Download Lysosomal Sequestration of Polyamine

[CANCER RESEARCH 58, 3883-3890.
September I, 1998]
Lysosomal Sequestration of Polyamine Analogues in Chinese Hamster Ovary Cells
Resistant to the S-Adenosylmethionine Decarboxylase Inhibitor, CGP-486641
Debora L. Kramer, Jennifer D. Black, Helmut Mett, Raymond J. Bergeron, and Carl W. Porter
ice Cancer Drug Center, Roswett Park Cancer Institute, Buffalo, New York 14263 ID. L. K., J. D. B., C. W. P.}; Research Lttboratories.
Grac
Novartis Pharmaceuticals
Pharmaceittic,
'-4002. Basel. Switzerland [H. M.¡;and Department of Medicinal Chemistry. University of Florida. J. Hillis Miller Health Center, Gainesville. Florida 326/0 [K. J. BJ
CH-4002
CGP-48664
ABSTRACT
CGP-48664,
an inhibitor
of the polyamine
biosynthetic
enzyme
\-
adenosylmethionine decarboxylase (AdoMetDC), is presently undergoing
Phase 1 clinical trials as an experimental anticancer agent. We have shown
previously (D. L. Kramer et al, J. Bici. Chem., 270: 2124-2132, 1995) that
Chinese hamster ovary (CHO) cells that are made resistant to the growth
inhibitory effects of the drug overexpress AdoMetDC because of a stable
gene amplification. Unexpectedly, these same cells (CHO/644) were found
to be insensitive to the growth inhibitory effects of Ai'^Vll-diethylnorspermine (DENSPM)—a polyamine analogue also undergoing Phase 1 clinical
trials—despite accumulating ~5 times more analogue than parental cells.
We now report that treatment of CHO/664 cells with DENSPM results in
the formation of numerous large cytoplasmic vacuoles, which on the basis
of electron microscopy and cytochemical staining seem to be lysosomal in
origin. A series of newly established CHO cell lines made differentially
resistant to 1, 3,10, 30, and 100 /IM CGP-4S664 by chronic exposure were
used to demonstrate that vacuole formation correlated with the accumu
lation of extremely high levels of DENSPM without increasing growth
inhibition. These same cells were used to show that AdoMetDC gene
overexpression as indicated by mRNA levels was unrelated to vacuole
formation; cells resistant to 100 /¿MCGP-48664 displayed a 170-fold
increase in AdoMetDC mRNA levels and formed vacuoles in response to
DENSPM, whereas those resistant to 10 JIM CGP-48664 displayed a
120-fold increase in AdoMetDC mRNA levels and failed to form vacuoles.
Despite accumulating to high intracellular levels, DENSPM was much less
effective than spermine at down-regulating ornithine decarboxylase and
polyamine transport activities in highly resistant cells. Similarly,
DENSPM was less able to induce spermidine/spermine
W'-acetyltransferase activity in cells that formed vacuoles than in those that did not.
Overall, natural polyamines failed to induce vacuoles and various ana
logues of DENSPM were used to probe the structural specificity of the
effect. The data are consistent with the probability that DENSPM is
sequestered to high concentrations in lysosomal vacuoles of CGP-48664resistant cells and is, therefore, not available to interact with polyamine
regulatory sites or to cytotoxically affect cell growth. In addition to
implicating the lysosome as a potential new site of CGP-48664 drug action
that could be involved in antitumor activity and/or host toxicities, the
findings also suggest a potential mechanism of cell resistance to analogues
such as DENSPM.
INTRODUCTION
As an alternative to anticancer and chemopreventive strategies
targeting the polyamine biosynthetic enzyme ornithine decarboxylase
(1, 2), investigators at Novartis Pharmaceuticals (Basel, Switzerland)
have developed analogues of MGBG,3 also known as methylGAG, as
potential inhibitors of the other key biosynthetic enzyme, AdoMetDC.
AG.,
(Fig. 1) was selected from a relatively large series of
MGBG analogues because of its potent and selective enzyme inhib
itory properties, its metabolic stability, and, most importantly, its
minimized antimitochondrial activity (3-5). The specificity of the
inhibitor in cell culture conditions is based on observed alterations in
polyamine pool perturbations that are characteristic of AdoMetDC
inhibition and on the finding that CHO cells that are made resistant
(CHO/664) to the growth inhibitory effects of CGP-48664 overex
press AdoMetDC (5, 6). On the basis of broad spectrum in vitro
antiproliferative activity and promising antitumor activity against a
variety of human tumor xenografts, CGP-48664 is presently under
going Phase 1 clinical trials (4, 5). Although subsequent studies (7-9)
failed to correlate antitumor activity with apparent polyamine meta
bolic effects, we have recently confirmed that, in a melanoma biopsy,
CGP-48664 acts as an inhibitor of AdoMetDC under conditions of
clinical use (10).
The CHO/664 cells mentioned above were derived by chronic
exposure to increasing concentrations of CGP-48664. They represent
the first cell line in which the AdoMetDC gene is overexpressed
because of gene amplification as indicated by genomic Southern blot
analysis and increased expression of AdoMetDC mRNA and enzyme
activity (6). The initial characterization of this cell line led to the
observation that these same cells were only cytostatically affected by
the polyamine analogue DENSPM (Fig. 1) despite accumulating at
least 5 times more analogue than parental cells. This observation was
unexpected inasmuch as DENSPM is known to have multiple sites of
action within cells in addition to the down-regulation of AdoMetDC.
