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ABSTRACT

THE RELATIONSHIP BETWEEN cis-Pt(II) AND HOST
IMMUNE RESPONSE IN A SYNGENEIC SYSTEM

BY
Douglas E. King

The exact mode of action of antitumor platinum com-
pounds has not been determined, although much research has
been devoted to this subject. One hypothesis, the enhanced
antigenicity hypothesis, suggests that these antineoplastic
agents [particularly cis-dichlorodiammineplatinum(II):
cis-Pt(II)] function by enhancing the expression of tumor
specific antigens on tumor cell surfaces, thereby allowing
the intact immune system to attack and destroy the tumor.
This study was undertaken to evaluate the relationship
between the host immune response and the antitumor activity
of cis-Pt(II).

Immunocompetent DBA/Z female mice were x-irradiated
with 700 rads whole-body x-irradiation (to suppress the

immune system), injected with 4 x 106

P388 lymphoma cells,
and treated with cis—Pt(II). Comparing the growth curve
of the P388 cells in this syngeneic system with that in
various control groups, e.g., mice with tumor alone, mice

with x-ray and tumor, and mice with tumor and cis-Pt(II),

Douglas E. King
insight into the relationship between the immune system and
cis-Pt(II) was obtained.

The results of this study indicate that the ability
of cis-Pt(II) to kill P388 tumor cells in this syngeneic
system is not undermined by suppression of the immune
response (as determined by the hemolytic plaque assay).
Tumor cells are destroyed with equal or increased efficiency
in cis-Pt(II)—treated, x-irradiated mice than in nonirradi-

ated, tumored mice treated with cis—Pt(II).

THE RELATIONSHIP BETWEEN cis-Pt(II) AND HOST

IMMUNE RESPONSE IN A SYNGENEIC SYSTEM

By
.8

kNWp .
Douglas E. King

A THESIS

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

MASTER OF SCIENCE

Department of Pathology

1977

Dedicated to Joan and Elizabeth

to whom I owe everything

ii

ACKNOWLEDGEMENTS

I wish to thank Dr. Barnett Rosenberg, my research
advisor, for his constant support of this research with
regard to the facilities and research materials provided.
His assistance and advice are greatly appreciated.

I cannot begin to express my appreciation to Loretta
VanCamp, not only for her expert technical advice and
assistance, but also for the many things she taught me
that were non-science related. She was thoughtful and
considerate of her fellow workers, and always managed a
smile even in the most distressing situations.

Special thanks to Martha Thomas, my academic advisor
and friend, for supplying some of the equipment for this
research, and for putting up with me in trying times.

Deep appreciation is due to Dr. John Dunkel, who was
always willing to unselfishly give of himself and his time
to listen to my problems.

Thanks also to Dr. Harold Miller for his advice con-
cerning the immunological aspects of this research, and
to Barbara Laughter for teaching me the hemolytic plaque
assay.

Appreciation is also expressed to Ann Grimes and Linda

Glover for their assistance with this research project.

iii

Special thanks and deep appreciation to my wife, Joan,

for her support and love in difficult times.

iv

TABLE OF CONTENTS

Page
INTRODUCTION. . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . 3

MATERIALS AND METHODS . . . . . . . . . . . . . . . 14

Experimental Animals . . . . . . . . . . . . 14
Drugs. . . . . . . . . . . . . . . . . 14
X- Irradiation. . . . . . . . . . . . . . . . 15
Tumor. . . . . . . . . . . . . 15
Tumor Cell Quantitation. . . . . . . . . . . 16
Experimental Scheme. . . . . . . . . . . . . l7

Hemolytic Plaque Assay . . . . . . . . . . . 18
RESULTS . . . . . . . . . . . . . . . . . . . . . . 20

Trial 1.. . . . . 20
The Growth Curve of P388 Cells
in DBA/Z Female Mice. . . . . 20

The Growth Curve of P388 Cells in

X- Irradiated DBA/Z Female Mice. . . 20
The Growth Curve of P388 Cells in

DBA/Z Female Mice Treated with

S x 1. 5 mg/kg cie- Pt(II). . . . 23
The Growth Curve of P388 Cells in

X- Irradiated DBA/Z Female Mice

Treated with S x 1.5 mg/kg cis-

Pt(II). . . . . . . . . . . . . . . 23

Trial 2.. . . . . 28
The Growth Curve of P388 Cells

in DBA/Z Female Mice. . . . . 28

The Growth Curve of P388 Cells in

X- Irradiated DBA/Z Female Mice. . . 28
The Growth Curve of P388 Cells in

DBA/2 Female Mice Treated with

5 x 1.5 mg/kg eta-Pt(II). . . . . . 35
The Growth Curve of P388 Cells in

X-Irradiated DEA/2 Female Mice

Treated with S x 1. 5 mg/kg cis-

Pt(II). . . 35
The Number of Plaque Forming Cells

in Spleens from DBA/Z Mice Sensi-

tized with Sheep Red Blood Cells. . 3S

Page
DISCUSSION. . . . . . . . . . . . . . . . . . . . . 46
CONCLUSIONS . . . . . . . . . . . . . . . . . . . . 50
REFERENCES. . . . . . . . . . . . . . . . . . . . . 52
VITA. . . . . . . . . . . . . . . . . . . . . . . . 56

vi

Table

II

III

IV

VI

VII

VIII

IX

LIST OF TABLES

Number of P388 cells in DBA/Z female mice:
Trial 1.. . . . . . . . . . . . .

Number of P388 cells in irradiated DBA/Z
female mice: Trial 1.. . .

Number of P388 cells in DBA/Z female mice
treated with 5 x 1. 5 mg/kg cis- Pt(II):
Trial 1. O O I O O O O O O O O 0

Number of P388 cells in irradiated DBA/Z
female mice treated with S x 1.5 mg/kg cis-
Pt(II): Trial 1 . . . . . . . . . . . .

Number of P388 cells in DBA/Z female mice:
Trial 2.. . . . . . . . . . . . . .

Number of P388 cells in irradiated DBA/Z
female mice: Trial 2. . . . . . . . . .

Number of P388 cells in DBA/Z female mice
treated with 5 x 1. 5 mg/kg. cis- Pt(II):
Trial 20 O O O O O O O O O 0

Number of P388 cells in irradiated DBA/Z
female mice treated with S x 1.5 mg/kg cis-
Pt(II): Trial 2 . . . . . . . . . . . .

Antibody response in spleens as measured
by the hemolytic plaque assay.

vii

Page

21

24

26

29

31

34

38

41

4S

Figure

10

11

12

LIST OF FIGURES

Growth curve of P388 cells in DBA/Z
female mice: Trial 1.. . . . .

Growth curve of P388 cells in irradiated
DBA/Z female mice: Trial 1.

