Download Inhibition of Cell Growth by Lovastatin Is Independent of ras Function

(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|methionine 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 mitogenesis 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-
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 COOHterminal 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 HMGCoA 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 farnesylation 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 doublestranded 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'-GGCCACCATGGGGAGTAGCAAGAGCAAGCCTAAGGACCCCAGCCAGCGCCGGATGACAGAATACA-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
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.
1The abbreviations used are: MVA, mevalonic acid; HMG-CoA. hydroxy
methylglutaryl coenzyme A; D-MEM. Dulbecco's modified Eagle's medium.
712
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LOVASTATIN-INHIBITED
CELL GROWTH AND raj FUNCTION
vector pGV16 (19), pBWl 160 (20), by ligation to vector linearized by
double digestion with Sacll and Xhol, which each cleave pBW1160
once. The resulting plasmid is designated pJDClOo.
Derivation and Culture of Cell Lines. Morphological transformation
was induced by calcium phosphate transfection of the NIH 3T3 (clone
7) line with the following plasmids: pBW'1423 for \-ras": pBW1631
for c-rtw" (21); pJDCl (22) for v-src, pJDClOo for the myristylated
ras; and MSV3611 proviral DNA for v-ra/(23). With the exception of
the v-ra/plasmid, each of the above plasmids contains neo as a linked
selectable marker. All cell lines except the v-ra/line were generated by
selection with G418 (600 MM)for 10-14 days, isolation of individual
transformed colonies with cloning cylinders, and expansion into cell
lines. The v-ra/line was generated by cloning an individual focus of
transformed cells and expansion into a cell line. Cells were cultured in
D-MEM supplemented with 10% fetal bovine serum (GIBCO) and 600
MMG418 where required.
Growth Inhibition Assays. On day 0, cells were trypsinized and plated
at 104/well in 8 triplicate sets in 24-well plates (Costar). On day 3, cells
effects of lovastatin
on these transformants
should distinguish
those of lovastatin in specifically blocking farnesylation of ras
protein from its other effects in ras-transformed cells. Cells
were also transformed with v-src and \-raf, which are ras
dependent and ras independent, respectively (4).
Fig. 1 represents photomicrographs of the cell lines. The
parental NIH 3T3 line exhibits a flat, nonrefractile morphology,
with fully contact-inhibited growth. In contrast, the v-ra/-transformed cells have an elongated, refractile appearance and tend
to pile on top of each other, as do the v-src-transformed cells,
although they have a rounded, refractile morphology. The cras, v-ras, and myristylated ras (myr-ras) transformed cells
display a morphology somewhat in between the \-raf- and the
v-src-transformed cells. Some of the cells are elongated, while
others are rounded (especially for v-ras), and all the ras-trans
formed lines lack contact inhibition and pile up extensively.
Thus
all of the oncogenes produce alterations in cell morphol
were fed with fresh medium containing either 0, 5, 10, or 15 MM
ogy and a loss of monolayer-restricted growth in NIH 3T3 cells.
lovastatin. After 48 h (day 5), one triplicate set of each concentration
In addition, the growth rates of the oncogene-transformed cells
was trypsinized and counted in a Coulter Counter. The other triplicate
sets were fed fresh medium with or without lovastatin at this time.
exceed that of the parental line (data not shown).
Two days later (96 h total treatment) the other triplicate sets were
Growth Inhibition by Lovastatin. To assess their relative sen
trypsinized and counted. For experiments in which MVA was included,
sitivity to lovastatin, the cell lines were treated with 5, 10, and
300 MMMVA was added together with 15 MMlovastatin at both day 3
15 AIMlovastatin for up to 96 h. This concentration range has
and day 5.
