Suppression of Poly(ADP-Ribose)

Home , Osamu Hayaishi

Proc. Nati. Acad. Sci. USA Vol. 81, pp. 7132-7136, November 1984 Cell Biology Induction of murine teratocarcinoma cell differentiation by suppression of poly(ADP-ribose) synthesis (NADW/retinoic acid/3-aminobenzamide/immunofluorescence) YASUHIRO OHASHI*t, KUNIHIRO UEDA*t, OSAMU HAYAISHI*§, KouICHI IKAI¶, AND OTSURA NIWAII Departments of *Medical Chemistry, ¶Dermatology, and "Experimental Radiology, Kyoto University Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606, Japan Contributed by Osamu Hayaishi, July 27, 1984

ABSTRACT Poly(ADP-ribose) synthesizing activity in present study, we report a marked decrease in nuclear po- mouse teratocarcinoma EC-Al cells decreased markedly dur- ly(ADP-ribose) synthesizing activity at a very early stage of ing differentiation induced by retinoic acid; the activities as- differentiation of teratocarcinoma cells induced by retinoic sayed in permeabilized cells decreased to 25% and 10% of the acid. Furthermore, we present evidence suggesting that ex- activity of control (uninduced cells) 2 and 3 days, respectively, ogenously added inhibitors of poly(ADP-ribose) synthetase after the addition of 0.1 ,uM retinoic acid to the culture medi- can induce the differentiation of these cells. um. This change preceded changes in morphology and DNA synthesis, which became prominent after 4 days. The decrease MATERIALS AND METHODS in poly(ADP-ribose) synthesizing activity appeared to be caused by a diminution of the synthetase protein and not by a Chemicals. [adenine-U-14C]NAD+ (266 Ci/mol; 1 Ci = 37 decrease in its catalytic activity, because the full activity dis- GBq) and [methyl-3H]dTTP (30 Ci/mmol) were purchased closed by DNase I treatment decreased in parallel, albeit at from Amersham. NAD+, deoxynucleoside triphosphates, about 20 times higher levels. When 8 mM 3-aminobenzamide and all-trans-retinoic acid were obtained from Sigma; bovine or 10 mM nicotinamide, specific inhibitors of poly(ADP-ri- pancreatic DNase I, from Worthington; and 3-aminobenza- bose) synthetase, was added to the culture medium, the cells mide and 3-aminobenzoic acid, from Tokyo Kasei. Fluores- underwent differentiation after 7-9 days. An analogue, 3- cein isothiocyanate-labeled swine antiserum to rabbit immu- aminobenzoic acid, which is not inhibitory to the synthetase, noglobulin (fluorescein isothiocyanate-to-protein molar ra- induced differentiation much less efficiently than did 3-amino- tio, 2.3) was purchased from DAKO-Immunoglobulins. benzamide, and the effect of 3-aminobenzoic acid appeared to Rabbit antiserum to poly(ADP-ribose) was prepared as de- be ascribable to its potent cytotoxicity. Immunohistochemical scribed previously (21). analysis using anti-poly(ADP-ribose) antibody confirmed the Cells and Cell Cultures. EC-Al cells (22), a subclone of marked reduction in poly(ADP-ribose) synthesizing activity in PCC4azal-derived EC-A murine teratocarcinoma cells (23, nuclei of the cells treated with retinoic acid or 3-aminobenza- 24), were cultured at 37°C in a modified Eagle's medium mide but not with 3-aminobenzoic acid. These results suggest (GIBCO) containing 10% fetal calf serum in a 5% CO2 hu- differen- midified atmosphere. Where specified, all-trans-retinoic that a decrease in poly(ADP-ribose) synthesis triggers acid dissolved in ethanol or 3-aminobenzamide (or 3-amino- tiation of teratocarcinoma cells. benzoic acid) dissolved in phosphate-buffered saline (0.15 M NaCl/10 mM sodium phosphate, pH 7.4) was added to the Retinoic acid and related compounds (retinoids) have been culture medium at a final concentration of 0.1 gM or 8 mM, shown to induce differentiation of several lines of murine respectively. The medium, together with drugs, was changed teratocarcinoma cells into endoderm epitheloid cells or fi- daily. Differentiation was examined with cells before con- broblast-like