These include suppression of ODC and polyamine transport activities
and the potent up-regulation of the polyamine catabolic enzyme SSAT
(11-13). In fact, recent studies with fibroblasts derived from transgenie animals that overexpress SSAT (14, 15) suggest strongly that
the induction of SSAT may be the most critically important of these
various regulatory responses in terms of depleting polyamine pools
and affecting cell growth (16).
It has also been observed that DENSPM treatment of CHO/664
cells leads to the appearance of numerous cytoplasmic vacuoles, and
the possibility has been suggested that the analogue is contained
within them (6). Herein, we provide morphological evidence to indi
cate that the vacuoles are lysosomal in origin, that intracellular ana
logue is sequestered in these vacuoles, and that their appearance in
response to DENSPM is not linked to overexpression of the
AdoMetDC gene. Because DENSPM exhibits promising antitumor
activity in several model systems (17-19) and is also undergoing
Phase 1 clinical trials, these studies may provide new insight into the
cellular effects of both DENSPM and CGP-48664, insight that could
Received 4/1/98; accepted 7/2/98.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by National Cancer Institute Grant ROI CA22153
prove to be clinically relevant to antitumor activity, tissue toxicity, or
drug resistance.
Roswell Park Cancer Institute Elm and Carlton Streets, Buffalo. NY 14263. Phone: (716)
845-3002: Fax: (716) 845-8857: E-mail: [email protected].
3 The abbreviations
used are: MGBG, methylglyoxal-bis(guanylhydrazone);
AdoMetDC, S-adenosylmethionine decarboxylase: dcAdoMet, decarboxylated 5-adenosylmethionine; CGP-48664, 4-amidinoindan-l-one
2'-amidinohydrazone; CHO. Chinese
CHO/3, Chinese hamster ovary cells resistant to 3 LIMCGP-48664; CHO/10. Chinese
hamster ovary cells resistant to 10 LIMCGP-48664; CHO/30, Chinese hamster ovary cells
resistant to 30 LIMCGP-48664: CHO/IOO, Chinese hamster ovary cells resistant to 100 LIM
CGP-48664; CHO/664. Chinese hamster ovary cells resistant to 100 LIMCGP-48664
derived previously (6): DENSPM. /V'.N1 '-diethylnorspermine [also known as BENSPM.
/V'./V1'-bis(ethyl)norspermine]; ODC, ornithine decarboxyla.se: SSAT, spermidine/sperm
ine /V'-acetyltransferase; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; PCA,
hamster ovary cells: CHO/1. Chinese hamster ovary cells resistant to 1 /AMCGP-48664:
perchloric acid.
(to C. W. P.) and Roswell Park Cancer Institute Core Grant CA16056 (to J. B.).
2 To whom requests for reprints should be addressed, at Grace Cancer Drug Center,
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LYSOSOMAL
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ANALOGUES
Cell Growth Studies. Exponentially growing resistant cells were placed in
the absence of CGP-48664 for at least 72 h before treatment with DENSPM
and related analogues. The doubling time for the parental CHO cells and each
resistant CHO/664 cell line was similar (—20h). Dose-response studies with
DENSPM
DENSPM were performed at concentrations from 0.01 to 100 JU.Mand the cell
lines were seeded at 1 X IO5 cells into T-25 flasks. Unless otherwise indicated,
all of the growth and biochemical assays were performed after analogue
treatment for 72 h. Cell growth was determined by electronic particle counting.
Light and Electron Microscopy. Cells were washed, fixed in situ in 3%
phosphate-buffered glutaraldehyde (pH 7.4), postfixed in 1% osmium tetroxide. embedded in Epon-Araldite plastic resin as described previously (23) and
sectioned at -500 nm for viewing by light microscopy after being stained with
toluidine blue and at —¿100nm for viewing in a Siemens 101 electron
CGP-48664
Fig. 1. Slniclures of the polyaminc analogue DENSPM and the AdoMelDC inhibitor
CGP-48664.
microscope after being stained with uranyl acetate and lead citrate. For
detection of lysosomal structures. CHO/100 cells were cultured for 3 days in
the absence of CGP-48664 before being plated on coverslips in the absence or
Table I Relame effects of DENSPM on growth and mornhotogv of CHO and
CHO/664 cells
presence of 10 /UMDENSPM and incubated for the time periods indicated.
After DENSPM treatment for 24 h, certain coverslips were exposed to l ¡J.M
of
an acidotropic dye (LysoSensor Green DND-153, Molecular Probes, Inc.) for
linesCHOCHO/664''Treatment
Cell
h)noneDENSPM
(10 /¿M,72
noneDENSPM%
control
growth10052
analogue"
(pmol/106
cells)none6.000
vacuolesnononoyesIntracellular
10078Cytoplasmic
none35,000
" Values represent the average of at least six separate determinations with SD < 20%.
* Grown in the absence of CGP-48664 for al least 72 h before treatment with
DENSPM.
MATERIALS
Materials.
