Effect of 6-aminonicotinamide and other protein synthesis inhibitors on formation of platinum-DNA adducts and cisplatin sensitivity

Article abstract of DOI:10.1124/mol.57.3.529

The present study was undertaken to examine the mechanistic basis for the recent observation that the pyridine nucleotide derivative 6- aminonicotinamide (6AN, NSC 21206) enhances the accumulation and resulting cytotoxicity of cisplatin in a variety of tumor cell lines. When A549 lung cancer cells or K562 leukemia cells were treated with 62.5 μM 6AN for 21 h and then pulse-labeled with [³⁵S]methionine for 1 h, increased labeling of five polypeptides, one of which corresponded to a M(r) ~78,000 glucose- regulated protein (GRP78), was observed. Two subsequent observations, however, suggested that up-regulation of these polypeptides was unlikely to explain the interaction between 6AN and cisplatin: 1) the concentration of 6AN required to induce GRP78 was 4-fold higher than the dose required to sensitize cells to cisplatin; and 2) simultaneous treatment of cells with 6AN and cycloheximide prevented the increase in GRP78 but not the sensitizing effect of 6AN. On the contrary, treatment with the protein synthesis inhibitors cycloheximide, anisomycin, or puromycin as well as prolonged exposure to the RNA synthesis inhibitor actinomycin D mimicked the biochemical modulating effects of 6AN on cisplatin action. Conversely, 6AN inhibited protein synthesis, whereas 18 6AN analogs that failed to enhance Pt-DNA adducts and cisplatin cytotoxicity failed to inhibit protein synthesis. These observations are consistent with a model in which 6AN and other inhibitors of protein synthesis act as modulating agents by increasing cisplatin accumulation, thereby enhancing the formation of Pt-DNA adducts and subsequent cisplatin-induced cell death.

Full text of DOI:10.1124/mol.57.3.529

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MOLECULAR PHARMACOLOGY, 57:529–538 (2000).
Effect of 6-Aminonicotinamide and Other Protein Synthesis
Inhibitors on Formation of Platinum-DNA Adducts and
Cisplatin Sensitivity
1
2
I. IMAWATI BUDIHARDJO, SCOTT A. BOERNER, STEVEN ECKDAHL, PHYLLIS A. SVINGEN, ROBERT RIOS,
MATTHEW M. AMES, and SCOTT H. KAUFMANN
Division of Oncology Research (I.I.B., S.A.B., P.A.S., R.R., M.M.A., S.H.K.) and Department of Laboratory Medicine (S.E.), Mayo Clinic and
Department of Molecular Pharmacology and Experimental Therapeutics (S.H.K., M.M.A.), Mayo Medical School, Rochester, Minnesota
Received August 13, 1999; accepted December 1, 1999
This paper is available online at http://www.molpharm.org
ABSTRACT
The present study was undertaken to examine the mechanistic cells with 6AN and cycloheximide prevented the increase in
basis for the recent observation that the pyridine nucleotide GRP78 but not the sensitizing effect of 6AN. On the contrary,
derivative 6-aminonicotinamide (6AN, NSC 21206) enhances treatment with the protein synthesis inhibitors cycloheximide,
the accumulation and resulting cytotoxicity of cisplatin in a anisomycin, or puromycin as well as prolonged exposure to the
variety of tumor cell lines. When A549 lung cancer cells or K562 RNA synthesis inhibitor actinomycin D mimicked the biochem-
leukemia cells were treated with 62.5 ␮M 6AN for 21 h and then ical modulating effects of 6AN on cisplatin action. Conversely,
35
pulse-labeled with [ S]methionine for 1 h, increased labeling of 6AN inhibited protein synthesis, whereas 18 6AN analogs that
five polypeptides, one of which corresponded to a M ϳ78,000 failed to enhance Pt-DNA adducts and cisplatin cytotoxicity
r
glucose-regulated protein (GRP78), was observed. Two subse- failed to inhibit protein synthesis. These observations are con-
quent observations, however, suggested that up-regulation of sistent with a model in which 6AN and other inhibitors of protein
these polypeptides was unlikely to explain the interaction be- synthesis act as modulating agents by increasing cisplatin ac-
tween 6AN and cisplatin: 1) the concentration of 6AN required cumulation, thereby enhancing the formation of Pt-DNA ad-
to induce GRP78 was 4-fold higher than the dose required to ducts and subsequent cisplatin-induced cell death.
sensitize cells to cisplatin; and 2) simultaneous treatment of
The nicotinamide analog 6-aminonicotinamide (6AN) is 6AN was observed to enhance the cytotoxicity of cisplatin in
undergoing preclinical evaluation as a potential agent for V79 Chinese hamster cells (Chatterjee et al., 1997) and a
modulating the cytotoxicity of antineoplastic drugs. Studies variety of human tumor cell lines in vitro (Budihardjo et al.,
performed by Berger and coworkers (1983, 1989) revealed
that 6AN sensitized L1210 murine leukemia cells to bis(2-
chloroethyl)nitrosourea in vitro and in a murine model. In
subsequent studies, a mixture containing the modulators
N-(phosphonacetyl)-L-aspartate, 6-methylmercaptopurine ri-
boside, and 6AN has been reported to enhance the efficacy of
ionizing radiation, 5-fluorouracil, doxorubicin, and paclitaxel
against spontaneous autochthonous breast cancers in mice
1
6
998). Collectively, these studies raised the possibility that
AN might enhance the effectiveness of various anticancer
agents in vitro and in vivo.
Despite these promising results, studies performed to eval-
uate the feasibility of using 6AN to modulate the cytotoxicity
of cisplatin in vivo have been disappointing (Walker et al.,
1
999). Optimal sensitization of A549 or K562 cells to cispla-
tin in vitro required pretreatment with 15 to 30 ␮M 6AN for
at least 10 h. Pharmacokinetic studies, on the other hand,
revealed that 6AN had a terminal serum half-life of only 20
to 30 min in mice (Walker et al., 1999). After bolus adminis-
tration of 10 mg/kg 6AN (one-fourth the single-dose LD50),
(
reviewed in Martin and Schwartz, 1997). More recently,
This work was supported in part by funds from the National Institutes of
Health (R01-CA67818, N01-CM57200) and the Jack Taylor Family Founda-
tion1.
