeling of control and lovastatin-treated cells with [15S|me- thionine or tritiated lipids revealed that 15 MM lovastatin blocked the processing of both endogenous ras and \-rti.\ proteins yet had no effect on the lipidation of myristylated ras proteins. Addition of 300 MM mevalonic acid overcame the inhibition induced by 15 MM lovastatin. Thus the inhibition of cell growth in vitro by lovastatin did not show specificity for cells the transformation of which is dependent upon iso prenylated ras protein. It is therefore likely that the inhibition of other pathways affected by lovastatin, such as cholesterol biosynthesis or the processing of other cellular proteins, are responsible for the growth inhibition by lovastatin. INTRODUCTION The ras group of genes, which are a highly conserved sub family of the small GTPases, encode closely related M, 21,000 protein products (p21s) (1, 2). In addition to serving an impor tant function in the control of normal eukaryotic growth and proliferation, mutated versions of ras proteins have been found in a wide variety of human and animal tumor systems (3). Studies in which neutralizing antibodies to ras proteins have been microinjected into NIH 3T3 cells have shown that mito- genesis of nontransformed cells, as well as lines transformed with the v-irc, \-fes, and \-fms oncogenes, is dependent on functional endogenous ras protein (4). Human cells derived from normal tissues also require raÃ-for DNA synthesis follow ing mitotic stimulation, and a few tumor cell lines without activated ras are still inhibited to the same degree by the antibody as are tumor lines with activated ras genes (5). Thus some human tumor cells can be described as ras dependent, although their genetic lesion(s) lie(s) outside of the ras genes. A critical requirement for ras function is the localization of raÃ-proteins to the cell plasma membrane from their initial synthesis in the cytoplasm (6). Membrane association occurs following posttranslational modifications that take place at the COOH terminus and include the covalent addition of lipid (7). Genetic studies indicated that the last four amino acids, espe cially an invariant cysteine, were required for posttranslational changes and membrane localization (8). Although it was origi- Received 8/6/90; accepted 11/1/90. 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. nally believed that palmitic acid represented the obligatory lipid, it has recently been shown that palmitylation is not an obliga tory step for some raÃ-proteins. Rather, the acylation of ras proteins involves the addition of a polyisoprenoid derivative of M VA,' which is an intermediate in the sterol-biosynthetic path way (9-11). The polyisoprenoid is probably a farnesyl residue (11). Following translation of p21 raÃ-,the farnesyl moiety is apparently added to the invariant cysteine residue, which is located four amino acids from the COOH-terminus; the three amino acids downstream of this cysteine are removed; and the carboxyl group of the farnesylated cysteine is methylated (12). At this point, cysteine residue(s) NH2-terminal to the farnesy lated cysteine, if present, may be palmitylated. This series of processing steps results in an increasingly hydrophobic COOH- terminal membrane anchor for the raÃ-proteins. The demonstration that proper processing of both normal and transforming raÃ-proteins is dependent on the availability of mevalonic acid-derived products has led to speculation that drugs such as compactin and lovastatin, which block the pro duction of M VA by inhibiting the rate-limiting enzyme HMG- CoA reducÃ-ase(13), may prove to be useful as chemotherapeutic agents against rai-dependent tumors (9, 14). However, since HMG-CoA reducÃ-aselies far upstream of the protein farnesy- lation step, blocking this enzyme may also interfere with other critical cellular processes (15). Furthermore, protein isopreny lation appears to be a common modification; in addition to raÃ-, a large number of other proteins are labeled by MVA-derived compounds (16-18). Thus the question of specificity arises as one of critical importance in assessing the potential efficacy of lovastatin in treating rai-dependent tumors. Using a series of cell lines transformed by defined oncogenes, the current study has sought to determine if lovastatin may have greater activity against rai-dependent cell lines than against rai-independent lines. Our in vitro studies demonstrate that under the culture conditions used here, the inhibition of cell growth by lovastatin was not selective for polyisoprenylated rai-dependent cell lines, which implies that the growth effects of lovastatin in these lines are not mediated principally through its inhibition of raÃ-protein function. MATERIALS AND METHODS Construction of Gene Encoding a Myristylated raÃ-Protein. A double- stranded oligonucleotide encoding the first 15 amino acids of the v-src protein and the first four amino acids of ras was synthesized (sense strand: S'-GGCCACCATGGGGAGTAGCAAGAGCAAGCCTAA- GGACCCCAGCCAGCGCCGGATGACAGAATACA-3'). For the annealed double stranded oligonucleotide, the 5' end encodes a Sad I site overhang, and the 3' end a HinDUl site overhang; the HindlU occurs naturally at amino acid 4 of v-rai". The main portion of the ras gene was derived from a Hind\\\/Xho\ fragment from the plasmid pBW1225. This fragment encodes amino acids 4-189 of the \-ras" protein including a cysteine-to-serine mutation at amino acid 186 which blocks the COOH-terminal posttranslational processing of raÃ-protein. These two fragments were inserted into a derivative of the expression 1The abbreviations used are: MVA, mevalonic acid; HMG-CoA. hydroxy methylglutaryl coenzyme A; D-MEM. Dulbecco's modified Eagle's medium. 712 Research. on December 14, 2021. © 1991 American Association for Cancer cancerres.aacrjournals.org Downloaded from