HOLECULAR EFFECTS OF TUMOR-INHIBETING
PLATENUM COMPOUNDS IN MAMMAUAN
{ZEUS Eli VITRO:

INVESTIGATEOHS ENTG THEIR POSSIBLE
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MOLECULAR EFFECTS OF TUMOR—INHIBITING PLATINUM COMPOUNDS

IN MAMMALIAN CELLS IN VITRO: INVESTIGATIONS INTO THEIR
POSSIBLE MECHANISMS OF ACTION

 

presented by

HAROLD CECIL HARDER

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Biophysics

 

 

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ABSTRACT
MOLECULAR EFFECTS OF TUMOR-INHIBITING PLATINUM COMPOUNDS
IN MAMMALIAN CELLS IN VITRO: INVESTIGATIONS INTO THEIR
POSSIBLE MECHANISM OF ACTION
BY

Harold Cecil Harder

Tumor-inhibiting, bacterial filament—inducing Cis—Platinum(II)
diamminodichloride (Cis—Pt(II)) was first shown to inhibit cell division
and also to induce giant cell formation in human amnion AV3 cells,
grown in culture.

Cis-Pt(II) was then shown to selectively inhibit DNA synthesis at
5 uM, measured by the incorporation of 3H~thymidine into an acid
insoluble material. At 25 uM the same compound only preferentially
inhibited DNA synthesis, for RNA and then protein syntheses as meas—
ured by the incorporation of 3H—uridine and 3H—leucine respectively,
were also inhibited but more slowly than DNA synthesis. Two other
tumor-inhibiting platinum compounds, Cis—Platinum(IV) diamminotetra—
chloride (Cis—Pt(IV)) and platinum(II) ethylenediaminedichloride
(Pt(II)en) were also demonstrated to preferentially inhibit DNA syn-
thesis at 25 uM. The relative ordering of inhibitory effects on DNA
synthesis was the same as the relative effectiveness in sarcoma 180
tumor-growth inhibition.

On the other hand, Trans—Platinum(IV) diamminotetrachloride

(Trans-Pt(IV)) and platinum(II) tetramminodichloride ([Pt(II)]+2),

 

 

 

 

 

Harold Cecil Harder
neither of which inhibits Sarcoma 180 growth in mice nor induces bac~
terial filaments, were shown to have no effect on DNA, RNA or protein
synthesis at 25 uM. The possibility these inhibitory effects on DNA
synthesis were a result of a platinum induced limitation of passage of
the labeled 3H—thymidine into the cell was eliminated by showing that
there was no effect on the uptake of the radioactive precursor into the
cell's acid soluble pools with 5 uM Cis—Pt(II). Hence these experiments
support the hypothesis that DNA synthesis is the primary target of tumor
inhibiting platinum compounds.

In order to determine how DNA synthesis is inhibited, experiments
were performed to determine whether Cis—Pt(II) acts as an antimetabolite,
as an inhibitor of enzymes directly involved with the replication of
and maintenance of DNA, or by reacting directly with DNA itself. The
effects of Cis—Pt(II) on the incorporation of 3H—deoxyuridine, 3H—cytidine,
3H—deoxycytidine, 3H—deoxyadenosine and 14C—methyl from 14C—formate into
DNA and RNA were shown to be similar to the inhibitory effects on
3H—thymidine and 3H—uridine uptake. However, the incorporation of
3H-thymidine was more efficiently inhibited than any other precursor.
This may indicate that the thymidine kinases are inhibited. More sig—
nificant was the finding that 14C-methyl from 14C—formate incorpora—
tion into RNA was inhibited to the same extent as its incorporation
into DNA at 5 uM. This was interpreted as evidence for a platinum—
induced inhibition of de novo purine synthesis. Nevertheless, the
extent of the reduction of purine synthesis was too small to account
for the magnitude of DNA synthesis inhibition. Furthermore, when
unlabeled deoxyribonucleosides were present in a sufficient quantity,
no reversal of the DNA synthesis—inhibitory effects were observed.
Hence it was concluded that platinum compounds do not act primarily

as antimetabolites.

 

 

 

 

Harold Cecil Harder
To determine whether platinum compounds affect enzymes involved in

the maintenance and replication of DNA, studies on the DNase and DNA
polymerase were performed. With both enzymes, a concentration—dependent
enhancement of activity was observed in extracts of AV3 cells treated
with between 50 and 500 uM Cis—Pt(II), Cis-Pt(IV) and Pt(II)en during
growth, except for Cis—Pt(II), which had no effect on the DNase activity.
However, all 3 of the above were shown to inhibit the DNase and DNA
polymerase activities when added to assays containing untreated cell

extracts. Hence platinum compounds cannot be directly activating these

 

enzymes by an allosteric interaction with them. [Pt(II)]+2 did not
inhibit DNA polymerase up to 240 uM. Also Cis-Pt(II) had no effect
on the purified enzyme, DNA dependent RNA polymerase from Pseudomonas
putida. The latter was taken as evidence that the effectiveness of a

given platinum compound strongly depends on its having the proper con—

 

figuration of ligands attached to it rather than simply being a heavy 3
metal poison effect. To account for both the enhanced DNase and DNA

polymerase activities in extracts of treated cells and the inhibition

of the same enzymes when assays containing untreated cell extract were

incubated with platinum compounds, it was suggested that a DNA repair

system was being activated by reactions of the DNA with platinum in

treated AV3 cells.

In summary, the molecular effects of tumor-inhibiting platinum com—
pounds were shown to mimic closely those of bifunctional alkylating
agents, especially HNZ, in terms of preferential inhibition of DNA syn—
thesis, giant cell formation, inhibition of de novo purine syntheses,
and enhanced DNase and DNA polymerase activities in extracts of treated
cells. Also paralleling the effects of bifunctional alkylating agents is

the ability of platinum compounds to induce lysogenized prophage

 

 

 

Harold Cecil Harder
(Reslova, 1970), and lower the melting temperature of DNA (Horacek and
Drobnik, 1970).

In contrast to bifunctional alkylating agents, tumor-inhibiting
platinum compounds inactivate both single and double—stranded DNA con—
taining bacteriophage with equal and high efficiency (Drobnik et aZ.,
1970); hence platinum compounds probably do not act by resting interstrand
cross—links. On the basis of the size of the platinum molecule, a
hypothesis was proposed that the mechanism of action of the tumor—
inhibiting platinum cmopounds involves the creation of platinum coor—
dinated intrastrand cross-links between 2 adjacent purines. Coordina—
tion could occur at either 1 or 2 sites of each adjacent purine.
Depending upon the strength of the ligand bonds, it is predicted
that the effects of such platinum coordinated purine dimers would
mimic the effects of UV induced pyrimidine dimers. On the basis of
this hypothesis, it would be predicted that Trans-platinum compounds
could not give rise to such dimers for steric reasons and that bulky
groups such as in bridged platinum compounds would make the reaction
more difficult to occur; however, Cis—Pt(IV) should be about as effect—
ive as Cis—Pt(II). Furthermore there would be no interstrand cross-link
type mutagenic effects such as dominant lethal mutations. There would
probably also be a repair mechanism for such platinum induced DNA
lesions. All of the presently known molecular effects of tumor—inhibiting
platinum compounds are consistent with the above predictions of this

hypothesis.

 

Drobnik, J., A. Krekulova, and A. Kubelkov (1970). Inactivation
of bacteriophages with Cis—dichlorodiamine—Platinum II. (manuscript in
preparation)

Horacek, P., and J. Drobnik (1970). Personal communication.

Reslova, S. (1970). Induction of lysogenic strains of Escherichia
coli by Cis—platinum(II) diamminodichloride. (submitted for publication)

 

 

 

 

 

 

 

 

MOLECULAR EFFECTS OF TUMOR-INHIBITING PLATINUM COMPOUNDS
IN MAMMALIAN CELLS IN VITRO: INVESTIGATIONS INTO THEIR

POSSIBLE MECHANISMS OF ACTION

By

Harold Cecil Harder

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Biophysics

1970

 

 

   
 

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Lg} Copyright by

HAROLD CECIL HARDER

1971

ii

 

 

 

 

 

 

 

 

 

Dedicated to my wife, son,

and to all those who have long hoped for better anti—cancer drugs

 

 

 

 

ACKNOWLEDGEMENTS

I wish to express my sincere gratitude to Linda, who patiently
endured 5 years as a graduate student's wife and who, along with my
son David, was always a source of encouragment in times of discouragement.

I would also like to acknowledge indebtedness to Dr. John
Boezi, whose stimulating lectures on biochemistry and molecular genetics
were responsible for my own interest in this field; to James Johnson
for the gift.of DNA dependent RNA polymerase; and to Dr. James E.
Trosko, without whose generosity in allowing me the use of his equip—
ment and without whose valuable assistance this research could not
have been performed. Finally, I wish to express my sincere thanks to
my thesis director, Dr. Barnett Rosenberg, for granting me a very wide
degree of freedom in pursuing this research project.

This research was supported by National Cancer Institute grant

CA 11349, and by both a grant and a predoctoral fellowship from Matthey—

Bishop Corporation.

iv

 

 

 

 

 

TABLE OF CONTENTS
Page
I. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . l

A. History of Biological Effects of Platinum

Compounds . . . . . . . . . l
B. Approach to the Problem: Clues to the

Possible Mechanism of Action. . . . . . . . . . .
C. Scope of the Thesis . . . . . . . . . . . . . . . 9

II. EXPERIMENTAL PROCEDURE . . . . . . . . . . . . . . . . . . . 11

A. Materials . . . . . . . . . . . . . . . . 11
1. Platinum Compounds . . . . . . . . . . . . 11
2. Other Drugs. . . . . . . . . . . . . . . . ll
3. Growth Media . . . . . . . . . . . . . . . ll

 

4. Radioisotopes. . . . . . . . . . . . . . . 12
B. Methods . . . . . . . . . . . . . . . . . . . . . 12
1. Measurement of DNA, RNA and Protein
Syntheses. . . . . . 12
2. Measurement of the Reversibility of
Platinum Treatment . . . . . . . . l4
3. Measurement of Membrane Effect . . . . . . 14
C. Enzyme Assays . . . . . . . . . . . . 15
1. Preparation of Enzyme Extracts . . . . . . 15
Protein Assay. . . . . . . 16

2
3 Acid Soluble (Exonuclease) Assay . . . . . l6
4. Endonuclease Assay . . . . . . . . . . . l7
5 DNA Polymerase . . . . . . . 18
6 DNA Dependent RNA Polymerase from

Pseudomonas putida . . . . . . . . . . 19

III. RESULTS. . . . . . . . . . . . . . . . . . . . . . . . . . . 21

A. Effects of Cis— —Pt(II)(NH3)2C12 on AV3 Cell

Growth, Division and Survival . . . . . 21
B. Effects of Cis—Pt(II)(NH3)2C12 on DNA, RNA,
and Protein Syntheses . . . . . 24

C. Comparison of the Effects of Three Effective
Tumor Inhibiting,Bacterial Filament— —forming
Platinum Compounds with Two Ineffective
Compounds . . .

D. Irreversibility of DNA Synthesis Inhibiting
after Platinum Treatment. . . .

E. Effect of Cis— —Pt(II)(NH3)2C12 on the Uptake

of 3H—thymidine into the Acid Soluble Pool. . . . 38

31

36

V

 

Page

Stability of Cis—Pt(II)(NH3)2C12 in MEM . . . . . 41
Comparison of the Inhibitory Effects of
Cis—Pt(II)(NH3)2C12 on the Incorporation of
Labeled Precursors into RNA and DNA . . . . . . . 41
Effect of Unlabeled Deoxyribonucleosides on
the Inhibition of DNA Synthesis by Cis—Pt(II) %
(NH3)2C12....... 46 ,_'
Effects of Platinum Compounds on the Ac1d
Soluble DNase and Endonuclease Activities in
Cell Free Preparations of AV3 Cells . . .

1. Characteristics of the DNase Activity.

2. Acid Soluble DNase and Endonuclease
Activity in Extracts of AV3 Cells
Treated with Cis—Pt(II)(NH3)2C12,
Cis—Pt(IV)(NH3)2Cl4 and Pt(II)(NH2)2
(CH2)2C12 During Growth. . . . . . . 51
Effects of the Presence of Cis-Pt(II) !,
(NH 3)2C12, Cis-Pt(IV)(NH3)2C14 and ‘
Pt(II) (NH2)2(CH2)2C12 in the Acid
Soluble DNase and Endonuclease Assays
Using Enzyme Extracts from Untreated

. . . . 56

Cells. . . . . . . . . . .

J. DNA Polymerase Studies. . . . . . . . . .
1. Enzyme Characterization. . . . . . . 6O
2 DNA Polymerase in Extracts of AV3 Cells
Treated with Tumor Inhibiting Platinum

0 a o o n o o o u

U 47 l
. 49 /

Compounds. . . . . . . . . . . . . . 6O
3. Effect of the Addition of Platinum Com—
pounds to the DNA Polymerase Assay Using
Untreated AV Cell Extract . . . . . 63
K. Effect of Cis—Pt(II§(NH3)2C12 on Purified DNA
Dependent RNA Polymerase from Pseudomonas
putidh. . . . . . . . . . . . . . . . . . . . . . 66
IV. DISCUSSION . . . . . . . . . . . 68
A. Platinum Induced Inhibition of Cell Divi51on. 68
B Inhibition of DNA Synthesis. A Primary
Target of Tumor Inhibiting Platinum Compounds . 69
C. Possible Evidence for a Metabolically Trans—
formed, Effective Platinum Moiety or an Irre—
versible Reaction of the Platinum with the
Target Molecules. . . . . . . 71
D Are Tumor Inhibiting Platinum Compounds Anti—
metabolites?. . . . . 74
E. Effects of Platinum Compounds on Enzymes Using
DNA as a Template or Substrate. . . . . . . . 76
l. Deoxyribonuclease Activity . . . . 76
2. DNA and RNA Polymerase Activities. . . . . 78
3. A Possible Involvement of DNA Repair
Enzymes. . . . . . . . . . . . . . . . . . 8O
. . . . . . . . . . . . 82

F. Summary . . . . .

A HYPOTHESIS OF THE MECHANISM OF ACTION OF TUMOR—
INHIBITING PLATINUM COMPOUNDS. . . . . . . 83

V.

vi

 

 

 

 

 

Page

87

EPILOGUE

VI.

88

o

BIBLIOGRAPHY

 

“n
V

 

 

 

Table

10

 

LIST OF TABLES

Page
Average distribution of Cis—lglPt(IV)(NH3)2Cl4 and
[1911’t(IV)C16]"2 among various bacterial cellular
components. . . . . . . . . . . . . . . . 3
A bibliography of tumor—inhibiting agents that
inhibit cell division and induce filament formation

7

in bacteria . . . . . . . . . . . . . . . . . . . . . .

Comparison of anti-tumor activity and bacterial fila—
ment forming abilities of five platinum compounds . . . . 32

Effect of Cis~Pt(II)(NH3)2C12 on the uptake of 3H-
thymidine into the acid soluble pool in AV3 cells . . . . 40

Effect of a 21 hour pre—incubation of Cis—Pt(II)(NH3)2C12
in MEM on its DNA synthesis- inhibiting activity in
AV3 cells measured by 3H— —thymidine incorporation. . . . . 42

A comparison of the inhibitory effects of Cis-Pt(II)
(NH3)2C12 on the relative incorporation of H— —thymidine,
H—uridine 3H- -deoxyuridine, 3H-cytidine, 3H— deo

cytidine, éH- ~deoxyadenosine and l4C—methyl from 1 C-

formate into RNA and DNA. . . . . . . . . . . . . . . 44

Summary of Table 6: The range of the inhibitory

effects of Cis—Pt(II)(NH3)2C12 on the relative incor—
poration of labeled precursors into DNA and RNA com-

pared with 3H—thymidine and 3H—uridine respectively . . . 45

Effect of the presence of unlabeled deoxyribonucleosides
on the relative inhibition of DNA synthesis by Cis-Pt(II)
(NH3)2C12 by 3H—thymidine incorporation . . . . . . . . 48

The effects of pH and the state of the DNA substrate

on the acid soluble DNase activity in CPM in extracts

of AV3 cells grown in Cis—Pt(IV)(NH3)2Cl4I using a

2—hour assay. . . . . . . . . . . . . . . . . . . 50

Characteristics of DNA polymerase in AV3 cell extracts. . 61

viii

 

  
   
  
  
 
 
    
 
  
  
    
     
    

 

Figure

l

2

 

LIST OF FIGURES

Page
Structures of platinum compounds. . . . . . . . . . . . . 6
Effect of Cis—Pt(II)(NH3)2C12 on cell Survival and
. . 22

cell division in AV3 cells. . . . . . . . . . . . . .