Present address: Department of Biochemistry, University of Texas South- serum 6AN levels peaked at ϳ90 ␮M but rapidly declined,
western Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235.
2
preventing prolonged exposure to concentrations that sensi-
tized cells to cisplatin in vitro. Conversely, a continuous
Present address: Department of Chemistry, University of Puerto Rico,
Mayaguez, PR 00681-5000.
ϩ
ABBREVIATIONS: 6AN, 6-aminonicotinamide; 6ANAD , the 6AN-containing analog of nicotinamide adenine dinucleotide; DRB, 5,6-dichloro-
␤-D-ribofuranosylbenzimidazole; GRP78, the M ϳ78,000 glucose-regulated protein; MeAIB, methylaminoisobutyrate; NEPHGE, nonequilibrium
1
r
pH gradient electrophoresis; PBS, calcium/magnesium-free phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; TCA, trichlo-
roacetic acid.
529

5
30
Budihardjo et al.
infusion strategy was capable of maintaining 6AN plasma (St. Louis, MO); HindIII from Life Technologies/BRL (Gaithersburg,
levels at 15 ␮M for 24 to 48 h but was uniformly lethal MD); proteinase K from Boehringer-Mannheim (Indianapolis, IN);
bicinchoninic acid from Pierce (Rockford, IL); enhanced chemilumi-
(
Walker et al., 1999), most likely as a consequence of the
nescence reagents (ECL) or Amplify (Amersham, Arlington Heights,
IL); pH 3.5 to 10 ampholytes from Pharmacia/LKB (Piscataway, NJ);
mouse monoclonal anti-GRP78 antibody from StressGen (Victoria,
British Columbia); and platinum standard from J.T. Baker (Phillips-
burg, NJ). RNase A (RAF grade; Worthington, Freehold, NJ) was
well-documented neurotoxicity of 6AN (Sternberg and Phil-
ips, 1958; Herken et al., 1969). These observations indicate
the need to identify a less toxic analog of 6AN, a process that
would be facilitated by a better understanding of the mech-
anism by which 6AN modulates the action of other agents.
Our previous study (Budihardjo et al., 1998) demonstrated
prepared as a 10 mg/ml solution in H O and boiled before storage at
2
Ϫ20°C. All chemicals for synthesis were of the highest available
that 6AN enhanced cisplatin-induced apoptosis but did not purity.
affect the cytotoxicity of etoposide, topotecan, 4-hydroperoxy-
6AN analogs C, D, F through J, L through P, and R were provided
cyclophosphamide, or chlorambucil, raising the possibility by the Pharmaceutical Resources Branch of the National Cancer
Institute (Rockville, MD). Compound E was synthesized by reacting
that 6AN might preferentially affect a drug-specific step in
1
.82 mmol of 6AN with 18.2 mmol of acetic anhydride in 5 ml of
the cell death pathway. Consistent with this hypothesis, 6AN
was observed to enhance cisplatin accumulation and the
subsequent formation of Pt-DNA adducts (Budihardjo et al.,
acetic acid on a steam bath for 30 min under N2 atmosphere. The
resulting precipitate was filtered, washed with acetic acid, and re-
crystallized from ethanol. Compound K was synthesized by reacting
.65 mmol of 6AN in acetic acid with 1 equivalent of hydrogen
peroxide (added as a 30% solution) at 75°C for 3 h. After the solvent
was evaporated under reduced pressure, the yellow residue was
1
998). Although the mechanisms responsible for cisplatin
3
accumulation are not completely understood, a current model
suggests that cisplatin uptake involves both passive diffusion
and carrier-mediated transport (Gately and Howell, 1993). recrystallized from ethanol. Compound Q was synthesized by react-
Accordingly, the observation that 6AN enhances intracellu- ing 1.46 mmol of 6AN with 1.54 mmol of benzoic anhydride in 0.2 ml
lar cisplatin levels raises the possibility that 6AN alters the of triethylamine on a steam bath overnight under N atmosphere.
2
expression or function of polypeptide(s) involved in cisplatin The resulting solid was filtered, rinsed with acetone, and recrystal-
lized from ethanol. 6AN and analogs B, C, D, F, H, J, L, M, O, and R
transport across the plasma membrane.
were prepared as 2.5 mM stocks in medium A. The remaining ana-
A different view of 6AN action has emerged from other
logs were prepared as 100 mM stocks in dimethyl sulfoxide with (E,
K, Q) or without (G, I, N, P) 1% acetic acid.
Cell Culture. A549 nonsmall cell lung cancer cells and K562
chronic myelogenous leukemia cells from American Type Culture
studies. In particular, the observation that 6AN enhances
expression of the glucose-regulated protein GRP78 (Chatter-
jee et al., 1995, 1997) has raised the possibility that GRP78
might somehow regulate cisplatin action. GRP78, however, is
a stress-inducible chaperone protein that resides in the en-
Collection (Manassas, VA) were cultured at 37°C in a humidified
atmosphere of 95% air, 5% CO in RPMI 1640 medium containing 5%
2
doplasmic reticulum, where it is hypothesized to function in heat-inactivated fetal bovine serum, 100 U/ml penicillin G, 100
the translocation of proteins from the cytosol and in the ␮g/ml streptomycin, and 2 mM L-glutamine (medium A). To ensure
correct assembly of proteins during early protein processing logarithmic growth, cultures were maintained at densities below 1 ϫ
6
1
0 cells/ml (K562) or 70 to 80% confluence (A549). Cells were fed on
(
1
Lee, 1992; Little et al., 1994; reviewed in Brostrom et al.,
995). A mechanistic link between 6AN-induced GRP78 ex-
the day before the start of each experiment.
5
Colony Forming Assays. Aliquots containing 3 to 5 ϫ 10 log
phase K562 cells in 1 ml of medium A were incubated with diluent or
a concentration (250 ␮M) of the various 6AN analogs for 18 h. A 1-␮l
aliquot of dimethyl sulfoxide containing the indicated final concen-
tration of cisplatin was added for 1 h. Cells were then sedimented at
pression and altered cisplatin action has not been estab-
lished.