Growth curve of P388 cells in DBA/Z female

mice treated with S x 1.5 mg/kg cis'Pt(II)2

Trial 1. O O O O O O 0

Growth curve of P388 cells in irradiated
DBA/Z female mice treated with S x 1.5
mg/kg gig-Pt(II): Trial 1 . . . . . .

Growth curve of P388 cells in DBA/Z female
mice: Trial 2 . . . . . . . . . .

Growth curves of P388 cells in DBA/Z female

mice: Trials 1 and 2.. . . . . . . .

Growth curve of P388 cells in irradiated
DBA/Z female mice: Trial 2. . . . . . .

Growth curves of P388 cells irradiated
DBA/Z female mice: Trials 1 and 2 . .

Growth curve of P388 cells in DBA/Z female
mice treated with S x 1. 5 mg/kg cis- Pt(II):
Trial 2.. . . . . . . . . . . . . . . .

Growth curves of P388 cells in DEA/2 female
mice treated with S x 1. 5 mg/kg cis- Pt(II):

Trials 1 and 2 . . . . . . . . . . . . .

Growth curve of P388 cells in irradiated
DBA/Z female mice treated with S x 1.5 mg/
kg cis-Pt(II): Trial 2. . . . . . . . . .

Growth curves of P388 cells in irradiated

DBA/2 female mice treated with S x 1. 5 .mg/
kg cis- Pt(II): Trials 1 and 2 . .

viii

Page

22

25

27

30

32

33

36

37

39

40

42

43

INTRODUCTION

The treatment of malignant disease by chemotherapy
has been used increasingly for the past several years.
Many effective chemotherapeutic agents with different
modes of action have been utilized efficiently against
different tumor types, and now are important tools in the
oncologist‘s armamentarium.

Experimental evidence indicates that coordination
complexes of platinum are potent antineoplastic agents. V
The most widely studied of these complexes, cis-dichloro-
diammineplatinum(II): ate-Pt(II), has been shown to be
a potent, broad spectrum antitumor compound in experi-
mental animals and human patients.

Although a great deal of time and effort has been
spent attempting to delineate the mode of action of these
complexes, the exact mechanism remains an enigma. One
hypothesis of particular interest suggests that the anti-
tumor activity of platinum compounds is related to their
ability to interact with the tumor-host immune system in
such a way that the expression and recognition of tumor
specific antigens is enhanced. This enhanced antigenicity v/
hypothesis was prOposed to interject specificity into the
mode of action of these compounds. This was needed

because, relative to nonneOplastic tissues (in viva),

1

2
there is no selective uptake of these platinum complexes
by neOplastic tissue.

This study was undertaken to examine the relationship
between the host immune system and the antitumor activity
of cis-Pt(II) in a syngeneic animal-tumor model. By modu-
lating the immunocompetence of host mice, it is hoped that
the validity of the enhanced antigenicity hypothesis can

be tested.

LITERATURE REVIEW

The biological activity of platinum complexes has
been known since its serendipitous discovery by Rosenberg
and VanCamp in 1965 (36). They were studying the electric
field effects on cultured Escherichia coli when they
noticed that the bacteria were growing into long, fila-
mentous rods, an indication that mitotic division had
ceased. Attempts to isolate the component(s) responsible
for the mitotic inhibition were successful (35), and it
was found that with low frequency alternating current
applied across the platinum electrodes, approximately 8
ppm of platinum went into solution in the steady state,
forming what was then thought to be the [PtCL6l' complex.

It was later observed (by L. VanCamp) that if solu-
tions of the complex were allowed to stand for a few days
their ability to induce filamentous growth in Escherichia
coli increased. That is, some sort of photochemical change
occurred which produced compounds more effective in inhibit-
ing mitotic division. Subsequently, it was shown that
when solutions of the doubly negative complex ([PtCL6]'),
which is actually a bacteriocide, were irradiated by light,
one or more ligand chloride ions was replaced by an ammonia
group from the bacterial medium (see reactions 1 and 2).
Replacement of one chloride ion produced the singly negative

3

4

[PtCl NH3]' complex, which has little or no effect on

S
E. coZi.y The neutral [PtCl4(NH3)2]0 complex, formed by g“
the replacement of two chloride ions is the most stable
form, and has no inhibitory effect on growth at concen—

trations where it completely inhibits cell division.

1) [PtCl6]-Z + NH4 -—h—"—> [Ptc1SNH31' + c1' + H"

- hv 0 - +
2) [PtClSNH3] + NH4 ———9 [PtCl4(NH3)2] + C1 + H

A variety of agents may cause filamentous growth in
Escherichia coli (16). Cytokinesis in bacteria subjected
to these agents, e.g., radiation, pressure, or temperature
changes, may be reinitiated by treatment with pantoyl
lactone, divalent cations or a temperature of 42° Centi-
grade. Rosenberg et a1. (33) showed that the inhibitory
effect of platinum on cytokinesis in bacteria could not
be reversed by treatment with these agents and only by
removal of the neutral platinum complex ([PtC14(NH3)2]O)
was cross-septation noticed. In addition, it was shown
that only Gram negative bacilli were sensitive to the
platinum complex with regard to its "antimitotic" activity.

Research to determine the antitumor activity of
platinum compounds was completed in 1969 when Rosenberg
et al. (34,37) reported that certain of the platinum com-
plexes were effective inhibitors of L1210 leukemia and
8-180 solid tumor in mice. Subsequent research indicated
that platinum complexes (particularly cis-tetrachloro-

diammineplatinum(IV) and cis-dichlorodiammineplatinum(II)

5
[cis-Pt(II)] were effective broad spectrum antitumor agents
in a variety of hosts, against virally induced, chemically
induced, and transplantable tumors. (See reference 30,
page 401, for a review of this antitumor activity.)

In addition to studies involving the above mentioned
tumors in experimental animals, numerous clinical trials
in human patients have been completed. These trials have
established the current mode of administration of cis-Pt(II)
as well as its toxic effects on nonneoplastic tissue.

While excellent results were obtained with cis-Pt(II)
against testicular tumors (11,12), a considerable number
of different tumor types have been shown to be sensitive
to the drug as well. Thus, as in experimental animals,
cia-Pt(II) has a wide range of antineOplastic activity in
human patients (20,30).

The relationship between the structure and activity
of platinum complexes has been studied by Cleare and
Hoeschele (6). They observed a marked dissimilarity in
the antitumor activity of cis— and trans-platinum analogues.
When trans isomers exist, they were found to be inactive
in comparison to the active cis isomer. The trans isomers
are more reactive chemically and the trans isomer of
[Pt(NH3)2C12] aquates four times faster and undergoes
ammonation thirty times faster than the cis form (7). The
authors suggested that the trans isomers apparently react
with a wider variety of molecules than the cis form, hence
decreasing the specificity of their action. This was

195m

suggested by Pt-labelled [Pt(NH3)2C1z] excretion

J'

6
studies which showed a higher initial excretion rate for
the cis isomer. The whole-body retention of both isomers
after five days was, however, comparable (approximately
20%) (6).

