Studies with hydroxyurea

48

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96734

STUDIES W I T H H Y D R O X Y U R E A T H E BIOLOGIC AND METABOLIC P R O P E R T I E S OF FORMAMIDOXIME

H E R B E R T S. R O S E N K R A N Z , R I C H A R D H J O R T H AND H O W A R D S. C A R R

Department o/Microbiology, College o/ Physicians and Surgeons, Columbia University, New York, N.Y. zoo32 (U.S.A.) (Received September 2ist, 197 o)

SUMMARY

Formamidoxime is a preferential inhibitor of bacterial and mammalian DNA synthesis. In bacteria, levels of formamidoxime up to 0.05 M are bacteriostatic while concentrations in excess of 0.05 M cause bacterial death. This lethal effect is accompanied by degradation of the cellular DNA. Inhibitors of protein and RNA synthesis and blockers of energy metabolism prevent formamidoxime-induced death. The two inhibitory effects of the chemical, viz. lethality and inhibition of DNA synthesis, are separable. A bacterial mutant deficient in DNA polymerase was much more sensitive to the lethal action of formamidoxime than its parent. The inhibitory effect of formamidoxime appears to be the result of its structural relationship to hydroxyurea.

INTRODUCTION

OH

Jr r

Hydroxyurea (H~N-C-N-OH) has been shown to possess promising anti-neoplastic and anti-viral properties z-3. In view of these unique features, the mechanism of action of this drug has been under intensive investigation for several years. Although it is now well established that hydroxyurea is a specific and reversible inhibitor of DNA synthesis 4-6, the mechanism whereby this relatively simple compound exercises its action has not been clarified unequivocally. Some investigators have presented evidence that the primary action of hydroxyurea was on nucleoside diphosphate reductase 7-9, others, however, using different cell systems have reached different conclusions 1°-14. In the meantime advantage is being taken of the unique properties of hydroxyurea to study the role of DNA synthesis in various biological phenomena (e.g. carcinogenesis, viral development, cellular differentiation, embryogenesis and genetic recombinations). In seeking to clarify further the metabolic reaction blocked specifically by hydroxyurea, a number of investigators have sought to gain a further understanding of the mode of action of this compound by examining the properties of a number of substances structurally related to hydroxyurea z5 35. The general concensus has been Biochim. Biopkys. Acta, 232 (1971) 48-60

49

BIOCHEMICAL PROPERTIES OF FORMAMIDOXIME

that the - N O H function was necessary for the unique properties of this group of substanceslT, 2~,,5. In addition, it was also possible to establish a relationship between the activity of hydroxyurea and that of its isomer isohydroxyurea.6,.7 (0-carbamoylO OH O

II

Ill

II

hydroxylamine, H2N-C-O-NH2) and N-carbamoyloxyurea (H2-N-C-N-O-C-NH2), an oxidation product of hydroxyurea ~,3°. It should be pointed out that neither of these compounds possesses an unsubstituted - N O H function. In an attempt to elucidate further the mode of action of hydroxyurea, a detailed study of the properties I-I

I

of formamidoxime (HzN-C~NOH), one of the simplest congeners of hydroxyurea, was undertaken. The present report describes some of the attributes of formamidoxime and compares them to those of hydroxyurea, isohydroxyurea and carbamoyloxyurea.

MATERIALS AND METHODS

Bacteria Escherichia coli C6oo is a derivative of E. coli KI2 which requires leucine, threonine and thiamine for growth. E. coli C6oo/HU, E. coli C6oo/isoHU and E. coli C6oo/HUN are bacterial strains resistant to hydroxyurea 1°, isohydroxyurea~e and N-hydroxyurethan .1, respectively. E. coli P347 8, a bacterial strain deficient in DNA polymerase 32 and the parent strain E. coli W3IIO were generously provided by Dr. John Cairns, Cold Spring Laboratory of Quantitative Biology. Techniques The procedures for growing and enumerating bacteria have been described 33 as have the metabolic techniques 2~,~t. Whenever radioactive thymidine was used, the medium was supplemented with uridine (366/zg/ml) to inhibit and repress thymidine phosphorylase 35. Previously described methods were used to determine sedimentation coefficients36, buoyant densities 37, and thermal helix-coil transition profiles~. Preparation o] ~H-labeled DNA The isolation of 3H-labeled DNA from embryonic chicks was described previously 3°. Ehrlich ascites cells Ehrlich ascites cells were propagated and used for metabolic experiments as previously described 39. Materials Formamidoxime was obtained from Aldrich Chemical Co., and hydroxyurea from Nutritional Biochemicals Corp. Only freshly prepared solutions of these compounds were used. Azauracil and SH-labeled compounds were purchased from Schwarz BioResearch, Inc., 14C-labeled compounds from New England Nuclear Corp., chlorampbenicol from Parke, Davis & Co., puromycin and calf thymus DNA from NnBiochim. Biophys. Acta, 232 (I971) 48-6o

