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Cytopathogenieity of Entamoeba histolytica: Trophozoite Homogenates Modulate DNA Synthesis in a Mammalian Cell Line. Salvador Said-Fernfindez and ...

Zeitschrift for

Parasitenkunde

Z Parasitenkd (1981)65 : 11 17

Parasitorogy Research

9 Springer-Verlag 1981

Cytopathogenieity of Entamoeba histolytica: Trophozoite Homogenates Modulate DNA Synthesis in a Mammalian Cell Line Salvador Said-Fernfindez and Rub6n Ldpez-Revilla* Departamento de Biologia Celular, Centro de Investigaci6n del IPN, Ap. Postal 14-740, M~xico 14, D.F., M6xico

Abstract. We examined the effect of total trophozoite homogenates from

four axenized strains of Entamoeba histolytica (HK9, HM1, HM2, and HM3) on the DNA synthesis of subconfluent cultures of Chinese hamster ovary (CHO) cells incubated at low (0.1%) serum concentration. HM1, HM2, and HM3 extracts increased [3H]thymidine incorporation to acidinsoluble material in CHO cells up to a maximum of 2.5, 1.5, and 1.5 times respectively, at doses of amebal protein ranging from 16 to 125 gg/ml, HM1 and HM2 extracts at doses higher than those causing maximal stimulation, and HM3 and HK9 extracts above 250 ~tg protein per ml, progressively inhibited [3H]thymidine incorporation by CHO cells at a strain-specific rate. The extracts with both the most potent stimulatory and inhibitory effects were those from HM1 and HM2, also the most virulent strains. This strain-specific ability of amebal products to modulate cell DNA synthesis may play a significant role in amebal virulence.

Introduction

Amebiasis, a parasitic disease affecting nearly one tenth of the world population (World Health Organization 1959), is characterized by necrotic lesions caused and circumscribed by Entamoeba histolytica trophozoites, whose virulence is strain-specific (Neal 1971 ; Mattern and Keister 1977). As in other invasive microorganisms, E. histolytica virulence appears to be a complex property determined by multiple unknown factors (L6pez-Revilla and Said-Fernandez 1980). It is only recently that several groups of workers have been able to detect various toxic activities of trophozoite extracts on animal cells (Bos 1979; Lushbaugh et al. 1979; Mattern et al. 1980; Ldpez-Revilla and Said-Fernfindez 1980) and are now trying to identify the corresponding amebal factors to define their involvement in virulence. * To whom offprint requests should be sent

0044-3255/81/0065/0011/$01.40

12

S. Said-Fern~mdez and R. L6pez-Revilla

T h e v i r u l e n c e o f Vibrio cholerae a n d e n t e r o p a t h o g e n i c Escherichia coli is primarily determined not by toxic factors but by substances called cytotonins ( K e u s c h a n d D o n t a 1975) b e c a u s e t h e y h a v e p l e i o t r o p i c m o d u l a t o r y e f f e c t s o n t h e f u n c t i o n , m e t a b o l i s m , 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 a n i m a l cells ( K e u s c h a n d D o n t a 1 9 7 5 ; G i l l 1 9 7 7 ; R i c h a r d s a n d D o u g l a s 1978). I n t h i s p a p e r we d e s c r i b e t h e d o s e - a n d s t r a i n - d e p e n d e n t m o d u l a t i o n t h a t E. histolytica t r o p h o zoite homogenates exert upon DNA synthesis in cultures of the Chinese hamster o v a r y ( C H O ) cell line, A t l e a s t p a r t o f t h i s e f f e c t a p p e a r s to b e c a u s e d b y c y t o t o n i c a m e b a l f a c t o r ( s ) t h a t m i g h t b e i n v o l v e d in a m e b a 1 v i r u l e n c e .

