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Experimental Therapeutics |
Department of Medicinal Chemistry, Janssen Research Foundation, Val De Reuil 27106, France [M. V., H. P., P. A., G. S.]; Department of Oncology, Janssen Research Foundation, Beerse B2340, Belgium [G. S., W. W.]; Johnson and Johnson Research Pty., Ltd., Strawberry Hills, New South Wales 2011, Australia [A. V. T., T. L. A., C. J. F.]; and Department of Oncology, Janssen Research Foundation, Spring House, Pennsylvania 19477 [D. W. E., S. S., A. D., C. B.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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75% were found to be sensitive
to R115777. The majority of sensitive cell lines had a wild-type
ras gene. Tumor cell lines bearing
H-ras or N-ras mutations were among the
most sensitive of the cell lines tested, with responses observed at
nanomolar concentrations of R115777. Tumor cell lines bearing mutant
K-ras genes required higher concentrations for
inhibition of cell growth, with 50% of the cell lines resistant to
R115777 up to concentrations of 500 nM. Inhibition of
H-Ras, N-Ras, and lamin B protein
processing was observed at concentrations of R115777 that inhibited
cell proliferation. However, inhibition of K-RasB
protein-processing could not be detected. Oral administration
b.i.d. of R115777 to nude mice bearing s.c. tumors at doses
ranging from 6.25–100 mg/kg inhibited the growth of tumors bearing
mutant H-ras, mutant K-ras, and wild-type
ras genes. Histological evaluations revealed
heterogeneity in tumor responses to R115777. In LoVo human colon
tumors, treatment with R115777 produced a prominent antiangiogenic
response. In CAPAN-2 human pancreatic tumors, an
antiproliferative response predominated, whereas in C32 human melanoma,
marked induction of apoptosis was observed. The heterogeneity of
histological changes associated with antitumor effects suggested that
R115777, and possibly farnesyl protein transferase inhibitors as a
class, alter processes of transformation related to tumor-host
interactions in addition to inhibiting tumor-cell proliferation. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The characterization of FPT and tetrapeptide inhibitors of the enzyme set off a search for low-molecular weight, nonpeptide inhibitors that could be developed as therapy-selective for tumors bearing ras mutations (9) . Initial studies with peptidomimetic compounds suggested that FTIs could, in fact, selectively inhibit the growth of ras-transformed cells (10, 11, 12) . However, as experience with FTIs increased, the role of ras proteins in mediating the antitumor effects of this class of agent became less certain (13) . As has been reported previously and in the current studies, FTIs have shown antiproliferative activity in tumor cell lines devoid of ras mutations (14) . Also, the different H-Ras, N-Ras, and K-RasB isoforms were found to behave quite differently in the presence of FTIs. With isolated enzyme, the K-RasB protein demonstrated a higher affinity for FPT, which decreased the potency of FTIs competitive for the CAAX binding site (15) . In intact tumor cells, the mutant, activated K-RasB and N-ras isoforms were associated with resistance to FTIs attributable to alternative processing by the parallel pathway of geranylgeranylation via GGPT I (16, 17, 18) . However, as will be described, R115777 and other inhibitors have shown antiproliferative and antitumor activity in tumor cell lines bearing K-ras mutations (19 , 20) . At the moment, there is discordance between the antitumor activity of FTIs and the biology of Ras proteins. Several molecular targets such as RhoB and centromere-associated proteins (21, 22, 23, 24) have emerged as possible downstream effectors for the FTIs. Treatment of cells with the FTI L-739,749 induced an alternative processing of RhoB via PGGT I, with a gain of geranylgeranylated RhoB and concomitant reduction of the farnesylated RhoB (21) . The accumulation of geranylgeranylated RhoB was associated with antiproliferative effects. Also in support of these observations, transfection of RhoB with CAAX motifs that restrict prenylation to geranylgeranylation reproduced many of the effects of FTI treatment, including reversal of the transformed phenotype as well as induction of apoptosis (23) . However, recent studies have shown that expression of either farnesylated RhoB or geranygeranylated RhoB produced similar tumor suppressive activity (25) . Thus, the role of RhoB in the effects of FTIs is uncertain. Additionally, inhibition of the farnesylation of the centromere-associated proteins CENP-E and CENP-F has been linked to a G2-M growth arrest observed in some tumor cells in vitro (24) . The current evidence suggests a role for multiple prenylated effectors in mediating the antitumor effects of FTIs. Involvement of multiple effectors might also be manifested as heterogeneous responses at the level of the tumor cell or intact tumors.
