[CANCER RESEARCH 49, 3369-3373, June 15, 1989]
Aberrant Mitochondria in Two Human Colon Carcinoma Cell Lines1
Josephine S. Modica-Napolitano,2 Glenn D. Steele, Jr., and Lan Bo Chen
Dana-Farber Cancer Institute, Department of Surgery, New England Deaconess Hospital, Harvard Medical School, Boston, Massachusetts 02115
ABSTRACT
Electron micrographs of CCL237 and FET cells (two slowly growing,
differentiated human colon carcinoma lines) revealed enlarged mitochon
dria with few cristae. Polarographic measurement of respiratory activity
in mitochondria isolated from these cell lines was compared to that of
C'V-I cells (a normal monkey kidney epithelial line) and MIP101 cells
(another human colon carcinoma line), both of which have mitochondria
with a "normar appearance. The respiratory control ratios of CCL237
and FET mitochondria were found to be considerably lower than those
of CV-1 and MIP101 mitochondria (approximately 3 as compared to
>10, respectively), indicating that in CCL237 and FET mitochondria the
processes of substrate oxidation and phosphorylation of ADP are only
loosely coupled. In intact cells, differences in radiolabeled tetraphenylphosphonium uptake showed that the mitochondria! membrane potential
in CCL237 and FET cells was less than that in CV-1 and MUM01 cells,
and that nigericin failed to hyperpolarize the mitochondria of CCL237
and FET cells. In addition, FET mitochondria exhibited significantly
lower ADP-stimulated and uncoupled respiratory rates than mitochondria
isolated from the other cell types, indicating that in the former, the
capacity for oxidative phosphorylation is somehow impaired. Selective
toxicity of FET cells was obtained by treatment with 2-deoxyglucose, an
inhibitor of glycolysis, suggesting the possibility of exploiting the phenotype of impaired oxidative metabolism for chemotherapy.
INTRODUCTION
Numerous pathological conditions are characterized by mi
tochondria that display unusual morphological features (for
review, see Ref. l). Various abnormalities have been described
with regard to the size and shape of this organdie, the number
and organization of its cristae, and the absence or presence of
"dense-granule" inclusions in the matrix. The tissues displaying
these unusual mitochondria include the brain, liver, cardiac and
skeletal muscle, kidney, and adrenal cortex. In most cases, these
ultrastructural peculiarities are associated with a particular
biochemical alteration, such as a defect in substrate oxidation
or ion transport systems, or a deficiency in enzyme levels or
cytochrome content. Whether these mitochondrial abnormali
ties are a cause or consequence of a given pathological state is
not always easily determined. However, since mitochondrial
occupy 15 to 50% of the total cytoplasmic volume of most
animal cells and participate in more metabolic functions than
any other organdÃ-es, it is apparent that their proper functioning
is essential to maintaining a normal cellular metabolism.
The fluorescent mitochondrial stain rhodamine 123 has been
used extensively in living cells to visualize mitochondrial mor
phology and monitor changes in mitochondrial function (2-4).
Over 200 different cell lines/types were screened in this labo
ratory for rhodamine 123 uptake. While the mitochondria of
most carcinoma cells exhibit increased uptake and retention of
the dye (5, 6), two slowly growing, differentiated human colon
carcinoma lines (CCL237 and FET) displayed unusually low
levels of rhodamine 123 uptake. The main objective of the
Received 9/23/88; revised 3/2/89; accepted 3/15/89.
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 has been supported by grants from the NIH (CA44704, CAI9589,
CA22427.GM38318).
2 Supported by postdoctoral training grants from the NIH to A. B. Pardee and
T. Benjamin.
present study was to determine the basis for this phenomenon.
Electron micrographs of FET and CCL237 cells revealed
mitochondria with striking morphological features; the organ
dies were enlarged and contained few cristae. Through meas
urement of various parameters of oxidative metabolism, includ
ing respiratory function in isolated mitochondria and TPP3
uptake under various conditions in whole cells, it has been
determined that these ultrastructural abnormalities are associ
ated with some biochemical impairment in respiratory function.
The possibility of exploiting this phenotype for chemotherapy
was investigated.