In the present study, the cellular effects of 6AN were
35
examined in greater detail. Labeling with [ S]methionine,
followed by two-dimensional nonequilibrium pH gradient
2
00g for 10 min, washed, diluted 1:500, and plated in 0.3% agar as
electrophoresis (NEPHGE)/SDS-polyacrylamide gel electro- described (Budihardjo et al., 1998; Walker et al., 1999). After a 10- to
phoresis (PAGE), was used to more completely define the 14-day incubation at 37°C, colonies containing Ն50 cells were
effects of 6AN on protein expression. Protein synthesis inhib- counted using an inverted phase-contrast microscope. Survival was
expressed relative to control cells incubated with the corresponding
itors were employed to examine the role of 6AN-induced
concentration of 6AN analog in the absence of cisplatin treatment.
Control plates typically contained 200 to 400 colonies.
Aliquots containing 300 to 500 A549 cells were plated in 35-mm
polypeptides on cisplatin accumulation and cytotoxicity. As-
says of methionine uptake and incorporation were performed
to assess the effect of 6AN on amino acid transport and
tissue culture plates containing 2 ml of medium A and allowed to
protein synthesis. Finally, 18 structural analogs of 6AN were
adhere for 12 to 14 h. After cells were treated with the indicated
tested for their ability to inhibit protein synthesis, increase
Pt-DNA adducts, and enhance cisplatin cytotoxicity. Results
concentration of 6AN or cycloheximide for 6 h, cisplatin was added to
the indicated final concentration. Following a 2-h incubation, cells
of these studies are consistent with a model in which 6AN- were washed twice with serum-free RPMI 1640 and incubated in
associated inhibition of protein synthesis prompts a compen- drug-free medium A for 7 days. The resulting colonies were stained
satory increase in amino acid and cisplatin accumulation, with Coomassie Brilliant Blue and counted manually. Control plates
generally contained 100 to 200 colonies.
Measurement of Whole Cell Cisplatin Accumulation and
Pt-DNA Adducts. For assessment of cellular cisplatin accumula-
with subsequent enhancement of Pt-DNA adducts and cis-
platin-induced cytotoxicity.
tion, duplicate 100-mm plates of A549 cells grown to 70 to 80%
confluence were incubated in medium A in the absence or presence of
Experimental Procedures
6AN, cycloheximide, puromycin, anisomycin, DRB, or actinomycin D
Materials. Reagents were purchased from the following suppli- for 6 to 24 h as indicated. Freshly prepared cisplatin was then added
ers: cisplatin, L-alanine, L-serine, 6AN, 6-aminonicotinic acid, nico- to a final concentration of 40 ␮M from a 1000ϫ concentrated stock.
tinamide, methylaminoisobutyrate (MeAIB), cycloheximide, aniso- After a 2-h incubation at 37°C, cells were washed once with ice-cold
mycin, puromycin, DRB, actinomycin D, and digitonin from Sigma PBS, briefly trypsinized, sedimented at 200g for 10 min, and washed

Protein Synthesis Inhibitors and Cisplatin Sensitivity
531
3
three times with ice-cold PBS. At the last wash step, cells were
6AN for 18 to 24 h. Cells were then treated with 1 ␮Ci/ml [ H]me-
resuspended in a total volume of 1 ml. A 100-␮l aliquot of cells was thionine in medium A for 5 min, washed five times with ice-cold PBS,
removed, solubilized in 100 ␮l of 1 M NaOH at 21°C overnight, and lysed in 0.5 ml of 0.5% SDS. Aliquots were subjected to scintil-
neutralized with 100 ␮l of 1 M HCl, and assayed for protein by the lation counting and protein measurement (Smith et al., 1985). To
bicinchoninic acid method (Smith et al., 1985). The remainder of the correct for nonspecific trapping of label, replicate aliquots were
3
cells were sedimented, reacted overnight at 21°C with 0.5 ml of treated with [ H]methionine for Ͻ10 s and handled in an identical
concentrated nitric acid, heated to 100°C for 5 min, diluted with 0.5
ml of 30% (w/w) H O , and heated again to 100°C for 5 min. Elemen-
fashion.
To measure methionine incorporation into macromolecules, A549
2
2
tal platinum in the lysates was assayed by the Metals Laboratory of cells were treated with 6AN, cycloheximide, puromycin, anisomycin,
the Mayo Clinic using inductively coupled plasma mass spectroscopy or actinomycin D for the indicated length of time. Radiolabeled
as recently described (Walker et al., 1999).
methionine was added at a final concentration of 1 ␮Ci/ml. After a
To measure Pt-DNA adducts, plates of 50 to 70% confluent A549 60-min incubation, the medium was removed and cells were washed
cells were incubated with 6AN, cycloheximide, or actinomycin D as once with ice-cold PBS. All further steps were performed at 4°C.
indicated. Cisplatin was then added to a final concentration of 40
␮M. Two h after addition of cisplatin, cells were washed with ice-cold
PBS, briefly trypsinized, sedimented at 200g for 10 min, washed
three times with ice-cold PBS, and lysed in 5 ml of TEN buffer [10
mM Tris-HCl (pH 7.4 at 21°C), 10 mM EDTA, 150 mM NaCl] sup-
plemented with 0.4% SDS and 1 mg/ml proteinase K. The lysates
were incubated at 50°C for 16 h. Highly purified DNA samples were
then prepared by phenol/CHCl3 and CHCl3 extraction, ethanol pre-
cipitation, RNase A treatment, phenol/CHCl and CHCl extraction,
3
3
and HindIII digestion as previously described in detail (Budihardjo
et al., 1998). After aliquots (2 ␮g of DNA) were subjected to electro-
phoresis on agarose minigels to confirm complete removal of RNA
and digestion of DNA, DNA was re-extracted with phenol/CHCl and
3
CHCl , ethanol-precipitated, resuspended in 750 ␮l of 0.6 M HCl,
3
and heated to 95°C for 30 min. The DNA concentration was esti-
mated by measuring absorbance at 260 nm, and elemental platinum
was assayed as described above. Similar techniques were used to
measure Pt-DNA adducts in K562 cells treated with 6AN or com-
pounds B through R.
Methionine Labeling, Protein Electrophoresis, and Immu-
noblotting. A549 cells were incubated in the absence or presence of
6
2.5 ␮M 6AN in medium A for 18 to 24 h. After culture medium was
removed, cells were briefly trypsinized and washed twice with RPMI
640 medium lacking methionine. Cells were resuspended in 1 ml of
methionine-free RPMI 1640 medium supplemented with 5% dialyzed
1
35
fetal bovine serum and 50 ␮Ci/ml [ S]methionine in the continued
absence or presence of 62.5 ␮M 6AN and incubated for 1 h at 37°C.
