Toxicology Letters, 59 (1991) 109-116 Q 1991 Elsevier Science Publishers B.V. 0378-4274/91/$3.50 109 TOXLET 02653 Effects of sinefungin and 5’-deoxy-S-S-isobutyl- adenosine on lipopolysac~harid~-induced proliferation and protein ~-methylation of arginyl residues in murine splenic lymphocytes Bok Ryang Kim* and Kyu Hwan Yang Department of Biological Science and Engineering, Korea Advanced Institute of Science and Technology, Seoul (Korea) (Received 6 May 1991) (Revision received 23 August 1991) (Accepted 26 August 1991) Key worak: Sinefungin; 5’-Deoxy-5’-S-isobutyl-adenosine; Lymphoproliferation; Protein N-methylation of arginyl residues; Protein methylase I SUMMARY The effects of sinefungin (SF) and S-deoxy-5’-9isobutyl-adenosine (SIBA) on lymphoproliferation and protein N-methylation of arginyl residues were investigated in splenic lymphocyte culture in vitro. Both SF and SIBA produced a dose-related inhibition of the lymphoproliferative response to lipopolysaccharide (LPS). The time-course of the inhibition indicated that SIBA had a more inhibitory effect than that of SF at short incubation periods, but SF showed more inhibition when the incubation was prolonged. The effects of SF and SIBA on the l~phoproliferative response corresponded with their effects on the protein N- methylation of arginyl residues by protein methylase I. Sinefungin (SF) and 5’-deoxy-5’-S-isobutyl-adenosine (SIBA) are inhibitors of most methyltransferases [l-7] due to their close structural similarity to S-adenosyl-L- *Present address: Department of Bi~hemist~, Wonkwang University School of Medicine, Iri, 570-749, Korea. Address for correspondence: Dr. K.H. Yang, Department of Biological Science and Engineering, Korea Advanced Institute of Science Technology, Cheongryang, P.O. Box 150, Seoul, Korea.
Effects of sinefungin and 5’-deoxy-S-S-isobutyl- adenosine on lipopolysac~harid~-induced proliferation and protein ~-methylation of arginyl residues in murine splenic lymphocytes
Bok Ryang Kim* and Kyu Hwan Yang
Department of Biological Science and Engineering, Korea Advanced Institute of Science and Technology,
Seoul (Korea)
(Received 6 May 1991) (Revision received 23 August 1991) (Accepted 26 August 1991)
Key worak: Sinefungin; 5’-Deoxy-5’-S-isobutyl-adenosine; Lymphoproliferation; Protein N-methylation of arginyl residues; Protein methylase I
SUMMARY
The effects of sinefungin (SF) and S-deoxy-5’-9isobutyl-adenosine (SIBA) on lymphoproliferation and protein N-methylation of arginyl residues were investigated in splenic lymphocyte culture in vitro. Both SF and SIBA produced a dose-related inhibition of the lymphoproliferative response to lipopolysaccharide (LPS). The time-course of the inhibition indicated that SIBA had a more inhibitory effect than that of SF at short incubation periods, but SF showed more inhibition when the incubation was prolonged. The effects of SF and SIBA on the l~phoproliferative response corresponded with their effects on the protein N- methylation of arginyl residues by protein methylase I.
Sinefungin (SF) and 5’-deoxy-5’-S-isobutyl-adenosine (SIBA) are inhibitors of most methyltransferases [l-7] due to their close structural similarity to S-adenosyl-L-
*Present address: Department of Bi~hemist~, Wonkwang University School of Medicine, Iri, 570-749, Korea.
Address for correspondence: Dr. K.H. Yang, Department of Biological Science and Engineering, Korea Advanced Institute of Science Technology, Cheongryang, P.O. Box 150, Seoul, Korea.
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homocysteine, which is the demethylated end-product of methyltransferases. SIBA was reported to be a selective inhibitor of protein methylase I (S-adenosyl-r-me- thionine:protein-arginine N-methyltransferase; EC 2.1 .1.23) from mammalian cells [1,2]. The enzyme was more sensitively inhibited by SF than by SIBA [2].