Replacement of the chloride ions in cis-[Pt(NH3)2ClZ]
with other anions greatly affected the toxic and antitumor
properties of the complex. Groups which were loosely bound
to platinum, e.g., NOE, H20, exhibited little or no anti-
tumor activity and were highly toxic. Ions, e.g., Cl-
and Br', with an intermediate lability (less readily
replaced by H20: reactions 3 and 4), demonstrated good
antitumor activity with minimal toxicity, while those
which were bound tightly to platinum, e.g., SCN', N02,
and CN', exhibited no activity whatsoever, and little
toxicity. It was suggested that readily replaced ligands
react rapidly and nonspecifically in vivo, leaving little
compound to react at sites where antitumor activity takes
place. Complexes with tightly bound groups which reach
the proper sites interact only minimally and have no anti-

tumor activity.
3) [Pt(II)(NH3)2C12]0 + H20 ——» [Pt(II)(NH3)2C1(HZO)]+ + c1'

4) [Pt(II)(Mupzmcfizon+ + H20 ...._. [Pt(II)(NH3)2(HZO)z]+++ (21‘

Variation of the ammine group in [Pt(II)(NH3)2C12]

modified the antitumor activity of the complex by virtue

7
of their differing steric, electronic and basic properties,
perhaps by interfering with its ability to hydrogen bond
to receptor biomolecules. This was suggested because
increased alkyl substitution of the hydrogens in NH3 led
to decreased antineoplastic activity.

The charge of the platinum complex greatly affects its
antitumor properties. To date, only neutral species have
demonstrated activity. This is probably related to mem-
brane transport functions and/or to the body's ability to
excrete charged molecules (increased H20 solubility) at a
faster rate than noncharged ones.

From the experimental work cited above, Cleare and
Hoeschele (5,6) have established a set of "empirical rules"
that are essential for antitumor activity of platinum com- /“
pounds. They are: (1) The complex should be neutral and
(2) contain a pair of cis leaving groups. (3) The "window
of liability" for the leaving groups should be centered
on the chloride ion. (4) The "carrier ligands" should be
strongly bonded, relatively inert ligands, preferentially
amines. (5) The complexes are square planar or octahedral
in configuration.

As early as 1970 data were being reported with regard
to the mode of action of antitumor platinum complexes.
Harder and Rosenberg (10) studied the effects of these
compounds on DNA, RNA, and protein syntheses by measuring

SH-thymidine, 3H-uridine, and 3H-L-

the incorporation of
leucine in human amniotic AV3 cells in tissue culture.

They showed that at a concentration of 5 uM cia-Pt(II)

8

3H-thymidine incorporation was inhibited, while 3H-uridine
and 3H-L-leucine incorporation was affected only minimally.
They stated that at concentrations of 5 uM cis-Pt(II) and
below, DNA synthesis was selectively inhibited. At 25 uM
(which is equivalent to a therapeutic dose of 8.0 mg/kg
in mouse-tumor systems), DNA synthesis was more rapidly
inhibited than RNA or protein synthesis: at 24 hours all
three were completely inhibited.

A selective and persistent inhibition of DNA synthesis ‘
by cis-Pt(II) was demonstrated by Howle and Gale, working

with Ehrlich ascites tumor cells (14). Based on the

3 3

incorporation rates of H-thymidine, 3H-uridine, and H-L-
leucine, they observed an initial inhibition of all three
labelled precursors with a maximum depression at six to
twelve hours after injection of cis-Pt(II). After this
time, however, the rates of RNA and protein synthesis
returned to normal levels, while the rate of DNA synthesis
remained inhibited for 96 hours. The authors suggested\‘
a conversion of the injected neutral platinum species
([cis-Pt(NH3)2C12]o) into a metabolically active complex,
which is responsible for the observed effects. I

The interaction of cis-Pt(II) with DNA has been
demonstrated by a number of researchers. Horacek and
Drobnik (13) observed a shift in the UV absorption maximum
of calf thymus DNA from 259 nm to 264 nm, after treatment
with cis-Pt(II). The shift was minimized by increasing

the concentration of chloride ions, hence decreasing the

concentration of positively charged aqua complexes as in

9
equations 3 and 4. This had the effect of decreasing the
affinity of the platinum complexes for DNA, by decreasing
the probability of nucleophilic attack. The authors showed
that the reaction of cis-Pt(II) facilitates renaturation
and suggested that it may be due to interstrand cross-
linking, an effect commonly observed with bifunctional
alkylating agents.

Roberts and Pascoe (27) pr0posed that the antitumor
activity of platinum complexes was the result of their
ability to form crosslinks between complementary DNA
strands. They developed an unequivocal method to demon-
strate the crosslinking ability of these complexes. They
reacted "heavy" single stranded DNA (made by growing cells
in the presence of S-bromo 2'—deoxyuridine:BUdR) with
"light" single stranded DNA in the presence of cis-Pt(II)
to produce "hybrid" double stranded DNA. After density
gradient centrifugation with cesium chloride, and spectro-
scopic analysis, the presence of the crosslinked hybrid
Species could be determined. Working with the DNA from
HeLa cells, they showed that hybridization did occur after
treatment with cis-Pt(II) in a fashion similar to mustard
gas, an agent known for its ability to crosslink DNA. By
radioactively labelling the "heavy" DNA, quantitation of-
the extent of crosslinking was accomplished. It was found
that the degree of crosslinking (by both cis- and trans-
isomers of dichlorodiammineplatinum(III)was directly
dependent on the dose of the platinum complex. The authors

suggested that the crosslinking occurred between the amino

10
groups of adjacent base pairs, particularly on guanine,
adenine, and cytosine.

Mansy et a1. (22) reported that guanosine, adenine,
and cytidine were the bases that react with platinum com-
pounds most readily. Based on UV spectrophotometric
analysis at varying pH's, they suggested that the cis
isomer (with two cis leaving groups) of dichlorodiammine-
platinum(II) forms a bidentate chelate between the 6-NH2
and either the N-l or N-7 of adenosine and between the
4-NHZ and N-3 of cytidine, while both isomers interact
monofunctionally with the N-7 of guanosine. Uridine and
thymidine were not bound by platinum. The distance between
the chloride leaving groups is 3.3 A (41), which is very
close to the base stacking distance of 3.4 A in the Watson
Crick DNA model. The authors concluded that when adenosine
and cytidine have the correct steric arrangement (as in
DNA) they will form bidentate chelates with cis-Pt(II).