been shown previously to inhibit the growth of cultured cell
Preparation of Lovastatin. Lovastatin was generously provided by lines, including NIH 3T3. As noted by others, a dramatic, doseAlfred Alberts of Merck, Sharp & Dohme Co. To convert the inactive
lactone form of lovastatin to the active form, the drug was dissolved in dependent rounding of the cells was observed after 24 h lovas
ethanol, heated at 50°Cin 0.1 N NaOH, neutralized with HC1, and
tatin treatment (28). This morphological effect, which was
stored unfiltered as a 4 mg/ml stock at -20°C.
especially pronounced at 15 /¿M
lovastatin, was similar in the
untransformed parental line and in the transformed lines (not
Metabolic Labeling and Immunoprecipitation of ras Proteins. Cells
were plated at a density of 2 x 106/75-cm2 flask (Costar). Labeling with
shown).
[35S]methionine was carried out in 2 ml of methioninc-free D-MEM
Following 48 h treatment with lovastatin, cell growth was
(Flow Laboratories) supplemented with 10% regular D-MEM, 2% fetal
inhibited by 25-45% for 5 ¿¿M
treatment and by 40-65% for 15
bovine serum, and 200 MCi/ml 15S-Trans-label (ICN). Labeling with
¿IMtreatment (Fig. 2A). All lines tested displayed a dose[3H]myristate or |'H|palmitate (NEN-Dupont) was carried out in reg
dependent inhibition over the concentration range tested. Fur
ular medium with 10% fetal bovine serum for 6 h with 0.5 and 2.0
thermore, the degrees of inhibition of the ras-transformed cell
mCi/ml, respectively. For the labeling experiments, lovastatin treat
lines, including two independently derived myr-ras transformment (15 MM)was for 24 h prior to cell lysis, including the labeling
ants,
were virtually identical and within 10% of the inhibition
period.
seen for the v-src- and the v-ra/-transformed lines. Only the
Cells were lysed as described (24). For analysis of "S-labeled cells,
lysates containing equal numbers of acid-precipitable counts were ana
parental NIH 3T3 line differed significantly in its response to
lyzed. The entire lysate from one flask was analyzed for the 'H-labeled
lovastatin; the inhibition of this line was less than for any of
cells. Lysates were immunoprecipitated with the ras-specific monoclo
the transformed lines (Fig. 2A). Ninety-six h of treatment of
nal antibody Y13-259 (25). Following a 2-h antibody incubation. 60 M' the cell lines with the same concentration range of lovastatin
of a 10% suspension of protein A-Sepharose coated with rabbit-anti rat
also resulted in a similar pattern of inhibition that was dose
IgG were added for 45 min. The immunoprecipitates were washed 4 dependent for each line. At this time point, the inhibition of
times with wash buffer as described (24), boiled in sodium dodecyl
the v-src, v-ras, and myr-ras lines was almost identical (Fig.
sulfate sample buffer, and loaded on 15% polyacrylamide gels.
2B), while the c-ras and v-ra/ lines were somewhat less inhib
ited, and again, the NIH 3T3 line was the least inhibited of the
RESULTS
lines tested. Furthermore, the inhibition of every line, at each
concentration, was greater at 96 h of treatment than at 48 h,
Morphological Properties of Transformed Cell Lines. To com
although the extent of inhibition and the variation among the
pare the effect of lovastatin on cell lines that were transformed
lines were greater at 96 h than after 48 h of treatment (Fig.
by oncogenes that are ras dependent or ras independent, NIH
3T3 cells, which require normal (c-ras) function for passage
IB). At 5 MM the inhibition ranged from 40 to 80%, while at
15 ¿IM
the range was from 70 to 90%. Thus growth inhibition
through the cell cycle, were transformed by a variety of onco
genes and expanded as cell lines. For lines that would be in these studies was both time and concentration dependent.