Giant cell formation in AV3 cells treated with 5 uM
Cis-Pt(II)(NH3)2C12 . . . . . . . . . . . . . . . . . . . 23

Effect of Cis—Pt(II)(NH3)2C12 on DNA synthesis in

AV3 cells as measured by the uptake of 3H—thymidine.

Both the platinum compound and 3H—thymidine were

applied to cells at time zero . . . . . . . . . . . . . . 25

Effect of Cis—Pt(II)(NH3)2C12 on RNA synthesis in

AV3 cells as measured by the uptake of 3H—uridine.

Both the platinum compound and 3H—uridine were added

at time zero. . . . . . . . . . . . . . . . . . . . . . . 27

Effect of Cis-Pt(II)(NH3)2C12 on protein synthesis
in AV3 cells as measured by uptake of H-leucine.
Both the platinum compound and 3H—leucine were

applied at time zero. . . . . . . . . . . . . . . . . . . 28

Relative effect of 25 uM Cis-Pt(II)(NH3)2C12 on DNA,
RNA and protein synthesis. The data from figure 2,
3, and 4 at 5 uM was transformed using the expression

cpm —cpmt cpmt —cpmt
[( t2 1)exp/( 2 1)control

cpmtl represent the counting rates for the incorpora—

1 where cpmtzand

tion of the labeled precursors at times t2 and t1 . . . . 29

Relative effect of 5 uM Cis—Pt(II)(NH3)2C12 on DNA,

RNA and protein synthesis. The data from figure 2,

3, and 4 at 25 uM was transformed using the expression
cpmt -cpmt cpmt —cpmt

[( 2 1)exp/( 2 1)control

and cpmt represent the counting rates for the incor—

poration of the labeled precursors at times t2 and t1 . . 30

] where cpmt2

ix

 

 

 

 

 

 

10

ll

12

l3

l4

 

 

Page

The relative effects of 25 pM Cis—Pt(II)(NH3)2C12,
CiS-Pt(IV)(NH3)2C14, Pt(II)(NH2)2(CH2)2C12, Trans-
Pt(IV)(NH3)2C14 and [Pt(II)(NH3)4]C12 on DNA synthe—

sis in AV3 cells measured by the uptake of H—

thymidine. Platinum compounds were applied at time

zero and cells were pulse labeled for only the last

2 hours of treatment at each time period. Results

are expressed relative to the control. . . . . . . . . . . 33

The relative effects of Cis—Pt(II)(NH3)2C12, Cis—Pt
(IV)(NH3)2C14, Pt(II)(NH2)2(CH2)2C12, Trans—Pt(IV)

(NH3)2C14 and [Pt(II)(NH3)4]C12 on RNA synthesis in

AV cells measured by the uptake of 3H—uridine.

Platinum compounds were applied at time zero and

cells were pulse labeled for only the last 2 hours

of treatment at each time period. Results are expressed
relative to the control. . . . . . . . . . . . . . . . . . 34

The relative effects of Cis—Pt(II)(NH3)2C12, Cis-Pt(IV)
(NH3)2C14, Pt(II)(NH2)2(CH2)2C12, Trans-Pt(IV)(NH3)2014

and [Pt(II)(NH3)4]Cl2 on protein synthesis in AV3

cells measured by the uptake of 3H—leucine. Plati-

num compounds were applied at time zero and cells

were pulse labeled for only the last 2 hours of

treatment at each time period. Results are expressed

relative to the control. . . . . . . . . . . . . . . . . . 35

The relative effect of a 4 hour pretreatment of AV

cells with Cis—Pt(II)(NH3) C12 on DNA synthesis

measured by the uptake of H—thymidine. Platinum

media was applied at time —4 hours, and decanted at

time zero. After the cells were washed 2 X with

Puck's saline D, fresh MEM was added and cells were

pulse labeled for only the last 2 hours of each

period. Results are expressed relative to the control . . 37

The relative effect of a 4 hour pretreatment of AV

cells with 5 uM Cis—Pt(II)(NH3)2C12, Cis—Pt(IV)(NH3)2

C14, Pt(II)(NH2)2(CH2)2C12 and 1 mM hydroxyurea on

DNA synthesis, measured by the uptake of 3H-thymidine.
Platinum media was applied at —4 hours and decanted

at time zero. After the cells were washed 2 X with

Puck's saline D fresh MEM was applied and cells were

pulse labeled for only the last 2 hours of each time

period. Results are expressed relative to the control . . 39

Acid soluble DNase activity in CPM in extracts of
AV3 cells treated with Cis—Pt(IV)(NH3)2Cl4. 18.6 pg
protein/assay. . . . . . . . . . . . . . . . . . . . .

  

   
 
 
 
   
 
   
    
  
 
   
 
   
 
 
  
 
 
 
   
  
 
 
 
 
 
   
 
 
 

 

 

 

 

 

 

15

l6

17

18

19

20

21

22

23

24

Endonuclease activity in CPM in extracts of AV cells
treated with Cis—Pt(IV)(NH3)2Cl4. 3.111g protein/
assay . . . . . . . . . . . . . . . .

Acid soluble DNase activity in CPM in extracts of
AV3 cells treated 5 hours with Cis—Pt(II)(NH3)2C12.
511g protein/assay. . . . . . . . . . .

Endonuclease activity in CPM in extracts of AV3 cells
treated 5 hours with Cis—Pt(II)(NH3)2C12.5 ug
protein/assay . . . . . . . . . . . . . . . .

Acid soluble DNase activity in CPM in extracts of
AV3 cells treated 6 hours with Pt(II)(NH2)2(CH2)2C12.
3. 3511g protein/assay . . . . . . . .

Effect of Cis—Pt(II)(NH3)2C12, Cis—Pt(IV)(NH3)2Cl4,
and Pt(II)(NH2)2(CH2)2C12 on the acid soluble DNase
activity in CPM in extracts from untreated AV3 cells.
2.511g protein/assay; 2 hours incubation at 370 C .

Effect of Cis—Pt(II)(NH3)2C12, Cis—Pt(IV)(NH3)2C14,

and Pt(II)(NH2)2(CH2)2C12 on the endonuclease activity

in CPM in extracts of untreated AV cells. 2.5 ug
protein/awsay; 2 hours incubation at 37° C; 19,100
CPM retained in assay lacking enzyme. . . . . . .

DNA polymerase activity in CPM in extracts of AV3
cells treated 6 hours with Cis—Pt(IV)(NH3)2C14 and

Pt<II)(NH2)2(CH2)2C12 . . . . . . .

DNA polymerase activity in CPM in extracts of AV
cells treated 5 hours with Cis—Pt(II)(NH3)2C12. .

Effect of Cis-Pt(II)(NH3)2C12, Cis—Pt(IV)(NH3)2C14,
Pt(II)(NH2)2(CH2)2C12 and [Pt(II)(NH3)4]C12 on DNA
polymerase activity in CPM in extracts of untreated
AV3 cells. 62.5 ug protein/assay . . . . . . . .

Effect of Cis— —Pt(II)(NH3)2012 on the purified DNA

dependent RNA polymerase from Pseudomonas putida. . .

xi

Page

53

54

55

57

58

59

62

64

65

67

 

 

 

 

 

I. INTRODUCTION

A. History of Biological Effects of Platinum Compounds

From its inception the study of the biological effects of platinum
compounds has been, to paraphrase Rosenberg, "a story in serendipity."
It started about 6 years ago when Rosenberg designed an experiment to
study the effects of alternating electric fields on bacterial cell
division. The very first time the current was turned on at 1000 cps
in the presence of a continuous culture of E. coli B, cell division was
inhibited. Microscopic examination revealed that the bacteria continued
to grow, forming very long filaments. Instead of being caused directly
by the electric field, however, these effects were found to be attribu-
table to various platinum species generated by the electrolysis of the
presumably—inert platinum electrodes (Rosenberg at al., 1965). At the
conclusion of this initial publication a number of questions were posed:
"Can these metal ions inhibit cell division in other bacteria and cells?
Where is the locus of action in the bacterial cell? How does the effect
of these metal ions relate to the actions of other causative agents of
filamentous growth——is there a weak link that all operate on? Finally,
what is the mechanism of action of these metal ions?"

In later studies the neutral platinum compound Cis—Pt(IV)(NH3)2C14
was identified as being a most effective inhibitor of cell division
and inducer of filaments (Rosenberg et aZ., 1967a). This was in sharp

contrast with the much less efficient trans isomer and the almost com-

 

plete lack of cell division inhibition by the single and double negative

1

 

 

 

 

 

 

IIIIIIIIIIIIIIIIIII:::::------------------------------------------—-wwr
2

species, [Pt(IV)NH3015]" and [Pt(IV)Cl6]=, although the latter was
found to be bactericidal at very low concentrations. Of the many ,
other group VIII—B transition metal complexes tested, only high con-
centrations of some rhodium and ruthenium compounds were able to induce
elongation in E. coli B significantly, but not to the extent nor to the
uniformity of the analogous platinum complexes (Rosenberg et al.,
1967b). The questions as to whether cell division and elongation are
produced in other bacteria by platinum compounds was found to be
affirmative with all tested gram negative bacilli; however, gram—
positive bacilli showed only slight elongation at near—toxic levels
(Rosenberg at al., 1967b).

By following the distribution of 191Pt labeled platinum compounds I\:?U

among various bacterial cellular components, Renshaw and Thomson (1967)
attempted to establish the locus of action of the active platinum com—
pounds. Their results, summarized in Table 1, showed that the

Cis—Pt(IV)(NH3)2Cl4 in elongated E. coli B was associated with metabolic

 

intermediates, nucleic acids and cytoplasmic proteins. On the other
hand, in (Pt(IV)C12)= growth inhibited E. coli B, the platinum was
bound only to cytoplasmic proteins. Since inhibition of cell division
did not occur when nearly all the platinum was bound to cytoplasmic
protein, it may be concluded that binding to cellular proteins per se
is not responsible for inhibition of cell division. Conversely, growth

inhibition was directly correlated with the binding of the platinum

 

cytoplasmic proteins. In similar experiments with l91Pt(IV)(NH3)ZClz,
treated Bacillus cereus and Staphylococcus aureus, in which neither
growth nor cell division were inhibited, the metal complex was bound

predominantly to metabolic intermediates. Significant binding still

 

 

 

 

 

Table 1. Average distribution of Cis—lglPt(IV)(NH3)2Cl4 and
[191Pt(IV)C16]— among various bacterial cellular

 

 

 

 

 

 

 

components
1
Percentage of Radioactivityb
Treatment Cis—l91Pt (IV) (N11,) .,01,La 191m; (1v)016
Bacterium B. cereus S. aureus "E.'c0li B E. coli B
Inhibitory effect on none none division growth
£116.91“
Metabolic intermedi-
ates 74 60 19 l
Lipids 2 l 6 3
Nucleic acids 19 20 30 1
Cytoplasmic proteins 5 19 45 96

 

aMade by UV irradiation of [lglPt(IV)C16]=, of which photo—
products Cis—lglPt(IV)(NH3)2Cl4 was the most predominant,
Trans—lgiPt(IV)(NH3)2Cl4 being only a minor component.

 

bData from Renshaw and Thomson (1967).

 

 

 

 

4
occurred with the nucleic acids, but to only two thirds of the extent
in elongated E. coli. The lack of a high degree of platinum binding
to cytoplasmic proteins and the lack of growth inhibition in B. serous
and S. aureus is consistent with the conclusion drawn above from
results with E. coll. In fact, the high degree of binding to metabolic
intermediates is indicative that this is a possible detoxification
mechanism, because the concentration oftheplatinum compound is far
too small to tie up a high enough fraction of most metabolites to be
responsible for either of these inhibitory effects. Therefore, the
results of Renshaw and Thomson provide some evidence that the binding
of platinum to nucleic acids plays a role in the induction of elongation.

In ultraviolet light induced E. coli filaments, cytokinesis
(cross septation) can be initiated by pantoyl lactone treatment or by
incubation at 42° C. (Adler and Hardigree, 1965b). Similarly in UV,
penicillin and D—amino acid induced Erwinia filaments, cytokinesis can
be induced by pantoyl lactone or divalent cation treatment (Grula and
Grula, 1962). However, Rosenberg et al. (1967b) found that cytokinesis
or cell division in platinum induced E. coliB filaments could not be
induced by any of these treatments. Reversal of the inhibition of
cell division could be achieved only by removal or decrease in the
platinum concentration. Therefore, in relating the effects of platinum
treatment with other agents that cause filamentous growth, they con—
cluded from these observations that platinum—induced inhibition of cell
division in E. coli B differs from the inhibition by other agents
mentioned above.

At this point, research had been published on all but the last

question stated in the first paragraph. Then it was discovered that

 

 

 

 

5
3 bacterial elongating compounds, Cis—Pt(II)(NH3)2ClZ (abbreviated
hereafter by Cis-Pt(II), Cis—Pt(IV)(NH3)2C14 (abbreviated Cis—Pt(IV)),
and Pt(II)(NH2)2(CH2)2C12 (abbreviated Pt(II)en) (see Figure l) were
potent inhibitors of Sarcoma 180 tumors in mice (Rosenberg at al., 1969).
More recently it has been shown that large, well-developed, solid 8—180
tumors in mice (Rosenberg and VanCamp, 1970), Dunning Ascites Leukemia
and intramuscular Walker 256 carcinosarcoma (Kociba et al., 1970) in
rats can be successfully regressed by Cis—Pt(II). Rapidly accumulating
experimental results now indicate that the platinum compounds are a new
class of broad spectrum antineoplastic drugs (Rosenberg, 1970; Haddow,
1970; Drobnik, 1970). The anti—tumor activity and the filamentous-
inducing activity of these platinum compounds now urgently require an
answer to the last question of the opening paragraph: "What is the
mechanism of action?" This is the question to which the research in

this thesis is addressed.

B. Approach to the Problem: Clues to the Possible Mechanism of Action

Since the effective platinum compounds are not structurally related
to other anti—neoplastic agents, this presented a problem as to where

to begin the investigation. Therefore, clues to possible mechanisms

of action of the tumor inhibiting platinum compounds were taken from
the mechanisms of action of other anti—tumor compounds which also induce
bacterial filamentous formation. These include ultraviolet light (and
ionizing radiation), nitrogen mustard, mitomycin C, hydroxyurea, and
azaserine (see Table 2).

Ultraviolet light in low doses affects cells primarily by the
induction of pyrimidine (primarily thymine-thymine) dimers (Setlow
at al., 1963). In this case the net effect is to stop DNA_synthesis

until the DNA is repaired. The nitrogen mustard, methylbis(2—ch10ro—

 

 

 

 

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8
ethy1)amine (NHZ) and all other bifunctional alkylating agents, react
with DNA, RNA and protein as well as with many metabolites of the cell.
Due to the lack of binding specificity, the only alkylation reaction
which could reasonably lead to cell death is that with DNA itself.
In that bifunctional alkylating agents are 50 to 100 times more effect—
ive than the corresponding monofunctional agents, it has been postulated
that the polyfunctional alkylating agents are interstrand cross-linking
agents; that is, they react at one point on each strand (Ross, 1962).
The net effect of cross-linking is to prevent the separation of the 2
strands which is necessary in semiconservative DNA replication. Never-
theless, regardless of the specific site of interaction, all bifunc-
tional alkylating agents result in the inhibition of DNA synthesis
(Ochoa and Hirschberg, 1967; Wheeler, 1962).

The antibiotic mitomycins are another class of tumor inhibitors
that also induce elongation in E. coli. Mitomycin C must be classified
as a bifunctional alkylating agent for it efficiently creates DNA
cross-links (Szybalski and Iyer, 1964) and causes radiomimetic type
damage in cells. Thus, as with the nitrogen mustards, Mitomycin C
inhibits DNA synthesis (Reich et al., 1961; Grula and Grula, 1962).

Azaserine is another tumor inhibiting, bacterial elongating anti—
biotic; however, in contrast to Mitomycin C, it is classed as an anti—
metabolite. By non—competitive binding to enzymatic sites normally
occupied by glutamine, azaserine primarily inhibits the amination of

N-formylglycine—amide ribonucleotide by the enzyme, L—glutamine—amido

 

 

 

 

 

9
ligase (ADP) in the de novo synthesis of purines (Hartman et al., 1955;
Skipper et al., 1954). Inhibition of purine biosynthesis by azaserine
eventually leads to inhibition of DNA synthesis (Hartman et al., 1955;
Krakoff and Karnofsky, 1958).

Finally, the bacterial elongating, tumor inhibiting antimetbolite
known as hydroxyurea has been shown to inhibit the production of
deoxyribonucleotides by inhibiting the enzyme ribonucleoside diphosphate
reductase (Krakoff et al., 1968). This chemotherapeutic agent, there—
fore, also results in the inhibition of DNA synthesis (Young and
Hodas, 1964; Young et al., 1967; Krakoff et al., 1968).