Materials and Methods Strains and Culture Methods. The axenic E. histolytiea strains used were HK9, HM1; IMSS (HM1), HM2:IMSS (HM2), and HM3:IMSS (HM3). The sources of HK9, HM2, and HM3 have been mentioned before (L6pez-Revilla and G6mez 1978; L6pez-Revilla and Said-Fernf.ndez 1980); HM1 (Diamond et at. 1972) was obtained from M. de la Torre (Centro de Estudios Sobre Amibiasis, IMSS, M+xico, D.F., M6xico). The CHO cell line derives from a clone obtained in our laboratory from strain CCL61 (American Type Culture Collection, Rockville, MD, USA), originally established in culture by Puck et al. (1958). Amebae were grown in Diamond's TPS-1 medium (Diamond 1968), with minor modifications (L6pez-Revilla and G6mez 1978). For cultures and experiments involving CHO cells, we used Eagle's minimum essential medium (MEM) prepared with Earle's saline and supplemented with non-essential amino acids (Eagle 1959) and dialyzed calf serum (Biocei, S.A., M6xico, D.F.) at the concentrations indicated.

Autoradiographic Analysis of [3H]Thymidine Incorporation. Five ml of MEM supplemented with 10% serum and containing 5• 10 s growing CHO cells were inoculated into 60x 15 mm tissue culture dishes (Falcon, Oxnard, CA, USA). Twelve hours later, when cells were attached to the dishes, the cultures were washed three times with phosphate saline buffer (PBS), and then 5 ml fresh MEM containing 5 gCi/ml of [methyl-3H]thymidine ([3H]dThd; 2 Ci/mmole, New England Nuclear, Boston, MA, USA) and variable serum concentrations were added to each dish. Arabinosyl cytosine hydrochloride (Ara C) at 30 ~tg/ml was also added to two plates in each experiment. The cultures were then placed 6 h at 37~ in a CO2 incubator, washed three times with PBS at the end of this period, fixed for 20 min with 2.5% glutaraldehyde (J.T. Baker Chemical Co., Phillipsburg, NJ, USA) in PBS, and washed again. After fixation, the cells were overlayed with AR-10 autoradiographic stripping film (Kodak Ltd., London, England) that was developed after six days. After staining with Glemsa, 200 cells from each dish were observed under the microscope and the proportion of [abeled ceils (i.e., ~those with more than four grains over the nucleus) was estimated.

Kinetics of [3H]dThd Incorporation by CHO cells. One ml of MEM supplemented with 10% serum and containing 6 • l0 s growing CHO cells was inoculated into 35 x 10 mm Falcon tissue culture dishes. Twelve hours later, the cultures were washed twice with PBS and then 1 ml fresh MEM containing 5 gCi/ml t3H]dThd and supplemented either with 0.1% or 10% serum was added to each plate. After variaNe incubation periods, 1 ml of 1% sodium dodecyl stllphate was added to stop incorporation and dissolve the cells, the dishes were scraped with a rubber policeman, their contents were placed into tubes to each of which 2 ml of 10% trichloroacetic acid (TCA) were added. After 10 min incubation in ice, the acid-insoluble precipitates were retained by filtration through 24 mm GF/C discs (Whatman Ltd., Maidstone Kent, England); the discs were washed three times with TCA and with ethanol, placed into scintillation vials and dried. To each vial 5 ml 0.6% 2,5-diphenyloxazole (Packard Instrument Co., Dowers Grove, IL, USA) in toluene were added, and the incorporated radioactivity was measured in a scintillation counter (Packard Tri-Carb, model 3003), adjusted to 43% efficiency for tritium. In our analysis of [3H]dThd incorporation, we included all the acid-precipitable material present in the culture medium and in the cells attached to the dishes. Incorporation was not due to contamination since in control dishes (incubated without CHO cells and included in every experi-

Modulation of Cell DNA Synthesis by E. histotytica

13

ment), it was never more than 1% that of untreated cultures. Among the controls we also included cultures treated with Ara C at a supramaximal inhibitory dose (data not shown) in order to ensure that we would be able to detect and measure a predictable inhibition of DNA synthesis.

Preparation of Cell-Free Extracts. Trophozoites were harvested at the end of the log phase by immersing the cultures for 10 rain in ice and centrifuging them for 9 rain at 600 x g and 4 ~ C. Pelleted trophozoites were washed by centrifugation twice with PBS, and suspended with two volumes of a sterile solution of isotonic sucrose (250 mM sucrose, 10 mM NaC1, 5 mM CaC12, 5 mM MgCI2, 5 mM 2-mercaptoethanol, 40 units/ml penicillin G, and 100 tag/ml streptomycin sulfate; pH 7.4). Suspended amebae were disrupted with 15 strokes of a Potter-Elvehjem homogenizer fitted with a teflon plunger and driven at 1,000 rev/min. Liver extracts were obtained from male Wistar rats weighing 150-250 g; the rats were killed by decapitation, their livers were rapidly excised and chopped, the liver fragments were washed three times with cold PBS, suspended in two volumes of isotonic sucrose, and homogenized as were amebae.