MATERIALS AND METHODS
Cell Cultures. CV-1 cells, a normal monkey kidney epithelial line,
were grown in DME medium (GIBCO) supplemented with 5% calf
serum (Hazelton). Human colon carcinoma cell lines FET, obtained
from Dr. M. Brittain (7), and MIP101, obtained from Dr. D. Zamcheck
(8), were grown in equal volumes of DME and RPMI 1640 (GIBCO)
supplemented with 5% calf serum and 5% Nu serum (Collaborative
Research). CCL237, another human colon carcinoma cell line, was
grown in RPMI 1640 supplemented with 10% fetal calf serum (Hazelton).
Fluorescence Microscopy. Cells grown on glass coverslips were ex
posed to rhodamine 123 (SO Mg/ml for 30 min; Eastman Organic
Chemicals, Rochester, NY), washed, and mounted on silicon rubber
chambers containing dye-free culture medium as previously described
(2). Fluorescence of rhodamine 123 was observed as previously de
scribed (2), using a Zeiss Photomicroscope III equipped with epifluorescence optics.
Electron Microscopy. Cell monolayers were fixed in situ for l h at
room temperature with 2.5% glutaraldehyde and postfixed for l h at
4"C in 1% OsO4 with 1% potassium ferricyanide. After washing,
monolayers were dehydrated in graded alcohols and embedded in Epon
812, and ultrathin sections were cut parallel to the surface by a Servali
MT2 microtome. After staining in uranyl acetate and lead citrate, grids
were examined by a Philips 300 electron microscope. Electron micros
copy was performed by Elizabeth Beaumont.
Isolation of Mitochondria. Mitochondria were isolated from cells in
culture as described previously (9). Typically, 5x10* cells were har
vested in culture medium, pelleted, and washed once with homogenization buffer (250 mM sucrose:! mM Tris-HCI:l mM EDTA:1 mg/ml
of bovine serum albumin, pH 7.4). The cells were resuspended to a
volume of 7 ml and homogenized in a Dounce tissue grinder until at
least 95% of the cells were disrupted (approximately 125 strokes). The
homogenate was centrifuged at 800 x g for 10 min at 4'C. The
supernatant was removed and saved, and the pellet was resuspended
and centrifuged again at 800 x g for 10 min. The supernatants were
then pooled and centrifuged at 9400 x g (10 min, -TV): the pellet was
resuspended, centrifuged again at 9400 x g, and finally resuspended to
a volume of about 5 mg/ml. Protein was determined by the Bio-Rad
protein assay.
Respiration. Oxygen consumption was measured polarographically
with a Clark electrode in a 1 ml water-jacketed chamber maintained at
30°C(10). The basic respiratory assay medium consisted of 225 mM
sucrose, 10 mM KC1, 1 mM EDTA, 10 mM K2HPO4-KH2PO4, 5 mM
MgCh, 10 mM Tris-HCl, and 1 mg/ml of bovine serum albumin, pH
7.4, to which additions were made in the following order: mitochondria
(0.18 to 0.27 mg of protein); rotenone (2 ¿ig/ml);succinate (10 mM);
ADP (120 nmol); and 2,4-dinitrophenol (80 MM).The ADP-stimulated
3 The abbreviations used are: TPP, tetraphenylphosphonium;
co's modified Eagle's; RCR, respiratory control ratio.
DME, Dulbec-
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ABERRANT COLON CANCER MITOCHONDRIA
respiratory rate was defined as the rate after ADP minus the rate before
ADP, while the uncoupled respirator)' rate was defined as the rate in
the presence of 2,4-dinitrophenol. The RCR was calculated by dividing
the rate of respiration obtained after ADP addition (State 3) by the rate
of respiration when ADP is limiting (State 4).