1 h to detect the intracellular localization of this fluorescent dye in relation to
vacuole formation. Cells were observed and photographed using a Zeiss
epifluorescence microscope equipped with appropriate optics and filter mod
ules. Cells were also photographed using a Leica phase contrast microscope
(Leitz, Model DMIL) with optics as indicated.
Polyamine and Polyamine Analogue Pools. Inlracellular polyamines
were extracted from cell pellets with 0.6 N PCA with a ratio of 2 X IO7 cells
AND METHODS
CGP-48664 was synthesi/.ed as described previously (3). The
polyamine analogue DENSPM was a gift from Warner Lambert Parke Davis
(Ann Arbor. MI). The polyamines spermidine and spermine were purchased
from Sigma (St. Louis. MO); norspermine was purchased from Aldrich (Mil
waukee. WI). All of the other spermidine and spermine analogues were
synthesi/.ed as described previously (20, 21). Tritiated spermidine and [32P]dCTP was obtained from DuPont NEN (Boston, MA). The pCM9 plasmid
construct containing the coding region for the cDNA of human AdoMetDC
(22) was kindly provided by Dr. Bruce Stanley (Hershey Medical Center,
Hershey. PA). The human GAPDH cDNA probe, used to quantitate Northern
blot loading differences, was obtained from Clontech Laboratories. Inc. (Palo
Alto. CA). The oligolabeling kit was obtained from Pharmacia LKB Biotech
nology (Piscataway, NJ). LysoSensor Green DND-153 was purchased from
Molecular Probes, Inc. (Eugene, OR) for detection of lysosomal acidic or-
per ml PCA, dansylated, and measured by reverse phase high-pressure liquid
chromatography as described previously with minor modifications (6).
Polyamine Enzyme Activities. After various cell treatments, ODC,
AdoMetDC, and SSAT activities were determined from cellular extracts pre
pared and measured as described by Porter et al. (13).
AdoMet, dcAdoMet, and CGP-48664 Pools. High-pressure liquid Chro
matographie analysis of intracellular AdoMet and metabolites was performed
with the same PCA extracts used for polyamine pool analysis. The Chromato
graphie conditions were adapted from Yarlett and Bacchi (24) with modifica
tions (6).
Spermidine Transport. Tritiated spermidine transport was evaluated by
Vmaxvalues determined as described previously (25). Cells were seeded into
six well plates at a density of 1 x IO4 cells per well and allowed to grow in
the presence or absence of 10 /J.MDENSPM for 72 h before performing the
assay.
ganelles.
Maintenance of CHO Cell Lines. The monolayer CHO cells, obtained
from American Type Culture Collection (CHO-K1 cells), were grown in
100
DMEM/F12 medium (Life Technologies. Grand Island. NY) containing 10%
PCS (Atlanta Biologicals. Norcross, GA.), 1% nonessential amino acids (Life
Technologies), I % sodium pyruvate (Life Technologies), and I mM aminoguanidine (Sigma). Cell cultures were maintained at 37°Cwith 5% CO2 in a
humidified incubator. Vented T-75 cm2 flasks (Falcon, Becton Dickinson,
Vacuoles
Bedford. MA) were used routinely for cell culture. CHO cells were detached
by trypsin (Life Technologies) with prompt removal of excess trypsin by
washing.
Derivation of CGP-48664-Resistant
CHO Cells. The parent CHO cell
line was chronically exposed to increasing levels of CGP-48664 for at least
o
u
eight passages beginning at 0.1 /J.Mdrug as described previously (6). Cell lines
were serially exposed to increasing concentrations until a panel of sublines was
obtained resistant to 1 (CHO/1). 3 (CHO/3), 10 (CHO/10), 30 (CHO/30), and
100 (CHO/100) P.M CGP-48664 for comparative studies, which initially fo
cused on AdoMetDC mRNA as an indication of gene overexpression and
presumably gene amplification (6). In terms of biochemical and molecular
characteristics, the CHO/100 cells, newly developed for these studies, were
found to be nearly identical to the original resistant line designated CHO/664
cells (5. 6).The cells were routinely cultured in DMEM/F12 described above
containing the appropriate amount of CGP-48664. Characterization studies
were performed with cells grown in the presence of the inhibitor or at various
times after removal of the inhibitor as indicated (typically, 72 h).
CHO
-•- CHO/664(+)
25
-n-
0.01
CHO/664(-)
0.1
1
10
100
DENSPM Treatment
Fig. 2. Comparison of growth sensitivity of CHO and CHO/664 lo increasing concen
trations of DENSPM during a 72 h incubation in the presence ( + ) and absence (-) of
CGP-48664. Noie thai in the absence of CGP-48664, CHO/664 cells form vacuoles and
are less sensitive than cells in the presence of CGP-48664 before and during DENSPM
treatment that do not form vacuoles.