cells (1, 2). The molecular mechanism of this fluency, because overgrown cultures occasionally produced induction of differentiation is not yet fully understood (3); foci of differentiated morphology in a nonspecific manner. De Luca (4), Wolf et al. (5), and Jetten and De Luca (6) re- The cells were counted, after trypsin treatment, by using a ported observations suggesting a role of retinyl phosphate hemocytometer. The viability of cells was judged by exclu- mannose in early cell surface changes, while Chytil and Ong sion of trypan blue. (7) and Jetten and Jetten (8) proposed an alteration in tran- Observation of Morphological Changes. Cells grown on a scription mediated by specific binding proteins. We became glass slide of a Lab-Tek chamber (Miles) were stained with interested in these cells in view of our and others' finding Wright and Giemsa solutions (Merck) and observed under a that poly(ADP-ribose) is closely related to cell differentia- light microscope. Polygonal cells with scarce cytoplasm and tion (9, 10). Poly(ADP-ribose) is a macromolecule synthe- flat cells with abundant cytoplasm were regarded as undif- sized from NAD in eukaryotic cells by a chromatin-bound ferentiated and differentiated cells, respectively. enzyme, poly(ADP-ribose) synthetase (11). This synthesis Assays. Poly(ADP-ribose) synthesizing activity was as- proceeds in covalent attachment, at one terminus of the sayed in permeabilized cells as described by Berger et al. polymer, to various protein acceptors such as histones, non- (25). Trypsin-treated cells (3 x 106 cells) were treated with a histone chromosomal proteins, and the synthetase itself (11). permeabilization buffer (10 mM Tris'HCl, pH 8.3/4 mM Accumulating evidence suggests that this polymer is in- MgCl2/1 mM EDTA/50 mM dithiothreitol/250 mM sucrose) volved in DNA repair (12-14), cell transformation (15, 16), (200 ,ul) for 45 min at 4°C. Under these conditions, up to 95% and cell differentiation (9, 10). Previously, we demonstrated of the cells became permeable as judged by retention of try- that the poly(ADP-ribose) synthesizing activity serves as a blue. the cell was with marker of differentiation of human granulocytes (17, 18), pan Then, suspension supplemented leukemic lymphocytes (19), and epidermal cells (20). In the tPresent address: Department of Biochemistry, Nara Medical Uni- versity, Kashihara, Nara 634, Japan. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" §Present address: Osaka Medical College, Takatsuki, Osaka 569, Ja- in accordance with 18 U.S.C. §1734 solely to indicate this fact. pan. 7132 Downloaded by guest on September 30, 2021 CellCellBiology:Biology:OhashiOhashietetaLal.~~Proc.Nati. Acad. Sci. USA 81 (1984) 7133 the poly(ADP-ribose) synthesis mixture (100,p1) and incubat- RESULTS ed for 30 min at 250C. The final concentrations of compo- Changes in Morphology of Teratocarcinoma Cells Treated nents in the synthesis mixture were 100 mM Tris-HCl at pH with Retinoic Acid. EC-Al cells cultured with no specific 8.0, 10 MM Mg9l2, 33.3 mM dithiothreitol, 667 AM EDTA, drug grew in tightly packed colonies (Fig. lA). In this state, 400 A.M [adenine-U-14C]NAD' (10 cpm/pmol), and 166 mMM 99.5% of the cells were polygonal and only 0.5% were flat; sucrose. Where indicated, 0.05% Triton X-100 and DNase I the latter cells represent spontaneous differentiation. The at 133,4g/ml were added to permeabilized cells. Poly(ADP- addition of 0.1 AM retinoic acid to the culture medium did ribose) synthesis was terminated by the addition of 2 ml of not produce any significant morphological change for 2 days; ice-cold 20% (wt/vol) Cl3CCOOH. The acidified mixture flat cells occupied 0.5% and 1.0% of the total cell population was sonicated for 10 sec at 240 W with a Branson sonifier. at days 1 and 2, respectively (Fig. 2). After 3 days, a margin- Acid-insoluble material was collected on a Whatman