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Effect of Cisplatin on the Plasma Membrane
Phosphatase Activities in Ascites Sarcoma-
180 Cells: A Cytochemical Study

presented by

Iran Niroomand-Rad

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MASTER OF SCIENCE . Zoology
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EFFECT OF CISPLATIN ON THE PLASMA MEMBRANE PHOSPHATASE ACTIVITIES

IN ASCITES SARCOMA-ISO CELLS: A CYTOCHEMICAL STUDY

By

Iran Niroomand-Rad

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Zoology

1982

ABSTRACT

EFFECT OF CISPLATIN ON THE PLASMA MEMBRANE PHOSPHATASE ACTIVITIES
IN ASICTES SARCOMA-IBO CELLS: A CYTOCHEMICAL STUDY

By

Iran Niroomand-Rad

In order to study the effects of cisplatin [cis-dichorodiammine-
platinum (11)] on the tumor cells in presence or absence of the immune
system, animals with ascites sarcoma-180 tumor burden were treated with
therapeutic dose levels (9 mg/kg). Similarly, ascites sarcoma-180 cells
were maintained in tissue culture media containing the same levels of the
drug. Cell samples were taken from the animals at 12 hour intervals for 3
days where as samples were drawn from the tissue cultures at 15, 30, 45,
60 minutes, 2, 3, 4 and 5 hour intervals. Treated and untreated cells
from in zitrg and in viva experiments when checked for alkaline-
phosphatase, 5'-nucleotidase, Ca2+FATPase and Na+FK+-ATPase show a
gradual decrease in activity on the plasma membrane. It takes about 60
minutes for inactivation of any enzyme in X3252 while it takes 2 days in
viva experiments. Quantitative analysis show alkaline phosphatase
activity drops from 9.7 to 4.9 nmoles in just 15 minutes further droping
to 0.79 nmoles after 2 hours. Inactivation of various plasma membrane
enzymes resulting in permeability changes are probably responsible for

cell death.

Dedicated to my parents and Masood
to whom I owe everything

ii

ACKNOWLEDGEMENTS

I wish to thank Dr. S. K. Aggarwal, my research advisor, for his
constant support of this research with regard to the facilities and research
materials provided. I would like to express my appreciation to him, not
only for his expert technical advice and assistance, but also for the many
things he taught me that were non-science related. His constant help and
advice are greatly appreciated.

Appreciation is due also to Drs. J. L. Edwards and S. L. Flegler, who
served as guidance committee members and assisted in the preparation of this
thesis.

The author also expresses appreciation to Dr. K. M. Brown for his

advice concerning experimental technique.

iii

LIST OF TABLES.

LIST OF FIGURES

INTRODUCTION.

MATERIALS AND METHODS
Cytochemical Studies

Biochemical Studies

RESULTS .

DISCUSSION

REFERENCES

TABLE OF

iv

CONTENTS

Page
IV

36

41

LIST OF TABLES
Table Page
1 Plasma membrane phosphatas activities before and after

cisplatin treatment in the Ascites sarcoma-180 cells
E Vitro. C 0 O C O I O O O O O O O O O O I O O O O O O O O O O 20

LIST OF FIGURES

Figure page
1-3 Light micrographs (Alkaline phosphatase). . . . . . . . . . . lO
4 Electron micrograph (Alkaline phosphatase). . . . . . . . . . 12

5 Electron micrograph (Alkaline phosphatase). . . . . . . . . . 14

6 Graph showing Alkaline phosphatase activity . . . . . . . . . 17

7 Electron micrograph (5'-Nuc1eotidase) . . . . . . . . . . . . 19

8-10 Light micrographs (CaZ+-ATPase) . . . . . . . . . . . . . . . 22

11 Electron micrograph (Ca2+eATPase) . . . . . . . . . . . . . . 24
12 Electron micrograph (Ca2+-ATPase) . . . . . . . . . . . . . . 26
13 Electron micrograph (CaZ+-ATPase) . . . . . . . . . . . . . . 28
14 Electron micrograph (Caz+¥ATPase) . . . . . . . . . . . . . . 30
15 Electron micrograph (CaZ+-ATPase) . . . . . . . . . . . . . . 32
16 Graph showing in viva and in 33532 (Ca2+-ATPase). . . . . . . 34

