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This is to certify that the

dissertation entitled

IMMUNE SYSTEM ACTIVATION AS A MECHANISM
OF TUMOR INHIBITION FOLLOWING NOVEL
PLATINUM DRUG ADMINISTRATION

presented by

Dale J. Telgenhofi‘

has been accepted towards fulfillment
of the requirements for

J’hj) degree in _Zoology__

 

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Date 9/1’01/

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LIBRARY
Michigan State
University

 

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE ' DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6/01 c:/CIRC/DateDue.p65—p.1s

IMMUNE SYSTEM ACTIVATION AS A MECHANISM OF TUMOR INHIBITION
FOLLOWING NOVEL PLATINUM DRUG ADMINISTRATION

By

Dale J. Telgenhoff

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Department of Zoology

2002

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ABSTRACT

IMMUNE SYSTEM ACTIVATION AS A MECHANISM OF TUMOR INHIBITION
FOLLOWING NOVEL PLATINUM DRUG ADMINISTRATION

By
Dale J. Telgenhoff

An effective method of destroying tumors in the body is through the activation of
the immune system. Activated macrophages seek out tumor cells, make contact with
cytoplasmic extensions, and transfer lysosomes to the tumor cell. Activated macrophages
also release various cytokines (IL-1, IL-2, TNFOL) which further activate the immune
system and cause tumor necrosis. Cisplatin is a potent anti-neoplastic agent which works
via three mechanisms: DNA crosslinking, inhibition of the mitotic spindle, and
macrophage activation. Other drugs have been developed which are more potent at
macrophage activation and much less toxic. These drugs, in particular Poly-plat, are
much larger in their molecular structure compared to cisplatin, with numerous branching
side chains radiating from a central platinum atom. In equal doses poly-plat has less
platinum than cisplatin and is more effective with less toxicities.

In vivo treatment, using platinum antineoplastic agents results in an increase in
macrophage activity, including an increase in extensions, cytolytic factors (cytokines),
and numbers. The increase in the number of macrophages is a result of monocyte
sequestration and differentiation. Activation of macrophages enhances the organism’s
ability to seek out and lyse tumor cells. Unfortunately, direct application of platinum
compounds to the organism causes stress to the animal due to the drugs, of which
gastrointestinal and nephrotoxicity are the dose limiting factors. By isolating leukocytes

from an animal, treating them with activating drugs, then returning them to the animal we

saw a sequestration and activation response similar to that of treating the animal directly
with the drug. Further, we did not see the toxicity caused by the antineoplastic agents
since the drug itself was not being introduced. A decrease in the harsh side effects is
beneficial to the patient, since they will not have to endure the pain and malaise

associated with chemotherapy.

Dedicated to my wife Kelly and my daughter Gwen, for enduring.

Ci

1'!

ACKNOWLEDGMENTS

I wish to thank my advisor, Dr. Surinder Aggarwal, for his guidance and support
throughout my years at Michigan State University. His patience and dedication
encouraged my development as a researcher and as an instructor. I would also like to
thank Drs. R. Neal Band, Will Kopachik, and Ashir Kumar for having served as
committee members. Their ideas and review of manuscripts has been critical in the
development of this dissertation.

I would also like to thank the Center for Advanced Microscopy, especially Drs.
Joanne Whallon and Shirley Owens for the use of their facility and technical assistance.
In addition I offer my thanks and appreciation to both the faculty and staff in the
Department of Zoology, who have been most helpful in my professional development.

Finally, I would like to thank all of the members of the laboratory who worked so
closely in developing research strategies, Drs. Ying Wang, Heather Meunchen, Brad
Johnson, and Mr. Travis Multhaupt; and my family, whose support has enabled me to

reach my goal.

L15

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TABLE OF CONTENTS

LIST OF TABLES ........................................................................ ix
LIST OF FIGURES ....................................................................... x
KEY TO ABBREVIATIONS ........................................................... xiii
INTRODUCTION ........................................................................ 1
References ......................................................................... 9
CHAPTER 1: Histochemical and morphological identification
of Kupffer cells activated by cisplatin ...................... 13
Summary ......................................................................... 14
Introduction ...................................................................... 15
Materials and Methods .......................................................... 17
Results ............................................................................ 20
Discussion ........................................................................ 28
Conclusion ....................................................................... 30
References ....................................................................... 3 1
CHAPTER 2: Kupffer cell activation after treatment with cisplatin
and its second generation Novel analogs SAP, SSP
and Poly-plat .................................................. 34
Summary ........................................................................ 35
Introduction ..................................................................... 37
Materials and Methods ......................................................... 38
Results ............................................................................ 39
Discussion ....................................................................... 47
References ....................................................................... 49

vi

CHAPTER 3: Effects of poly-plat on tumor cells in vitro ................ 50

Summary .......................................................................... 5 1
Introduction ....................................................................... 52
Materials and Methods .......................................................... 54
Results ............................................................................. 58
Discussion ........................................................................ 68
References ........................................................................ 70
CHAPTER 4: Immune cell activation and tumor cell interaction
following Poly-plat treatment ................................ 73
Summary .......................................................................... 74
Introduction ....................................................................... 75
Materials and Methods ........................................................... 77
Results ............................................................................. 8 1
Discussion ........................................................................ 95
Conclusion ........................................................................ 98
References ........................................................................ 99
CHAPTER 5: Macrophage activation as an anti-tumor mechanism
following administration of platinum antineoplastic
agents ............................................................ 101
Summary .......................................................................... 102
Introduction ....................................................................... 103
Materials and Methods .......................................................... 105
Results ............................................................................. 109
Discussion ........................................................................ 1 l7

vii

References ......................................................................... 121

CONCLUSION ............................................................................ 124

References ......................................................................... 129

viii

CHAPTER 2:

Table I

Table 2

CHAPTER 3:

Table 1

CHAPTER 4:

Table 1

Table 2

Table 3

Table 4

CHAPTER 5:

Table 1

Table 2

LIST OF TABLES

Changes in weight of rats following platinum
drug administration ............................................. 41

Kupffer cell extension formation and non-specific
esterase staining in treated and untreated rat liver .......... 46

Table of human fibrosarcoma (HT1080) cells after
treatment with cisplatin or Poly-plat .......................... 63

Changes in mouse 3T3 fibroblast cell count following
treatment with cisplatin or Poly-plat .......................... 88

Table of human fibrosarcoma (HT1080) cells after
treatment with cisplatin or Poly-plat .......................... 91

Table of human fibrosarcoma (HT1080) cells co-
cultured with macrophages after treatment with
cisplatin or Poly-plat ............................................ 92

Table of human fibrosarcoma (HT1080) cells
exposed to macrophage supernatant from macrophages
treated with cisplatin or Poly-plat ............................. 93

Table showing leukocyte numbers from three rats
per treatment with cisplatin, Poly-plat, or normal saline .. 112

IL-2 levels in animals which received either normal

saline, cisplatin, Poly-plat, Leukocytes from donor

animals, leukocytes from donor animals treated with
cisplatin, or leukocytes from donor animals treated

with Poly-plat .................................................... 113

ix

INTRODUCTION:
Figure 1

Figure 2

Figure 3

CHAPTER 1:

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

CHAPTER 2:

Figure 1

Figure 2

Figure 3

LIST OF FIGURES

Structure of cis-diamminedichloroplatinum H (cisplatin) . 6

Main adducts formed in the interaction of cisplatin
with DNA ......................................................... 7

Structure of novel anti-neoplastic agents SAP, SSP,
and Poly-plat ..................................................... 8

Light micrograph showing esterase staining within
the hepatic sinusoid of the control tissue and the
cisplatin treated tissue .......................................... 22

Normal Kupffer cell in the sinusoidal lumen ............... 23

Cisplatin activated Kupffer cell along the endothelial
Lining ............................................................ 24

After one day post-cisplatin treatment the Kupffer cells
on average exhibited one or more extensions ............... 25

Hepatic sinusoid six days post-cisplatin treatment
showing interaction between activated Kupffer cell
and natural killer cell ........................................... 26

Graph showing the proportional changes in the liver
Kupffer cells following cisplatin treatment .................. 27

Circulating monocyte levels following treatment with
cisplatin or Poly-plat ............................................ 42

Gross morphology of animals following platinum
drug administration .............................................. 43

Kupffer cells from control, cisplatin, and Poly-plat
treated rat liver ................................................... 44

Figure 4 Light micrographs showing esterase staining in the
liver of rats which received no treatment or treatment

with cisplatin, SAP, SSP, or Poly-plat ....................... 45
CHAPTER 3:

Figure 1 Graph showing the effects cisplatin or Poly-plat

treatment on WRC-256 cell growth .......................... 60
Figure 2 Graph showing the effects Poly-plat treatment on

HT1080 cell growth ............................................ 61
Figure 3 Light micrographs showing HT 1080 cells after

48 hours ......................................................... 62
Figure 4 Blots stained using Ponceau S staining solution to

visualize protein bands on nitrocellulose membrane ...... 64
Figure 5 PCNA and p27 levels in HT1080 cells at 24 and 48

hours after treatment with Poly-plat ......................... 65

Figure 6 PCNA levels in HT1080 cells following Poly-plat
treatment ......................................................... 66

Figure 7 p27 levels in HT1080 cells following Poly-plat

treatment ......................................................... 67
CHAPTER 4:
Figure 1 Graph showing IL-2 release in the supernatant of
macrophages treated with cisplatin or Poly-plat
at various times .................................................. 84

Figure 2 Mouse 3T3 IL-2 assay showing IL-2 production in
cultured mouse fibroblasts treated with cisplatin,

Poly-plat, or normal saline ..................................... 85
Figure 3 Light micrographs showing macrophages at 0 hours

and 48 hours in normal medium, and 48 hours after

cisplatin and Poly-plat treatments ............................. 86
Figure 4 Light micrographs showing mouse 3T3 cells at 0 hours

and 48 hours in normal medium, and 48 hours after
cisplatin and Poly-plat treatments ............................. 87

xi

CH.

 

 

 

 

Figure 5

Figure 6

Figure 7

CHAPTER 5:

Figure 1

Figure 2

Figure 3

Figure 4

Dose optimization for IL-2 release from cisplatin
treated macrophages ........................................... 89

Dose optimization for IL-2 release from Poly—plat
treated macrophages ........................................... 90

HT1080 cells and macrophages cultured at opposite
ends of a glass slide ............................................ 94

Graph showing IL-2 levels in treated leukocytes
following treatment with cisplatin, Poly-plat, or
normal saline in vitro .......................................... lll

Enzyme histochemistry for succinate dehydrogenase
and alkaline phosphatase of liver and kidney ............... 114

Graph showing changes in Kupffer cell number as
seen via peroxidase staining following various

treatments ........................................................ 1 15

Tumor regression of tumors induced in the right hip
of rats via injection of WRC cells ............................ 116

xii

CDDP
CdK
DAB
DMSO
ELISA
FBS
G1

62

HS

IL- 1
IL-2
KOH
LPS
MEM
PBS
NaCl
NF-KB
PCNA
PMA

Poly-plat

RPMI

S

KEY TO ABBREVIATIONS
Cisplatin
Cyclin Dependent Kinase
Diaminobenzidine
Dimethyl Sulfoxide
Enzyme Linked Immunosorbant Assay
Fetal Bovine Serum
Growth Phase 1
Growth Phase 2
Horse Serum
Interleukin-1
Interleukin-2
Potassium Hydroxide
Lipopolysaccharide
Minimal Essential Medium
Phosphate Buffered Saline
Sodium Chloride
Nuclear Factor K B
Proliferating Cell Nuclear Antigen
Phorbol Myristate Acetate

(Poly-[(trans-l,2—diaminocyclohexane) platinum]
carboxyamylose)

RPMI Medium

Interphase

xiii

S.D.

SAP

SDS

SSP

TEM

TNFa

TUNEL

v/v

w/v

X8

Standard Deviation

(4-Hydroxy-a-sulfonylphenylacetato [trans 1,2
diaminocyclohexane] platinum II)

Sodium Dodecyl Sulfate
(5-sulfosalicylato-trans-[l,2-diaminocyclohexane] platinum)
Transmission Electron Microscopy

Tumor Necrosis Factor (1

TdT Mediated dUTP Nick-end Labeling

Volume/Volume

Weight/Volume

Gravity

xiv

WTRODUCTION

Cisplatin (cis-Diamminedichloroplatinum H) is an inorganic water soluble
platinum containing complex which has seen wide use as an anti-cancer agent (Figure 1)
(Goodman et al. 1996). Since its development in 1965, it has been used to treat testicular,
head, neck, bladder, and ovarian cancer with varying degrees of success (Abrams and
Murrer 1993), (B051 et al. 1980). Cisplatin’s major mechanisms of action include
inhibition of DNA synthesis by interstrand and intrastrand crosslinking, inhibition of
cytokinesis, induction of apoptosis (Nehme et al. 1997), (Ferreira et al. 2000), and
macrophage activation (Abrams and Murrer 1993), (Fichtinger-Schepman et al. 1986).
Cisplatin appears to enter the cell by diffusion, where the chloride ions are lost by
hydrolysis leaving two active ligand sites which are able to bind DNA forming
interstrand and intrastrand crosslinks (Figure 2), which ultimately arrest the cell cycle at
G1, S, or G2 (Fichtinger-Schepman et al. 1986) (Sundaralingam et al. 1985) (Gonzalez et
al. 2001). These platinum-DNA adducts also alert DNA damage sensors (ATM, DNA-
PKcs, and ATR), which phosphorylate p53, ultimately resulting in the activation of the
caspase cascade and apoptosis (Porter et al. 1997).

Although cisplatin has been shown to be effective in treating various types of
cancers (Abrams and Murrer 1993) drug resistance is a major problem, especially with
repeated doses of the drug (Welters et al. 1998); (Beale et al. 2000). Factors affecting
resistance include an increase in drug efflux, a decrease in drug influx, increases in
glutathione levels, DNA repair, and drug tolerance (Henkels and Turchi 1999), (Beale et
al. 2000), (Nishimura et al. 1996). Cisplatin is administered intravenously and has an

initial half-life in the plasma of 25 to 50 minutes (Goodman et al. 1996).

 

 

 

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Cisplatin activates the immune system by inducing macrophages to form
cytoplasmic extensions which seek out and phagocytose tumor cells (Palma and
Aggarwal 1994). Activated macrophages also release various cytokines (IL-1, TNFCX),
which further activate the immune system and cause tumor necrosis (Basu et al. 1991;
Muenchen et al. 1997). The activation of macrophages is probably accomplished through
the release of nuclear factor kappa B (NF-KB) from its sequestration in the cytoplasm by
IKB (Maldonado et al. 1997). NF-KB is a protein which, when released from the IKB
complex, migrates to the nucleus and is responsible for the transcription of inflammatory
cytokines (Brach et al. 1993), (Stacey et al. 1996).

One of the inflammatory cytokines also produced by activated macrophages is
interleukin-2 (IL-2) (Burkitt and Aggarwal 2000). IL-2 is primarily secreted by activated
T lymphocytes, but has been shown to be released in great quantities by fibroblasts and
macrophages following treatment with platinum anti-cancer agents (Burkitt and
Aggarwal 2000), (Telgenhoff and Aggarwal 2000). IL-2 activates cells with 1L-2
receptors (T and B lymphocytes, natural killer cells, and macrophages) to proliferate
(Sharon 1998) and differentiate into cells with increased immune system activity (Collins
and Oldham 1995), (Ding et al. 1988). IL-2 has also been shown to induce monocyte
differentiation into macrophages through activation of the macrophage colony
stimulating factor gene (Brach et al. 1993), (Zhu et a1. 1993). The use of IL—2 as a
treatment for cancer has been shown to induce regression of metastatic cancers in
selected patients that had failed on other chemotherapies (Rosenberg et al. 1988).

Cisplatin has been shown to activate Kupffer cells by increasing their number and

cytoplasmic extensions (Telgenhoff and Aggarwal 1998). The Kupffer cells are the

resident macrophages in the liver, comprising the largest population of fixed tissue
macrophages in the body (Xu et al. 1984). They are generally stellate in appearance, and
reside in the sinusoids, interdigitating their filipodia in the endothelial lining Wisse
1974). Kupffer cells constitute 15% of the total liver cell population in number but only
2.5% of the overall protein content (Kuiper et al. 1994). During episodes of hepatic
stress, however, they have been shown to greatly increase in size and number (Toge et al.
1981), (W isse 1974). Although self-replication has been observed with chronic hepatic
stimulation, population increases during acute stimulation are generally attributed to
monocyte differentiation (Bouwens and Wisse l982),(van der Rhee et al. 1979a). Due to
the increase in both size and number, the Kupffer cell population is better equipped to
deal with hepatic insult and return the body to a state of homeostasis (McCuskey and
McCuskey 1990), (Shi et al. 1996).

Other drugs have been developed which are more potent at macrophage activation
and much less toxic (Fiebig et al. 1996), (Drees et al. 1995). Three novel second
generation platinum containing antineoplastic agents have been developed; SAP (4-
Hydroxy-a-sulfonylphenylacetato [trans 1,2-diaminocyclohexane] platinum H), SSP (5-
sulfosalicylato-trans-[l,2-diaminocyclohexane] platinum), and Poly-plat (Poly-
[(trans-l,2-diaminocyclohexane) platinum]-carboxyamylose) (Andrulis
Pharmaceuticals, Bethesda, MD) (Figure 3) which have been shown in initial studies to
be more effective in the treatment of cancer and less toxic than cisplatin (Muenchen et al.
1997). These drugs, in particular Poly-plat, are much larger than cisplatin, with
numerous branching side chains radiating from a central platinum atom. In equal doses

Poly-plat has less platinum than cisplatin, which is a possible reason for the decrease in

 

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toxicity. Poly-plat has been shown to activate macrophages to produce inflammatory
cytokines in greater quantities than cisplatin (Muenchen and Aggarwal 1998).

Typically macrophages have been activated by treating the animal with the drug
(in vivo). In vivo treatment of macrophages with platinum antineoplastic agents results in
an increase in macrophage activity, including an increase in extensions, cytolytic factors
(cytokines), and numbers (Telgenhoff and Aggarwal 1998), (Bahadur et a1. 1984), (Gupta
and Sodhi 1987). The increase in the number of macrophages present is a result of
monocyte sequestration and differentiation (van der Rhee et al. 1979b), (Tanner et al.
1982). Activation of macrophages enhances the organisms ability to seek out and lyse
tumor cells (Adams and Hamilton 1988), (Bucana et a1. 1976).

The objectives of the following research were to examine how platinum
compounds affect an organism in vitro and in vivo, to understand the mechanisms of
action of less toxic platinum compounds, to determine the ability of these drugs to
enhance the immune response, and to use the stimulation of the immune system to treat
tumor burdened animals. The first group of experiments involved examining the ability
of cisplatin to induce changes in Kupffer cell morphology, with the aim of understanding
the effectiveness of this compound at stimulating immune cells. Next we compared the
activation of Kupffer cells by SAP, SSP, and Poly-plat to that seen following cisplatin
treatment. We then examined the effects of treating both tumor cells and macrophages
directly with Poly-plat. Based on the data from these experiments we hypothesized that
immune cells treated with Poly-plat were better equipped to scavenge tumor cells. To
show this we activated immune cells in vitro and used them to treat tumor burdened

animals.

 

 

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The following thesis builds the premise for cancer treatment which entails
activating immune cells outside of the body and then returning them to the tumor
burdened patient. In this way the patient never encounters the toxic effects of the drug.
While adoptive transfer is not a novel concept (Wang et al. 1986), (Herscowitz et al.
1981), it has not been performed with platinum agents with known immunomodulatory
effects. It is our hope the techniques developed here will be pursued to a greater degree

in cancer immunotherapy.

Figure 1:

C1 NH3

Pt

\/
/\

C1 NH3

Structure of cis-diamminedichloroplatinum II (cisplatin)

 

Figure 2: Main adducts formed in the interaction of cisplatin with DNA. (a),
interstrand cross-link. (b), 1,2-intrastrand cross-link. (c), 1,3-intrastrand
cross-link, (d), protein-DNA cross-link. (Gonzalez et al. 2001)

‘Poly-plat'

CH20H
“0 ‘0 CH—O
/ / \
- 0 HC ('3H- 0- CH CH—O’
COOH COO COO / H
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Poly-[(rrans-t ,2-diaminocydohexane)platinuml-camoxyamylose

5-sultosalicylato— trans-(1 .2-diaminocydohexane)p|atinum

SAP

Cn::::> <:__ —>c~ {0M

4-hydroxyoa-sultonytphenytaoetato(trans
1 .2-diaminocyctohexane)platinum(ll)

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(Muenchen et al. 1997)

 

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REFERENCES

Abrams MJ, Murrer BA (1993) Metal compounds in therapy and diagnosis. Science
261:725-30

Adams D, Hamilton T (1988) Activation of macrophages for tumor cell kill: Effector
mechanisms and regulation. In Heppner G, Fulton A, eds. Macrophages and Cancer.
Boca Raton, CRC Press, 39

Bahadur A, Sarna S, Sodhi A (1984) Enhanced cell mediated immunity in mice after
cisplatin treatment. Pol J Pharmacol Pharm 36:441-8

Basu S, Sodhi A, Singh SM, Suresh A (1991) Up-regulation of induction of lymphokine
(IL-2)-activated killer (LAK) cell activity by FK-565 and cisplatin. Immunol Lett 27: 199-
204

Beale PJ, Rogers P, Boxall F, Sharp SY, Kelland LR (2000) BCL—2 family protein
expression and platinum drug resistance in ovarian carcinoma. BrJ Cancer 82:436-40

Bosl G, Lange P, Franley E (1980) Vinblastine, bleomycin and cis-
diamrninedichloroplatinum in the treatment of ovarian and testicular cancer. American
Journal of Medicine 68:492

Bouwens L, Wisse E (1982) On the dual origin of the Kupffer cell. In Knook DL, Wisse
E, eds. Sinusoidal Liver Cells. Leiden, Elsevier, 165-172

Brach MA, Arnold C, Kiehntopf M, Gruss HJ, Herrmann F (1993) Transcriptional
activation of the macrophage colony-stimulating factor gene by IL-2 is associated with
secretion of bioactive macrophage colony-stimulating factor protein by monocytes and
involves activation of the transcription factor NF-kappa B. J Immunol 150:5535-43.