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LYSOSOMAL
SEQUESTRATION
OF POLYAMINE
ANALOGUES
Fig. 3. Light microscopy of the CHO parental cells
treated with 10 ¿IM
DENSPM for 72 h (A), and newly
derived panels of CHO/10 (ß),CHO/30 (O. and
CHO/100 (O) cells grown out of CGP-48664 for 72 h
and treated with 10 /IM DENSPM for 72 h. The
differences between these cell lines in growth re
sponse (as shown in Fig. 2) are not reflected here
because regions of similar cell density were chosen
for photography. Treated CHO/100 cells (D) were
washed, placed in drug-free medium, and allowed to
recover for 24 h (E) and 48 h (F). Notice the gradual
disappearance of vacuoles. Vacuoles were noi ob
served in CHO/100 cells grown in the presence of
CGP-48664 before and during 10 /¿M
DENSPM treat
ment (C) nor in CHO/100 cells removed from CGP48664 (as above) and treated with 10 /IM spermine
for 72 h (H). XI200.
Northern Blot Analysis. Methods follow those reported previously in
greater detail (26). Total RNA was extracted with guanidine isothiocyanate and
purified by a CsCI gradient centrifugation from the parental CHO cells and
each of the sublines CHO/1, CHO/3. CHO/10, CHO/30, CHO/100 grown
continuously in the presence of CGP-48664. Each sample (10 /ig) was electrophoresed onto a 1.5% agarose gel, transferred to Duralon membrane and
probed with the Sacl/Xba fragment (~1(KX) bp) cut from the pCM9 plasmid
containing the human AdoMetDC cDNA coding region (23). A GAPDH
cDNA probe was used to adjust quantitation for loading differences. Modifi
cations specific for probing with AdoMetDC cDNA were noted previously (6).
Autoradiograms were scanned and bands of RNA quantitated using a Molec
ular Dynamics densitometer with Image Quant Software (Molecular Dynam
ics, Sunnyvale. CA).
RESULTS
CHO/664 cells containing an amplified AdoMetDC gene were
derived originally (6) by chronic exposure to increasing concentra
tions of CGP-48664 up to 100 ¡JLM.
When those cells were grown in
the absence of CGP-48664 for up to 3 weeks, the gene amplification
was found to be stable, they were cross-resistant to other known
AdoMetDC inhibitors, and they were found to be less sensitive to
DENSPM despite accumulating ~5-fold more of the analogue than
the parental line (6). In this subsequent detailed characterization.
CHO/664 (also designated CHO/100) cells were found again to ac
cumulate extremely high intracellular levels of DENSPM (Table 1),
unexpectedly accompanied by much less of an effect on cell growth
(Fig. 2). A prominent feature of the treated cells was that they
contained numerous cytoplasmic vacuoles. Interestingly, when the
CHO/664 cells were grown in the presence of CGP-48664 up to and
during treatment with DENSPM, vacuole formation was not observed,
and these cells were growth-inhibited and contained levels of
DENSPM similar to the parental CHO cells (Fig. 2). Possible reasons
for this differential response to DENSPM in the absence and presence
of CGP-48664 will be discussed later.
One goal of the present study was to determine the origin of the
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LYSOSOMAL
SEQUESTRATION
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ANALOGUES
Fig. 4. CHO/HX) cells cultured on coverslips in the
absence (A) and Ihe presence (Bl of 100 U.Mchloroquine for 3 h or (O with K) /IM DENSPM for 24 h, and
then siained for l h wiih l /IM lysosensor lluorescent
acidotropic dye (Lysosensor Green DND-153. Molec
ular Probes. Inc.). Observations using a fluorescence
microscope showed thai the same cells in B and C
containing numerous large vacuoles (left pam-I) were
siained more intensely with the lysosome-specific dye
(right panel) compared with untreated cells. In panel
D. lysosensor dye, added I h before DENSPM treat
ment, prevented the formation of large vacuoles, bar
(jiitnt't A), 10 /im.
cytoplasmic vacuoles that appeared in DENSPM-treated CHO/664
cells. To gain insight into the nature of the vacuoles, CHO/664
(hereafter designated as CHO/100) cells were removed from CGP-
exaggerated form (not shown). Examination of CHO/100 cells treated
with 10 JUMDENSPM indicated that cytoplasmic vacuolation was
well established by 24 h (Fig. 4C) and, when treated CHO/100 cells
that had contained vacuoles for 48 h were placed in drug-free medium,
48664 for 3 days, treated for 72 h with 10 JUMDENSPM. and
the vacuoles shrank (Fig. 3, £and F) and cell growth resumed.
