EXPERIMENTAL
CELL
RESEARCH
201,
log-112 (19%)
High Sensitivity of Cultured Human Trophoblasts to Ribosome-Inactivating Proteins MARIA GIULIABATTELLI,’ Department
of Experimental
Pathology
VERA MONTACUTI,* ANDFIORENZOSTIRPE
and *Department
of Obstetrics and Gynecology,
Many plant proteins possessing abortifacient activities were identified as ribosome-inactivating proteins (RIPS). The effect of several ribosome-inactivating proteins (saporin 6, dianthin 32, pokeweed antiviral protein from seeds, gelonin, bryodin-R, and momordin) on primary cultures of human trophoblasts and human embryonal fibroblasts and on choriocarcinoma (JAR and BeWo) and ovarian carcinoma (TG) cell lines was studied. Protein synthesis of human trophoblasts and BeWo cells was lowered by RIPS more than that of other cells. The trophoblastic receptors for estradiol were not affected by treatment of the cells with momordin. The binding and uptake of saporin 6 and momordin by BeWo and HeLa cells were not correlated to cell toxicity. 0 1992 Academic Press, Inc.
INTRODUCTION Radix trichosanthis is a traditional Chinese drug extracted from the root tuber (Tian-hua-fen) of the Cucurbitacea Trichosanthes kirilowii Maxim. It was used during the 16th century to induce menstruation and expulsion of fetal membranes (review in [l, 21). The active principle of the drug is a protein, trichosantin, which is used to induce midtrimester abortion in official Chinese medicine [l]. It causes necrosis of the placental syncytiotrophoblast, followed by circulation hindrance [ 31. It was also reported to be active against hydatidiform mole, malignant mole, choriocarcinoma, and ectopic pregnancy (review in [4]). Trichosanthin induces midterm abortion in mice, rabbits, and monkeys [5] (review in [l]), but not in rats and hamsters [5], suggesting a differential response in different species toward this drug. The in vivo inhibition of mouse embryo implantation was obtained by intraperitoneal injection of trichosanthin and of a-momorcharin (corresponding to the major isoform of momor-
’ To whom correspondence and reprint requests should dressed at the Dipartimento di Patologia Sperimentale, Via coma 14, I-40126 Bologna, Italy.
be adS. Gia-
University
of Bologna, Bologna, Italy
din [6], from the seeds of the Cucurbitacea Momordica charantia) [ 71. The in vitro study of the effect of trichosanthin and a-momorcharin on mouse embryos showed that the syncytiotrophoblast was always preferentially affected [7]. Two human choriocarcinoma cell lines (JAR and BeWo) were also more sensitive to trichosanthin than other cultured human cells [8]. The abortifacient proteins trichosantin and ol-momorcharin and ,&momorcharin (another isoform of momordin) are ribosome-inactivating proteins (RIPS) and, consistently, all other RIPS tested (pokeweed antiviral protein, saporins, and gelonin) showed abortifacient activity [9]. Ribosome-inactivating proteins are a group of similar and almost ubiquitous plant enzymes existing in nature in two forms: type 1 and type 2, the latter consisting of two subunits, an A chain with ribosome-inactivating activity and a B chain with galactose-specific lectin activity (review in [lo-131). Through their enzymatic activity the A chains arrest eukaryotic protein synthesis by cleaving the N-glycosidic bond of a specific adenine residue in 28s rRNA (A4324 of rat liver rRNA [14] ). This change in the 60s ribosome subunit prevents the binding of the elongation factor 1 [15] or 2 [16] needed for the translocation of the nascent polypeptidic chain. RIPS type 1 have the same enzymatic activity as the A chains of RIPS type 2 and like these have a relatively low toxicity, missing the lectin moiety that makes toxins enter easily into the cell. The aim of the present research was to investigate if the abortifacient activity of RIPS could be due to a higher sensitivity of trophoblasts to these proteins. To this purpose the sensitivity to several type 1 RIPS of primary cultures of human trophoblasts was compared to those of human embryonal fibroblasts and JAR and BeWo (from choriocarcinoma) and TG (from ovary carcinoma) cell lines. The possibility that the abortifacient activity of momordin was due to interference with the production of receptors for estradiol by trophoblasts was investigated. The toxicity to BeWo and HeLa cells of saporin 6 and momordin was also compared with their binding to and uptake by these cells.