All of the above 5 bacterial elongating, antineoplastic agents
have one ultimate effect in common, the inhibition of DNA synthesis,
although by different mechanisms. Therefore, it is logical to hypothe—
size that the antitumor platinum compounds also inhibit DNA synthesis
regardless of the specific mechanism of action (see Table 2 for a
summary). Accordingly, this hypothesis was employed as the starting

point for this dissertation research.

C. Scope of the Thesis

The overall purpose of this research is to elucidate the mechan—
ism(s) of action of tumor—inhibiting platinum compounds.

The first major objective was to determine the primary target of
the platinum compounds in terms of general cellular function. This was
accomplished by measuring the effects on overall DNA, RNA and protein
syntheses. Hence this part of the research will prove whether or not
the hypothesis of inhibition of DNA synthesis is correct. Because
initial results showed that DNA synthesis is indeed inhibited (Harder

and Rosenberg, 1969), the second major question to which this research

 

 

 

 

 

 

10
is addressed is, "How do the platinum compounds inhibit DNA synthesis?"
That is, is the tumor inhibiting activity of platinum compounds caused
by a platinum induced inhibition of the production of the deoxyribo-
nucleotide triphosphates as antimetabolites, or is it caused by a
direct interaction of the platinum compound with the cellular DNA (as

is the case for alkylating agents and certain antibiotics)?

 

 

II. EXPERIMENTAL PROCEDURE

A. Materials

1. Platinum Compounds. All platinum compounds were prepared in
our laboratory by Mr. Thomas Krigas as described elsewhere (Lededinskii
and Korabel'nik, 1947;Kauffman, 1963), except for Pt(II)(NH2)2(CH2)2C12,
which was purchased from Alpha Inorganics (Beverly, Mass.) and recrystal—
lized before use. Crystalline platinum compounds were purified by
repeated recrystallization and sent to Galbraith Laboratories, Inc.
(Knoxville, Tenn.) for analytic tests. The sum of the deviations from
the predicted values was less than 0.5%. Stored in the dark in the
crystalline state, platinum compounds were freshly dissolved into media
immediately before use to avoid unnecessary photochemical and hydrolytic

decomposition of these compounds.

2. Other Drugs. Hydroxyurea was purchased from Nutritional Bio—

chemicals Corporation.

3. Growth Media. Human Amnion, type AV3, tissue culture cells
were obtained from American Type Culture Collection (Rockville, Md.)
and grown in glass bottles at 37° C. in a humidified atmosphere sup—
plemented with 5% C02. The cells were cultured in Eagle's minimal
essential medium (MEM) with Earl's salts and supplemented with 10% calf
serum, all purchased from either Grand Island Biological Company,
Grand Island, N.Y., or Flow Laboratories, Rockville, Md. The growth

11

 

 

 

 

 

 

 

 

 

rrrs

12
medium also contained 100 units/ml penicillin G plus 100 ug/ml Strep—
tomycin sulfate obtained from Eli Lilly, Indianapolis, Ind. Trypsin
(0.25%) from Difco Laboratories, Detroit, Mich., in Hank's Balanced

Salt Solution (HBSS) was used to transfer the cells.

4. Radioisotopes. All radioisotopes were purchased from Swartz

Bioresearch, Inc., Orangeburg, N.J.

 

B. Methods

1. Measurements of DNA, RNA and Protein Syntheses. Human Amnion

 

AV3 cells from several growth bottles were harvested by trypsin treat—
ment, pooled, and then inoculated in equal numbers into 5 cm diameter
Falcon disposable tissue culture petri dishes. The plates, containing
2-3x106 cells as determined by a standard hemocytometer count at the
beginning of the experiment, were incubated overnight at 37° C. to allow

the cells to attach. DNA synthesis was specifically measured by the

 

incorporation of [methyl-3H]-thymidine (0.5 to 1.0 uC/ml, specific
activity 1.9 C/mmole) and by [6-3H]-deoxyuridine (2 uC/ml, specific
activity 24 C/mmole). RNA was specifically measured by the uptake of
[5-3H]—uridine (2 uC/ml, specific activity 20 C/mmole). DNA and RNA
syntheses together were followed by the incorporation of [5—3H]—cytidine
(2 uC/ml, 25.9 C/mmole); [5—3H]—deoxycytidine (2 uC/ml, 27 C/mmole);
[8-3H]—deoxyadenosine ( pC/ml, 20.3 C/mmole); and [14C]—sodium formate
(1.0 uC/ml, 52.4 mC/mmole). Protein synthesis was measured by the
incorporation of L—[4,5—3H]—leucine (2 uC/ml, 2.5 mC/mmole). All of

the above concentrations and specific activities represent the concen-

tration and specific activity in MEM at the time it is applied to the

cells in MEM.

 

 

13
In earlier experiments, both the labeled precursor and the platinum
compound freshly dissolved in MEM and filter sterilized were applied
to the plates at the beginning of the experiment (3 ml/plate). To assess
more accurately the effects of treatment over a 24-hour interval, later
experiments were performed by applying media containing only platinum
(2.9 ml/plate) at time zero. Then 2 hours prior to analysis after a

specific exposure period, the appropriate radioactive precursor (0.1

 

ml) was added. By this method, all plates were tested for incorporation

activity over an identical pulse period and isotopic dilution was

minimized. In all experiments incorporation was determined at various .
times up to 24 hours of treatment by setting the plates on ice to stop

the uptake of the labeled precursor, sonicating the monolayered cells

for 10 sec. in 3 m1 Puck's saline D with a Branson 140C sonifier, fol—

lowed by applying 0.10 ml of well agitated sonicate onto each of 2

Whatman 3 MM, 2.3 cm diameter filter discs. After being washed 3 X

in iced 5% trichloroacetic acid (TCA), 2 X in absolute ethanol and

 

once in acetone, the discs were dried and counted in 5 ml 2,5—bis—2—
(5—tert—Butylbenzoazolyl)-Thiophene (BBOT) in toluene (4 g/l) with
Packard Tri Carb Liquid Scintillation Spectrometer. The effective
background in all experiments was determined by applying the radioactive
precursors to prechilled plates which were then analyzed as described
above. The counting efficiency was 35 to 40% for 3H and 75 to 80%

for 14C determined by the channel's ratio method. Each data point
consists of the average of the counts from duplicate plates and dupli—
cate samples from each plate less the background. This is a modifica—

tion of procedures described elsewhere (Bollum, 1966; Regan and Chu,

1966).

 

 

 

 

 

 

 

 

 

 

 

14

In experiments in which RNA and DNA.were simultaneously labeled,
the RNA was separated from the DNA by the following procedure: The
radioactive media from 2 sets of plates was decanted and the plates
were placed on ice. After 3 iced saline rinses, the plates were incu—
bated 1 hour with 3 ml of 0.2 N HClO4 on ice to fix the cells. Follow—
ing 2 additional rinses with HBSS to neutralize the acid, 2 m1 of
40 ug/ml Worthington bovine pancreas RNase A (heat activated by incu—
bation at 80 C. for 15 minutes) in HBSS was added to one set of plates.
To another set of plates, 2 ml of HBSS was added. After both sets were
incubated 1 hour at 37 C, all plates were set on ice and an additional
1 ml of saline was added to each plate to give a total of 3 ml/plate.

Finally the plates were sonicated and analyzed as described above.

2. Measurement of the Reversibility of Platinum Treatment. The
reversibility of the effects of treatment with platinum compounds was
measured by the recovery of DNA synthesis from inhibition. The pro—
cedure used was to preincubate all plates 4 hours in MEM containing a
given platinum compound followed by washing 2 X in Puck's saline D and
adding 2.5 ml fresh MEM. Two hours prior to the termination of incuba-
tion 0.5 m1 of 3H-thymidine (3H—TdR) in MEM was added to yield a total
of 0.5 uC/ml. The zero hour points, i.e., 4 hours after application
of the test media, were obtained by adding the 3H—TdR to the plates 2
hours after application of the test media, and stopping the incorpora-

tion at time zero. The remaining procedure was as described above.

3. Measurement of Membrane Effects. To determine the amount of
3H—TdR penetrating treated cells, AV3 cells on 10 cm Falcon plates were

pretreated at 37° C. with 9.5 m1 MEM containing Cis—Pt(II)(NH3)2C12 for

 

 

 

 

15

6 or 22 hours and then 0.50 ml of 10 uC/ml 3H—TdR was added. After a

2-hour labeling period at 37° C, the culture dishes were chilled on ice,
rinsed 3 X with ice-cold 0.10 mM thymidine in Hank's balanced saline
solution (HBSS) and 2 X in iced HBSS. After the dishes were drained
thoroughly, 5 m1 of ice-cold 0.2 N HC104 was placed onto each. After

15 minutes the HC104 solution was collected and the dishes were rinsed

with an additional 2 ml 0.2 N HC104. The combined HC104 solutions from

each dish were centrifuged 5 minutes at about 1500 X g in a clinical

centrifuge. The O.D.260 of the supernatant was measured and the radioac—

tivity in two 0.5 m1 fractions/plate was determined by liquid scintil-
lation spectrometry using 10 ml of the dioxane fluor described above.
The acid fixed cells were dissolved in 5 ml of 0.5 N KOH by heating

30 minutes at 50° C. Using Worthington calf thymus DNA as a standard,

the concentration of DNA was measured by the method of Ceriotti (1952).

The acid insoluble radioactivity was determined by neutralizing 0.5 ml

 

of the alkaline extract with .25 ml 1N HCl, placing 0.10 on each of 2

2.3 cm filter papers and following the same procedure as described above.

C. Enzyme Assays

1. Preparation of Enzyme Extracts. For all deoxyribonuclease

(DNase) measurements, enzyme extracts of AV3 cells served as the source

of enzymes to be studied. After incubation with a given concentration

of a platinum compound for up to 24 hours, about 107 AV3 cells were
collected by EDTA treatment, centrifuged, washed 2 X with saline D and

resuspended in 0.5 m1 saline D. The extracts were prepared by the quick

freeze—thaw method, using an acetone dry ice slurry repeated 4 times.

Finally the lysate was centrifuged at 20,000 X g for 15 minutes and

 

 

 

 

16

the supernatant transferred to another test tube. This extract was
stored at ~20° C. and diluted before use with saline D.

A different procedure was found to be necessary for preparation
of the enzyme extract for DNA polymerase measurements. After treatment,
approximately 5 X 107 AV3 cells were collected by scraping the plates
with a rubber policeman in saline D. The cells were spun down and the
supernatant decanted. Following a very quick rinse in 2 ml iced 10
mM Tris-HCl, pH 8.1 containing 1 mM MgC12, which caused the cells to
swell to about 2—3 X their initial size, the cell pellet was resuspended
and incubated in the same iced buffer. The hypotonic and osmotic
shock treatment resulted in a very efficient breakage of the cell walls.
The cell nuclei remain intact in this buffered solution for up to 1-
abOut 2 hours and are free of cytoplasmic debris. Hence this is an
excellent way to isolate nuclei. However, for this purpose, the nuclei
and cell debris were spun down before they lysed after about 1 hour
of incubation at 0° C. Although the integrity of the nuclear membrane
is temporarily maintained, the DNA polymerase apparently passes through
it into the supernatant because extracts of these nuclei had no DNA
polymerase activity. The supernatant was centrifuged a second time at

30,000 X g for 1 hour at 4° C. and stored in the refrigerator.

2. Protein Assay. The protein content in all extracts was measured

by the method of Lowry et al. (1951).

3. Acid Soluble DNase (Exonuclease) Assay. The acid soluble DNase
assay used here was adapted from the E. coli exonuclease II assay of
Lehman and Richardson (1964). The assay measures the conversion of

DNA with a 3H-TdR label to trichloracetic acid (TCA) soluble fragments.

 

 

 

 

17

The incubation mixture (0.25 ml) contained 20 umole of sodium acetate
buffer, pH 4.80, lllmole MgClz, 10 ug 3H—DNA (prepared by phenol extrac-
tion from E. coli 15 T' cultured in 411g/m1 3H—TdR) 2 to 20 ug enzyme
extract and water to yield a total volume of 0.25 ml. The reaction

was started by adding the enzyme extract to the rest of the assay mix—
ture (chilled) and mixing by gently swirling the reaction test tube.
After removing a zero time aliquot, the reaction was quickly started

by swirling the assay tube in a 37° water bath. Thereafter at regular
time intervals 25 ul aliquots were removed and placed in iced test tubes
containing 0.2 m1 100 ug/ml heat denatured salmon sperm DNA, followed
by the addition of 0.5 ml iced 5% TCA and vigorous shaking. After at
least 5 minutes additional incubation in ice, each assay aliquot was
centrifuged 5 minutes at 20,000 X g and the supernatant was trans-
ferred to another test tube. Following 4 ethyl ether extractions, 0.60
ml of the aqueous fraction was counted in a polyethylene scintillation

vial with dioxane fluor containing 3 g BBOT plus 150 G naphthalene

per liter.

4. Endonuclease Assay. The nitrocellulose disc method of measur—
ing single stranded DNA as described by Geiduschek and Daniels (1965)
was used for determining endonuclease activity. Although this method
is most sensitive to endonucleases, it might also be sensitive to
induced excision repair enzymes, if they are present in sufficient
amounts or nonspecific. The incubation mixture contained 20 umoles of
sodium acetate buffer at pH 4.8, l umole MgClz, 10 pg native E. coli
3H—DNA (see previous paragraph), 3 to 10 ug enzyme extract, and water

to make a total volume of 0.25 ml. The reaction was initiated in the

same way as described above. At various times thereafter, 25 ul aliquots

 

 

 

 

 

 

18
were removed from the reaction tube and placed into another test tube
containing 1 ml of iced solution of 4 mM NaH2P04, 7 mM NazHPO4, 1 mM
disodium ethylenediaminetetraacetate, pH 7.06, to stop the reaction.
Next the remaining DNA was denatured by heating 6 minutes in boiling
water followed by quick cooling in an ice bath. After further diluting
the mixture with 15 m1 of a 0.5 M KCl, 10 mM tris-HCl, pH 7.5, solution,
and vigorous mixing on a vortex stirrer, the remaining DNA was col—
lected on a nitrocellulose filter (24 mm, Scheicher and Schuell,
Keene, N.H., membrane filter, type B—6) that had been presoaked in
the same tris—KCl solution at least 15 minutes. Then the assay tube
and filter (in a millipore apparatus) were washed with a total of 75
ml of the same solvent and finally by 5 m1 of 100% ethanol. After dry-

ing, the filters were counted in 5 ml of BBOT—Toluene.

5. DNA Polymerase Assay. The DNA polymerase assay was essentially

 

the same as that described for measuring the DNA polymerase from Micro—

 

coccus lysodeikticus as described by Zimmerman (1966). The assay mix-
ture contained 25 umoles Tris—HCl buffer, pH 7.6; 0.5 umoles MgClZ;

0.4 umoles 2—mercaptoethanol; 50 ug heat—denatured calf thymus DNA
(Worthington) as primer; 10 mumoles each of deoxyribonucleotide tri—
phosphate (dCTP, dGTP, dTTP, dATP) including either 14C-dATP (10 mC/
mmole) or 3H-dATP (25 mC/mmole), 30 to 300 ug of enzyme extract, and
water, with or without platinum compounds, to yield a total volume of
0.25 ml. The reaction was initiated by adding the enzyme extract (see
above extraction procedure) to prewarmed assay tubes containing the assay
mixture. Following 1 to 2 hours incubation at 37° C, the label incorporated
into DNA was measured by adding 0.5 ml iced 7% HC104 to stop the reaction.

After incubation 10 minutes in ice, the acid precipitated samples were

 

W 1 FW‘?

19
diluted with 2 m1 of cold 0.0 1M Na4P207 followed by collection of the
DNA on moistened 2.4 cm glass filter paper (Whatman GF/C). After the
reaction tube and filter were washed with 10 m1 of cold 0.7% HC104
in 3 portions, the disc was rinsed with 1 ml of 100% ethanol, dried,

and counted in a glass vial with 5 ml of BBOT—Toluene.

6. DNA Dependent RNA Polymerase from Pseudbmonas putida. Several
attempts were made to establish the conditions necessary to measure
DNA dependent RNA polymerase activity in extracts of AV3 cells, without
success. Since the purified enzyme from Pseudomonas putida was readily
available (due to a generous gift from J. Johnson of the Department of
Microbiology and Public Health at Michigan State University),this puri—
fied bacterial DNA dependent RNA polymerase was used instead. The
assay mixture contained 5 umoles Tris acetate buffer, pH 8.0; 0.5 umole
Mg(CHSCOO)2; 0.125 umole Mn(CH3COO)2; 0.10 umole each of ATP, GTP, UTP,

and 3H—CTP (2500 cpm/mumole); 25 ug heat denatured calf thymus DNA;

 

4 to 17 ug peak 11 RNA polymerase (purified about 1000 X, with about
1000 units activity/m1, 1 unit being defined as the incorporation of
l mumole CTP/mg/hour), and glass distilled water with or without freshly ‘ 1
dissolved platinum compounds to yield a total assay volume of 0.165 ml
(Johnson at al., 1971). The reaction was initiated by adding the

enzyme to prewarmed test tubes with the rest of the assay mixture.