Uptake of TPP. Uptake of radiolabeled TPP was monitored accord
ing to the procedure of Davis et al. (11). Briefly, cells were grown in
their appropriate medium on 12-mm glass coverslips (Bradford Scien
tific Co., Epping, NH) with a seeding density of approximately 5 x IO4
cells per coverslip. Cells were incubated with [3H]tetraphenylphosphonium chloride (specific activity, 4.3 Ci/mol; final concentration, 1 to 3
nM; Dupont-New England Nuclear, Boston, MA) in either low K*
medium [137 mM NaCl:3.6 rriM KC1:0.5 IHMMgCl2:1.8 mM CaCl2:4
mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic
acid:l mg/ml of
dextrose: 1% modified Eagle's medium-amino acid solution (lOOx;
GIBCO), pH 1.2} or high K+ medium (137 mM KC1:3.6 mM NaCI:other
components same as low K* medium). At specific time points, coverslips were removed from the medium and rinsed several times with
phosphate-buffered saline. Radioactivity was determined by liquid scin
tillation counting. The uptake ratio was calculated by dividing the total
cellular uptake in cpm by the cpm in a volume of external medium
equal to the volume of cells. Estimates of cell volume were obtained by
centrifugation of cell suspensions in a graduated tube. Results presented
in Figs. 3 and 4 are the mean of 2 to 4 separate determinations.
Cytotoxicity Assay. Cells were seeded at 5 x IO5 cells/60-mm dish
and incubated at 37°Cin 5% CO2. 2-Deoxyglucose (2.5 mM) was
applied the same day and, at specified intervals during continuous drug
exposure, cells were trypsinized and counted by hemocytometer. The
exclusion of trypan blue was used as an indicator of live cells.
RESULTS
Rhodamine 123 Staining. Living cells stained with rhodamine
123 (Fig. 1) show that the mitochondria of CV-1 cells (normal
epithelial) take up much more of the dye and fluorescence much
more brightly than those of CCL237 and FET cells (2 human
colon carcinomas). This is unusual since most carcinoma cells
exhibit an increased uptake and prolonged retention of rhoda
mine 123 and other lipophilic cations compared to CV-1 cells
(3,5,9, 11).
Ultrastructure. Ultrathin sections were cut from embedded
glutaraldehyde-fixed monolayers of cells. Electron micrographs
(Fig. 2) of these sections show that, while CV-1 and MIP101
cells contain mitochondria with no unusual structural charac
teristics, FET and CCL237 cells contain enlarged mitochondria
with few cristae.
Mitochondria! Respiration. Polarographic measurements of
respiratory activity in mitochondria isolated from FET and
CCL237 cells were compared to those from CV-1 and MIP101
cells, both of which contain "normal" looking mitochondria.
The RCRs of mitochondria isolated from FET and CCL237
cells are significantly lower than those of mitochondria isolated
from CV-1 and MIP101 cells (Table 1). The RCR is a criterion
used to assess the extent of coupling between mitochondrial
respiration and ATP synthesis. While RCR values greater than
10 are indicative of well-coupled mitochondria, much lower
values, resulting from an increase in State 4 respiration, are
associated with an abnormal leak of protons through the inner
membrane in the absence of ADP and are indicative of a "loose
coupling" of the processes of substrate oxidation and ATP
synthesis. In addition, Table 1 shows that FET mitochondria
exhibit much lower ADP-stimulated and uncoupled respiratory
rates than mitochondria isolated from the other cell types. This
suggests that, in FET cells, the capacity for mitochondrial
respiration is somehow impaired.
TPP Uptake. The permeant cationic compound TPP was
used to monitor membrane potential in whole cells. Since the
Fig. 1. Mitochondrial staining by rhodamine 123 in living cells. Cells were
stained with rhodamine 123 (SO fig/ml) for 30 min and left in dye-free culture
medium for 1 h before micrographs were taken A, CV-1; A, CCL237; and C, FET
cells.
plasma membrane potential of epithelial cells is thought to be
primarily a K+ diffusion potential (12), a medium high in K+
concentration should depolarize the plasma membrane, and
therefore differences in TPP accumulation may be attributed
solely to the mitochondria membrane potential. Fig. 3 depicts
approximately 3- to 5-fold greater accumulation of TPP in high
K* medium in MIP101 and CV-1 cells than in either FET or
CCL237 cells. This suggests that the mitochondrial
membrane
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ABERRANT COLON CANCER MITOCHONDRIA
*
Fig. 2. Electron micrographs of FET (A) (X
15,228), CCL237 (B) (X 37,989), MIP101 (C)
(x 14,555), and CV-1 (D) (x 12,820).