vi

INTRODUCTION

Amminoplatinum (II) complexes have attracted considerable attention
as potential chemotherapeutic agents (2,3,46,48) against a broad spectrum
of chemically-induced, virally-induced and transplantable tumors both in
animals (49) and in humans (27). Of these, cis-dichlorodiammineplatinum
(II) (cisplatin, DDP) is now available for the treatment of testicular
(54) and ovarian cancers (28). However, the drug is not free from its
toxic side effects on the kidney (proteinuria and shedding of the renal
enzymes in the urine, together with changes in renal morphology),
intestine (emesis, anorexia and diarrhea), and toxicity to the
hematopoietic organs (bone marrow aplasia, splenic atrophy, atrophy of
thymus and lymph nodes) (3). Renal toxicity remains the most limiting
factor in its use. A second generation of platinum coordination
complexes with little or no nephrotoxicity is currently being tested in
clinical trials (16). Ways are still being sought to combat the toxic
side effects of the available compound through the use of various
diuretics and antagonists without rendering the drug ineffective against
tumor systems (46).

Although various symptoms due to use of the drug have been well
classified since its first discovery, the mechanisms of its action in
tumor regression or its various toxicities have not yet been fully
understood (42,47). Inhibitory effects of some platinum compounds on
DNA, RNA and protein synthesis have been demonstrated in vitrg using
mammalian cells (41,25). The interaction of cisplatin with DNA seems to

l

2
be through the cross-linking of the complementary strands of DNA (42,60).
If the DNA has already been replicated, cisplatin has been shown to
inhibit the process of karyokinesis (1). Again, if the nucleus has
already replicated the process of cytokinesis has been shown to be
arrested, probably through the depolymerization of microfilaments (1).
Further possible enhancement of the antigenicity of tumor cells (48) and
regression of the tumor through the immune system have been prOposed
(3,7,45).

In order for cells to perform their essential metabolic functions,
they must be able to transport ions and molecules against a concentration
gradient. Present investigations were undertaken to study various plasma
membrane enzymes such as Ca+2 ATPase, Na+-K+-ATPase, Alkaline
phosphatase and 5'-nucleotidase that are involved with the transport of
ions across the membrane, better to understand the mechanism of action of

cisplatin in tumor regression and the cause of its various toxicities.

MATERIALS AND METHODS

In Vitro Studies

 

Highly inbred Swiss Webster female mice 4-5 weeks old (Spartan
Research Animals, Haslett, Michigan) were implanted intraperitoneally
with ascites sarcoma-180 according to protocols of the Cancer
Chemotherapy National Service Center (50). The day of tumor implant was
taken as day 0. The animals were sacrificed by cervical dislocation on
day 8 of the tumor implant. Ascites fluid was removed with the help of a
disposable pipette and transferred to culture flasks containing Eagle's
Basal Medium (BME) with Eagle's Salt (Gibco, Grand Island, N.Y.)
containing 2x amino acids with glutamine supplemented with 10% calf
serum. Cultures were maintained at 37°C. Cell counts were made at the
start of each experiment using a Coulter counter model 281. A
concentration of 107 cells/ml was a standard for each experiment.

Cisplatin (Johnson Matthey Research Laboratories, Sonning Commons,
Reading, U.K.) was added to the cultures as a freshly prepared solution
(stock solution 9 mg/lO m1 of 0.752 sterile saline) at a concentration of
0.9 mg/100 ml of the culture. Aliquotes of cells were withdrawn at 0,
15, 30, 45 and 60 minutes, 2, 3, 4 and 5 hours and were fixed in 1%
buffered glutaraldehyde (0.05 M cacodylate buffer, pH 7.3) for 10 minutes
at 4°C. After fixation, cells were washed twice in buffered sucrose
(3.5% sucrose in 0.1 M cacodylate buffer, pH 7.3) and stored at 4°C until

use.