GF/C al morphological change was observed (Fig. 1B), and there- filter disc and washed three times with 20% C13CCOOH, after, flat cells resembling endodermal epitheloid cells (Fig. twice with 5% C13CCOOH, and twice with 95% (vol/vol) 1C) emerged and gradually increased in number. At days 4 ethanol; then the radioactivity was determined in a toluene- and 5, flat cells were 26% and 97%, respectively, of total based scintillation fluid as previously described (26). DNA cells. In parallel to this morphological change, the cells pro- synthesizing activity was also assayed in permeabilized duced an increasing amount of plasminogen activator (data cells; the cells (3 x10 cells in 50 Al), trypsinized and per- not shown), as previously reported by Strickland and Mah- meabilized as above, were supplemented with 0.05% Triton davi (1) and Jetten et al. (2). In the absence of retinoic acid, X-100 and the reaction mixture for DNA synthesis (25 ,ul). only 1% of the cells became flat after 5 days. The final concentrations of components in the reaction mix- Changes in DNA Synthesizing Activity During Differentia- ture were 100 mM Hepes-NaOH at pH 7.8, 20 mM MgCI2, tion. Another phenotypic change examined was the DNA 33.3 mM dithiothreitol, 667 AM EDTA, 210 mM NaCl, 15 synthesizing activity. The activity decreased almost in paral- mM ATP, 300 AM each of dATP, dGTP, dCTP, and [methyl- lel to the morphological change; for 1 day after the addition 3H]dTTP (50 cpm/pmol), and 166 mM sucrose. Incubation of retinoic acid, the activity remained unchanged, and then it was for 30 min at 370C and was terminated by the addition of decreased slightly (8-10%) for 2 days, followed by a more 2 ml of ice-cold 10% C13CCOOH containing 2% Na4P2O7. marked decrease thereafter. The activities in differentiation- The mixture was briefly sonicated, and acid-insoluble radio- induced cells after 4 and 5 days were about 55% and 30%, activity was collected on a Whatman GF/C filter disc and respectively, compared with control (uninduced) cells. quantified as described above. Plasminogen activator was Changes in Poly(ADP-ribose) Synthesizing Activity During assayed by the fibrin-agar overlay method of Jones et al. Differentiation. In the course of differentiation of EC-Al (27). cells, the activity of poly(ADP-ribose) synthesis changed Immiunohistochemical Analyses. Cells grown on a glass dramatically (Fig. 3). The activity fell to 50% within 1 day slide of a Lab-Tek chamber with or without specific drugs after the addition of retinoic acid and further decreased to were washed with and cultured in a fresh medium with no 25% and 10% after 2 and 3 days. The activity remained un- drug for 5 hr. This procedure was used to remove any residu- changed thereafter at this low level despite the ongoing mor'- al drugs from the cells. The cells were fixed with cold 95% phological change of the cells (Fig. 2). When the assay was (vol/vol) ethanol for 10 min at 0OC and then incubated for 30 performed in the presence of DNase I, a similar and remark- min at 250C with a mixture containing 100 mM Tris-HCI at able decrease in poly(ADP-ribose) synthesizing activity was 10 MM 1 mM and 200 observed; the activities after 2 and 3 days were 42% and pH 8.0, MgCI2, dithiothreitol, 1fAM 10%, respectively, of the initial level. These values were par- NAD'. After the incubation, the cells were treated for 30 allel to those obtained without DNase I treatment, although min at 250C with rabbit antiserum to poly(ADP-ribose) dilut- the activities were about 20-fold higher. Sinice the DNase I ed 1:10 with phosphate-buffered saline. The control was in- treatment promotes full activation of poly(ADP-ribose) syn- cubated with preimmune serum. The cells were washed with thetase by nicking and cutting DNA strands (25), this result phosphate-buffered saline and then treated for 30 min at suggested a decrease in the amount, rather than the catalytic 250C with fluorescein isothiocyanate-labeled swine antise- activity, of the enzyme. rum to rabbit immunoglobulin diluted 1:10 with phosphate- Changes in Morphology After Treatment with 3-Aminoben- buffered saline. The cells were washed as above and viewed zamide or 3-Aminobenzoic Acid. The foregoing results with a Nikon photomicroscope equipped with an epiillumin- prompted us to examine whether the inhibition of poly(ADP- ation system (21). ribose) synthesis by inhibitors could induce the differentia-

