Inhibition of proliferation and differentiation of mouse erythroleukemia cells by hydroxamic acids

Leukemia Research Vol. 9, No. 4, pp. 463-.469, 1985. Printed in Great Britain.

0145-2126/8553.00 + .00 Pergamon Press Ltd.

PRELIMINARY

REPORT

INHIBITION OF PROLIFERATION AND DIFFERENTIATION OF MOUSE ERYTHROLEUKEMIA CELLS BY HYDROXAMIC ACIDS CHRISTOF U. ANDERS*, THOMAS V. KREYBIG~ and GEBnARO KOCH* *Abteilung Molekularbiologie, Universit~t Hamburg, Grindelallee 117, D-2000 Hamburg 13, F.R.G.; and tAbteilung for Zahn-, Mund- und Kieferchirurgie, Nordwestdeutsche Kieferklinik der Universit~it Hamburg, Martinistr. 52, D-2000 Hamburg 20, F.R.G. Abstract--The effect of several hydroxamic acids on cell growth and differentiation was studied in vitro in cultures of Friend erythroleukemia cells, line F4-6. Terminal differentiation in F4-6 cells can be induced by exposure to a variety of structurally unrelated compounds or to conditions which inhibit cell growth. Hydroxamic acids do not induce erythroid differentiation but interfere with both cell growth of F4-6 cells and the induction of differentiation by DMSO in these cells. DMSO-induced terminal differentiation is inhibited even when F4-6 cells are pretreated for 24 h with hydroxamates followed by removal of the hydroxamates and transfer to fresh medium containing 1~0 DMSO. Reduction of cell growth by hydroxamates is completely and immediately reversible upon removal. In contrast, the inhibition of DMSO inducibility is not reversible within 24 h. Cell pretreated with hydroxamates for 24 h prior to a 96 h-exposure to DMSO show the same reduction in synthesis of hemoglobin as cells simultaneously exposed to DMSO and hydroxamates.

Key words: Hydroxamic acids, cell differentiation, cell proliferation Friend cells.

INTRODUCTION MANY hydroxamic acids are found as naturally occuring compounds [6]. One member of the group, hadacidin (HA) which is produced by Penicillium aurantioviolaceum [I 5], has been used as an anti-tumor agent (for review see [6]). Complex hydroxamic acids, which are referred to as siderophores, mediate iron transport in bacteria and mammalian cells [13]. Certain other hydroxamic acids, e.g. desferal (the methane sulfonate derivate of deferriferrioxamine B) inhibit iron transport and synthesis of D N A and to a lower extent RNA and protein synthesis in H e L a cells [19]. In addition, hydroxamic acids are efficient inhibitors o f ribonucleotide reductase [22] and of urease [9] and are, therefore, used in the treatment of hepatic coma in man [1]. H A induces sporulation in the bacterium Bacillus subtilis, probably by interference with the purine nucleotide pathway [7]. Therefore, it was of interest to analyze the effects o f hydroxamates on growth and differentiation in an eukaryotic cell system. Friend virustransformed mouse erythroleukemic cells were chosen since they have been used as an in vitro model system for studies of induced erythroid differentiation. Friend cells are morphologically similar to proerythroblasts and presumably are arrested in this stage of differentiation by the virus-induced transforming event [21]. The Friend cell line F4-6 was established from spleens o f mice infected with Friend virus by Ostertag et al. [16]. DMSO (dimethyl sulfoxide) induces heme synthesis and erythroid differentiation to the reticulocyte stage in Friend cells in vitro [8]. During terminal differentiation an increase was observed in iron transport [8], Friend spleen focus forming virus release [5] .and synthesis of globin m R N A [20] and o f globin [16]. Abbreviations: FH, formydroxamic acid; HA, hadacidin; AH, acetohydroxamic acid; MAH, N-methylacetohadroxamin acid; 6-N-Arg, 6-N-hydroxy-arginine; FG, N-Formyl glycine; FCS, fetal calf serum. Correspondence to: Dr. G. Koch, Abteilung Molekularbiologie, Universit~t Hamburg, Grindelallee 117, D-2000 Hamburg 13, F.R.G. 463