Animal Studies

 

Swiss Webster female mice 4-5 weeks old with ascites sarcoma-180 were
injected with sterile cisplatin solution (9 mg/kg) as intraperitoneal
injection on day 8 of the tumor implant. The animals were sacrificed at
12 hour intervals (6 in each group) for a period of 8 days and the cells
were collected using a disposable pipette and processed for

Ca2+¥activated ATPase studies.

Cytochemical Studies

 

Ascites sarcoma-180 cells after various intervals of drug treatment
and fixation were incubated in one of the several media as described
below.

Alkaline Phosphatase (AP) activity was detected after incubation of

 

cells at 37°C for 60 minutes in a medium containing 0.2 M Tris-maleate
buffer (pH 8.2), 1.2% sodium-B -glycerophosphate, 0.2 mM magnesium
chloride, 1% lead nitrate according to the method of Hugon and Borgers
(29). The control incubation medium contained in addition 0.5 mM
levamisole hydrochloride (13) or 50 mM L-phenylalanine (33) as the
inhibitors of AP.

5'-Nucleotidase (5'-N). For the visualization of 5'-N, the cell

 

suspension was incubated at 37°C for 60 minutes according to the
technique of Uusitalo and Karnovsky (52). The medium consisted of 0.1 M
Tris-maleate buffer (pH 7.3), 1.4 M adenosine-5'-monophosphate (AMP), 1%
lead nitrate, 10 mM magnesium sulfate and 5% sucrose. The control medium
contained in addition SOLIM ofa,,3 ~methy1ene adenosine diphosphate (ADP)
(43) or else the substrate AMP was omitted.

Ca2+-activated ATPase (Ca2+-ATPase) was detected by

 

incubating the cell suspensions in a medium containing 0.1 M Tris-maleate

5
(pH 7.3), 5% sucrose, 2 mM lead nitrate, 1 mM adenosine triphosphatase
(ATP), 1 mM calcium chloride, 0.2 mM Magnesium chloride (17,43). The
incubation lasted for 60 minutes at 37°C. The control incubation medium
contained in addition 15 ug/ml quercetin (an inhibitor of Ca2+eATPase
(19)) or the ATP was omitted from the incubations.

Na+-K+-activated ATPase (Na+-K+-ATPase) activity was

 

determined by incubating the cell suspension in a medium consisting of
0.1 M Tris-maleate buffer (pH 7.3), 1 mM ATP, 1% lead nitrate, 10 mM
magnesium sulfate, 5% sucrose and 100 mM sodium chloride (36). Controls
were incubated in the same medium but without sodium chloride or ATP.
Incubations were performed at 37°C for 60 minutes.

Thick (lum) frozen sections of normal mouse liver and kidney were
used to check for the reactivity of the various incubation media while
also incubating various sarcoma-180 cell samples. All cell samples from
various incubation media were washed in ice-cold corresponding buffer
identical to the one used for incubation. Half of the cells from each
sample were stained with 1% light ammonium sulfide and mounted in
glycerine jelly after proper washings. Such slides were viewed using a
Zeiss Photomicroscope II. Photomicrographs were prepared of random areas
from the various samples. The other half of the cell samples were fixed
in buffered 1% 0304 (pH 7.3) for 1 hr at 4°C and processed for routine
electron microscopy. Ultrathin sections were cut on an LKB ultratome III
ultramicrotome and were viewed in a Hitachi-HUllE electron microscope
with or without uranyl acetate and lead citrate staining. The enzyme
reaction product was visualized as an electron dense deposit on the

surface of the cells. Records were made from the electron micrographs of

6
very high response (++++), moderate response (+++), low response (++), or

a very poor response (+) and compared for the effects of the drug.