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FIG. 1. Morphological changes of EC-Al teratocarcinoma cells induced by retinoic acid. The cells were cultured with no addition for 2 days (A) or 0.1 AiM retinoic acid for 3 days (B) or 5 days (C). (Wright/Giemsa stain; x410.) Downloaded by guest on September 30, 2021 7134 Cell Biology: Ohashi et aL Proc. NatL. Acad. Sci USA 81 (1984)

O" I I I II o 400 0 _80 E G 300 v60 40 A ° 200 0 20 o (X 1/20) 0Co1000 0~ 0 1 2 3 4 5 6 7 8 9 .0I Time ( days ZO FIG. 2. Morphological changes of EC-Al teratocarcinoma cells 0 1 2 3 4 5 treated with various drugs. Cells cultured for the indicated periods Time ( days with 0.1 AM retinoic acid (o), 8 mM 3-aminobenzamide (A), 8 mM 3- aminobenzoic acid (A), or no addition (e) were stained with Wright/ FIG. 3. Changes in poly(ADP-ribose) synthesizing activity in Giemsa and examined for differentiated morphology. At least 200 EC-Al teratocarcinoma cells treated with retinoic acid. After culti- cells from two separate chambers (Lab-Tek) were scored. vation with 0.1 MAM retinoic acid for indicated periods, the cells were permeabilized and examined for their activity in incorporating ['4C]ADP-ribose in the absence (e) or presence (o) of DNase I. The tion. When 3-aminobenzamide (8 mM), a potent inhibitor of values obtained with DNase I are plotted on a 1/20th reduced scale. poly(ADP-ribose) synthetase (28), was added to the culture medium, the morphology of the cells started to change on on both growth and viability, 3-aminobenzoic acid is very day 4 (Fig. 2), and about 80% of the cells became flat after 9 cytotoxic to EC-Al cells. days (Fig. 4). Another inhibitor, nicotinamide (10 mM), also Immunohistochemical Analyses. The in situ activity of po- induced differentiation of the cells in 7-9 days (data not ly(ADP-ribose) synthesis was examined by immunofluores- shown). In contrast to these inhibitors, 3-amtinobenzoic acid cence staining of the polymer produced by preincubation (8 mM), an analogue with no inhibitory action on the synthe- with NAD+ (Fig. 6). In the control (untreated) cells, intense tase, induced differentiation much less efficiently than did 3- and freckly immunofluorescence was seen diffusely over the aminobenzamide (Fig. 2). nuclei (Fig. 6A). In contrast, weaker immunofluorescence Effects of Retinoic Acid, 3-Aminobenzamide, and 3-Amino- was detected in restricted areas of the nuclei of flat cells benzoic Acid on Cell Growth. Retinoic acid (0.1 AM), 3-amin- induced to differentiate by retinoic acid treatment (Fig. 6B). obenzoic acid (8 mM), and 3-aminobenzamide (8 mM) were The nuclei of flat cells induced to differentiate by treatment inhibitory in this order to the growth of EC-Al cells (Fig. 5). with 3-aminobenzamide were also immunostained weakly On the other hand, the viability of the cells was affected in and unevenly (Fig. 6C), whereas the nuclei of many cells the order of 3-aminobenzoic acid, retinoic acid, and 3-amino- treated with 3-aminobenzoic acid were immunostained in- benzamide at the same concentrations as above; the viabili- tensely and diffusely (Fig. 6D). No immunofluorescence was ties on day 7 were 81%, 92%, and 95%, respectively, of the observed in either type of cells when the antibody was re- value of a control culture. As judged from the strong effects placed by control (preimmune) serum (data not shown).