After labeling, the cells were sedimented at 800g for 10 min at 4°C;
washed twice with ice-cold PBS containing 5 mM unlabeled methi-
onine; sonicated in SDS sample buffer consisting of 4 M urea, 2%
(
w/v) SDS, 62.5 mM Tris-HCl (pH 6.8), and 5% (v/v) ␤-mercaptoetha-
nol; and heated to 70°C for 30 min. Aliquots containing equal
amounts of trichloroacetic acid-perceptible radiolabel (500,000 cpm)
were mixed with four volumes of IEF buffer [9.5 M urea, 8% (w/v)
Nonidet P-40, 2% (w/v) pH 3.5 to 10 ampholytes, and 5% (w/v)
␤
-mercaptoethanol] and subjected to NEPHGE in the first dimension
followed by SDS-PAGE in the second dimension as previously de-
scribed (O’Farrell et al., 1977; Kaufmann and Shaper, 1984). After
the second dimension, gels were impregnated with Amplify and
exposed to Kodak X-Omat AR film for 4 days at Ϫ70°C in the
presence of intensifying screens as specified by the supplier.
To perform immunoblotting, subconfluent A549 cells were treated
with increasing 6AN concentrations for 18 h at 37°C, washed twice
with ice-cold PBS, released by brief trypsinization, diluted with
medium A, sedimented at 200g, washed twice with ice-cold PBS,
lysed in 6 M guanidine hydrochloride under reducing conditions, and
prepared for SDS-PAGE as previously described (Kaufmann, 1989).
Aliquots containing 50 ␮g of protein, as determined by the bicincho-
ninic acid assay (Smith et al., 1985), were heated to 65°C for 20 min,
subjected to SDS-PAGE on gels with 5 to 15% (w/v) acrylamide
gradients, and transferred to nitrocellulose. Blots were probed using
murine monoclonal anti-GRP78 or chicken anti-lamin B1 (Kauf-
mann, 1989) as primary antibodies.
Methionine Uptake and Incorporation. To assess methionine
uptake, A549 cells in 24-well plates were treated with 0 to 1000 ␮M
Fig. 1. Structures of 6AN analogs used in this study. 6AN is depicted as
compound A.

5
32
Budihardjo et al.
Cells were scraped into 10% (w/v) trichloroacetic acid (TCA). After
the lysates were sedimented at 12,000g for 5 min, the pellets were
washed once with 10% TCA and three times with acetone. The
pellets were then solubilized in 0.5 M NaOH, heated to 70°C for 30
min, and neutralized with 0.5 M HCl. After an aliquot was removed
for determination of protein (Smith et al., 1985), the remainder was
subjected to scintillation counting. Similar techniques were applied
to K562 cells except that analogs B through R were substituted for
6AN and radiolabeled cells were collected by sedimentation at 3200g
for 1 min before lysis in 10% TCA. Similar results were obtained
35
3
using either S-labeled or methyl- H-labeled methionine.
Results
Evaluation of the Effects of 6AN Analogs. Previous
experiments demonstrated that the niacin derivative 6AN
enhances cisplatin-induced Pt-DNA adducts and cytotoxicity
in a variety of human tumor cell lines (Budihardjo et al.,
1998). To search for structural analogs that might have sim-
ilar effects, 18 6AN derivatives (Fig. 1) were evaluated for
their ability to increase Pt-DNA adducts and cisplatin toxic-
ity in K562 cells. Results of these experiments are summa-
rized in Fig. 2.
In the absence of cisplatin, 6AN was cytostatic but not
cytotoxic during the 24-h exposure period. In contrast, the
analogs were neither cytostatic nor cytotoxic by themselves.
When cisplatin was added, 6AN was found to be relatively
unique among these agents in its ability to enhance the
cytotoxicity of cisplatin (Fig. 2, A and B). In particular, mod-
ification of the side chain amide (compound A) to a carboxylic
acid (compound B), ethyl ester (compound C), ketone (com-
pound P), or methyl group (compound D) completely abol-
ished the activity of 6AN. Likewise, oxidation of the pyridine
nitrogen (compound K) abolished activity. Finally, most mod-
ifications of the amino group at the 6-position, including
methylation (compound O) and amidation with acetic or ben-
zoic acid (compounds E and Q, respectively), abolished activ-
ity. The inability of these analogs to enhance the cytotoxicity
of cisplatin (Fig. 2, A and B) paralleled their inability to
enhance formation of Pt-DNA adducts (Fig. 2C). Further
studies were undertaken in an attempt to better understand
the relatively unique effects of 6AN on cisplatin action.
6AN Selectively Increases the Expression of Five
Polypeptides. A549 cells were used for most of the subse-
quent studies because the effects of 6AN on cisplatin sensi-
tivity were somewhat greater in this cell line (Budihardjo et
al., 1998). All major findings presented below, however, were
also replicated in K562 cells to confirm that the results were
not unique to one line.
The previous observation that the effect of 6AN on forma-
tion of Pt-DNA adducts requires exposure of cells for 6 to 10 h
Fig. 2. Effects of 6AN and its analogs. A, K562 cells were incubated for
1 h with 250 ␮M 6AN or 250 ␮M analogs D, F, H, L, M, and R. Cisplatin
was then added at the indicated final concentration. After a 1-h incuba-
2
(
Walker et al., 1999) raised the possibility that the effect of
tion with cisplatin, cells were washed and plated in drug-free agar to 6AN might reflect the synthesis of one or more polypeptides.
allow colonies to form. Data are expressed as the ratio of the number of
To examine this possibility, A549 cells were incubated in the
colonies in samples treated with the 6AN analog ϩ cisplatin to the
absence or presence of 62.5 ␮M 6AN for 21 h, then pulse-
number of colonies formed in samples treated with the 6AN analog alone.