The current studies describe the preclinical pharmacology of R115777, a novel imidazole FTI that has entered Phase III clinical trials. Our findings will be discussed in relationship to the ras status of tumor cell lines as well as the heterogeneity of tissue responses observed in three human tumor xenografts after in vivo treatment with R115777.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals.
Female nu/nu immunodeficient nude mice (42 days old) were purchased
from Charles River Laboratories (Wilmington, MA). Mice were housed five
per cage in microisolator cages placed in laminar flow shelving to
maintain sterility. All bedding, food, water, and cages were
autoclaved. Animals were handled within the sterile confines of a
laminar flow cabinet. The mice were otherwise maintained under standard
vivarium conditions. Tumor studies were conducted under a protocol
approved by the Institutional Animal Care and Use Committee.
Isolation of FPT and PGGT 1.
FPT and PGGT I were isolated from Kirsten virus-transformed human
osteosarcoma cell tumors essentially as described by Reiss et
al. (8
, 26)
. Tumors were excised and immediately
homogenized in buffer (3.0 ml/tumor) containing 50
mM Tris, 1 mM EDTA, 1
mM EGTA, and 0.2 mM
phenylmethylsulfonylfluoride (pH 7.5). The homogenate was centrifuged
100,000 x g for 60 mi, and a 30–50%
ammonium sulfate precipitate was prepared from the supernatant. After
dialysis, FPT and PGGT 1 activity were further isolated by ion exchange
chromatography on Fast Q Sepharose. FPT was measured using the Amersham
Scintillation Proximity Assay using the lamin B peptide substrate
(Biotin-YRASNRSCAIM) provided with the assay. In some studies,
the K-rasB peptide (0.25 µM) was
substituted for the lamin B peptide. PGGT 1 assays were performed using
a modification of the Amersham Scintillation Proximity Assay methods. A
biotin-YRASNRSCAIL peptide substrate was incubated with
[1-[3
H](n)]geranylgeranylpyrophosphate for
120 min at 37°C. After terminating reactions, samples were
equilibrated overnight before counting.
Cell Proliferation Assays.
Trypsinized cell suspensions were inoculated into six-well cluster
dishes at an initial density of 200,000 cells/well in 3 ml of complete
growth medium. R115777 was added at concentrations ranging from
0.5–500 nM in 3 µl of DMSO. Cells were allowed to
proliferate to high saturation densities beyond confluence for 4–7
days. Cell numbers were quantified by detaching cell monolayers in 1 ml
of trypsin and counting cell suspensions on a Coulter particle counter.
Analysis of Activating Mutations in ras Genes.
DNA was prepared from human tumor cell monolayers using the DNAzol
Reagent (Life Technologies, Inc.). An RFLP-PCR strategy was used to
screen for activating mutations within K-ras,
N-ras and H-ras (27)
. Exons 1 and 2
of all three ras genes were simultaneously amplified in a
single multiplex reaction and an aliquot was used for a second round of
PCR. Resistance to cleavage at natural or primer-induced restriction
enzyme sites in second-round amplicons indicated the presence of a
mutation that had abolished the site at the loci being analyzed.
Restriction enzymes for the analysis of specific loci were
BstN I (K-ras codon 12), Bsl I
(K-ras codon 13, N-ras codons 12 and 13),
Msc I (H-ras codon 61; N-ras codon 61,
positions 1 and 2), HaeIII (K-ras codon 61,
position 1), Bfa I (N-ras codon 61, position 3),
and Tru9 I (K-ras codon 61, positions 2 and 3;
H-ras intron D, position 2719). Reactions were digested
overnight and PCR products were analyzed by gel electrophoresis. The
correlation of ras mutation status versus
sensitivity to R115777 were analyzed by a 
2
test with sensitivity defined as 50% inhibition of cell proliferation
at concentrations of 
100 nM.
Analysis of Ras Protein Processing in Intact Cells in Culture.