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Al. j 'v AO FIG. 4. Morphology of EC-Al teratocarcinoma cells treated with 0.01% ethanol (a vehicle for retinoic acid) for 2 days (A), 0.1 MiM retinoic acid for 7 days (B), 8 mM 3-aminobenzamide for 9 days (C), or 8 mM 3-aminobenzoic acid for 9 days (D). (Wright/Giemsa stain; x410.) Downloaded by guest on September 30, 2021 Cell Biology: Ohashi et aL Proc. NatL Acad. Sci. USA 81 (1984) 7135 6), while the other is the nucleus, where retinoic acid (or retinol) carried by cellular binding proteins (7, 8) binds to chromatin (29, 30) and leads to alteration in gene expression (31). A possible relationship to protein kinases has also been suggested by recent findings of cooperative actions of aden- osine 3',5'-cyclic monophosphate (32) or phorbol esters (33) with retinoids in embryonal teratocarcinoma cells. Our present study showed that retinoic acid gave rise to a 10 dramatic decrease in poly(ADP-ribose) synthesizing activity in teratocarcinoma cells prior to the onset of morphological changes and that the suppression of poly(ADP-ribose) synthesis by exogenously added inhibitors of poly(ADP-ribose) 100 synthetase induced the differentiation. These results argue 0~~~~~~ strongly that the nuclear action of retinoids to induce differentiation is mediated by suppression of poly(ADP-ribose) synthesis. The effect of retinoic acid on the synthetase activity is indirect, because the retinoid per se did not affect the 10 enzyme activity in vitro (unpublished results). The observations that DNase I-stimulated and unstimulated activities of poly(ADP-ribose) synthesis changed in parallel (Fig. 3) and that poly(ADP-ribose) synthetase itself was the main accep- tor of the polymer in both differentiated and undifferentiated 1 cells (unpublished results) support the view that the decrease 0 1 2 3 4 5 6 7 8 in poly(ADP-ribose) synthesizing activity in retinoic acid- Time ( days ) treated cells is caused by a decrease in the amount of synthetase protein and not in the catalytic activity of the enzyme or FIG. 5. Effects of various drugs on the growth of EC-Al terato- in the availability of other acceptors of poly(ADP-ribose). carcinoma cells. z~, 3-Aminobenzamide (8 mM); *, 3-aminobenzoic The cell line that we employed in this study, EC-Al (a acid (8 mM); o, retinoic acid (0.1 1M); *, no addition. subclone of PCC4azal), is not very stable in the undifferentiated phenotype; an overgrowth of the culture, slight acidifi- DISCUSSION cation (pH < 6) of the culture medium, or a treatment with cytotoxic agents such as cytosine arabinonucleoside or N- Since the finding by Strickland and Mahdavi (1) that embry- methyl-N'-nitro-N-nitrosoguanidine induces nonspecific dif- onal teratocarcinoma stem cells can be induced to differenti- ferentiation of a part of the cells (unpublished results). These ate by retinoic acid, this system has been investigated by observations suggest that a weak differentiation-inducing ef- embryologists, cell biologists, and biochemists as a model fect of 3-aminobenzoic acid (Fig. 2) may be ascribable to its for exploring the control mechanisms of cell differentiation potent cytotoxicity (Fig. 5), although a possibility remains to and proliferation (3). Evidence so far obtained suggests two be tested that 3-aminobenzoic acid is metabolized to a com- possible sites of action of retinoids; one is the plasma mem- pound with an inhibitory effect on poly(ADP-ribose) synthe- brane, where retinol (rather than retinoic acid) participates tase in the cell. in glycosyl transfer and thus alters cell surface structures (4- Possible involvement ofpoly(ADP-ribose) in cell differen-