Bucana C, Hoyer L, Hobbs B (1976) Morphological evidence for translocation of
lysosomal organelles from cytotoxic macrophages into the cytoplasm of tumor target
cells. Cancer Research 36:4444

Burkitt K, Aggarwal SK (2000) Immune system activation by CDDP and "Poly-plat".
Anticancer Res 20:2729-37

Collins RA, Oldham G (1995) Effect of recombinant bovine m-l and IL-2 on B cell
proliferation and differentiation. Vet Irnmunol Irnmunopathol 44: 141-50.

Ding AH, Nathan CF, Stuehr DJ ( 1988) Release of reactive nitrogen intermediates and
reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of
activating cytokines and evidence for independent production. J Irnmunol 141:2407-12

 

 

 

 

 

 

 

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Drees M, Dengler WM, Hendriks HR, Kelland LR, Fiebig HH (1995) Cycloplatam: a
novel platinum compound exhibiting a different spectrum of anti-tumour activity to
cisplatin. Eur J Cancer 32356-61

Ferreira CG, Tolis C, Span SW, Peters GJ, van Lopik T, Kummer AJ, Pinedo HM,
Giaccone G (2000) Drug-induced apoptosis in lung cancer cells is not mediated by the
Fas/FasL (CD95/APOl) signaling pathway. Clin Cancer Res 6:203-12

Fichtinger-Schepman AM, Lohman PH, Berends F, Reedijk J, van Oosterom AT (1986)
Interactions of the antitumour drug cisplatin with DNA in vitro and in vivo. IARC Sci
Publ 78:83-99

Fiebig H, Dress M, Ruhnau T, Misra H, Andrulis P, Hendriks H (1996) GB-21, a novel
platinum complex with antitumor activity in human renal and mammary xenografts.
Proceedings of the American Association of Cancer Research 37:297

Gonzalez VM, Fuertes MA, Alonso C, Perez JM (2001) Is cisplatin-induced cell death
always produced by apoptosis? Mol Pharmacol 591657-63.

Goodman LS, Gilman A, Hardman JG, Gilman AG, Limbird LE (1996) Goodman &
Gilman's the pharmacological basis of therapeutics, 9th / ed. New York, McGraw-Hill
Health Professions Division

Gupta P, Sodhi A (1987) Increased release of interleukin-1 from mouse peritoneal
macrophages in vitro after cisplatin treatment. Int J Irnmunopharmacol 9:385-8

Henkels KM, Turchi JJ (1999) Cisplatin-induced apoptosis proceeds by caspase-3-
dependent and -independent pathways in cisplatin-resistant and -sensitive human ovarian
cancer cell lines [published erratum appears in Cancer Res 2000 Feb 15:60(4):1150].
Cancer Res 59:3077-83

Herscowitz HB, Holden HT, Bellanti JA, Ghaffar A (1981) Manual of Macrophage
Methodology. In Rose N, ed. Immunology Series. New York, Marcel Dekker, Inc, 531

Kuiper J, Brouwer A, Knook DL, van Berkel TJC (1994) Kupffer and Sinusoidal
Endothelial Cells. In Arias IM, Boyer JL, Fausto N, Jakoby WB, Schachter DA, Shafritz
DA, eds. The Liver: Biology and Pathobiology, Third Edition. New York, Raven Press,
Ltd., 791-818

Maldonado V, Melendez-Zajgla J, Ortega A (1997) Modulation of NF-kappa B, and Bcl-
2 in apoptosis induced by cisplatin in HeLa cells. Mutat Res 381:67-75.

McCuskey RS, McCuskey PA (1990) Fine structure and function of Kupffer cells. J
Electron Microsc Tech 142237-46

 

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Muenchen HJ, Aggarwal SK (1998) Immune system activation by cisplatin and its analog
'Poly-plat': an in vitro and in vivo study. Anticancer Drugs 9:93-9

Muenchen HJ, Aggarwal SK, Misra HK, Andrulis PJ (1997) Enhanced
immunostimulation by novel platinum anticancer agents. Anti-Cancer Drugs 8:323-328

Nehme A, Baskaran R, Aebi S, Fink D, Nebel S, Cenni B, Wang JY, Howell SB,
Christen RD (1997) Differential induction of c-Jun NH2-terminal kinase and c-Abl
kinase in DNA mismatch repair-proficient and -deficient cells exposed to cisplatin.
Cancer Res 57:3253-7

Nishimura T, Newkirk K, Sessions RB, Andrews PA, Trock BJ, Rasmussen AA,
Montgomery EA, Bischoff EK, Cullen KJ (1996) Irnmunohistochemical staining for
glutathione S-transferase predicts response to platinum-based chemotherapy in head and
neck cancer. Clin Cancer Res 2: 1859-65

Palma JP, Aggarwal SK (1994) Cisplatin and carboplatin mediated release of cytolytic
factors in murine peritoneal macrophages in vitro. Anticancer Drugs 5:615-22

Porter AG, Ng P, J anicke RU (1997) Death substrates come alive. Bioessays 19:501-7

Rosenberg SA, Schwarz SL, Spiess PJ (1988) Combination immunotherapy for cancer:
synergistic antitumor interactions of interleukin-2, alfa interferon, and tumor-infiltrating
lymphocytes. J Natl Cancer Inst 80: 1393-7

Sharon J (1998) Basic Immunology. Baltimore, Williams and Wilkins

Shi J, Fujieda H, Kokubo Y, Wake K (1996) Apoptosis of neutrophils and their
elimination by Kupffer cells in rat liver. Hepatology 24: 1256-63

Stacey KJ, Sweet MJ, Hume DA (1996) Macrophages ingest and are activated by
bacterial DNA. J Irnmunol 157:2116-22

Sundaralingam M, Rubin JR, Rao ST (1985) X-ray studies on the interaction of the
anticancer agent cis- [Pt(NH3)2c12] to tRNAphe. A mechanism for the formation of the
intrastrand cross-link to adjacent guanines in DNA. Prog Clin Biol Res :175-84

Tanner A, Keyhani A, Arthur M, Wright R (1982) Evidence for a sequence of
macrophage activation during recruitment into the liver. In Knook DL, Wisse E, eds.
Sinusoidal Liver Cells. Leiden, Elsevier Biomedical Press, 405-412

Telgenhoff D, Aggarwal S (2000) Immune stimulation by macrophages and fibroblasts
following exposure to platinum anticancer agents. In Histochemical Society Slst Annual
Meeting. New Orleans, 111

 

 

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Telgenhoff DJ, Aggarwal SK (1998) Kupffer cell activation after treatment with cisplatin
and its second generation novel analogs SAP, SSP, and "Poly-plat". In Collery P, Bratter
P, Negretti de Bratter V, Khassanova L, Etienne J-C, eds. Metal Ions in Biology and
Medicine. Neuherberg, Germany, John Libbey Eurotext, 646-648

Toge T, Nakanishi K, Yamada Y, Yanagawa E, Hattori T (1981) Scanning electron
microscopic studies on the surface structure of activated macrophages and on their
interaction with tumor cells. Gann 72:305-9

van der Rhee HJ, van der Burgh-de Winter CP, Daems WT (1979a) The differentiation of
monocytes into macrophages, epithelioid cells, and multinucleated giant cells in
subcutaneous granulomas. I. Fine structure. Cell Tissue Res 1972355-78

van der Rhee HJ, van der Burgh-de Winter CP, Daems WT (1979b) The differentiation of
monocytes into macrophages, epithelioid cells, and multinucleated giant cells in
subcutaneous granulomas. H. Peroxidatic activity. Cell Tissue Res 197:379-96

Wang BS, Lumanglas AL, Durr FE (1986) Immunotherapy of a murine lymphoma by
adoptive transfer of syngeneic macrophages activated with bisantrene. Cancer Research
46:503-506

Welters MJ, Fichtinger—Schepman AM, Baan RA, Flens MJ, Scheper RJ, Braakhuis BJ
(1998) Role of glutathione, glutathione S-transferases and multidrug resistance-related
proteins in cisplatin sensitivity of head and neck cancer cell lines. Br J Cancer 77:556—61

Wisse E (1974) Kupffer cell reactions in rat liver under various conditions as observed in
the electron microscope. J Ultrastruct Res 46:499-520

Xu ZL, Bucana CD, Fidler U (1984) In vitro activation of murine Kupffer cells by
lymphokines or endotoxins to lyse syngeneic tumor cells. Am J Pathol 117:372-9

Zhu HG, Zollner TM, Klein-Franke A, Anderer FA (1993) Activation of human
monocyte/macrophage cytotoxicity by m-2/IFN gamma is linked to increased expression
of an antitumor receptor with specificity for acetylated mannose. Immunol Lett 38: 1 1 1-9.

 

 

 

 

CH7-

 

CHAPTER 1: HISTOCHEMICAL AND MORPHOLOGICAL
HDENTIFICATION OF KUPFFER CELLS ACTIVATED BY
CISPLATIN

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SUMMARY

Cisplatin is a potent anti-cancer agent which has been shown to activate Kupffer
cells. These activated macrophages demonstrate an increase in cytoplasmic extensions,
lysosomes, and peroxisomes elevating their anti-tumor activity. Wistar rats were treated
with cisplatin (9 mg/kg) and sections of liver were excised for light and electron
microscopic (T EM) analysis at l, 6, 15, and 30 days post treatment. Non-specific
esterase staining was used to differentiate Kupffer cells using light microscopy, while
morphologic criteria were used for TEM analysis. Liver sections taken 6 days post
cisplatin treatment showed the greatest number of activated macrophages, with the

highest degree of activation.

14

 

 

 

 

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INTRODUCTION

In inflammatory reactions the body utilizes a number of different mechanisms to
restore homeostasis. In the case of organ inflammation the lymphoid system induces
monocytes to enter the infected organ and differentiate into macrophages (Cohn 1978),
(Ogawa et al. 1978), (van der Rhee et al. 1979a). These macrophages, while maintaining
many of their monocyte characteristics, undergo a number of changes to make them more
efficient scavengers of invading agents (Karnovsky and Lazdins 1978), (Ogawa et al.
1978), (Toge et al. 1981). An important change which occurs is an increase in the
number of peroxisomes (van der Rhee et al. 1979b), which increase tumor cytotoxicity
(Singh and Sodhi 1988).

The Kupffer cells are the resident macrophages in the liver, comprising the largest
population of fixed tissue macrophages in the body (Xu et al. 1984). They are generally
stellate in appearance, and reside in the sinusoids, interdigitating their filipodia in the
endothelial lining (Wisse 1974a). Kupffer cells constitute 15% of the total liver cell
population in number but only 2.5% of the overall protein content (Kuiper et al. 1994).
During episodes of hepatic stress, however, they have been shown to greatly increase in
size and number (Toge et al. 1981), (W isse 1974a). Although self-replication has been
observed with chronic hepatic stimulation, population increases during acute stimulation
are generally attributed to monocyte differentiation (Bouwens and Wisse l982),(van der
Rhee et al. 1979a). Due to the increase in both size and number, the Kupffer cell
population is better equipped to deal with hepatic insult and return the body to a state of

homeostasis (McCuskey and McCuskey 1990), (Shi et al. 1996).

 

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Cisplatin is a broad spectrum anti-cancer agent. Since its development in 1965, it
has been used to treat testicular, head, neck, bladder, and ovarian cancer with varying
degrees of success (Abrams and Murrer 1993), (Bosl et al. 1980). Cisplatin’s major
mechanisms of action include inhibition of DNA synthesis by interstrand and intrastrand
crosslinking, inhibition of cytokinesis, and macrOphage activation (Abrams and Murrer
1993), (Palma et al. 1992). Cisplatin activates the immune system by inducing
macrophages to form cytoplasmic extensions which seek out and phagocytose tumor cells
(Palma and Aggarwal 1994). More recently, cisplatin has been shown to activate Kupffer
cells by increasing their number and cytoplasmic extensions (Telgenhoff and Aggarwal
1998).

In the present study electron microscopic analysis was utilized to examine the
cytoplasmic extensions in greater detail. Morphological characteristics were used to
determine Kupffer cell number and size, and these data were compared with light
microscopic studies in order to obtain a better understating of cisplatin’s effect on the

Kupffer cell population.

16

MATERIALS AND METHODS

Animals. Six male Wistar rats (Charles Rivers Laboratories, Wilmington MA)
weighing 160-180 grams were kept on a twelve hour light, twelve hour dark cycle with
free access to feed and water. All rats were weighed two days prior to the first injection
and each day thereafter until they were sacrificed on day eight for light microscopic
studies. The rats received an intraperitoneal injection of cisplatin (1.8 mg/kg with .85%
(w/v) normal saline as the vehicle of injection) for five consecutive days beginning on
day three and continuing through day seven. Controls received only the normal saline.

Tissue Preparation. On day eight the rats were euthanized using carbon dioxide,
followed by decapitation using the guillotine. The liver was excised and cut into small
blocks (1 cm3) and mounted on a cryostub in OCT mounting media at -20° C (Miles
Scientific, Elkhart, DJ). The frozen tissue was sectioned at -20° C using a slow fluid
motion. Two sections (7-10 pm) of each treatment were collected on glass coverslips.
The sections were then air dried. They were immersed in a fixative consisting of 1%
(V/v) glutaraldehyde, 5% (w/v) sucrose in 0.5M cacodylate buffer, pH 7.2, for 30-60

Seconds at 4° C. Following fixation, the coverslips were washed in sodium cacodylate
buffer.

Esterase Staining. A diazonium staining method was employed with Napthol AS-
LC-Acetate (Sigma, USA) as the substrate at a pH of 7.1. The sections were immersed in
the enzyme incubation medium for 40 minutes at 37° C. Following incubation, the
Sections were washed briefly in distilled water, then mounted onto glass slides using

glycerin jelly.

 

 

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The sections were examined for non-specific esterase staining using a Zeiss IH
photomicroscope. The population of macrophages in each of the sections were
quantitatively rated based on the number of stained macrophages present within the
tissue. These cells were counted per unit area using the same magnification with each
section. Cell morphology was also examined by focusing through the 10 um section.

Electron Microscopy. For electron microscopy studies nine male Wistar rats were
treated as described but were followed through a thirty day post-treatment period. On
days 8, 13, 22, and 37 the cisplatin treated rats (days 8 and 37 for the control) were
injected with Equithesin and opened via abdominal incision. A 22 gauge catheter
attached to a 0.85% (w/v) normal saline (room temperature, 70 cm) perfusion apparatus
Was inserted into the left ventricle. A bag containing 2% (v/v) paraformaldehyde in
Phosphate buffer (room temperature, 70 cm) was attached to the apparatus via a distal
Port. The perfusion was started and the right atrium was cut to allow drainage. After 90
Seconds the saline line was closed and the paraformaldehyde line was opened. After 15
minutes of perfusion fixation the liver was excised and transferred to a petri dish. The
Periportal region was excised and transferred to another dish containing 2% (v/v)
glutaraldehyde in PBS (pH 7.3). The liver was diced into 1 mm segments and washed in
COld PBS for peroxidase staining, using the DAB method. The tissues were then post-
fixed in 1% (w/v) OsO4 in PBS (pH 7.3) for 60 minutes at 4° C, followed by three
washings in 0.1 M sodium acetate. The tissues were then washed in a graded acetone

Series and embedded in araldite.

 

 

 

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The sectioning was perfomted on the LKB Ultramicrotome and collected on
copper mesh grids, which were then stained with uranyl acetate and lead citrate. The

tissues were viewed on the Phillips CM-lO TEM.

l9

RESULTS

Light Microscopy. The esterase stained control sections showed distinct

 

macrophage staining within the hepatic sinusoid (Figure 1a). The cells appeared discoid
in shape, without cytoplasmic extensions radiating from the cell body. Sections observed
from the cisplatin treated animals (Figure lb) showed a ten fold increase in the
macrophage population, compared to the controls (Table 1). Also, the degree of
cytoplasmic extensions radiating from the cell body were greatly increased (Figure lb,
insert). In transmission electron microscopic studies, normal Kupffer cells are visualized
spanning the sinusoidal lumen with extensions interdigitating into the Space of Disse
(Figure 2). Identification of the Kupffer cells was based on peroxidase staining using the
DAB method, which created electron dense deposits in the Kupffer cells, enabling
differentiation from other sinusoidal cell types. In cases where the DAB did not penetrate
thoroughly, morphologic characteristics unique to Kupffer cells were used for
identification. These included location in the sinusoid, nuclear shape, vermiforrn bodies,
and annulate lamellae. The organelles visible in the cytoplasm include mitochondria,
golgi apparatus, lysosomes, peroxisomes, and endoplasmic reticulum.

Electron Microscopy. Morphologically, the cisplatin treated liver showed an

 

increase in the size and number of Kupffer cell extensions compared to the control
(Figure 3). The cytoplasmic to nuclear ratio was twice as much as in the control tissue.
Also noted was a three-fold increase in organelles in the treated cells over the controls
(Table 1). This included an increase in mitochondria, lysosomes, peroxisomes, and
various other vacuoles. Extensions, which increased one day post cisplatin treatment

(Figure 4a) were more prevalent in Kupffer cells at day six (Figure 4b), and decreased in

20

at days fifteen (Figure 4c) and thirty (Figure 4d). Other signs of increased activation such
as increase in Kupffer cell numbers, mitochondria, electron lucent and dense organelles,
and vermiform bodies were much more prevalent in day six tissues over any other time.
Also seen in tissue six days post treatment was cell to cell interaction between the
Kupffer cells and natural killer (pit) cells (Figure 5). This type of interaction was not

seen in any other treatment mode.

 

Figure 1

Light micrograph showing esterase staining within the hepatic sinusoid of
the control tissue (A) and the cisplatin treated tissue (B). Note the increase
in Kupffer cells (arrows) and increase in extensions (insert, 2x) following

cisplatin treatment. (Bar = 50 pm).

22

 

 

 

 

Figure 2 Normal Kupffer cell (KC) in the sinusoidal lumen (S). Very few
extensions (E) are exhibited. N, nucleus; A, annulate lamellae; H,
hepatocyte (Bar = 1.0 um).

23

 

Figure 3 Cisplatin activated Kupffer cell (KC) along the endothelial lining.
Extensions (E) can be seen branching out from the center. An
increase in lysosomes and peroxisomes (*) is also noted. N, nucleus;
H, hepatocyte (Bar = 3.0 pm).

24

 

Figure 4

 

After one day post-cisplatin treatment (A) the Kupffer cells on average
exhibited one or more extensions. Six days post—cisplatin treatment (B)
showed the highest increase in numbers, extensions, and cytoplasmic
organelles. These signs of activation were decreased by day fifteen (C),
and returned to normal by day thirty (D). .KC, Kupffer cell; H,
hepatocytes; En, endothelial cell.

(Bar: A = 1.5 urn, B = 1.9 tun, C = 2.6 pm, and D =1.5 um)

 

i
\

 

 

 

 

 

 

Figure 5 Hepatic sinusoid six days post-cisplatin treatment showing interaction
between activated Kupffer cell (KC) and natural killer cell (NK) (Bar = 1.5

um).

26

 

 

 

10 - ‘
I I Cisplatin Treated

l El Control

  

 

 

  

 

 

 

Number Extensions Cytoplasm Organelles

 

 

 

 

 

 

 

 

 

Treatment Average Number Average Cytoplasm to Number of

of Cells per Number of Nuclear Ratio Organelles
Viewing Area Extensions

Normal 2.2 i 0.4 0.7 i 0.5 l 2.2:1 13 i 2.5

Saline

Cisplatin (9 19.8 :1: 4.0 3.2 i- 1.1 4.1:1 27 i- 6.8

Ins/kg)

Figure 6 Graph showing the proportional changes in the liver Kupffer cells

following cisplatin treatment. The number of Kupffer cells increased
nearly ten-fold (based on esterase staining). Other changes include
increases in extension formation, cytoplasm, and cellular organelles (based
on TEM images). Actual changes are recorded in table below graph and
are averages based on 50 areas viewed per treatment.