T24F1 or CAPAN-2 cells were grown as monolayers in T75 tissue culture
flasks in 25 ml of complete growth medium. The monolayer cultures were
treated with R115777 for 72 h. Then the growth medium was removed
and the monolayers were washed once with 5 ml of PBS. Cells were
harvested by scraping into ice-cold PBS and collected by centrifugation
(100 x g for 5 min). Total cellular Ras
processing was analyzed in particulate and soluble fractions of cells
as described by Yan et al. (28)
. Detection of
K-Ras immunoreactivity required immunoprecipitation with
v-H-Ras antibody conjugated to agarose before electrophoresis and
immunoblotting procedures. Protein determinations were performed on 5-
to 10-µl samples of pellets and supernatants (29)
.
Samples were normalized such that equal amounts of protein were added
to Laemmli sample buffer and separated on 10–20% gradient SDS
polyacrylamide slab gels. After transfer to polyvinylidene
difluoride membranes, samples were incubated overnight at 4°C
with primary antibodies. The immunostained antigens were visualized
using horseradish peroxidase-conjugated secondary antibodies and
Amersham enhanced chemiluminescence detection reagents.
Tumor Studies in Nude Mice.
Tumor cell lines maintained as monolayer cultures were detached by
trypsinization. Tumor cell suspensions were pooled and trypsin was
inactivated by the addition of serum-containing medium. Cells were
collected by centrifugation and washed once in HBSS. Cell suspensions
were adjusted to a final concentration of 1 x 106 cells/0.1 ml of HBSS. Mice were inoculated
with a single s.c. injection of 0.10 ml of tumor cell suspension in the
inguinal region of the thigh. Mice were housed five per cage, with 15
mice randomly assigned to treatment groups. Three days after tumor
inoculation, treatment with R115777 was initiated. R115777 was
administered b.i.d. by oral gavage in a 20% ß-cyclodextrin vehicle
as a volume of 0.10 ml of solution/10 g body weight. Control groups
received the same dosage/volume of the 20% ß-cyclodextrin vehicle.
Body weight and tumor size as determined by caliper measurements were
monitored weekly. At the end of study, mice were sacrificed by
CO2 asphyxiation. Tumors were excised, weighed,
and fixed immediately in 4% paraformaldehyde. ANOVA, mean values for
treatment groups, and SE for in vivo parameters were
calculated using IMSL subroutines compiled by R. W. Johnson of the
Pharmaceutical Research Institute, Science Information Department, on a
VAX computer. A value of P <0.05 was considered
significant.
Preparation of Tumors for Histology.
Fixed tumors were cut into thin fragments (approximately 10 x
10 x 3 mm). The tissues were rinsed overnight in 0.1
M phosphate buffer (pH 7.4). After dehydration in acetone,
LoVo and CAPAN-2 tumors were infiltrated and embedded in Technovit 8100
(Kulzer, Wehrheim, Germany). C32 melanoma tumors were dehydrated in
ethanol/xylol and infiltrated in paraffin block. For Technovit
embedding, infiltration and embedding were performed under a nitrogen
atmosphere at 0°C. Sections (3 µm) were cut with a Leica Jung
Autocut and mounted onto glass slides by drying at 50°C for 2 days.
Paraffin sections were mounted with Biobound-coating (British Biocell
International, Cardiff, United Kingdom). After staining with
erythrosine B and hematoxylin, the slides were mounted with Pertex (LED
Techno, Hechtel, Belgium).
Immunocytochemistry.
Sections were treated with a 1:10 dilution of Target Unmasking Fluid
(Sanbio, Uden, the Netherlands) by heating to 90°C in a microwave
oven. Slides were incubated with 0.5% trypsin solutions for 30 min at
37°C. The slides were extensively rinsed, and then endogenous
peroxidase was blocked by incubating with 1% peroxide in methanol for
15 min. Nonspecific antibody-binding sites were blocked by incubation
with 0.5% lysozyme for 60 min. Incubations with the primary antibody
were performed at room temperature at various times optimized for each
antibody. Staining with the primary antibody was visualized using the
biotin-avidin peroxidase method (Dako, Glostrup, Denmark) using Sigma
Fast DAB (Sigma, St. Louis, MO) At the end of the procedure, the slides
were counterstained with 0.25% methyl green. To monitor apoptosis,
ISEL was performed according to the directions of the TACSTM1 Klenow
Kit (Trevigen, Gaithersburg, MD).