examined by light and electron microscopy. By light microscopic
As further confirmation of the apparent lysosomal nature of the
preview, 95% of the cells were observed to contain a large number of
cytoplasmic vacuoles (Figs. 3D and 4C) that were not apparent in analogue-induced vacuoles. CHO/100 cells were treated with 10 /XM
analogue-treated CHO parental cells (Fig. 3/4). A nearly identical
DENSPM for 24 h and then stained with 1 p.M lysosensor green (a
morphological effect was obtained with chloroquine (Fig. 4ß),a fluorescent dye specific for lysosomes; Ref. 30), 1 h before exami
well-known lysosomotropic agent (27-29). Ultrastructural studies re
nation by fluorescence microscopy (Fig. 4). The cytoplasm of
DENSPM-treated CHO/100 was seen to contain large, brightly fluo
vealed that virtually every cell contained swollen cytoplasmic vacu
oles with electron-dense material typical of secondary lysosomes as rescent bodies that corresponded to the swollen vacuoles observed in
well as numerous tiny vesicles presumed to be endosomal in origin
the same field of cells with phase optics (Fig. 4C). The response of
these cells to 100 /MMchloroquine for 3 h showed similar swollen and
(Fig. 5). In many cases, the vacuoles appeared to be fusing with one
another. All of the other organdÃ-es including mitochondria, endoplasfluorescent vacuoles (Fig. 4B). The cytoplasm of untreated CHO/100
mic reticulum, and nuclear membrane appeared normal and unswolcells (Fig. 4/4) contained much smaller fluorescent bodies, which were
len. Ultrastructural studies confirmed light microscopic indications
identical to those seen in untreated CHO cells (not shown) and
that vacuoles did not form in the parental CHO cells even in a less
presumed to be lysosomes. Interestingly, exposure to the fluorescent
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LYSOSOMAL
SEQUESTRATION
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ANALOGUES
B
Fig. 5. Electron micrograph of untreated CHO/
100cells (A) and those Ireated with 10 fiM DENSPM
for 72 h (B and O showing the presenceof numerous
cytoplasmic vacuoles containing electron dense ma
terial and numerous small vesicles. Note that mito
chondria and other cellular organdÃ-esare unaffected
by analogue treatment, bar. I firn.
dye for only l h before DENSPM treatment fully prevented vacuole
formation (Fig. 4D). Taken together, the light and electron micro
scopic data indicated that the vacuoles were most likely lysosomal in
origin and suggested that DENSPM may possess lysosomotropic
activity in CHO/100 cells similar to that reported for certain other
polyamine analogues in LI210 cells (23).
Because the lysosomal vacuolation only appeared in DENSPMtreated CHO/100 cells, two additional goals of the study were: (a) to
determine whether it was a consequence of AdoMetDC gene overexpression; and (b) whether there exists a possible relationship between
vacuole formation and DENSPM accumulation. For these purposes, a
panel of CHO cells that were differentially resistant to CGP-48664
was derived by chronically treating cells with the drug beginning at
concentrations of 0.1 JU.Mand increasing to a maximum of 100 /AM
over the course of several months. Cells were grown for a minimum
of at least eight passages before increasing the drug concentration.
Adapted cells were removed from the common culture at 1,3, 10, 30,
and 100 JAMCGP-48664 and maintained separately at those concen
trations. The cells were then subjected to biochemical and molecular
characterization. The CHO/100 cells were found to be remarkably
similar to the originally derived CHO/664 cells, which were also
resistant to 100 /J.M CGP-48664. AdoMetDC mRNA levels were
measured by Northern blot analysis of resistant cells growing in the
presence of CGP-48664 (Fig. 6). Quantitation of AdoMetDC mRNA
levels relative to untreated CHO parental cells and normalized to
GAPDH message, indicated an 11- to 15-fold increase for cells
adapted to 1 and 3 JU.MCGP-48664, respectively, and an increase of
120- to 170-fold in cells adapted to 10 /J.Mand 100 /U.MCGP-48664
(Fig. 6; Table 2). These same cells were then compared for vacuole
formation and analogue accumulation after treatment for 72 h with
DENSPM (Fig. 3; Table 2). Two findings emerged: (a) vacuole
formation seemed unrelated to gene overexpression inasmuch as it
occurred in CHO/100 cells and to some degree in CHO/30 cells but
not in CHO/10 cells, which, according to AdoMetDC transcript level,
displayed a similar level of AdoMetDC gene overexpression. This
suggests that, at sufficiently high drug levels, CGP-48664 apparently
alters lysosomal membranes and/or contents. In this regard, it is
relevant that vacuole formation did occur in CHO/100 cells when they
were not removed from CGP-48664 before treatment with DENSPM
(Fig. 3G), which suggests that, as with lysosensor green (Fig. 4D),
there are direct effects on the lysosome; and (b) major increases in
intracellular DENSPM levels correlated with the appearance of vacu
oles (Table 2) suggesting that the analogue is sequestered within the
vacuoles.
mRNA
_kb_
3.4
AdoMetDC
2.1
GAPDH
x11
x15
x120
x125
x170
x1
Fold Increase of AdoMetDC / GAPDH mRNA
Fig. 6. Northern hint analysis of AdoMetDC mRNA from CHO cells for comparison
with the CHO cell panel designated CHO/1. CHOß. CHO/10. CHO/30. CHO/100
described in "Materials and Methods." Note the presence of two delectable AdoMetDC
transcripts of 3.4- and 2. l-kb lengths. The fold-increase values are based on the ratio of
the 3.4-kb AdoMetDC transcript to the GAPDH mRNA to adjust for loading.