27

 

 

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DISCUSSION

The staining pattern observed in light microscopy was the result of the non-
specific esterases found in the tissue. The mammalian liver has been shown to exhibit
one of the highest concentrations of these esterases compared to other organs, thus
making esterase staining a valuable tool for the observation of the various cell types
(Smedsrod et al. 1985). Specifically, cells of monocyte lineage can be identified due to
the abundance of histochernically detectable esterases within the cytoplasm (ten Hagen et
al. 1996). These cells are differentiated from the positively staining hepatocytes due to
their location in the sinusoid. Difficulty arises when trying to quantify the number of
Kupffer cells using this method in non-perfused tissue, as circulating monocytes will also
stain positive (van der Rhee et al. 1977). Peroxidase staining can be used to differentiate
Kupffer cells from monocytes, but hepatocytes do stain, making difficulties in counting
(Bouwens et a1. 1986), (Wisse 1974b). Antibodies against Kupffer cells and in-situ
hybridization techniques are also effective ways to visualize and quantify these cells, but
these techniques vary in reproducibility and can be very expensive (Bodenheimer et al.
1988), (ten Hagen et al. 1996).

Electron microscopic visualization is the most effective method of differentiation
due to the unique morphological qualities of the Kupffer cells. Their location, size, and
overall appearance are fairly constant in both scanning and transmission electron
microscopy (Toge et al. 1981), (W isse 1977). More specifically, they contain organelles
found exclusively in Kupffer cells; bristle coated micropinocytic vesicles and vermiform
bodies (W isse 1974b). Using these morphologic characteristics we were able to

determine the Kupffer cell population did not increase as dramatically as previously

28

described (Johnson and Aggarwal 1996). In fact, in tissues one day post-treatment there
is no noticeable increase in Kupffer cells numbers compared to the control. The previous
estimation was due to positive esterase staining in circulating monocytes, contributing to
the overall number. These monocytes migrated to the liver, but had not differentiated
into macrophages (Tanner et a1. 1982).

It is not until six days post-treatment that we see an overall increase in the number
of Kupffer cells. On average, each section of liver revealed a three-fold increase in
Kupffer cells over the control. Self proliferation of Kupffer cells is impossible due to the
inhibition of cell division in cisplatin treated tissue (Rosenberg et a1. 1965), therefore the
increase in numbers are solely the result of monocyte differentiation. These Kupffer cells
are more active in phagocytosing invader cells, due to their, increase in cytoplasmic
extensions (T oge et al. 1981). These extensions have been shown to seek out and either
phagocytose invader cells, or present them to natural killer cells for destruction (Malter et
al. 1986).

Macrophage activation is a rapid event, usually occurring within 1-2 days after
initial administration of the activating agent (Cohen et a1. 1986), (Drysdale et al. 1983),
(Malter et al. 1986). Monocytic differentiation into macrophages, however, takes a
longer period of time (van der Rhee et al. 1979a). This is in part due to the need to
migrate to the area of differentiation, then develop the attributes of the specific tissue
macrophage. For cisplatin treated tissues, we have shown this differentiation occurs
between one and six days post-treatment. After the six day peak, the size and number of

Kupffer cells begins to return to pre-treatment levels, and by day thirty return to normal.

29

CONCLUSION

Kupffer cells are activated by the anti-cancer agent Cisplatin. Activation entails
two events, each working independently. These events are macrophage activation and
monocyte recruitment. Macrophage activation occurs rapidly, with noticeable changes in
shape and function occurring within one day post treatment (T oge et al. 1981). Monocyte
recruitment is a more lengthy process (van der Rhee et al. 1979a). Monocyte migration
occurs within one day post-treatment, but differentiation takes up to five days post-
treatment before the optimal number of Kupffer cells have been produced. The liver, in
an effort to restore homeostasis, regulates the number of Kupffer cells, reducing them by
day fifteen and returning them to their pre-treatment numbers by day 30. The exact
mechanism of this regulation is unknown.

Due to the increase in numbers and cytoplasmic extensions, the Kupffer cell
population is better equipped to deal with hepatic insult. Activated Kupffer cells,
efficient in phagocytosing invader cells, are effective scavengers of invading organisms in
the bloodstream due to their location in the sinusoid (Malter et al. 1986), (Ohnishi et al.
1982). An increased understanding of the mechanisms controlling Kupffer cell size and

number can lead to more effective treatment of liver distress.

30

REFERENCES

Abrams MJ, Murrer BA (1993) Metal compounds in therapy and diagnosis. Science
2612725-30

Bodenheimer HC, Jr., Faris RA, Charland C, Hixson DC (1988) Characterization of a
new monoclonal antibody to rat macrophages and Kupffer cells. Hepatology 8: 1667-72

Bosl G, Lange P, Franley E (1980) Vinblastine, bleomycin and cis-
diamrninedichloroplatinum in the treatment of ovarian and testicular cancer. American
Journal of Medicine 68:492

Bouwens L, Baekemand M, Wisse E (1986) A balanced view on the origin of Kupffer
cells. In Kim A, Knook DL, Wisse E, eds. Cells of the Hepatic Sinusoid. Leiden,
Elsevier, 7-12

Bouwens L, Wisse E (1982) On the dual origin of the Kupffer cell. In Knook DL, Wisse
E, eds. Sinusoidal Liver Cells. Leiden, Elsevier, 165-172

Cohen SA, Werner M, von Muenchhausen W, Dembinski W, Nolan JP (1986) Role of
interferon and cytotoxin in the natural cytotoxitiy of isolated murine nonparenchymal
liver cells. In Kim A, Knook DL, Wisse E, eds. Cells of the Hepatic Sinusoid. Leiden,
Elsevier Biomedical Press, 405-406

Cohn ZA (1978) Activation of mononuclear phagocytes: fact, fancy, and future. J
Immunol 121:813-6

Drysdale BE, Zacharchuk CM, Shin HS (1983) Mechanism of macrophage-mediated
cytotoxicity: production of a soluble cytotoxic factor. J Immunol 131:2362-7

Johnson BN, Aggarwal SK (1996) The effects of cisplatin, taxol, and cisplatin plus taxol
on non-specific esterase and alkaline phosphatase activity in rat Kupffer cells. In Bailey
GW, Corbett JM, Dimlich RVW, Michael JR, Zaluzec NJ, eds. Microscopy and
Microanalysis 1996. Minneapolis, MN, San Francisco Press, Inc., 788-789

Karnovsky ML, Lazdins JK (1978) Biochemical criteria for activated macrophages. J
Immunol 121:809-13

Kuiper J, Brouwer A, Knook DL, van Berkel TJC (1994) Kupffer and Sinusoidal
Endothelial Cells. In Arias HVI, Boyer JL, Fausto N, Jakoby WB, Schachter DA, Shafritz
DA, eds. The Liver: Biology and Pathobiology, Third Edition. New York, Raven Press,
Ltd., 791-818

Malter M, Friedrich E, Suss R (1986) Liver as a tumor cell killing organ: Kupffer cells
and natural killers. Cancer Res 46:3055-60

31

McCuskey RS, McCuskey PA (1990) Fine structure and function of Kupffer cells. J
Electron Microsc Tech 14:237-46

Ogawa T, Koerten HK, Daems WT (1978) Peroxidase activity in monocytes and tissue
macrophages of mice. Cell Tissue Res 188:361-73

Ohnishi R, Yanai K, Sugawa-Katayama Y, Chin K (1982) SEM observations of Kupffer
cells with special reference to phagocytosis and the cytoskeleton. In Knook DL, Wisse E,
eds. Sinusoidal Liver Cells. Leiden, Elsevier Biomedical Press, 127-138

Palma JP, Aggarwal SK (1994) Cisplatin and carboplatin mediated release of cytolytic
factors in murine peritoneal macrophages in vitro. Anticancer Drugs 5:615-22

Palma JP, Aggarwal SK, Jiwa A (1992) Murine macrophage activation after cisplatin or
carboplatin treatment. Anticancer Drugs 31665-76

Rosenberg B, Van Camp L, Krigas T (1965) Inhibition of cell division in Escherichia coli
by electrolysis products from a platinum electrode. Nature 205:698-699

Shi J, Fujieda H, Kokubo Y, Wake K (1996) Apoptosis of neutrophils and their
elimination by Kupffer cells in rat liver. Hepatology 24:1256-63

Singh SM, Sodhi A (1988) Interaction between cisplatin-treated macrophages and
Dalton's lymphoma cells in vitro. Exp Cell Biol 56:1-11

Smedsrod B, Pertoft H, Eggertsen G, Sundstrom C (1985) Functional and morphological
characterization of cultures of Kupffer cells and liver endothelial cells prepared by means
of density separation in Percoll, and selective substrate adherence. Cell Tissue Res
241:639-49

Tanner A, Keyhani A, Arthur M, Wright R (1982) Evidence for a sequence of
macrophage activation during recruitment into the liver. In Knook DL, Wisse E, eds.
Sinusoidal Liver Cells. Leiden, Elsevier Biomedical Press, 405-412

Telgenhoff DJ, Aggarwal SK (1998) Kupffer cell activation after treatment with cisplatin
and its second generation novel analogs SAP, SSP, and "poly-plat". In Collery P, Bratter
P, Negretti de Bratter V, Khassanova L, Etienne J-C, eds. Metal Ions in Biology and
Medicine. Neuherberg, Germany, John Libbey Eurotext, 646-648

ten Hagen TL, van Vianen W, Bakker-Woudenberg 1A (1996) Isolation and

characterization of murine Kupffer cells and splenic macrophages. J Immunol Methods
193281-91

32

Toge T, Nakanishi K, Yamada Y, Yanagawa E, Hattori T (1981) Scanning electron
microscopic studies on the surface structure of activated macrophages and on their
interaction with tumor cells. Gann 72:305-9

van der Rhee HJ, de Winter CP, Daems WT (1977) Fine structure and peroxidatic activity
of rat blood monocytes. Cell Tissue Res 185: 1-16

van der Rhee HJ, van der Burgh-de Winter CP, Daems WT (1979a) The differentiation of
monocytes into macrophages, epithelioid cells, and multinucleated giant cells in
subcutaneous granulomas. I. Fine structure. Cell Tissue Res 197:355-78

van der Rhee HJ, van der Burgh-de Winter CP, Tijssen JG, Daems WT (1979b)
Comparative study on peroxidatic activity in inflammatory cells on cutaneous and
peritoneal implants. Cell Tissue Res 197:397-412

Wisse E (1977) Ultrastructure and function of Kupffer cells and other sinusoidal cells in
the liver. In Wisse E, Knook DL, eds. Kupffer cells and other sinusoidal lining cells.
Amsterdam, Elsevier, 33-60

Wisse E (1974a) Kupffer cell reactions in rat liver under various conditions as observed
in the electron microscope. J Ultrastruct Res 46:499-520

Wisse E (1974b) Observations on the fine structure and peroxidase cytochemistry of
normal rat liver Kupffer cells. J Ultrastruct Res 46:393-426

Xu ZL, Bucana CD, Fidler I] (1984) In vitro activation of murine Kupffer cells by
lymphokines or endotoxins to lyse syngeneic tumor cells. Am J Pathol 117:372-9

33

CHAPTER 2: KUPFFER CELL ACTIVATION AFTER TREATMENT
WITH CISPLATH‘J AND ITS SECOND GENERATION
NOVEL AN ALOGS SAP, SSP AND POLY-PLAT

34

SUMMARY

SAP (4-Hydroxy-a-sulfonylphenylacetato [trans 1,2-diaminocyclohexane]
platinum II), SSP (5-sulfosalicylato-trans-[1,2-diaminocyclohexane] platinum), and
Poly-plat (Poly-[(trans-l,2-diaminocyclohexane) platinum]-carboxyamylose) are
second generation analogs of cisplatin (CDDP) with higher efficacy than cisplatin. In
vitro studies using these compounds have shown activation of peritoneal macrophages in
terms of various cytokines secreted. The present study was undertaken to examine the
effects of these new compounds on Kupffer cell activation and their role in

detoxification.

Wistar rats (100-120g) were given intraperitoneal injections of either cisplatin (9
mg/kg), SAP (10 mg/kg), SSP (10 mg/kg), or Poly-plat (10 mg/kg) in 0.9% (w/v) normal
saline over 5 days. Controls received equal amounts of 0.9% normal saline. On day 6 the
animals were sacrificed and the livers fixed in 2% (v/v) glutaraldehyde in 0.1 M
cacodylate buffer (pH 7.3). Liver tissues were post-fixed in 1% (w/v) osmium tetroxide,
dehydrated, and embedded for electron microscopy. Thick sections (1 mm) were placed
in KOH for 15 minutes to remove araldite, and stained with hemotoxylin and eosin for
light microscopy.

Light microscopy studies revealed an increase in the non-parenchymal cells in the
treated tissues compared to the normal. Also evident under light microscopy was an
increase in extension formation. The most obvious increases occurred with all three
analogs with respect to cisplatin. Transmission electron microscopy disclosed increases
in size, extensions, and cytoplasmic contents in treated tissues compared to the control.

Extension formation was greatest in the Poly-plat treated tissues, showing more

35

C\

ilk

 

extensions per Kupffer cell on average than any other treatment. There was also a
corresponding increase in cytoplasm, lysosomes, and mitochondria beyond other
treatments.

These results show SAP, SSP, and Poly-plat exhibit an increased ability to
activate the immune system compared to the widely used antineoplastic drug cisplatin.
Of the three compounds, Poly-plat appears to be the most promising analog for immune

system activation.

36

INTRODUCTION

An effective method of destroying tumors in the body is through the activation of
the immune system (Bahadur et al. 1984), (Zacharchuk et al. 1983). Activated
macrophages seek out tumor cells, make contact with cytoplasmic extensions, and
transfer lysosomes to the tumor cell (Palma and Aggarwal 1995). Cisplatin is a potent
anti-neoplastic agent which works via macrophage activation (Abrams and Murrer 1993).
Other drugs have been developed which are more potent at macrophage activation and
much less toxic (Fiebig et a1. 1996), (Drees et al. 1995). Three novel second generation
platinum containing antineoplastic agents have been developed; SAP (4-Hydroxy-a-
sulfonylphenylacetato [trans 1,2-diaminocyclohexane] platinum H), SSP (5-
sulfosalicylato-trans-[1,2-diaminocyclohexane] platinum), and Poly-plat (Poly-
[(trans-l,2-diaminocyclohexane) platinum]-carboxyamylose) (Andrulis
Pharmaceuticals, Bethesda, MD) which have been shown in initial studies to be more
effective in the treatment of cancer and less toxic than cisplatin (Muenchen et al. 1997).
These drugs, in particular Poly-plat, are much larger than cisplatin, with numerous
branching side chains radiating from a central platinum atom.

The present study was undertaken to examine the effects of these new compounds
on Kupffer cell activation in the liver as well as non-specific esterase activity in the
hepatocytes. Circulating monocyte levels were also examined to correlate increases in

resident macrophages with monocyte levels.

37

MATERIALS AND METHODS

Animals. Wistar rats (125-135 grams) were kept on a twelve hour light, twelve
hour dark cycle with free access to feed and water. All rats were weighed two days prior
to the first injection and each day thereafter until they were sacrificed. Three rats each
were given intraperitoneal injections of either cisplatin (9 mg/kg), SAP (10 mg/kg), SSP
(10 mg/kg). or Poly-plat (10 mg/kg) in 0.9% (w/v) normal saline over 5 days. Controls
received equal amounts of 0.9% normal saline. Tail vein blood draws were performed
each day throughout the experiment. 22 gauge needles were inserted into the tail veins of
the rats and 0.5 ml was withdrawn. Blood was smeared onto slides and air-dried.
Monocyte levels were determined morphologically by Giemsa staining of air—dried blood
smear slides. The number of monocytes per viewing area were counted and averaged for
25 areas per treatment.

Tissue Prengration. On day 6 the animals were anesthetized with carbon dioxide
and the animal was opened via abdominal incision. The livers were removed and either
fixed in 2% (v/v) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) for TEM analysis or
were placed in OCT compound and frozen using dry ice and acetone for light
microscopy. Tissue fixed in glutaraldehyde was post-stained in 1% (w/v) osmium
tetroxide, dehydrated through a graded acetone series, and embedded in araldite for
transmission electron microscopic observation. Sections were cut on the ultrarnicrotome,
stained with uranyl acetate and lead citrate, and examined on the Phillips CM-lO TEM.
Frozen tissue was cut at 10 um thickness on the cryotome and stained for the
demonstration of non-specific esterase using the azo dye method [3]. Stained sections

were viewed and staining intensity determined on the Zeiss laser scanning microscope.

38

RESULTS
Weight Changes. Animals given only the vehicle of injection continued to gain
weight throughout the course of the experiment, with an average weight gain of 35
grams. Animals treated with platinum compounds gained weight initially, but reached a
plateau three days after treatment. On average these animals only gained 20 grams.
Cisplatin treated rats gained weight initially, but after day three steadily lost weight, for

an average weight gain of only 5 grams (Table 1).

Circulating Monocyte Levels. Monocyte levels following treatment with cisplatin
or Poly-plat decreased following treatment with the platinum agents, possibly due to
sequestration of the monocytes into resident macrophages (Figure 1). In the Poly-plat
treated rats this decrease was followed by a sharp increase indicating an immune
response to the decrease. Cisplatin treatment caused a more lengthy suppression of
circulating monocyte levels. Monocyte levels in SAP and SSP treated animals were
similar to the control (not shown).

Gross Morphology. Prior to euthanasia animal behavior was monitored and

 

recorded. Animals receiving cisplatin appeared listless after the third day of injection,
which continued until the end of the experiment. Cisplatin treated animals ate very little
food, and had loose, infrequent stool. All other animals treated appeared normal, with
food intake similar to the untreated rats. After euthanasia the internal organs were
removed and examined (Figure 2). The rats treated with cisplatin had bloated stomachs,
filled with undigested food. The intestines had a slippery feel, with a slime-like coating
not seen in other rats. The kidneys of the cisplatin treated rats were slightly smaller than

untreated rats, and had a grainy appearance on the surface. All other platinum

39

Comt‘

exttt‘.
ettcr
slmll.
pltllll‘
Quarf
norm.
to to."
in at
4b). 8
the lit
SlllUSt‘
Signili

alllOUl.

 

compounds had a morphology similar to the untreated rats.

Morphology. In the normal liver Kupffer cells were round and exhibited few

 

extensions (Figure 3a). Cisplatin treatment demonstrated an increase in Kupffer cell
extensions (Figure 3b). Extension formation following SAP and SSP treatment was
similar to CDDP (Not Shown). Poly-plat treatment, however, revealed an even more
pronounced activation with a greater increase in extension formation (Figure 3c).
Quantification of Kupffer cells was performed using non-specific esterase staining. The
normal liver exhibited a moderate amount of esterase staining, and was used as a standard
to compare the drug treated against (Figure 4a). The CDDP treated liver had an increase
in esterase staining in the sinusoids, and a significant decrease in the hepatocytes (Figure
4b). SAP treatment showed decreased staining in the sinusoids and increased staining in
the hepatocytes (Figure 4c). SSP treatment had decreases in staining in both the
sinusoids and the hepatocytes (Figure 4d). Poly-plat treatment demonstrated a
significantly higher degree of staining in the sinusoids than CDDP, and a moderate

amount of staining in the hepatocytes, comparable to normal liver (Figure 4e).

40

Table ‘

 

Weight Changes Following Platinum Drug Administration

Days of Treatment

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment 1 2* 3* 4* 5* 6* 7 8
Control 135 139 143 148 155 162 164 170
Cisplatin 130 139 143 150 146 142 140 135
SAP 132 137 142 145 146 149 152 154
SSP 132 138 140 141 143 141 148 150
Poly-plat 131 136 146 150 152 154 153 155
Table 1: Changes in weight of rats following platinum drug administration. The

values listed are the averages of three rats per treatment group. Asterisks
denote days of treatment.

41

Flgurc

 

Figure 1:

Monocytes per Viewing Area Following Platinum
Drug Treatment

 

4.5 ~——-————l +N0rmal .
4 «7 ———, +Cisplatin
3.5 + ~———— l-t-Poly-plat

 

 

 

 

 

 

 

N
u:

 

 

p—g
O

1

O
OUIv—‘Ui
(\

 

 

Monocytes per Viewing Area
N

 

 

 

Circulating monocyte levels following treatment with cisplatin (10 ug/ml)
or Poly—plat (10 ug/ml). Note the decrease in monocytes following
treatment with the platinum agents, followed by a sharp increase
indicating an immune response to the decrease. Cisplatin treatment
caused a more lengthy suppression of circulating monocyte levels.

42

Fi

 

Qure

 

Figure 3: Kupffer cells from control (A), cisplatin (B), and Poly-plat (C) treated rat
liver. Note the large extensions evident in the Poly-plat treated liver. Bar
= 2 urn. '

 

 

 

Fight:

 

Figure 4:

 

-v_
s
1..