Quantitative image analysis was performed using a Zeiss Axioplan microscope fitted with CCD cameras interfaced to a Silicon Graphics Indy R5000 Unix-based workstation. Image analysis routines were written in C under the SCIL-Image software package (SCIL Image, Version 1.3; TNO-TPD, Delft, the Netherlands). For evaluation of BrdUrd-labeling and apoptosis, images were taken with a black and white CCD camera (MX5, Adimec, the Netherlands). For quantification of cell proliferation, the total number of cells was obtained by imaging the methyl green staining through a 650-nm broadband interference filter. To obtain images of the nuclei labeled with BrdUrd, the same microscopic fields were viewed with a 450-nm broadband interference filter. For quantifying Factor VIII and VEGF-staining, 24-bit RGB color images were acquired with a cooled CCD-camera (Sony DXC-930 P) and transformed to HSI-space. The immunocytochemical signal was quantified as a percentage of the area in a field. For measurements of BrdUrd incorporation and apoptosis, a x20 objective was used; whereas, for VEGF- and Factor VIII-staining, a x40 objective was used. The total areas on each slide evaluated for BrdUrd incorporation and Ki-67 staining was 4 mm2 . Apoptosis was evaluated in an 8-mm2 area. The total number of cells quantified ranged from 8,000–25,000 cells/slide. Areas evaluated for Factor VIII- and VEGF-staining varied from 1–8 mm2 . Five tumors were evaluated from each treatment group.
Data from quantitative image analysis were analyzed by the Wilcoxon Mann-Whitney test. For proliferation and apoptosis, data were expressed as the percentage of positive cells/section and calculated as the mean ± SD for each tumor. For Factor VIII and VEGF staining, data were expressed as the percent area with staining signal versus the total section area viewed. These data were calculated as medians ± SE for each tumor.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Consistent with this difference in potency for the two peptide
substrates, kinetic studies revealed that R115777 was a competitive
inhibitor for both the K-ras and lamin B1 peptide substrates
(Ki = 0.5
nM) and noncompetitive for the
farnesylpyrophosphate substrate (data not shown). PGGT I activity was
virtually insensitive to R115777, with only 40% inhibition of enzyme
activity observed at 50 µM R115777.
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The farnesylation of Ras protein was studied in T24
H-ras-transformed NIH3T3 cells by Western blot analysis with
a pan-Ras antibody. Cells treated with 0.5–50 nM
R115777 displayed a concentration-dependent accumulation of Ras
immunoreactivity in the cytosol with a concomitant reduction of
processed, particulate Ras immunoreactivity (Fig. 2)
. Visually obvious responses were obtained within the concentration
ranges that inhibited cellular proliferation. However,
antiproliferative and morphological effects were observed at R115777
concentrations lower than the 10-nM concentration
that was required to deplete prenylated Ras. Therefore, depletion of
fully processed, activated Ras protein was not required for the
cellular effects of R115777.
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are the results from examining the substrate-dependent proliferation of
53 human tumor cell
lines.4
DNA prepared from each of the cell lines was analyzed for the presence
of mutations in N-ras and K-ras at codons 12, 13,
and 61 and in H-ras at codons 12 or 61 and position 2719 of
intron D. Overall, 75% of the cell lines were sensitive to the
antiproliferative effects of R115777 irrespective of ras
gene mutations. Sensitivity was operationally defined as a 50%
reduction in cell counts at concentrations of 100
nM. Cell lines bearing H-ras or
N-ras mutations were among the most sensitive to R115777,
with IC50s for inhibition of proliferation <10
nM. No evidence for resistance to R115777 was
observed in N-ras mutated cell lines, as has been suggested
previously (18)
. However, 50% of the cell lines bearing
K-ras mutations were resistant to R115777. Cell lines
bearing K-ras mutations that did respond to R115777 required
higher concentrations, with IC50s ranging from
10–100 nM. Approximately 80% of the cell lines
with wild-type ras were sensitive to R115777, which was
consistent with previous data (14)
. Some clustering of
sensitivity to R115777 was observed for tumor cell lines from similar
tissue origins. For example, human bladder tumor, rhabdomyosarcoma, and
melanoma cell lines were among the most sensitive to R115777, with
cells bearing wild-type ras or ras mutations
virtually indistinguishable in their responses (Table 3)
. Data for lung and pancreatic tumor cell lines are also summarized in
Table 3
to exemplify the resistance observed in cell lines bearing
K-ras mutations. In contrast to previous publications, A549
cells were relatively insensitive to R115777 (24
, 30)
.