-A
ft
B
r\
\
D
•¿v
Table 1 Respiratory function of mitochondria isolated from cells grown in culture
Mitochondria! respiratory rate (nanoatoms of oxygen/min/mg)
3106.5
±4.7°
CV-1
MIP101
88.6
CCL237
140.7
FETState
60.6
* Mean ±SE of
stimulated70.6
43.8
±3.6
±0.8
±3.5
±27.7
57.6 ±16.9
2.8 ±0.4 90.5 ±17.0 >10
±31.7
70.9 ±16.0
51.4 ±4.6 103.5 ±19.3
2.7
±12.3Alii' 21.7 ±1.0State 23.5 ±8.7Uncoupled107.8
32.5 ±5.5RCR>10
2.9
at least 3 separate determinations.
potential in FET and CCL 237 cells is significantly less than
that found in MIP101 or CV-1 cells.
We also measured TPP uptake in low K+ medium, a condition
which more closely resembles the normal cellular environment
than does high K+ medium. Fig. 4 shows that, in the presence
of 5 Mg/ml of nigericin, a K*/H+ exchange ionophore which
dissipates the pH gradient across the mitochondrial inner mem
brane while compensatorily increasing the mitochondrial mem
brane potential, there is a significant increase in TPP uptake
over control values in both CV-1 and MIP101 cells, yet no
significant effect on TPP uptake in FET and CCL237 cells.
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ABERRANT COLON CANCER MITOCHONDRIA
about 10% of the original treated population), whereas the total
number of control cells after 10 days of growth was 5.95 x IO4,
representing an approximate 6-fold increase in cell number over
the original untreated population. In contrast, 10 days of con
stant exposure to 2.5 mM 2-deoxyglucose resulted in a 60-fold
increase in MIP101 cells over the original treated population,
while the number of control cells increased by 120-fold, a 50%
decrease in the rate of cell growth attributable to 2-deoxyglu
cose. This compound also inhibited the rate of cell growth of
CV-1 cells, while having a minimal effect on CCL237 cells
(data not shown).
DISCUSSION
Fig. 3. TPP uptake by CV-1, MIP101, FET, and CCL237 cells in high K*
medium.
MIP101
Nigericin
a
a
-
100
FET
CCL 237
80
60
40
20
Hours
Fig. 4. TPP uptake by CV-1, MIP101, FET, and CCL237 cells in low K*
medium in the presence (•)and absence (•)of nigericin at 5 »ig/ml.
Effect of 2-Deoxyglucose. We tested the effect of an inhibitor
of glycolysis, 2-deoxyglucose, on the growth of cultured FET
versus MIP101 cells. Fig. 5 shows that, after 10 days of constant
exposure to 2.5 mM 2-deoxyglucose, the total number of viable
FET cells was determined to be 6.3 x IO2 (representing only
Certain unusual morphological features have been character
ized previously for tumor mitochondria in situ. Hruban et al.
(13) noted that some tumor cell lines exhibit differences in the
size of their mitochondria relative to normal controls; mito
chondria from rapidly growing tumors tended to be smaller
with fewer cristae, whereas those from slowly growing tumors
were larger with characteristics more closely resembling those
of normal cells. Although both FET and CCL237 are slowly
growing, it is apparent that the mitochondria of these cell lines
do not resemble normal mitochondria. Isolated tumor mito
chondria have been shown to display abnormal shapes, unusual
cristae, helical structures, or inclusions (14-16; see Ref. 17 for
review). Most of these studies, however, involved chemically
induced hepatomas. CCL237 and FET may be the first human
colon carcinoma cell lines demonstrated to have aberrant mi
tochondria! morphology.
We investigated whether these ultrastructural abnormalities
are associated with impairments in mitochondria! respiratory
function. Polarographic measurements of respiratory activity
in mitochondria isolated from FET and CCL237 cells were
compared to values obtained for mitochondria isolated from a
normal epithelial cell type (CV-1) and another colon carcinoma
line where this organdie displayed no unusual morphological
characteristics (MIP101). As Table 1 illustrates, mitochondria
isolated from FET cells exhibit significantly lower ADP-stimulated and uncoupled respiratory rates when compared to mi
tochondria from other cell types. These data appear to be
indicative of an impaired capacity for mitochondria! oxidative
phosphorylation, a phenomenon first described by Warburg
more than 50 yr ago (18). In addition, the low RCR values
exhibited by CCL237 and FET mitochondria indicate that, in
these cells, the processes of substrate oxidation and phospho
rylation of ADP are only loosely coupled. Interestingly, this is
not the first time that a pathological condition has been found
to be associated with this particular mitochondrial abnormality.