Treatment of CHO Cultures with Extracts. One ml of MEM supplemented with 10% serum and containing 3.5 x 105 growing CHO cells was inoculated into 35 x 10 mm tissue culture dishes. Twelve hours later, the cultures were washed twice with PBS, 1 ml fresh MEM containing 5 gCi [3H]dTdh/ml and supplemented with 0.1% serum was added to each dish, and the cultures were incubated for 2 h ("preincubation" period) before adding the extracts to them. At the end of preincubation, 200 gl of predetermined dilutions of the extracts (in isotonic sucrose) were added to each culture and incubation followed for another 8 h. Protein in the extracts was measured with the method of Lowry et al. (1951) and with bromsulphthalein (Said-FernS.ndez and Lfpez-Revilla 1976). Normalization and Statistical Analysis of [3H]dThd Incorporation. We performed two independent experiments with extracts obtained from rat livers and the four amebal strains. In each experiment, eight untreated cultures were included; at the end of preincubation, [3H]dThd incorporation was stopped in four of them while 200 gl isotonic sucrose were added to each of the remaining four that were incubated for an additional 8 h, as were the treated cultures. From the incorporation of cultures incubated during the I0 h that the experiments lasted, we substracted that occurring during preincubation in order to estimate net incorporation, i.e., that which occurred during the 8 h treatment with the extracts. To the average net incorporation of untreated cultures in a given experiment a value of 1.00 was assigned to normalize the activity of treated cultures. The normalized activities of each dose of each extract tested in the two experiments were averaged, and the statistical significance of the maximal stimulation was calculated by means of one-tailed Student's t test. The mean (i.e., 50%) inhibitory dose of each strain was obtained by graphic interpolation in Fig. 2.

Chemicals. Inorganic salts, toluene, and TCA, were reagent grade and purchased from J.T. Baker S.A. (M~xico, D.F.); the organic components of MEM, Ara C, 2-mercaptoethanol, sucrose, and sodium dodecyl sulfate were from Sigma Chemical Co. (St. Louis, MO, USA) ; penicillin G (sodium salt), and streptomycin sulfate were from Farmacduticos Lakeside (M~xico, D.F.). Results

Effect o f Serum on [ 3 H ] d T h d Incorporation by C H O Cells. S i n c e a m e b a l e x t r a c t s might cause either stimulation or inhibition of DNA synthesis, we reasoned t h a t b o t h e f f e c t s c o u l d b e d e t e c t e d o n l y in C H O c u l t u r e s h a v i n g s u b m a x i m a l rates of DNA replication, a condition that could be attained by decreasing t h e s e r u m c o n t e n t in t h e c u l t u r e m e d i u m . T h u s , t h e e f f e c t o f s e r u m o n [ 3 H ] d T h d i n c o r p o r a t i o n b y C H O cells w a s a n a l y z e d a u t o r a d i o g r a p h i c a l l y . A t all s e r u m c o n c e n t r a t i o n s t e s t e d , t h e cells r e m a i n e d in g o o d m o r p h o l o g i c c o n d i t i o n , a n d the radioactive label was a l m o s t exclusively i n c o r p o r a t e d into the nuclei. The h i g h e s t p r o p o r t i o n o f l a b e l e d cells ( 0 . 2 0 - 0 . 2 4 ) o c c u r r e d w i t h e i t h e r 1 % o r 1 0 %

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S. Said-FernS.ndez and R. L6pez-Revilla

Table 1. Serum-dependence of [3H]dThd incorporation to C H O nuclei Additions to culture medium

Fraction of cells with labeled nucle?