Shooter et al. (38) were able to show that interstrand
platinum crosslinks between complementary DNA strands were
of little importance in the inactivation of T7 bacterio-
phage. Munchausen (25), working with transforming DNA in
Hemophilus influenzae,concluded that interstrand cross-
links with platinum complexes were too infrequent to
account for the loss of transforming ability. However,
she added that the integration of platinum-bound trans-
forming DNA into the host genome was inhibited at a

rate parallel to the loss of transforming ability. Hence,

11
the ability to form intrastrand crosslinks may play a role
in the activity of platinum compounds as suggested by
Thomson (40), Harder (9), and Rosenberg (32).

Guanosine has been implicated as the preferential
binding site for platinum complexes (21,28,39). Recently,
data obtained by x-ray photoelectron spectroscopy was
presented which showed the involvement of the 0-6 of
guanine in cis-Pt(II) binding (23). It was stated that
the specificity of cis-Pt(II) could be explained by the
bidentate chelate formed between the N-7 and 0-6 of
guanine. Following DNA replication, this could lead to
a base substitution mutation, and mispairing of bases in
subsequent DNA strands.

An earlier hypothesis proposed by Rosenberg (29,30,
31,32) implicated the host immune response in the anti-
tumor activity of platinum complexes. That is, because
there is no selective uptake of these complexes by neo-
plastic tissues (42), their specific antineoplastic
activity could not be explained. By imposing the host
immune response into the mechanism of action, specificity
could be obtained by virtue of the inherent specificity
of immunocompetent cells for tumor specific antigens.

This "enhanced antigenicity" hypothesis has been
suggested by data from viral transformation and immuno-
logical studies. Resolva (26) demonstrated that antitumor
platinum complexes were potent inducers of lysogenic
bacteria. That is, after incorporation of viral DNA

(which was subsequently represSed) into the host genome,

12
treatment with cis-Pt(II) derepresscd the viral genome,
causing production of viral particles, and cell lysis.
This was similar to other agents, e.g., radiation, x-rays,
chemical carcinogens, etc., which likewise caused derepres-
sion of viral DNA and cytolysis. Vonka et al. (43) also
showed that treatment of cultured human lymphocytes
(transformed by Epstein-Barr virus) with cis-Pt(II)
resulted in increased numbers of new virally-coded pro-
teins on the cell surfaces.

In immunological studies (8) injecting hydrocortisone
(a nonspecific immunosuppressant) into Swiss white mice
with S-180 solid tumors (an allogeneic system) decreased
the number of cures by 50% over controls after treatment
with cis-Pt(II). In a syngeneic system (S-180 tumors in
BALB/c mice) injection of Zymosan (a nonspecific immuno-
stimulant) prior to treatment with cis-Pt(II) resulted
in a 50% cure rate. The cure rate for mice without
Zymosan stimulation was 0%.

Ironically, most platinum complexes with antitumor
activity have demonstrated immunodepressant characteris-
tics. cis-Pt(II) decreased the number of plaque forming
cells in spleens removed from mice sensitized with sheep
red blood cells (1,17). .Brambilla et a1. (3) observed a
significant depression in skin allograft rejection (graft-
vs-host) in mice after treatment with therapeutic levels
of cis-Pt(II). Maximum effect was observed when the drug
was administered 24 hours prior to grafting. Other research

has shown thatcis-Pt(II)inhibits nucleic acid synthesis

13
in lymphocytes stimulated with phytohemagglutinin (15)

and suppresses lymphocyte blastogenesis in man (18).

MATERIALS AND METHODS

Experimental Animals

 

In all phases of this experiment, 9 week old female
DBA/Z mice were used. They were obtained from Jackson
Laboratory (Bar Harbor, Maine) and maintained in the
laboratory of Dr. Barnett Rosenberg in the Biophysics
Department. During the testing, eight mice were housed
per cage and given food and water as required. To increase
the life span of the mice following x-irradiation, acidified-
chlorinated water was supplied to all mice. This was pre-
pared by adding 4.0 ml concentrated hydrochloric acid and

4.5 m1 bleach to 3.78 liters water.

Drugs
The cis-dichlorodiammineplatinum(II) [cis-Pt(II)]

used in this experiment was made available by the Biophysics
Department at Michigan State University. It was prepared
for injection by mixing it with sterile physiological

saline not more than 20 hours prior to the final injec-
tions. The mice were weighed and the cis-Pt(II) was
administered by intraperitoneal injections (i.p.) on a
multiple dose schedule, consisting of 1.5 mg/kg cis-Pt(II)
every five hours for 25 hours. The total cis-Pt(II) con-

centration per mouse was 7.5 mg/kg body weight.

14

15

X-Irradiation

 

The source of x-irradiation was supplied by Dr. Ulreh
Mostosky of the Veterinary Clinical Center, Michigan State
University. Whole-body x-irradiation equivalent to 700
rads was applied to the mice by using 1 mm copper at 300
mVolts and 200 mAmps. The total dose was divided into four
segments of 175 rads each, with an exposure time of 8.9
minutes per segment. After each time interval the mice
were rotated in such a way as to insure uniform irradia-

tion from mouse to mouse.

111121;

The P388 tumor is a lymphoma originally induced in
1956 with methylcholanthrene. It grows in the ascites
form, and is syngeneic at the H-2 locus with DEA/2 mice
(4). The tumor was obtained from the A.D. Little Company
in Cambridge, Massachusetts, and maintained in female
DEA/2 mice by transfer every eight days. The transfer
mice were killed by cervical dislocation eight days after
injection of the tumor. The ascitic fluid was removed
under sterile conditions, was mixed with sterile physio-
logical saline (0.85% NaCl), and centrifuged at 500 rpm
for five minutes at 4°C. The supernatant fluid was dis-
carded to remove undesirable material, e.g., connective

*
tissue and red blood cells. The tumor cells were washed

 

* I O

Mice w1th exce551vely bloody asc1tes were not used
as transfer animals. Another mouse was selected and
sacrificed.

16
repeatedly by this method until no red blood cells remained.
The remaining P388 cells were then suspended in sterile
saline, and a hemacytometer was used to determine their
concentration in the suspension. The cells were then
diluted (again with sterile saline) to a concentration of

4x106

cells in 0.2 ml. Two-tenths milliliter of the
suspension was injected (i.p.) into the DBA/Z mice after
cleansing the injection site with 70% ethyl alcohol.

All mice deve10ped the tumor, as evidenced by the profuse;

ascites observed eight days later.

Tumor Cell Quantitation

On given days, randomly selected mice from all experi-
mental and control groups were sacrificed, and the ascitic
fluid removed. Physiological saline was used to thoroughly
wash the peritoneal cavity, i.e., until the washings were
clear. The total volume of fluid was noted and an aliquot
was diluted (1:500) in an autodiluter. This diluted speci-
men was then used for counting in a Coulter counter (model
B).* The total number of cells present in the ascitic
fluid was determined by multiplying the number of cells
counted per milliliter of sample times the dilution factor
times the total ascitic fluid volume. To establish the
total number of P388 cells per mouse, a drop of the fluid
was smeared on albuminized microscope slides, was fixed

with 95% ethyl alcohol, and stained by the Papanicolaou

 

*
Coulter Electronics Corporation, Hialeah, Florida.