7
Si-
Fig. 5. Effect of 2-deoxyglucose at 2.5 mM
on the growth of two human colon carcinoma
cell lines. ,4, FET; B, MIP101 cells.
E 4
¿
= 3
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ABERRANT COLON CANCER MITOCHONDRIA
In 1962 Luft et al. (19) described the case of a 35-yr-old woman
with severe hyperthermia and muscle myopathy. Electron mi
croscopic examination of a muscle biopsy revealed abnormally
large mitochondria with tubular cristae and rod-like inclusions.
Measurements of mitochondrial respiratory function indicated
normal State 3 respiratory rates (which occur in the presence
of substrate, P¡,and ADP), yet abnormally high rates of State
4 respiration (which occur when ADP is limiting). Luft et al.
attributed the patient's symptoms to the "loose coupling" of
muscle mitochondria.
We then considered whether these differences in bioenergetic
function would be expressed by mitochondria in situ. Fig. 3
illustrates that the mitochondrial membrane potential in
CCL237 and FET cells is much lower than that found in CV-1
and MIP101 cells. This accounts for the unusually low uptake
of rhodamine 123 observed in CCL237 and FET cell lines. In
addition, we show that, while nigericin hyperpolarizes the mi
tochondria of CV-1 and MIP101 cells, there is no such hyperpolarization in either CCL237 or FET cell lines (Fig. 4). This
can be explained by the fact that, while nigericin mediates the
electroneutral exchange of K+ ions for protons, hyperpolarization can occur only at the expense of an existing pH gradient
(as is the case for CV-1 and MIP101 ). However, if mitochondria
have an impaired respiratory capacity and/or are only loosely
coupled (as is the case for CCL237 and FET cells), there is no
substantial pH gradient and therefore no hyperpolarization
upon addition of nigericin. These results are significant in that
they confirm those obtained for isolated mitochondria and
establish the fact that the low RCR values of isolated FET and
CCL237 mitochondria do not result from damage to the organelles during the course of isolation.
Mitochondrial oxidative phosphorylation and glycolysis are
the two major pathways for cellular ATP production. It may
therefore be possible to obtain selective killing of those cells
exhibiting an impairment in oxidative metabolism by blocking
their alternative energy supply. We tested the effect of 2deoxyglucose, an inhibitor of glycolysis, on the growth of cul
tured FET cells (with aberrant mitochondria) and MIP101 cells
(with normal mitochondria). While the assay medium contained
glucose as its primary fuel source, it also contained oxidizable
substrates such as pyruvate, lipids, and various amino acids. As
shown in Fig. 5, 10 days of constant exposure to 2.5 mM 2deoxyglucose exert a potent cytotoxic effect on FET cells,
whereas in MIP 101 cells, it merely inhibits the rate of cell
growth. Interestingly, 2-deoxyglucose was found not to be cy
totoxic to CCL237 cells (data not shown), suggesting that it is
not the degree of coupling but rather the capacity for mitochon
drial respiration that is the determining factor for selective
killing by this compound.
The incidence of colon cancer in the United States remains
high. There are 160,000 cases reported annually, with 60,000
deaths per year being attributed to this disease. Most traditional
forms of treatment for this disease have been aimed at inhibition
of DNA metabolism. We appear to have identified a distinct
class of human colon carcinomas that display impairments in
mitochondrial bioenergetic function. The fact that selective
cytotoxicity of FET cells was obtained with 2-deoxyglucose is
important in that it suggests the possibility of exploiting this
phenotype as a target for chemotherapy. The frequency with
which an impairment in mitochondrial function occurs in hu
man colon carcinomas and the efficacy of 2-deoxyglucose in
the treatment of this class of cancer remain to be determined.
ACKNOWLEDGMENTS
We are indebted to Dr. June R. Aprille and Dr. Samuel Davis for
helpful discussions and review of the manuscript.
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3373
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Aberrant Mitochondria in Two Human Colon Carcinoma Cell
Lines
Josephine S. Modica-Napolitano, Glenn D. Steele, Jr. and Lan Bo Chen
Cancer Res 1989;49:3369-3373.
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