35
B, summary of the effects of all 18 analogs on cisplatin sensitivity in labeled with [ S]methionine for 1 h. Compared with cells
clonogenic assays performed as indicated in A. Results are expressed as
treated with diluent alone, 6AN-treated cells displayed in-
the ratio of the LD90 in absence of the analog to the LD90 in the presence
of the analog. Results are the means of two to six experiments with
analogs B through R. C, summary of the effects of all 18 analogs on
35
creased incorporation of [ S]methionine into a small subset
of polypeptides (cf. Fig. 3, A and B), including species at Mr
formation of Pt-DNA adducts. Data are expressed as the ratio of Pt-DNA ϳ45,000 (pI ϳ9.1), M ϳ66,000 (pI ϳ8.9), M ϳ78,000 (pI
r
r
adducts in the presence of the analog to Pt-DNA adducts in the absence
of any analog. Results are the means of 17 experiments with 6AN (com-
pound A, included as a control in each experiment) and two to four
experiments with analogs B through R.
ϳ5.3), and M ϳ90,000 (pI ϳ7.1). The mobility of the Mr
r
ϳ78,000 (pI ϳ5.3) polypeptide was consistent with the re-
ported mobility of GRP78 (Patierno et al., 1987), a polypep-

Protein Synthesis Inhibitors and Cisplatin Sensitivity
533
tide previously shown to be up-regulated in 6AN-treated sure. Treatment with 250 ␮M 6AN for 6 h resulted in a 4.2-
hamster cells (Chatterjee et al., 1995, 1997). Immunoblotting Ϯ 1.5-fold (n ϭ 3) increase in Pt-DNA adducts (Fig. 5A).
with an anti-GRP78 antibody confirmed that GRP78 Addition of cycloheximide 30 min before 6AN did not prevent
polypeptide content increased 3- to 4-fold in 6AN-treated this increase. On the contrary, treatment with cycloheximide
A549 cells (Fig. 4B, inset lanes 5 to 7). The M ϳ45,000 (pI alone for 6.5 h enhanced the formation of Pt-DNA adducts
r
ϳ9.1) polypeptide comigrated with a major Coomassie Bril- 2.9- Ϯ 0.9-fold (n ϭ 3, Fig. 5A). Consistent with these results,
liant Blue-stained polypeptide that was identified as the pretreatment with cycloheximide also failed to diminish the
translation elongation factor EF-1␣ by microsequencing. The effects of 6AN on cisplatin sensitivity in colony forming as-
identity of the other three 6AN-induced polypeptides is cur- says (Fig. 5B). A 6-h pretreatment with 6AN decreased the
rently unknown.
cisplatin LD90 by 3.5- Ϯ 1.5-fold (n ϭ 6). This effect was not
To examine the potential relationship between increased prevented by adding cycloheximide 30 min before 6AN. In-
GRP78 and enhanced cisplatin sensitivity, the effects of var- stead, treatment with cycloheximide alone resulted in a 2.3-
ious 6AN concentrations on these processes were compared. Ϯ 0.2-fold decrease in the LD90, and the combination of
Interestingly, the 7.8 to 31.2 ␮M concentrations of 6AN that cycloheximide plus 6AN resulted in a 5.2- Ϯ 0.4-fold decrease
reproducibly sensitized A549 cells to cisplatin (Fig. 4A) in the LD90 (n ϭ 3). In other words, cycloheximide mimicked
caused only small changes in GRP78 levels (Fig. 4B, inset the effects of 6AN when applied alone and augmented the
lanes 3 and 4) relative to those seen at higher 6AN concen- effects of 6AN when combined with this agent. Interestingly,
trations, raising the possibility that up-regulation of GRP78 all the changes observed after the 6-h incubation occurred
and enhancement of cisplatin sensitivity might be two inde- without any change in GRP78 (Fig. 5B, inset), providing
pendent 6AN-induced processes.
Protein Synthesis Inhibitors Mimic the Effects of effects of 6AN on cisplatin action can be dissociated.
AN on Formation of Pt-DNA Adducts and Cisplatin These effects were not limited to cycloheximide. Treatment
Sensitivity. To further examine the potential role of the of cells for 24 h with actinomycin D, a classical inhibitor of
AN-induced polypeptides in sensitizing cells to cisplatin, RNA synthesis, also enhanced the formation of Pt-DNA ad-
additional support for the view that induction of GRP78 and
6
6
A549 cells were treated with 6AN or diluent in the presence ducts (Fig. 5C). This effect occurred at the same actinomycin
of 107 ␮M cycloheximide, a concentration that inhibited pro- D concentrations that caused inhibition of protein synthesis
tein synthesis in A549 cells by Ͼ90%, before cisplatin expo- as assessed by a 1-h methionine pulse at the end of the
Fig. 3. 6AN treatment is accompanied by enhanced expression of multiple cellular polypeptides. Two-dimensional gels of A549 cells were treated with
35
buffer (A) or 62.5 ␮M 6AN (B) for 21 h then pulsed with [ S]methionine for 1 h before lysis. NEPHGE (right to left) was followed by SDS-PAGE (top
3
to bottom). Numbers at left are molecular weights of standard proteins (run on the edge of the gel) ϫ 10 . Numbers at top are pH values of
corresponding sections of blank NEPHGE gel run in parallel. Circled areas are species that increase in 6AN-treated cells. Results are representative
of three experiments.

5
34
Budihardjo et al.
pretreatment (Fig. 5C). In additional experiments, a 24-h to 80% (Fig. 7B), providing further support for the relation-
DRB exposure likewise inhibited protein synthesis and in- ship between inhibition of protein synthesis and enhance-
creased formation of Pt-DNA adducts.
ment of cisplatin action.