17

technique. The slides were then examined and a differen-
tial cell count performed. In this manner the relative
number of P388 cells was determined. This number, when
expressed as a percentage and multiplied by the total
number of cells present in the ascitic fluid, equals the
total number of tumor cells present.

Ascitic fluids with detectable numbers of red blood
cells after dilution were treated with Zap-Isoton, the
standard red cell lysing agent used for Coulter counting.

It had no effect on the tumor cells.

Experimental Scheme

 

Randomly selected female DBA/Z mice were arbitrarily
divided into two groups: irradiated and nonirradiated.
The irradiated group received 700 rads whole-body
x-irradiation on day -1. The following day (day 0), both

6 P388 cells. On

groups of mice were injected with 4 x 10
the day following tumor cell injection (day 1) both groups
(irradiated and nonirradiated) were again divided (randomly)
with roughly half of the irradiated and nonirradiated

groups each getting multiple injections of cis-Pt(II).

Thus, four groups of animals were produced. The first
group (untreated control) was injected with only 4 x 106
P388 cells. The second group was x-irradiated and injected

with 4 x 106

6

P388 cells. Group three was injected with

4 x 10 P388 cells, and 5 x 1.5 mg/kg cis-Pt(II). The

fourth group was x-irradiated and injected with 4 x 106

P388 cells and 5 x 1.5 mg/kg cis-Pt(II) (experimental group).

18
Beginning on day 1 (after three cis-Pt(II) injec-
tions) three animals from each group were sacrificed (by
cervical dislocation) and the ascitic fluid removed. The
total number of P388 tumor cells was determined as described
above, and the values averaged for the three mice. This

was continued until the mice were depleted.

Hemolytic Plaque Assay

 

The hemolytic plaque assay (modified Jerne plaque
assay) was used to determine the efficiency of the
x-irradiation to eradicate the immune response in the
host mice. A set of mice other than those used in the
actual experiments was randomly divided into two groups:
irradiated and nonirradiated. The irradiated group was
again divided into two groups. One group received 700
rads whole-body x—irradiation, while the other received

700 rads x-irradiation, 4 x 106

P388 cells (i.p.) and

five injections (i.p.) of 1.5 mg/kg cis-Pt(II).. The
nonirradiated DBA/Z mice were separated into three groups,
one of which served as a normal control. Of the two
remaining groups, one was injected with 4 x 106 P388 cells
(i.p.) and five injections of 1.5 mg/kg cis-Pt(II), while

the other received only 4 x 106 P388 cells. Several hours

8

following injection of the P388 cells, 5 x 10 sheep red

blood cells (SRBC's) were injected into the lateral tail

 

a
The same scheme of x-irradiation followed by tumor

cells followed by cis-Pt(II) that was used for the experi-

mental mice was employed for the hemolytic plaque assay.

19
vein of the mice. Four days following injection of the
SRBC's, all mice were sacrificed, their spleens were

removed, and the assay performed (19).

RESULTS

Trial 1

The Growth Curve of P388 Cells
in DBA72 Female Mice

Nine week old female DBA/Z mice were injected (i.p.)
with 4 x 106 P388 lymphoma cells on day 0. Beginning on
day l and on each day thereafter, 3 mice were sacrificed
and the average number of tumor cells in each mouse quanti-
tated as described above.. The average number of P388 cells
per day is presented in Table I. A plot of these data as
a function of time is shown in Figure 1.

The mean day of death for this group of mice was
10.1»days.
The Growth Curve of P388 Cells in
XiIrradiated'DBA/Z Female Mice

Seven hundred rads whole-body x-irradiation was
administered to nine week old female DBA/Z mice on day -1.

On day 0 they received 4 x 106

P388 cells by i.p. injection.
The mean day of death for these mice was 6.3 days,
which is consistent with the results of other x-irradiation
studies (24). No animal died from tumor burden, even though

sizable ascitic fluids were observed in all mice. Death

was probably due to x-irradiation and subsequent bacterial

20

21

 

 

Table I. Number of P388 cells in DBA/Z female mice:
Trial 1
Time in Avg. number tumor Standard
days cells in mice deviation
1 5.53 x 106 2.69 x 105
2 2.06 x 107 2.65 x 105
3 6.68 x 107 2.23 x 107
4 2.35 x 108 4.76 x 107
5 3.06 x 108 4.36 x 107
6 5.25 x 108 5.97 x 107
7 6.09 x 108 5.71 x 107
8 8.47 x 108 3.75 x 107

 

IOOO-

IOOP

IO-

Number of tumor cells x lo6

 

'iesz. swerve
Timeindoys

Figure 1. Growth curve of P388 cells in DBA/Z female
mice: Trial 1.

@00-

IO-

Number of tumor cells x IO6

 

'iésa ééiééTo
Timeindoys

Figure 1. Growth curve of P388 cells in DBA/Z female
mice: Trial 1.

IOOO-

soop
loo--
“’9
X
=-“’.. 50-
CD
(9
E
"5
E:
5 IO-
:3
Z
5..

 

'ié34 54556.6
Timeindoys

Figure 1. Growth curve of P388 cells in DBA/Z female
mice: Trial 1.

23
infection, although the exact cause of death was not
determined.
The average number of P388 cells per day is presented
in Table 11. These data are shown graphically in Figure 2.
The Growth Curve of P388 Cells in

DEA/2 Female Mice Treated with
52x 1.5'mg/kg cis-Pt(II)

 

 

Nine week old female DBA/Z mice were injected (i.p.)

with 4 x 106

P388 cells on day 0 and 5 x 1.5 mg/kg cis-Pt(II)
on day l.

The mean day of death for these animals was 24.9 days.
No mouse in this group died before day 17, and the last
three mice died on day 30. No cures were observed, and
most of the mice eventually showed evidence of ascites by
day 15.

The therapeutic effectiveness of cis-Pt(II) on the
P388 lymphoma can be established by calculating the
increased life span (ILS) of the treated animals as com-
pared with the untreated animals. Thus, the ILS for this
tumor-treatment system was 146%.

The average number of P388 cells per day is presented
in Table III. A plot of the average number of P388 cells
as a function of time is shown in Figure 3.