After incubation for 30 minutes at 30° C, the incorporation was terminated
by adding 3 ml of iced 10% TCA, placing the reaction mixture in ice 10
minutes and adding a few drops of carrier DNA (denatured salmon sperm).
Each sample was collected on a nitrocellulose filter,presoaked in water

and prewashed with 10 ml iced 10% TCA immediately before use. Then

 

 

 

 

I’—————7 7 "TT‘I

20
the filter was rinsed with 15 m1 of iced 10% TCA in 3 portions, dried

and counted in a glass vial with 5 ml BBOT—Toluene.

 

 

 

 

III. RESULTS

A. Effects of Cis-Pt(II)(NHq)7C17 on AVQ Cell Growth, Division, and
Survival

The effect of Cis—Pt(II)(NH3)2Cl2 on cell division and survival
was measured simply by counting the number of cells remaining attached
on plates as a function of time after treatment. Figure 2 shows the
effects of a 4 and 24 hour treatment of AV3 cells with 0, l, 5, and
25 uM Cis—Pt(II)(NH3)2C12. Initially all plates had the same number
of cells. After the 4 or 24 hour treatment period, the platinum con—
taining media was decanted, the plates were rinsed twice with Puck's
saline D and 3 ml fresh MEM added to the plates. Each point represents
the average of the number of cells remaining on duplicate plates de—
termined by duplicate hemocytometer counts (2X2 slides) of each plate.
A concentration dependent, duration of treatment dependent, inhibition
of cell division was shown to occur in Figure 2. Furthermore, the
inhibition of cell division persists up to at least 4 days after treat—
ment at 1 or 5 uM without a marked apparent loss of cell survival.

In contrast to this inhibitory effect on cell division, it was
noted that 3 to 4 days after AV3 cells were initially treated, a majority
of the surviving population had grown into giant cells many times the
diameter of untreated cells. Figure 3 shows the treatment duration
dependence of giant cell formation. Only 2 days after AV3 cells were
treated four hours with 5 uM Cis—Pt(II), the cells appeared larger
(Figure 3b) than untreated cells (Figure 3a). By 86 hours after

21

 

 

Cells /plote (mo—6)

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22

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Control

   

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,'

 

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5pM—24hrs

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24 48 96 hrs

Incubation time at 37°C after start of
treatment

0.

Figure 2. Effect of Cis—Pt(II)(NH3)2C12 on cell survival and cell
division in Av. cells.

 

 

 

 

 

 

Figure 3

Giant cell formation in AV3 cells treated with 5 uM Cis—Pt(II)(NH3)2012.

3a: Untreated AV3 cells at t = 98 hrs.

3b: AV3 cells treated 4 hrs at t = 48 hrs; cells slightly
larger than untreated cells.

3c: AV3 cells treated 24 hrs at t = 98 hrs; giant cells
up to 200 uM in size.

3d: AV3 cells treated 4 hrs at t = 84 hrs; cells larger than
in 3b and micronucleated.

3e: AV3 cells treated 24 hrs at t = 98 hrs; many normal size
nuclei in one cell.

3f: AV3 cells treated 4 hrs at t = 98 hrs; possible cross—
septation of giant cells.

Time t is the time after the Cis—Pt(II)(NH3)2C12 containing medium was
initially applied to the cells. All plates initially had the same cell
density. After 4 or 24 hours of treatment with 511M Cis—Pt(II)(NH3)2C12,
the platinum—containing medium was decanted and the plates were rinsed

2 X with Puck's saline D. Then fresh MEM was applied and the plates were
incubated at 37° C. Pictures were taken with an inverted phase Nikon
microscope, and the pictures were taken of live cells in MEM through a
dark green filter. The total magnification is 300 X in all pictures.

 

 

 

 

21(11) (113)2(12.

I15 slightly

: ant cells

1 ls larger than

.ny normal size

.sib le cross-

ing medium was
(1 the same cell

—Pt (11) <NH3>2C121
es were rlnse
1d the Plates were
-d phase Nikon
1 MEM through a
all pictures.

 

 

 

 

 

24

treatment initiation, the cells appeared even larger (Figure 3d).
Cells that were incubated 24 hours of 511M Cis—PT(II) grew up to 200
11M in diameter (Figure BC) on the plates in contrast to an average
lO-2511M in diameter for untreated cells (Figure 3e). Some giant cells
appeared perfectly normal (Figure 3c); some contained from 2 to 6 nor—
mal sized nuclei (Figure 3e); while others had several micronuclei
(Figure 3d). The ability of these giant cells to divide to normal
sized cells was not quantitatively determined, although preliminary
studies indicated that 7 to 10 days after treatment a large fraction
of the surviving pOpulation had returned to normal size. Figure 3f
shows what appears to be cytokinesis occurring in giant cells contain-
ing several normal sized nuclei. Note also that the cells in Figure
3f, at 98 hours postinitiation of treatment, are smaller than the cells
in Figure 3d at 86 hours after a 4—hour treatment. Hence Cis—Pt(II)
(NH3)C12 inhibits AV3 cell division over a long period of time while

not apparently affecting total cell growth.

B. Effects of Cis-Pt(II)(NH3_)__2_Cl_2 on DNA, RNA and Protein Syntheses.

 

 

Figure 4 shows the concentration dependent effect of Cis-Pt(II)
(NH3)2012 on DNA synthesis as measured by the incorporation of 3H—TdR
in human amnion AV3 cells over a 24-hour period. DNA synthesis was
inhibited to a small degree at concentrations as low as 0.50 uM. At
2511M, which is approximately equivalent to a single injection of 8 mg/
kg, the best therapeutic dose in Swiss white mice for treatment of
S-180, assuming a uniform distribution of the compound over the entire
body, there is almost complete inhibition after 6 to 10 hours of treat-
ment. The decrease in total 3H-TdR incorporated between 10 and 24

hours at 125 uM is probably the result of the degradation of previously

 

 

 

 

 

 

 

 

 

25

 

 

 

 

30 -
control
f,“ 27 ‘
Q
" 24 ' 05 M
E ' J”
5"
v 2| -
to ID pM
.E
E l8 ~
E 2.5}JM
I.- I5 -
I
.3"
F l2t‘ 5.0}JM
C3
.5 9 r
E
C) 6 -
3 1, 25.}JM
C)
L)
E 3 t
'25.}JM
0 2 4 6 IO 24hrs
Incubation Time in 3H-Thymidine 8: Cis-—Pt(II)(NH,,)zCl2

Media

Figure 4. Effect of Cis~Pt(II)(NH§)2C12 on DNA synthesis in
AV3 cells as measured by the uptake of H—thymidine. Both the platir
num compound and 3Hmthymidine were applied to cells at time zero.

 

 

 

 

26
synthesized, labeled DNA caused by release of degradative enzymes in
dead or dying cells.

Figures 5 and 6 show the effect of Cis-Pt(II) on RNA and protein
synthesis as measured by the uptake of 3H—uridine (3H—UR) and 3H—leucine
respectively. In contrast to the inhibition of the incorporation of
3H—TdR, there was very little inhibition of 3H—UR and 3H—leucine incor—
poration at 5 uM.

In order to facilitate the comparison of these effects on DNA,

RNA and protein syntheses, the data from charts 4, 5, and 6 were
replotted as the incorporation activity relative to the control, i.e.,
[cpth—cpmtl]exp./[cpmt2—cpmtl]control. From this transformation
Figures 7 and 8 are obtained showing the relative effects of Cis—Pt(II)
on the uptake of isotopically labeled precursors into DNA, RNA and
protein at 25 and 5.0 uM. At 25 MM, Figure 7 shows that all processes ' 1
were completely inhibited after 24 hours; however, DNA synthesis was

inhibited much more quickly. For example, after 6 hours the relative

incorporation rates are 0.17, 0.49 and 0.67 of the control for the uptake

of 3H—TdR, 3H—UR, and 3H—leucine respectively. Figure 8 shows that the

incorporation of 3H-TdR at 5 uM is almost selectively inhibited, there

being little effect on RNA and protein synthesis. The 24—hour point

inFigure 8 for DNA synthesis is higher than the 10~hour point due to

isotopic dilution in the control plates after the first 10 hours of

incubation. In later experiments with 5 uM Cis-Pt(II), in which plates

were pulse labeled for just 2 hours prior to sonication, no such increase

in the relative incorporation OCCurred between the 10th and the 24th

hour of treatment.

 

 

 

 

 

 

 

 

 

 

 

27
I8-
«7‘
1 I6" 0.5}1M
9 control
x
a.
8
up '2 '
.5
IE
5 lo-
I
I
n 8‘
u—
o
.5 6' 25. pM
‘6
5
a 45
L.
o
o
.E 2‘
l|7.pM
() 1 I 1 1 11
0 2 4 IO 24hrs

Incubation Time in Cis-Pt(II)(NH3)ZCI2 8
sH-Uridine Media
Figure 5. Effect of Cis—Pt(II)(NH3)2C12 on RNA syntheei: in AV3

cells as measured by the uptake of 3H—uridine. Both the platinum
compound and 3H—uridine were added at time zero.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

28
'4 ' 0.5}1M
control
I2 - 5.0 MI
".77
Q
X
E5 IC) '
CL
8
C
2 8 r
(3
L
C)
CL
5
o 6 ”
E
G)
.E
:1:
l0
2 —
.1 4 l25.}.IM
l l J l J
o 2 4 6 IO 24hrs
Incubation Time in z’H-Leucine 8: Cis-Pt(II)(NH3)ZCI2
Media

Figure 6. Effect of Cis~Pt(II)(NH3)2012 on protein synthesis in
AV3 cells as measured by uptake of H—leucine. Both the platinum com—
Pound and 3H-leucine were applied at time zero.

 

 

 

 

 

 

 

 

 

29

I20 '

LOO
c .80 '
.9
E _
o
a.
8
o .60 ~ 3
E H—Leucine
o
.2 '
E2
6'3 .40 ~

- 3H-Uridine
.20 *
3 . .
H-Thymldlne
O 2 4 6 IO 24hrs
Incubation Time in labeled precursor 8r
25pM ClS-P1(H)(NH3)2C|2 Media
Figure 7. Relative effect of 25 uM Cis-Pt(II)(NH3)2012 on DNA,

RNA and protein synthesis. The data from figureSZ, 3, and 4 at 5 uM

was transformed using the expression I(cpmtz—cpmtl)exp/(cpmt2_cpmtl)contr31]

where cme and cpmt represent the counting rates for the incorporation
of the laagled precursors at times t2 and t1.

 

 

 

 

 

 

 

 

3O

 

 

 

 

 

 

I.2C)~
IKDC)
3H-Leucine
g; 13C) 3 ..
3: H-Urrdine
o
L-
o
o.
8..
8 o
15 6
o
.2:
E
a:
a: .4C)
3H-Thymidine
.2C)
C) L I 1 l
O 2 4 6 IO 24hrs

Incubation Time in 5.0 pM Cis-Pt(II)(NH )zCl2 Media
Figure 8. Relative effect of 5 uM CiS"Pt(II)(N§§) 012 on DNA, RNA
and protein synthesis. The data from figuresZ, 3, and at 25 uM was

transformed using the eXPression [(Cpmt2"Cpmt1)exe/(Cpmt2_Cpmtl)contr01]

where cpmt and cp-mt represent the counting rates for the incorporation
Of the labeled precuisors at times t2 and t1.

 

 

 

 

 

 

31

C. Comparison of the Effects of Three Effective Tumor-Inhibiting,
Bacterial Filament—Forming Platinum Compounds with Two Ineffective

Platinum Compounds

In this section, the effects of the tumor inhibiting, bacterial
filament-forming platinum compounds on DNA, RNA and protein syntheses are
compared with the effects of 2 non—tumor inhibiting, non-bacterial fila~
ment forming platinum compounds in an attempt to establish effects common
to all platinum, anti—tumor compounds. Table 3 gives the Test/Control
ratios for S-l80 tumor inhibition and the ability of 5 platinum compounds
to induce filaments in E. coli. Clearly, the first 3 compounds, Cis—Pt(II),
Cis—Pt(IV), and Pt(II)en, exhibit some degree of anti—tumor and bacterial
filament forming activity, whereas Trans—Pt(IV) and [Pt(II)(NH3)4]+2
exhibit very little or no effect on tumor growth and bacterial elonga—
tion. Figure 9 shows the effects of these compounds on the incorpora—
tion of 3H—TdR into DNA at 25 uM, expressed relative to untreated cells.
In this and all of the remaining experiments cells were pulse labeled
with the appropriate isotopically labeled precursor for only the last
2 hours of treatment. Thereby the results could be directly compared
at each time period without the problem of isotopic dilution. Cis—Pt(II),

Cis—Pt(IV), Pt(II)en inhibit DNA synthesis at 25 uM almost completely

 

after 10 hours of treatment and in the order of the anti—tumor activity
shown in Table 3. However, 25 uM Trans—Pt(IV) and [Pt(II)(NH3)4]+2
Show very little inhibition of DNA synthesis. In fact, in earlier
studies, no effect of 375 uM [Pt(IV)(NH3)4C12]C12 could be found on
DNA synthesis (Harder and Rosenberg, 1969).

Figures 10 and 11 show the effects of these compounds on incorpora—
tion of 3H—uridine and 3H—leucine into RNA and protein respectively.
Once again, both figures show that Cis-Pt(II), Cis—Pt(IV), and Pt(II)en

at 2511M inhibit RNA and protein synthesis progressively over a 24—hour

 

 

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LC)
.9 '

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.55

33 .8 "

E5

2‘

p. .7 '

I
.2: 6 _ o 25 pM [Pt(II)(NH3)4]Cl2

“5' ' A 25).:M Trans-Pt(IE)(NH3)2CI4
C U 25pM Pt(IIXNH2)2(CH2)2Cl2
.9 .5 ' A 25pM Cis- Pt(II)(NH3)2CI4
L-

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15 .3 r

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3: .2 '

£2

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Incubation Time in Platinum Media
Figure 9. The relative effects of 2511M Cis—Pt(II)(NH3)2Cl?, Cis—Pt(lv)
(bH3)2Cl4, Pt(II)(NH2)2(CH2)2C12, Trans—Pt(IV)(NH3)2C14 and Et<f1)(nn3)4]
C12 on DNA synthesis in AV3 cells measured by the uptake of H-thymidine.
Platinum compounds were applied at time zero and cells were pulse labeled
for only the last 2 hours of treatment at eaCA time period. Results are
expressed relative to the control.

 

 

 

 

 

 

 

34
H ”
A A 25pM [Pt(II)(NH3)4]C|2
'0 ‘FV "
Q “ IOOpm Trans-Pt(11)(NH3)2Cl4
.9 - - 0
g o
312
5 .8-
' 25JJM Pl (H)(NH2)2(CH2)2C|2
”I .7- ° °
0:.—
o
c .6 "
.9
E _
o .5
o.
L
o
o .4 '
E
o
.2 .3-
E
g ‘2 _ 25W CiS-Pi(N)(NH3)ZCI4
.l _
25pM Cis—Pt(II)(NH3)ZC|2
() all,ri 1 1 1
O 2 4 6 l0 24hrs
Incubation Time in Platinum Media
Figure 10. The relative effects of Cis—Pt(Il)(NH3)2C12, Cis—Pt(IV)

 

(NH3)2C14, Pt(II)(NH2)2(CH2)2C12, Trans—Pt(IV)(NH3)2Cl4 and £Pt(ll)(NH3)4]
C12 on RNA synthesis in AV3 cells measured by the uptake of H—uridine.
.latinum compounds were applied at time zero and cells were pulse labeled
for only the last 2 hours of treatment at each time period. Results are
exgrecsed relative to the control.

 

 

 

 

 

 

 

 

    
  
 
  
  
 

 

i:
25 pM Trans—Pt(IY)(NH3)2Cl4

 

 

lOOpM [Pt(Il)(NH3)4]Cl2

25 pM Pt(II)(NH2)2(CH2)2CI2

lOOpM Trans-Pt(ISZ)(NH3)2C|2
.