4ft."

if)

3 57?“ j

”if W ' "Tfi-z

‘7-
r

Light micrographs showing esterase staining in the liver of rats which
received no treatment (A) or treatment with cisplatin (B), SAP (C), SSP
(D), or Poly-plat (E). Black arrows denote Kupffer cells. Note the
increase in the number of Kupffer cells in the cisplatin and Poly—plat
treated tissues. The SAP and SSP treatments were decreased below
normal. The hepatic esterase (background staining) was decreased
following cisplatin and SSP treatment. Hepatic esterase was normal in
SAP and Poly-plat treated tissues. Bar = 50 um.

45

 

 

Table

 

Summary of The Effects of Platinum Agents

 

 

 

 

 

 

 

 

 

 

 

 

Treatment Extension Formation Sinusoidal Esterase Hepatic Esterase

Normal None + + + +

Cisplatin Increased + + + +

SAP Increased + V2 + + +

SSP Increased + +

Poly-plat Large Increase + + + + + +

Table 2. Kupffer cell extension formation and non-specific esterase staining in

treated and untreated rat liver. Staining intensity was measured visually
and an average score was obtained from 10 slides per treatment using
these ratings: (-) None (negative control), (+) Low, (+ +) Moderate, (+++)
High, (++++) Very High. Data collected for extension formation from 50
TEM images per treatment group. Sinusoidal and hepatic esterase data
collected from 25 light microscopy images per treatment group.

46

 

tiger:
d.l
set c
sulp‘
enzj.
ktc.

rear

Clfl;
incrc
ener
the r

bond

 

 

SSP,
Woul
Unkn
hydr
SSP
ktel
aCllL

C5“ r

 

DISCUSSION

Stimulation of the immune system is a major function of many antineoplastic
agents. Cisplatin stimulates the immune system through macrophage activation (Palma et
al. 1992), making the macrophages more efficient in phagocytosing tumor cells. The
severe toxic side effects evident in cisplatin treatment seem to be due to the disruption in
sulphydryl containing mitochondrial dehydrogenase enzymes. The disruption of these
enzymes prevents calcium uptake thereby reducing calcium levels in the cell to cytotoxic
levels (Aggarwal 1993). SAP, SSP, and Poly-plat show no such disruption, and as a

result have been shown to be much less toxic.

Due to low toxicity SAP, SSP, and Poly-plat would appear to be alternatives to
CDDP. SAP and SSP both activate macrophages, however there is no significant
increase in Kupffer cell numbers after SAP and SSP treatment, as shown by sinusoidal
esterase staining. Poly-plat not only increases Kupffer cell activation, but also increases
the number of Kupffer cells. Non-specific esterases catalyze the breakdown of ester
bonds, which is a major mechanism of detoxification in the liver (Williams 1985). SAP,
SSP, and Poly-plat all contain ester bonds, and a decrease in hepatocellular esterase
would result in increased circulating concentrations of unhydrolyzed drugs. It is
unknown whether these drugs are active in their natural or biotransformed state. If ester
hydrolysis is required for drug activation, then the decreased levels of esterase found in
SSP treated liver may result in a reduced pharmacological effect. Conversely, the normal
levels of esterase found in Poly-plat treated hepatocytes may explain the increased
activation seen in Kupffer cells after Poly-plat administration. It is unclear why Kupffer

cell numbers are low in SAP treated livers, yet hepatic cell esterase levels are higher than

47

 

 

 

 

 

IOU!
civ

Cist

 

found in the normal tissue. These results show that Poly-plat is more effective than
cisplatin, SAP, or SSP in macrophage activation, with less toxic side effects compared to

cisplatin.

48

 

 

 

Abt
201'

 

REFERENCES

Abrams MJ, Murrer BA (1993) Metal compounds in therapy and diagnosis. Science
261:725-30

Aggarwal SK (1993) A histochemical approach to the mechanism of action of cisplatin
and its analogues. J Histochem Cytochem 41:1053-73

Bahadur A, Sarna S, Sodhi A (1984) Enhanced cell mediated immunity in mice after
cisplatin treatment. Pol J Pharmacol Pharm 36:441-8

Drees M, Dengler WM, Hendriks HR, Kelland LR, Fiebig HH (1995) Cycloplatam: a
novel platinum compound exhibiting a different spectrum of anti-tumour activity to
cisplatin. Eur J Cancer 3:356-61

Fiebig H, Dress M, Ruhnau T, Misra H, Andrulis P, Hendriks H (1996) GB-21, a novel
platinum complex with antitumor activity in human renal and mammary xenografts.
Proceedings of the American Association of Cancer Research 37:297

Muenchen HJ, Aggarwal SK, Misra HK, Andrulis PJ (1997) Enhanced
immunostimulation by novel platinum anticancer agents. Anti-Cancer Drugs 8:323-328

Palma JP, Aggarwal SK (1995) Cisplatin and carboplatin-mediated activation of murine
peritoneal macrophages in vitro: production of interleukin-1 alpha and tumor necrosis
factor-alpha. Anticancer Drugs 6:311-6

Palma JP, Aggarwal SK, Jiwa A (1992) Murine macrophage activation after cisplatin or
carboplatin treatment. Anticancer Drugs 3:665-76

Williams F (1985) Clinical significance of esterases in man. Clinical pharmacokinetics
10:392-403

Zacharchuk CM, Drysdale BE, Mayer MM, Shin HS (1983) Macrophage-mediated

cytotoxicity: role of a soluble macrophage cytotoxic factor similar to lymphotoxin and
tumor necrosis factor. Proc Natl Acad Sci U S A 80:6341-5

49

 

 

 

Cll

 

 

CHAPTER 3: EFFECTS OF POLY-PLAT ON TUMOR CELLS IN VITRO

50

SUMMARY

The action of poly-[(trans-l,2-diaminocyclohexane)platinum]-carboxyamylose
(Poly-plat) on tumor cells was examined using human fibrosarcoma (HT1080) and
Walker rat carcinoma (W RC-256) in culture. These cells were treated with Poly-plat (10
ug/ml) or cis-dichlorodiamrnine platinum (cisplatin) (10 ug/ml) for 2-5 days. Apoptosis
assays (morphology and TdT-mediated dUTP nick-end labeling) were used to detemtine
cytotoxic effects of drugs on tumor cells. Cell protein extracts were collected and
analyzed for the cell cycle regulatory proteins p27 (an inhibitor of cell cycle progression)
and Proliferating Cell Nuclear Antigen (a marker of cell proliferation) via Western blot
analysis. The results demonstrate the ability of Poly-plat to arrest the growth of tumor

cells but not induce apoptosis in these cell lines.

51

 

D.\
phi
ape
tre;
prrv

204

&p( tr

0th:

actlt

 

WTRODUCTION

Cisplatin, a potent anti-neoplastic agent, works via inhibition of DNA replication
(Fichtinger-Schepman et al. 1986), inhibition of the cytokinesis (Abrams and Murrer
1993), induction of apoptosis (Nehme et al. 1997); (Ferreira et al. 2000), and macrophage
activation (Palma et al. 1992). Cisplatin exerts its cytotoxicity via the formation of
interstrand and intrastrand crosslinks, which ultimately arrests the cell cycle at G1, S, or
G2 (Fichtinger-Schepman et al. 1986) (Sundaralingam et al. 1985). These platinum-
DNA adducts also alert DNA damage sensors (ATM, DNA-PKcs, and ATR), which
phosphorylate p53, ultimately resulting in the activation of the caspase cascade and
apoptosis (Porter et al. 1997). Although cisplatin has been shown to be effective in
treating various types of cancers (Abrams and Murrer 1993) drug resistance is a major
problem, especially with repeated doses of the drug (Welters et al. 1998); (Beale et al.
2000). Factors affecting resistance include an increase in drug efflux, a decrease in drug
influx, increases in glutathione levels, DNA repair, and drug tolerance (Henkels and
Turchi 1999); (Beale et al. 2000) (Nishimura et al. 1996).

The interactions between cisplatin and DNA are believed to be the main cause of
apoptosis in cisplatin mediated cell death (Gong et al. 1999) (Henkels and Turchi 1999).
Other platinum based drugs have been developed which are more potent at macrophage
activation and are less toxic (Fiebig et al. 1996) (Drees et al. 1995). Poly-plat (Poly-
[(trans-1,2-diaminocyclohexane) platinum]-carboxyamylose) is one such compound
(Fiebig et al. 1996). Poly-plat has been shown to activate macrophages to produce

inflammatory cytokines in greater quantities than cisplatin (Muenchen and Aggarwal

52

1998). Poly-plat is less toxic than cisplatin in equal doses of the drugs (Muenchen et al.
1997).

Two cell cycle regulatory proteins, Proliferating Cell Nuclear Antigen (PCNA)
and p27, have important roles in the cell cycle. PCNA is required for DNA replication
during the S-phase of the cell cycle (Kurki et al. 1986). Increased levels of PCNA in cell
cultures indicate an increase in proliferation of the cells (Giordano et al. 1991). p27 is a
protein belonging to the family of CDK inhibitors, which inhibit progression through the
cell cycle (Luo et al. 1995), (Sgambato et al. 2000). An increase in p27 levels in rapidly
proliferating cell types following treatment with antineoplastic agents is a good marker
for cell cycle inhibition (Oshita et al. 2000). Specifically, p27 binds to Cdk4/cyclin D

complexes resulting in cell cycle arrest at G, the growth phase prior to DNA replication.

53

MATERIALS AND METHODS

Cell Cultures. The HT1080 human fibrosarcoma cells were a gift from the

 

Michigan State University Carcinogenesis Laboratory (East Lansing, MI). WRC-256
Walker rat carcinoma cells were purchased from American Type Cell Culture (Rockville,
MD). Cell lines were kept in MEM supplemented with 10% (v/v) horse serum and 1%
(w/v) antibiotic [penicillin G (10,000 U/ml) and streptomycin sulfate (10,000 ug/ml)] and
stored in T25 flasks at 37° C in a 5% CO; incubator.

Treatments. Cisplatin and Poly-plat were dissolved in 0.1 ml dimethyl

 

formamide, followed by the addition of MEM for a final concentration of 1 mg/ml. Drug
solutions were prepared fresh daily. Twelve flasks of HT 1080 and WRC-256 cells were
used per treatment. Cells were treated with cisplatin (10 ug/ml), Poly-plat (10 ug/ml), or
drug solvent alone in the T25 flasks for five days. Three flasks per treatment were
removed and the supernatant was poured off. Cultures were rinsed with MEM and 1 ml
trypsin/EDTA (0.25g trypsin/0.1g EDTA in 0.22% (w/v) NaCl) was added for five
minutes to remove adherent cells. Horse serum (5 ml) was added to each flask to inhibit
trypsin and the entire solution was poured into a 50 ml conical polypropylene tube. Cells
were centrifuged at 1000 g for five minutes, washed once with MEM and resuspended in
fresh medium. Cell counts were performed using an American Optical hemacytometer
via dye exclusion with 0.2% (w/v) trypan blue. This was repeated 1, 2, and 5 days after
treatment.

Apoptosis Assg HT1080 cells were seeded onto 18 mm2 glass coverslips at l X

106 cells/ml and placed in 35 mm plastic Petri dishes. Cells were incubated for 4 hours at

37° C in a 5% CO; incubator. Fresh MEM containing 10% horse serum (5 ml) was

54

added to the Petri dishes. HT1080 cells were then treated with cisplatin (10 ug/ml), Poly-
plat (10 ug/ml), or drug solvent for two hours. The cells were rinsed with MEM and
cultured in normal medium. Coverslips were removed from culture after 48 hours, fixed
in 2% (v/v) glutaraldehyde in phosphate buffered saline (PBS), and stained by the In situ
cell death detection kit, using double strand DNA breaks by the TdT-mediated dUTP
nick-end labeling (T UNEL) reaction (Sanders and Wride 1996). In addition to the
TUNEL assay coverslips were fixed in 2% glutaraldehyde in PBS and stained with Harris
hematoxylin (Kieman 1999). All coverslips were dehydrated in graded ethanol, cleared
in xylene, and mounted on glass slides with Perrnount mounting medium. Slides were
viewed on the Zeiss LSM 210 confocal microscope and in transmitted light. TUNEL
slides were also viewed in fluorescence with an excitation wavelength of 488 nm and
emission of >520 nm. The experiments described above were prepared in triplicate.
PCNA and p27 Analysis. Cultures of HT1080 cells were grown to 75%
confluence in twelve T75 flasks. Six flasks were treated with Poly-plat (10 ug/ml,
prepared as listed above), the remaining six with the vehicle only. After two hours the
treatment was removed and the flasks were rinsed with PBS. Fresh medium was added to
the flasks and they were returned to the incubator. Three flasks from each treatment
group were removed at 24 and 48 hours post treatment and the medium was aspirated.
Cell lysing solution was added to each flask (300 mM urea and 2% (w/v) SDS in PBS)
and the flasks were scraped with plastic scrapers. The solution was pipetted into screw
cap 2 ml tubes and sonicated for 1 minute with a tip sonicator. The tubes were then
placed in a boiling water bath for five minutes. The extracts were then frozen at -20° C

until all extracts were collected.

55

Protein Separation. The protein extracts were removed from the freezer and
thawed at room temperature. Three 10 ul aliquots were taken from each tube and the
protein concentration for each sample was determined from an average of the three using
the BCA protein assay and the spectrophotometer. 15 ug of protein from each sample
was mixed with loading buffer [glycerolz B-mercaptoethanol: bromphenol blue (1 mg/ml
in water) in a 3:1:0.5 ratio, to total 30 ul] and loaded on a 10-20% tricine gel. Four
identical gels were prepared. A marker protein ladder was added in lane 1. The gels
were run at 55 mA for 4 hours, until the dye front approached the bottom. The gels were
then electroblotted onto a nitrocellulose membrane for 90 minutes at 100 volts. The blots
were stained with ponceau S for 2 minutes, rinsed briefly in PBS, and photographed for
transfer of protein. The protein marker ladders were marked with ink and the blots were
rinsed in PBS to remove the ponceau S. The blots were then blocked in blocking buffer
(7.5% (w/v) milk and 0.1% (v/v) Tween-20 in PBS) for one hour at room temperature.

Antibodies. Mouse anti-p27 and mouse anti-PCNA antibodies were prepared
separately in 1:500 dilutions in blocking buffer. The four blots were placed in plastic
bags and two were filled with mouse anti-p27 antibody solution, two with mouse anti-
PCNA antibody solution. The bags were sealed and placed at 4° C overnight. Following
day the blots were rinsed with PBS three times for ten minutes each. The blots were
again placed in bags and sealed with goat anti-mouse HRP conjugated secondary
antibody (1:1000 dilution in blocking buffer) for 2 hours at room temperature. The blots
were rinsed three times with PBS for ten minutes each.

Visualization of Protein Bands. The blots were placed on parafilm and excess

PBS was removed. ECL working detection reagent (Amersham) was applied in the dark

56

and the blots were covered with parafilm for five minutes. Excess ECL reagent was
removed and the blots were placed between sheets of plastic and taken to the dark room.
The blots were exposed to film for times ranging from 5-30 seconds. The film was
developed and scanned with a Microtek scanner. Banding intensity was determined

using Image Pro Plus imaging software.

57

RESULTS

Cell Growth Inhibition. As measured by trypan blue exclusion untreated WRC-
256 cells increased in numbers from 2.1 X 105 i 0.3 to 3.5 X 105 i 0.2 after 24 hours and
6.8 X 105 i 1.1 after 48 hours (Figure l). WRC-256 cells treated with cisplatin (10
ug/ml) or Poly-plat (10 ug/ml) were inhibited to a cell growth rate nearly one half that of
the normal cells. Untreated HT1080 cells also showed a rapid growth rate, from 2.0 X
105 i 0.3 at the beginning of the experiment to 1.2 X 10‘5 i 1.1 after five days. HT1080
cells treated with Poly-plat (10 ug/ml) showed growth inhibition at 24 and 48 hours
(Figure 2). However after five days of treatment the HT 1080 cells returned to a normal
growth rate. Between 24 and 48 hours no growth was seen in the Poly-plat treated
HT1080 cell cultures.

Apoptosis Assay. HT 1080 cells stained with hematoxylin after 48 hours in
culture and examined by transmission microscopy appear large and show multiple mitotic
figures (Figure 3 A-C; Table 1). Apoptosis can be visualized in untreated HT1080 cells
after 48 hours in culture by nuclear fragmentation (Figure 3 D-F) at a rate of 1.1% (T able
1), or 2.1% using the TUNEL assay (Figure 3 G, H). The TUNEL assay detected cells
with morphological signs of apoptosis (DNA fragmentation) and cells which appeared
normal histologically (cells with DNA strand breaks but no morphological aspects of
apoptosis). No mitotic figures were seen in the cisplatin and Poly-plat treated cells after
48 hours. The Poly-plat treated cells had an apoptotic rate similar to the untreated cells.
Only the cisplatin treated cells had a marked increase in apoptosis at 48 hours after

treatment.

58

PCNA and p27. PCNA and p27 were examined for their role in progression
through the cell cycle. The nitrocellulose blots were photographed after Ponceau S
staining to show equivalence of protein loading in the gels (Figure 4). PCNA levels after
24 hours of Poly-plat treatment were roughly equal to that of the untreated cells. By 48
hours the PCNA levels had nearly tripled (Figures 5 and 6). p27 levels were higher than
the control at 24 hours after Poly-plat treatment and continued to increase after 48 hours

(Figures 5 and 7).

59

Figure 1:

Walker Rat Carcinoma Cell Growth Inhibition

 

 

8 -

7 El Untreated
6 _ ! ICisplatin

5 _ l IPoly-plat

 

Cells per flask x 10"5

 

 

 

 

Hours After Treatment

Graph showing the effects cisplatin (10 ug/ml) or Poly-plat (10 ug/ml)
treatment on WRC-256 cell growth. Note the inhibition of cell growth in
cisplatin and Poly-plat treated cultures after 24 and 48 hours. Cells
numbers were measured by trypan blue dye exclusion and standard
deviation calculated from triplicate runs of experiment.

60

Cells per flask x 10"5

Figure 2:

Human Fibrosarcoma Cell Growth Inhibition

 

 

 

 

 

 

 

 

 

 

 

 

 

2 - I ClUntreated
o l ‘
8 l I Poly-plat
6 J
A
”I :i
0 *— r r
0 1 2 5

Days After Treatment

Graph showing the effects Poly-plat (10 ug/ml) treatment on HT1080 cell
growth. Note the inhibition of cell growth in Poly-plat treated cultures
after 1 and 2 days. Cell growth returns to normal by day 5. Cells numbers
were measured by trypan blue dye exclusion and standard deviation
calculated from triplicate runs of experiment.

61

Figure 3:

 

Light micrographs showing HT1080 cells (large arrows) after 48 hours.
Untreated cells stained with hematoxylin shown not undergoing mitosis
[A], in prophase (determined by condensation of the nucleus, white arrow)
[B], and metaphase (determined by chromosomes lined up at the equator,
white arrow) [C]. Neither cisplatin (10 rig/ml) nor Poly-plat (10 ug/ml)
treated cells showed any mitotic activity after 48 hours of treatment.
Untreated cells stained with hematoxylin also showed very little apoptotic
activity [D]. Apoptosis was increased slightly in cells treated with Poly-
plat (10 ug/ml) after 48 hours [B] and can be seen with hematoxylin
staining as fragmentation of the nucleus into smaller membrane bound
vesicles (white arrows). An increase in HT1080 cells undergoing
apoptosis is seen after cisplatin treatment (10 ug/ml) after 48 hours [F]
(fragmented nucleus, white arrows). The TUNEL assay was also
performed and apoptotic cells were seen either via fluorescence [G;
untreated HT1080 cell undergoing apoptosis after 48 hours] or
transmission [H; Poly-plat treated cells (10 ug/ml) after 48 hours
treatment]. Bar = 20 ttm (A-G); 80 um (H).

62

HT1080 Cells Following Treatment

 

 

 

Treatment MitoticI ApoptosisT Apoptosis2 Viable Cells3
Figures (Hematoxylin) (TUNEL) (X 105 )
Untreated 2.0 i 0.2 1.1 i 0.3 2.7 i- 1.8 6.8 i 0.5
Cisplatin 0 18 i 2.0 26 i 2.7 3.2 i 0.8
Poly-plat O 3 i- l.1 4 i 1.3 4.6 i 0.6
Table 1: Table of human fibrosarcoma (HT1080) cells after treatment with cisplatin

(10 ug/ml) or Poly-plat (10 ug/ml).

' Visualized in transmitted light from hematoxylin stained slides.

2 Apoptosis TUNEL assay examined in fluorescence using 488 nm
excitation and > 520 nm emission.

3 Viable cells counts x 105 (1- S.D.) calculated via hemacytometer using
trypan blue dye exclusion. Beginning cell count was 2.1 X 105 for all
treatments.

* Values for mitotic figures and apoptosis given in percent 1- 3D. of 250
total cells counted per treatment.

63

Poly-plat, 24 Hours
Poly-plat, 48 Hours

if:
:r
0
~-
in
fi
N
'23
E
c:
0

Control, 48 Hours

 

Figure 4: Blots stained using Ponceau S staining solution to visualize protein bands
on nitrocellulose membrane. Note the protein bands in each lane are
similar in size and position indicating equal loading of protein extracts.