5°

H.S.

ROSENKRANZ

et al.

tritional Biochemicals Corp., NaN 3 and sodium arsenate (Na2HAsOa) from Fisher Scientific Co., minimal essential medium with H a n k ' s salts and phosphate-buffered saline from Grand Island Biological Co.

RESULTS

E//ect o//ormamidoxime on the growth o / E . coli Exposure of E. coli to o.o5 M formamidoxime resulted in a bacteriostatic effect which was maintained for as long as 24 h of contact with the drug (Table I). When cells were exposed to formamidoxime concentrations in excess of 0.05 M they were killed, after an initial period of bacteriostasis (Table I). When drug levels of o.I M TABLE

I

E F F E C T OF FORMAMIDOXIME ON THE GROWTH OF

E. coli

Bacteria (E. coli C 6 o o ) i n m e d i u m H A ( r e f . 3 3 ) w e r e b r o u g h t t o t h e e x p o n e n t i a l at which time portions of the cultures were distributed into flasks containing amounts of Iormamidoxime. At intervals portions of the cultures were removed mination of turbidity (45 ° n m ) and to enumerate viable bacteria.

Time (h )

Molarity o[ [ormamidoxime

Viable bacteria/ml

Turbidity

o 2 4 24

o o o o

i.i • lO s 9 . 6 . lO s 1. 3 - l O 9 3 . 8 " lO 9

0.28 0.67 o.73 1.55

2 4 24

0.05 0.05 0.05

8. 4 - l O 7 1.2. IO s 2.2. lO s

0.50 0.53 0.95

2 4 24

o.o8 0.o8 0.08

7.0. lO 7 3.2" lO 7 1.9" lO 5

o.42 o.48 o.47

2 4 24

o. i o o.Io o.io

6.0- lO 7 7.1-1o ~ 1.6- lO s

0.36 0.37 0.42

2 4 24

0.20 0.20 0.20

1.2 • l O 8 3.0. lO 7 1.I • lO 4

o.31 o.31 0.29

growth phase premeasured for the deter-

and less were employed it was observed that although cell multiplication ceased, the bacterial mass continued to increase as evidenced by the rise in turbidity of treated cultures (Table I). Microscopic examination of such cultures revealed that treated bacteria had become elongated (Fig. I). When bacteria were treated with formamidoxime levels exceeding o.I M, the bacterial mass no longer increased but cell death persisted.

E]/ect o[ metabolic inhibitors on/ormamidoxime-induced death NAN3, a blocker of oxidative metabolism, protected cells from the detrimental action of formamidoxime (Table II) however as exposure to formamidoxime was Biochim. Biophys. Acta,

232 (1971) 48-6o

BIOCHEMICAL PROPERTIES OF FORMAMIDOXIME

51

Fig. I. P h o t o g r a p h s of b a c t e r i a i n c u b a t e d for 60 rain in a m e d i u m c o n t a i n i n g 0.08 IV[ f o r m a m i d o x i m e . A. T r e a t e d culture. ]3. Control culture. TABLE II EFFECT

OF

METABOLIC

INHIBITORS

ON

THE

LETHA.L

ACTION

OF

FORMAMIDOXIME

The e x p e r i m e n t a l p r o c e d u r e s used were e s s e n t i a l l y i d e n t i c a l t o t h e ones d e s c r i b e d i n t h e l e g e n d of T a b l e I. F i n a l d r u g c o n c e n t r a t i o n s : azauracil, 3o0 # g / m l ; c h l o r a m p h e n i c o l , 3 ° p g / m l ; f o r m a m i d oxime, o.I M, h y d r o x y u r e a , o.2 M; N a N a o.oi M; p u r o m y c i n , 9o/ ~ g/ ml ; a r s e n a t e o.oi .-~.