The Growth Curve of P388 Cells in
YiIrradiated DBA72‘Female_Mice

Tfeatedfiwith 5 x 1.5 mg7kg
cis-Pt(II)

 

X-irradiated DEA/2 mice injected with 4 x 106 P388

lymphoma cells (day 0) and treated with 5 x 1.5 mg/kg

24

Table II. Number of P388 cells in irradiated DEA/2 female
mice: Trial 1

 

 

Time in Avg. number tumor Standard
days cells in mice deviation
1 3.40 x 106 4.55 x 105
2 7.86 x 106 3.60 x 106
3 1.91 x 107 7.00 x 106
4 9.76 x 107 6.46 x 107
5 1.18 x 108 4.63 x 107
6 1.68 x 108 1.87 x 107
7 -- --
3 -- -_

 

.25

IOOO-

lOO~

(N
O
l

 

IOP

Number of tumor cells x IO6

 

 

 

 

'3234 56789IO
Timeindoys

Figure 2. Growth curve of P388 cells in irradiated
DBA/Z female mice: Trial 1.

 

26

Table III. Number of P388 cells in DBA/Z female mice
treated with 5 x 1.5 mg/kg cis-Pt(II):

 

 

Trial 1

Time in Avg. number tumor Standard
days cells in mice deviation
1 6.41 x 106 2.29 x 106

2 4.21 x 106 1.32 x 106

3 2.89 x 106 8.52 x 105

4 2.51 x 106 8.50 x 105

s 2.12 x 106 1.06 x 106

6 2.36 x 106 9.47 x 105

7 2.29 x 106 1.34 x 106

8 2.11 x 106 5.37 x 105

 

27

 

IOOO-

SOO-

loo-
(0
9
X

g 50-
0
O
25
E
2
“5
2

E '0’
:3
Z

5..

I n L 1 A

 

 

 

I234 5678 9 o
Timeindoys

Figure 3. Growth curve of P388 cells in DBA/Z female
mice treated with 5 x 1.5 mg/kg cis-Pt(II): Trial 1.

28
cis-Pt(II) had a mean day of death of 5.5 days. This value
was similar to that for irradiated tumored mice without
cis-Pt(II). No deaths were caused by tumor.
The average number of P388 cells per day is presented

in Table IV. These data are shown graphically in Figure 4.

Trial 2
The experimental scheme used in Trial 1 was repeated
exactly for Trial 2, and the results of this trial are
presented below.

The Growth Curve of P388 Cells
in’DBA/ZDFEmaIe—Mice

 

 

The mean day of death of DBA/Z mice injected with

4x106

P388 cells (day 0) was 9.8 days, with a range of
8 to 12 days. Table V shows the average number of P388
cells per day. A graphic representation of these data

is shown in Figure 5. For comparison's sake, Figure 6 is
a composite of the graphs in Figures 1 and 5.

The Growth Curve of P388 Cells in
X-Irradiated DBAIZ’Female Mice

 

X-irradiated DBA/Z female mice injected with 4 x 106

P388 cells had a mean day of death of 6.5 days. Two
animals died on day 5, three died on day 6, and nine mice
died on day 7. As in Trial 1, no animals died from tumor
burden, even though ascites was prominent.

The average number of P388 cells per day is presented

in Table VI. A plot of these data as a function of time

29

Table IV. Number of P388 cells in irradiated DBA/Z female
mice treated with 5 x 1.5 mg/kg cis-Pt(II):

 

 

Trial 1

Time in AVg. number tumor Standard
days cells in mice deviation
1 4.94 x 106 1.08 x 106
2 2.23 x 106 1.01 x 106
3 8.91 x 105 2.90 x 105
4 3.01 x 105 1.87 x 105
5 2.71 x 105 2.59 x 105
6 2.11 x 105 1.67 x 105

7 -- --

3 -- --

 

30

IOO -

so.

5

C”
I

Number of tumor cells x IO6

0.5 1-

 

 

 

 

 

?: 3 4 5 6 i 8 9 IO
Timeindoys

 

DJ ‘|

Figure 4. Growth curve of P388 cells in irradiated
DBA/Z female mice treated with 5 x 1.5 mg/kg cis-Pt(II):
Trial 1.

31

 

 

Table V. Number of P388 cells in DBA/Z female mice:
Trial 2

Time in Avg. number tumor Standard

days cells in mice deviation

1 1.12 x 107 1.58 106

2 3.25 x 107 1.07 107

3 1.10 x 108 6.23 107

4 2.76 x 108 5.23 107

5 4.56 x 108 5.46 107

6 6.57 x 108 1.09 108

7 9.12 x 108 1.55 108

8 1.04 x 109 1.33 109

 

32

IOOO

500 -

 

 

«o IOO-
9 1
x
(0
fi 50-
o is
3
E
3
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«‘1':
.n
g IO-
:2
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'I2 34 5 678912)
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Figure 5. Growth curve of P388 '
mice: Trial 2. cells 1n DBA/Z female

33

IOOO -

/

lOOb
“’9
X
é’ 50-
d)
0
3
E
5:!
"5
.8
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3
Z
5 D
OTriol I
ATria|2

 

 

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Figure 6. Growth curves of P388 cells in DEA/2
female mice: Trials 1 and 2.

34

Table VI. Number of P388 cells in irradiated DBA/Z female
mice: Trial 2

 

 

 

Time in Avg. number tumor Standard
days cells in mice deviation
1 7.29 x 106 4.44 x 106
2 1.59 x 107 4.53 x 106
3 6.21 x 107 8.41 x 106
4 2.58 x 108 6.16 x 107
5 3.51 x 108 7.91 x 107
6 5.17 x 108 3.22 x 107
7 5.01 x 108 3.41 x 108

 

35
is shown in Figure 7. Figure 8 is a composite of Figures
2 and 7.

The Growth Curve of P388 Cells in
DEA/2 Female Mice Treated with'

5 x 1.5 mg/kg cis-Pt(II)

 

 

The mean day of death of mice injected with 4 x 106

P388 cells and treated with 5 x 1.5 mg/kg cis-Pt(II) was
22.2 days. This value, as well as the calculated ILS of
127%, is consistent with the results of Trial 1.

The average number of P388 cells per day is presented
in Table VII, and a graph of these data is shown in Figure
9. Figure 10 is a composite of Figures 3 and 9.

The Growth Curve of P388 Cells in
Xllrradiated DBA/Z‘Female Mice

Tieated with 5 xlTSngEg
cis-Pt(II)

6 P388

X-irradiated DBA/Z mice injected with 4 x 10
cells and treated with S x 1.5 mg/kg cis-Pt(II) had a mean
day of death of 6.0 days. It is consistent with the mean
day of death reported for Trial 1. Again, no animal died
from tumor burden.

The average number of P388 cells per day is presented
in Table VIII. These data are presented graphically in
Figure 11, and a composite of this graph and Figure 4 is
shown in Figure 12.

The Number of Pla ue Formin Cells in
SpIeens from DBA7§ Mice Sensitizédi

wifh Sheep_Red'BIood’Cells

 

 

DBA/Z mice, treated as described in the materials and

methods section, were injected with S x 108 sheep red blood

 

36

IOOO-

 

lOO -

50-

Number of tumor cells x IO6

 

 

 

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Figure 7. Growth curve of P388 cells in irradiated
DBA/Z female mice: Trial 2.