Protein methylase I methylates the guanidino group of arginyl residues of protein. It was found that the intracellular level of the enzyme closely paralleled the rate of cell proliferation in regenerating adult rat liver, rapidly growing hepatoma, fetal brain, fast growing hepatoma, and continuously dividing HeLa S-3 cells [8]. Enzyme activity was also elevated during the stimulatory process of splenic lymphocytes by pokeweed mitogen and Staphylococcus aureus Cowan I [9].
In this study, the effects of SF and SIBA on the lymphoproliferative response to LPS were investigated in murine splenic lymphocytes. Studies were also performed to elucidate the relationship between the lymphoproliferative response and the cellular protein N-methylation of arginyl residues by protein methylase I.
N”,hlG-DMA and E-N-monomethyllysine (MML) were obtained from Sigma Chemi- cal Co. (St. Louis, MO), NG-monomethylarginine (MMA) and &-N-trimethyllysine (TML) from Calbiochem-Behring Diagnostics (La Jolla, CA). &-N-dimethyllysine (DML) from Bachem Feinchemikalien AG (Bubendorf, Switzerland), S-adenosyl-r- [methyl-14C]methionine (spec. act. 57 mCi/mmol) from Amersham (Buck- inghamshire, U.K.), and L-[methyl-3H]methionine (spec. act., 15 Wmmol) from ICN Radiochemicals (Irvine, CA) were used.
Preparation and culture of cells Spleen cells from female BALB/c mice, 68 weeks old, were obtained according to
the method of Yang et al. [lo]. The cells (5.0~10~ cells/ml) were suspended in RPM1 1640 medium with 20% heat-inactivated fetal calf serum and incubated at 37°C for 90 min. Non-adherent cells were collected and lymphocytes were isolated with Ficoll- Hypaque. After 2 washes, the cells (1.0~10~ cells/ml) were suspended in RPM1 1640 medium supplemented with 10% fetal calf serum, 15 mM HEPES buffer, 50 &ml of gentamycin, and 2 mM glutamine.
LPS-induced lymphoproliferative response Cell suspensions (0.2 ml) were dispensed in 96-well plates and incubated with vari-
ous concentrations of SF and SIBA for indicated periods in the presence of LPS. Each set of cells received a 12 h terminal pulse with 1 ,uCi of [3H]thymidine (TdR) and then was harvested onto glass filters using a multiple sample harvester.
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Analysis of methylated amino acid derivatives Lymphocytes were cultured alone, with LPS (100 pg/ml), LPS + SIBA (100 PM),
or LPS + SF (100 ,uM) for 48 h at 37°C in a humidified 5% CO,/95% air atmosphere; 24 h before incubation ended, 2 ,Ki/ml of L-[methyl-3H]methionine was added and the cells (2.0x10’) were harvested by centrifugation. After removing nucleic acids and phospholipids, the residual protein was hydrolyzed in 6 N HCl for 24 h at 110°C in vacua. After removing HCl, the hydrolyzate was dissolved in 100 ,~l of water. Before injection, internal standards of methylated amino acids (5 nmol of each fl-MMA, NG,N’G-DMA, No-NG-DMA, MML, DML and TML) were added to the hydro- lyzate. The analysis was performed by a HPLC system (Beckman, San Ramon, CA). A Sepherogel Amino Acid column (4.6~250 mm) was equilibrated with a mixture of 0.33 M citrate buffer (pH 3.28) and 0.25 M sodium nitratea. M borate buffer (pH 8.5) (v/v=1:9); 20 ~1 of the sample was loaded. Elution was performed with 0.25 M sodium nitrate-0.16 M borate buffer (pH 8.5) for 85 min and then with 0.25 M sodium nitrate&l. 16 M borate buffer (pH 9.2) at a flow rate of 0.4 ml/min. Derivatiza- tion of the separated amino acids was done with o-phthalaldehyde at 0.4 ml/min. The temperatures of the column and reactor were maintained at 50°C. Each peak of the methylated amino acids amplified by internal standards was pooled separately and the radioactivity was analyzed.