d dditions None Formamidoxime Formamidoxime + Chloramphenicol Formamidoxime + Azauracil Formamidoxime + Puromycin Formamidoxime + NaN 8

Viable bacteria/ml oh

24h

3 . 8 . lO s

5,2

chloramphenicol

5.2

puromycin NaN 3 2.1

• 10 8

12~a N 3

None Forrnamidoxime Formamidoxime + arsenate Arsenate

• 10 8

4.8 • lO 6 4.2 • lO 8 4.4 " l°T 3.7" l os 3 . 7 ' 106 3.7" 1oS

Azauracil

None Formamidoxime Formamidoxime + hydroxyurea Hydroxyurea F o r m a m i d o x i m e + ~ aN 3

• lO 9

6. 4 • lO 6 1.9 • IOs

9.2

• IO 7

3.5 5.4 4.3 4.4

lO9 IO~ lO3 zo5

2. 7

lO 5

2.6

IO v

7 . 6 . lO 9 3.0. io s I.I

•

I.O

• IO 8

10 8

Biochim. Biophys. Acta, 232 (1971) 48-60

52

H . S . ROSENKRANZ e[ al.

prolonged (e.g. 24 h) the extent of the salutary effect on NaN 3 was diminished. Arsenate, on the other hand, an agent which blocks oxidative as well as fermentative processes 4°-42 was very efficient in blocking the lethal effects of formamidoxime (Table II). Simultaneous exposure of cells to formamidoxime and chloramphenicol - an inhibitor of protein production -- resulted in a greatly increased survival rate. Puromycin, another blocker of protein synthesis, and azauracil, an RNA antagonist, also had some protective effect (Table II). Hydroxyurea, a potent inhibitor of DNA synthesis a3,44, was synergistic with formamidoxime.

Metabolic e#ects o/]ormamidoxime Exposure of growing cultures of E. coli C6oo to o.o8 M formamidoxime revealed (Fig. 2) that DNA synthesis was most sensitive to repression by formamidoxime.

x

5

Formam idoxime

N 10 "tJ

,

,

11- F o r m a m i d o x i m e

OL,~ 4r"

0

,

,

5

10

l

,

15 20 Time (rain)

2~5

30

Fig. 2. Effect of f o r m a m i d o x i m e on i n c o r p o r a t i o n of specific p r e c u r s o r s into m a c r o m o l e c u l a r c o n s t i t u e n t s . B a c t e r i a (E. coli C6oo) were b r o u g h t to t h e e x p o n e n t i a l g r o w t h p h a s e a t w h i c h t i m e t h e c u l t u r e s were s u p p l e m e n t e d w i t h r a d i o a c t i v e p r e c u r s o r s [aH]leucine (8.9. IO -s 1~, 0.87 /*C/ml) for proteins; [5-SH]uridine (2.o. lO -4 M, 2. 4/~C/ml) for R N A a n d [ S H ] t h y m i d i n e (7.4" IO-S M, 0. 5 # C / m l ) a n d non-labeled u r i d i n e (366/~g/ml) for D N A ) . A t t h e end of 4.5 m i n of i n c u b a t i o n p o r t i o n s from each c u l t u r e were d i s t r i b u t e d into p r e - w a r m e d flasks c o n t a i n i n g f o r m a m i d o x i m e (final c o n c e n t r a t i o n 0.08 M). S a m p l e s were r e m o v e d a t i n t e r v a l s for t h e d e t e r m i n a t i o n of radioa c t i v i t y i n c o r p o r a t e d into acid-insoluble form. 0 , control; ®, 0.08 M f o r m a m i d o x i m e .

RNA and protein synthesis were quite refractory to inhibition by the drug. Chloramphenicol, which prevents formamidoxime-induced killing, did not inhibit formamidoxime-caused inhibition of DNA synthesis (Table III).