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ANALOGUES
Table 2 Comparison of CHO cells that are differentially resistant to CGP-48664
were unable to induce the formation of vacuoles in the CHO/100 cells
and did not accumulate to higher levels in CHO/IOO cells than in the
h)fcCclls/CGP-48664resistant
parental CHO cells (Table 5). On the other hand, all of the mono- and
di-alkylated spermidine and spermine analogues behaved similarly to
i-lliil.uCGP-48664"(pmol/IO6
AdoMettnRNA"1IIIS120125170Vacuoleformation(% DENSPM in their ability to induce vacuoles except for DMSPM and
cells)<53060IÃŽ32211350Relative
cells)6,5458,6507,6256,21513,69532,200
cells)000n4896IntraccllularDENSPM'(pmol/106
(/IM)CHO
A/'-MESPM. In every case in which vacuoles were observed, ana
logue levels were 3- to 5-fold higher (range, 13-31 nmol/106 cells)
parentalCHO/ICHO/3CHO/
10 ;ÃŒM
DENSPM-trealed(72
than levels of analogue that failed to produce vacuoles. Unlike DM
SPM, the two other dimethyl-analogues, A'1,Ai1'-dimethylnorspermine
and Ar',/V'4-dimethylhomospermine, did induce vacuoles, which indi
II)CHO/30CHO/IOOliin.i.
cated that intra-amine carbon distances may play a role in lysosomal
recognition. Thus, alkylation of the terminal amines seemed to be
important, but not the only, structural determinant for triggering the
lysosomotropic response.
" Values determined on cells maintained in the presence of CGP-48664.
* Cells grown in the absence of CGP-48664 for 72 h before DENSPM treatment.
' Values represent averages of six separate determinations with SD < 20%.
The relative metabolic effects of differential amplification of the
AdoMetDC gene are compared in Table 3. Because AdoMetDC
activity is inhibited in the presence of CGP-48664, we measured
enzyme activity, dcAdoMet pools, and polyamine pools on cells
grown in the absence of drug for 72 h. We observed that enzyme
activity did not seem to correlate closely with mRNA, in part because
of compensatory responses to drug withdrawal and also because of the
well-known posttranscriptional regulation of the enzyme activity (31).
Nonetheless, enzyme activity was clearly increased in cells resistant to
10 JAMCGP-48664 and higher. These increases were paralleled by
rises in dcAdoMet pools from <10 pmol/106 cells in CHO, CHO/1,
and CHO/3 cells up to >6000 pmol/106 cells in CHO/IOO cells. In the
CHO/IOO cells. AdoMetDC activity did not closely parallel the rise in
pools. One possible explanation for this is that, although undetectable,
residual CGP-48664 in the cell extract partially inhibits the enzyme
under in vitro assay conditions. AdoMet pools were unaffected (data
not shown). Shifts in polyamine pools favored spermine synthesis for
the CHO/10, CHO/30, and CHO/IOO cells in which dcAdoMet pools
were accumulated to levels that were no longer rate-limiting. Thus,
despite the dramatic shift in the polyamine pool profile including an
80% decrease in spermidine pools, the cells remained viable with a
similar growth rate. The similar polyamine profile in the CHO/10 and
CHO/IOO cells would argue against vacuole formation being depend
ent on the shifts in the pools created with CGP-48664 withdrawal.
As further indication that DENSPM was contained within the
vacuoles, we compared how effective the analogue was at mediating
the various regulatory responses involving ODC, SSAT, and transport
activities between the CHO. CHO/10, and CHO/IOO cells. The cells
were removed from CGP-48664 for 72 h and cultured for an addi
tional 72 h in the presence or absence of 10 /XMDENSPM. In CHO
and CHO/10 cells, ODC activity was reduced by 98% whereas in
CHO/IOO it decreased by only -40% (Table 4). A similar trend was
seen with spermidine transport activity as indicated by DENSPMmediated decreases of >80% in Vm.lxin CHO and CHO/10 but not
CHO/IOO cells. Notice that both ODC and transport were downregulated similarly in all of the three lines treated with spermine. The
lack of regulatory response was most evident with SSAT activity
where the analogue caused ~120- and 200-fold induction in enzyme
activity in CHO and CHO/10 cells, respectively, but only a —¿6-fold
DISCUSSION
The present findings extend our earlier characterization of the CHO
cell line resistant to CGP-48664, the only cell line known to contain
an amplified AdoMetDC gene (6). Grown in the absence of CGP48664, in addition to being cross-resistant to other AdoMetDC inhib
itors, these cells were unexpectedly found to be insensitive to the
antiproliferative activity of DENSPM. It seemed relevant that treat
ment with the analogue resulted in the formation of numerous cytoplasmic vacuoles. We now report that these vacuoles are part of the
lysosomal system and that they contribute to the massive accumula
tion of DENSPM and account for the growth insensitivity of these
cells by compartmentalizing the analogue.
Morphological evidence strongly suggests that the vacuoles are
lysosomal in origin. By electron microscopy, the swollen vacuoles
were seen to contain electron-dense material and endosomes typical of
secondary lysosomes. In addition, the vacuoles were selectively
stained by the supravital dye lysosensor green, which is known to
accumulate specifically in that organelle (30). Finally, pretreatment of
the cells with the dye interfered with vacuole formation by the
analogue. In terms of light microscopy and dye staining, the vacuoles
were found to be nearly identical to those produced by chloroquine, a
well-known lysosomotropic agent (27-29). Other organdÃ-es includ
ing the endoplasmic reticulum, the Golgi apparatus, the mitochondria,
and the nuclear membrane were unaffected by the drugs, which
indicates that the effect was not due to some generalized osmotic or
toxic effect. It is interesting that CGP-48664 resistance should be
associated with a lysosomal effect inasmuch as the compound from
which it was conceptually derived, MGBG, is well known for its
selective effect on mitochondria leading to a cytoplasmic vacuolation
that at the light microscopic level appears to be nearly identical to the
one observed here (32).