None 0.1% 1.0% 10.0% 10.0%

0.00 0 . t l +0.04 0.20 + 0.07 0.24 + 0.04 0.00

serum serum serum serum plus Ara C b

Determined autoradiographically in subconfluent monolayer cultures incubated 6 h in medium containing 5 gCi [3H]dThd per ml. Values are the m e a n + SD of two experiments u Arabinosyl cytosine, 30 l~g/ml

dbJ '

'

'

'

'

~

,

0_ o

_a

Z O

2E

,

~I

i-

u- N 1'5

20.5 O

O"

9

Z

al FA"-'--'-A

_^

z~ o ~I.0

bJ Z 0.5

Ig T "3-

z

O0

1

l

2

,

4

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6

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8

l

I0

INCUBATION TIME (HOURS)

l

12

o

2

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d

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; i ~

2 0.4 O. AMEBAL PROTEINS(mg/ml)

Fig. 1. Kinetics of [3H]dThd incorporation by C H O cells incubated with 0.1% (o) and 10% serum (o) Fig. 2. Effect of extracts on [3H]dThd incorporation by C H O cells incubated with 0.1% serum. Average incorporation of untreated cultures fluctuated between 8.5 x 104 and 3.6 x l0 s cpm. The symbols represent the m e a n of two independent experiments performed with extracts from E. histolytica strains H K 9 ([]), HM1 (e), H M 2 (o), and H M 3 (u), and with rat liver extracts (zx)

serum and decreased to half (0.11) with 0.1% serum, while no label above the background was detected in cultures incubated without serum, nor in cultures with 10% serum to which Ara C was also added (Table 1). Therefore, D N A synthesis by nuclei of C H O cells appeared to occur at around half the maximal rate in cultures with 0.1% serum. We next determined the kinetics of [3H]dThd incorporation for 12 h in cultures incubated with 0.1% and 10% serum. Incorporation was linear with 0.1% serum, while it increased exponentially with 10% serum, being quantitatively similar at both serum concentrations only during the first 2 h of incubation (Fig. 1).

Modulation of Cell DNA Synthesisby E. histolytica

15

Effect of Extracts on [3H]dThd Incorporation by CHO cells Incubated with 0.1% Serum. Amebal extracts caused either stimulation or inhibition of [3H]dThd incorporation, depending on the dose used, as shown in Fig. 2. Stimulation occurred at doses smaller than those causing inhibition. Rat liver extracts, at protein concentrations comparable to those of amebae, did not have a significant effect on D N A synthesis. Both maximal stimulation and the dose causing it, were specific for each amebal strain. HM1 extracts produced the highest stimulation (2.4 times the incorporation of untreated cultures, p H M 2 > HK9 > HM3. Discussion At low doses, total homogenates of three of the four amebal strains analyzed by us progressively stimulated [3H]dThd incorporation by CHO cells up to a maximum, while at large doses, extracts from all strains caused inhibition (Fig. 2). The degree of maximal stimulation and the dose of extract causing it, as well as the inhibitory potency were strain-specific. This modulatory activity also appears to be species-specific since rat liver extracts did not have a significant effect upon D N A synthesis by CHO cells (Fig. 2). According to the pattern of their activities, two types were found among the strains tested: (0 one strain was only inhibitory (HK9), and (i/) three caused both stimulation and inhibition (HM1, HM2, HM3). A positive correlation appears to exist between virulence and potency in the extracts since HM1 and HM2, the two most virulent strains (Mattern and Keister 1977; A MartinezPalomo personal communication) provided the most potent extracts both to stimulate and to inhibit [3H]dThd incorporation. During their exposure to amebal extracts, CHO cells were maintained in medium supplemented with low serum to keep them in a state of submaximal rate of D N A synthesis under which stimulation as well as inhibition of D N A synthesis could be detected. After 10 h of incubation, cultures with 10% serum incorporated 1.7 times more [3H]dThd than those with 0.1% serum (Fig. 1), while autoradiographically no difference in the proportion of labeled nuclei was found between cells incubated with 1% and 10% serum (Table 1). CHO