This may reflect differences in experimental parameters as the former
studies were conducted under conditions of anchorage-independent
growth, which is an important consideration when assessing FTIs
(31)
. Overall, the sensitivity of tumor cell lines to the
FTI R115777 were consistent with data reported for cell line
sensitivity to the FTI L-744,832
(14)
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. The majority of the pan-Ras immunoreactivity shifted to
the cytosol was cross-reactive with an N-ras antibody (Fig. 3B)
. No H-Ras immunoreactivity was detected in
CAPAN-2 particulate or soluble fractions. Immunoprecipitation before
Western blot analysis was required to detect the low levels of K-Ras
immunoreactivity in particulate fractions of CAPAN-2 cells. Although an
increase in K-Ras immunostaining was noted after treatment with
R115777, no increase in soluble K-Ras immunoreactivity (Fig. 3C)
was noted. R115777 did not alter the
distribution of Rap1a immunoreactivity, a geranylgeranylated protein,
or RhoB immunoreactivity, a protein that can be farnesylated or
geranylgeranylated (Fig. 3A)
. The results were consistent
with the selectivity of R115777 observed in studies with isolated
enzyme.
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. A b.i.d. dosing schedule for R115777 was determined from plasma
pharmacokinetics in mice (data not shown). Oral administration of
R115777 at doses of 6.25, 12.5, and 25 mg/kg (b.i.d.) to nude mice
bearing T24 tumors suppressed the growth of tumors during the 14 days
of administration. Tumor growth measured as postmortem tumor weight was
inhibited by 56%, 84%, and 86% at the three respective dose levels.
Consistent with the in vitro cell proliferation data, higher
doses of R115777 were required to inhibit the growth of tumors bearing
K-ras mutations. In vitro, R115777 inhibited the
proliferation of LoVo human colon tumor cells bearing a
K-ras mutation with an IC50 of 16
nM. In nude mice bearing LoVo tumors, doses of 50
and 100 mg/kg were again required to significantly inhibit tumor growth
by 68% and 81%, respectively, as determined from postmortem tumor
weights after 32 days of treatment. Similar results were obtained for
the CAPAN-2 tumors. The C32 melanoma was selected as a representative
wild-type ras tumor for in vivo evaluation.
In vitro, R115777 inhibited the proliferation of C32
melanoma with an IC50 of 6
nM (Table 3)
. Oral administration of R115777 at
doses of 25, 50, and 100 mg/kg, b.i.d. inhibited the growth of C32
tumors by 48%, 76%, and 90% respectively. In all tumor studies, the
weekly measurements of tumor area were consistent with the effects of
R115777 on postmortem tumor weights (data not shown). Significant
changes in body weight were not observed for R115777 treatment (data
not shown). Analysis of protein prenylation was attempted in postmortem
tumor samples but was not successful because of the presence of mouse
antigens with apparent molecular masses of 60 kDa and 25 kDa, which
crossreacted with our antimouse IgG secondary antibodies used in
Western blot analysis.
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. In CAPAN-2 tumors, an antiproliferative effect was observed as
statistically significant reductions of BrdUrd-labeling at doses of 50
and 100 mg/kg. A modest but significant increase in apoptosis was also
observed at the 100 mg/kg dose level. By using combined Factor VIII
staining with ISEL, apoptotic cells were found to be relegated to the
endothelial cells of the CAPAN-2 tumor vasculature (Fig. 4)
. Despite this apparent antiangiogenic event, no significant changes in
markers related to angiogenesis (Factor VIII or VEGF) could be detected
in CAPAN-2 tumors. In contrast, a marked reduction in the endothelial
cell Factor VIII-staining was observed in LoVo human colon tumors, with
reductions of 45% and 60% observed at 25 and 50 mg/kg, respectively.