Biochemical assays

 

Detailed biochemical quantitations were carried out in the case of
alkaline phosphatase only. Cells from various intervals of cisplatin
treatment were homogenized in carbonate-bicarbonate buffer (pH 10.0) (1:5
w/v) using 16 full strokes of a Potter-Elvehyem homogenizer fitted with a
teflon pestle rotating at 2,000 rpm (57). 0.3 ml of the homogenate was
added to 0.6 ml of carbonate-bicarbonate buffer (pH 10.0) and mixed with
0.03 ml of 100 mM magnesium sulfate. The mixture was incubated at 30°C
for 10 minutes before adding 0.07 ml of 87.6 mM disodium-p-nitrophenyl
phosphate dissolved in 0.1 ml of the same buffer to start the reaction
(32). An increase in the absorption at 400 mu was recorded as a result
of the release of inorganic phosphate (P1) (34). The controls consisted
of incubation medium without the cell sample or of medium and cells to
which 0.5 mM levamisole had been added (13). Increase in absorption at
400 mu was measured using a Beckman 25 spectrophotometer with recorder.
The cuvettes used had a 1 cm path length and a 1 ml capacity. Enzyme
activity was further calculated using Beer's law and expressed as nM

nitrophenyl/min/mg protein (53).

Protein determination

 

Standard protein solutions containing 1.0-10.0 ug of bovine serum
albumin (Sigma Chemical Co., St. Louis, M0) were prepared in 50 ul of
Tris-buffered saline and 950 ul of the dye reagent (39). A standard

curve was prepared from an absorption at 595 my to determine the protein

concentrations in unknown samples.

7
It was found necessary to further dilute the above cell homogenates
by a factor of 20x in order for the readings to fall within the standard
curve. 50 ul of the diluted samples were added to 950 (a of the dye

reagent and read for absorption at 595 mu for 2 minutes.

RESULTS

Various effects of cisplatin on cellular morphology have already
been reviewed (8) and therefore will not be repeated here. Since no
attempt was made to synchronize the cells in a certain phase of the cell
cycle, the following observations are based on random cells in various

stages of the cell cycle.

Alkaline Phosphatase

 

Alkaline phosphatase activity as depicted by the reaction product
(r.p.) after 1 hr of incubation appears as dense deposits only on plasma
membranes (Fig. 1). It may be pointed out that not all the cells in a
random section show positive r.p. As many as 10-20% of the cell total
may be negative. The number of the dense deposits decreases as the
length of exposure of the cells to cisplatin is increased (Fig 2). The
activity totally disappears after 45 minutes of such an exposure (Fig. 3
and Table I). At the electron microscope level, the reaction product
appears as a thin, continuous, electron dense layer interspersed with
dense patches on the outer surface of the cells (Fig. 4). The undulating
segments of the plasma membrane, together with the microvilli, show the
highest amount of deposits. The number of electron dense patches
gradually decreases after cisplatin treatment, until, after 2 hours of
treatment one can hardly see any r.p. on the cell surface (Fig. 5).
Similarly no r.p. could be observed in cells incubated in a medium

containing either levamisole or L-phenylalanine and without cisplatin

treatment .

Fig. 1 Localization of alkaline phosphatase around normal
sarcoma-180 cells. Note the uniform distribution of
reaction product (r.p.) on the plasma membrane (arrows).
Original magnification x3600. Bar = 511m.

Fig. 2 Light mirograph taken with Nomarski Optics showing the
r.p. in patches (arrow) after 30 minutes of cisplatin.
Original magnification x1600. Bar = lOle.

Fig. 3 No r.p. can be observed after 45 minutes of cisplatin
treatment. Photomicrograph taken with Nomarski Optics.
Original magnification x1600. Bar = 1011m.

10

 

 

 

11

Fig. 4 Electron micrograph depicting alkaline phosphatase
(arrows) on the surface of sarcoma-180 cells. N, nucleus.
Original magnification x12000. Bar = 111m.

 

12

 

 

Fig. 5

13

No r.p. on the surface of cisplatin (2 hrs) treated cell.
N, nucleus; Nu, nucleolus. Original magnification x8600.

 

14

 

 

 

15

The visual cytological observations are further confirmed by
biochemical determination of AP activity (Fig. 6). The treated cells
show a change in alkaline phosphatase activity from 9.7 to 4.9 nmoles
after just 15 minutes of exposure to cisplatin. These values drop to
0.79 nmoles after about 2 hours. Since observation ceased after 3 hours,
it is not known if the value ever drops to zero.
_521

As in the case of alkaline phosphatase, the cytochemical reaction
for 5'-nuc1eotidase is mainly localized on the plasma membrane (Fig. 7).
The reaction product is more abundant on the microvilli and on the
undulations than on the nonundulated regions of the plasma membrane.
Enzyme activity is vastly affected by cisplatin over a time period of 2

hrs (Table I). Again no r.p. is observed after incubation in a medium

containing B-methyl ADP, or when the substrate is omitted.