16

S. Said-Fernf.ndez and R. L6pez-Revilla

cultures incubated with both 1% and 10% serum attained a similar (maximum?) rate of DNA synthesis, while those with 0.1% serum had a submaximal rate and were selected for treatment with amebal extracts. The incorporation of [3H]dThd in the cultures stimulated maximally by HM2 and HM3 extracts reached the level of untreated cultures incubated with 10% serum, while this level was surpassed by a factor of 1.5 with HM1 extracts. Therefore, under our experimental conditions, HM2 and HM3 extracts equalled and HM1 extracts exceeded the stimulatory effect of 10% serum on DNA synthesis by CHO cells. The mechanism underlying stimulation by amebal extracts may include an increase in thymidine uptake or DNA polymerization, or both. Stimulation occurred with the lowest doses of amebal extracts and was not accompanied by microscopic damage or cell detachment. On the other hand, the inhibitory effect appeared to be less specific, because it required much larger doses of extracts and was accompanied by cytolysis, cell rounding, and detachment of the monolayers, cytopathic effects previously described (Bos 1979; Lushbaugh et al. 1979; Mattern etal. 1980; L6pez-Revilla and SaidFerngndez 1980) and confirmed by us (data not shown). These results lead us to believe that the increase in [3H]dThd incorporation may have been caused by amebal cytotonic factor(s) different from those responsible for the inhibition. However, we cannot at present exclude the possibility that both effects are due to the same factor(s). If the amebal factor(s) that we have shown here to modulate [aH]dThd incorporation by CHO cells were produced in tissues infected by amebae, the ensuing cytotonic activities could play a significant role in the pathogenesis of amebiasis. Acknowledgments. We thank Herminia G. Martinez for the CHO cells and valuable help, and Hugo Ar6chiga and Walid Kuri-Harcuch for reviewing tile manuscript. This work was supported in part by grant PCSANAL 790272 from Programa Nacional de Salud (CONACYT, M6xico) awarded to R.L.-R., and by a CONACYT predoctoral fellowship awarded to S.S.-F.

References Bos HJ (1979) Entamoeba histolytica: Cytopatbogenicity of intact amebae and cell free extracts; isolation and characterization of an intracellular toxin. Exp Parasitol 47 : 36%377 Diamond LS (1968) Techniques of axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and E. histolytica-like amebae. J Parasitol 54:1047 1056 Diamond LS, Mattern CFT, Bartgis IL (1972) Viruses of Entamoeba histolytica. J Virol 9:326-341 Eagle H (1959) Amino acid metabolism in mammalian cell cultures. Science 130:432-437 Gill MD (1977) Mechanism of action of cholera toxin. Adv Cyclic Nucleotide Res 5:85-118 Keusch FT, Donta ST (1975) Classification of enterotoxins on the basis of activity in cell culture. J Infect Dis 131 : 58-69 L6pez-Revilla R, G6mez R (1978) Entamoeba histolytica, E. invadens, and E. moshkovskii: Fluctuations of the DNA content of axenic trophozoites. Exp Parasitol 44:243 248 L6pez-Revilla R, Said-FernS.ndez S (1980) Cytopathogenicity of Entamoeba histolytica: Hemolytic activity of trophozoite homogenates. Am J Trop Med Hyg 29:209 212 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275 Lushbaugh WB, KairalIa AB, Cantey JR, Hofbauer AF, Pittman FE (1979) Isolation o f a cytotoxinenterotoxin from Entamoeba histolytica. J Infect Dis 139:9 17

Modulation of Cell DNA Synthesis by E. histolytica

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Mattern CFT, Keister DB (1977) Experimental amebiasis. II. Hepatic amebiasis in the newborn hamster. Am J Trop Med Hyg 26:402-411 Mattern CFT, Keister DB, Natovitz PC (1980) Entamoeba histoIytica " t o x i n " : fetuin neutralizable and lectin-like. Am J Trop Med Hyg 29:26-30 Neal RA (1971) Pathogenesis of amebiasis. Bull NY Acad Med 47:46~468 Puck TF, Cieciura SJ, Robinson A (1958) Genetics of somatic mammalian cells. III. Long-term cultivation of euploid cells from human and animal subjects. J Exp Med 108:945 955 Richards KL, Douglas SD (1978) Pathophysiological effects of Vibrio cholerae and enterotoxigenic Escherichia coli and their exotoxins on eucaryotic cells. Microbiol Rev 42:592-613 Said-Fernfindez S, L6pez-Revilla R (1976) Electrophoretic characterization of proteins from four axenic Entamoeba strains. In: B Sepfilveda, LS Diamond (eds) Proceedings of the International Conference on Amebiasis. Instituto Mexicano de1 Seguro Social, Mexico City, p 104~111 World Health Organization (1959) Amoebiasis. WHO Technical Report No. 421

Received June 24, 1980