464

CHRISTOF U. ANDERS, THOMAS V. KREYBIGand GEBHARD KOCH

H y d r o x a m i c a c i d s induce specific t e r a t o g e n i c effects in rats. In a d d i t i o n to the p h a r m a c o l o g i c a l effects described a b o v e , t h e y are used in m o d e l studies on early m o r p h o logical a l t e r a t i o n s l e a d i n g to m a l f o r m a t i o n , e.g. cleft o f lip, alveolus a n d p a l a t e [17, 18]. C o m p l e m e n t i n g b i o c h e m i c a l studies h a v e n o t yet been p e r f o r m e d . O u r s t u d y o n the action o f h y d r o x a m i c acids on g r o w t h a n d d i f f e r e n t i a t i o n in tissue c u l t u r e cells m a y o p e n the w a y for f u r t h e r analysis o f in vivo effects o f h y d r o x a m i c acids.

MATERIALS AND METHODS Chemicals Formhydroxamic acid (FH), N-formylglycinie(FG) and N-methyl-acetohydroxamic (MAH) acid were obtained from NATEC, ln~titut fiir Naturwissenschaftlich-Technische Dienste GmbH, Hamburg, FRG; hadacidin (HA) was obtained from Merck, Sharp & Dohme, Rahway, N. J., U.S.A.; 6-N-hydroxy-arginine(6-N-Arg)was supplied by Professor Viswanatha, University of Waterloo, Ontario, Canada. All other chemicals were obtained from Merck & Co., Darmstadt, F.R.G. Cell cultures . " The Friend virus transformed murine erythroleukemia cell-line F4-6 originally established by Ostertag et al. [16] was propagated in our laboratory. Cells were grown in suspension in Joklik's minimal essential medium (Flow laboratories. U.K.) buffered at pH 7.4 with 25 mM HEPES and supplemented with 10070fetal calf serum (FCS) (Boehringer Mannheim, F.R.G.) [10]. Cells from logarithmically growing cultures were used for the experiments described below. Cell growth was followed by counting aliquots of two samples in a hemocytometer. For hemoglobin assay, cell extracts were prepared by treatment with 0.1% NP-40 and sonication. Extracts were centrifuged and stored frozen or used immediately. Hemoglobin content in the cell extract was measured spectrophotometrically in duplicate samples with the benzidine procedure described by Luftig et al. [14]. The variability observed was __+7% for the cell number and ±5% in the hemoglobin assay.

RESULTS H y d r o x a m a t e i n d u c e d inhibition o f p r o l i f e r a t i o n o f F r i e n d cells T h e p r o l i f e r a t i o n o f F r i e n d cells is d r a s t i c a l l y i n h i b i t e d b y increasing c o n c e n t r a t i o n s o f several h y d r o x a m i c acids. A d d i t i o n o f 50-100 p.g/ml o f M A H a n d o f 100 ~ g / m l o f A H to a F4-6 c u l t u r e reduces the g r o w t h r a t e by m o r e t h a n 95 % within 48 h. F H a n d H A s h o w similar b u t less severe effects (Table 1). A l l f o u r h y d r o x a m a t e s i n h i b i t the g r o w t h r a t e a f t e r a d d i t i o n to cells, b u t cell-growth recovers r a p i d l y a f t e r r e m o v i n g h y d r o x a m i c acids ( T a b l e 2). T h e h y d r o x a m a t e related c o m p o u n d s F G a n d 5 - N - A r g d o n o t i n t e r f e r e with g r o w t h o f F4-6 cells u n d e r o u r a s s a y c o n d i t i o n s . I n h i b i t i o n o f D M S O induced d i f f e r e n t i a t i o n o f F r i e n d cells b y h y d r o x a m i c acids I n c u b a t i o n o f F r i e n d cells with 2 0 - 1 0 0 ~tg/ml o f the i n d i c a t e d h y d r o x a m i c acids for 96 h does not alter the h e m o g l o b i n c o n t e n t o f cells, which r e m a i n e d at 4 p.g/10 s cells. C l e a r l y the h y d r o x a m i c acids d o n o t induce e r y t h r o i d d i f f e r e n t i a t i o n o f F r i e n d cells. H o w e v e r , interference with D M S O i n d u c e d d i f f e r e n t i a t i o n b y h y d r o x a m i c acids b e c o m e s a p p a r e n t after s i m u l t a n e o u s a d d i t i o n o f D M S O a n d h y d r o x a m i c acids. A l l h y d r o x a m i e acids decrease the h e m o g l o b i n c o n t e n t o f F r i e n d cells in a d o s e d e p e n d e n t m a n n e r a f t e r a 96 hi n d u c t i o n with 1% D M S O (Fig. 1). F G does n o t alter the h e m o g l o b i n c o n t e n t in D M S O induced cells. E x p e r i m e n t s designed to d e t e r m i n e the effect o f h y d r o x a m i c acids on D M S O i n d u c e d e r y t h r o i d d i f f e r e n t i a t i o n were p e r f o r m e d in p a r a l l e l cultures with a n d w i t h o u t D M S O . Inc u b a t i o n o f F4-6 cells with h y d r o x a m a t e s (50 p,g/ml) for v a r i o u s time intervals d u r i n g ind u c t i o n o f d i f f e r e n t i a t i o n with 1% D M S O r e v e a l e d i n t e r f e r e n c e with the i n i t i a t i o n p h a s e o f d i f f e r e n t i a t i o n . W h e n h y d r o x a m a t e s are a d d e d at a later time a f t e r D M S O they increase the h e m o g l o b i n c o n t e n t o f i n d u c e d cells (Fig. 2). P r e i n c u b a t i o n o f F r i e n d cells for 24 h with 50 ~tg/ml o f the respective h y d r o x a m a t e s inhibits h e m o g l o b i n p r o d u c t i o n i n d u c e d by D M S O to the s a m e extent as does a d d i t i o n o f h y d r o x a m a t e s d u r i n g the first 24 h o u r s after D M S O a d d i t i o n ( T a b l e 3).