Relative Incorporation of 3H-Leucine

 

 

 

.4 — 25w Cis-Pt(N)(NH3)ZCI4
.3 ”
.2 t
.| .
25pM Cls- Pt(IIXNH3)ZCl2
O 41 r L 1 1
O 2 4 6 IO 24hrs
Incubation Time in Platinum Media
Figure ll. The relative effects of Cis—Pt(Il)(NH3)2C12, Gig—Pt(iv)
(NH3)2C14, Pt(Il)(NHZ)2(CH2)2CL2, Trans-I’t(IV)(NH3)2Cl4 and [Pt(II)(N1l3)4]
C12 on protein synthesis in AV3 cells measured by the uptake of 3H-leucine.
Platinum compounds were applied at time zero and cells were pulse labeled

 

for only the last 2 hours of treatment at each time period. Results are
expressed relative to the control.

 

 

 

 

 

 

 

 

 

36

period, whereas Trans-Pt(IV) and [Pt(II)(NH3)4]+2 at 2511M do not
inhibit RNA synthesis at all. Higher concentrations of the latter com—
pounds were used to determine if any inhibitory effect could be estab—
lished at a higher concentration. Trans—Pt(IV) at lOOlJM inhibits pro—
tein synthesis to 50% of control.

Comparing the effects of Cis—Pt(II), Cis—Pt(IV), and Pt(II)en at
25 uM in Figures 9, 10 and 11, one will observe that DNA synthesis is
most rapidly inhibited, followed by RNA and protein syntheses, as in
Figures 7 and 8. Therefore, there appears to be a good correlation
between the effectiveness of a platinum compound's oncostatic activity
toward Sarcoma—180 tumor growth (see Table 3) and the inhibitory effect
on DNA, RNA and protein syntheses as measured by the uptake of labeled
thymidine, uridine and leucine. On the other hand, Trans—Pt(IV) and
[Pt(II)(NH3)4]+2, platinum compounds known to be ineffective against
S—180 growth, do not inhibit DNA, RNA and protein syntheses at 25 pM.
Only 100 uM Trans-Pt(IV) shows a significant inhibition of protein syn—

thesis, but not of RNA synthesis.

D. Irreversibility of DNA Sypthesis Inhibition After Platinum Treatment
It is desirable to establish whether or not the effects of the
platinum compounds are reversible in terms of the recovery of DNA syn—
thesis following 4 hours of treatment. As before, the cells were pulse
labeled with 3H—thymidine for only the last 2 hours of treatment prior
to sonication. Figure 12 displays the effects of a 4—hour pretreatment
with from 0.10 to 2511M Cis-Pt(II)(NH3)2C12 on DNA synthesis. There is
no recovery of DNA synthesis over a 24—hour period following a 4—hour
pretreatment with as low as 0.10 uM. Similarly, it is shown in Figure

13 that all 3 tumor inhibiting platinum compounds irreversibly inhibited

 

 

 

 

 

 

 

 

 

 

 

37

 

 

 

 

 

LO
Q)
.E
-- .8"
E. O.IO ,uM
J:
'T l-
SF

.6”
s.—
(D
E " 0.50 pM
S
8
Q .4-
L-
(D
{J
£5 r
‘3
1: ~ 5.0 M
2 .2 )1
d)
m 1—

250nm
O 1 1 1 1 J
-4 O 2 6 IO 24hrs

Incubation Time after Removal of Pt Media
Figure 12. The relative effect of a 4 hour pretreatment of AV3 cells
with Cis—Pt(ll)(NH3)2Cl on DNA synthesis measured by the uptake of 3H~
thymidine. Platinum media was applied at time -4 hours, and decanted at
time zero. After the cells were washed 2 X with Puck's saline D, fresh
MEM was added and cells were pulse labeled for only the last 2 hours of
each period. Results are expressed relative to the control.

 

 

 

 

 

 

 

 

 

38
DNA synthesis when the cells were incubated only 4 hours at 5 pM.

To compare the irreversibility of inhibition of DNA synthesis by
these platinum compounds with a reversible type of inhibition, the
effect of a 4—hour pretreatment with 1 mM hydroxyurea, which is known
to reversibly inhibit DNA synthesis (Kim et al., 1967; Young and Hodas,
1964) is included in Figure 13. The recovery of DNA synthesis from
hydroxyurea treatment starts immediately after the removal of hydroxy—
urea and is complete 6 hours later. DNA synthesis continues to be
inhibited to a greater extent in the platinum pretreated cells even up
to 6 hours after the platinum media is removed. This comparison further
illustrates the irreversibility of the inhibition of DNA synthesis over
a 24—hour period in human amnion AV3 cells pretreated 4 hours with Cis-
Pt(II), Cis—Pt(IV), and Pt(II)en at 5 uM. Since the DNA synthesis
inhibitory effects of these 3 tumor inhibiting platinum compounds are
all irreversible, there can be no correlation of the relative effective—
ness with irreversibility of inhibition; only the extent of inhibition
can be correlated with anti-tumor activity.

E. Effect of Cis—Pt(II)(NHq)2Cl7 on the Uptake of 3H—Thymidine into
The Acid Soluble Pool.

 

Table 4 shows that there was no inhibition of 3H—TdR uptake into
the acid soluble pool after 6 hours of treatment at 25 uM. 0n the con-
trary, there appears to be a small degree of accumulation of 3H—TdR in
the acid soluble pool. At the same time, the average Specific activity
of the DNA measured by the incorporation of thymidine during the last
2 hours of treatment dropped to 40 and 14% of the control for a 6~hour
treatment with 5 and 25 uM Cis-Pt(II) respectively. However, after 24

hours of treatment when the average specific activity was 11 and 3%

 

 

 

 

39

 

 

 

 

 

 

 

H t

.0 f‘\
g .9
'E' 8 _ lmM Hydroxyurea
>~ .
.c
I.—
I .7 t
I
'0
”5 .6 -
c
.9 5 _
E} 4.t
o .
o
E .
g _3 I 511M Gus—Pt(ISZ)(NH3IZCl
E
e '2'

5 pM Cis-Pt(11)(NH3)2Cl2
.l “
O l 1 L g 1
-4 O 2 6 IO 24hrs
Incubation Time after Removal of Experimental Media
Figure 13. The relative effect 4hour pretreatment of AV3 cell:

with 5 uM Cis— —Pt(lI)(NH3)2C12, Cis-Pt(IV)(NH3)2C14, Pt(II)(NBZ)2(CH2)2C12
and 1 mM hydroxyurea on DNA synthesis, measured by the uptake of H—
thymidine. Platinum media was applied at —4 hours and decanted at time
zero. After the cells were washed 2 X with Puck's saline D fresh MEM was
applied and cells were pulse labeled for only the last 2 hours of eac.
time period. Results are expressed relative to the control.

 

 

40

Table 4. he effect of Cis-Pt(II)(NH3)2012 on the uptake of
H—thymidine into the acid soluble pool in AV3 cells

 

 

 

 

 

6 hr treatmenta 24 hr treatmenta
CPM/0D260 CPMb /u g CPM/on260 CPMb /u g
(x10‘3) (x10'3)
in acid soluble of DNA in acid soluble of DNA
extract extract
Control 182 4312 217 4501
Control 184 5021 218 4304
5 pM 227 2025 168 510
5 MM 219 1729 161 487
25 uM 193 658 132 116
25 uM 184 615 121 132

 

 

a3H—thymidine was present during only the last 2 hours of treat—
ment. The data for the 6 and 24 hour treatments are from 2 separate
experiments.

bThe counts per minute (cpm) were based on the amount of DNA
applied to the filters.

T—m‘

41

of the control, the average amount of 3H—thymidine in the acid soluble
pool was reduced to 73 and 58% of the control respectively. Neverthe—
less the amount of this decrease in the uptake of 3H-thymidine into the
cell's acid soluble pool is insufficient to account for the extent of

the decrease in the incorporation of 3H-TdR into DNA.

F. Stability of Cis—Pt(II)(NHq)7017 in MEM

The stability of the DNA synthesis inhibiting effects of Cis—Pt(II)

 

(NH3)2C12 was measured by incubating a 25 uM solution of Cis—Pt(II)
dissolved in MEM 21 hours at 37° C. before applying it to the AV3 cells.
The effects of the day—old Cis—Pt(II) solution were contrasted with the
inhibitory effects of a 25 uM Cis—Pt(II) solution freshly dissolved in
MEM that was also preincubated 21 hours at 37° C. AV3 cells were then
tested every 2 hours up to 6 hours for DNA synthesis by the incorpora-
tion of 3H—TdR during the last 2 hours of treatment in the usual manner.

The results which are given in Table 5 were expressed relative to

 

untreated cells having 21—hour preincubation MEM on them. Although the
21—hour preincubation at 37° C. in MEM reduces the extent of the DNA
synthesis inhibition, the difference in the amount of inhibition is
small enough that the stability of Cis-Pt(II) (or the active species)
in MEM would not be a problem before freshly prepared platinum contain—
ing media is added to the cultures or during early hours of treatment.
G. Comparison of the InhibitoryfiEffects of Cis-Pt(II)(NH3)2912 on

the Incorppration of Labeled Precursors into RNA and DNA

It has now been established that tumor inhibiting platinum compounds
result in the inhibition of cell division but not of growth in vitro
in human amnion AV3 cells. It has also been demonstrated that a

Primary target of these compounds is DNA synthesis and that this

42

Table 5. The effect of a 21 hr pre—incubation of Cis-Pt(II)(NH3)2C12
in MEM on its DNA synthesis-inhibitory activity in AV3 cells
measured by 3H—thymidine incorporation

 

 

 

Duration of AV3 Treatment (hrs)
4

 

Control 1.00 1.00 1.00

25 uM Cis—Pt(II)-MEM

incubated 21 hrs .86 .44 .17 i
Fresh 25 uM
Cis—Pt(II)—MEM .84 .30 .10

 

 

43
inhibitory phenomenon is not caused by a platinum induced limitation
of the uptake of labeled thymidine into the acid soluble pools of the
cells. Therefore, the next problem to be investigated is the mechanism
of inhibition of DNA synthesis. Among other possibilities, three general
ways in which DNA synthesis can be interrupted will be considered: First,
by inhibiting the production of the precursor deoxynucleoside triphos—

phates as with the antimetabolites; second, by reacting with enzymes

 

directly responsible for replication and maintenance of DNA; third, by
reacting directly with the DNA itself, as with the alkylating agents,
such that the DNA can no longer serve as a primer for replication.

One way to determine if a DNA synthesis—inhibiting drug acts as
an antimetabolite is to examine its effects on the incorporation of a .
variety of different labeled precursors into DNA. The results of a , Ni
series of experiments on the incorporation of 6 nucleosides, 3H—thymidine
(3H—Tdr), 3H—cytidine (3H—CR), 3H-deoxycytidine (3H—CdR), 3H—uridine
(3H—UR), 3H—deoxyuridine (3H—UdR), 3H—deoxyadenosine (3H-AdR), and 140-
methyl from l4C—sodium formate into both DNA and RNA following 6 and 24
hours of treatment with 5 and 25 UM Cis—Pt(II)(NH3)2C12 are Shown in
Table 6. These results were obtained by incubating the AV3 cells with
the labeled precursor for only the last 2 hours treatment. One set of
plates labeled with 3H—CR, 3H—CdR, 3H—AdR, and l4C—formate were treated
with RNase to degrade labeled RNA as described under experimental methods.

The difference between the counts incorporated in the RNase treated

 

plates and those not treated with RNase was taken for the amount of
the label incorporated into DNA. The RNase treatment removes all but
4% of the counts from 3H—UR incorporation but leaves 95% of the counts
due to 3H—TdR incorporation.

The data in Table 6 are best summarized in Table 7, in which the

range of the relative inhibitory effects of Cis—Pt(II)(NH3)2C12 on the

 

 

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mumEHOMIUqH Eoum Hzcuoaloqa

as. .manmoamsmsxoos-sm .mansausosxoos-mm .maasnuao-em .mansnusaxoma-sm .msnsaus-sm .msnsaassu-sm

mo coaumuomuoonfi m>fiumawu new so NHUNAmmZVAHHVumeHQ mo wuoommo kuouwnflcdfi who mo domflummaou <

.0 mHQMH

 

 

 

 

45

Table 7. A summary of Table 61 The range of the inhibitory effects
of Cis-Pt(II)(NH3)2C12 on the relative incorporation of
labeled precursors into DNA and RNA compared with
3H—thymidine and 3H—uridine respectively

 

 

 

 

 

DNA1 RNA1
6 hr 24 hr 6 hr 24 hr
3H—TdR 3H-UR
5 M 0 30 0 ll 5 M 0 88 0 87
25 M 0.13 0.00 25 M 0.48 0.01
Other Other
Precursors Precursors
5 M 0.55-0.74 0.55—0.77 5 M 0.88-1.21 0.62-0.88
(0.54)4 (0.48)4
25 M 0.28-0.39 0.11—0.05 25 M 0.44—0.66 0.00—0.02
(0.06)4

 

 

1All results are expressed relative to untreated cells.

ZIncluding 3H—UdR, 3H—CR, 3H—Cdr, 3H—AdR and lac—formate.

3Same as 2 excluding 3H—UdR.

4The figures in parentheses represent values for l4C-formate
which were considered outside of the range of the results achieved with
the nucleosides.

 

 

 

 

 

46
incorporation of the labeled precursors into DNA are compared with
3H—TdR incorporation, and the range of the relative inhibitory effects
on the precursor incorporation into RNA are compared with 3H—UR incor—
poration. There are a number of points to be noted: First, there is
a dosage dependent inhibition of incorporation of all the other labeled
precursors into both DNA and RNA, as with 3H—TdR and 3H—UR. Second,
the nucleoside incorporation into DNA is more efficiently inhibited
than nucleoside incorporation into RNA, as occurs when these measure-
ments are made using 3H—TdR and 3H—UR. However, there are a number of
differences: First, the degree of inhibition of DNA synthesis as
measured by the uptake of precursors other than 3H—TdR is less than
the inhibition of the uptake of 3H—TdR at both 5 and 2511M. Second,
the inhibition of DNA synthesis at 511M appears to have either leveled
off after 6 hours of treatment or to have recovered to the 6 hour rate
On the other hand, the inhibition of 3H—TdR incor—

of incorporation.

poration into DNA progressively increases with time at 5 uM. Third,

 

the extent of the inhibition of the 14C methyl from 14C—formate into
RNA is as great as the inhibition of its incorporation into DNA. Never—
theless, these results generally demonstrate that Cis—Pt(II) inhibits
the incorporation of all tested nucleosides into DNA and RNA, the only
major difference being the greater inhibition of 3H—TdR incorporation
over that of the other precursors.

B. Effect of Unlabeled Deoxynucleosides on the Inhibition of DNA

§ynthesis by Cis—Pt(II)(NH3)2C12
There is another way to determine if the mechanism of action of a

DNA synthesis—inhibiting drug involves the inhibition of the production

of the precursors of DNA synthesis. That is to look for a recovery of

 

 

 

 

 

47
DNA synthesis when a sufficient supply of the necessary precursors is
applied to treated cultures. Accordingly, an experiment was performed
in which, after AV3 cells were preincubated 4 hours with 5 uM Cis—Pt(II)
at 37° then rinsed 2 X with Puck's saline D and MEM containing 10 uM each
of unlabeled deoxyadenosine, deoxycytidine and deoxyguanosine was added
to one set of treated plates while just MEM was added to the other set
of treated plates. Then the effects of the Cis—Pt(II) pretreatment
were measured by the incorporation of 3H—TdR during the last 2 hours
of treatment up to 6 hours after the removal of Cis—Pt(II) and the
addition of the deoxynucleosides. The results in Table 8 provide good
evidence that the addition of the deoxynucleosides even after the plati—
num is removed does not reverse the inhibition of DNA synthesis. There—
fore, it may be concluded that the Cis—Pt(II) induced inhibition of
DNA synthesis is not brought about by limiting deoxyribonucleoside

production at 511M.

 

I. Effects of Platinum Compounds on the Acid Soluble DNase and Endo-
nuclease Activities in Cell Free Preparations of AVq Cells

The previous results strongly indicate that the mechanism of the
platinum compounds, and specifically Cis—Pt(II)(NH3)2C12, does not pri—
marily involve the inhibition of deoxyribonucleoside production. There—
fore, the following experiments were performed to examine the possible
effects on those enzymes which use DNA as a primer or substrate. First,
the effects of the platinum compounds on the acid soluble deoxyribonu—
clease (DNase) and endonuclease activity were studied. AV3 cells were
incubated for a given period of time in MEM containing a given platinum
compound at a certain concentration. Then cell free extracts of both

treated and untreated cells were prepared as described under Methods.