Figure 5:

P01} -plat, 48 Hours

Control 24 Hours
Control, 48 Moms
Pol} -plnt, 24 Hours

- “" ‘-‘ I j PCNA

4 a E. i
= z ., 3
a a: N

_- _ é a
e e a '9'.
E E a: b
u o a. r3

" :~ k-JI ,I p27

PCNA and p27 levels in HT1080 cells at 24 and 48 hours after treatment
with Poly-plat. Note the large increase in PCNA levels in the Poly-plat
treated cells after 48 hours, indicating an increase in proliferation. p27
levels are elevated at 24 hours after Poly-plat treatment, but remain

constant over the following 24 hours.

65

Figure 6:

PCN A Levels in HT1080 Cells after Poly-plat Treatment

 

 

 

 

 

 

 

 

 

":8: 3.5 [

5:5: 3 “i DUntreated —“‘ e “
E 2': flfil IPoly-plat I...» __ _
E; 1.5

t 1 —

g 0.5

E o

 

 

   

 

 

 

24 Hours 48 Hours

Hours after Treatment

PCN A levels in HT1080 cells following Poly-plat treatment. Levels
determined via three Western Blots of protein extracts of treated cells, and
analysis of band optical density performed using Image Pro Plus, taking an
average of the three blots.

66

p27 Levels in HT1080 Cells Following Poly-plat Treatment

 

3

2.5 H EIUntreated

1 . IPoly-plat

 

 

   

 

 

 

Bands

 

 

 

 

 

 

Integrated Optical Density of

 

 

 

 

24 48

Hours After Treatment

Figure 7: p27 levels in HT1080 cells following Poly-plat treatment. Levels
determined via three Western Blots of protein extracts of treated cells, and
analysis of band optical density performed using Image Pro Plus, taking an
average of the three blots.

67

DISCUSSION

The safe and effective use of chemotherapy in clinical practice requires a
thorough understanding of the drug's actions as well as its associated toxicity. Cisplatin
has been used to treat testicular, ovarian, head, neck, and bladder cancers with a high
degree of success (Palma and Aggarwal 1995). It is believed to enter the tumor cell by
diffusion and disrupt the DNA double helix by interstrand and intrastrand crosslinking
(Roberts and Pascoe 1972) (Goodman et al. 1996). The major toxicity caused by
cisplatin is impairment of renal tubular function (Walker and Gale 1981). Irreversible
kidney damage can occur at higher doses or repeated courses (Goodman et al. 1996). In
addition to other toxicity, marked nausea and vomiting occur in almost all patients
(Abrams and Murrer 1993); (Goodman et al. 1996). For some, this can be a dose-limiting
factor.

Other less toxic platinum analogs have been examined for their ability to
stimulate the immune response (T elgenhoff and Aggarwal 1998). Of these, Poly-plat
appears to be the most effective at immune cell activation (Muenchen et al. 1997). In this
study we examined Poly-plat’s effectiveness at inhibiting tumor cell growth. Both WRC-
256 and HT 1080 cells were inhibited by Poly-plat treatment. This inhibition was shown
to be nearly equal to that of treating with cisplatin using the WRC-256 cells. HT1080
cell growth was inhibited between days one and two, however the growth rate returned to
normal by day five. This data coupled with the lack of apoptosis in these cells when
treated with Poly-plat alone could indicate that these cells are arrested in the cell cycle

until repair mechanisms can be activated.

68

Further evidence of cell cycle arrest was apparent from the Western blot data.
Levels of p27 were elevated in Poly-plat treated cells 24 and 48 hours after treatment.
An elevation of p27 has been shown to indicate cell cycle arrest in tumor cells following
platinum drug treatment (Oshita et al. 2000). This data correlates well with the data from
figure 2, which shows a growth stasis between 24 and 48 hours after Poly-plat treatment.
PCNA levels were near normal levels 24 hours after Poly-plat treatment. A large
increase in PCNA levels 48 hours after treatment indicates a push for the cell to continue
through the cycle. PCNA is present during all phases of the cell cycle, increasing in late
G. and reaching a peak in S phase (Kurki et al. 1986). Increased levels of this protein
following Poly-plat treatment may indicate the cells are recovering from the insult and
are upregulating proteins required for cell cycle progression. Growth resumes and
approaches near normal levels five days after treatment, presumably due to cell damage
repair mechanisms in the tumor cell.

From these studies it can be assumed that Poly-plat alone in vitro is ineffective at
inducing apoptosis in tumor cells. Further it has been shown that tumor cells treated with
Poly-plat recover from treatment within five days. Since Poly-plat has been shown to be
effective against certain tumors in viva, it is possible that Poly-plat’s effectiveness lies
not in it’s ability to kill tumor cells, but rather in it’s ability to slow tumor cell growth
until an effective immune resonse can be mounted. It may also be seen that the main

effect of Poly-plat is the induction of immune system activation.

69

REFERENCES

Abrams MJ, Murrer BA (1993) Metal compounds in therapy and diagnosis. Science
261:725-30

Beale PJ, Rogers P, Boxall F, Sharp SY, Kelland LR (2000) BCL-2 family protein
expression and platinum drug resistance in ovarian carcinoma. BrJ Cancer 821436-40

Drees M, Dengler WM, Hendriks HR, Kelland LR, Fiebig HH ( 1995) Cycloplatam: a
novel platinum compound exhibiting a different spectrum of anti-tumour activity to
cisplatin. Eur J Cancer 32356-61

Ferreira CG, Tolis C, Span SW, Peters GJ, van Lopik T, Kummer AJ, Pinedo HM,
Giaccone G (2000) Drug-induced apoptosis in lung cancer cells is not mediated by the
Fas/FasL (CD95/APOl) signaling pathway. Clin Cancer Res 6:203-12

Fichtinger-Schepman AM, Lohman PH, Berends F, Reedijk J, van Oosterom AT (1986)
Interactions of the antitumour drug cisplatin with DNA in vitro and in vivo. IARC Sci
Publ 78:83-99

Fiebig H, Dress M, Ruhnau T, Misra H, Andrulis P, Hendriks H (1996) GB-21, a novel
platinum complex with antitumor activity in human renal and mammary xenografts.
Proceedings of the American Association of Cancer Research 37:297

Giordano M, Danova M, Pellicciari C, Wilson GD, Mazzini G, Conti AM, Franchini G,
Riccardi A, Romanini MG (1991) Proliferating cell nuclear antigen (PCNA)/cyclin
expression during the cell cycle in normal and leukemic cells. Leuk Res 15:965-74

Gong JG, Costanzo A, Yang HQ, Melino G, Kaelin WG, Jr., Levrero M, Wang JY
(1999) The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced
DNA damage [see comments]. Nature 399:806-9

Goodman LS, Gilman A, Hardman JG, Gilman AG, Limbird LE (1996) Goodman &
Gilman's the pharmacological basis of therapeutics, 9th / ed. New York, McGraw-Hill
Health Professions Division

Henkels KM, Turchi JJ (1999) Cisplatin-induced apoptosis proceeds by caspase-3-
dependent and -independent pathways in cisplatin-resistant and -sensitive human ovarian
cancer cell lines [published erratum appears in Cancer Res 2000 Feb 15;60(4):1150].
Cancer Res 59:3077-83

Kieman JA (1999) Histological and histochemical methods : theory and practice, 3rd ed.
Oxford ; Boston, Butterworth Heinemann

Kurki P, Vanderlaan M, Dolbeare F, Gray J, Tan EM (1986) Expression of proliferating
cell nuclear antigen (PCNA)/cyclin during the cell cycle. Exp Cell Res 1662209-19.

70

Luo Y, Hurwitz J, Massague J (1995) Cell-cycle inhibition by independent CDK and
PCNA binding domains in p21Cip1. Nature 375: 159-61.

Muenchen HJ, Aggarwal SK (1998) Immune system activation by cisplatin and its analog
'Poly-plat': an in vitro and in vivo study. Anticancer Drugs 9293-9

Muenchen HJ, Aggarwal SK, Misra HK, Andrulis PJ (1997) Enhanced
immunostimulation by novel platinum anticancer agents. Anti-Cancer Drugs 82323-328

Nehme A, Baskaran R, Aebi S, Fink D, Nebel S, Cenni B, Wang JY, Howell SB,
Christen RD (1997) Differential induction of c-Jun NH2-terminal kinase and c-Abl
kinase in DNA mismatch repair-proficient and -deficient cells exposed to cisplatin.
Cancer Res 57:3253—7

Nishimura T, Newkirk K, Sessions RB, Andrews PA, Trock BJ, Rasmussen AA,
Montgomery EA, Bischoff EK, Cullen KJ (1996) Immunohistochemical staining for
glutathione S-transferase predicts response to platinum-based chemotherapy in head and
neck cancer. Clin Cancer Res 2: 1859-65

Oshita F, Kameda Y, Nishio K, Tanaka G, Yamada K, Nomura I, Nakayama H, Noda K
(2000) Increased expression levels of cyclin-dependent kinase inhibitor p27 correlate
with good responses to platinum-based chemotherapy in non- small cell lung cancer.
Oncol Rep 7:491-5.

Palma JP, Aggarwal SK (1995) Cisplatin and carboplatin-mediated activation of murine
peritoneal macrophages in vitro: production of interleukin-1 alpha and tumor necrosis
factor-alpha. Anticancer Drugs 62311-6

Palma JP, Aggarwal SK, Jiwa A (1992) Murine macrophage activation after cisplatin or
carboplatin treatment. Anticancer Drugs 3:665-76

Porter AG, Ng P, Janicke RU (1997) Death substrates come alive. Bioessays 19:501-7

Roberts JJ, Pascoe JM (1972) Cross-linking of complementary strands of DNA in
mammalian cells by antitumour platinum compounds. Nature 235:282-4

Sanders EJ, Wride MA (1996) Ultrastructural identification of apoptotic nuclei using the
TUNEL technique. Histochem J 28:275-81.

Sgambato A, Cittadini A, Faraglia B, Weinstein IB (2000) Multiple functions of
p27(Kipl) and its alterations in tumor cells: a review. J Cell Physiol 183:18-27.

Sundaralingam M, Rubin JR, Rao ST (1985) X-ray studies on the interaction of the

anticancer agent cis- [Pt(NH3)2c12] to tRNAphe. A mechanism for the formation of the
intrastrand cross-link to adjacent guanines in DNA. Prog Clin Biol Res 2175-84

71

Telgenhoff DJ, Aggarwal SK (1998) Kupffer cell activation after treatment with cisplatin
and its second generation novel analogs SAP, SSP, and "Poly-plat". In Collery P, Bratter
P, Negretti de Bratter V, Khassanova L, Etienne J-C, eds. Metal Ions in Biology and
Medicine. Neuherberg, Germany, John Libbey Eurotext, 646-648

Walker EM, Jr., Gale GR (1981) Methods of reduction of cisplatin nephrotoxicity. Ann
Clin Lab Sci 11:397-410

Welters MJ, Fichtinger-Schepman AM, Baan RA, Flens MJ, Scheper RJ, Braakhuis BJ

(1998) Role of glutathione, glutathione S-transferases and multidrug resistance-related
proteins in cisplatin sensitivity of head and neck cancer cell lines. Br J Cancer 77:556-61

72

CHAPTER 4: IMMUNE CELL ACTIVATION AND TUMOR CELL
WTERACTION FOLLOWH‘IG POLY-PLAT TREATMENT

73

SUMMARY

Rat peritoneal macrophages and mouse fibroblasts were treated with cisplatin (10
ug/ml) or Poly-plat (10 [lg/ml) for 2 hours and allowed to grow in normal medium for 48
hours. Supematants from treated cells were collected at 0, 4, 18, 24, and 48 hours and
lL-2 levels were examined using the enzyme linked immunosorbant assay (ELISA)
technique. Optimal dosing of Poly-plat was determined via IL—2 production from
macrophages treated with varying doses. Cultures of peritoneal macrophages were
treated with cisplatin (10 ug/ml) or Poly-plat (5 [lg/ml) for two hours and allowed to
grow in normal medium for 24 hours. Cytotoxicity of these supematants was examined
using HT1080 and WRC-256 cells. Apoptosis assays were used to determine cytotoxic
effects of macrophage supematants on tumor cells. Macrophage movement following
treatment with Poly-plat was also examined. These results demonstrate the ability of

Poly-plat to activate macrophages to induce apoptosis in these cell lines.

74

INTRODUCTION

Activated macrophages seek out tumor cells, make contact through cytoplasmic
extensions, and transfer lysosomes to the tumor cell (Palma and Aggarwal 1995),
(Muenchen and Aggarwal 1998). Activated macrophages also release various cytokines
(IL-1, TNFor), which further activate the immune system and cause tumor necrosis (Basu
et al. 1991), (Muenchen et al. 1997). One of the inflammatory cytokines also produced
by activated macrophages is interleukin-2 (H.-2) (Burkitt and Aggarwal 2000).

IL-2 is primarily secreted by activated T lymphocytes, but has been shown to be
released in great quantities by fibroblasts and macrophages following treatment with
platinum anti-cancer agents (Burkitt and Aggarwal 2000), (T elgenhoff and Aggarwal
2000). H.-2 activates cells with H.-2 receptors (T and B lymphocytes, natural killer cells,
and macrophages) to proliferate (Sharon 1998) and differentiate into cells with increased
immune system activity (Collins and Oldham 1995), (Ding et al. 1988). H.-2 has also
been shown to induce monocyte differentiation into macrophages through activation of
the macrophage colony stimulating factor gene (Brach et al. 1993), (Zhu et al. 1993).
The use of IL-2 as a treatment for cancer has been shown to induce regression of
metastatic cancers in selected patients that had failed on other chemotherapies
(Rosenberg et al. 1988).

Poly-plat has been shown to activate macrophages to produce inflammatory
cytokines in greater quantities than cisplatin (Muenchen and Aggarwal 1998). The
purpose of this study was to examine the effects of Poly-plat on activating macrophages
and other cell types to release IL-2, and to determine the optimal dose for Poly-plat based

on H-2 production. The ability of Poly-plat treated macrophages to induce apoptosis in

75

tumor cells was also examined, as well as the ability of macrophages to seek out tumor

cells in vitro.

76

MATERIALS AND METHODS

Cell Cultures. Mouse 3T3 fibroblasts were obtained from Dr. Peter Davidson

 

(Michigan State University) and cultured in Minimal Essential Medium (MEM) with
10% (v/v) Fetal Bovine Serum (FBS). Rat peritoneal macrophages were isolated from
methoxyflurane anesthetized 8 week old male Wistar rats (Charles River, MA) via
peritoneal injection of 10 ml chilled RPMI Medium (Gibco, NY) following institutional
guidelines (Palma et al. 1992). After gently massaging the abdominal wall, cells were
aspirated and placed in conical centrifuge tubes and centrifuged at 1000x g for five
minutes. The supernatant was removed and the cells were resuspended in RPMI with
10% (v/v) horse serum (Atlanta Biologicals, GA). A cell count was performed via
hemacytometer using the trypan blue dye exclusion method (Sigma, MO) and 3 x 105
cells were placed in each T25 flask and incubated at 37° C for 2 hours, after which cells
were washed with RPMI to remove non-adherent cells and fresh RPMI with 10% (v/v)
horse serum was added. The HT1080 human fibrosarcoma cells were a gift from the
Michigan State University Carcinogenesis Laboratory (East Lansing, MI). Macrophage
and HT1080 cell lines were kept in RPMI supplemented with 10% (v/v) horse serum and
1% (w/v) antibiotic [penicillin G (10,000 U/ml) and streptomycin sulfate (10,000 ug/ml)]
and stored in T25 flasks at 37° C in a 5% C02 incubator.

Treatments. Cisplatin and Poly—plat were dissolved in 0.1 ml dimethyl
formamide, followed by the addition of MEM for a final concentration of 1 mg/ml. Drug
solutions were prepared fresh daily. Six flasks of 3T3 cells and macrophages were

treated with either cisplatin (10 ug/ml), Poly-plat (10 [Lg/ml), or the drug vehicle alone

for 2 hours at 37° C. The cells were rinsed in PBS and fresh MEM with 10% (v/v) fetal

77

bovine serum was added to the 3T3 cells, RPMI with 10% (v/v) horse serum was added
to the macrophages. Supematants were obtained from each culture at 0, 4, 18, 24, and 48
hours. ELISA kits (Pharrningen, CA) were used to analyze m-2 levels. The method
utilized the multiple antibody sandwich principle, where a monoclonal antibody to rat IL-
2 was used to capture rat H.-2 present in samples. A biotinylated polyclonal antibody
binding the captured IL-2 was added and unbound material was washed out. Peroxidase
conjugated avidin was used to bind these biotin tagged complexes, resulting in a color
change. Standard curves were generated with H.-2 (0-200 pg/ml) provided in the kits.
The reaction was stopped by acidification and absorbance was read at 450 nm using an
Umax kinetic spectrophotometer microplate reader (Molecular Devices, CA). 3T3 cells
were trypsinized and counted via hemocytometer at 24 and 48 hours. Duplicate
macrophage and 3T3 cultures were grown on coverslips and treated as described above.
Coverslips were fixed in buffered formalin and viewed using phase contrast optics on the
Zeiss 210 laser Scanning microscope.

Optimal Dose Determination. In order to determine the optimal dose for

 

induction of H44 release from macrophages activated with Poly-plat macrophages were
obtained as previously described and cultured in 60 mm culture plates in RPMI with 10%
(v/v) horse serum. Plates were treated with either control medium, cisplatin (2, 5, 10, 25,
50 or 100 rig/ml), or Poly-plat (2, 5, 10, 25, 50, or 100 ug/ml); two plates for each
treatment dose. Plates were treated for 2 hours at 37° C. The media was aspirated, the
plates were rinsed with PBS, and fresh RPMI with 10% (v/v) horse serum was added.
The plates were returned to the incubator and 500 [.11 of the medium was removed from

each plate at 0, 4, 24, and 48 hours after treatment and replaced with 500 pl of normal

78

media. The supematants were frozen at —20° C until all were collected. The ELISA IL-2
assay was performed on the supematants as previously described.

Apoptosis Assay. HT1080 cells were seeded onto 18 mm2 glass coverslips at 1 x

 

106 cells/ml and placed in 35 mm plastic Petri dishes. Cells were incubated for 4 hours at
37° C in a 5% CO; incubator. Fresh RPMI containing 10% (v/v) horse serum (5 ml) was
added to the Petri dishes. Macrophages (1 x 105) were obtained as previously described
and co-cultured with half of the HT1080 cell cultures for an additional 2 hours.
Macrophages were also cultured alone in normal medium. All three culture types
(HT1080, macrophages, and HT1080 + macrophages) were then treated with cisplatin
(10 rig/ml), Poly-plat (5 jig/ml), or drug solvent for two hours. The cells were rinsed with
RPMI and cultured in RPMI with 10% (v/v) horse serum. HT1080 and HT1080 +
macrophages coverslips were removed from culture after 48 hours, fixed in 2% (v/v)
glutaraldehyde in PBS, and stained by the In situ cell death detection kit (Boehringer-
Mannheim, CA), using double strand DNA breaks by the TdT-mediated dUTP nick-end
labeling (T UNEL) reaction (Sanders and Wride 1996). The supematants from the treated
macrophages were removed after 24 hours and placed in Petri dishes containing untreated
HT1080 cells (1 x 106). The cells were incubated at 37° C for an additional 48 hours.
Coverslips were removed, fixed, and stained using the TUNEL reaction. In addition to
the TUNEL assay coverslips from all three cultures (HT1080, HT1080 + macrophages,
and HT1080 + macrophage supernatant) were fixed in 2% (v/v) glutaraldehyde in PBS
and stained with Harris hematoxylin. All coverslips were dehydrated in graded ethanol,

cleared in xylene, and mounted on glass slides with Permount mounting medium. Slides

79

were viewed on the Zeiss LSM 210 confocal microscope and in transmitted light. The
experiments described above were prepared in triplicate.

Macrophage Movement. Macrophages and HT 1080 cells obtained as previously
described. 1x 104 Macrophages were suspended in 1 ml of RPMI with 10% (v/v) horse
serum and placed as a single droplet on one end of a glass slide. 1 x 104 HT 1080 cells
were also suspended in 1 ml of RPMI with 10% (v/v) horse serum and placed as a single
droplet on the opposite end of the slide, so that neither droplet was touching. The slides
were placed in an incubator for six hours at 37° C with 5% C02. The slides were then
rinsed to remove non-adherent cells and placed in Petri dishes containing RPMI with
10% (v/v) horse serum. Twelve slides were prepared in this way and were divided into
four groups of three slides each. The groups were treated with either phorbol myristate
acetate (PMA, 10 ng/ml, dissolved in DMSO and prepared in RPMI with 10% (v/v) horse
serum), lipopolysaccarhide (LPS, 10 ug/ml prepared in RPMI with 10% (v/v) horse
serum), Poly-plat (5 rig/ml, prepared as previously described), or normal medium plus
DMSO (3 til/ml) as a control. These slides remained in culture for 48 hours in the
incubator. The slides were then rinsed in PBS and fixed with 2% (v/v) glutaraldehyde in
PBS. Peroxidase staining was performed to visualize macrophages using the DAB
method (Kieman 1999), and slides were counterstained with 1% (w/v) mentanil yellow.
The slides were dehydrated in a graded series of ethanol, cleared in xylene, and
coverslips were applied with Permount mounting medium. Slides were viewed on the

Zeiss 210 laser scanning microscope.