Thus far, the profound lysosomal vacuolation seen with DENSPM
only occurs in CHO cells resistant to CGP-48664. Because such
vacuoles do not form in parental cells, they must represent an adap
tation to treatment with CGP-48664 during derivation of the resistant
increase in the CHO/IOO cells. Importantly, DENSPM accumulated to
much higher levels in CHO/IOO cells than in either CHO or CHO/10
cells. Overall, the data are consistent with the interpretation that in
CHO/30 and CHO/IOO cells, DENSPM is sequestered inside the
vacuoles and is unavailable to interact at various polyamine regulatory
sites located elsewhere in the cell.
Finally, we examined whether natural polyamines and certain an
alogues of DENSPM could induce vacuoles in CHO/100 cells. Data in
Fig. 3 indicate that the natural polyamines, as well as norspermine.
Table 3 Characterization
of metabolic shifts in CHO cells that are differentially
resistant to CGP-48664
Polyamine pools* (pmol/106 cells)
Cells/CGP-48664
AdoMetDC''
resistance" (JÕM) (nmol/h/mg)
dcAdoMet''
(pmol/lO6 cells)
Pulrescine
Spermidine
Spermine
CHO parental
<IO<10140020406100270
0.700.35353522<10
241520652305001602510
3605352533253430
CHO/1CHO/3CHO/10CHO/30CHO/IOO0.75
115854560752340
" These cells were grown in the absence of CGP-48664 for 72 h.
'' Values represent at least three separate determinaiions with SD < 20%.
3888
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LYSOSOMAL SEQUESTRATIONOF POLYAMINE ANALOGUES
Table 4 DENSPM regulation of enzyme and transpon
activities in CHO ceux that are differentially rexisttwt to CGP-48664
DENSPM"
Cell/CGP-48664
cells)6,5457.62532.200average
(pmol/106
(nmol/h/mg)0.850.020.03
(JIM)CHOCHO/IO''CHO/100''"
resistance
(10
h)NoneDENSPMSPMNoneDENSPMSPMNoneDENSPMSPMIntracellular
JIM. 72
1.150.060.01
1.100.500.03with
Values represent the
of at least two separate determinations performed in duplicate
' Resistant cells wereTreatment
grown in the absence of CGP-48664 for 72 h before treatment.ODCactivity"
line. The possibility was examined that vacuole formation might
somehow be related to overexpression of the AdoMetDC gene as has
been shown to be the case in CHO/664 cells (6). It is possible, for
example, that cells that overexpress AdoMetDC have metabolic per
turbations, such as overproduction of spermine, that lead to adaptive
responses intended to minimize resulting toxic effects. We examined
this possibility by deriving and characterizing a panel of CHO cells
that were differentially resistant to CGP-48664. Although on the basis
of transcript level the AdoMetDC gene was differentially overexpressed in all of the CHO cell variants chronically exposed to CGP48664. DENSPM-induced vacuole formation only occurred in the
CHO/100 cells and to a lesser degree in CHO/30 cells. Because
vacuolation did not occur in CHO/10 cells with a polyamine-pool
profile similar to that in the CHO/30 and CHO/100 cells, related
metabolic effects such as a possible rapid accumulation of spermine
could not account for the lysosomal response to DENSPM.
On the other hand, we did not encounter cells that formed vacuoles
but did not overexpress AdoMetDC. Thus, although AdoMetDC
overexpression alone can account for low level CGP-48664 resist
ance, it is not clear whether lysosomal changes are required for
high-level resistance or whether they are simply a consequence of the
higher levels of drug exposure. Because CGP-48664 is a basic com
pound, it is possible that high levels of drug exposure result in the
interaction of the drug with the lysosomal system without producing
the swelling seen with the analogue. When the CHO/100 and CHO/30
cells are continuously grown in CGP-48664 before and during
transport"
(pmol/min/mg)3,700415150
2.63020095
3.1601.39090SSAT
activity"
(pmol/min/mg)784510
71.43022
95010
SD < 10%.Spermidine
DENSPM treatment, vacuole formation is not observed and DENSPM
is only accumulated to levels similar to those seen in CHO and
CHO/10 cells. This suggests that CGP-48664 is bound at intracellular
sites, thus preventing analogue accumulation into the lysosome.
The fact that the level of DENSPM accumulation in cells that form
vacuoles (CHO/100) is ~5 times higher than in cells that do not form
vacuoles (CHO/10) suggests that analogue may actually be contained
in the swollen organelles. Further evidence for this is provided by a
comparison of growth inhibitory and gene regulatory responses in
CHO/100 cells treated with either the analogue or the natural poly
amine spermine. Despite accumulating to extremely high levels,
DENSPM is not particularly more growth inhibitory and is much less
effective than spermine at down-regulating ODC and transport. The
two are known to behave almost identically in this regard (13, 25),
including the CHO cells shown here (Table 4). Likewise, although
DENSPM accumulated to higher levels in CHO/100 cells than in
CHO/10 cells, induction of SSAT by the analogue was less apparent
in the former cells. Both induction of SSAT and down-regulation of
ODC are regarded as largely posttranscriptional events and thus
should occur in the cytosol (33, 34). Thus, the overall data are
consistent with the likelihood that most intracellular DENSPM is
sequestered in the vacuoles and unable to interact at various regula
tory sites represented elsewhere within the cell.