Protein Synthesis Inhibitors Increase Cisplatin Ac-
In a final series of experiments, cells were incubated with
cumulation. Previous observations suggested that the effect 6AN in the presence of a 10-fold excess of nicotinamide.
of 6AN on Pt-DNA adducts is mediated by a 6AN-induced Previous observations indicated that excess nicotinamide in-
ϩ
increase in cisplatin accumulation that is partially inhibited hibits conversion of 6AN to its active metabolite 6ANAD
by the nonmetabolizable amino acid MeAIB (Budihardjo et and abolishes the effect of 6AN on cisplatin sensitivity (Budi-
al., 1998). To determine whether protein synthesis inhibitors hardjo et al., 1998). As shown in Fig. 7C, excess nicotinamide
might be modulating the action of cisplatin by a similar abrogated the effects of 6AN on both protein synthesis (Fig.
mechanism, A549 cells were incubated for 24 h with 107 ␮M 7C, left) and formation of Pt-DNA adducts (Fig. 7C, right).
cycloheximide, 100 ␮M puromycin, or 38 ␮M anisomycin. These results not only suggest that 6AN must be metabolized
ϩ
Even though each of these agents inhibited protein synthesis to 6ANAD to inhibit protein synthesis but also provide
by Ͼ90%, cell viability as determined by trypan blue exclu- additional support for the relationship between inhibition of
sion remained Ն98% after 24 h. When these cells were as- protein synthesis and enhanced cisplatin action.
sayed for cisplatin accumulation, treatment with each of the
protein synthesis inhibitors was accompanied by increased
accumulation of cisplatin (Fig. 6A). In other experiments, a
2
0- to 24-h exposure to actinomycin D or DRB had the same
effect. Addition of 20 mM MeAIB at the same time as cispla-
tin partially reversed the effect of the protein synthesis in-
hibitors just as it partially reversed the effect of 6AN (Fig. 6B
and data not shown). In contrast, serine had no effect on
cisplatin accumulation (data not shown). Taken together,
these data indicate that 6AN and protein synthesis inhibitors
increase cisplatin accumulation, possibly through a mecha-
nism that involves a neutral amino acid transporter.
6
AN Inhibits Protein Synthesis at the Concentra-
tions That Increase Cellular Cisplatin Accumulation.
Because cycloheximide, puromycin, and anisomycin pro-
duced effects similar to those of 6AN, we next investigated
the possibility that 6AN might be capable of inhibiting pro-
tein synthesis. When A549 cells were incubated with increas-
ing concentrations of 6AN for 18 h, a dose-dependent de-
crease in incorporation of radiolabeled methionine into TCA-
precipitable macromolecules was observed (Fig. 7A).
Decreased methionine incorporation was evident at 8 ␮M
6
␮
AN and reached a maximum of 70 to 80% inhibition at 62.5
M. To rule out the possibility that these results reflect
decreased methionine uptake rather than diminished protein
synthesis, uptake of radiolabeled methionine into cells was
measured at time points ranging from 30 s to 5 min. In 10
separate experiments, 6AN enhanced methionine uptake by
a factor of 1.8- Ϯ 0.5-fold (e.g., inset, Fig. 7A). These results
argue against the possibility that 6AN is inhibiting methio-
nine uptake and instead suggest that 6AN is inhibiting pro-
tein synthesis. Furthermore, the effects of 6AN on protein
synthesis (Fig. 7A), cellular cisplatin accumulation (Budi-
hardjo et al., 1998), and cisplatin cytotoxicity (Fig. 4) were
observed at similar concentrations (half-maximal effects at
1
6 ␮M 6AN in all cases), suggesting that these changes were
related.
To confirm that these results were not unique to the A549
cell line, K562 cells were treated with varying concentrations
of 6AN before measurement of methionine incorporation. Fig. 4. Effect of 6AN on GRP78 expression and cisplatin cytotoxicity in
A549 cells. A, A549 cells were treated with the indicated concentration of
Once again, the dose-response curves for the effect of 6AN on
protein synthesis and cisplatin LD90 were essentially super-
6
2
AN for 18 h. The indicated concentration of cisplatin was then added for
h. At the completion of the incubation, cells were washed and incubated
imposable (data not shown). Additional experiments revealed for 7 days to allow colonies to form. B, from the data in A, the cisplatin
that 6AN was unique among the 18 6AN analogs in its ability LD90 was calculated at each 6AN concentration. Inset, immunoblotting
for GRP78 and lamin B . A549 cells were incubated for 18 h with the
1
to inhibit protein synthesis. Although analog G inhibited
protein synthesis modestly and irreproducibly, 6AN was the
indicated concentration of 6AN, harvested for SDS-PAGE, and probed
with monoclonal anti-GRP78 (top). Lamin B (bottom) served as a loading
1
only niacin derivative that inhibited protein synthesis by 70 control. All results are representative of at least three experiments.

Protein Synthesis Inhibitors and Cisplatin Sensitivity
535
Fig. 6. Effect of protein synthesis inhibitors on cisplatin accumulation. A,
effect of a 24-h treatment with 250 ␮M 6AN, 107 ␮M cycloheximide, 100
␮
M puromycin, or 38 ␮M anisomycin on cellular accumulation of cisplatin
by A549 cells. Error bars, range of duplicate samples in this experiment.
In multiple experiments, cisplatin accumulation was enhanced 2.6- Ϯ
0
.7-fold (mean Ϯ S.D.) by cycloheximide, 3.6- Ϯ 0.6-fold by puromycin,
.8- Ϯ 0.8-fold by anisomycin, and 3.8- Ϯ 0.3-fold by actinomycin D. B,
2
effect of MeAIB on the 6AN- and cycloheximide-induced increase in
cisplatin accumulation. A549 cells were treated for 24 h with 250 ␮M 6AN
or 107 ␮M cycloheximide. MeAIB was then added to a concentration of 20
mM, followed 30 min later by addition of 40 ␮M cisplatin. After a 2-h
incubation with cisplatin, samples were harvested for determination of
cell-associated platinum. Error bars, range of duplicate samples in this
experiment. In three independent experiments, MeAIB diminished the
effect of 6AN by 48 Ϯ 8% and the effect of cycloheximide by 81 Ϯ 18%.