 

 

 

 

 

48

Table 8. Effect of the presence of unlabeled deoxynucleosides on the
relative inhibition of DNA synthesis by Cis—Pt(II)(NH3)2012
by 3H—TdR incorporation

 

 

Time After Removal of 5 uM Cis—Pt(II)—MEMa

 

 

2 hrs 4 hrs 6 hrs
Control 1.00 1.00 1.00 1.00
4 hr pretreatedb 0.59 0.30 0.16 0.11
4 hr pretreated and
deoxynucleosidesC 0.59 0.31 0.22 0.16

 

aAV3 cells were preincubated 4 hours at 37° C. in 5 uM Cis—Pt(II)
(NH3)2012, then rinsed twice with Puck's saline D and fresh MEM added.
To half of the plates, MEM having 10 uM e ch of deoxyadenosine, deoxy—
guanosine, and deoxycytidine was added. -thymidine was present during
the 2 hours of incubation.

bRelative to untreated cells without deoxynucleosides.

CRelative to untreated cells with deoxynucleosides.

 

 

 

——

49

1. Characteristics of the DNase Activity. The optimum assay con—

 

ditions for the acid soluble DNase activity in AV3 cell extracts were
first determined. Table 9 shows that with an extract from untreated
cells, pH 4.80 (acetate buffer) was the optimum pH for releasing acid
soluble deoxyribonucleotides. A pH 9.00 assay (Tris buffer) also showed
a small amount of activity, whereas essentially no activity was observed
in a pH 7.54 phosphate buffer. The table also shows that a native
labeled DNA substrate was preferred over a heat denatured labeled DNA

by a factor of 3 to 4. Also included in Table 9 are the results of a
parallel study using extracts of AV3 cells which had been treated with
50 and 50011M Cis—Pt(IV) for 24 and 12 hours respectively; the data
show that the enzyme extract from treated cells was also most active

at pH 4.80 with a native DNA substrate. However, at pH 9.00, denatured
DNA was the preferred substrate. A similar study demonstrated that

the endonuclease activity in AV3 cell extracts was also most active
with a native DNA substrate at pH 4.80.

No additional efforts were attempted to further characterize the
acid soluble DNase and endonuclease activities, as, for example, to
determine the nature of the products by chromatographic separation or
to determine the molecular weight of the products of the endonuclease
activity. Nevertheless, the general kinetics of the activity, the pH
optimum and high preference for native DNA parallel the characteristics
of mammalian DNase II, a spleen endonuclease which can yield mono—

nucleotides (Sugar and Sierakowski, 1967).

 

 

50

Table 9. The effect of pH and the state of the DNA substrate on the
acid soluble DNase activity in CPM in extracts of AV3 cells
grown in Cis—Pt(IV)(NH3)2Cl4 using a 2-hour assay

 

 

 

 

pH = 4.80 pH = 7.54 pH = 9.00

Extract Native Denatured Native Denatured Native Denatured
(3.1 ug/assay) DNA DNA DNA DNA DNA DNA
Control 5,465 1,460 88 107 179 196
50 uM
Cis—Pt(IV)
24 hr
treatment 7,262 1,867 34 66 142 662
500 uM
Cis—Pt(IV)
12 hr

treatment 12,009 3,795 96 50 172 1,202

 

 

 

/—"‘"T’ _ ”——---—
. ._ _ ._-.- -——-—-

 

 

 

 

—' T

51
2. Acid Soluble DNase and Endonuclease Activity in Extracts of
923 Cells Treated with Cis—Pt(II)(NHq)9C19, Cis—Pt(IV)(NH3)2§14
and Pt(II)(NH7)9(CH7)jCl) During Growth.

The acid—soluble DNase activity was measured in extracts of AV3
cells treated with 50 and 500 pM Cis—Pt(IV)(NH3)2Cl4 for 24 and 2 hours
respectively as a function of assay incubation time at 37° C. 18.6 ug
protein—enzyme extract was added to each reaction tube to start the
assay. The results, shown in Figure 14, demonstrate a marked concen—
tration—dependent enhancement of the DNase activity. The acid soluble
counts released into the assay medium by the extract from 500 uM treated
cells was higher than that by the control extract by a factor of approxi-
mately 3 after 120 minutes of incubation. Similarly when the same
extracts were used for measuring endonuclease activity, using only 3.1
pg enzyme extract per reaction tube, an enhancement of the endonuclease
activity was also observed (Figure 15). In this assay, activity is
measured by the rate of loss of acid precipitable 3H—DNA. The maximum

enhancement was a factor of 2 with the extract from the 500 uM treated

AV3 cells. However, this enhancement was limited to the early minutes

 

of the reaction, for after 60 minutes of incubation, the extent of the
enhanced endonuclease activity was greatly reduced. Perhaps this
apparent rapid decline in the endonuclease activitywas due to substrate
limitation.

The results of similar studies using the enzyme extract from AV3
cells treated 5 hours with O, 50, 150, and 500 uM Cis—Pt(II)(NH3)2C12
(511g protein reaction tube) are shown in Figures 16 and 17. Clearly

Cis—Pt(II)(NH 012 does not induce an enhancement of either acid soluble

3)2
DNase or endonuclease activity. On the other hand, in a parallel experi—

ment using extracts of AV3 cells treated 6 hours with O, 50, 150 and

500 uM Pt(II)(NH2)2(CH2)2C12 (3.35 pg protein per assay tube) a dosage

(CPM)

Acid Soluble 3H label released

 

52

 

11

 

Control

  

 

50 11M Ci3"Pi(l\/)(NH3)ZCI4
(24 hr.)

    

./
/ 500 yM gIs-PHM(NH3)ZCI4
(2 hr.)

    
  

 

 

l I I l
15 3O 60

9a 120
Incubation Time (MIN)

Figure 14. Acid soluzle DNase activity in CPM in extracts of
AV3 cells treated with Cis—?t(IV)(NE3)2Cl4- 18.6 Mg protein/assay.

 

 

 

 

53

 

5‘

  
   

5
T

 

DNA retained (CPM x IO'S)

   
 

  
 
   
 

 

 

 

8 _
Control
4 _ 50 )1M
CI:-Pt(lV)(NH3)2C
(24hr)
500 JIM CiS‘PNlVXNH ) CI
~ (2 hr.) 3 2 4
I I I I

Incubation Time (MIN)

Figure 15.1:1sdonuclea e activity in CPM in extracts of AV3
c 115 treated with Cis—-t(IV)(.' H3)2C14. 3. 1 ug protein/assay.

 

54

 

50004

etl

4000-

  
 

30001 Po Control
A 5011M Cis-PtlIDiNH3)2Cl2
[3 "5C, H II ll 11 u u
.lfHDCMl u ll ll ll n

 

2000~

Acid soluble 3H-Iabel released (cpm)

O

O

O
l

a
/

 

 

 

o . r . 4 a
0 I5 30 60 90 I20 min
Incubation time at 37°C

Figure 16. Acid soluble DNase activity in CPM in extracts of

AV3 cells treated 5 hours with Cis—Pt(ll)(NH3)2012. 5 ug protein/
assay.

 

 

 

 

 

 

6000

5000‘

55

 

C)

 

4000

DNA retalned (cpm)

 

3000‘

2CKK¥

1000 l

 

O

0 Control

A 50}JM CiS'Pl(H)(NH3)2Cl2
[3 |5()Il 11 ll ll 0 u

‘. ESCND 11 ll ll n ll

kn
O\9m§
A o

 

 

0

Figure 17.

T1

30

Endonuclease activity in CPM in extracts of AV3
treated 5 hours with Cis—Pt(lI)(NH3)2012. 5 pg protein/assay.

éo $0 Tao min
Incubation time at 37°C

 

 

Cgllfi

 

56
dependent enhancement of acid soluble DNase was measured (Figure 18).

However, no enhancement of endonuclease activity was observed as with

extracts from cells treated as with Cis—Pt(II)(NH3)2C12.

3. Effects of the Presence of Cis-Pt(II)(NH3)_§1n, Cis-Pt(IV)
(NH322C1A or Pt(II)(NH222(CH2)2C12 in the gcid Soluble DNase

and Endonuclease Assays Using Enzyme Extracts from Untreated
Cells

The previous experiments established a dosage dependent enhance—

ment of nuclease activities in extracts of cells that were treated

during growth with Cis—Pt(IV). This raises the question as to whether

this enhancement is caused by a direct p1atinum.induced activation of
the nucleases or due to a platinum induced activation of nuclease pro—

duction such that a higher concentration of such enzymes would be pres-

ent. The following experiments were performed to test the former pos—

sibility. The three tumor inhibiting platinum compounds Cis-Pt(II),
Cis—Pt(IV) and Pt(II)en were added directly to the assay mixtures before
adding the enzyme extract from untreated AV3 cells. Figure 19 shows

a rather steep, concentration—dependent inhibition of acid soluble

DNase activity. Pt(II)en was the most effective inhibitor followed by

Cis—Pt(IV) and then Cis-Pt(II). Similarly towards endonuclease activity,

Pt(II)en was the most potent inhibitor (Figure 20) followed by Cis-Pt(II);

however, Cis-Pt(IV) showed almost no effect on the endonuclease activity

from untreated cells. These 2 experiments provide sufficient evidence

to conclude that the platinum compounds do not directly activate the
nuclease activities by an allosteric interaction with these enzymes

because the platinum compounds inhibit their activity.

 

 

 

 

5000

 

4500 <

4000-

C3

“35004 0

nn

3000‘

a a
C) (D
C? 9

(71
o
0

Acid soluble3H label released (cp

 

1000- A
/o 0 Control
/ A 5011M PI(1D(NH2)2(CH2)20IZ

500‘ £1500 ll n ll n

lice/o ol50 .. .. .. ..

 

 

 

O ' ' ' f r
0 I5 30 60 9O |20 I80
Incubation time at 37°C

 

Figure 18. Acid soluble DNase activity in CPM in extracts of

AV3 cells treated 6 hours with Pt(II)(NH2)2(CH2)2C12. 3.35 ug
protein/assay.

 

 

 

 

released

3H label

Acid soluble

4000 1

3000 ‘

2000 ‘

|000 ‘

0

Figure 19.

58

 

   

 

 

 

 

 

o
\.
o o
o
Cis-Pt(]1)(NH3)2 Clz
o o_o
U \0/0

Cis-PI(IY)(NH3)2C|2

o\l>l(11)(NH2)2(Cllzlzcl2
C]

 

0

30 lio 150 240 250 360 11M
Concentration ocfl Platinum
compoun

Effect of Cis~Pt(11)(NH3) 012, Cis—Pt(1v)(Nn3)zc14

and Pt(II)(NH2)2(CH2)2012 on the acid solu 1e DNase activity in CPM
in extracts from untreated AV3 cells. 2.5 ug protein/assay; 2 hours
incubation at 37° C.

 

 

 

 

 

 

59

 

l0,000 -
9000 ‘ U PI(ID(NH2)2(CH2)2C|2

8000 -

)

V
o
o
o

O C IS -Pt(II)(NH3)2CI2

(
m
o
o
o

f
/
.://:

w..i§~.

39
80

DNA retained

<

2000‘

I000 —

 

 

 

O I T ‘f I V

0 50 I00 I50 200 250 llM

Concentratlon of PIatInum
compound

Figure 20. Effect of Cis—Pt(11)(NH3)2012, Cis—Pt(IV)(NH3)2014 and
Dt(+I)(NH2)2(CH2)2C12 on the endonuclease activity in CPM in extracts
Of untreated AV3 cells. 2. 5 ug protein/assay; 2 hours incubation at
37°C; 19,100 CPM retained in assay lacking enzyme.

 

 

60

J. DNA Polymerase Studies

 

1. Epzyme Characterization. The DNA polymerase obtained from
extracts of AV3 cells prepared as described under Methods was character—
ized as summarized in Table 10. First it was noted that the DNA
polymerase had considerable preference for a heat denatured DNA primer
over a native one. It was also shown that a phosphate buffer of the
same concentration and pH greatly inhibited the activity. To demon—
strate that the incorporated or that the measured radioactivity was
being incorporated into DNA, various controls were performed. For
example, it was shown that enzymatic activity was required for the
incorporation of 3H—dATP because when the incubation was done at 0° C,
essentially no counts were incorporated. Likewise, the DNA primer
was an absolute requirement, as was the presence of both pyrimidine and
purine deoxynucleotide triphosphates for maximum incorporation. The
dependence of the activity on the protein content, or on the amount of
enzyme extract added to the assay, was found to be linear up to more
than 300 pg. Finally, the DNA polymerase activity was found to be

linear with time up to and including 2 hours of incubation at 37° C.

 

Therefore, assay incubation periods of 1 to 2 hours were normally
employed to maximize the incorporation. Hence even though this DNA
polymerase was only in the form of a crude extract, it behaved quite

as well as a DNA polymerase preparation for these studies.

2. DNA Polymerase in Extracts of AV3 Cells Treated with Tumor

Inhibiting Platinum Compounds.

In extracts of AV3 cells preincubated 6 hours at 37° C. in MEM

containing Cis—Pt(IV) and Pt(II)en the DNA polymerase activity as a

function of protein content per 1 hour assay is shown in Figure 21.

 

Table 10. Characteristics of DNA polymerase in AV3 cell extracts

 

 

3H—dATP incorporated
into DNA (CPM)

 

1. Complete assay plus 62.5 pg enzyme extract,

incubated 2 hours at 37° C. 2745
2. Complete but incubation 2 hours at 0° C. 127
3. Complete, minus DNA, 2 hours at 37° C. 147
4. Complete, minus dCTP 2431
5. Complete, minus dCTP and dTTP 917
6. Complete, minus dCTP, dTTP, and dGTP 389

7. Complete, but native instead of heat denatured DNA 1584

8. Complete, but phosphate buffer instead of tris-
HCl, same pH 154

 

 

 

 

 

62

 

 

4000-

' 3000..

(cts/mlnl

 

__ 50C1|H
2000 018 g'w

 

l000 B

DNA Polymerase Activity

 

 

1 1
0 I00 200 300 J19

Protoln Content

 

Figure 21. DNA polymerase activity in CPM in extracts of AV
cells treated 6 hours with Cis—Pt(IV)(NH3)2C14 and Pt(II)(NH2)2(CH2)2C12.

 

 

 

63

Except for the 50 pM Pt(II)en extract, there was a concentration—
dependent enhancement of the DNA polymerase, which was especially
large in the extract prepared from 500 pM Cis—Pt(IV) treated cells.

In addition, this figure shows that the effect is greater in Cis—Pt(IV)
than in Pt(II)en extracts. In a parallel study using extracts prepared
from AV3 cells preincubated 5 hours with Cis—Pt(II), the DNA polymerase
activity was also greater (Figure 22). By comparing Figures 21 and

22, one will see that the enhancement by Cis—Pt(II) was about the same
as that by Pt(II)en.

3. Effect of the Addition of Platinum Compounds to the DNA
Polymerase Assay Using Untreated AV3 Cell Extract.
Once again this platinum enhanced DNA polymerase activity might

be considered to arise by a direct activation of the enzyme or by the
stimulation of a higher production rate of DNA polymerase. Accordingly,
experiments were performed in which platinum compounds were added
directly to the assay mixture to which an untreated enzyme extract was
added. The results in Figure 23 show that there was a concentration
dependent inhibition of the DNA polymerase activity by Pt(II)en,
Cis-Pt(IV) and Cis—Pt(ll) in that order. Therefore, platinum compounds
cannot directly activate the DNA polymerase activity by binding to the
polymerase molecules. However, absolutely no inhibition of the DNA
polymerase activity was observed by [Pt(II)(NH3)4]C12 up to 250 pM,

the highest concentration tested. This latter result is consistent

with the lack of inhibition of DNA synthesis by [Pt(II)(NH3)4]C12. The
possible implications of this finding may be of great importance, as
discussed later. On the other hand, the relative ordering of the inhi-

bition of DNA polymerase by the 3 active compounds is opposite to the

 

 

 

 

6000f

5 000

4:.
CD
C)
Q

ol
0
o
9

C]

DNA Polymerase activity (cpm)

h)
o
0
Q

 

 

IOOO- 0 Control
0 50pM Cis- Pt(II)(NH3)zC|2
D 500" II II II

01

 

40 80 I20 léo 200 240 280119
Protein content

Figure 22. DNA polymerase activity in CPM in extracts of AV3 cells
treated 5 hours with Cis—Pt(II)(NH3)2C12.

   

 

 

 

 

 

 

4000 .—
‘~f . :
«ll
ll-
3000— \A .\
O\A o
cts/mln o \
A O
z \.
E 2000 —
o
< A
o \
(0
o
8 A
E o
_>.‘ \
< ' Ci: Pt"lNH3)2CI2
0 I l _L(. 4 l
0 30 60 I2 ISO 240 ,uM
Concentration of Pt in assay
Figure 23. Effect of Cis—Pt(II)(NH3)2C12, Cis—Pt(IV)(NH )

Cl

2 ’
Pt(II)(NH2)2(CH2)2012 and [Pt(II)(NH3)4]C12 on DNA polymerase actiéity

in CPM in extracts of untreated AV3 cells.

 

62.5 pg protein/assay.

 

 

 

66

ordering of the relative effectiveness of both the inhibition of DNA

synthesis and S—180 tumor growth.