dependent upon exogenous ras, cells were transformed by muLovastatin Blocks Processing of ras Proteins. To confirm that
ialiona IK activated v-ras or by overexpression of c-ras. We also lovastatin was inhibiting the processing of the authentic (nonmyristylated) endogenous and exogenous ras proteins, cells
took advantage of the ability of ras protein to be transforming
when its membrane association is driven by myristylation at its were labeled with ['5S]methionine, ['H]myristate, or ['HjpalmiNH2 terminus in the absence of COOH-terminal posttranslatate, in the presence or absence of lovastatin, and lysates of cell
tional changes by inducing transformation with a nonfarnesyextracts were immunoprecipitated with the anti-ras monoclonal
antibody Y13-259 (Fig. 3). In the absence of lovastatin, the ras
lated, myristylated transforming ras protein the COOH termi
nus of which had been mutated to prevent posttranslational
proteins detected under these conditions represent fully proc
processing (26, 27). Since the transforming activity of the essed forms, which migrate approximately 2-3 kDa faster than
myristylated ras protein is independent of farnesylation, the an unprocessed primary ras translation product. Normal NIH
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LOVASTATIN-INHIBITED
CELL GROWTH AND ras FUNCTION
'
Fig. I. Phase-contrast photomicrographs of the cell lines used in this study. A. normal NIH 3T3: B. v-ro/-transformcd line; C v-.wr-transformed line; I), c-ra.stransformed line; E. v-ra.v-transformed line; F. myr-rai-transformed line 4-3; 6", myr-ro.i-transformed line 4-4. Cells were plated at 8 x lO'/oO-mm dish 2 days prior
to photomicroscopy. Original magnification. X-85.
3T3 cells labeled with [15S]methionine exhibited a low level of
endogenous ras proteins at M, 21,000-22,000 (Fig. 3A. Lane
/). As expected, the \-ras protein migrated as a doublet (29);
the upper band represents the viral protein that is phosphorylated at Thr-59 and the lower band represents the nonphosphorylated form (Fig. 3A, Lane 2). The two myr-ra.v lines, since
they also contain Thr-59, express a doublet of M, 26,000 and
28,000; their slower migration rate results from the additional
15 amino acids at their NH2 terminus and their lack of COOH
terminal processing (Fig. 3A, Lanes 3 and 4). In the presence
of 15 ßMlovastatin. the endogenous ras doublet was replaced
by a single band (M, 22,000) (Fig. 3.-1,Lanes 5 and 6). while
both the phosphorylated and nonphosphorylated v-ras proteins
migrated approximately 2 kDa slower, indicating a lack of
farnesylation, proteolytic cleavage, and carboxymethylation. In
contrast, no effect on the migration of the myr-ras protein was
seen (compare Lanes 4 and 6). We conclude that lovastatin has
blocked the posttranslational modifications of newly synthe
sized c-ras and \-ras proteins but has not altered the myr-ras
proteins.
When the cells were labeled with ['Hlmyrislate
(Fig. 3B), as
expected no label was incorporated into the endogenous ras or
the \-rax proteins (Fig. 35, Lanes I and 2). The myr-ras proteins
did, however, incorporate label in the presence or absence of
lovastatin, indicating that they were myristylated, and no
change in the migration of m\r-ras protein was seen in the
lovastatin-treated cells (Fig. 3fi. Lanes 3-5). The slight reduc
tion in label in Lane 5 compared to Lane 4 probably reflects
the smaller number of cells in the flasks treated with lovastatin.
Labeling of cells in the presence or absence of lovastatin
showed that lovastatin blocked the processing of newly synthe
sized endogenous ras and \-ras proteins, but no effect was noted
on the processing of the myr-ras proteins. The cycling of
palmitatc on and off previously processed ras proteins contin
ued in the presence of lovastatin, as shown by metabolic labeling
with ['H]palmitate (data not shown).