109
0014.4827/92
$5.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
110
BATTELLI,
TABLE Ribosome-Inactivating
MONTACUTI,
1 Proteins
Used Activity” b
Source
Name
Caryophyllaceae Saponarin officinalis seeds (soapwort) Dianthus caryophyllus leaves (carnation) Phytolaccaceae Phytolacca americana seeds (pokeweed) Euphorbiaceae Gelonium multifirum seeds Cucurbitaceae Momordica charantia seeds Bryonia dioica roots (white bryony)
Saporin 6 Dianthin 32
0.04 0.12
PAP-S
0.04
Gelonin
0.40
Momordin Bryodin-R
0.06 0.12
‘From [13] for all RIPS except bryodin (171. b Concentration giving 50% inhibition of protein bit reticulocyte lysate.
synthesis by a rab-
AND
STIRPE
1 ml cold buffered saline solution, and extracted for 1 h at 37°C with 0.5 ml absolute ethanol. Extracted radioactivity was counted in a liquid scintillation counter. Preparation of ‘25Z-labeled RIPS. RIPS were labeled with Nan51 (Amersham International) by the method of Fraker and Speck [19], using the Iodogen reagent (Pierce UK Ltd., Chester, UK). A specific activity of 0.6-2 X lo6 cpm/pg protein was obtained. Binding and uptake of RIPS. Cells (2 X 10’) were seeded into each well of 24-well plates in RPM1 1640 complete medium. The following day the medium was substituted by serum-free RPM1 1640 buffered with 20 mM Hepes, pH 7.4, containing 20 jtM iZ51-labeled RIPS. Nonspecific binding was checked by the addition of 0.5 mg/ml bovine serum albumin. The cells were incubated for various times at 0 or 37°C for binding or uptake assay, respectively. At the end of the incubation time the cells were washed five times with 0.5 ml of cold buffered saline solution and extracted for 1 h at room temperature with 0.1 M KOH. Extracted radioactivity was counted in a gammacounter. Statistical analysis. Results are given as means f SE. Student’s t test was used for statistical comparisons. Concentrations giving 50% inhibition of protein synthesis (I&,) were calculated by the linear regression analysis. The estradiol binding capacity and the receptor dissociation constant were calculated by Scatchard analysis.
RESULTS MATERIALS
AND METHODS
Ribosome-inactivating proteins. Ribosome-inactivating proteins used were dianthin 32, saporin 6, momordin (major isoform), gelonin, pokeweed antiviral protein from seeds (PAP-S) [13], and bryodin-R [17]. The proteins were prepared as described earlier [18], and their characteristics are summarized in Table 1. Primary cell cultures. Human trophoblasts were obtained from chorionic villi sampling at lo-12 weeks of gestation. Villi were dissected, the obtained fragments were placed in culture flasks containing Chang complete medium (Hana Biologicals, Alameda, CA) supplemented with glutamine and antibiotics and were incubated at 37°C in a humidified atmosphere of 5% CO,/95% air. Subcultures were obtained by trypsin treatment of primary cultures. Human fibroblasts were obtained from embryonal tissues after termination of pregnancy and were cultured as described for trophoblasts. Cell lines. BeWo and JAR (from choriocarcinoma) and TG (from ovary carcinoma) cell lines were maintained in culture flasks, with RPM1 1640 complete medium supplemented with 10% fetal calf serum (Biological Industries, Beth HaEmek, Israel), glutamine, and antibiotics. Cell protein synthesis. Cells from confluent culture were trypsinized, counted, and seeded into 24-well plates (0.5-l X 105/well). On the following day the medium was substituted with 0.5 ml of serumfree RPM1 1640 medium containing various amounts of RIPS. After 18 h the medium was changed to 0.25 ml of leucine-free RPM1 1640 containing 0.5 &i/ml of [3H]leucine (Amersham International, Amersham, Bucks, UK) (45 Cilmmol). After 2 h protein synthesis was arrested with 10% trichloroacetic acid and the acid-soluble material was washed away. The acid-insoluble material was dissolved with 0.1 M KOH and the incorporated radioactivity was measured in a scintillation counter. Estrogen receptor assay. Trophoblasts were seeded into 24-well plates in Chang complete medium. At confluence the medium was substituted by serum-free RPM1 1640 medium either with no additions or containing 0.64 PM momordin. After 18 h the medium was replaced by serum-free RPM1 1640 medium containing six different concentrations (0.2-1.7 nM) of 17P-[3H]estradiol (NEN Research Products, Boston, MA) (102 Ci/mmol). Nonspecific binding was determined by the addition of 200-fold excess 17P-estradiol. Cells were. incubated for 2 h at 37”C, then placed on ice, washed three times with
Inhibition
of Protein
Synthesis by Cells
The effects of RIPS on protein synthesis by cells are reported in Fig. 1. Marked differences were observed both in the effect on various cells and in the action of different RIPS on a given cell type. Primary trophoblast cultures and the BeWo cell line were generally more sensitive to all RIPS than other cells, with IC,,s always below 0.3 PM. At the other extreme, primary fibroblasts
41
Dianthin 32
41
Saporin 6
Momordin
41
Gelonin
18 (II
JA TC R
HL EU
RMJATCRHLEU
41
Bryodin-R
PAP-S
n
FIG. 1. Effect of RIPS on cell protein synthesis.
w
JA m
m HL
EU
IC,, is the concentration of the indicated RIP giving 50% inhibition of cell protein synthesis. [3H]Leucine incorporation of untreated cells was: trophoblasts (TB), 6000 dpm; BeWo cells (BW), 5800 dpm; JAR cells (JA), 7200 dpm; TG cells (TG), 12,600 dpm; fibroblasts (FB), 2700 dpm. Bars represent standard error. The asterisk indicates an IC, significantly higher than that of trophoblasts (P < 0.005). Values of IC, for HeLa (HL) and EUE (EU) cells are as reported [lo, 131.