Formamidoxime and pulse-labeled R N A Formamidoxime did not decrease significantly the amount of pulse-labeled RNA made when the drug and the specific precursor were added simultaneously. When, however, cells were pretreated with formamidoxime for 9 rain there was a Biochim. Biophys. Acta, 232 (1971) 4 8 - 6 o

BIOCHEMICAL PROPERTIES OF FORMAMIDOXlME

53

TABLE III CHLORAMPHENICOL

AND

FORMAMIDOXIME-INDUCED

INHIBITION

OF

DNA

SYNTHESIS

A culture of growing bacteria (E. coli C6oo) was distributed into flasks containing premeasured a m o u n t s of f o r m a m i d o x i m e and chloramphenicol. Each culture was subdivided f u r t h e r into two equal p a r t s one of which was s u p p l e m e n t e d with a m i x t u r e of [3H]thymidine (2.8 • lO -l° M, 0.83 #C/ml) and uridine (366/zg/ml) at once and the other 2 h later. After 0.5 h of incubation in the presence of ESH]thymidine replicate i-ml portions were w i t h d r a w n from each culture and processed for the d e t e r m i n a t i o n of radioactivity incorporated into acid-insoluble form.

Additions

Radioactivity incorporated (counts~rain per ml)

None Chloramphenicol, 4 °/~g/ml F o r m a m i d o x i m e , 0.o8 M Chloramphenicol + f o r m a m i d o x i m e

o-o.5 h

2.o-2.5h

2883 2059 988 691

2339 446 1122 10o9

68 % reduction in the amount of pulse-labeled RNA (Table IV). Addition of the drug to cells after the initial pulse of [3Hluridine revealed (Fig. 3) that the rapidly labeled RNA was quite stable (i.e. it was not degraded as in actinomycin D-treated cells45-aT). TABLE IV EFFECT

OF FORMAMIDOXlME

ON THE SYNTHESIS

OF PULSE-LABELED

R~N~A

Bacteria (E. coli C6oo) in m e d i u m H A (ref. 33) a t 3 °0 were b r o u g h t to the exponential g r o w t h phase at which time i-ml portions of the culture were distributed into flasks containing prem e a s u r e d a m o u n t s of [SH]uridine (5-3 " lO-1° IV[, 2.1 ,uC/ml) and formamidoxime. After 0. 5 min of incubation the cultures were s u p p l e m e n t e d with trichloroacetic acid (final concentration 5 %) and processed for d e t e r m i n a t i o n of incorporated radioactivity. Those cultures which were pret r e a t e d with f o r m a m i d o x i m e were handled as above, except t h a t [3H]uridine was added 9 min after the drug. The results listed in the table are the averages of five separate determinations.

Conditions

No additions [sH]Uridine and f o r m a m i d o x i m e (0.05 M) added simultaneously F o r m a m i d o x i m e (0.05 M) added at t = --9 min [SH]uridine added at t = o

[SHi Uridine incorporated during 3o-sec pulse Counts/rain per ml

°/o o/control

2o 155

IOO

18 124

9°

6 409

32

Ribosomes and/ormamidoxime The ribosomes which existed at the time of addition of formamidoxime as well as those that were made subsequently were normal with respect to amount and composition of their subunits (Fig. 4), this can be deduced from the superimposition of the absorbance (mainly pre-existing ribosomes) and radioactivity (newly synthesized ribosomes) curves. Similarly no differences were observed when ribosomes were analysed in buffer containing o.oi M Mg~+ (unpublished results). Finally no change was observed in pre-existing ribosomes by these same criteria when they were extensively labeled with E3H]uridine prior to exposure to formamidoxime (unpublished results). Biochim. Biophys. Acta, 232 (I97 x) 48-60

54

H . S . ROSENKRANZ et

al.

Control OL

E

Q3

Q2

9

× .E

X

E

E Ol =

8

0

"~ I

0

o '

'

FormamidoxJmetreoted

'

3 .Q

o2

8

~ 1

(31 0

._o

4

&

$

~b

Time (rain)

0

o

lo

20

30

0

Fraction No.