Mevalonic Acid Overcomes the Effects of Lovastatin. Since
lovastatin blocks the enzyme HMG-CoA reducÃ-ase,which con
verts HMG to MVA, MVA can overcome effects of lovastatin
that arc due to its inhibition of HMG-CoA reducÃ-ase. Rcpre714
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LOVASTATIN-INHIBITED
CELL GROWTH AND ras FUNCTION
A
Lov:
-
-
29 —
ras
B
123456
Lov: — — — — +
29 —
18.4 —
14.3 —
12345
Fig. 3. Expression of ras proteins in the presence or absence of lovastatin as
detected by radiolabeling and immunoprecipitation. In A, cells were labeled for
20 h with |"S|methionine, with or without 24 h lovastatin treatment (15 MM)(top
abscissa). Lane I, NIH 3T3; Lanes 2 and 5, v-rai-transformed line; Lane 3, myrras-transformed line 4-3; lanes 4 and 6, myr-rai-transformed line 4-4. In B, cells
were labeled for 6 h with ['Hjmyristate with or without lovastatin treatment.
Arrow, migration of the myr-raa proteins. Lane I, NIH 3T3; Lane 2, v-rastransformed line; Lane 3, myr-ras-transformed line 4-3; Lanes 4 and 5. myr-raitransformed line 4-4. Immunoprecipitation was carried out as described in
"Materials and Methods." Left ordinate, migration of molecular weight standards
(x IO3).
Lovastatin
Fig. 2. Inhibition of cell growth by 48- or 96-h lovastatin treatment. In A, cells
were treated for 48 h with lovastatin at the concentrations indicated, and the
number of cells obtained were presented as the percentage of untreated controls.
In B, cells were treated for 96 h.
this pathway, may specifically inhibit the growth of cells which
are dependent on ras function for their growth. Our resulls
demonstrated, in accordance with earlier reports (9-11), thai
under ihe in vitro cullure condilions used here, lovaslalin
blocked Ihe processing of both endogenous ras and \-ras pro
teins. As expected, no effecl was seen by lovaslalin on the
processing of a control transforming ras protein that became
membrane associated via myristylation, which is a form of
acylation lhal does noi depend on the sterol pathway. Despite
these biochemical differences, lovastatin induced similar, dosedependent, biological changes (morphological changes and
growth inhibition) in iransformed lines, regardless of whelher
the transforming ras protein was prenylated. A similar degree
of growih inhibition was also observed for lines Iransformed by
\-src and \-raf, allhough transformation by v-raf has been
shown to be independent of endogenous ras. Since it has been
shown that transforming ras genes can provide the essential
function of endogenous ras (30), and the myristylated ras gene
should also provide this function, it is unlikely that the lack of
specificity for growth inhibition seen here is due to the inhibi-
sentative lines were therefore treated with lovastatin in the
presence of MVA to determine the extent to which the effects
of lovastatin on the cell lines resulted from HMG-CoA reduc
Ã-aseinhibition (Fig. 4). When 300 /UMMVA was added together
with 15 ßM
lovastatin, cell growth was restored to within 15%
of untreated controls, at both 48 and 96 h. In addition, no cell
rounding was observed for cells treated simultaneously with
MVA and lovastatin (data not shown). We conclude that the
major effects of lovastatin on the cell lines depend upon its
inhibition of HMG-CoA reducÃ-ase.
DISCUSSION
Based on the recent observation that proper processing of ras
proteins depends on products derived from the sterol-biosynthetic pathway, we have here investigated the possibility lhat
the compound lovastalin, which blocks ihe rate-limiling step in
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LOVASTATIN-INHIBITED
CELL GROWTH AND raj FUNCTION
Effect of MVAon Growth Inhibition by Lovastatin
by this compound may be overly optimistic, although it remains
possible that specificity might be seen under different in vitro
culture conditions or in tumorigenicity studies carried out in
animals.
The lack of specificity by lovastatin demonstrated here should
not restrict further attempts to pharmacologically block the
membrane localization of ras protein. However, it may be more
appropriate to focus on targets downstream from HMG-CoA
reducÃ-asewhich are more directly involved in the posttranslational processing of ras, such as the farnesylation step itself
(35). Even such inhibitors of downstream targets may also affect
macromolecules in addition to ras. As antagonists of farnesy
lation, carboxymethylation, or related events are identified,
their degree of specificity for ras transformation may be readily
evaluated by comparison with their ability to inhibit myr-ras
transformed cells.