K. Effect of Cis—Pt(II)(NHq)9C12 on Purified DNA Dependent RNA
Polymerase from Pseudbmonas putidh

 

A similar set of experiments dealing with the effects of platinum
compounds on DNA dependent RNA polymerase from AV3 cells had been

planned. However, when I was unable to establish the proper assay con-

 

ditions and extraction technique, I decided to try to use the purified
DNA dependent RNA polymerase instead. Thus when Cis—Pt(II) was added
to the assay mixture in the presence of native DNA and the assay mixture
was allowed to incubate 30 minutes at 30° C, the results in Figure 24

were obtained. Absolutely no inhibition of the RNA polymerase activity

 

occurred up to 160 pM, the highest concentration tested. Hence,
Cis-Pt(Il) has no effect on a purified bacterial DNA dependent RNA

polymerase, whereas it inhibits both AV3 DNA synthesis and DNA polymer—

 

ase when added to extracts of AV3 cells.

 

 

67

 

 

 

 

3: 8 T

E

3

<1

3

to 4th-

3

e

>~

'5

O.

‘2‘ o 1_ 1 1 1
'1'- 0 4o 80 I20 l60 11M

0 Is PtulN H3)2Cl2 Concentratlon

Figure 24. Effect of Cis-Pt(II)(NH3)2C12 on the purified DNA
dependent PM polymerase from Psaudomonas putida.

 

 

 

 

 

IV. DISCUSSION

A. Platinum Induced Inhibition of Cell Division

Cis-Pt(II)(NH3)2C12 induced inhibition of cell division was demon—
strated to occur in Human Amnion AV3 cells, as shown in Figure 2. The
finding that Cis-Pt(II) also induces giant cell formation 3 to 4 days
after treatment (Figure 3), in conjunction with the observed inhibition
of cell division, closely parallels its effects in E. coli B, i.e.,
cell division inhibition leading to filament formation (Rosenberg at
al., 1967a). Following exposure to Cis-Pt(II) or other tumor inhi-
biting platinum compounds, cytokinesis was observed to occur uniformly
throughout the filamentous E. coli B after removal of the Cis—Pt(II).
The resulting daughter cells appeared to be normal in every reapect.
On the other hand, it is not yet known whether giant AV3 cells regain
the ability to complete mitotic division successfully and give rise to
competent progeny cells. However, preliminary observations indicate
that at least a given fraction of giant cells begin to divide 4 to 10
days after an initial 4 hour, 511M Cis-Pt(II) treatment.

Other anti—neoplastic drugs also induce giant mammalian cells.
These include the nitrogen mustards, as HN2 (Wheeler, 1962; Levis at
al., 1963) and Mitomycin C (Shatkin at al., 1962). Perhaps the most
important conclusion which may be inferred from giant cell formation is
that RNA and protein synthesis is not being inhibited. This conclusion

must be qualified somewhat to state that at least those proteins and

their respective messenger RNA which are required for cell growth are

68

 

___
I
u

 

 

 

69

not being inhibited a few days after treatment. Hence the above does

not necessarily apply to those proteins employed for mitosis.

B. Inhibition of DNA Synthesis: A Primary Target of Tumor Inhibit-
ing Platinum Compounds

The results shown in Figure 4 clearly demonstrate a dosage-
dependent inhibition of DNA synthesis in AV3 cells. Furthermore, Figure
9 shows that all 3 tested platinum anti—neOplastic compounds, Cis~Pt(II),
Cis-Pt(lV) and Pt(II)en inhibit DNA synthesis in these cells, whereas
Trans—Pt(IV)(NH3)2C14 and [Pt(II)(NH3)4]C12, which have no anti—
neoplastic activity against S-180 growth, did not inhibit DNA synthesis
at 2511M. In 1969, Harder and Rosenberg reported that Pt(II)(NH3)4Cl2
had no effect on DNA synthesis up to 375 pM.

DNA synthesis was examined extensively by measuring the inhibition
of 3H—TdR incorporation into a TCA insoluble product. Table 7 shows
that Cis-Pt(II) also inhibits the incorporation of the nucleosides,
3H—UdR, 3H—CdR, 3H—CR, 3H-AdR as well as the 14c—methy1 from 14C—formate
into DNA.

The possibility that this inhibitory effect was caused by a plati—
num induced reduction of the permeability of the cell membrane to the
nucleosides was eliminated by the findings that there was essentially
no effect at 511M on the uptake of 3H-TdR into the acid soluble pool.
Although there was a measurable reduction in the uptake of 3H—TdR into
the cell pool by a 24—hour treatment with 2511M Cis—Pt(II), the extent
of this reduction was far too small to account for the degree to which
its incorporation into DNA was reduced. The relative effect of Cis—Pt(II)
on the specific activity of 3H-TdR incorporation in this same experiment

agrees very well with the results on the inhibition of DNA synthesis

obtained in the usual manner. The validity of measuring DNA synthesis

 

 

 

 

 

 

70

in the way most frequently employed was therefore substantiated.

Figures 5 and 6 show the Cis—Pt(II) concentration-dependent
inhibition of RNA and protein syntheses as measured by the incorpora—
tion of 3H—UR and 3H-L-leucine respectively. However, it was shown
that at 5 pM, and lower concentrations, DNA synthesis was selectively
inhibited (Figure 8). Further, DNA synthesis was inhibited more
rapidly than RNA synthesis, which in turn was inhibited more rapidly
than protein synthesis at the higher concentration of 25 pM (Figure‘
7). The same effects on RNA and protein synthesis in relation to DNA
synthesis occurred in AV3 cells treated with Cis-Pt(IV) and Pt(II)en
(Figures 10 and 11). Similarly 25 pM Cis-Pt(II) inhibited the incor—
poration of the nucleosides 3H-CR, 3H-CdR, 3H-AdR into RNA but not as
rapidly as the same nucleosides were inhibited from incorporation into
DNA (Table 7). With all precursors except 14C-formate and 3H—TdR the
extent of inhibition of incorporation into DNA and RNA was very similar.
Thus all of these studies on the inhibitory effects of platinum com—
pounds toward the incorporation of nucleosides into DNA and RNA pro—
vide evidence in support of the hypothesis that DNA synthesis is the
primary target of the tumor—inhibiting platinum compounds.

In further support of this hypothesis are the results of Howle and
Gale (1970), who found that DNA synthesis was selectively and persist—
ently inhibited in Ehrlich Ascites tumor cells removed periodically
from rats up to 4 days after treatment with a single injection of 10
mg/kg of Cis-Pt(II). RNA and protein synthesis were also inhibited
initially, but these processes recovered after 4 days. When Ehrlich

ascites tumor cells were treated in vitro with high concentrations of

 

 

 

 

 

71

Cis-Pt(II), (lOOllM), DNA, RNA and protein syntheses were inhibited

in that order, in agreement with the results reported here on AV3 cells.
Unfortunately, the effects of Cis-Pt(II) at lower concentrations were
not reported so that a more thorough comparison of the results with the
Ehrlich ascites cells with the effects in human amnion cells is not
possible.

Additional support for the above conclusion is given by the find-
ing that sufficient platinum is deposited or sequestered in S—180 tumors
in mice to inhibit DNA synthesis in AV3 cells. When mice having well
developed S-l80 tumors were given a single injection of 8 mg/kg
Cis-Pt(II), the optimum therapeutic dose, a maximum of about 1.511g/mg
of platinum was found in the tumor within 24 hours of the injection
(Toth—Allen, 1970). This concentration is roughly equivalent to 511M,
assuming a gram of tissue is equivalent to a gram of water. Actually
because only about 70% of the body is water (Hirshaut, at al., 1969),
the above figure is obviously an underestimate, but it will serve as
a conservative estimate. Since at concentrations of 511M and below,
DNA synthesis was selectively inhibited in AV3 cells, one may infer
from this that the mechanism of action of these platinum compounds may ulti—

mately involve the inhibition of DNA synthesis in vivo as well as

in vitro.

C. Possible Evidence for a Metabolically Transformed, Effective
Platinum Moiety or an Irreversible Reaction of Platinum with the

Target Molecules

 

In contrast to hydroxyurea, which is also a tumor—inhibitor and
induces filaments in E. coli, platinum compounds inhibit DNA synthesis
at a relatively slow rate in the therapeutic dose range. For example,

at a'l mM concentration of hydroxyurea, DNA synthesis in HeLa S—3 cells

 

 

 

 

72
is reduced to about 10% of the control in 2 hours (Kim et al., 1967;
Pfeiffer and Tolmack, 1967; Young at al., 1967), whereas in our studies
DNA synthesis is still about 82% of the control in human amnion AV3
cells treated 2 hours with 25 pM Cis-Pt(II). Further,hydroxyurea does
not inhibit RNA or protein synthesis in vitro at 1 mM as do the platinum
compounds at 2511M.

One possible explanation for the slow rate of inhibition by p1ati-
num compounds is that the applied platinum compound is itself inactive
and must be converted intracellularly to an active species, by means
of a one or a many step reaction. Howle and Gale, 1970, suggest a 2
stage transformation, the first yielding a product inhibitory in general
to DNA, RNA and protein syntheses, and the second inhibiting only DNA
synthesis. The results given here support a different interpretation
of this posulate: Ptd+Pti1Pt2. Here PtO represents the initial platinum
Species and Ptl is a slowly increasing metabolically changed platinum
species to which DNA synthesis is more sensitive than RNA and protein
synthesis. Another possibility is that Ptl inhibits only DNA synthe—
sis and a second cellularly transformed platinum Species, Ptz, is
responsible for RNA and protein synthesis inhibition. The latter sug—
gestion would account for the slow rate of DNA synthesis inhibition,
assuming a slow rate of Pt1 formation. Furthermore a low equilibrium
constant for Ptz production would account for the selective DNA synthe—
sis inhibition at low concentrations and the slower rate of RNA and pro—
tein synthesis inhibition at higher concentrations. This proposal also
accounts for the results of Howle and Gale (1970). Incidentally,
Mitomycin C (Szybalski and Iyer, 1964) and cyclophosphamide (Bosen and
Davis, 1969) are 2 examples of other tumor-inhibitors that must be

enzymatically activated.

 

 

 

73
However, a far better explanation of the sequential inhibition of

_DNA, RNA and protein synthesis is that the active platinum Species
binds directly to DNA such that at low concentrations, only DNA synthe-
sis is affected. Then at high concentrations, a higher frequency of
platinum induced lesions would lead to a measurable inhibition of
messenger RNA production which in turn would eventually result in a
reduction of protein synthesis. Such a mechanism would lead to irre—
versible inhibitory effects unless and until such damage would be
repaired. Figures 12 and 13 show that tumor inhibiting platinum com—
pounds irreversibly inhibit DNA synthesis.

Cross-linking DNA is, in fact, the mechanism proposed for the
bifunctional alkylating agents (Wheeler, 1962). Bifunctional alkylating
agents in general, including HN2, have been shown to inhibit DNA synthe-
sis without much effect on RNA and protein synthesis in both E. coli
and mammalian cells in vivo and in vitro (Wheeler, 1962; Harold and
Ziporin, 1959; Brewer at aZ., 1961; Lawey and Brooks, 1965; Smith and
Busch, 1964). Similarly, Mitomycin C selectively and irreversibly
cross—links and inhibits DNA synthesis without concomitant effects on
RNA or protein synthesis in mammalian and bacterial cells (Magee and
Miller, 1962; Shiba at al., 1959; for an excellent review, see
Szybalski and Iyer, 1964). Therefore, the effects of the tumor inhibit—
ing platinum compounds on DNA, RNA, and protein syntheses in mammalian
cells very closely mimic the effect observed with bifunctional alky—
lating agents!

A third possible explanation for the slow rate of inhibition is
that the diffusion or active uptake of platinum compounds is the rate
limiting step. Included in this category is a possible time dependent

interaction of a platinum compound with the lipids of the cell membrane

 

 

 

 

 

74

which may render it more or less permeable to either the drug or labeled
precursor. The absence of inhibition of uptake of 3H-TdR into the acid
soluble pools after 6 hours of treatment with Cis—Pt(II) provides con—
clusive evidence against the possibility that a membrane effect is
responsible for the observed inhibition of DNA synthesis initially.
However, the dose dependent decrease in uptake after 24 hours indicates
that there may be a very slow membrane effect which plays a role in
long duration measurements of DNA synthesis inhibition. Thus the irre—
versible inhibition of DNA synthesis by Cis-Pt(II), Cis—Pt(IV) and
Pt(II)en might be explained by the gradual limitation of the passage
of 3H-TdR into cells and a slow diffusion of the original or cellularly

transformed platinum compounds out of the cell, such that 2 rinses would

not efficiently remove them.

D. Are Tumor Inhibiting Platinum Compounds Antimetabolites?

Now that the inhibition of DNA synthesis has been shown to be a
primary target of tumor inhibiting platinum compounds, the next question
to which this thesis is addressed is whether or not the mechanism of
this DNA synthesis inhibition is by the inhibition of the production
of the precursors of DNA synthesis. This possibility was investigated
by measuring the inhibitory effects of the platinum compounds towards
the incorporation of various nucleosides and the l4C-methyl from
14C—formate into DNA and RNA. The results are given in Table 6 and
summarized in Table 7 for the effects of Cis-Pt(II).

Interference with enzymes catalyzing the reduction of ribonucleo-
sides to deoxyribonucleosides appears to be excluded because the incor-
poration into DNA of labeled 3H—deoxycytidine and 3H—cytidine were

inhibited to almost the same degree. Further evidence that ribonucleoside

%

 

 

 

 

75
diphOSphate reductase is not inhibited was provided by the finding
that the addition of sufficient unlabeled deoxynucleosides did not
reverse the inhibition of DNA synthesis (Table 8). In contrast to this,
the addition of deoxyribonucleoside to HeLa cells being treated with
hydroxyurea, which is known to inhibit this enzyme, results in the par—
tial protection from the inhibition of 3H—TdR incorporation into DNA
(Young at al., 1967).

By a similar argument, the interference with the one carbon trans-
fer in the pathway from deoxyuridilic acid to thymidylic acid seems
unlikely because the incorporation into DNA of the labeled 3H—UdR was
inhibited to no greater extent than the labeled 3H-TdR incorporation.
The same conclusion is supported by the absence of a greater degree of
inhibition 0f lAC—methyl from l4C-formate into DNA.

However, the finding that 3H—TdR incorporation into DNA is most
effectively inhibited compared to the other tested deoxyribonucleosides
is evidence that the thymidine kinases may be inhibited. Likewise the
finding that the incorporation of the label from 14C-formate into RNA
is inhibited to a far greater extent than the inhibition of the incor-
poration of the label from the other precursors (Table 7) is indicative
of a platinum—induced inhibition of da novo ribonucleoside synthesis.
It is most interesting that HN2, and other alkalating agents, have also
been shown to inhibit the dc nova synthesis of ribonucleosides by fol-
lowing the incorporation of 14C from 14C formate into the purines of
DNA both in vivo and in vitro (Wheeler and Alexander, 1964b). Further—
more, it was shown that this inhibitory effect was less in HN2 resistant
tumors than in HNZ sensitive ones. Their conclusion was based on the
inhibition of incorporation of 14C from l4C-formate into RNA in the

absence of the inhibition of ribonucleoside incorporation into RNA.

 

 

 

 

 

l-._.\...—‘——

76
Therefore Cis—Pt(II) again very closely mimics the effects of HN2 in
this respect.

Nevertheless, the finding that the incorporation of exogenously
applied ribo— and deoxyribonucleosides into RNA and DNA is also inhibited
provides evidence against a mechanism of action based primarily on the
inhibition of de novo ribonucleoside synthesis. This conclusion applies
to Cis—Pt(II) as well as to HN2. Thus with the possible exception of
the deoxynucleoside kinases, these results suggest that the tumor in-
hibiting platinum compounds are not interfering with the metabolism of
an individual base, but rather that they react with either DNA per se
or enzymes for which DNA is a primer or a substrate.

E. Effects of Platinum Comppunds on Enzymes Using DNA as a Template
or Substrate

 

l. Deoxyribonuclease Activity. At this point it is quite diffi—
cult to draw any valuable conclusions regarding the studies of the
effects of platinum compounds on the stimulation of DNase activity in
platinum treated AV3 cells because Cis-Pt(II) has no enhancing activity.
However, it should be emphasized that the different results on the DNase
activities in extracts of AV3 cells treated with Cis-Pt(II) and
Cis—Pt(IV) represent the only major difference in the molecular effects
of Cis-Pt(II) and Cis—Pt(IV). This may be an interesting point to
pursue in further research. The results from experiments using
Cis—Pt(IV) on AV3 cells are consistent with earlier bacterial studies.
These established that both pure Cis—Pt(IV) and UV irridiated
KZPt(IV)C12, the major photo-product being Cis—Pt(IV), induced a simi—

lar enhancement of endonuclease activity in E. coli B (Harder and

Zimmerman, 1968).