SENSITIVITY
OF TROPHOBLASTS
and HeLa and EUE cells were the most resistant cells, the I&s ranging from 0.6 to more than 3 PM. JAR and TG cell lines had intermediate sensitivity to ribosomeinactivating proteins, though TG cells were highly sensitive to saporin and JAR cells to gelonin. Saporin 6 was the RIP generally most toxic to cells. However, considerable variations, with IC,,s ranging up to more than 20fold, were observed in the effects of each RIP on different cells. Data available in the literature about the cytotoxicity observed after exposure to RIPS for a period of time close enough to 18 h are reported in Table 2. These include 14 different human cell lines in which protein synthesis, cloning efficiency, or cell viability were examined. Effect of RIPS on Estrogen Receptors of Trophoblasts The binding capacity for estradiol was determined on whole trophoblasts. At saturation, 14.4 fmol of estradiol was bound per lo6 cells, with a dissociation constant equal to 1.3 nM. The treatment of trophoblasts with 0.64 PM momordin for 18 h did not affect the binding capacity for estradiol, which was 12.8 fmol/106 cells, with Kd = 0.7 nM. Binding
of RIPS to, and Uptake by, Cells
The binding of RIPS to, and their uptake by, cells was investigated using lz51-labeled RIPS. These experiments were performed with BeWo (high sensitivity) and HeLa
TABLE
I
b
60
40
:.ii__::
C
1
i2ow .c m
4
SO
60
30
40
d
2op 30
20
Time (mid
Time (mid
FIG. 2. Binding of RIPS to, and uptake by, cells. Uptake by HeLa (a) and by BeWo (b) cells and binding to HeLa (c) and to BeWo (d) cells of momordin (0) and saporin 6 (m).
(low sensitivity) [ 131 cells, and with saporin 6 (high cytotoxicity) and momordin (low cytotoxicity). No saturable binding sites were found on BeWo or on HeLa cells by time-course experiments up to 1 h in the presence of 20 PM saporin 6 or momordin (Fig. 2). No differences were observed either in the absence or in the presence of 0.5 mg/ml bovine serum albumin (results not shown). Each cell took up 0.5-l X lo3 molecules of RIP/min (Fig. 2). DISCUSSION
2
Toxicity to Cells of Ribosome-Inactivating
111
TO RIPS
Proteins
Saporin 6 ‘J&o’= (PM)
Momordin TC,” (PM)
Gelonin TG,” (PM)
>3.3*J >1.7*.4 >1.7*J o.2*v9
>3.3b,2 >3.3*z5
l.ObJ 23.3b.6 >3.3’,48 0.W LO+ o.lc*‘o 2.0dJ’ >3.3”‘2 >3.3dJ3 2.04”
’ Concentration toxic to 50% of the cells. Cytotoxicity was assessed by: 3nhibition of cell protein synthesis, ‘cell cloning inhibition, ‘loss of cell viability. Cells used: ‘LoVo/Dx doxorubicin-resistant colon carcinoma cell line [24]; 2U266 multiple myeloma cell line [25]; 3Ramos Burkitt’s lymphoma cell line [26]; ‘U937 histiocystic lymphoma cell line [27, 281; 5RAJI Burkitt’s lymphoma cell line [25, 281; ‘Priess lymphoblastoid cell line [26]; ‘HL60 promyelocytic cell line [27]; ‘Namalwa B cell line [29, 301; ‘K562 hemopoietic cell line [27]; “Rex T cell line [28]; ?a23 Namalwa mutant cell line [30]; ‘*J5rR6 Namalwa mutant cell line [30]; 13J5rRll Namalwa mutant cell line [30]; 14J5rR12 Namalwa mutant cell line [30].