50.0% 40.0% 30.0% 20.0% -
v-ras tMVA
myr v-ras 3
myr v-ras 3 +MVA
10.0% -
myr v-ras 4
myf v-ras 4 +MVA
Day
Fig. 4. Effect of M VA on growth inhibition induced by lovastatin. The indicated
cell lines were plated on day 0 and treated at day 3 with IS fiM lovastatin alone
or together with 300 ^M MVA. Cell growth was expressed as in Fig. 2.
tion of endogenous ras. The biological effects of lovastatin on
the ras-transformed lines could be overcome by simultaneous
treatment with MVA, which strongly implies that lovastatin
was acting primarily via its inhibition of HMG-CoA reducÃ-ase
(13). The results indicate that the lines transformed by HMGCoA reductase-dependenl ras protein were not more sensitive
to the growth-inhibitory effects of lovastatin than were lines
transformed by an oncogene the activity of which was HMGCoA reducÃ-aseindependent. We therefore conclude that, under
the culture conditions tested here, the inhibitory growth effects
of lovastatin on these lines are not mediated primarily via the
effects of the drug on ras processing. Growth of NIH 3T3 cells
transformed by a myristylated c-ras gene (27) is also inhibited
when the cells are treated with compacting which also blocks
HMG-CoA reducÃ-ase.This result further supports the conclu
sion that ras-independent activities mediate Ihe growth inhibi
tion observed when Ihis slep is blocked.
Although we failed to detect any specificity of lovastatin
toward cells the transformation of which depended upon farnesylated ras protein, rapidly growing, transformed lines were
more sensitive to the growth-inhibitory effects of lovastatin
than was the parental NIH 3T3 line, which exhibits a longer
doubling time. Thus lovastatin may inhibit tumor cells by
nonspecifically blocking critical cellular processes which are
especially important during rapid cell proliferation.
The finding that growth inhibition by lovastatin is not specific
for ras-transformed cells is perhaps not surprising. In studies
where cells have been treated with compactin (which also blocks
HMG-CoA reducÃ-ase), then labeled with [3H]MVA, over 40
proteins have been shown, by 2-dimensional electrophoresis, to
incorporate the label (16). These proteins include the nuclear
lam ins A and B (18, 31, 32), which may play an essential role
in the progression through the cell cycle, as well as several M,
20,000-25,000 proteins, which, like ras protein, may regulate
critical functions involved in cellular growth control. Further
more, MVA-derived products also include cholesterol, a regu
lator of membrane fluidity, as well as dolichols, and the ubiquinones (14, 33, 34). In view of the wide variety of cellular
processes that may be disrupted by lovastatin treatment, the
hope that ras-transformed cells could be specifically inhibited
2 A. Cox and J. Buss, personal communication.
REFERENCES
1. Marshall. C. J. The H4S oncogenes. J. Cell. Sci. (Suppl.), 10:, 157-169,
1988.
2. Barbacid. M. ras genes. Annu. Rev. Biochem.. 56: 779-827, 1987.
3. Bos, J. L. The ras gene family and human carcinogenesis. Mutât.Res., 195:
255-271, 1988.
4. Smith, M. R., DeGudicibus, S. J., and Stacey, D. W. Requirement for c-ras
proteins during viral oncogene transformation. Nature (Lond.), 320: 540543, 1986.
5. Rung, H.-F., Smith, M. R., Bekesi, E., Manne. V.. and Stacey, D. W. Reversal
of transformed phenotype by monoclonal antibodies against Ha-ra.çp2l
proteins. Exp. Cell Res., 162: 363-371. 1986.