Hydroxamic acids and cell metabolism TABLE 1. INHIBITIONOF CELL-GROWTH BY HYDROXAMATES

Concentration in ~g/ml

0-24 h

24-48 h

48-72 h

72-96 h

FH 20 50 100

78 42 15

73 39 15

43 46 43

75 50 36

HA 20 50 100

76 55 26

69 42 27

21 59 31

36 45 88

AH 20 50 100

87 62 50

55 27 5

84 24 4

89 41 9

MAH 20 50 100

87 62 56

33 21 9

77 12 3

64 7 1

HA 20 50 100

68 47 21

69 43 21

15 43 46

38 45 54

+ FG + 20 + 50 + 100

Hydroxamate mediated inhibition of cell-growth was determined at the indicated intervals. Ceils were harvested from logarithmically growing cultures and seeded at a density of 2 × 10~/ml. The extent of inhibition by FH, HA, AH, M A H and the combination of H A and FG is listed in e/0 of the growth of control cells.

TABLE 2. RECOVERYOF CELL-GROWTH

Cells × 106/ml (growth rate) Start

48 h

96 h

144 h

Control

0.2

1.81(9.0) 3.12(2.2) 6.61(2.1)

FH

0.2

0.48(2.4) 2.5 (5.2) 6.03(2.4)

HA

0.2

0.57(2.8) 3.1 (5.4) 5.86(1.8)

FG

0.2

1.71(8.5) 3.18(2.4) 6.6 (2.0)

The effect on Friend F4-6 cell growth in the presence of I g/0 DMSO of a 48 h exposure to FH, HA and FG in concentration of 100 ~g/ml was measured. Cell samples were taken from a logarithmically growing culture and seeded at a density o f 2 x 105/ml. At 4 8 h , 9 6 h a n d 144h the growth of the ceils was determined by counting with a hemocytometer. Medium (Joklik MEM + 10°7o FCS) was changed every 48 h (growth rate = ratio cell densities).

465

CHRISTOF U. ANDERS, THOMAS V. KREYBIG a n d GEBHARD KOCH

466

1 0 0 ~

~ $0 o

1

--

20

I

I

50 concenlrotioninjug Iml

I00

FIG. 1. Inhibition of DMSO induced differentiation by hydroxamic acids. Cells from a logarithmically growing culture were seeded at a concentration of 2 x l0 s cells/ml in parallel cultures in medium containing 1°70 DMSO and hydroxamates in concentrations of 0, 20, 50, 100 lig/ml. FG ( = Lx), HA ( = O), FH ( = ~), MAH (= U2) and AH ( = X). At 96h hemoglobin content was determined spectrophotometrically in cell extracts by the method of Luftig et al. [14]. The data are listed in % of control.