The present work reports the effects of RIPS on human trophoblasts and on choriocarcinoma-derived cell lines and compares these effects to those of RIPS on other normal and tumor-derived human cells. We also report a summary of the results available in the literature on the toxicity of RIPS to human cells, limited to those comparable to ours in the time of exposure of cells to RIPS (Table 2). Taken together our results confirm and extend previous observations about the generally low cytotoxicity of RIPS and the high sensitivity of trophoblasts to trichosanthin [ 11. It is noteworthy that gelonin, which has the lowest inhibitory activity on cell-free systems (Table l), is the RIP most active on JAR cells. The high sensitivity of the tumor-derived cell lines to one or two RIPS only is consistent with the sensitivity to trichosanthin of a B16 melanoma-derived cell line [8], which is comparable to that of trophoblasts. To ascertain if the abortifacient activity of ribosomeinactivating proteins could be correlated to a decrease of cellular estrogen receptors due to the inhibition of protein synthesis, the binding capacity of trophoblasts for estradiol was determined. Our results support the no-
112
BATTELLI,
MONTACUTI,
tion that the hormonal derangement observed in choriocarcinoma cells after treatment with trichosanthin and momordin is mainly due to loss of cells [8]. The possibility was considered that the high sensitivity to RIPS of trophoblasts could be due to the high pynocytic activity of these cells, which consequently might take up proteins more easily. To verify this hypothesis, the binding of RIPS to, and their uptake by, cells was investigated. Our results suggest that no specific binding sites for RIPS are present on the cell surface. Furthermore, there was no correlation between cell uptake and sensitivity to a given RIP, suggesting that differences in the internalization process are not the reason for the differences observed in the cell sensitivity to RIPS. The different sensitivity to RIPS of various cells, and in particular that of Bewo and JAR cells, both derived from a choriocarcinoma, is unexplained, but could be at least in part due to differences in the strain. Similar differences in the inhibition of protein synthesis by various cells were observed with other recently purified RIPS [20]. It is possible, however, that RIPS are taken up through different intracellular routes and that in some cells RIPS are directed to an intracellular compartment where they may be inactivated. It should also be recalled that RIPS have different activity on ribosomes from different organisms [21] and that ricin-resistant cell mutants were isolated with ribosomes unaffected by the toxin [22, 231. Thus a different sensitivity to RIPS of ribosomes or lack of some cofactor(s) necessary for RIP activity in the cells used in present experiments cannot be excluded. In conclusion, if the evidence we report at the cellular level is not enough to definitely clarify the in uiuo effects of RIPS, nevertheless our results support the hypothesis that the abortifacient activity of RIPS could be due to their high toxicity to trophoblastic cells. The work was supported nale delle Ricerche, Rome, logie e Biostrumentazione, Ricerca Scientifica, by the Cancro, and by the Pallotti’s
by a contract from the Consiglio Naziowithin the Progetto finalizzato Biotecnoby the Minister0 dell’Universit& e della Associazione Italiana per la Ricerca sul Legacy for Cancer Research.
AND
5. Chang, M. C., Saksena, S. K., Lau, I. F., and Wang, Y.-H. (1979) Contraception 19, 175-184. 6. Barbieri, L., Zamboni, M., Lorenzoni, E., Montanaro, L., Sperti, S., and Stirpe, F. (1980) Biochem. J. 186,443-452. 7. a. 9. 10. 11. 12. 13. 14.
Received December 4, 1991 Revised version received February
17, 1992
Yeung, H. W., Li, W. W., Feng, Z., Barbieri, L., and Stirpe, F. (1988) Znt. J. Pept. Protein Res. 31, 265-268. Barbieri, L., and Stirpe, F. (1982) Cancer Surveys 1,489-520. Jimenez, A., and Vazquez, D. (1985) Annu. Rev. Microbial. 39, 649-672. Roberts, W. K., and Selitrennikoff, C. P. (1986) Biosci. Rep. 6, 19-29. Stirpe, F., and Barbieri, L. (1986) FEBS Z&t. 195, l-8. Endo, Y. (1988) in Immunotoxins 89, Kluwer Academic Publishers,
(Frankel, Boston.
A. E., Ed.), pp. 75-
Nilsson, L., Asano, K., Svensson, B., Poulsen, F. M., and Nygird, 0. (1986) Biochim. Biophys. Actu 868, 62-70. 16. Montanaro, L., Sperti, S., Mattioli, A., Testoni, G., and Stirpe, F. (1975) Biochem. J. 146,127-131.
17. Stirpe, F., Barbieri, danza, A., Lorenzoni,
L., Battelli, M. G., Falasca, A. I., AbbonE., and Stevens, W. A. (1986) Biochem. J.
240,659-665. 18.
Barbieri,
L., Stoppa, C., and Bolognesi,
A. (1987) J. Chromatogr.
408,235-243. 19.
20.
21. 22.
Fraker, P. J., and Speck, J. C., Jr. (1978) Biochem. Biophys. Res. Commun. 80,849-857. Bolognesi, A., Barbieri, L., Abbondanza, A., Falasca, A. I., Carnicelli, D., Battelli, M. G., and Stirpe, F. (1990) Biochim. Biophys. Acta 1087,293-302. Cenini, P., Carnicelli, D., and Stirpe, F. (1990) Camp. Biochem. Physiol. B 96,581-584. Ono, M., Kuwano, M., Watanabe, K.-I., and Funatsu, G. (1982) Mol. Cell. Biol. 2, 599-606. Sallustio, S., and Stanley, P. (1990) J. Biol. Chem. 265,582-588.
23. 24. Dinota,
25.
26. 27.
831-840.
Law, L. K., Tam, P. P. L., and Yeung, H. W. (1983) J. Reprod. Fertil. 69, 597-604. Tsao, S. W., Ng, T. B., and Yeung, H. W. (1990) Toxicon 28, 118331192.