80

RESULTS

IL-2 Release. There was an increase in H.-2 levels detected in the supernatants of

 

the treated macrophages with Poly-plat (10 ug/ml) and cisplatin (10 ug/ml) for the
various times tested (Figure 1). The levels of H.-2 released in the cisplatin treated
macrophages was above normal after 12 hours, and remained high even after 48 hours.
The Poly—plat treated macrophages demonstrated a very high level of H.-2 throughout the
duration of the experiment compared to the normal or cisplatin treated cultures. H.-2
release from Poly-plat treated fibroblasts was also much higher than cisplatin or the
untreated (Figure 2).

Cell Morphology. Untreated macrophages appear discoid in shape (Figure 3a),
gradually becoming longer and flattened after 48 hours (Figure 3b). Cisplatin or Poly-
plat treated macrophages show a large increase in extensions and cytoplasmic content
after 48 hours (Figures 3c and 3d). The extensions formed in Poly-plat treated
macrophages were generally longer and more in number (Figure 3e). In addition, the rate
of fibroblast growth appeared increased in Poly-plat treated cultures possibly due to the
elevated release of IL-2 (Figure 4). Fibroblasts experienced growth inhibition after
cisplatin treatment (Table 1). The was no significant increase in fibroblast growth in the
Poly-plat treated cultures compared to the control. The appearance of increased growth
(Figure 4) could be due possibly to the increase in size of the fibroblasts.

Dose Determination. Prior to conducting further experiments, a dose response
curve was generated for cisplatin and Poly-plat with the intention of discovering the
optimal dose for H-2 production in cultured macrophages. For the cisplatin treated

macrophages the optimal dose ranged between 5 and 10 ug/ml, with the greatest response

81

at 48 hours in the cells treated with 10 ug/ml (Figure 5). In the Poly-plat treated
macrophages the largest response was at 5 rig/ml, at the 24 hour mark (Figure 6).
Average IL-2 levels declined after this point.

Apoptosis Assay. As reported in chapter 3, apoptosis can be visualized in

 

untreated HT 1080 cells after 48 hours in culture by nuclear fragmentation at a percent
apoptotic of 1.1% (Table 2), or 2.1% using the TUNEL assay. No mitotic figures were
seen in the Cisplatin and Poly-Plat treated cells after 48 hours. The Poly-plat treated cells
had an apoptotic index similar to the untreated cells. Only the Cisplatin treated cells had
a marked increase in apoptosis at 48 hours after treatment. HT1080 cells which were
cultured with macrophages and left untreated had an apoptosis percent increase twice that
of HT1080 cells alone after 48 hours (Table 3). Cells co-cultured and treated with
Cisplatin showed no appreciable difference in the rate of apoptosis. HT1080 cells co-
cultured with macrophages and treated with Poly-plat had a large increase in the
apoptotic rate, nearly nine times that of treating the cells with Poly-plat alone. The
addition of untreated macrophage supernatant to the HT1080 cells had no effect on cell
division or apoptosis (Table 4). The addition of Cisplatin treated macrophage
supernatant to the HT1080 cells increased the apoptotic rate nearly six times (in both
assay systems), but when Poly-plat treated macrophage supernatant was added the
apoptotic rate for the HT1080 cells was even greater than that of the cells treated with the
cisplatin treated macrophage supernatant.

Macrophage Movement AssaL To examine the extent of movement
macrophages were co-cultured with HT1080 cells at opposite ends of a glass slide. Both

cell populations were treated with agents known to induce macrophage activation (PMA,

82

LPS) and Poly-plat. HT1080 cells (Figure 7a) and macrophages (Figure 7b) cultured at
opposite ends of a glass slide have a tendency to stay separated when left untreated for 48
hours. When treated with macrophage activating agents LPS (Figure 7c) and PMA
(Figure 7d) the macrophages are found in conjunction with the tumor cells. Poly-plat
treatment (Figure 7e) yields a response similar to that seen with PMA. Individual

macrophages (Figure 7f) are also much more spread out following Poly-plat treatment.

83

1L-2 Production From Treated Macrophages

 

 

 

140 ~-
120 —
E, 100 —
E:
g 80 --
8
{3’ 60 -
8
n.
(\'| 40 +
:3 .
20 + 4'\.
0 h ‘— fi
0 4 18 24 48
Hours after treatment
+ Normal -I- Cisplatin + Poly-plat
Figure 1: Graph showing H.-2 release in the supernatant of macrophages treated

with cisplatin (10 ug/ml) or Poly-plat (10 ug/ml) at various times. Note
the increase in IL-2 production in Poly-plat treated macrophages.
Standard deviations were calculated from triplicate analysis of
supematants.

84

80

7O

60

40

IL-2 Levels (pg/ml)

20

10

Figure 2:

IL—2 Production from Treated Fibroblasts

 

 

50

 

 

+ Normal
-I- Cisplatin
+ Poly-plat ;
I...
l
r \f
l
o 4 24 48
Hours Post Treatment
Mouse 3T3 H.-2 assay showing H_.-2 production in cultured mouse

fibroblasts treated with cisplatin (10 mg/L), Poly-plat (10 mg/L), or
normal saline. Poly-plat was the only treatment which resulted in an
increase in IL-2 production. Standard deviations were calculated from

triplicate analysis of supematants.

85

 

Figure 3:

 

Light micrographs showing macrophages at 0 hours (A) and 48 hours (B)
in normal medium, and 48 hours after cisplatin (C) and Poly-plat (D)
treatments. Note the increase in cytoplasmic extensions and lysosomes
formation following cisplatin and Poly—plat treatment. Poly-plat
macrophages in general show a greater degree of extension formation (E).
Bar = 5 pm (A), 2.5 pm (B-E).

86

 

Figure 4: Light micrographs showing mouse 3T3 cells at 0 hours (A) and 48 hours
(B) in normal medium, and 48 hours after cisplatin (C) and Poly-plat (D)
treatments. Bar = 2.5 pm.

87

Mouse 3T3 Fibroblast Cell Counts Following Treatment with Platinum Compounds

 

 

 

 

 

 

 

 

 

 

Treatment 0 Hours 24 Hours 48 Hours
Untreated 8.1 x 10’I 1.23 x 105 2.02 x 105
Cisplatin -- 9.50 x 10“ 4.80 x 104
Poly-plat -- 1.12 x 105 2.32 x 105
Table]: Changes in mouse 3T3 fibroblast cell count following treatment with

cisplatin (10 rig/ml) or Poly-plat (10 ug/ml). Cisplatin caused a reduction
in overall cell count, while Poly-plat increased the number of cells slightly
above the untreated count. Counts are averages of three experiments.

88

Effects of Varying doses of cisplatin on H.-2 Release

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

140
-°-Normal ’
120 a C' . l)< ____
'0- 1splat1n (2) /
100 , *Cisplatin (5) __ -
Q -X-Cisp]atin (10) A
go 80 “ -X-Cisplatin (25) M— _ _
S: .0-Cisplatin (50)
. 60 - _
:3 -+-Cisp1atin (100)
40
20
/
o — ' , , v
0 4 24 48
Hours
Figure 5: Dose optimization for H.-2 release from cisplatin treated macrophages.

Doses shown are in rig/ml. Only doses lower than 10 ug/ml showed any
increase in H.-2 levels, with the maximal release at 10 ug/ml.

89

 

Effects of Varying Doses of Poly-plat on H.-2 Release

 

 

 

 

 

 

 

 

 

 

 

300 -°-Normal
'CI-Poly-plat (2) [
250 l ~~#— ——~ A » he- . .zfi
.0-Poly-plat (5) 5
A 200 -X-Poly-p]at (10) 7-7 __
g) ' -X-Poty-p1at (25) g
53: 150 -O-P : x h
N oly-plat (50) A
1:] 100 -+-Po]y-plat(100) 'A‘
50 ~
0 2
Hours
Figure 6: Dose optimization for H..-2 release from Poly-plat treated macrophages.

Doses shown are in jig/ml. The maximal response was seen in the cells
treated with 5 ug/ml after 24 hours in culture.

90

 

 

HT1080 Cells Following Treatment

 

 

 

Treatment MitoticI Apoptosisl Apoptosis2 Viable Cells:fi
Figures (Hematoxylin) (TUNEL) (x 105)
Untreated 2.0 i 0.2 1.1 i 0.3 2.7 i 1.8 6.8 i 0.5
Cisplatin 0 18 i 2.0 26 i 2.7 3.2 i 0.8
Poly-plat 0 3 i 1.1 4 i 1.3 4.6 i- 0.6
Table 2: Table of human fibrosarcoma (HT1080) cells after treatment with cisplatin

(10 jig/ml) or Poly-plat (10 ug/ml).
' Visualized in transmitted light from hematoxylin stained slides.

2 Apoptosis TUNEL assay examined in fluorescence using 488 nm

excitation and > 520 nm emission.

3 Viable cells counts x 105 (1 SD.) calculated via hemacytometer using
trypan blue dye exclusion. Beginning cell count was 2.1 X 105 for all

treatments.

* Values for mitotic figures and apoptosis given in percent i SD. of 250

total cells counted per treatment.

91

 

 

HT1080 cells co-cultured with rat macrophages

 

 

 

Treatment MitoticI ApoptosisI Apoptosis2 Viable Cells3
Figures (Hematoxylin) (TUNEL) (x 10 5 )
Untreated 1.3 i 0.2 1.9 i 0.2 4.6 i 1.0 2.0 i 0.5
Cisplatin 0 22 i 2.8 28 i 9.2 0.3 i 0.8
Poly-plat 0 18 i 1.6 35 i- 3.3 0.8 i- 0.6
Table 3. Table of human fibrosarcoma (HT1080) cells co-cultured with

macrophages after treatment with cisplatin (10 ug/ml) or Poly-plat (10
its/ml)-

' Apoptosis visualized in transmitted light from hematoxylin stained
slides.

2 Apoptosis TUNEL assay examined in fluorescence using 488 nm
excitation and > 520 nm emission.

3 Viable cells counts x 105 (:1: SD.) calculated via hemacytometer using
trypan blue dye exclusion. Beginning cell count was 2.1 X 105 for all
treatments.

Values for mitotic figures and apoptosis given in percent i SD. of 250
total cells counted per treatment.

92

 

HT 1080 cells treated with macrophage supernatant

 

Treatment Mitotic Apoptosis Apoptosis Viable Cells

Figures (Hematoxylin) (T UNEL) (X 10 )

 

Untreated

Macrophage 2.2 i 0.4 2.1 i- 0.3 2.4 i 0.8 2.4 i l
supernatant

Cisplatin

Macrophage 1.8 i 0.3 12 i- 3 18 : 3.3 1.3 i 0.6
supernatant

Poly-plat

Macrophage 2.7 i- 0.4 19 i 4 21 i 4.5 1.0 i 0.6
supernatant

 

Table 4.

Table of human fibrosarcoma (HT1080) cells exposed to macrophage
supernatant from macrophages treated with cisplatin (10 rig/ml) or Poly-
plat (10 ug/ml).

Visualized in transmitted light from hematoxylin stained slides.
2 Apoptosis TUNEL assay examined in fluorescence using 488 nm
excitation and > 520 nm emission.
3 Viable cells counts x 105 (1 SD.) calculated via hemacytometer using
trypan blue dye exclusion. Beginning cell count was 2.1 X 105 for all
treatments.
Values for mitotic figures and apoptosis given in percent i SD. of 250
total cells counted per treatment.

93

 

Figure 7:

 

HT1080 cells (A) and macrophages (B) cultured at opposite ends of a
glass slide have a tendency to stay separated when left untreated for 48
hours. When treated with macrophage activating agents LPS (C) and
PMA (D) the macrophages are found in conjunction with the tumor cells.
Poly-plat treatment (B) yields a response similar to that seen with PMA.
Individual macrophages (F) are also much more spread out following
Poly-plat treatment. (Arrows indicate macrophages stained for
peroxidase). Bar = 5 pm (A, C-F); 10 run (B).

94

DISCUSSION

IL-2 is an inflammatory cytokine, typically associated with T lymphocytes,
inducing clonal expansion and producing tumor infiltrating lymphocytes which play a
pivotal role in tumor regression. Macrophages treated with cisplatin have been shown to
seek out and lyse tumor cells, and release cytokines such as TNF-or and m-la (Palma et
al. 1992). This study reveals another mechanism whereby activated macrophages are
immune system stimulators through the release of high levels of H.-2. Poly-plat treated
macrophages release even greater quantities of H.-2 than cisplatin, and reveal an increase
in morphologic characteristics of activation. Non-immune cells are also stimulated to
release IL-2, as is seen by the 3T3 cells. These cells rapidly divide in culture, but this
division is inhibited following cisplatin treatment. Poly-plat treatment however, does not
inhibit fibroblast division, and may actually increase cell division possibly through the
high amounts of H.-2 released. Therefore it may be that Poly-plat’s antitumor effects rely
not on its ability to stop cell division, but rather on its enhancement of immune cells to
seek out and lyse the tumor cells. Further experimentation is required to determine the
effect of blocking H32 in the supernatant with IL-2 antibodies.

Poly-plat is a large compound, with a molecular weight of 707. Compared to
cisplatin, which has a molecular weight of 132, in equal doses its molarity is nearly one-
seventh. This could account for the decreased toxicity of Poly-plat. For consistency in
past experiments the dose of Poly-plat has been kept the same as that of cisplatin, but in
order for Poly-plat to be effective its dose-response relationship needed to be determined.
Since the goal of the current research is to find more effective ways of enhancing the

immune system function, and since increased IL-2 levels have been shown to correlate

95

 

with macrophage activation, we chose H.-2 as a marker for dose response (Burkitt and
Aggarwal 2000), (Basu et al. 1991). Although 10 ug/ml of Poly-plat has been shown to
be effective in the past at inducing IL-2 release, our studies show the maximal release of
IL-2 to be from Poly-plat at a dose of 5 rig/ml. This dose is used through the remainder
of the experimentation with Poly-plat.

In this study we examined Poly-plat’s effectiveness at inhibiting tumor cell
growth through the activation of the immune response. Poly-plat alone is unable to
induce apoptosis in HT1080 cells. However, when co-cultured with macrophages there is
a tremendous increase in the apoptotic rate for these cells. This is even true for HT1080
cells which were not co-cultured with macrophages, but which were treated with
macrophage supernatant from macrophages which had been treated with Poly-plat. This
increase in apoptosis may be caused by various cytokines (H.401 and TNF-OL) which have
been shown in previous studies to increase dramatically in macrophages following
treatment with Poly-plat (Muenchen and Aggarwal 1998). Macrophages in this study
have also been shown to seek out tumor cells when treated with Poly-plat, moving across
an entire glass slide. Morphologically the macrophages appear different, but in addition
the tumor cells must be releasing some factor following treatment which allows the
macrophage to find them. Similar chemotactic responses have been seen in viva by
immune cells (Springer and Anderson 1986),(Fumarulo et al. 1980).

The development of a tumor is caused by an aberrant cell that escapes detection
by the immune system (Sharon 1998). By activating immune cells with these drugs we
hope to empower immune cells to seek out and destroy tumor cells. This study has

shown growth inhibition in Poly-plat treated tumor cells. These tumor cells are able to

96

recover from treatment with Poly-plat, either through repair mechanisms or loss of drug
stability. The addition of immune cells, as are found in vivo, also causes these cells to go
apoptotic. The two mechanisms (cell growth inhibition and apoptosis) may be the cause

of Poly-plat’s effectiveness in treating animals burdened with tumors.

97

CONCLUSION

The ability of cisplatin to effectively treat tumors relies in part on its ability to
bind to the DNA and to activate the immune system. Poly-plat activates immune system
cells to a greater extent than cisplatin, and has fewer toxic side effects. Activation of
immune cells has been shown through an increase in cell size and content, an increase in
IL-2 production, and an increase in chemotactic movement. Although it does not appear
to cause apoptosis or inhibit cell division, its immunomodulatory effects need to be
explored to gain a better understanding of its role in the immune system. Previous
studies have shown its effectiveness at treating various cancers in viva. To further
explore its potential as an antineoplastic agent its effects will need to be explored in
animals with a depleted or deficient immune response. Through an increased
understanding of how the immune system is modulated by chemicals such as Poly-plat

we can gain insight into more efficient ways to harness its power.

98

REFERENCES

Basu S, Sodhi A, Singh SM, Suresh A (1991) Up-regulation of induction of lymphokine
(H-2)-activated killer (LAK) cell activity by FK-565 and cisplatin. Immunol Lett 27:199-
204

Brach MA, Arnold C, Kiehntopf M, Gruss HJ, Herrmann F (1993) Transcriptional
activation of the macrophage colony-stimulating factor gene by BIZ is associated with
secretion of bioactive macrophage colony-stimulating factor protein by monocytes and
involves activation of the transcription factor NF-kappa B. J Immunol 150:5535-43.

Burkitt K, Aggarwal SK (2000) Immune system activation by CDDP and "Poly-plat".
Anticancer Res 20:2729-37

Collins RA, Oldham G (1995) Effect of recombinant bovine H-1 and H.-2 on B cell
proliferation and differentiation. Vet Immunol Irnmunopatho] 44:141-50.

Ding AH, Nathan CF, Stuehr DJ (1988) Release of reactive nitrogen intermediates and
reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of
activating cytokines and evidence for independent production. I Irnmuno] 141:2407-12

Fumarulo R, Giordano D, Riccardi S, Aresta M (1980) Modification of macrophages
chemotaxis caused by cis-Pt(NH3)2C12. Proc Soc Exp Biol Med 164: 164-6

Kieman JA (1999) Histological and histochemical methods : theory and practice, 3rd ed.
Oxford ; Boston, Butterworth Heinemann

Muenchen HJ, Aggarwal SK (1998) Immune system activation by cisplatin and its analog
'Poly-plat': an in vitro and in vivo study. Anticancer Drugs 9:93-9

Muenchen HJ, Aggarwal SK, Misra HK, Andrulis PJ (1997) Enhanced
immunostimulation by novel platinum anticancer agents. Anti-Cancer Drugs 8:323-328

Palma JP, Aggarwal SK (1995) Cisplatin and carboplatin-mediated activation of murine
peritoneal macrophages in vitro: production of interleukin-1 alpha and tumor necrosis
factor-alpha. Anticancer Drugs 6:311-6

Palma JP, Aggarwal SK, Jiwa A ( 1992) Murine macrophage activation after cisplatin or
carboplatin treatment. Anticancer Drugs 3:665-76

Rosenberg SA, Schwarz SL, Spiess PJ (1988) Combination immunotherapy for cancer:
synergistic antitumor interactions of interleukin-2, alfa interferon, and tumor-infiltrating
lymphocytes. J Natl Cancer Inst 80: 1393-7

Sanders EJ, Wride MA (1996) Ultrastructural identification of apoptotic nuclei using the
TUNEL technique. Histochem J 28:275-81.

99

Sharon J (1998) Basic Immunology. Baltimore, Williams and Wilkins

Springer TA, Anderson DC (1986) The importance of the Mac-1, LFA-l glycoprotein
family in monocyte and granulocyte adherence, chemotaxis, and migration into
inflammatory sites: insights from an experiment of nature. Ciba Found Symp 118: 102-26

Telgenhoff D, Aggarwal S (2000) Immune stimulation by macrophages and fibroblasts
following exposure to platinum anticancer agents. In Histochemical Society Slst Annual
Meeting. New Orleans, 111

Zhu HG, Zollner TM, Klein-Franke A, Anderer FA (1993) Activation of human
monocyte/macrophage cytotoxicity by H.-2/H7N gamma is linked to increased expression
of an antitumor receptor with specificity for acetylated mannose. Irnmuno] Lett 38:111-9.

CHAPTER 5: MACROPHAGE ACTIVATION AS AN ANT I-TUMOR
MECHANISM FOLLOWING ADMH‘IISTRATION OF
PLATH\IUM ANTWEOPLASTIC AGENTS

10]

SUMMARY

Activated macrophages seek out tumor cells, make contact with cytoplasmic
extensions, and transfer lysosomes to the tumor cell. Activated macrophages also release
various cytokines (E-l, H32, TNFOL) which further activate the immune system and cause
tumor necrosis. Typically macrophages have been activated by treating the animal with
the immune stimulating drug (in viva). Unfortunately, direct application of platinum
compounds, such as cisplatin or Poly-plat, to the organism causes stress due to the
toxicity of the drugs. The goal of the following research was to examine the effects of
cisplatin and Poly-plat on activating the immune cells in vitro and returning them to
another animal in order to examine the degree of immunostimulation. These results were
compared to treating the animal directly with cisplatin or Poly-plat. It was found that the
blood cells from the donor animals were activated by the drugs and were able to cause an
immune response in the recipient animal, which was seen in the increase of serum
interleukin-2 levels and resident macrophages (Kupffer cells). Although the response
was not as great as that of treating the animal with the drugs directly, it was much less
toxic to the animal as evidenced by histochemical staining for the enzymes succinate
dehydrogenase and alkaline phosphatase. This treatment method was then applied to
tumor burdened animals and resulted in tumor regression nearly equal to that of treating

with the platinum drugs.