Ca2+-ATPase

 

Cells that have been incubated in a medium for Ca2+-ATPase activity,

when viewed under the light microscope, show r.p. on the cell surface as
dark granules (Fig. 8). This granulation decreases when the cells are
incubated in a culture medium containing cisplatin (Fig. 9). The r.p.
completely disappears after 120 minutes of exposure to cisplatin (Fig
10). At the electron microscope level, the r.p. appears on the surface
of almost all the cells as electron dense granules (in both the resting
and the actively dividing cells) (Figs. 11-12). At times, r.p. can also
be observed inside some vacuoles and around lipid globules. The r.p.
decreases gradually from the surface as the time of treatment with

cisplatin is increased from.0 to 120 minutes (Figs. 13-15 and Table I).

Quercetin exposed cells show no reaction product.

 

16

Fig. 6 Phosphatase activity (n moles;)-nitrophenol/min/mg
protein) in ascites sarcoma-180 cell homogenates before
and after cisplatin treatment (0.9 mg/100 ml).

 

Alkallne phosphatase acllvlly (nmoles/min/mg protein)

lO-1

8..

b.

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17

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Time after ciSplatin treatment (minutes)

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18

Fig. 7 5'-N r.p. (arrows) distributed on the plasma membrane.
N, nucleus; Nu, nucleolus. Original magnification
x20,000. Bar = l um.

 

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O .TTI. .III. III: .TTI.
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8 I H ++ ++
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-Trr+ a <mn< manosm ammonwos
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somenn<o «moanwos

Fig. 8

Fig. 9

Fig. 10

21

Reaction product (arrows) depicting CaZ+-ATPase on
the surface of untreated sarcoma-180 cells. Original
magnification x3000. Bar = 511m.

Ascites sarcoma-180 cells after 30 minutes of cisplatin
treatment showing only patches of reaction product
indicative of Ca +-ATPase activity. Original
magnification x3000. Bar = 514m.

Ascites sarcoma—180 cells after 2 hrs of ciSplatin
treatment showing no r.p. for CaZ+-ATPasé.- Original
magnification x3600. Bar = 511m.

 

22

 

Fig. 11

23

Electron micrograph show Ca2+-activated ATPase r.p.
(arrows) on the surface of a sarcoma-180 cell during
interphase. M, mitochondria; N, Nucleus. Original
magnification x25000. Bar = 0.511m.

24

 

25

Fig. 12 Ascites sarcoma-180 cell in metaphase showing
Ca2+-ATPase r.p. (arrows) on the surface. CH,
chromosomes. Original magnification x22500. Bar = llJm.

26

awn Emma.

.1

 

I... ”... ..r ..‘N
u meramkrslfia 4.x. . a

. I... win

 

27

Fig. 13 Interface between two ascites sarcoma-180 cells showing
Caz+-ATPase r.p. (arrows) after exposure to cisplatin
for 30 minutes. N, nucleus. Original magnification
x25,000. Bar = 1 pm.

 

 

29

Fig. 14 Interface between two ascites sarcoma cells showing
Ca2+-ATPase r.p. after 45 minutes of cisplatin
treatment (arrow). Note the sparse distribution of
reaction product. N, nucleus. Original magnification

x18000. Bar = l Um.

3O

 

 

31

Fig. 15 Ascites sarcoma-180 cell in metaphase eXposed to
cisplatin for 2 hrs showing no r.p. for Ca2+-ATPase.
Compare with Fig. 12 for normal reaction. CH,
chromosomes. Original magnification x25,000. Bar = 1 pm.

32

 

33

Fig. 16. Graph showing changes in the Ca2+-activated ATPase as
depicted by the concentration of reaction product of
various incubations in ascites sarcoma-180 ig_vitro and

in vivo after cisplatin treatment.