15.
REFERENCES The Second Laboratory, Shanghai Inst. Exp. Biol. (1976) Scientia Sin. 19,811-827. Wang,Y., Quian, R.-Q., Gu, Z.-W., Jin, S-W., Zhang, L.-Q.,Xia, Z.-X., Tian, G.-Y., and Ni, C.-Z. (1986) Pure Appl. Chem. 58, 789-798. Cheng, K.-F. (1982) O&et. Ginecol. 59,494-498. Tsao, S. W., Yan, K. T., and Yeung, H. W. (1986) Tonicon 24,
STIRPE
28.
29.
30.
A., Tazzari, P. L., Michieli, M., Visani, G., Gobbi, M., Bontadini, A., Tassi, C., Fanin, R., Damiani, D., Grandi, M., Pileri, S., Bolognesi, A., Stirpe, F., Baccarani, M., Tsuruo, T., and Tura, S. (1990) Cancer Res. 50,4291-4294. Dinota, A., Barbieri, L., Gobbi, M., Tazzari, P. L., Rizzi, S., Bontadini, A., Bolognesi, A., Tura, S., and Stirpe, F. (1989) Br. J. Cancer 60,315-319. Wiels, J., Junqua, S., Dujardin, P., Le Pecq, J.-B., and Tursz, T. (1984) Cancer Res. 44, 129-133. Gasperi-Campani, A., Zauli, G., Roncuzzi, L., Valvassori, L., Vitale, L., Gaggioli, L., and Bagnara, G. P. (1989) Exp. Hematol. 17, 755-759. Goldmacher, V. S., Scott, C. F., Lambert, J. M., McIntyre, G. D., Blattler, W. A., Collinson, A. R., Stewart, J. K., Chong, L. D., Cook, S., Slayter, H. S., Beaumont, E., and Watkins, S. (1989) J. Cell. Physiol. 141, 222-234. Goldmacher, V. S., Blattler, W. A., Lambert, J. M., McIntyre, G., and Stewart, J. K. (1989) Mol. Phurmucol. 36, 818-822. Goldmacher, V. S., Anderson, J., Schulz, M.-L., Bliittler, W. A., and Lambert, J. M. (1987) J. Biol. Chem. 262,3205-3209.
CELL
RESEARCH
201,
log-112 (19%)
High Sensitivity of Cultured Human Trophoblasts to Ribosome-Inactivating Proteins MARIA GIULIABATTELLI,’ Department
of Experimental
Pathology
VERA MONTACUTI,* ANDFIORENZOSTIRPE
and *Department
of Obstetrics and Gynecology,
Many plant proteins possessing abortifacient activities were identified as ribosome-inactivating proteins (RIPS). The effect of several ribosome-inactivating proteins (saporin 6, dianthin 32, pokeweed antiviral protein from seeds, gelonin, bryodin-R, and momordin) on primary cultures of human trophoblasts and human embryonal fibroblasts and on choriocarcinoma (JAR and BeWo) and ovarian carcinoma (TG) cell lines was studied. Protein synthesis of human trophoblasts and BeWo cells was lowered by RIPS more than that of other cells. The trophoblastic receptors for estradiol were not affected by treatment of the cells with momordin. The binding and uptake of saporin 6 and momordin by BeWo and HeLa cells were not correlated to cell toxicity. 0 1992 Academic Press, Inc.
INTRODUCTION Radix trichosanthis is a traditional Chinese drug extracted from the root tuber (Tian-hua-fen) of the Cucurbitacea Trichosanthes kirilowii Maxim. It was used during the 16th century to induce menstruation and expulsion of fetal membranes (review in [l, 21). The active principle of the drug is a protein, trichosantin, which is used to induce midtrimester abortion in official Chinese medicine [l]. It causes necrosis of the placental syncytiotrophoblast, followed by circulation hindrance [ 31. It was also reported to be active against hydatidiform mole, malignant mole, choriocarcinoma, and ectopic pregnancy (review in [4]). Trichosanthin induces midterm abortion in mice, rabbits, and monkeys [5] (review in [l]), but not in rats and hamsters [5], suggesting a differential response in different species toward this drug. The in vivo inhibition of mouse embryo implantation was obtained by intraperitoneal injection of trichosanthin and of a-momorcharin (corresponding to the major isoform of momor-
’ To whom correspondence and reprint requests should dressed at the Dipartimento di Patologia Sperimentale, Via coma 14, I-40126 Bologna, Italy.