100- (a)

loo

~!i!i!i!i :::::2 :'S-S" S{':':

!5o

S':'X" ,S'S-: S'S-Z-

.02

:i:!:i:i:! :.s,x-

50o

_N 'i

ii!i!i!i! !i!?!i!i S'S'2 :,:-:.:,:, :':':'S

-S'S-:

i!i i i !i

O-2t, 2t,-46 t.8-72 72-96 h time ot exposureto formhydmxnmic~d

O-2t, 2t,-t,8 68-7272-96h timeof exposureto hodocidin

FIG. 2. Effect of duration of exposure to hydroxamic acids on hemoglobin synthesis in DMSO induced Friend cells. Friend F4-6 cells were seeded at a concentration of 2 x 10~cells/ml, incubated in the presence of 1% DMSO for 96 h and the hemoglobin content was then measured spectrophotometrically (see Fig. 1). The data are listed in % of control. Hydroxamic acids (50p.g/ml) were added from 0-24, 24-48, 48-72 and 72-96 h. The medium in all cultures was renewed every 24 h. (a) Formhydroxamic acid; (b) Hadacidin.

DISCUSSION Table 4 summarizes known pharmacological properties, tetratogenic effects of hydroxam a t e s a n d t h e i r i n f l u e n c e o n g r o w t h a n d d i f f e r e n t i a t i o n o f F4 cells as o b s e r v e d in this s t u d y . A s p e c i f i c p r o p e r t y o f h y d r o x a m i c a c i d s is t h e c a p a c i t y to i n d u c e t h e s y n d r o m e o f c l e f t o f lip, a l v e o l u s a n d p a l a t e in r a t s , in 1 0 - 5 0 % o f t h e o f f - s p r i n g w h e n h y d r o x a m i c a c i d s a r e a p p l i e d o n d a y 12 o f g e s t a t i o n [11, 12]. H y d r o x a m a t e s at a c o n c e n t r a t i o n o f 2 0 - 1 0 0 ~ g / r n l i n t e r f e r e w i t h t h e g r o w t h o f s e v e r a l t i s s u e c u l t u r e cell lines in vitro. H i g h e r

467

H y d r o x a m i c acids and cell metabolism TABLE 3. COMPARISON OF THE EFFECT OF HYDROXAMIC ACIDS EXERTED BY PREEXPOSURE AND BY EXPOSURE AT DIFFERENT TIME INTERVALS DURING DMSO INDUCTION TO HYDROXAMIC ACIDS OF HEMOGLOBIN SYNTHESIS IN FRIEND CELLS

Time of exposure to hydroxamic acids

F o r m h y d r o x a m i c acid Hadacidin Acetohydroxamic acid N-Methylacetohydroxamic acid C o m b i n a t i o n of hadacidin and N-formylglycin

°70 of induction -24-0 h

0-24 h

0-96 h

51 73 68 77

50 61 80 69

23 48 20 21

59

58

26

Friend cells at a density of 2 x 10' cells/ml were incubated for 24 h with 50 p_g of hydroxamic acids. The inducibility of these cells, determined as described in the legend to Fig. 1, was compared to that of unexposed cells (control) and cells exposed either during the first 24 h or during 96 h. All cell cultures were incubated from 0-96 h in the presence of DMSO.

doses o f hydroxamates are toxic for cells. However, the mechanism of the cytotoxic effects has not been analysed. In previous studies hydroxamates (20-200 ~tg/ml) have been found to inhibit ribonucleotide reductase isolated and partially purified from N o v i k o f f h e p a t o m a cells [22]. The mechanism for either hydroxamates induced growth inhibition, or interference with induction of erythroid differentiation is unknown. The use of agents which interfere with iron transport on inhibiting Friend cell differentiation is novel and may offer a new a p p r o a c h to study events in Friend cell differentiation especially if mutants blocked in differentiation are used to define whether the block is an early or a late event following D M S O addition. A n u m b e r of studies have revealed other agents that can interfere with erythroid differentiation in Friend cells. Interferon [14], several local anesthetics, e.g. lidocaine, xylocaine [2], and amphotericin B [4] inhibit DMSO induced differentiation, yet the precise mechanism of this inhibition is also unkown. Certain nucleoside analogs [3, 10] also interfere with the induction o f terminal differentiation. Nucleoside analogs such as 5-bromodeoxyuridine induce their inhibitory effect only in TK + cells where they are incorporated into D N A [10]. H y d r o x a m a t e s are only effective blocking differentiation when present up to 24 h before D M S O addition or during the first 24 h together with DMSO that is prior to the c o m m i t m e n t phase of differentiation. When hydroxamic acids are added at later times the synthesis o f hemoglobin is increased. The inhibition of differentiation by hydroxamates does not seem to be due to generalized cytotoxicity since the growth inhibitory effect o f a 24 h-exposure of cells to hydroxamates is readily reversible (Table 2). Previous experiments with 5-bromo- and 5-ethyldeoxyuridine indicated that incorporation of the analogs into D N A at an early time prior to commitment resulted in inhibition of erythroid differentiation [10] presumably by inhibition ofglobin m R N A transcription [20]. The addition of 5-ethyldeoxyuridine after c o m m i t m e n t to differentiation resulted i/r increased hemoglobin synthesis. The ability of the hydroxamic acids to inhibit ribonucleotide reductase suggests that hydroxamic acids may alter the cellular pools of ribo- and deoxyribonucleotides. We are presently involved in the analysis of nucleotide pools after the onset o f erythroid differentiation and subsequent to exposure to hydroxamic acids.