There is a biological precedent for storage of polyamines in vacu
oles. Davis and colleagues have shown (35, 36) that under normal cell
culture conditions, as much as 25% of the total spermidine of Neu
rospora cells resides specifically within the cytoplasmic vacuole
(often compared with the animal lysosome) of these organisms where
Table 5 Comparison of polyamine and polyamine analogue acctinndiiiion and
induction of cytoplasmic vacuoles in CHO and CHO/100 cells"
it complexes with polyphosphates. In addition, we have shown pre
viously (23) that certain polyamine analogues that are structurally
cells'*VacuolesnononoyesyesyesyesyesyesyesnoyesnoAccumulation'(nmol/106
cellsTreatment(10
CHO
dissimilar and many that are structurally similar to DENSPM (Table
5) can also behave as lysosomotropic agents. Depending on the net
'nononononononononononononoAccumulation*(nmol/106
cells)5427435444432CHO/100
cells)345311821262727137154
h)SpermidineSpermineNorspermineDESPDN'-MESPDN8-MESPDDENSPMDESPMDEHSPMDMNSPMDMSPMDMHSPMN'-MESPMVacuoles
JIM. 72
pK.t of a molecule, compounds such as chloroquine (27-29) enter
lysosomes and become irreversibly trapped in the organelle by pro-
"DESPD.
W'A^-diethylspermidine:
W'-MESPD.
W'-monoethylspermidine;
MESPD, /V^-monoethylspermidine;
DESPM, /v'./Vl2-diethylspermine:
DEHSPM,
Af',JVl4-dietnylhomospermine;
DMNSPM,
Nl V-dimethylnorspermine:
DMSPM,
¿V'JVl2-dimethylspermine: DMHSPM. AT1
JV14-dimelhylhomosperniinc: /v'-MESPM. /V1monoethylspermine.
* Vacuoles were visualized under a phase contrast microscope using L40/0.50 Phaco
1a optics.
' Values represent the average of three separate determinations
with SD < 20%.
Cells were grown in the absence of CGP-48664 for 72 h before analogue treatment.
tonation. The continued accumulation of the drug leads to lysosomal
swelling and the eventual formation of visible vacuoles identical to
those we have described here. Because in the case of DENSPM,
vacuole formation has not been observed to occur in other cell types,
we presume that the lysosomal properties of CHO/KX) cells have been
altered by CGP-48664 in such a way that the analogue can either enter
the lysosome more readily or is more effectively protonated once
inside. In this regard, drug accumulation into lysosomes has been
attributed to alterations in the intralysosomal milieu (37). Changes in
lysosomal pH or membrane permeability brought about by previous
exposure to CGP-48664 represent two possibilities. As noted above,
these vacuoles do not seem to form in cells treated with spermine and,
therefore, probably do not represent a drug-mediated exaggeration of
a biological mechanism for processing or storing naturally occurring
polyamines. Although the pK, of the molecule may be one character-
3889
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LYSOSOMAL
SEQUESTRATION
istic responsible for the vacuole formation in the CHO/100 cells, this
does not seem to be the case inasmuch as Bergeron et al. (38) reported
that the pK., values for the four charged nitrogens of spermine (pA"a
values = 7.95, 8.82, 10.05 and 10.86) are similar to those of
DENSPM (pA"a values = 7.68, 8.51, 9.91 and 10.83). Studies with
DENSPM analogues (Table 5) indicated that vacuolation may be a
function of terminal alkylation as well as intra-amine carbon dis
tances. Thus, the overall mechanism remains to be defined.
It would seem that vacuole formation represents a potential means by
which cells can become resistant to the growth inhibitory effects of
DENSPM. As shown here, cells that form vacuoles (i.e., CHO/100 cells)
are less sensitive to growth inhibition by the analogue than those that do
not (i.e., CHO/10 and parental cells). The unusual feature of this form of
resistance is that the cells actually accumulate more of the analogue rather
than less, unlike the more classical form of drug resistance involving
defects in polyamine transport (39). The findings also have obvious
therapeutic relevance for CGP-48664 inasmuch as they seem to indicate
another site of action of the drug that could have greater relevance under
in vivo conditions. At least two groups have reported (7-9) that the
magnitude of the inhibitor effects on tumor polyamine pools does not
seem to correlate with the substantial antitumor activity seen in certain in
vivo systems. The present finding provides an additional or alternative
possible mechanism for in vivo antitumor activity and/or host toxicities.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the technical assistance
Diegelmun. John Miller. Deborah Ogden, and Edward Hurley.
of Paula
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3890
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Lysosomal Sequestration of Polyamine Analogues in Chinese
Hamster Ovary Cells Resistant to the S-Adenosylmethionine
Decarboxylase Inhibitor, CGP-48664
Debora L. Kramer, Jennifer D. Black, Helmut Mett, et al.
Cancer Res 1998;58:3883-3890.
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