LoVo tumors from animals treated with the 100 mg/kg dose of R115777
were too small to prepare for histology. Modest reductions in VEGF
staining were also observed, but these did not achieve statistical
significance. No significant changes in apoptosis or BrdUrd labeling
were observed in LoVo tumors from R115777-treated mice. In the C32
melanoma tumors, yet another profile of responses to R115777 was
observed. R115777 induced a marked, dose-related increase in tumor cell
apoptosis. Apoptosis increased from 4.4% in vehicle-treated controls
to 22% at 25 mg/kg. At the highest tested doses of 50 and 100 mg/kg,
the response appeared to reach a maximum of 41–44% incidence of
apoptosis. Staining for the proliferation marker Ki67, VEGF, and Factor
VIII were not affected by treatment with R115777 in the C32 melanoma
tumors.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To a degree, the loss of potency with K-RasB at the level of the enzyme was reflected in studies in intact cells in vitro. Cell lines with N-ras or H-ras mutations responded to lower concentrations of R115777 than did the cell lines bearing K-ras mutations. These findings are consistent with previously published studies, wherein tumors bearing mutant H-ras appear to be far more sensitive to FTIs than tumors bearing K-ras mutations (9 , 10 , 19 , 20 , 35 , 36) . Additionally, the presence of K-ras mutations correlated with resistance to R115777. The latter observation was consistent with resistance to FTIs being conferred by K-RasB protein geranylgeranylation via PGGT I when the farnesyl protein transferase pathway is inhibited (16, 17, 18) . At the biochemical level, the involvement of K-RasB with the cellular effects of R115777 could not be substantiated. CAPAN-2 human pancreatic tumor cells responded to R115777 in vitro and in vivo. However, unprocessed K-RasB could not be detected at concentrations of R115777, which inhibited the prenylation of lamin B and N-Ras. The data suggested that the mutant K-RasB protein underwent alternative prenylation, but the postulated resistance to the FTI R115777 was absent. The findings support a role for other farnesylated targets mediating the response to the FTI R115777, as has been suggested previously (16, 17, 18 , 37) . Similar results were recently reported for viral-K-ras transgenic mice wherein the FTI L-744,832 produced antitumor effects in the K-RasB-driven tumors without effects on K-RasB prenylation (38) . The activity of R115777 in Raf-transformed NIH 3T3 fibroblasts lends additional support to this concept. Despite the conflicting data, gain of geranylgeranylated RhoB remains an attractive hypothesis to explain discordance between Ras processing and the effects of FTIs (21 , 22 , 33) . Although the Ras proteins may not be the primary farnesylated protein required for the activity of FTIs, expression of the different mutant Ras isoforms, in particular K-RasB, does appear to influence the relative sensitivity of tumor cells to this class of compound. Whether this reflects an interaction of farnesylated K-RasB signaling pathway with geranylgeranyl RhoB or the function of centromere-associated proteins is an interesting area for additional research. A point of convergence could be the high-affinity binding sites for prenylated K-RasB found in microtubules (39) .
In studies of Ras-processing in the T24 H-ras-transfected NIH3T3 cells, the levels of Ras immunoreactivity consistently decreased. In contrast, the levels of other endogenous farnesylated proteins were noted to increase with the increase derived from the accumulation of unfarnesylated protein. This was also apparent in the immunostaining for K-Ras, which remained as a single membrane-ssociated band in CAPAN-2 cells treated with R115777. Although it has not been systematically explored, the findings suggest the possibility of a feedback to protein expression involving protein trafficking or prenylation. Consistent with the present studies, such a mechanism would not be expected to regulate expression from transfected ras genes, which carry engineered promoters.
In four tumor models, R115777 demonstrated significant antitumor effects when administered b.i.d. by the oral route. Although the sensitivity of the cell lines to the antiproliferative effects of R115777 in vivo mirrored the relative sensitivity of tumors in vitro, histological studies revealed that the responses elicited by R115777 in xenografts involved far more than antiproliferative effects. Computer-assisted quantitative image analysis revealed that R115777 treatment produced predominantly an antiproliferative effect in CAPAN-2 pancreatic tumors, which was accompanied by an induction of apoptosis relegated to the host endothelial cells of the tumor vasculature. A prominent antiangiogenic effect was observed in LoVo colon tumors, whereas a marked induction of apoptosis was noted in C32 melanoma tumors. The latter effect could not be observed in C32 melanoma cells cultured as monolayers in vitro (data not shown). The effects of R115777 in C32 melanoma are reminiscent of the report that FTI L-739,749 could produce apoptotic effects under conditions of anchorage-independent growth but not in monolayer cultures (40) . Either blockade of Ras signaling via the PI3 kinase and Akt pathway or gain of geranygeranylated RhoB could account for the induction of apoptosis in vivo (23 , 41) . The emergence of additional antitumor activities in test systems more complex than monolayer culture is consistent with FTIs acting to revert to the transformed phenotype, because transformation encompasses survival and proliferation in tumor xenografts.