35

Na+-K+-ATPase

 

The plasma membrane distribution patterns for Na+-K+-ATPase are
exactly similar to those for Ca2+-ATPase, both before and after
cisplatin treatment, and are summarized in Table 1.

In ighzitgg studies most of the above enzymes on the cell surfaces
are totally inactivated within 2 hours of their exposure to cisplatin.
However, in the in Kilo studies it takes about 2 days to achieve the same

results (Fig. 16).

DISCUSSION

In the present study we have investigated the effects of cisplatin
on plasma membrane enzymes because of their importance in the homeostasis
of the cell. Control of plasma membrane functions such as transport of
metabolites (23), cell growth (25), synthesis and breakdown of cyclic
nucleotides (24) and the process of mitosis (6) are known to be affected
by cisplatin.

Alkaline phosphatase has been demonstrated to be mostly associated
with the plasma membrane (40). In the liver, the enzyme is present
predominantly in the plasma membranes of the bile canaliculi and does not
extend appreciably to the basal or the lateral membranes beyond the tight
juctions (20).

The discovery of alkaline phosphastase in the proximal convoluted
tubules of the kidney, the intestinal microvilli, and the follicular
epithelium of the ovary suggests some role for this enzyme in active
transport of substances across the membrane (18). It is known that this
enzyme is needed for the hydrolysis of extracellular phosphate esters, so
that the reaction products such as orthophosphate and organic residues
can then be transported into the cell (56). Further evidence for a
transport function of alkaline phosphatase comes from the finding that
some mutant strains of E, 221$.Wh1Ch lack alkaline phosphatase are unable
to grow in phosphate-containing media (22). Since alkaline phosphatase

is able to transfer phosphate groups from one alcohol to another, it is

36

37
possible that its physiological function depends on its transferase
activity, rather than its hydrolase activity (55). Thus,
membrane-associated alkaline phosphatase activities are closely related
to, if not identical with, Ca2+-dependent ATPase (11).

A parallel can be found between the activity of alkaline phosphatase
and DNA synthesis in synchronized HeLa cells, in that high specific
activity of AP has been used as a marker for the mitotic plasma membranes
(35). However, just the contrary has been demonstrated using certain
aliphatic monocarboxylates that inhibit cell division in HeLa cells, yet
alkaline phosphatase activity is increased on the plasma membrane (53).
Similar conclusions have been drawn concerning cultured human
fibroblasts, which, when treated with DNA inhibitors like hydroxyurea and
methothrexate, show elevated levels of alkaline phosphatase (58).
However, using cisplatin, which is a known inhibitor of DNA synthesis and
a mitotic inhibitor, we have observed a distinct drop in the enzyme
activity in both inuziggg and in Xilg studies. In the in zi££2_system it
takes about 60 minutes for an inactivation of the enzyme, whereas in the
in_vixg system it takes about 2 days using the same ascites sarcoma-180
cells. This is probably due to the fact that the levels of cisplatin in
the in XEEEE system are much higher than in the animal. In the animal
system, 70-90% of the injected drug is usually excreted in the initial
minutes of its injection (45) and thus the amount available to the cells
for interaction is very small. Inhibition of DNA by cisplatin has been
determined to occur through an intrastrand and interstrand crosslinking
with nuclear DNA (59,30). In either case, the prime interaction is with
the purine and pyrimidine bases. Inhibition of DNA synthesis and

increase or decrease of alkaline phosphatase activity therefore, do not

seem to be interrelated.

38

A direct inhibition of various enzymes after cisplatin treatment has
been reported (21), however, the drug concentrations used in such assay
systems are much higher than those normally found in cells. Alkaline
phosphatase activity in the urine of cisplatin-treated rats shows a
50-70% increase with a corresponding decrease in the kidney homogenates,
indicating a stripping of the enzyme from the brush borders (5). A
similar stripping effect of cisplatin has been demonstrated on sialic
acid in normal splenocytes and transformed lymphocytes in culture (38).

Patients suffering from carcinoma of the testis when treated with
cisplatin show a 75% decrease in plasma sialyltransferase that
corresponds with a relative decrease in the size of the tumor (31).
However, the enzyme by itself is again unaffected by cisplatin in the
assay system.