37

 

 

 

 

 

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.L

IOO-
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g 50-
8
25
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5- eTriol I
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Figure 8. Growth curves of P388 cells in irradiated
DEA/2 female mice: Trials 1 and 2.

37

 

 

 

 

 

IOOO-
500-
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X
g 50L
8
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Figure 8. Growth curves of P388 cells in irradiated
DBA/Z female mice: Trials 1 and 2.

37

 

 

 

 

 

IOOO-
500»
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Q ,
X
.L-‘-.-’ 50L
8
25
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a
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I
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Figure 8. Growth curves of P388 cells in irradiated
DBA/Z female mice: Trials 1 and 2.

38

Table VII. Number of P388 cells in DBA/Z female mice
treated with 5 x 1.5 mg/kg cis-Pt(II):

 

 

Trial 2
Time in Avg. number tumor Standard
days cells in mice deviation
1 6.47 x 106 1.96 x 106
2 9.21 x 106 4.65 x 106
3 7.22 x 106 3.29 x 106
4 5.25 x 106 6.10 x 105
5 4.59 x 106 2.72 x 106
6 3.61 x 106 8.34 x 105
7 4.22 x 106 3.41 x 106
8 2.76 x 106 1.80 x 106

 

 

39

 

 

 

 

 

 

IOOO-

500P

IOOP
“’2
x

g 50-
m
o
3
E
3
“5
3

E IO-
:3
Z

5..

.1
l
1 L J
l

I 2 3 4 6 6 7 8 9 IO
Timeindoys

Figure 9. Growth curve of P388 cells in DBA/2 female
mice treated with 5 x 1.5 mg/kg cis-Pt(II): Trial 2.

40

IOOO-
500- 0 Trial I
A TriolZ
I005
19
Q
X
g 50r-
o
o
3
E
P.
“6
:2";
E IO!-
:1
Z

 

s. 4
“In

I I I I I LIJ
'12 34 5 6 7 8910
Timeindays

Figure 10. Growth curves of P388 cells in DEA/2

female mice treated with 5 x 1.5 mg/kg cis-Pt(II): Trials
1 and 2.

 

 

 

41

Table VIII. Number of P388 cells in irradiated DBA/Z
female mice treated with 5 x 1.5 mg/kg
cis-Pt(II): Trial 2

 

 

Time in Avg. number tumor Standard
days cells in mice ' deviation
1 5.45 x 106 8.73 x 105

2 4.79 x 106 7.93 x 105

3 1.95 x 106 9.36 x 105

4 7.00 x 105 2.90 x 105

5 6.01 x 105 2.10 x 105

6 6.75 x 105 9.29 x 104

7 3.45 x 105 1.41 x 105

 

42

IOO-

IO-

Number of tumor cells 11 IO6

0.5 -

 

0' L l L l l l l l L J

' l 2 3 4 5 6 7 8 9 IO
Timelndoys

FigurelJ” Growth curve of P388 cells in irradiated

DBA/Z female mice treated with 5 x 1.5 mg/kg cis-Pt(II):
Trial 2.

 

l00-

5

Ch
I

Number of tumor cells 11 IO6

0.5 -

 

 

43

d

 

0 Trial l
ATriol 2

J

 

 

0.l ‘ ‘

12345

67691

Time in days

Figure 12. Growth curves of P388 cells in irradiated
DBA/Z female mice treated with 5 x 1.5 mg/kg cis-Pt(II):

Trials 1 and 2.

 

44

cells on day 0. They were sacrificed on day 4 and their
spleens were removed. The hemolytic plaque assay was
performed and the number of plaque forming cells (PFC)
per spleen was determined. The results of this assay are
shown in Table IX.

The difference in numbers of plaque forming cells
between control and nonirradiated groups was not

significant.

 

45

Table IX. Antibody response in spleens as measured by
the hemolytic plaque assay

 

 

Experimental. Number of Number of direct plaque
group mice forming cells/spleen

Control 4 2.44 x 103 i 1.34 x 103

X-irradiation 6 0

X-irradiation +

P388 cells + 6 0

cis-Pt(II)

P388 cells + 5 1.21 x 103 e 4.94 x 102

cis-Pt(II)

P388 c6115 3 3.92 x 103 t 1.09 x 103

 

 

DISCUSSION

In a syngeneic host-tumor system such as the one used
in this study, one would expect a "linear" increase in the
number of tumor cells as a function of time, and no spon-
taneous cures due to H-Z incompatibility. Figure l, the
standard growth curve for the P388 lymphoma in immunocom-
petent DBA/Z female mice, indicates that this mouse-tumor
system is usable as a positive control for further studies.

Figure 2 demonstrates the effect of prior x-irradiation
on the growth of P388 cells. While the absolute number of
tumor cells per day in this group of mice is consistently
lower than for corresponding days in Figure 1 (for an unde-
termined reason), the slopes of both curves are approxi-
mately the same. This indicates that 700 rads x-irradiation
when administered prior to injection of tumor cells has
little effect on their rate of proliferation.

The efficiency of cis-Pt(II) to destroy P388 cells in
this syngeneic system is shown in Figure 3. On day 1, an

6 P388 cells were present. This number

average of 6.41 x 10
decreases steadily until the fourth day, when the number
of tumor cells plateaus. Comparing this graph with those
in Figures 1 and 2, it is evident that a dosage of 1.5

mg/kg cis-Pt(II) when administered in 5 i.p. injections

46

47
over a period of 25 hours is effective against the P388
lymphoma.

As reported in the results section of this thesis,
the ILS for this group of mice was 146%.

As shown in Table IX, mice which received 700 rads
whole-body x-irradiation were devoid of the ability to
produce antibodies to a challenge of sheep red blood cells.
Figure 4 demonstrates graphically the ability of cis-Pt(II)
to eradicate tumor cells in immunologically depressed
mice. In this group of mice there is a parallel if not
greater effect of cis-Pt(II) on the number of P388 cells
per day than in the previous group of immunocompetent
mice (Figure 3). In fact, a more precipitous decrease in
the number of tumor cells can be seen in Figure 4. As
reported by Zak (44), when 700 rads x-ray were administered
prior to cis-Pt(II), there was a slight additive effect of
their toxicities. This additive effect could account for
the greater decrease in the number of P388 cells in these
mice.

Examining the graphs of the data for Trial 2 (Figures
5, 7, 9 and 11) and comparing these with the graphs for
Trial 1 indicates that there is a high degree of reproduci-
bility in the numbers of tumor cells per day per group of
mice. This reproducibility suggests that the methods used
in this study are reliable, and that the conclusions drawn
from the data are significant.