In conclusion, R115777 is a p.o.-active FTI that demonstrated antitumor effects at nontoxic doses in mice. The variety of histological responses produced by treatment with R115777 suggested that modification of aspects of the malignant phenotype concerned with host-tumor interactions might be an important component of FTI effects.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Department of Oncology, Janssen Research Foundation,
Spring House, PA 19477. Phone: (215) 628-5974; Fax: (215) 628-5047;
E-mail: dend{at}prius.jnj.com 
2 The abbreviations used are: FPT, farnesyl
protein transferase; FTI, farnesyl protein transferase inhibitor; GGPT
I, geranygeranyl protein transferase type I; R115777,
(B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3chlorophenyl)-1-methyl-2(1H)-quinolinone;
ISEL, in situ end labeling; VEGF, vascular endothelial
cell growth factor; b.i.d., twice daily.
3 D. W. End, M. G. Venet, P. R. Angiband, and G.
C. Sanz, the synthesis will be published elsewhere and can be found in
patent WO 9716443 A1.
4 The complete set of data can be obtained from
the National Auxiliary Publications Service, c/o Microfiche
Publications, P.O. Box 3513, Grand Central Station, New York, NY
10163-3513. Phone: (516) 481-2300; Fax:(516) 481-6213.
Received 5/23/00. Accepted 11/ 1/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit. Cell, 66: 429-434, 1991.
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J.-L. Harousseau, J. E. Lancet, J. Reiffers, B. Lowenberg, X. Thomas, F. Huguet, P. Fenaux, S. Zhang, W. Rackoff, P. De Porre, et al. A phase 2 study of the oral farnesyltransferase inhibitor tipifarnib in patients with refractory or relapsed acute myeloid leukemia Blood, June 15, 2007; 109(12): 5151 - 5156. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Qiu, X. Liu, W. Zou, P. Yue, S. Lonial, F. R. Khuri, and S.-Y. Sun The Farnesyltransferase Inhibitor R115777 Up-regulates the Expression of Death Receptor 5 and Enhances TRAIL-Induced Apoptosis in Human Lung Cancer Cells Cancer Res., May 15, 2007; 67(10): 4973 - 4980. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Fenaux, A. Raza, G. J. Mufti, C. Aul, U. Germing, H. Kantarjian, L. Cripe, R. Kerstens, P. De Porre, and R. Kurzrock A multicenter phase 2 study of the farnesyltransferase inhibitor tipifarnib in intermediate- to high-risk myelodysplastic syndrome Blood, May 15, 2007; 109(10): 4158 - 4163. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Schafer-Hales, J. Iaconelli, J. P. Snyder, A. Prussia, J. H. Nettles, A. El-Naggar, F. R. Khuri, P. Giannakakou, and A. I. Marcus Farnesyl transferase inhibitors impair chromosomal maintenance in cell lines and human tumors by compromising CENP-E and CENP-F function Mol. Cancer Ther., April 1, 2007; 6(4): 1317 - 1328. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Raponi, J.-L. Harousseau, J. E. Lancet, B. Lowenberg, R. Stone, Y. Zhang, W. Rackoff, Y. Wang, and D. Atkins Identification of Molecular Predictors of Response in a Study of Tipifarnib Treatment in Relapsed and Refractory Acute Myelogenous Leukemia Clin. Cancer Res., April 1, 2007; 13(7): 2254 - 2260. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. M. Hayes, P. E. Carrigan, and L. J. Miller Serine-Arginine Protein Kinase 1 Overexpression Is Associated with Tumorigenic Imbalance in Mitogen-Activated Protein Kinase Pathways in Breast, Colonic, and Pancreatic Carcinomas Cancer Res., March 1, 2007; 67(5): 2072 - 2080. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. G. Papatsoris, M. V. Karamouzis, and A. G. Papavassiliou The power and promise of "rewiring" the mitogen-activated protein kinase network in prostate cancer therapeutics Mol. Cancer Ther., March 1, 2007; 6(3): 811 - 819. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-P. Armand, A. K. Burnett, J. Drach, J.-L. Harousseau, B. Lowenberg, and J. San Miguel The Emerging Role of Targeted Therapy for Hematologic Malignancies: Update on Bortezomib and Tipifarnib Oncologist, March 1, 2007; 12(3): 281 - 290. |