 

 

77

In comparison with inhibitory effects on DNA synthesis, the results
show that the significant enhancement of DNase activity occurred only
when AV3 cells were treated with extremely high concentrations (5001JM)
of Cis—Pt(IV). Therefore, enhanced DNase activities measured by these
techniques would seem to be ruled out as being involved in the mechanism
of action of tumor inhibiting platinum compounds. The lack of enhanced
DNase activities in extracts from Cis—Pt(II) treated cells supports this
conclusion.

The same argument would appear to apply to the results of experi—
ments in which the inhibition of the DNase activities were measured by
the addition of platinum compounds to assays containing untreated cell

extracts. However, it must be recalled that the inhibition of DNA

 

synthesis was a rather slow process. This fact suggests that a greater
degree of inhibition might have been observed if the measurement were
performed over a 4— to 6—hour incubation period instead of just 2 hours.
On the other hand, it is questionable if these nucleases could remain
active that long at 37° C. A completely different method of measuring
the effects of platinum compounds on overall DNase activities is there—
fore suggested for future research. This would be to prelabel mammalian
cells with 3H-TdR, chase with unlabeled TdR, and follow both the increase
in acid soluble 3H—nucleotides and the decrease in the amount of 3H—label
incorporated into the DNA after the beginning of treatment.

Other tumor inhibiting agents also cause an elevation in DNase
activities in HeLa cells. These include Mitomycin C, HN2, hydroxyurea,
and FUdR (S—fluorouricil-deoxyriboside) (Studsinski and Cohen, 1966a).
Thus it would appear that these agents might function by depolymerizing

cellular DNA. But Studzinski and Cohen (1966b) also demonstrated that

the DNA content of Mitomycin C—inhibited cells was not decreased and

78
acid soluble deoxyribose compounds did not accumulate to any great
extent. Consequently it was concluded that the enhanced DNase activity
played a doUbtful role in the mechanism of the inhibitory action of
Mitomycin C. Nevertheless, this result does not eliminate the possi—
bility that the elevated DNase levels have a function. One example of

this would be in the repair of the Mitomycin C-alkylated DNA.‘

2. DNA and RNA Polymerase Activities. The same reasoning that
was given for the enhanced DNase activities also applies for the
enhanced DNA polymerase activities which were observed in extracts of
cells treated with all 3 tumor inhibiting platinum compounds. In other

words, a significant enhancement of DNA polymerase activity occurs at

 

only very high concentrations (500 uM) compared to concentrations
required to inhibit DNA synthesis. Therefore, enhancement of DNA
polymerase activity may be excluded as a possible mechanism of action

of the tumor inhibiting platinum compounds. This conclusion must be
made a priori because a DNA synthesis—inhibitory platinum effect on

DNA polymerase could conceivably occur only by the inhibition of its
activity. This, of course, is exactly what was observed when tumor
inhibiting platinum compounds were added to the DNA polymerase assay in
which untreated AV3 cell extracts were used as the enzyme preparation
(Figure 23). However, the degree of inhibition is very low compared to
concentrations at which DNA synthesis inhibition is observed. It must
be recalled, however, that inhibition of DNA synthesis is rather slow,
and after 2 hours at 25 UM it is still about 80% of the control (Figure 9).
Therefore, it is suggested that DNA be incubated with platinum compounds

for various time periods and reisolated before use in the DNA polymerase

 

 

79

assay. Measuring the effect on the template activity using DNA which
has been isolated from platinum treated cells would be another valuable
experiment to be performed in the future. Such experiments could pro—
vide valuable evidence for biologically important reactions of platinum
compounds with DNA.

Other drugs also have an effect on DNA template activity using
the DNA polymerase assay. DNA isolated from Lettré—Ehrlich ascites
cells treated in vitro with HN2 served as a better primer for DNA
polymerase than DNA from untreated cells; yet the opposite result

was obtained when cells were treated in viva (Goldstein and Putman,

1964).

Both Mitomycin C and Porforomycin induced enhanced DNA polymerase

uh...

activities in extracts of treated HeLa cells (Bach and Magee, 1962;
Magee and Miller, 1962). On the other hand, the primer activity of
Mitomycin~treated DNA is reduced for DNA polymerase (Pricer and
Weissbach, 1964) but is not reduced for DNA dependent RNA polymerase.
Therefore, the antineoplastic platinum compounds closely mimic the
effects of alkylating agents in these properties as well.

The finding that [Pt(II)(NH3)4]Clz has no effect on either DNA,
RNA, protein synthesis (Figures 9, 10, and 11) or on DNA polymerase
(Figure 23) at very high concentrations is significant because it shows

that the inhibitory effects of the platinum compounds cannot be

 

attributed to a nebulous, heavy-metal, poison effect. On the con—
trary, this indicates that the inhibitory effects of the platinum
compounds are very specific and strongly depends upon the ligands sur-

rounding the platinum atom. Moreover, this conclusion is substantiated

 

 

80

by the lack of inhibition of DNA dependent RNA polymerase (Figure 24)

by Cis-Pt(II)(NH3)2C12.

3. A Possible Involvement of DNA Repair Enzymes. How, then,

 

might the enhanced DNA polymerase activity in extracts of platinum
treated cells be explained? There is one very probable explanation
which could account for both the enhanced DNA polymerase and DNase
activities——an activation of DNA repair systems induced by a platinum
reaction with the cellular DNA. Assuming platinum reacts with DNA,

and the cell recognizes this as an insult to its genetic apparatus, the
normal response to this or any DNA damage would be to attempt to repair
the damage. This would involve the excision of the defective bases

and the repair of the gap in the DNA by replication using the opposite
strand as a template. At this point, the order of the process is
unimportant.

The relevance of the repair of chemically induced DNA damage to
the effectiveness of a given tumor inhibiting agent is just now becom—
ing apparent. For example, Lawley and Brooks (1965) demonstrated that
incubation of HN2 resistant E. coli, after alkylation, resulted in the
excision of bifunctionally alkylated guanine moieties from the DNA,
whereas a sensitive strain of E. coli was deficient in this ability.
Yet the DNA from both resistant and sensitive strains were alkylated
to the same extent initially. Therefore, Lawley and Brooks (1965)
proposed that the cytotoxic action of nitrogen mustards is due to the
creation of interstrand cross-links in DNA, which would interfere with
DNA replication but not transcription.

Similarly, HeLa cells (Roberts et al., 1968; Crathorn and Roberts,

1966) and mouse L cells (Reid and Walker, 1969) have been shown to

 

 

 

81

excise both mono- and bifunctionally alkylated DNA products after
sulfur mustard alkylation. Wheeler and Alexander (1964b) showed both
in vivo and in vitr0*that HN2 alkylates the DNA in sensitive and
resistant plastocytomas equally. Therefore, they concluded that the
extent of gross alkylation of DNA is not related to the anti-tumor
activity. However, their conclusion is probably invalid because with
both in vivo and in vitro experiments, no time was allowed for repair
after the administration of HN2. This latter point is borne out by
some of the very recent results in viva. ‘When analine mustard was
administered to both a resistant and sensitive tumor, Connors and
Double (1970) showed that resistant tumors were alkylated to a greater
extent than DNA from the sensitive tumor 12 hours after injection.
However, 2 days after administration, the amount of drug still bound
to the resistant-cell DNA was greatly reduced in contrast to that still
bound to sensitive~tumor DNA. Thus a repair mechanism was invoked to
explain the difference.

The tumor inhibiting properties of bifunctional alkylating agents
have been attributed to the cross—linking ability partially because
monofunctional alkylating agents lack anti-tumor activity (Bosen and
Davis, 1969; Ochoa and Hirschberg, 1967). Nevertheless it is interest—
ing to note that bacteria can also repair alkylated DNA caused by methyl
methanesulfonate, MMS, a monofunctional alkylating agent (Reiter and
Strauss, 1965), although by a different method. Furthermore, there is
good evidence that a repair mechanism exists for MMS—induced DNA damage

in mouse spermatozoa at certain stages of development (Cumming and

Walton, 1970).

Am

 

 

e.— "

82
F. Summary
In general the findings of this research show that platinum com-

pounds mimic the effects of the alkylating agents. These include giant
cell formation; the persistent inhibition of cell division; selective
inhibition of DNA synthesis; inhibition of de novo purine synthesis;
enhanced DNase and DNA polymerase activities in extracts of treated
cells; and the inhibition of the same enzymes when added to assays with
untreated cell extracts. Recently Reslova (1970) showed that Cis—Pt(II)
but not Trans—Pt(II) has the ability to induce lysogenic strains of

E. coli. Similarly HN2 (Endo at al., 1963), UV (Endo at al., 1963;
Witkin, 1967), Mitomycin C (Heinemann and Howard, 1964), azaserine
(Price et al., 1964), but not hydroxyurea (Heinemann and Howard, 1964)
induced lysogenized bacteria. This makes the parallel of the platinum

effects with the effects of alkylating agents even more striking.

 

 

 

 

 

V. A HYPOTHESIS OF THE MECHANISM OF ACTION OF
TUMOR—INHIBITING PLATINUM COMPOUNDS

If the mechanism of action of tumor-inhibiting platinum com-
pounds is indeed shown to involve a direct interaction with DNA as
proposed above, how and where do platinum compounds react with DNA?
Cross-linking of DNA by platinum compounds would appear, at first, to
be unlikely because the 2 chlorine ligands in Cis—Pt(II) are separated
by only about 3.3 A (Milburn and Truter, 1966). Yet for the most
probable reaction which would occur between 2 guanine residues at the
N—7 (imidizole nitrogen) position, a minimum of about 8 A must sepa—
rate the 2 functional groups in a bifunctional alkylating agent in
order to bridge the interstrand gap. HN2 has been shown to decrease
the melting temperature (Tm) of DNA (Wheeler and Stephen, 1965). How-
ever, the monofunctional alkylating agent, MMS, also causes a decrease
in the Tm of the synthetic, double—stranded DNA, polyA—polyU (Ludlum
et al., 1964). In addition, copper (II) causes a large Tm decrease
(Eickhorn and Clark, 1965). Both the MMS and Cu(II) effects have been
interpreted as evidence for a direct reaction at those sites directly
involved in hydrogen bonding. Specifically, the N—6 nitrogen of
adenine in the MMS example, or C-6—keto oxygen of guanine. Preliminary
results by Rahn (1970) and Horacek and Drobnik (1970) indicate that
Cis-Pt(II) also lowers the Tm of calf thymus DNA.

Taking both this and the size of Cis-Pt(II) into consideration,

one may hypothesize that the reaction of Cis—Pt(II) occurs by

83

 

 

84

substitution at both the N-6 and/or N-7 position of 2 adjacent adenines
on the same strand of DNA. substitution at the C-6—keto oxygen and

N—7 position of guanine might also occur. Thus, depending upon whether
2 or all 4 ligands are displaced, the following intrastrand cross-

links might occur: [Pt(II)(AdR)2], [Pt(II)(AdR)(GdR)], [Pt(II)(GdR)2],
[Pt(II)(AdR)2(NH3)2], [Pt(II)(AdR)(GdR)(NH3)2], and [Pt(II)(GdR)2(NH3)2].
On the other hand, one may hypothesize that Trans—Pt(II) can react with
only one base, just as the monofunctional alkylating agents. This
hypothesis is primarily based on the reactions of Cis— and Trans—Pt(II)
(NH3)2C12 with the adenine analogue, 6-mercaptopurine (Hzmp): at neutral
pH, Cis—Pt(II) forms a Pt(II)(Hmp)2 complex, whereas Trans—Pt(II) forms
a [Trans-Pt(II)(NH3)2(H3mp)]+4 complex with 6 mercaptopurine (Grinberg
at al., 1968). However, intrastrand purine cross—links should not be
possible with Trans—Pt(II) because the 2 chlorine ligands are separated
by 4.6 A (Milburn and Truter, 1966), whereas the bases are separated

by about 3.4 A in DNA. It is further speculated that substitution or
reaction with 2 bases is required in order to create a sufficiently rigid
structure to sterically interfere with the replication process. This is
indicated by the lack of anti—tumor activity and lack of DNA synthesis-
inhibition by monofunctional alkylating agents as MMS (Wheeler, 1962).
The effects of such platinum—coordinated, purine dimers would be
expected to be similar to the effects of UV induced pyrimidine dimers
(Setlow at al., 1963), depending on the stability of the platinum bond—
ing to the bases.

In addition to the effects on T this hypothesis of the mechanism

In,
of action is also consistent with the following known biological

effects of the platinum compounds. First, it predicts that the Trans—

platinum compounds would be ineffective, or far less effective

 

 

 

 

 

85
inhibitors of DNA synthesis and therefore less effective tumor inhi—
bitors. Second, in experiments on ¢X 174, a bacteriOphage having
single stranded DNA, and therefore in which no interstrand cross-
linking can occur, monofunctional alkylating agents have been found
to be just as efficient as bifunctional agents in the inactivation of
this virus. However, with bacteriophage containing double-stranded DNA,
bifunctional alkylating agents were more effective than their mono-
functional counterparts (Loveless, 1966). Recent results of Drobnik
at al. (1970) show that Cis-Pt(II) inactivates both single and double
stranded DNA containing bacteriophage with high and equal efficiency.
Hence this is evidence against an interstrand cross-linkage mechanism
but the intrastrand reaction mechanism proposed above is consistent with
this finding. Moreover, if the reaction would occur predominantly with
2 adjacent adenines, one Would predict a greater inhibition of thymidine
incorporation into DNA than any of the other deoxynucleosides, which is
in agreement with the results reported here.

Due to the similarity to UV induced pyrimidine dimers, a repair
mechanism that is similar if not the same as that for UV dimers would
be expected to exist for platinumrcoordinated purine dimers. This
prediction agrees well with the interpretation of the experiments
reported here on the activation of the DNases and DNA polymerase.
Furthermore, an activation of a repair mechanism stimulated by platinum
coordinated DNA lesions could very well lead to the excision or the
activation of the virus genome while repairing the DNA of the viral
genome. Hence because HN2, Mitomycin C, and UV also cause DNA lesions
and stimulate repair processes, a generalized theory of prophage induc-

tion could be developed on this basis.

 

 

 

86

From the type of DNA damage proposed above, one would predict
absence of damage caused by interstrand cross-links. Dominant lethal
mutations, which are caused by interstrand cross—links, in fact, do
not occur in Cis-Pt(II)(NH3)2C12 treated Drosophila (Wilson, 1970).
However, genetic damage due to effects on individual bases, as point
mutations, would be expected to occur.

Finally, the preposed mechanism of the platinum coordination in
between 2 purines with Cis—Pt(II)(NH3)2012 is equally applicable to
Cis-Pt(IV)(NH3)2C14 because the axis between the extra 2 chlorines
would also lie in between the 2 coordinated purines. On the other
hand, this hypothesis would predict that Pt(II)(CH2)2(NH2)2C12 and

platinum compounds with more bulky bridges would not create a bipurine

 

purine coordination complex as easily due possibly to steric hindrance
of the ethylenediamine bridge with the bases. Once more, the data on
the tumor—inhibiting and DNA synthesis—inhibiting properties of these
platinum compounds confirm these predictions.

As far as the author knows, this hypothesis of the mechanism of
action is consistent with all of the presently known effects of the

tumor inhibiting platinum compounds in vivo and in vitro.

 

 

 

VI. EPILOGUE

Seven years ago Furst (1963) made the following predictions in
discussing the design of new anti-cancer compounds:

Compounds without chelating groups, active against
at least one experimental tumor should be modified,
keeping the following spacing in mind: between two
atoms of carbon, or between one atom of carbon and
one of N, O, S, P, there should be the following
functional groups: NHZ, =NH, C=N, C=O, C—OH, C=S,
C—SH, N—OH. These will give rise to representative
combinations like those in CX [X=halides]. Bi-
functional molecules may be better than monofunc-
tional ones. The groups should be close enough
spatially to complex the same metal:
N—C-C—N, N-C-C—S, O~C-C-O, S=C—C—O, S=C—C—N,
O=C—C—N, etc.

A few random examples may be suggested.

.. Perhaps platinum or palladium derivatives
should be made. Since thioethers have a strong
tendency to unite with these metals, sulfur
mustards [H82] and chelates may prove useful.

A reaction between K2Pt014 with sulfur mustard
would yield three isomers [two of which are]:
cis-Pt(II)(H82)C12 and trans-Pt(II)(HSZ)2C12.
(Furst, 1963)

 

It is most amusing that although Furst first predicted the anti-
tumor activity of platinum compounds, the structurally simpler Cis—
Pt(II)(NH3)2C12 behaves like, and appears to be at least as effective

as other bifunctional alkylating agents.

87

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