468

CHRISTOF U. ANDERS, THOMAS V. KREYBIG a n d GEBHARD KOCH TABLE 4. PHARMACOLOGICAL PROPERTIES OF HYDROXAMIC ACIDS

Molecular LD~o weight (mg/kg) Formhydroxamic acid (FH): O OH II / H--C--N \ H Hadacidin (HA): O OH IJ / H--C--N \

TD* (mg/kg)

Teratogenic effects

G-ID~o~ (~tg/ml)

D-ID~0:~ (p.g/ml)

61

570

500-550 Induces C L A P § Brachygnathia

69

20

119

5000

1000-3000 Induces C L A P Brachygnathia

71

46

74

2000

*

CH2COOH Acetohydroxamic acid (AH): O OH ~1 / CH3--C--N \ H N-Methyl-Acetohydroxamic Acid (MAH): O OH II / CH3--C--N \ CH,

87

Hydroxy-urea (HU): O OH II / NH2--C--N \ H N-Formylglycin (FG): O H U / H--C--N \ CH~COOH

800

Several

40

34

200-400

Several

28

29

Induces C L A P Brachygnathia

Not tested

Not tested

No effect on growth

No effects on differentiation

No effect on growth

Not tested

76

4700

750

103

3000

No teratogenic effects

194

Not known

Not known

6-N-Hydr oxy-ar~;inine (6-N-Arg):

H

I

Not known

OH

/

HOOC--C--CH2--CH2CH2--N NH I \ // NH2 C \ NH2 *TD: Dose to induce teratogenic effects. ~'G-ID,0: Concentration, that inhibits cell growth after 96 h to 50%. :]:D-ID,0: Concentration, that inhibits differentiation (hemoglobin content of cells) after 96 h to 50%. §CLAP: Cleft o f lip, alveolus and palate.