Our observations show 5'-nucleotidase activity mostly on the plasma
membrane of the ascites sarcoma-180 cells. S'-Nuc1eotidase has been
extensively studied as an ectoenzyme and is often bound to the brush
borders of various cells (43). 5'-Nuc1eotidase is one of the enzymes
directly involved in the metabolism of adenosine and indirectly in the
synthesis of ATP and cyclic AMP and also in the breakdown of the latter
(53). Plasma membranes are permeable to nucleosides whereas nucleotides
are unable to traverse the membrane intact. Hence 5'-nucleotidase is
involved in the dephosphorylation of nucleoside monophosphate and the
nucleoside product may then be internalized (44,40). Thus inhibition of
this enzyme would inhibit further DNA synthesis because of unavailability
of various nucleotides needed. Cisplatin indeed does affect the enzyme
activity at all stages of the cell cycle, therefore interfering with cell

metabolism and replication.

39
Ca2+-ATPase is involved in the transport of Ca2+ ions across
the plasma membrane and in regulating the level of these ions inside the
cell (44). Ca2+ plays a key role in regulating cell metabolic
processes by stimulating or inhibiting key enzymes (14). In general,

2+

cells must keep the intracellular Ca concentration at a very low

level in order to maintain Na+FK+-ATPase in an active state. Even a

small increase in internal Ca2+

would therefore act as a powerful
messenger of extracellular membrane stimuli (44).

A correlation has been proposed between Ca2+-ATPase activity and
the chromosomal cycle to a point that the enzyme activity has been shown
to be increased as the cell prepares for mitosis (37). However, we have
not observed any significant increase or decrease in Ca2+-ATPase
activity on the plasma membrane of the interphase or actively dividing
sarcoma-180 cells. Cisplatin knocks out all ATPase activity from the
plasma membrane irrespective of the stage of cell cycle. Cisplatin has

2+-ATPase activity from

also been shown to cause an inactivation of Ca
the brush border of the proximal tubule cells (4). A parallel has
further been established in the inactivation of CaZ+- ATPase
activity and Ca2+ ions within mitochondria (8,12). If the internal
generation of Pi is prevented, Ca2+ is spontaneously released from
the mitochondria (15). Lowering the internal concentration of Pi with
the use of apprOpriate inhibitors leads to the release of accumulated
Ca2+, which is related to the permeability of the membrane.

Various platinum coordination complexes including cisplatin have
been shown to stimulate state 4 respiration which is similar to

uncoupling of oxidative phosphorylation (8,12), again causing effux of

Ca2+ from the mitochondria. The membrane permeability changes have

40
been implicated in the release of various hydrolytic enzymes from the
lysosomes, thus inducing damage to the cell. Various compounds that are
competitors or inhibitors of Ca2+ sites (26) and block Ca2+
transport mechanisms have also been proven to be potent antitumor agents
(9).

Using frog skin as a model, it has further been demonstrated that
cisplatin increases the permeability of the cellular pathway (10), an
observation common to other antineoplastic agents such as adriamycin

2+ are known to shut down

(51). Increased levels of intracellular Ca
Na+-K+-ATPase which is an integral glycoprotein of the plasma

membrane. Guarino SE 213 (23) have demonstrated an inactivation of
Na+—K+-ATPase in isolated kidney tubules under the influence of
cisplatin. They have implicated this inactivation of ATPase to be
responsible for the kidney toxicity so very prominent after cisplatin
treatment.

From the above considerations, we conclude that cisplatin is
responsible for the inactivation of various plasma membrane associated
enzymes that are very important in the normal metabolic processes of the
cell, such as transport, synthesis, breakdown of cyclic nucleotides and
mitosis. The drug does not seem to be selective in its action as it
affects normal cells and tumor cells alike. However, the tumor cells are
known to be metabolically more active, and are probably destroyed faster
than they have time to recover the various membrane enzymes. Thus, in
addition to the inhibition of DNA synthesis by crosslinking the

complementary strands, cisplatin also functions to alter the surface

properties of cells to cause antitumor or cytotoxic effects.

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