102

INTRODUCTION

An effective method of destroying tumors in the body is through the activation of
the immune system (Bahadur et al. 1984), (Zacharchuk et al. 1983). Activated
macrophages seek out tumor cells, make contact with cytoplasmic extensions, and
transfer lysosomes to the tumor cell (Palma and Aggarwal 1995), (Muenchen and
Aggarwal 1998). Activated macrophages also release various cytokines (l—l, H34,
TNFOt) which further activate the immune system and cause tumor necrosis (Muenchen et
a]. 1997), (Basu et al. 1991). Cisplatin is a potent anti-neoplastic agent, which works via
three mechanisms: DNA crosslinking, inhibition of the mitotic spindle, and macrophage
activation (Abrams and Murrer 1993), (Palma et a1. 1992). Other drugs have been
developed which are more potent at macrophage activation and much less toxic (Fiebig et
al. 1996), (Drees et al. 1995). These drugs, in particular Poly-plat (Poly-[(trans-1,2-
diaminocyclohexane) platinum]-carboxyamylose) (Andrulis Pharmaceuticals, Bethesda,
MD), are much larger than cisplatin, with numerous branching side chains radiating from
a central platinum atom. In equal doses Poly-plat has less platinum than cisplatin, which
is a possible reason for the decrease in toxicity.

Typically macrophages have been activated by treating the animal with the drug
(in viva). In viva treatment of macrophages with platinum antineoplastic agents results in
an increase in macrophage activity, including an increase in extensions, cytolytic factors
(cytokines), and numbers (Telgenhoff and Aggarwal 1998), (Bahadur et al. 1984), (Gupta
and Sodhi 1987). The increase in the number of macrophages present is a result of
monocyte sequestration and differentiation (van der Rhee et a]. 1979), (Tanner et a].

1982). Activation of macrophages enhances the organisms ability to seek out and lyse

103

 

tumor cells (Adams and Hamilton 1988), (Bucana et a]. 1976). Unfortunately, direct
application of platinum compounds to the organism causes stress due to the toxicity of the
drugs, of which gastrointestinal and nephrotoxicity are the dose limiting factors (Walker
and Gale 1981), (Wang and Aggarwal 1997).

By isolating white blood cells from an organism, treating them with macrophage
activating drugs, then returning them to the animal we should see a sequestration and
activation response similar to that of treating the animal directly with the drug. Further,
we will not see the toxicity caused by the antineoplastic agents since the drug itself is not
being introduced. A decrease in the harsh side effects is beneficial to the patient, since

they will not have to endure the pain and malaise associated with chemotherapy.

MATERIALS AND METHODS

Blood Cells. White blood cells were isolated from nine adult male Sprague-

 

Dawley rats via cardiac puncture. The rats were first anesthetized with methoxyflourane
and kept unconscious using a methoxyflourane mask. The chest and abdomen were
sterilized with betadine and an 18-gauge needle was inserted into the heart. The blood
obtained was placed in tubes containing EDTA to prevent coagulation, and the white
blood cells were separated by centrifugation on a Ficoll gradient. The isolated leukocytes
(which were composed of granulocytes and mononuclear leukocytes) from each rat were
placed in three separate sterile T-25 flasks and treated with either cisplatin (10 ug/ml),
Poly-plat (5 jig/ml), or normal saline for two hours. The drugs were removed and the
cultures were rinsed with phosphate buffered saline. Supematants were collected at 0, 4,
18, 24, and 48 hours, and H.-2 levels were measured using ELISA kits. In addition,
duplicate plates of cells were treated as above, rinsed to remove the drug, and remained
undisturbed in culture for 48 hours. After this time the cultures were treated with 10%
(v/v) trypsin for 5 minutes to remove adherent cells. The cells were then collected in
normal saline, stained with tryphan blue, and their viable numbers were determined via
hemocytometer using the dye exclusion method.

Immune Cell Trangplant. Fifteen 10-week-old healthy untreated Spague-Dawley
rats were used as blood donors. Each day for five consecutive days three rats were placed
in methoxyflourane chambers for two minutes. Upon removal, they were placed face up
and their chest was scrubbed with betadine. An 18-gauge needle was then inserted and 8-
10 ml of blood was withdrawn via cardiac puncture, after which the rats were euthanized.

The blood was then placed in sodium heparin tubes. The leukocytes were separated by

105

 

centrifugation for 10 minutes at 2000x g. The white cells were then placed in sterile
tubes and each was treated with either normal saline, cisplatin (10 ug/ml), or Poly-plat (5
rig/ml). The tubes were then placed on a rotator for two hours to prevent cell adhesion.
The tubes were then centrifuged for 10 minutes at 4000x g. The supernatant was
removed and the cells were rinsed in phosphate buffered saline to remove any of the drug.
They were again centrifuged; the supernatant discarded, and rinsed a second time. The
cells from each treatment group were counted via hemacytometer and separated into three
equal portions in normal saline. The cells were then injected into the peritoneal cavity of
nine 10-week-old Sprague—Dawley rats that came from the same breeding line as the
donor rats. This treatment regimen was repeated each day for five consecutive days. Six
additional rats from the same breeding line were treated directly with either cisplatin (10
mg/kg), Poly-plat (5 mg/kg), or normal saline via intraperitoneal injection over a five day
period. On the seventh day the rats were euthanized with methoxyflourane. A cardiac
puncture was performed on each rat to collect blood, which was placed in serum separator
tubes for serum H.-2 analysis. The H.-2 analysis was performed three times using an
ELISA kit. The liver and kidney of each rat was removed and flash frozen in OCT
compound for enzyme analysis.

Eng/me Analysis. Frozen sections from each rat were cut at 10 um on a cryotome
and air-dried on coverslips. Triplicate sections from each animal were stained for either
peroxidase, succinate dehydrogenase, or alkaline phosphatase activity using
histochemical methods (Kieman 1999). Following staining the coverslips were

dehydrated through a graded series of ethanol, cleared in xylene, and mounted on

106

 

microscope slides using Permount mounting medium. Enzyme levels were examined on
the Zeiss 210 laser scanning microscope.

Tumor Cells. Ten T-75 flasks of Walker rat carcinoma (W RC) cells were kept in

 

MEM supplemented with 10% (v/v) horse serum and 1% (w/v) antibiotic [penicillin G
(10,000 U/ml) and streptomycin sulfate (10,000 ug/ml)] at 37° C in a 5% C02 incubator.
The cells were given fresh medium daily until they reached 90% confluency. The cells
were then rinsed with phosphate buffered saline and removed exposed to 3 ml of trypsin
EDTA solution for five minutes at room temperature. Flasks were tapped to dislodge
adherent cells and 10 ml of horse serum was added. The cells were collected into 50 ml
centrifuge tubes and centrifuged at 4000 rpm to pellet the cells for 5 minutes. The
supernatant was removed and the cells were rinsed with phosphate buffered saline and
resuspended. The centrifuge and wash was repeated twice more.

Tumor Burdened Animals. Twenty-four lO-week-old healthy untreated Spague-
Dawley rats were given injections using an 18 gauge needle of l x 106 WRC cells from
cultures described above. These injections were given subcutaneously in the right hip
region, which had been shaved and scrubbed with betadine prior to the injection. These
injections were repeated for two additional days. After five days only nine rats developed
measurable tumors in the region. The animals which did not develop tumors were
euthanized.

Tumor Treatments. Treatments began when all tumor burdened animals

 

developed tumors larger than 1 cm as measured by linear calipers. Three rats were
treated with either norrna] saline, cisplatin (10 mg/kg), or Poly-plat (5 mg/kg) over a five

day period. The remaining six rats were divided into groups of two and treated for five

107

 

consecutive days with leukocytes isolated from donor rats and treated with phosphate
buffered saline, cisplatin, or Poly-plat as previously described. The treated leukocytes
were injected subcutaneously at the site of the tumor using an 18 gauge needle.
Measurements of tumor growth were performed daily throughout the treatments and for
six days after treatment. At day eleven all animals were euthanized using

Methoxyflourane. Tissues were collected and frozen in OCT mounting media at -20° C.

108

 

RESULTS

Blood Cells. The treated cells increased in number, presumably through release of

 

H32 H.-2 levels were increased in the Poly-plat treated cell supematants (Figure 1).
Cisplatin H32 levels decreased below the levels of the untreated cells. The cells were
initially plated at 2.2 x 105 cells. After 24 hours the untreated cells remained at 2.2 x 105.
The cells treated with cisplatin dropped to 1.86 x 105 cells, and Poly-plat treated cells also
dropped to 1.98 x 105. After 48 hours in culture the untreated cell count was 2.8 x 105.
The cisplatin count plummeted to 0.9 x 105. The Poly—plat count showed the most
dramatic increase in cell numbers, rising to 3.6 x 105 (Table 1).

Serum IL-2. In order to determine potential benefits of transferring immune cells,

 

serum 1L-2 levels from animals given immune cells treated in vitro was examined. The
animals which were treated with normal saline had the lowest H.-2 serum levels (29.1
pg/ml), followed closely by the animals which were treated with saline treated blood
(29.5 pg/ml). Those receiving cisplatin and Poly-plat directly had the highest levels of
serum H-Z (51.2 and 67.8 pg/ml, respectively). The animals which received blood that
had been treated with cisplatin (35.0 pg/ml) or Poly-plat (54.5 pg/ml) did not have levels
as high as the direct treatment, but higher than the untreated (T able 2).

Enzyme Assays. Each of the animals that received donor blood had enzyme
levels of SDH and AP that were similar to that of the control animals (Figure 2). The
cisplatin treated rats had enzyme levels which were significantly lower than the control,
indicating a high degree of toxicity. The Poly-plat treated rats also showed a decrease in
the two enzymes, though not to the extent of the cisplatin treated. Resident macrophages

in the liver (Kupffer cells) show a high degree of peroxidase staining, and therefore this

enzyme was used as a marker to examine macrophage numbers. Both cisplatin and Poly-
plat treated rats showed an increase in Kupffer cell numbers over that of the control (2.5
and 3 times as many) (Figure 3). The animals treated with white blood cells that had
been exposed to these drugs showed on average twice as many Kupffer cells as either the
normal or the rats treated with blood alone.

Tumor Regression. Of the nine rats which developed tumors only two of the

 

animals showed no signs of regression, the untreated control and the rat receiving
leukocytes which were not treated (Figure 4). All other treatments caused a regression in
tumor size as measured by linear calipers. On day eleven the tumors were measured and
the rats were euthanized with methoxyfluorane. Upon examination of the internal organs
it was discovered that tumor metastasis had occurred to the lung, spleen, and kidney in
the control animal. Macrophage metastasize was also seen in the rat treated with Poly-
plat in the lung and the spleen. No other metastasis was seen in any of the other treatment

groups.

110

 

IL-2 (pg/ml)

Figure 1:

H.-2 Levels in Leukocyte Supematants

 

 

 
  

 

 

     
   
 

 

 

 

 

80
70 T—o—Normal .
60 gig-D—Cisplatin
I-A—Poly-platj
50 + I
40 7‘
30 + l
l .
20 +
10
0 if e , ,
o 4 18 24 48
Hours After Treatment
Graph showing H34 levels in treated leukocytes following treatment with

cisplatin (10 ug/ml), Poly-plat (5 ug/ml), or normal saline in vitro. Note
the increase in H.-2 levels following Poly-plat treatment. [Average of

three repetitions i SD]

111

Leukocyte Counts Via Hemacytometer
Following Treatment

 

 

 

 

 

 

 

 

 

 

Treatment 0 Hours 24 Hours 48 Hours
Control 2.2 x 105 2.2 x 105 2.8 x 105
Cisplatin 2.2 x 105 1.86 x 105 0.9 x 105
Poly—plat 2.2 x 105 1.98 x 105 3.6 x 105
Table 1: Table showing Leukocyte numbers from three rats per treatment group

following treatment with cisplatin (10 ug/ml), Poly-plat (5 jig/ml) or
normal saline. Counts of viable cells performed via hemacytometer and
averages of ten counts given as total cell count per flask.

112

 

Table 2:

H.—2 Levels in Serum of Treated Rats

 

 

 

 

 

 

 

 

 

 

IL-2 (pg/ml)
Control (2) 29.1 +/- 2.7
Cisplatin (2) 51.2 +/- 9.8
Poly-plat (2) 67.8 +/- 5.5
Untreated Leukocytes (3) 29.5 +/- 5.7
Cisplatin Treated 35.0 +/- 8.2
Leukocytes (3)
Poly-plat Treated 54.5 +/- 8.1
Leukocytes (3)

 

H.-2 levels in animals which received either normal saline (2 rats),
cisplatin (10 ttg/ml, 2 rats), Poly-plat (5 pg/ml, 2 rats), Leukocytes from
donor animals (3 rats), leukocytes from donor animals treated with
cisplatin (10 ug/ml, 3 rats), or leukocytes from donor animals treated with
Poly-plat (10 pig/ml, 3 rats). The H..-2 levels from the animals treated
directly with the platinum drugs was the highest, however the leukocytes
which were treated with the platinum drugs elicited a release of H.-2 much
greater than the control as well.

113

Normal

Cisplatin

Poly-plat

Untreated
Leukocytes

Cisplatin
Treated
Leukocytes

Poly-plat
Treated
Leukocytes

Figure 2:

SDH — Liver SDH — Kidney AP — Live AP — Kidney

k...
‘

     
    

. - ~ I; :1 :1, 1'7“ ; 3“ v. ,' ‘
{iii ‘ :23 ' " I ’ .’ . R*:'. (“If . ,. i ‘

Enzyme histochemistry for succinate dehydrogenase (SDH) and alkalih

phosphatase (AP) of liver and kidney from control animals (A, G, M, S) or
animals treated with cisplatin (B, H, N, T), Poly-plat (C, I, O, U),
untreated leukocytes (D, J, P, V), leukocytes treated with cisplatin (E, K,
Q, W), or leukocytes treated with Poly-plat (F, L, R, X). The enzyme
levels in the cisplatin treated tissues showed the greatest decrease. Bar =

50 um.

114

Changes in Kupffer Cell Number per Viewing Area

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

12

3?

O

S 10

8

18

E" 8

3

.2

> 6

8.

g 4

° I I

3

.. I I I I

0 _
Untreated Cisplatin Poly-plat Control Cisplatin Poly-plat
Leukocytes Treated Treated
Lekocytes Leukocytes
Treatments
Figure 3: Graph showing changes in Kupffer cell number as seen via peroxidase

staining following various treatments. Averages and standard deviations
were obtained from viewing twenty areas in the sections from each rat, for
a total of 40 (untreated, cisplatin, Poly-plat) or 60 (leukocyte treatments)
data sets. Note the increase in Kupffer cells from treating rats with
leukocytes exposed to Poly-plat (5 gig/ml) is nearly equal to that of treating
directly with cisplatin (10 pg/ml) or Poly-plat (5 ug/ml).

115

Tumor Regression Following Platinum Drug Treatments

 

Tumor size (cm)

 

 

l 2 3 4 5 6 7

 

 

Days
-0-Control -D-Cisp]atin l
-n-Po]y-plat -X-Untreated Leukocytes ._
-X-Cisplatin Treated Leukocytes -O-Poly-plat Treated Leukocytes A
Figure 4: Tumor regression of tumors induced in the right hip of rats via injection of

WRC cells. Data shown from rats which developed tumors 2 1 cm in
diameter. Three rats were treated with either normal saline, cisplatin (10
mg/kg), or Poly-plat (5 mg/kg) on days 1-5. The remaining six rats were
divided into groups of two and treated for five consecutive days with
leukocytes isolated from donor rats and treated with either normal saline,
cisplatin (10 pg/ml), or Poly-plat (5 rig/ml). Only two groups showed no
regression of tumors with treatment, the control and the untreated
leukocyte treated animal.

116

 

DISCUSSION

The safe and effective use of chemotherapy in clinical practice requires a thorough
understanding of the drugs actions as well as its associated toxicity. Cisplatin has been
used to treat testicular, ovarian, head, neck, and bladder cancers with a high degree of
success (Palma and Aggarwal 1995). It is believed to enter the tumor cell by diffusion
and disrupt the DNA double helix by interstrand and intrastrand crosslinking (Roberts
and Pascoe 1972). The major toxicity caused by cisplatin is impairment of renal tubular
function. Irreversible kidney damage can occur at higher doses or repeated courses
(Goodman et al. 1996). In addition to other toxicity, marked nausea and vomiting occur
in almost all patients (Abrams and Murrer 1993), (Goodman et al. 1996). For some, this
can be a dose-limiting factor. The goal of our research has been to find ways to deliver
this and other highly effective drugs without the associated side effects.

One concept we have been studying is the ability of cisplatin to activate the
immune system. We have seen cisplatin’s effects on immune cells and believe this to be
an important part of it effectiveness (Telgenhoff and Aggarwal 1998). We have also
examined other less toxic platinum analogs for their ability to stimulate the immune
response. Of these, Poly-plat appears to be the most effective at immune cell activation
(Muenchen et a]. 1997). The development of a tumor is caused by an aberrant cell that
escapes detection by the immune system. By activating immune cells with these drugs
we hope to empower immune cells to seek out and destroy tumor cells selectively without
the associated toxicity of directly treating the animal with the drug.

Only in recent times have we seen the rise of a new type of therapy for treating

cancers, which in some instances has shown to be as effective as surgery, radiation, and

117

chemotherapy. This new therapy, referred to as biological therapy, is defined as cancer
treatments that act primarily through natural host defense mechanisms or by the
administration of natural mammalian substances (Rosenberg 1991). Adoptive
immunotherapy, which is the transfer of immune cells that have antitumor activity to a
tumor-bearing host, is a type of biological therapy. Dr. Steven Rosenberg at the National
Institutes of Health has been a leader in adoptive immunotherapy. His initial studies
involved using lymphocytes that had been exposed to the tumor either in vitro or in viva.
These cells were grown in culture and returned to the tumor-bearing host (Rosenberg
1991). He also used H.-2 to induce the clonal expansion of T lymphocytes (Lotze et al.
1990). H.-2 is an inflammatory cytokine, typically associated with T lymphocytes,
inducing clonal expansion and producing tumor infiltrating lymphocytes which play a
pivotal role in tumor regression (Lotze et al. 1990). Rosenberg’s most recent work in the
field has been with combination adoptive immunotherapy. He has used H.-2, which does
show antitumor activity by itself, in conjunction with alpha interferon and tumor
infiltrating lymphocytes (Rosenberg et a]. 1988). The difference in our study in
comparison to Dr. Rosenberg’s is that we use immune cells that have not been exposed to
a specific tumor. The immune cells in our studies are activated by a platinum compound
that results in a more generalized activation. This generalized activation has been shown
to be effective in various tumor models, including Walker rat carcinoma, human
fibrosarcoma, murine sarcoma 180, and human ovarian teratocarcinoma cells (Palma et
a1. 1992) (Burkitt and Aggarwal 2000).

Macrophages treated with cisplatin have been shown to seek out and lyse tumor

cells, and release cytokines such as TNF-OL and E-la (Palma et al. 1992). This study

118

reveals another mechanism whereby activated macrophages are immune system
stimulators through the release of high levels of H2 Poly-plat treated macrophages
release even greater quantities of H_.-2 than cisplatin, and show an increase in
morphologic characteristics of activation. Through this generalized form of activation we
have seen immune cells returned to the animal and create an immune response which,
while not on the same level as treating with the drugs directly, shows promise due to the
increase in circulating levels of H34 and the lack of toxicity.

The Walker rat carcinoma is an aggressive tumor, which has been shown to
readily metastasize to the lung and other organs (Bellamy and Hinsull 1978). On treating
the tumor burdened animals with activated immune cells we saw a regression of the
localized tumor and no metastasis. Animals which were not treated showed a steady
growth in the size of the tumor, and metastasis occurred in two of the animals. It is
unclear why the Poly-plat treated animal developed secondary tumors while the initial
tumor regressed. In previous studies, Poly-plat was shown to slow tumor cell growth
without inducing apoptosis (unpublished data). Poly-plat may be arresting the tumor cells
replicative cycle, but is ineffective at preventing metastasis. A larger treatment group
will need to be utilized to determine Poly-plat’s effectiveness.

In determining the effectiveness of treating leukocytes in vitro and using those to
cause tumor cell regression we have developed a system of adoptive immunotherapy
which avoids the use of cultured macrophages (which are difficult to obtain in large
numbers) and that could be applied clinically with minimal discomfort or risk to the

patient. Further studies are required to determine the effectiveness of this treatment

119

method on spontaneous tumors, which are generally more difficult to treat than

transplanted tumors (Rockwell and Moulder 1990).

120

REFERENCES

Abrams MJ, Murrer BA (1993) Metal compounds in therapy and diagnosis. Science
261:725-30

Adams D, Hamilton T (1988) Activation of macrophages for tumor cell kill: Effector
mechanisms and regulation. In Heppner G, Fulton A, eds. Macrophages and Cancer. Boca
Raton, CRC Press, 39

Bahadur A, Sarna S, Sodhi A (1984) Enhanced cell mediated immunity in mice after
cisplatin treatment. Pol J Pharmacol Pharm 36:441-8

Basu S, Sodhi A, Singh SM, Suresh A (1991) Up-regulation of induction of lymphokine
(H.-2)-activated killer (LAK) cell activity by FK-565 and cisplatin. Immunol Lett 27:199-
204

Bellamy D, Hinsull SM (1978) Influence of lodgement site on the proliferation of
metastases of Walker 256 carcinoma in the rat. Br J Cancer 37:81-5.