Fig. 3. Effect of f o r m a m i d o x i m e on the longevity of pulse-labeled RNA. Bacteria growing at 3 °0 were exposed to [SH]uridine (5"IO-Z° M, I #C/ml) for 3 ° sec, w h e r e u p o n the culture was s u p p l e m e n t e d w i t h all excess of unlabeled uridine (8.2.1o -4 M), half of the culture was w i t h d r a w n and added to a p r e - w a r m e d flask containing f o r m a m i d o x i m e (final concentration 0.08 M). At intervals samples were w i t h d r a w n for radioactivity determination. 0 , control; ®, o.08 M formamidoxime. Fig. 4. Properties of ribosomes isolated from f o r m a m i d o x i m e - t r e a t e d cells. Bacteria in early exponential g r o w t h phase (2. lO 8 cells/ml) were divided into t w o equal p o r t i o n s (500 ml each) one of which received f o r m a m i d o x i m e (0.o8 M), 0. 5 h later the cultures each received [SH]uridine (0. 5 #C/ml). After incubation for a n o t h e r 0. 5 h, the cells were processed for extraction of ribomes 5=. Ribosomes were analysed in linear gradients of sucrose (5-20 %) in o.oi M Tris buffer (pH 7.0) containing I . lO -4 Mg ~+. Centrifugations were carried out in the SW-5o r o t o r of the Spinco Model L-2 ultracentrifuge at 35 ooo r e v . / m i n for 3 h.

Formamidoxime and cellular DNA Exposure of cells pre-labeled with [3HJthymidine to formamidoxime resulted in degradation of the cellular DNA to acid-soluble products. This depolymerization occurred only after a delay (Table V, Expt. I). It was not prevented by metabolic inhibitors. On the contrary, chloramphenicol actually stimulated solubilization of the cellular DNA (Table 5, Expt. II). Isolated DNA and/ormamidoxime Exposure of radioactive DNA to the drug for long periods did not lead to depolymerization of DNA, even when the reaction was carried out at an elevated temperature (56°) (Table VI). Nor could an effect of formamidoxime on purified DNA be demonstrated when the more sensitive methods of physical chemistry were used. Thus formamidoxime had no effect on the sedimentation coefficient of treated DNA (Table vii). When thermally denatured DNA was analysed, the treated specimen did not exhibit a greater decrease in sedimentation coefficient than the control, J~iochim. Biophys. Acta, 232 (i97 I) 48-60

BIOCHEMICAL PROPERTIES OF FORMAMIDOXIME TABLE

55

V

~FFECT OF FORMAMIDOXIME

O N STABILITY OF C E L L U L A R D N A

Bacteria (E. coli C6oo) in m e d i u m H A (ref. 33) containing [t4C]thymidine (8 • lO - n M, o.2/~C/ml) and uridine (366 #g/ml) were b r o u g h t to the beginning of the exponential g r o w t h phase whereu p o n t h e y were washed, resuspended in fresh p r e w a r m e d medium, incubated w i t h aeration for 3° min and distributed into flasks containing formamidoxime. A t intervals p o r t i o n s from each culture were w i t h d r a w n and processed for d e t e r m i n a t i o n of radioactivity remaining acid-insoluble.

Expt. I

It

Time (h)

Additions

Radioactivity retained (counts/rain per ml)

o i 2 4 24 i 2 4 24

None

o 24 24 24 24 24 24 24 24

None None . o.i M f o r m a m i d o x i m e F o r m a m i d o x i m e + a z a u r a c i l (30o/,g/ml) Azauracil (3oo #g/ml) F o r m a m i d o x i m e + c h l o r a m p h e n i c o l (4 ° / , g / m l ) Chloramphenicol (4 ° / z g / m l ) F o r m a m i d o x i m e + o . o i IV[ 1qaNz o.oi M N a N 3

3 188 3 474 3 503 3 414 3 17o 3 247 3 214 2 923 2 182

o.i M f o r m a m i d o x i m e

15 429 15 554 9 147 8 5o8 14 602 5 743 18 03 I 9 539 15 413

TABLE VI EFFECT

OF FORMAMIDOXIME

ON THE STABILITY

OF PURIFIED

DNA

A solution of [SH]DNA was s u p p l e m e n t e d with o.i 1~{ f o r m a m i d o x i m e and incubated either at 37 or 560 , at intervals p o r t i o n s of the solutions were r e m o v e d and processed for determination of radioactivity remaining acid-insoluble