6. Willumsen, B. M.. Christensen, A.. Hubbert, N. L., Papageorge, A. G., and
Lowy, D. R. The p21 ras terminus is required for transformation and
membrane association. Nature (Lond.), 310: 583-586, 1984.
7. Sefton. B. M.. Throwbridge, I. S., Cooper, J. A., and Scolnick, E. M. The
transforming proteins of Rous sarcoma virus. Harvey sarcoma virus and
Abelson sarcoma virus contain tightly bound lipid. Cell, 31: 465-474, 1982.
8. Willumsen, B. M., Norris, K.. Papageorge, A. G., Hubbert, N. L., and Lowy,
D. R. Harvey murine sarcoma virus p21 ras protein: biological and biochem
ical significance of the cysteine nearest the carboxy terminus. EMBO J., 3:
2581-2585, 1984.
9. Schafer, W. R., Kim, R., Sterne, R., Thorner, J.. Kim, S.-H.. and Rine, J.
Genetic and pharmacological suppression of oncogenic mutations in A' I.S
genes of yeast and humans. Science (Washington. DC), 245: 379-385. 1989.
10. Hancock. J. F.. Magee, A. I.. Childs, J. E., and Marshall, C. C. All ras
proteins are polyisoprenylated but only some are palmitoylated. Cell, 57:
1167-1177. 1989.
11. Casey, P. J.. Solski, P. A., Der, C. J., and Buss, J. E. p21 raj is modified by
a farnesyl isoprenoid. Proc. Nati. Acad. Sci. USA, 86: 8323-8327, 1989.
12. Gutierrez, L., Magee, A. I., Marshall, C. J., and Hancock, J. F. Posttranslational processing of p21 raÃ-is two step and an involves carboxyl-methylation and carboxy-terminal proteolysis. EMBO J.. 8: 1093-1098, 1989.
13. Alberts, A. W. Discovery, biochemistry' and biology of lovastatin. Am. J.
Cardiol.. 62: IOJ-15J, 1988.
14. Rine, J.. and Kim. S.-H. A role for isoprenoid lipids in the localization and
function of an oncoprotein. New Biol.. 2: 219-226, 1990.
15. Lowy, D. R., and Willumsen. B. M. New clue to Ras lipid glue. Nature
(Lond.), 341: 384-385. 1989.
16. Schmidt, R. A.. Schneider, C. J., and Glomset, J. A. Evidence of posttranslational incorporation of a product of mevalonic acid into Swiss 3T3 cell
proteins. J. Biol. Chem., 259: 10175-10180. 1984.
17. Maltese, W. A., and Sheridan, K. M. Differentiation of neuroblastoma cells
induced by an inhibitor of mevalonate synthesis: relation of neurite outgrowth
and acetylcholinesterase activity to changes in cell proliferation and blocked
isoprenoid synthesis. J. Cell. Physiol., 125: 540-558, 1985.
18. Beck, L., Hosick, T. J., and Sinensky, M. Incorporation of a product of
mevalonic acid metabolism into proteins of Chinese hamster ovary cell nuclei.
J. Cell Biol., 107: 1307-1316, 1988.
19. Jhappan. C.. Vande Woude, G. P., and Robins, T. Transduction of host
cellular sequences by a retroviral shuttle vector. J. Virol., 60: 750-753. 1986.
20. Willumsen, B. M., Papageorge A. G., Kung. H.-F., Bekesi, E., Robins, T.,
Johnsen, M., Vass, W. C.. and Lowy, D. R. Mutational analysis of a raÃcatalytic domain. Mol. Cell. Biol.. 6: 2646-2654. 1986.
21. Stone, J. C, Vass, W. C., Willumsen, B. M., and Lowy, D. R. pli-ras
effector domain mutants constructed by "cassette" mutagenesis. Mol. Cell.
Biol., A.-3565-3569, 1988.