Bucana C, Hoyer L, Hobbs B (1976) Morphological evidence for translocation of
lysosomal organelles from cytotoxic macrophages into the cytoplasm of tumor target
cells. Cancer Research 36:4444

Burkitt K, Aggarwal SK (2000) Immune system activation by CDDP and "Poly-plat".
Anticancer Res 20:2729-37

Drees M, Dengler WM, Hendriks HR, Kelland LR, Fiebig HH (1995) Cycloplatam: a
novel platinum compound exhibiting a different spectrum of anti-tumour activity to
cisplatin. Eur J Cancer 3:356-61

Fiebig H, Dress M, Ruhnau T, Misra H, Andrulis P, Hendriks H (1996) GB-21, a novel
platinum complex with antitumor activity in human renal and mammary xenografts.
Proceedings of the American Association of Cancer Research 37:297

Goodman LS, Gilman A, Hardman JG, Gilman AG, Limbird LE (1996) Goodman &
Gilman's the pharmacological basis of therapeutics, 9th / ed. New York, McGraw-Hill
Health Professions Division

Gupta P, Sodhi A ( 1987) Increased release of interleukin-1 from mouse peritoneal
macrophages in vitro after cisplatin treatment. Int J Irnmunopharmacol 9:385-8

Kieman J A (1999) Histological and histochemical methods : theory and practice, 3rd ed.
Oxford ; Boston, Butterworth Heinemann

121

 

Lotze MT, Finn OJ, Cetus Corporation (1990) Cellular immunity and the immunotherapy
of cancer : proceedings of a Cetus, Immunex, and Triton Biosciences-UCLA Symposia
Colloquium held at Park City, Utah, January 27-February 3, 1990. New York, N.Y.,
Wiley-Liss

Muenchen HJ, Aggarwal SK (1998) Immune system activation by cisplatin and its analog
'Poly-plat': an in vitro and in vivo study. Anticancer Drugs 9:93-9

Muenchen HJ, Aggarwal SK, Misra HK, Andrulis PJ (1997) Enhanced
immunostimulation by novel platinum anticancer agents. Anti-Cancer Drugs 8:323-328

Palma JP, Aggarwal SK (1995) Cisplatin and carboplatin-mediated activation of murine
peritoneal macrophages in vitro: production of interleukin-1 alpha and tumor necrosis
factor-alpha. Anticancer Drugs 6:311-6

Palma JP, Aggarwal SK, Jiwa A (1992) Murine macrophage activation after cisplatin or
carboplatin treatment. Anticancer Drugs 3:665-76

Roberts JJ, Pascoe JM (1972) Cross-linking of complementary strands of DNA in
mammalian cells by antitumour platinum compounds. Nature 235:282—4

Rockwell S, Moulder JE (1990) Hypoxic fractions of human tumors xenografted into
mice: a review. Int J Radiat Oncol Biol Phys 19:197-202.

Rosenberg SA (1991) Irnmunotherapy and gene therapy of cancer. Cancer Res 51:5074s-
5079s

Rosenberg SA, Schwarz SL, Spiess PJ (1988) Combination immunotherapy for cancer:
synergistic antitumor interactions of interleukin-2, alfa interferon, and tumor-infiltrating
lymphocytes. J Natl Cancer Inst 80: 1393-7

Tanner A, Keyhani A, Arthur M, Wright R (1982) Evidence for a sequence of
macrophage activation during recruitment into the liver. In Knook DL, Wisse E, eds.
Sinusoidal Liver Cells. Leiden, Elsevier Biomedical Press, 405-412

Telgenhoff DJ, Aggarwal SK (1998) Kupffer cell activation after treatment with cisplatin
and its second generation novel analogs SAP, SSP, and "Poly-plat". In Collery P, Bratter
P, Negretti de Bratter V, Khassanova L, Etienne J-C, eds. Metal Ions in Biology and
Medicine. Neuherberg, Germany, John Libbey Eurotext, 646-648

van der Rhee HJ, van der Burgh-de Winter CP, Daems WT (1979) The differentiation of

monocytes into macrophages, epithelioid cells, and multinucleated giant cells in
subcutaneous granulomas. H. Peroxidatic activity. Cell Tissue Res 197:379-96

122

Walker EM, Jr., Gale GR (1981) Methods of reduction of cisplatin nephrotoxicity. Ann
Clin Lab Sci 11:397-410

Wang Y, Aggarwal SK (1997) Effects of cisplatin and taxol on inducible nitric oxide
synthase, gastrin and somatostatin in gastrointestinal toxicity. Anticancer Drugs 8:853-8

Zacharchuk CM, Drysdale BE, Mayer MM, Shin HS (1983) Macrophage-mediated
cytotoxicity: role of a soluble macrophage cytotoxic factor similar to lymphotoxin and
tumor necrosis factor. Proc Nat] Acad Sci U S A 80:6341-5

123

 

CONCLUSION

The safe and effective use of chemotherapy in clinical practice requires a
thorough understanding of the drugs action’s as well as their associated toxicities. The
goal of our research has been to find ways to deliver highly effective drugs without the
associated side effects. One concept we have been studying is the ability of platinum
based compounds to activate the immune system. We have seen cisplatin’s effects on
immune cells and believe this to be an important part of it effectiveness (Telgenhoff and
Aggarwal 1998). Stimulation of the immune system is a major function of many
antineoplastic agents. Cisplatin stimulates the immune system through macrophage
activation (Palma et al. 1992), making the macrophages more efficient in phagocytosing
tumor cells. The effects of chemotherapeutic agents on modulating host immune
defenses have been identified in many studies. Matheson et al. demonstrated that low
doses of methotrexate and 5-fluorodeoxyuridine augmented natural killer cell activity
(Matheson et al. 1983). The activation of macrophages to destroy tumor cells was seen in
rats treated with mitomycin C (Ogura et al. 1982) and in mice treated with adriamycin
and cyclophosphamide (Stoychkov et al. 1979). Macrophage activation is considered to
be an important part of the organisms response to chemotherapy (Ohanian et al. 1980).
Macrophage activation occurs rapidly, with noticeable changes in shape and function
occurring within one day post treatment (T oge et al. 1981). Monocyte recruitment, an
event which increases the number of available macrophages to counter insult, is a more

lengthy process (van der Rhee et al. 1979).
The severe toxic side effects evident in cisplatin treatment seem to be due to the

disruption in sulphydryl containing mitochondrial dehydrogenase enzymes (Aggarwal

124

1993). The disruption of these enzymes prevents calcium uptake thereby reducing
calcium levels in the cell to cytotoxic levels. SAP, SSP, and Poly-plat are novel platinum
agents which show no such disruption, and as a result have been shown to be much less
toxic (Muenchen and Aggarwal 1998), (Telgenhoff and Aggarwal 1998). Due to low
toxicity SAP, SSP, and Poly-plat would appear to be alternatives to CDDP. SAP and
SSP both activate macrophages, however, there is no significant increase in Kupffer cell
numbers after SAP and SSP treatment, as shown by sinusoidal esterase staining. Poly-
plat not only increases Kupffer cell activation, but also increases the number of Kupffer
cells present. In this research we examined Poly-plat’s effectiveness at inhibiting tumor
cell growth. Both WRC-256 and HT 1080 cells were inhibited by Poly-plat treatment.
This data coupled with the lack of apoptosis in these cells when treated with Poly-plat
alone could indicate that these cells are arrested in the cell cycle until repair mechanisms
can be activated. Poly-plat activates immune system cells to a greater extent than
cisplatin, and has fewer toxic side effects. Activation of immune cells has been shown
through an increase in cell size and content, an increase in H.-2 production, and an
increase in chemotactic movement. Poly-plat treated macrophages release even greater
quantities of H-Z than cisplatin, and show an increase in morphologic characteristics of
activation.

Chemotherapy is a widely used treatment for cancer, but the mechanisms of drug
action are still being discovered. While early studies (Rosenberg and VanCamp 1970),
(Roberts and Pascoe 1972) focused on chemotherapeutic agent’s abilities to crosslink
DNA and prevent cell cycle progression, more recent experiments were designed to

examine the role of inducing apoptosis in tumor cells (Jiang et al. 1999), (Nehme et al.

125

 

 

1997). One of the main problems with this type of research is that drugs exert their
effects differently depending on the cell type used, and may even react differently to the
same type of tumor in different subjects. In one study cisplatin mediated apoptosis by
down-regulating inhibitory proteins of the Fas/FasL signalling pathway in sarcoma cells
(Kinoshita et al. 2000), in another study lung cancer cells exposed to cisplatin underwent
apoptosis without utilizing the Fas/FasL pathway (Ferreira et a]. 2000). Cells from the
same lineage can also become resistant to cisplatin and fail to undergo apoptosis either
due to a decrease in cisplatin uptake or through a change in DNA repair ability
(Blommaert et al. 1998), (Fink et al. 1997).

Based on the studies conducted with cisplatin, our goal was to examine the effects
of Poly-plat on inducing apoptosis. From these studies it can be assumed that Poly-plat
alone in vitro is ineffective at inducing apoptosis in tumor cells. Further it has been
shown that tumor cells treated with Poly-plat recover from treatment within five days.
Since Poly-plat has been shown to be effective against certain tumors in viva, it is
possible that Poly-plat’s effectiveness lies not in it’s ability to kill tumor cells, but rather
in it’s ability to slow tumor cell growth until an effective immune resonse can be
mounted. Further evidence of cell cycle arrest following Poly-plat treatment was
apparent from the levels of p27 and PCNA. p27 was elevated in Poly-plat treated cells
24 and 48 hours after treatment. An elevation of p27 has been shown to indicate cell
cycle arrest in tumor cells following platinum drug treatment (Oshita et al. 2000). A
large increase in PCNA levels 48 hours after treatment indicates a push for the cell to

continue through the cycle. Increased levels of this protein following Poly-plat treatment

126

may indicate the cells are recovering from the insult and are upregulating proteins
required for cell cycle progression.

Adoptive immunotherapy, which is the transfer of immune cells that have
antitumor activity to a tumor-bearing host, is a type of biological therapy (Rosenberg
1990). In most studies involving immunotherapy the cytotoxic T-cell is the effector of
choice (Basu et al. 1991), (Rosenberg et al. 1988), Wang and Rosenberg 1996). Several
studies have examined the effects of various agents on inducing macrophages to kill
tumor cells (Han et a]. 1999), (Johnson et al. 1986), (Ogura et al. 1982). Very few
studies treated macrophages with an immune stimulating drug and returned the
macrophages to tumor burdened animals with the effect of alleviating the tumor (Wang et
al. 1986), (Di Luzio et al. 1976). The possible reason for the lack of interest in this
method is the difficulty in obtaining large populations of macrophages for treatment in a
clinical setting. In addition, macrophages do not survive well in culture, and rarely
proliferate due to already being terminally differentiated cells (Cohn 1978), (Herscowitz
et al. 1981).

In our studies macrophages were activated by cisplatin or Poly-plat which
resulted in a generalized form of activation. Activation of immune cells was shown
through an increase in cell size and content, an increase in H..-2 production, and an
increase in chemotactic movement. Poly-plat treated macrophages released even greater
quantities of l-2 than cisplatin, and showed an increase in morphologic characteristics
of activation. This generalized activation was shown to be effective in various tumor
models, including Walker rat carcinoma, human fibrosarcoma, murine sarcoma 180, and

human ovarian teratocarcinoma cells (Palma et al. 1992) (Burkitt and Aggarwal 2000).

127

In order to develop an adoptive immunotherapy system utilizing Poly-plat clinically we
had to use immune cells which were readily obtainable and which would result in an
immune response similar to that seen with macrophages in vitro. We chose to use
peripheral circulating leukocytes for the adoptive transfer technique. Leukocyte
populations contain lymphocytes which have been shown in previous studies to be good
effectors of tumor cell kill (Cole et al. 1994) (Rosenberg et al. 1988), and monocytes
which differentiate into macrophages when activated (Kleinerman et a]. 1980), (Sutton
and Weiss 1966).

On treating the tumor burdened animals with leukocytes activated with cisplatin
and Poly-plat we saw a regression of the localized tumor and no metastasis. Animals
which were not treated showed a steady growth in the size of the tumor, and metastasis
occurred in two of the animals. In addition, we saw none of the associated toxicities of
treating the animal directly with the drugs. Due to the ease of obtaining large numbers of
leukocytes from an organism this treatment method can easily be applied in the clinical
setting. Leukocytes can even be obtained from the tumor burdened individual (for non
leukemia or lymphoma cancers), treated, and returned to the same individuals without
cross reactivity. The treatment could be repeated many times with little to no side effects
for the patient. A decrease in harmful side effects is not only easier for the patient to
tolerate, but also decreases damage to the kidney and liver. The immune system can be a
powerful tool in combating disease, but occasionally cancer cells evade detection.
Through an increased immune response following adoptive immunotherapy it may be
possible to boost the systems awareness to the tumor and mount a greater, more efficient

response.

128

 

REFERENCES

Aggarwal SK (1993) A histochemical approach to the mechanism of action of cisplatin
and its analogues. J Histochem Cytochem 41:1053-73

Basu S, Sodhi A, Singh SM, Suresh A (1991) Up-regulation of induction of lymphokine
(H-2)-activated killer (LAK) cell activity by FK-565 and cisplatin. Immunol Lett 27:199-
204

Blommaert FA, Floot BG, van Dijk-Knijnenburg HC, Berends F, Baan RA, Schomagel
JH, den Engelse L, Fichtinger-Schepman AM (1998) The formation and repair of
cisplatin-DNA adducts in wild-type and cisplatin-resistant L1210 cells: comparison of
immunocytochemical determination with detection in isolated DNA. Chem Biol Interact
1082209-25

Burkitt K, Aggarwal SK (2000) Immune system activation by CDDP and "Poly-plat".
Anticancer Res 20:2729-37

Cohn ZA (1978) Activation of mononuclear phagocytes: fact, fancy, and future. J
Immunol 1212813-6

Cole DJ, Taubenberger J K, Pockaj BA, Yannelli JR, Carter C, Carrasquillo J, Leitman S,
Steinberg SM, Rosenberg SA, Yang YC (1994) Histopathological analysis of metastatic
melanoma deposits in patients receiving adoptive immunotherapy with tumor-infiltrating
lymphocytes. Cancer Immunol Immunother 382299-303

Di Luzio NR, McNamee R, Jones E, Cook JA, Hoffman EO (1976) The inhibition of
Shay leukemia tumor in rats following treatment with glucan-induced peritoneal
macrophages. In Fink MA, ed. The Macrophage in Neoplasia. New York, Academic, 181

Ferreira CG, Tolis C, Span SW, Peters GJ, van Lopik T, Kummer AJ, Pinedo HM,
Giaccone G (2000) Drug-induced apoptosis in lung cancer cells is not mediated by the
Fas/FasL (CD95/APOl) signaling pathway. Clin Cancer Res 62203-12

Fink D, Zheng H, Nebel S, Norris PS, Aebi S, Lin TP, Nehme A, Christen RD, Haas M,
MacLeod CL, Howell SB (1997) In vitro and in vivo resistance to cisplatin in cells that
have lost DNA mismatch repair. Cancer Res 57: 1841-5

Han X, Wilbanks GD, Devaja O, Ruperelia V, Raju KS (1999) H.-2 enhances standard
IFNgamma/LPS activation of macrophage cytotoxicity to human ovarian carcinoma in
vitro: a potential for adoptive cellular immunotherapy. Gynecol Oncol 75:198-210.

Herscowitz HB, Holden HT, Bellanti JA, Ghaffar A (1981) Manual of Macrophage
Methodology. In Rose N, ed. Immunology Series. New York, Marcel Dekker, Inc, 531

129

Jiang S, Song MJ, Shin EC, Lee MO, Kim SJ, Park JH (1999) Apoptosis in human
hepatoma cell lines by chemotherapeutic drugs via Fas-dependent and Fas-independent
pathways [see comments]. Hepatology 292101-10

Johnson WJ, Steplewski Z, Matthews TJ, Hamilton TA, Koprowski H, Adams DO
(1986) Cytolytic interactions between murine macrophages, tumor cells, and monoclonal
antibodies: characterization of lytic conditions and requirements for effector activation. J
Immunol 13624704-13

Kinoshita H, Yoshikawa H, Shiiki K, Hamada Y, Nakajima Y, Tasaka K (2000) Cisplatin
(CDDP) sensitizes human osteosarcoma cell to Fas/CD95-mediated apoptosis by down-
regulating FLIP-L expression [In Process Citation]. Int J Cancer 882986-91

Kleinerrnan ES, Zwelling LA, Muchmore AV (1980) Enhancement of naturally occurring
human spontaneous monocyte-mediated cytotoxicity by cis-
diamminedichloroplatinum(II). Cancer Res 4023099-102

Matheson DS, Green B, Hoar D1 (1983) The influence of methotrexate and thymidine on
the human natural killer cell function in vitro. Journal of Immunology 13121619-1621

Muenchen HJ, Aggarwal SK (1998) Immune system activation by cisplatin and its analog
'Poly-plat': an in vitro and in vivo study. Anticancer Drugs 9293-9

Nehme A, Baskaran R, Aebi S, Fink D, Nebel S, Cenni B, Wang JY, Howell SB,
Christen RD (1997) Differential induction of c-Jun NH2-terminal kinase and c-Abl
kinase in DNA mismatch repair-proficient and -deficient cells exposed to cisplatin.
Cancer Res 57:3253-7

Ogura T, Shindo H, Shinzato O, Namba M, Masuno T, Inoue T, Kishimoto S, Yamamura
Y (1982) In vitro tumor cell killing by peritoneal macrophages from mitomycin C treated
rats. Cancer Immunology and Irnmunotherapy 132112-117

Ohanian SH, Borsos T, Schlager SI ( 1980) Enhancement of immune killing of tumors
following treatment with chemotherapuetic drugs. In Israel L, Lagrange P, Salomon JC,
eds. Cancer Immunology and Parasite Immunology. New York, Marcel Dekker, 199-211

Oshita F, Kameda Y, Nishio K, Tanaka G, Yamada K, Nomura I, Nakayama H, Noda K
(2000) Increased expression levels of cyclin-dependent kinase inhibitor p27 correlate
with good responses to platinum-based chemotherapy in non- small cell lung cancer.
Oncol Rep 72491-5.

Palma JP, Aggarwal SK, Jiwa A (1992) Murine macrophage activation after cisplatin or
carboplatin treatment. Anticancer Drugs 3:665-76

Roberts JJ, Pascoe JM (1972) Cross-linking of complementary strands of DNA in
mammalian cells by antitumour platinum compounds. Nature 235:282—4

130

Rosenberg B, VanCamp L (1970) The successful regression of large solid sarcoma 180
tumors by platinum compounds. Cancer Res 3021799-802

Rosenberg SA (1990) Adoptive immunotherapy for cancer. Sci Am 262:62-9

Rosenberg SA, Schwarz SL, Spiess PJ (1988) Combination immunotherapy for cancer:
synergistic antitumor interactions of interleukin-2, alfa interferon, and tumor-infiltrating
lymphocytes. J Natl Cancer Inst 80:1393-7

Stoychkov JN, Schultz RM, Chirigos MA, Pavlidis NA, Goldin A (1979) Effects of
adriamycin and cyclophosphamide treatment on induction of macrophage cytotoxic
function in mice. Cancer Research 39:3014-3017

Sutton JS, Weiss L (1966) Transformation of monocytes in tissue culture into
macrophages, epithelioid cells and multinucleated giant cells. Jouma] of Cell Biology
292303

Telgenhoff DJ, Aggarwal SK (1998) Kupffer cell activation after treatment with cisplatin
and its second generation novel analogs SAP, SSP, and "Poly-plat". In Collery P, Bratter
P, Negretti de Bratter V, Khassanova L, Etienne J-C, eds. Metal Ions in Biology and
Medicine. Neuherberg, Germany, John Libbey Eurotext, 646-648

Toge T, Nakanishi K, Yamada Y, Yanagawa E, Hattori T (1981) Scanning electron
microscopic studies on the surface structure of activated macrophages and on their
interaction with tumor cells. Gann 72:305-9

van der Rhee HJ, van der Burgh-de Winter CP, Daems WT (1979) The differentiation of
monocytes into macrophages, epithelioid cells, and multinucleated giant cells in
subcutaneous granulomas. I. Fine structure. Cell Tissue Res 1972355-78

Wang BS, Lumanglas AL, Durr FE (1986) Irnmunotherapy of a murine lymphoma by
adoptive transfer of syngeneic macrophages activated with bisantrene. Cancer Research
462503-506

Wang RF, Rosenberg SA (1996) Human tumor antigens recognized by T lymphocytes:
implications for cancer therapy. J Leukoc Biol 602296-309

131