be adS. Gia-
University
of Bologna, Bologna, Italy
din [6], from the seeds of the Cucurbitacea Momordica charantia) [ 71. The in vitro study of the effect of trichosanthin and a-momorcharin on mouse embryos showed that the syncytiotrophoblast was always preferentially affected [7]. Two human choriocarcinoma cell lines (JAR and BeWo) were also more sensitive to trichosanthin than other cultured human cells [8]. The abortifacient proteins trichosantin and ol-momorcharin and ,&momorcharin (another isoform of momordin) are ribosome-inactivating proteins (RIPS) and, consistently, all other RIPS tested (pokeweed antiviral protein, saporins, and gelonin) showed abortifacient activity [9]. Ribosome-inactivating proteins are a group of similar and almost ubiquitous plant enzymes existing in nature in two forms: type 1 and type 2, the latter consisting of two subunits, an A chain with ribosome-inactivating activity and a B chain with galactose-specific lectin activity (review in [lo-131). Through their enzymatic activity the A chains arrest eukaryotic protein synthesis by cleaving the N-glycosidic bond of a specific adenine residue in 28s rRNA (A4324 of rat liver rRNA [14] ). This change in the 60s ribosome subunit prevents the binding of the elongation factor 1 [15] or 2 [16] needed for the translocation of the nascent polypeptidic chain. RIPS type 1 have the same enzymatic activity as the A chains of RIPS type 2 and like these have a relatively low toxicity, missing the lectin moiety that makes toxins enter easily into the cell. The aim of the present research was to investigate if the abortifacient activity of RIPS could be due to a higher sensitivity of trophoblasts to these proteins. To this purpose the sensitivity to several type 1 RIPS of primary cultures of human trophoblasts was compared to those of human embryonal fibroblasts and JAR and BeWo (from choriocarcinoma) and TG (from ovary carcinoma) cell lines. The possibility that the abortifacient activity of momordin was due to interference with the production of receptors for estradiol by trophoblasts was investigated. The toxicity to BeWo and HeLa cells of saporin 6 and momordin was also compared with their binding to and uptake by these cells.
109
0014.4827/92
$5.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
110
BATTELLI,
TABLE Ribosome-Inactivating
MONTACUTI,
1 Proteins
Used Activity” b
Source
Name
Caryophyllaceae Saponarin officinalis seeds (soapwort) Dianthus caryophyllus leaves (carnation) Phytolaccaceae Phytolacca americana seeds (pokeweed) Euphorbiaceae Gelonium multifirum seeds Cucurbitaceae Momordica charantia seeds Bryonia dioica roots (white bryony)
Saporin 6 Dianthin 32
0.04 0.12
PAP-S
0.04
Gelonin
0.40
Momordin Bryodin-R
0.06 0.12
‘From [13] for all RIPS except bryodin (171. b Concentration giving 50% inhibition of protein bit reticulocyte lysate.
synthesis by a rab-
AND
STIRPE
1 ml cold buffered saline solution, and extracted for 1 h at 37°C with 0.5 ml absolute ethanol. Extracted radioactivity was counted in a liquid scintillation counter. Preparation of ‘25Z-labeled RIPS. RIPS were labeled with Nan51 (Amersham International) by the method of Fraker and Speck [19], using the Iodogen reagent (Pierce UK Ltd., Chester, UK). A specific activity of 0.6-2 X lo6 cpm/pg protein was obtained. Binding and uptake of RIPS. Cells (2 X 10’) were seeded into each well of 24-well plates in RPM1 1640 complete medium. The following day the medium was substituted by serum-free RPM1 1640 buffered with 20 mM Hepes, pH 7.4, containing 20 jtM iZ51-labeled RIPS. Nonspecific binding was checked by the addition of 0.5 mg/ml bovine serum albumin. The cells were incubated for various times at 0 or 37°C for binding or uptake assay, respectively. At the end of the incubation time the cells were washed five times with 0.5 ml of cold buffered saline solution and extracted for 1 h at room temperature with 0.1 M KOH. Extracted radioactivity was counted in a gammacounter. Statistical analysis. Results are given as means f SE. Student’s t test was used for statistical comparisons. Concentrations giving 50% inhibition of protein synthesis (I&,) were calculated by the linear regression analysis. The estradiol binding capacity and the receptor dissociation constant were calculated by Scatchard analysis.