Assay for protein methylase I Cultured cells (2.0x10’) were washed and suspended in 500 ~1 of ice-cold buffer
consisting of 5 mM sodium phosphate (pH 7.2). 1 mM dithiothreitol, and 0.1 mM EDTA. The cells were then homogenized by a sonicator. Protein methylase I activity was assayed according to the method described by Durban et al. [l 11. The enzyme activity was expressed as the amount of S-adenosyl-L-[methyl-i4C]methionine used/ min/mg enzyme protein.
0 50 100 150 200
Concentration (NM)
Fig. 1. Effect of SF and SIBA on LPS-induced lymphoproliferation. Splenic lymphocytes were cultured with various concentrations of SF (0) or SIBA (0) in the presence of LPS (100 pg/ml). Cultures were harvested at 48 h after a 12 h pulse with [%I-TdR. Results are expressed as a percentage of the LPS-treated
group. Each value represents mean f SD of quadruplicate cultures.
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24 46 72 96
incubation Period (hr)
Fig. 2. Time-course of inhibition by SF and SIBA of LPS-induced lymphoproliferation. Splenic lymphocy- tes were cultured with 100 ,f#rnI of LPS (@!), LPS + 100 PM SF (a), or LPS + 100 pM SIBA (m). At the times indicated, incorporation of [%JTdR was measured. Results are expressed as mean + SD of quadru-
plicate cultures. *Si~ifi~nti~ different from the IJ?%reated group (PcO.05).
Student’s f-test was used to evaluate the statistical si~ifican~ of the results.
RESULTS
The effects of SF and SIBA on the lymphoproliferative response to LPS following 48 h of exposure were assessed (Fig, 1). Both compounds produced a dose-related inhibition of the response. However, each profile was slightly different. SF showed significant inhibition from 5 PM and produced 54 f 4% inhibition at 200 ,uM. SIBA had no effect at 5 and 25 J&I, but the inhibitory effect increased rapidly above 50 ,&I and reached almost 100% at 200 PM. The viability of the cultured cells treated with the highest ~on~n~ation of SF and SIBA was similar to control, indicating that the inhibition was not due to the direct cytotoxicity of SF and SIBA on splenic lymphocy- tes (data not shown).
Splenic lymphocytes were incubated for various periods with 100 ,&I SF or 100 ,uM SIBA in the presence of LPS (Fig. 2). The degree of inhibition by SF and SIBA was different with incubation periods; SIBA showed more inhibition than SF up to 48 h, but SF showed more inhibition than SIBA at 96 h.
Figure 3 shows the HPLC profile for HCl-hydrolyzate of cellular protein methyla- ted with ~-t3H]methioni~e. Each methylated arginine derivative was separated as a single peak ~
The results in Table I show that the total specific amount of methylated arginine derivatives assayed at 48 and 96 h of incubation in the LPS-treated group was 3.3 and 1.8 times higher than the control value, respectively. SF was less effective on the inhibition of the protein N-methylation of arginyl residues than SIBA at 48 h, but the inhibitor effect of SF became more potent than that of SIBA at 96 h.
The activity of protein methylase I in splenic lymphocytes was investigated at
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0 40 60
Elution time (min)
120 160
Fig. 3. Separation of HCl-hydrolyzate of cellular protein methylated with L-[‘Hlmethionine by HPLC. Peak 1 = TML; peak 2 = DML; peak 3 = MML + lysine; peak 4 = ammonia; peak 5 = p,fl-DMA; peak 6 =
NG,WG-DMA; peak 7 = MMA; peak 8 = arginine.
various incubation periods with LPS, LPS + SF, or LPS + SIBA (Fig. 4). Enzyme activity increased with time in the LPS-treated group. A significant suppression of enzyme activity was observed in the SIBA-treated group at 12 h. Meanwhile, SF produced a significant inhibition from 36 h.