Hydroxamic acids and cell metabolism

469

A c k n o w l e d g e m e n t s - - T h i s work was supported by the Stiftung Volkswagenwerk and by the Deutsche Forschungsgemeinschaft. We thank Dr. J. A. Bilello and Dr. W. Ostertag for stimulating discussions and hclp in the preparation of the manuscript. REFERENCES I. AOYAGI T. & SUMMERSKILL W. H. J. (1966) Inhibition by acetohydroxamic acid of h u m a n mucosal and faecal urease-specific activity. Lancet 1,296. 2. BERNSTEIN A., BOYD A., CRICHLE¥ V. & LAMB V. (1976) Induction and inhibition of Friend leukemic cell differentiation: the role o f m e m b r a n e active c o m p o u n d s . In Biogenesis and Turnover o f Membrane Macromolecules (Cook J., Ed.) p. 145. Raven Press, New York. 3. BICK M. D. & CULLEN B. R. (1976) Bromodeoxyuridine inhibition of Friend leukemia cell induction by butyric acid: time course o f inhibition, reversal, and the effect of other analogs. Somatic Cell Gen. 2, 545. 4. DUBES. K., GAEDICKEG., KLUGEN., WEIMANB. J., MELERIS H., STEINHEIDERG., CROZIERT., BECKMANN H. & OSTERTAC W. (1973) Hemoglobin synthesizing mouse and h u m a n erythroleukemic cell lines as modes systems for the study of differentiation and control of gene expression. In Proc. 4th Int. Syrup. Princess Takanmatsu Cancer Res. Fund T o k y o (NAKAHARA et al., Eds.) p. 99. University of Tokyo Press, Tokyo. 5. DUBES. K., PRAGNELL I. B., KLUGE N., GAEDICKE G., STEINHEIDER G. & OSTERTAG W. (1975) Induction of endogenous and spleen focus-forming viruses during dimethylsulfoxide-induced differentiation of mouse erythroleukemia cells transformed by spleen focus-forming virus. Proc. Natn. Acad. Sci. U.S.A. 72, 1863. 6. EMERY T. (1971) Hydroxamic acids o f natural origin. Adv. Enzymol. 35, 135. 7. FREESE E., LOPEZ J. & FREESE E. (1979) Initiation o f bacterial and yeast sporulation by partial deprivation of guanine nucleotides. In Regulation o f Macromolecular Synthesis by L o w Molecular Weight Mediators (KOCH G. & RICHTER ~ . , Eds.), p. 127. Academic Press, New York. 8. FRIEND C., SCHER W., HOLLAND J. G. & SATO T. (1971) Hemoglobin synthesis in murine virus-induced leukemic cells in vitro: Stimulation of erythroid differentiation by dimethyl sulfoxide. Proc. Natn. Acad. Sci. U.S.A. 68, 378. 9. KOBASHI K., TAKEnE S., TERASHIMA N. & HASE J. (1975) Inhibition of urease activity by hydroxamic acid derivates of a m i n o acids. J. Biochem. 77, 837. 10. KOCH G., WARNECKE G., KOHNE J., BILELLO J. A. (1979) Effect of base analogs upon the induction of differentiation in Friend erythroleukemia cells. Adv. OpthalmoL 38, 222. 11. KREYBIG T. v. (1976) Zur Teratologie der Hydroxams~iuren. Med. Klin. 71,844. 12. KREYBIGT. v., PREUSSMANN R. & SCHMIDT W. (1968) Chemische Konstitution und teratogene Wirkung bei der Ratte. Carbons~iureamide, Carbons~.urehydrazide und Hydroxams~iuren. Arzneimttelforsch. (Drug. Res.) 18, 645. 13. LEONG J. & NEILANDS J. B. (1976) Mechanism of siderophore iron transport in enteritic bacteria. J. Bacteriol. 126, 823. 14. LUFTIG R. B., CONSCIENCE J. F., SKOULTCHIA., MCMILLAN P., REVEL M. & RUDDLE F. H. (1977) Effect of interferon on dimethylsulfoxide-stimulated Friend erythroleukemic cells: ultrastructural and biochemical study. J. Virol. 23, 799. 15. NEtLANDS J. B. (1967) Hydroxamic acids in nature. Sophisticated ligands play a role in iron metabolism and possibly in other processes in microorganisms. Science 156, 1443. 16. OSTERTAG W., CROZIER T., KLUGE N., MELDERIS H. & DUBE S. K. (1973) Action of 5-bromodeoxyuridine on the induction of haemoglobin synthesis in m o u s e leukemia cells resistant to 5-BUdR. Nature 243, 203. 17. PREI~ER G. & KREYI31GT. v. (1973) !Slber die Atiologie der Lippen-Kieferspaltformen und Gaumenspalten beim Menschen und im Tierversuch. Sep. Experientia 29, 225. 18. PFEIVER G., SCHLOTE H. H. & KREYBIG T. V. (1984) Clefts of the face in animal experiments. J. max. fac. Surg. 2, 230. 19. ROBBINS E., FANT J. & NORTON W. (1972) Intracellular iron-binding macromolecules in HeLa cells. Proc. Natn. Acad. Sci. U.S.A. 69, 3708. 20. Ross J., GEILEN J., PACKMAN S., IKAWA Y. & LEDER P. (1974) Globin gene expression in cultured erythroleukemic cells. J. Mol. BioL 87, 697. 21. SATO T., FRIEND C. & DEHARVEN E. (1971) Ultrastructural changes in Friend erythroleukemia cells treated with dimethyl sulfoxide. Cancer Res. 31, 1403. 22. VAN'T RIET B., KIER L. B. ELFORD H. L. (1980) Structure-activity releationships of benzohydroxamic acid inhibitors of ribonucleotide reductase. J. Pharm. Sci. 69, 856.