22. De Clue, J. E.. and Martin, G. S. Linker insertion-deletion analysis of the virc gene: isolation of host- and temperature-dependent mutants. J. Virol.,
63: 542-554, 1989.
23. Rapp, U. R.. Goldsborough, M. D.. Mark, G. E., Bonner, T. I.. Groffen, J.,
Reynolds F. H. J.. and Stephenson J. Structure and biological activity of v-
716
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research.
LOVASTATIN-INHIBITED
CELL GROWTH AND ras FUNCTION
raf, a unique oncogene transduced by an oncogene. Proc. Nati. Acad. Sci.
USA. 80: 4218-4222. 1983.
24. Papageorge, A. G., Lowy. D. R.. and Scolnick, E. M. Comparative biochem
ical properties of p21 ras molecules coded for by viral and cellular ran genes.
J. Viro!., «.-509-5I9, 1982.
25. Furth. M. E.. Davis. L. J.. Fleurdelys. B.. and Scolnick. E. M. Monoclonal
antibodies to the p2l products of the transforming gene of Harvey murine
sarcoma virus and of (he cellular ras gene family. J. Virol.. 43: 294-304,
1982.
26. Lacal. P. M.. Penninglon. C. Y.. and Lacal. J. C. Transforming activity of
raj proteins translocated to the plasma membrane by a myristoylation se
quence from the src gene product. Oncogene, 2: 533-537. 1988.
27. Buss, J. E.. Solski. P. A.. Schacffcr. J. P.. MacDonald. M. J.. and Der. C. J.
Activation of the cellular proto-oncogene product p2lras by addition of a
myristylation signal. Science (Washington DC). 243: 1600-1603. 1989.
28. Fairbanks. K. P.. Barbu. V. D.. Witte, L. D.. Weinstein. I. B., and Goodman.
D. S. Effects of mcvinolin and me\alonate on cell growth in several trans
formed cell lines. J. Cell. Physiol.. 127: 216-222. 1986.
29. Shih, T. Y., Stokes. P. E., Smythers. G. W., Dhar, R., and Oroszlan, S.
Characteri/ation of the phosphorylation sites and the surrounding aminoacid
sequences of the p21 transforming proteins coded for by the Harvey and
Kirsten strains of murine sarcoma viruses. J. Biol. Chem., 275: 1176711773. 1982.
30. Papageorge. A. G.. Willumsen. B. M.. Johnsen. M.. Rung. H.-F., Vass, W.
C., and Lowy. D. R. A transforming ras gene can provide an essential
function
ordinarily
1843-1846.
1986." supplied by an endogenous ras gene. Mol. Cell. Biol.. 6:
31. Wolda. S. L.. and Glomset, J. A. Evidence for modification of lamin B by a
product of mevalonic acid. J. Biol. Chem., 263: 5997-6000, 1988.
32. Vorburger, K.. Kitten. G. T.. and Nigg, E. A. Modification of nuclear lamin
proteins by a mevalonic acid occurs in reticulocyte lysates and requires the
cysteine residues of the C-Ierminal CXXM motif. EMBO J.. 8: 4007-4014.
1989.
33. Goldstein. J. 1... and Brown. M. S. Regulation of the mevalonatc pathway.
Nature (Lond.). .143: 425-430. 1990.
34. Glomset. J. A.. Gelb. M. H., and Farnsworth, C. C. Premi proteins in
cukarvotic cells: a new type of membrane anchor. Trends Biochem. Sci.. 15:
139-142, 1990.
35. Reiss, Y.. Goldstein, J. L.. Scabra. M. C.. Casey. P. J.. and Brown, M. S.
Inhibition of purified p2lras farncsyl-protcin transferase by Cys-AAX tetrapeptidcs. Cell. 62: 81-88. 1990.
717
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research.
Inhibition of Cell Growth by Lovastatin Is Independent of ras
Function
Jeffrey E. DeClue, William C. Vass, Alex G. Papageorge, et al.
Cancer Res 1991;51:712-717.
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