Time

(h)

o 2 4 24 48

TABLE

Radioactivity retained (counts]rain per ml) 37 °

56 °

Control

o.z M ]ormamidoxime

Control

o.x M [ormamidoxime

14 823 15 048 16 321

15 14 15 14 14

14 14 15 14 15

14 608 16 057 16 039 16 o39 15 o70

211 662 299 442 792

809 880 202 721 265

VII

EFFECT OF FORMAMIDOXIME

O N T H E PHYSICAL C H E M I C A L P R O P E R T I E S O1~ PURIFIED D N A

Solutions of calf t h y m u s D N A (o.5 m g / m l of O.Ol 5 IV[ N a C I containing o.ool 5 i~ s o d i u m citrate) were incubated at .56° with or without o.I ~f f o r m a m i d o x i m e for 72 h. T m is the midpoint of the thermal helix-coil transition curve.

Physical determination

Control

Formamidoximetreated

Sedimentation coefficient (S) S e d i m e n t a t i o n coefficient (S) after t h e r m a l d e n a t u r a t i o n B u o y a n t d e n s i t y (g/cm 3) Tm

18. 3 14.8 1.7o 4 84.4 °

17. 9 15.o 1.7o 5 83.9 °

Biochim. Biophys. Acta, 232 (1971) 48-60

56

H.S.

ROSENKRANZ

et

al.

untreated, sample, thus indicating that the treated DNA did not contain "hidden breaks" (in the phosphodiester backbone). The thermal helix-coil transition curve of the treated DNA differed only slightly from that of the control and the same was found to be the case for buoyant density values (Table vii).

Formamidoxime and a bacterial strain lacking DNA polymerase E. coli P3478, a bacterial strain lacking DNA polymerase 3~ was much more sensitive to the lethal action of formamidoxime, than E. coli W3IiO, the parent strain. The lethal action of the drug on the m u t a n t strain could not be reversed by chloramphenicol (Table v i i i ) TABLE

VIII

E F F E C T OF FORMAMIDOXIME ON BACTERI AL STRAIN LACKING D N A

POLYMERASE

Bacteria in medium HA (ref. 33) containing thymine (5/~g/ml) were brought to the exponential growth phase and distributed into flasks containing premeasured amounts of formamidoxime and/or chloramphenicol. The number of viable bacteria was determined by plating serial dilutions (o.I ml) of cultures on agar containing medium HA (ref. 33) and thymine.

Time

Additions

(h)

Viable bacteria/ml

o 4 4 24 24

None None o.08 M formamidoxime None 0.08 M formamidoxime

o 24 24 24 24

None None 0 . 0 8 B{ f o r m a m i d o x i m e Formamidoxime+chloramphenicol Chloramphenicol (20 12g/ml)

Parent strain (E. coli W3zzo)

Mutant strain (E. coli P3478)

1. 3 4.2 1.8 i.o 1.8

1. 3 1.2 2. 4 1.6 2.1

• • • • •

2. 3 1. 4 1. 3 1.2 i.o

• 10 7

•

• • •

108 lO 9 lO 8 i o z0 lO 8

• • • •

107 Io 9 io s lO 9 lO 4

lO 9 lO 4 lO 3 lO 7

Formamidoxime and mutant bacterial strains A bacterial strain resistant to hydroxyurea (E. coli C6oo/HU) was found to exhibit cross-resistance to formamidoxime (Table IX), on the other hand strains resistant to isohydroxyurea (E. coli C6oo/isoHU) and to hydroxyurethan (E. coli TABLE IX E F F E C T OF FORMAMIDOXIME ON SEVERAL

Expt.