Scrutinizing the composite graphs (Figures 6, 8, 10

and 12) demonstrates an interesting feature. Note that in

48
Figure 6 the number of P388 cells per day for_Trial l is
consistently below that for corresponding days in Trial 2.
The same phenomenon can be seen in all subsequent composite
figures, and it is of particular significance in Figure 10.
(The ILS for Trial 2 is also lower than that for Trial 1:
127% and 146%, respectively.) The number of tumor cells

in this group of mice on day l is 6.47 x 106

6

, and 9.21 x
10 on day 2. This modest increase in the number of cells
suggests that there may be a relationship between the
total absolute number of tumor cells injected on day 0
and the dosage of cis-Pt(II) required to substantially
reduce their numbers. That is, is the initial effective—
ness of the drug dependent upon tumor mass (numbers of
tumor cells)? While research examining this relationship
must be completed for verification, I propose that a
larger number of P388 cells was injected on day 0 in

Trial 2 (compared with Trial 1) and that perhaps this
could explain the transient increase in the number of
tumor cells (in this and other studies) shortly after
treatment with cis-Pt(II).

With regard to the syngeneic animal tumor system that
was used in this study, it is unlikely that the antitumor
activity of cis-Pt(II) is related to enhancement of tumor
specific antigens (TSA's). By choosing a syngeneic experi-
mental system, bias was placed in favor of the enhanced
antigenicity hypothesis. That is, by not using an allo-
geneic tumor-mouse system the possibility of interference

by H-Z histocompatibility antigens was diminished. Hence,

 

49
if immune enhancement by cis-Pt(II) were to occur, it would

be directed at TSA's and not H-2 antigens.

 

49
if immune enhancement by cis-Pt(II) were to occur, it would

be directed at TSA's and not H-Z antigens.

 

 

CONCLUSIONS

By comparing the growth curves of P388 cells in
untreated control mice with the growth curve with P388
cells in x-irradiated mice treated with cis-Pt(II), insight
into the relationship between the host immune system and
the antitumor activity of cis-Pt(II) was obtained. Based
upon the data presented in this study, it is clear that
the antineoplastic prOperty of this compound is not related
to its ability to enhance expression and recognition of
tumor specific antigens as proposed by the enhanced anti-
genicity hypothesis.

It has been shown that cis-Pt(II) is effective in
reducing the number of P388 cells in immunocompetent DBA/Z
mice (Figure 10). If this activity is dependent upon an
intact host immune response (which can be modulated by
x-irradiation: Table IX), then the growth curve of P388
cells in x-irradiated cis-Pt(II) treated mice (Figure 12)
should resemble that of control groups (Figures 6 and 8).
What was observed instead was a more rapid decline in the
number of tumor cells per day. Thus, there is no dependence
of cis-Pt(II) on the immune response to effect initial
tumor cell death in this system.

This does not imply that in immunocompetent animals
cis-Pt(II) does not function by reducing the tumor burden

50

 

51
to such a level that the immune response can "handle" the
tumor load and effect a cure. In fact, it would be sur-
prising if this were not the case.

It has been suggested (2) that an increase in the
number of tumor cells following cis-Pt(II) injection was
related to host intervention, thus implicating the immune
system as inherent in the mode of action of cis-Pt(II).

It is conceivable, however, that these transient increases
in the number of tumor cells can be explained by the pos-

sible relationship between initial tumor mass (burden) and

 

dose of cis-Pt(II). This is suggested as an alternative
explanation. To examine this proposition, a study similar
to this one must be done on an allogeneic mouse-tumor
system using different quantities of tumor cells relative
to the dose of platinum. This could establish a dose-
response relationship and could provide insight into the
reason for the transient increase in cell numbers as

described above.

REFERENCES

 

10.

REFERENCES

Berenbaum, M. C. Immunosuppression by Platinum
Diammines. Brit. J. of Cancer 25:208-211, 1971.

Brace, L. C. Host Intervention and Eradication of
the Ascites Sarcoma-180 in Mice Treated with Platinum
Compounds. M.S. Thesis, 1975.

Brambilla, G., Cavanna, M. and Maura, A. Effect of
cis-Diamminedichlor0platinum on the Allograft Reac-
tion in Mice. Canc. Chemo. Reports, Part I, 58(5):
633-636, 1974.

Catalogue of Marine Immune Related Culture and Tumor
Cell Lines. The Salk Inst. for Biol. Studies, 2nd
edition, San Diego, 1974.

Cleare, M. J. and Hoeschele, J. D. Antitumor Platinum
Compounds - Relationship Between Structure and
Activity. Plat. Metals Rev., l7(1):2-l3, 1973.

Clear, M. J. and Hoeschele, J. D. Studies on the
Antitumor Activity of Group VIII Transition Metal
Complexes. Part I. Pt(II) Complexes. Bioinorganic
Chem., 2:187-210, 1973.

Colvin, C. B., Gunther, R. 6., Hunter, L. D., McLean,
J. A., Tucker, M. A. and Martin, D. 8. Kinetics of
Ammonation of the Chloro-Ammine Series of P1atinum(II)
Complexes. Inorg. Chim. Acta 2:487-489, 1968.

Conran, P. B. The Role of Host Defenses in cis-
Dichlorodiammineplatinum(II) Mediated Regressions of
Sarcoma 180 in Mice. PhD Thesis, 1973.

Harder, H. C. Molecular Mechanisms of Platinum Anti-
tumor Agents at the Cellular Level. 168th Amer. Chem.
Soc. Ann. Mtg., Sept. 1974.

Harder, H. C. and Rosenberg, B. Inhibitory Effects
of Antitumor Platinum Compounds of DNA, RNA and Pro-
tein Synthesis in Mammalian Cells in vitro. Intl. J.
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52

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VITA

The author was born in Battle Creek, Michigan, on
June 27, 1949. He received his primary and secondary
education at Gull Lake Community School in Hickory Corners,
Michigan. He continued his education at Michigan State
University, where he graduated in 1971 with a Bachelor of
Science degree in Zoology.

After attending summer classes at the Kellogg Biologi-
cal Station in Hickory Corners, Michigan, he enlisted in
the U.S. Army. In 1973 he was honor graduate of the U.S.
Army School of Cytotechnology (ASCP) and was subsequently
stationed at Tripler Army Medical Center (TAMC), Honolulu,
Hawaii. While assigned at TAMC, he worked in the clinical
cytology laboratory and was responsible for teaching
cytology to resident pathologists and staff personnel.

Upon returning to the mainland as a civilian in
August, 1975, he enrolled in the Clinical Laboratory
Science program at Michigan State University as a Master
of Science candidate. He was lead instructor of cyto-
technology at the Lansing Community College School of
Cytotechnology from September, 1975, to June, 1977.

The author married Joan DeVregud on the 29th of
December, 1972, and their first child, Elizabeth Marie,
was born on January 11, 1977.

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