Fig. 5. Effect of cycloheximide on platinum-DNA adduct formation and
cisplatin cytotoxicity. A, A549 cells were incubated for 6 h with 250 ␮M
6
AN or 6.5 h with 107 ␮M cycloheximide. Cisplatin (40 ␮M) was then
were harvested for immunoblotting with monoclonal anti-GRP78 anti-
body. Both 6AN and cycloheximide enhance Pt-DNA adducts (A) and
cytotoxicity of cisplatin (B) without increasing GRP78 (inset) over the
time course of this exposure. C, effect of actinomycin D on cisplatin
accumulation and protein synthesis. A549 cells were incubated for 22 h
with the indicated concentration of actinomycin D. Cisplatin (40 ␮M) was
then added, and the incubation was continued for an additional 2 h before
cells were harvested for measurement of Pt-DNA adducts (●). Alterna-
tively, incorporation of radiolabeled methionine into TCA-precipitable
material was determined (⅜). Results in each panel present an experi-
ment that is representative of three to six separate experiments.
added, and the incubation was continued for an additional 2 h before cells
were harvested for measurement of Pt-DNA adducts. Error bars, range of
duplicate determinations in this experiment. The text summarizes re-
sults of multiple independent experiments. B, effect of 6AN and cyclo-
heximide on A549 colony formation. A549 cells were incubated for 6 h
with 250 ␮M 6AN or 6.5 h with 107 ␮M cycloheximide (CHX) before the
addition of the indicated concentration of cisplatin. After a 2-h incubation
with cisplatin, cells were washed and allowed to form colonies in drug-
free medium for 7 days. Inset, in a parallel experiment, A549 cells treated
with 250 ␮M 6AN or 107 ␮M cycloheximide for 6 or 6.5 h, respectively,

5
36
Budihardjo et al.
Discussion
Recent studies have demonstrated that 6AN sensitizes
cells to cisplatin in vitro (Chatterjee et al., 1997; Budihardjo
et al., 1998). Because 6AN demonstrated neurotoxicity in
animals in vivo (Sternberg and Philips, 1958; Walker et al.,
1999) as well as unfavorable pharmacokinetics (Walker et
al., 1999), studies were initiated to investigate the mecha-
nism by which 6AN modulates the action of cisplatin and to
search for analogs that might have similar effects but lack
the neurotoxicity of 6AN. These experiments have led to a
series of novel observations.
Previous studies have indicated that 6AN causes a variety
of biochemical changes, including inhibition of 6-phosphoglu-
conate dehydrogenase and depletion of products downstream
from this enzyme, notably 5Ј-phosphoribosyl-1Ј-pyrophos-
phate and NADPH, in intact cells (Herken et al., 1969; Keller
et al., 1976; Varnes, 1988; Street et al., 1996). Depletion of
5
Ј-phosphoribosyl-1Ј-pyrophosphate in turn leads to the de-
ϩ
creased synthesis of NAD observed in 6AN-treated cells
Hunting et al., 1985; Berger et al., 1987; Martin and
(
Schwartz, 1997), whereas depletion of NADPH contributes to
glutathione depletion (Varnes, 1988; Budihardjo et al., 1998).
In addition, 6AN has been shown to directly inhibit poly-
ϩ
(
ADP-ribose) polymerase (Sims et al., 1982), an NAD -con-
suming nuclear enzyme implicated in DNA repair (reviewed
in de Murcia et al., 1997). Our earlier study not only con-
firmed that 6AN had these effects but also ruled out these
biochemical changes as potential explanations for 6AN-in-
duced enhancement of cisplatin toxicity (Budihardjo et al.,
1998). In particular, 6AN enhanced cisplatin accumulation
and formation of Pt-DNA adducts, whereas other agents that
ϩ
depleted glutathione, diminished NAD levels, or inhibited
poly(ADP-ribose) polymerase did not.
Experiments performed in the present study examined the
possibility that effects on formation of Pt-DNA adducts might
be due to 6AN-induced changes in expression of one or more
polypeptides. After 6AN treatment for 16 to 24 h, increased
labeling of at least five polypeptides was observed (Fig. 3).
One of these comigrated with GRP78, a glucose-regulated
heat shock family member that is localized in the lumen of
the endoplasmic reticulum (Lee, 1992). While this work was
in progress, Berger and coworkers reported that two different
GRP78 inducers, 6AN and 2-deoxyglucose, each enhanced
cisplatin sensitivity (Chatterjee et al., 1997). These data
prompted the suggestion that GRP78 levels might affect cis-
platin-induced DNA crosslinks and cytotoxicity (Chatterjee
et al., 1997). Although it is conceivable that enhanced GRP78
expression and increased cisplatin sensitivity might be mech-
Fig. 7. Effect of 6AN and 6AN analogs on protein synthesis in A549 and anistically linked in some cell lines, several observations
K562 cells. A, effect of 6AN on methionine incorporation into TCA-
suggest that this is not the case in A549 cells. First, dose-
response curves indicate that cells are sensitized to cisplatin
precipitable material in A549 cells. Cells were incubated for 18 h with
increasing concentrations of 6AN, then pulsed with radiolabeled methi-
onine for 1 h before lysis in 10% TCA. Inset, effect of 6AN on radiolabeled at 6AN concentrations that have small effects on GRP78
methionine uptake in A549 cells. Cells were treated with the indicated
levels (Fig. 4). Second, treatment of cells with high 6AN
concentration of 6AN for 22 h, then exposed to radiolabeled methionine
concentrations (e.g., 250 ␮M) results in increased cisplatin
for 5 min. B, summary of the effects of 18 6AN analogs on methionine
accumulation and cytotoxicity beginning in ϳ6 h (Fig. 5) even
incorporation into TCA-precipitable material in K562 cells. Cells were
incubated with the indicated 6AN analog (see Fig. 1 for key) at 250 ␮M for though GRP78 is not elevated at this time (Fig. 5, inset).
1 h. Radiolabeled methionine was then added for 1 h. At the completion
2
of the incubation, TCA precipitates were collected and counted. Results of
a single experiment in which all 18 analogs were tested simultaneously
are presented. Similar results were obtained in two to three independent
experiments. C, effect of nicotinamide on 6AN-inhibited protein synthesis
and formation of Pt-DNA adducts. A549 cells were incubated for 21 h
with 250 ␮M 6AN in the absence or presence of 2.5 mM nicotinamide.
Aliquots were then pulsed for 1 h with radiolabeled methionine or treated
with 40 ␮M cisplatin for assessment of Pt-DNA adducts. In three sepa-
rate experiments, nicotinamide restored protein synthesis to 86 Ϯ 17% of
control levels and decreased Pt-DNA adducts from 560 Ϯ 180% of control
(6AN alone) to 90 Ϯ 10% of control (6AN ϩ nicotinamide).