RESULTS MATERIALS
AND METHODS
Ribosome-inactivating proteins. Ribosome-inactivating proteins used were dianthin 32, saporin 6, momordin (major isoform), gelonin, pokeweed antiviral protein from seeds (PAP-S) [13], and bryodin-R [17]. The proteins were prepared as described earlier [18], and their characteristics are summarized in Table 1. Primary cell cultures. Human trophoblasts were obtained from chorionic villi sampling at lo-12 weeks of gestation. Villi were dissected, the obtained fragments were placed in culture flasks containing Chang complete medium (Hana Biologicals, Alameda, CA) supplemented with glutamine and antibiotics and were incubated at 37°C in a humidified atmosphere of 5% CO,/95% air. Subcultures were obtained by trypsin treatment of primary cultures. Human fibroblasts were obtained from embryonal tissues after termination of pregnancy and were cultured as described for trophoblasts. Cell lines. BeWo and JAR (from choriocarcinoma) and TG (from ovary carcinoma) cell lines were maintained in culture flasks, with RPM1 1640 complete medium supplemented with 10% fetal calf serum (Biological Industries, Beth HaEmek, Israel), glutamine, and antibiotics. Cell protein synthesis. Cells from confluent culture were trypsinized, counted, and seeded into 24-well plates (0.5-l X 105/well). On the following day the medium was substituted with 0.5 ml of serumfree RPM1 1640 medium containing various amounts of RIPS. After 18 h the medium was changed to 0.25 ml of leucine-free RPM1 1640 containing 0.5 &i/ml of [3H]leucine (Amersham International, Amersham, Bucks, UK) (45 Cilmmol). After 2 h protein synthesis was arrested with 10% trichloroacetic acid and the acid-soluble material was washed away. The acid-insoluble material was dissolved with 0.1 M KOH and the incorporated radioactivity was measured in a scintillation counter. Estrogen receptor assay. Trophoblasts were seeded into 24-well plates in Chang complete medium. At confluence the medium was substituted by serum-free RPM1 1640 medium either with no additions or containing 0.64 PM momordin. After 18 h the medium was replaced by serum-free RPM1 1640 medium containing six different concentrations (0.2-1.7 nM) of 17P-[3H]estradiol (NEN Research Products, Boston, MA) (102 Ci/mmol). Nonspecific binding was determined by the addition of 200-fold excess 17P-estradiol. Cells were. incubated for 2 h at 37”C, then placed on ice, washed three times with
Inhibition
of Protein
Synthesis by Cells
The effects of RIPS on protein synthesis by cells are reported in Fig. 1. Marked differences were observed both in the effect on various cells and in the action of different RIPS on a given cell type. Primary trophoblast cultures and the BeWo cell line were generally more sensitive to all RIPS than other cells, with IC,,s always below 0.3 PM. At the other extreme, primary fibroblasts
41
Dianthin 32
41
Saporin 6
Momordin
41
Gelonin
18 (II
JA TC R
HL EU
RMJATCRHLEU
41
Bryodin-R
PAP-S
n
FIG. 1. Effect of RIPS on cell protein synthesis.
w
JA m
m HL
EU
IC,, is the concentration of the indicated RIP giving 50% inhibition of cell protein synthesis. [3H]Leucine incorporation of untreated cells was: trophoblasts (TB), 6000 dpm; BeWo cells (BW), 5800 dpm; JAR cells (JA), 7200 dpm; TG cells (TG), 12,600 dpm; fibroblasts (FB), 2700 dpm. Bars represent standard error. The asterisk indicates an IC, significantly higher than that of trophoblasts (P < 0.005). Values of IC, for HeLa (HL) and EUE (EU) cells are as reported [lo, 131.
SENSITIVITY
OF TROPHOBLASTS
and HeLa and EUE cells were the most resistant cells, the I&s ranging from 0.6 to more than 3 PM. JAR and TG cell lines had intermediate sensitivity to ribosomeinactivating proteins, though TG cells were highly sensitive to saporin and JAR cells to gelonin. Saporin 6 was the RIP generally most toxic to cells. However, considerable variations, with IC,,s ranging up to more than 20fold, were observed in the effects of each RIP on different cells. Data available in the literature about the cytotoxicity observed after exposure to RIPS for a period of time close enough to 18 h are reported in Table 2. These include 14 different human cell lines in which protein synthesis, cloning efficiency, or cell viability were examined. Effect of RIPS on Estrogen Receptors of Trophoblasts The binding capacity for estradiol was determined on whole trophoblasts. At saturation, 14.4 fmol of estradiol was bound per lo6 cells, with a dissociation constant equal to 1.3 nM. The treatment of trophoblasts with 0.64 PM momordin for 18 h did not affect the binding capacity for estradiol, which was 12.8 fmol/106 cells, with Kd = 0.7 nM. Binding
of RIPS to, and Uptake by, Cells
The binding of RIPS to, and their uptake by, cells was investigated using lz51-labeled RIPS. These experiments were performed with BeWo (high sensitivity) and HeLa
TABLE
I
b
60
40
:.ii__::
C
1
i2ow .c m
4
SO
60
30
40
d
2op 30
20
Time (mid
Time (mid
FIG. 2. Binding of RIPS to, and uptake by, cells. Uptake by HeLa (a) and by BeWo (b) cells and binding to HeLa (c) and to BeWo (d) cells of momordin (0) and saporin 6 (m).