II

Time

(h)

Additions

E. coli

STRAINS

Viable bacteria/ml C6oo

C6oo/HU

C6oo/isoHU

o 24 24

None None o.i M formamidoxime

2.o • lO 8 7 . 5 " lO9 4.2 • lO 6

3 . 5 " lO8 5 . 3 " lO9 9 . 8 • IO '~

1.9 • lO s 4 . 0 ' lO9 2.0 • lO 3

o 24 24

None None o.I M formamidoxime

6.1 - lO 7 5 . 5 " IO° 2 . 0 • lO 2

1.8 • lO 8 4 . 9 " lO8 2. 7 • i o e

1. 4 - l O 8 5 . 4 " lO9 3.8 • lO s

Biochim. Biophys. Acta,

232 (1971) 48-60

C6oo/HUN

3.1 • lO 7 2 . 9 • zo o 4.0 • lO 9

BIOCHEMICAL PROPERTIES OF FORMAMIDOXlME

57

C6oo/HUN), which also show cross-resistance to hydroxyurea ~6 were found to be exceedingly sensitive to the lethal action of formamidoxime (Table IX). It has been found previously that E. coli C6oo/isoHU possessed very unique properties ~. Thus it is the only organism studied thus far in which hydroxyurea does not inhibit the synthesis of DNA, rather, in this special strain hydroxyurea blocked protein production. This special metabolic effect of hydroxyurea was mimicked by formamidoxime (Table X), even though E. coli C6oo/isoHU is exquisitely sensitive to the lethal action of formamidoxime (Table IX) while it is resistant to hydroxyurea-induced killing 26. TABLE

X

EFFECT OF FORMAMIDOXIME ON THE METABOLISM OF E. coli RESISTANT TO ISOIIYDROXYUREA B a c t e r i a (E. coli C6oo/isoHU) were b r o u g h t to t h e e x p o n e n t i a l g r o w t h p h a s e a t w h i c h t i m e port i o n s of t h e c u l t u r e s were d i s t r i b u t e d into flasks c o n t a i n i n g p r e m e a s u r e d a m o u n t s of f o r m a m i d o x i m e a n d of t h e specific precursors. [ a H ] t h y m i d i n e (2.8 • lO -l° IV[, 0.83 #C/ml) (and u n l a b e l e d u r i d i n e to repress a n d i n h i b i t t h y m i d i n e p h o s p h o r y l a s e 8") for D N A ; [5-aH]uridine (2.0 • lO -4 M, 2. 4 tiC/m]) for 1RNA a n d [3H]lysine for proteins. A t t h e e n d of 3o rain of i n c u b a t i o n d u p l i c a t e i - m l p o r t i o n s f r o m each c u l t u r e were w i t h d r a w n a n d processed for t h e d e t e r m i n a t i o n of incorporated radioactivity.

Metabolic process

Concentration/or 5 ° °/o inhibition (M)

1RNA s y n t h e s i s DNA synthesis Protein synthesis

o.oo18 o.o81 o.o13

/ ~ R N A F~otein~ 120 1OC

"6 6O 4O 2O 0 10-5

DNA ~ , i 10-4 10-3 I0-2 Formamidoxime concn. (M)

Fig. 5. Effect of f o r m a m i d o x i m e on t h e m e t a b o l i s m of E h r l i c h ascites cells. S u s p e n s i o n s of cells p r e p a r e d as described in t h e t e x t were i n c u b a t e d w i t h f o r m a m i d o x i m e a n d w i t h specific p r e c u r s o r s ([SH]thy midine, 7,4"IO-* M, 0.5/~C/ml; [5-3H]uridine, 2.O.lO -41V[, 2. 4 /,C/ml; a n d [3H]lysine, 5.4" lO-5 M, 2/zC/nll). T h e cells were i n c u b a t e d at 37 ° for I h w h e r e u p o n d u p l i c a t e i - m l p o r t i o n s were w i t h d r a w n a n d r a d i o a c t i v i t y i n c o r p o r a t e d w a s d e t e r m i n e d . ®, IRlqA; O , D N A ; × , proteins.

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H . S . ROSENKRANZ et

al.

Formamidoxime and the metabolism o] mammalian cells In mammalian (Ehrlich ascites) cells, the inhibitory action of formamidoxime was also directed at DNA synthesis (Fig. 5). As a matter of fact, blockage of DNA synthesis in these cells resulted in an enhanced RNA and protein production.