TABLE I
FORMATION OF VARIOUS METHYLATED ARGININE DERIVATIVES IN THE CELLULAR PROTEIN OF SPLENIC LYMPHOCYTES FROM BALB/c MICE
Treatment Methylated arginine derivatives
MMA NG,NG-DMA p A+-DMA ’ (cpm of [‘H]methyVmg cellular protein)
Assayed at 48 h Control LPS
LPS + SF LPS + SIBA
917 f 48 2812 f 365 214 + 49 2818 ?r 419* 9587 f 1029* 643 +_ 78’ 2467 z!z 351* 5515 + 654’ 432 + 72+ 1709 +_ 246* 4010 f 437* 398 + 64*
Assayed at 96 h Control 810 f 92 2346 f 318 245 f 37 LPS 1221 f 272* 4379 f 513f 526 f 51* LPS + SF 903 * 155 2218 f 281 301 f 47 LPS + SIBA 1085 f 173* 2937 f 354* 361 f 50*
Splenic lymphocytes were cultured alone (Control), with 100 @ml of LPS, LPS + SF (lOOpM), or LPS + SIBA (100 PM). Cultures were harvested at time indicated after a 24 h terminal pulse with Ljmethyl- ‘I-Ilmethionine. Results are expressed as mean + SD of quadruplicate determinations. *Significantly differ- ent from the control group (PcO.05).
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Incubation Period (hr)
Fig. 4. Effect of SF and SIBA on activity of protein methylase I in splenic lymphocytes. Splenic lymphocy- tes were cultured with 100 @ml of LPS (m), LPS + 100 PM of SF (0), or LPS + 100 PM of SIBA (0). At the times indicated, the cells were harvested and homogenized. The homogenate was assayed for protein methylase I activity. Each value represents mean f SD of quadruplicate determinations. *Significantly
different from the LPS-treated group (PcO.05).
DISCUSSION
SF and SIBA have been used as chemical probes to elucidate the physiological role of several methyltransferases [4-61 including protein methylase I [ 1,3,7]. Exposure of SF and SIBA to splenic lymphocytes inhibited the lymphoproliferative response to LPS. The cellular protein N-methylation of arginyl residues was also inhibited by the addition of SF and SIBA. This suggested that methylation by protein methylase I might be involved in the immune response of splenic lymphocytes.
If the inhibitory effects of SF and SIBA on the lymphoproliferative response were due to inhibition of protein methylase I, the degree of inhibition by SF and SIBA should be similar both in the lymphoproliferative response and in the in vitro enzyme activity. SF is known to have a greater inhibitory effect on this in vitro activity of protein methylase I than the effect of SIBA [2]. However, the response to LPS and protein N-methylation of arginyl residues was inhibited more by SIBA than by SF at 48 h. These results are consistent with the results reported by Vedel et al. [l] and Paolantonacci et al. [3]. They investigated effects of SF and SIBA on the transfor- mation of chick embryo fibroblasts and the multiplication of some protozoan parasi- tes, but failed to show any positive correlation between suppression of cellular re- sponses and inhibition of protein methylase I activity. Based on these findings, they ruled out the possibility of involvement of protein methylase I in the cellular response process.
However, the negative correlation in these studies might be due to insufficient exposure of SF and SIBA so as to exert their toxicity. Since SIBA is hydrophobic and SF is relatively hydrophilic, the transport rate of SF across the cellular membrane might be considerably slower than that of SIBA. Some evidence for this assumption is shown in Figures 2 and 4. SF showed a greater inhibitory effect on the lymphopro-
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liferative response to LPS than did SIBA at shorter incubation periods (24 and 48 h). On the other hand, SIBA showed more inhibition when the incubation periods were prolonged (72 and 92 h). Also, SIBA showed rapid inhibition of protein methylase I within 12 h, whereas the effect of SF gradually increased with incubation periods greater than 24 h. Work to compare the cellular transport of SF and SIBA is currently under way.
In conclusion, in order to observe the effect of SF on culture of splenic lymphocy- tes, the cells must be exposed to SF for more than 48 h. Under this condition, the effects of SF and SIBA on the lymphoproliferative response and methylation of cellu- lar protein-bound arginyl residues would correspond with their in vitro affinities for protein methylase I.
ACKNOWLEDGEMENTS
This work was supported by a Korea Science and Engineering Foundation re- search grant.
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11 Durban, E., Lee, H.W., Kim, S. and Paik, W.K. (1978) Purification and characterization of protein methylase I (Sadenosylmethionine:protein-arginine methyltransferase; EC 2.1.1.23) from calf brain. In: G. Stein, J. Stein and L. Kleinsmith (Eds.), Methods in Cell Biology, Vol. 19, Academic Press, New York, pp. 5947.