(low sensitivity) [ 131 cells, and with saporin 6 (high cytotoxicity) and momordin (low cytotoxicity). No saturable binding sites were found on BeWo or on HeLa cells by time-course experiments up to 1 h in the presence of 20 PM saporin 6 or momordin (Fig. 2). No differences were observed either in the absence or in the presence of 0.5 mg/ml bovine serum albumin (results not shown). Each cell took up 0.5-l X lo3 molecules of RIP/min (Fig. 2). DISCUSSION
2
Toxicity to Cells of Ribosome-Inactivating
111
TO RIPS
Proteins
Saporin 6 ‘J&o’= (PM)
Momordin TC,” (PM)
Gelonin TG,” (PM)
>3.3*J >1.7*.4 >1.7*J o.2*v9
>3.3b,2 >3.3*z5
l.ObJ 23.3b.6 >3.3’,48 0.W LO+ o.lc*‘o 2.0dJ’ >3.3”‘2 >3.3dJ3 2.04”
’ Concentration toxic to 50% of the cells. Cytotoxicity was assessed by: 3nhibition of cell protein synthesis, ‘cell cloning inhibition, ‘loss of cell viability. Cells used: ‘LoVo/Dx doxorubicin-resistant colon carcinoma cell line [24]; 2U266 multiple myeloma cell line [25]; 3Ramos Burkitt’s lymphoma cell line [26]; ‘U937 histiocystic lymphoma cell line [27, 281; 5RAJI Burkitt’s lymphoma cell line [25, 281; ‘Priess lymphoblastoid cell line [26]; ‘HL60 promyelocytic cell line [27]; ‘Namalwa B cell line [29, 301; ‘K562 hemopoietic cell line [27]; “Rex T cell line [28]; ?a23 Namalwa mutant cell line [30]; ‘*J5rR6 Namalwa mutant cell line [30]; 13J5rRll Namalwa mutant cell line [30]; 14J5rR12 Namalwa mutant cell line [30].
The present work reports the effects of RIPS on human trophoblasts and on choriocarcinoma-derived cell lines and compares these effects to those of RIPS on other normal and tumor-derived human cells. We also report a summary of the results available in the literature on the toxicity of RIPS to human cells, limited to those comparable to ours in the time of exposure of cells to RIPS (Table 2). Taken together our results confirm and extend previous observations about the generally low cytotoxicity of RIPS and the high sensitivity of trophoblasts to trichosanthin [ 11. It is noteworthy that gelonin, which has the lowest inhibitory activity on cell-free systems (Table l), is the RIP most active on JAR cells. The high sensitivity of the tumor-derived cell lines to one or two RIPS only is consistent with the sensitivity to trichosanthin of a B16 melanoma-derived cell line [8], which is comparable to that of trophoblasts. To ascertain if the abortifacient activity of ribosomeinactivating proteins could be correlated to a decrease of cellular estrogen receptors due to the inhibition of protein synthesis, the binding capacity of trophoblasts for estradiol was determined. Our results support the no-
112
BATTELLI,
MONTACUTI,
tion that the hormonal derangement observed in choriocarcinoma cells after treatment with trichosanthin and momordin is mainly due to loss of cells [8]. The possibility was considered that the high sensitivity to RIPS of trophoblasts could be due to the high pynocytic activity of these cells, which consequently might take up proteins more easily. To verify this hypothesis, the binding of RIPS to, and their uptake by, cells was investigated. Our results suggest that no specific binding sites for RIPS are present on the cell surface. Furthermore, there was no correlation between cell uptake and sensitivity to a given RIP, suggesting that differences in the internalization process are not the reason for the differences observed in the cell sensitivity to RIPS. The different sensitivity to RIPS of various cells, and in particular that of Bewo and JAR cells, both derived from a choriocarcinoma, is unexplained, but could be at least in part due to differences in the strain. Similar differences in the inhibition of protein synthesis by various cells were observed with other recently purified RIPS [20]. It is possible, however, that RIPS are taken up through different intracellular routes and that in some cells RIPS are directed to an intracellular compartment where they may be inactivated. It should also be recalled that RIPS have different activity on ribosomes from different organisms [21] and that ricin-resistant cell mutants were isolated with ribosomes unaffected by the toxin [22, 231. Thus a different sensitivity to RIPS of ribosomes or lack of some cofactor(s) necessary for RIP activity in the cells used in present experiments cannot be excluded. In conclusion, if the evidence we report at the cellular level is not enough to definitely clarify the in uiuo effects of RIPS, nevertheless our results support the hypothesis that the abortifacient activity of RIPS could be due to their high toxicity to trophoblastic cells. The work was supported nale delle Ricerche, Rome, logie e Biostrumentazione, Ricerca Scientifica, by the Cancro, and by the Pallotti’s
by a contract from the Consiglio Naziowithin the Progetto finalizzato Biotecnoby the Minister0 dell’Universit& e della Associazione Italiana per la Ricerca sul Legacy for Cancer Research.
AND
5. Chang, M. C., Saksena, S. K., Lau, I. F., and Wang, Y.-H. (1979) Contraception 19, 175-184. 6. Barbieri, L., Zamboni, M., Lorenzoni, E., Montanaro, L., Sperti, S., and Stirpe, F. (1980) Biochem. J. 186,443-452. 7. a. 9. 10. 11. 12. 13. 14.
Received December 4, 1991 Revised version received February
17, 1992
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