DISCUSSION

Formamidoxime and DNA synthesis The initial observations (Table I) of a continued rise in the turbidity of bacterial cultures exposed to o.o5-o.lo M formamidoxime, in the absence of an increase in the number of viable bacteria indicates that this is presumably another example of "unbalanced growth", i.e. continued synthesis of RNA and proteins with concomitant blockage of DNA production ~, indeed the metabolic experiments summarized in Figs. 2 and 5, support this conclusion. Although unbalanced growth is usually accompanied by cellular death the data of Table I indicate that this is not necessarily so when bacteriostatic levels of formamidoxime (i.e. 0.0 5 M) are used. Such a situation has also been found to exist when cells are treated with hydroxyurea ~ or novobiocin 49. When bacteria were treated with formamidoxime levels exceeding o.I M, unbalanced growth was abolished (Table I) but this did not lead to rescue of the treated bacteria. This finding suggested that the lethal action of formamidoxime was not related directly to unbalanced growth (and DNA synthesis). It is perhaps significant that studies on the mode of action of hydroxyurea had led earlier to the postulation 1°,~ that the hydroxyurea-induced inhibition of DNA synthesis was distiuct from the lethal effect of hydroxyurea which is seen when exposure to hydroxyurea is unduly prolonged 5°. In support of these separate effects of formamidoxime was the finding that chloramphenicol, which prevented formamidoximeinduced cellular devitalization (Table II) did not inhibit formamidoxime-caused inhibition of DNA synthesis (Table III). Basis o/ the bactericidal e//ect It is probable that the cause of the lethal action is an effect of formamidoxime on the cellular DNA. The basis of this interaction is not known at present, but current studies on the effect of formamidoxime on deoxynucleosides and deoxynucleotides m a y provide an answer. It is certain, however, that the degradation of the DNA that was seen in treated cells was not a direct effect of formamidoxime. Rather it would appear to reflect an attempt b y the cells to excise and repair the damaged DNA. DNA polymerase has been implicated as the enzyme responsible for this repair 32,51 and it is significant that a bacterial mutant deficient in DNA polymerase was much more sensitive to the lethal action of formamidoxime (Table viii). It is interesting that chloramphenicol, one of the best inhibitors of formamioxime-caused death (Table II), actually stinmlated the degradation of cellular DNA (Table V). This suggests that protection of formamidoxime-treated cells by metabolic inhibitors involves a number of sequential steps: (I) formamidoxime-induced damage to cellular DNA, (2) attempts by the cell to excise portions of the damaged genome, (3) repair of the excised DNA, and (4) cellular replication. It is probable t h a t when ce[Biochim. Biophys. Acta, 232 (1971) 48-6o

BIOCHEMICAL PROPERTIES OF FORMAMIDOXIME

59

lular replication begins before repair has been completed cells will be devitalized (see also ref. 31). Chloramphenicol (and the other inhibitors as well) by preventing premature duplication while allowing continued excision may then protect formamidoxime-treated cells. Indeed cells unable to repair damaged DNA, are not protected (Table viii) from the lethal effects of formamidoxime by chloramphenicol, since delay of cellular duplication would be of no advantage to them. In support of this multi-step scheme is the finding (unpublished results) that the DNA of E. colt C6oo/ HU, a strain resistant to the lethal action of formamidoxime (Table IX), was also degraded by formamidoxime (and presumably repaired). Forrnamidoxime and hydroxyurea The synergism between formamidoxime and hydroxyurea (Table II) and the fact that both agents inhibit DNA synthesis suggested that they have similar mechanisms of action. This conclusion is supported by the finding that bacteria resistant to hydroxyurea were also resistant to formamidoxime (Table IX) and by the similarity in the action of the drugs towards E. colt C6oo/isoHU (Table X). A scheme seeking to explain some of the known relationships of hydroxyurea and its derivatives has been proposed ~7. Current studies are designed to determine the place of formamidoxime in the postulated series of interconversions. The present data suggest that the in vitro degradation that is observed when DNA is exposed to hydroxyurea for prolonged periods at elevated temperatures3° is not related to the in vivo degradation of cellular DNA induced by this same agent as formamidoxime which possesses the latter property does not affect isolated DNA to a significant extent.

ACKNOWLEDGMENTS

This investigation was aided by the Annie R. Masch Memorial Grant for Cancer Research from the American Cancer Society. One of the authors (H.S.R.) is a Research Career Development Awardee of the Division of General Medical Sciences, U.S. Public Health Service (5-K3-GM-29 024). REFERENCES I 2 3 4

5 6 7 8 9 io II 12 13 14 15 16 17

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