Protein Synthesis Inhibitors and Cisplatin Sensitivity
537
Finally, cycloheximide prevents 6AN-induced increases in synthesis and increased formation of Pt-DNA adducts, pro-
expression of GRP78 (I.I.B. and S.H.K., unpublished obser- viding a means of reconciling the results of Chatterjee et al.
vations) but does not prevent the effect of 6AN on formation (1997) with the present model. The observation that other
of Pt-DNA complexes (Fig. 5A) or cytotoxicity (Fig. 5B).
protein synthesis inhibitors, notably sparsomycin, cinnama-
The observations with cycloheximide and other protein ldehyde, anguidine, and L-histidinol, also enhance the cyto-
synthesis inhibitors are difficult to reconcile with the hypoth- toxicity of cisplatin (Hromas and Yung, 1986; Dornish et al.,
esis that any of the 6AN-induced polypeptides (Fig. 3) play a 1989; Hofs et al., 1995; Warrington et al., 1996) is likewise
critical role in altering the accumulation and cytotoxicity of consistent with the model presented in Fig. 8, although the
cisplatin in A549 cells. Instead, the present results are con- effects of the latter agents on Pt-DNA adducts remain to be
sistent with a model (Fig. 8) in which 6AN treatment results evaluated.
in inhibition of protein synthesis; inhibition of protein syn-
Third, as indicated in the inset to Fig. 7A, we observed that
thesis causes enhanced uptake of a variety of compounds, 6AN treatment resulted in increased uptake of methionine
including cisplatin; and enhanced accumulation of cisplatin (which is imported by a neutral amino acid transporter) as
results in increased formation of Pt-DNA adducts, which in well as enhanced cisplatin accumulation. The possibility that
turn results in increased cytotoxicity. This model is sup- protein synthesis inhibitors can alter membrane transport
ported by a variety of observations.
processes is well established. Treatment of 3T3-L1 adipo-
First, we observed that 6AN inhibits protein synthesis by cytes with cycloheximide causes a 7-fold increase in 2-deoxy-
0 to 80% (Fig. 7A) at concentrations that sensitize cells to glucose transport without any alteration in the plasma mem-
7
cisplatin (Fig. 4). In contrast, 18 inactive 6AN analogs failed brane levels of the glucose transporter (Clancy et al., 1991;
to inhibit protein synthesis by more than 30% (Fig. 7B). The Harrison et al., 1992). Likewise, cycloheximide rapidly stim-
effects of 6AN on protein synthesis, like the effects on Pt- ulates leucine uptake on the system L amino acid transporter
DNA adducts, were antagonized by nicotinamide (Fig. 7C). in cultured vascular smooth muscle cells (Low et al., 1994).
All of these observations provide support for a link between Because the polypeptide corresponding to the system L
inhibition of protein synthesis and modulation of cisplatin amino acid transporter has not been identified, it is not
action. Although the suggestion that 6AN inhibits protein known whether levels of this polypeptide are altered by cy-
synthesis (Fig. 7A) appears to conflict with the enhanced cloheximide. Nonetheless, these examples provide precedent
expression of a subset of cellular polypeptides (Fig. 3), there for alterations in transport as a consequence of treatment
is ample precedent for this observation. Studies examining that inhibits protein synthesis. In both cases, the authors
stress-induced proteins have indicated that a number of cel- postulated that cycloheximide inhibited the synthesis of a
lular stresses (e.g., heat shock) simultaneously up-regulate short-lived polypeptide that suppressed the activity of the
expression of certain genes (e.g., heat shock protein genes) transporters under basal conditions (Clancy et al., 1991; Har-
and inhibit protein synthesis (Lindquist and Craig, 1988). rison et al., 1992; Low et al., 1994).
The net effect on cellular levels of these polypeptides reflects
In view of previous claims that diminished accumulation is
the increased abundance of message encoding these polypep- a potentially important cause of cisplatin resistance (Gately
tides vis a´ vis the diminished overall rate of translation. 6AN and Howell, 1993), it is tempting to speculate that the addi-
appears to exert a similar effect, increasing the expression of tion of protein synthesis inhibitors to platinating agents
genes such as GRP78 (Chatterjee et al., 1995) while simul- might provide one means of overcoming cisplatin resistance.
taneously decreasing overall protein synthesis (Fig. 7A).
Of the anticancer drugs that are currently in clinical use,
Second, our studies establish a correlation between inhibi- actinomycin D and L-asparaginase are capable of inhibiting
tion of protein synthesis and increased Pt-DNA adducts. In protein synthesis. Each of these agents, however, causes a
addition to 6AN, four different protein synthesis inhibitors variety of toxicities in vivo (Chabner and Loo, 1996); and
had an effect similar to that of 6AN (Figs. 5 and 6). Actino- neither is routinely combined with cisplatin. Accordingly,
mycin D inhibits protein synthesis by inhibiting transcrip- further preclinical studies are required to determine whether
tion; anisomycin inhibits the transpeptidation step; cyclohex- the mechanism of biochemical modulation outlined above can
imide inhibits the peptidyl transferase reaction on be developed into a regimen suitable for testing in the clinical
ribosomes; and puromycin causes premature chain termina- setting.
tion (Moldave, 1985; Pain, 1986). The demonstration that all
four of these compounds exert similar effects suggests that
inhibition of protein synthesis is the common feature of their
action. Additional experiments (IIB, unpublished observa-
Acknowledgments
This paper is dedicated to the memory of Mette Strand, who
directed the Graduate Program in Pharmacology at the Johns Hop-
tions) have demonstrated that a 22-h treatment of A549 cells kins University School of Medicine during the period of this thesis
with 10 mM 2-deoxyglucose also causes decreased protein project. We gratefully acknowledge the kind gift of 6AN analogs from
Fig. 8. Proposed mechanism by which 6AN enhances cisplatin cytotoxicity.

5
38
Budihardjo et al.
cell growth, poly(ADP-ribose) synthesis and nucleotide metabolism. Biochem
Pharmacol 34:3999–4003.
the National Cancer Institute, advice of Alex Adjei regarding the
methionine transport assay, provocative discussions with Daniel S.
Martin and Nathan Berger, secretarial help of Deb Strauss, and
assistance of the Metals Laboratory of the Mayo Clinic (Thomas P.
Moyer, Director) with the platinum assays.
Kaufmann SH (1989) Additional members of the rat liver lamin polypeptide family:
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Polypeptides of the internal nuclear matrix. Exp Cell Res 155:477–495.
Keller K, Kolbe H, Herken H and Lange K (1976) Glycolysis and glycogen metabo-
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