FIG. 6. Poly(ADP-ribose) synthesis in EC-Al teratocarcinoma cells treated with medium alone for 2 days (A), 0.1 "iM retinoic acid for 7 days (B), 8 mM 3-aminobenzamide for 9 days (C), or 8 mM 3-aminobenzoic acid for 9 days (D). The cells were preincubated with NAD+ and stained with rabbit antiserum to poly(ADP-ribose) and fluorescein isothiocyanate-labeled swine antiserum to rabbit immunoglobulin. (x410.) Downloaded by guest on September 30, 2021 7136 Cell Biology: Ohashi et aL Proc. Natl. Acad. Sci. USA 81 (1984)

tiation has been well documented. For example, Caplan and 11. Ueda, K., Kawaichi, M. & Hayaishi, 0. (1982) in ADP-Ribosy- co-workers demonstrated a close correlation between chon- lation Reactions: Biology and Medicine, eds. Hayaishi, 0. & drocytic differentiation of embryonal chicken limb mesen- Ueda, K. (Academic, New York), pp. 117-155. chymal cells and their poly(ADP-ribose) synthesizing activi- 12. Ogata, N., Kawaichi, M., Ueda, K. & Hayaishi, 0. (1980) Bio- ty (34) or the content of poly(ADP-ribose) (35). This correla- chem. Int. 1, 229-236. 13. Shall, S. (1982) in ADP-Ribosylation Reactions: Biology and tion was confirmed recently by Nishio et al. (36), who Medicine, eds. Hayaishi, O. & Ueda, K. (Academic, New showed augmentation of chondrocytic differentiation of cul- York), pp. 477-520. tured embryonal chicken limb bud cells by inhibitors of po- 14. Ohashi, Y., Ueda, K., Kawaichi, M. & Hayaishi, 0. (1983) ly(ADP-ribose) synthetase. A decrease in poly(ADP-ribose) Proc. Natl. Acad. Sci. USA 80, 3604-3607. synthesizing activity during differentiation has also been 15. Miwa, M., Oda, K., Segawa, K., Tanaka, M., Irie, S., Yama- found in other types of cells; Pekala et al. (37), Morioka et guchi, N., Kuchino, T., Shiroki, K., Shimojo, H., Sakura, H., al. (38), and Kanai et al. (39) reported a decrease in the activ- Matsushima, T. & Sugimura, T. (1977) Arch. Biochem. ity of differentiation-induced mouse preadipocytes, murine Biophys. 181, 313-321. erythroleukemia 16. Hirai, K., Ueda, K. & Hayaishi, 0. (1983) Cancer Res. 43, cells, and human leukemic promyelocytes, 3441-3446. respectively. Furthermore, the latter two researcher groups 17. Ikai, K., Ueda, K., Fukushima, M., Nakamura, T. & Hayai- succeeded in inducing differentiation of cells with various shi, 0. (1980) Proc. Natl. Acad. Sci. USA 77, 3682-3685. inhibitors of poly(ADP-ribose) synthetase (38, 40, 41). We 18. Ikai, K., Ueda, K. & Hayaishi, 0. (1982) in ADP-Ribosylation also demonstrated previously the disappearance of poly- Reactions: Biology and Medicine, eds. Hayaishi, 0. & Ueda, (ADP-ribose) synthesis in the course of differentiation and K. (Academic, New York), pp. 339-360. maturation of granulocytes (17), leukemic promyelocytes 19. Nakao, Y., Matsuda, S., Kimoto, H., Matsui, T., Kobayashi, (18), leukemic lymphocytes (19), epidermal cells (20), and N., Kishihara, M., Fujita, T., Watanabe, S., Ueda, K. & Ito, intestinal epithelial cells (16). All these results are in accord Y. (1982) let. J. Cancer 30, 687-695. with the notion obtained from the present study that a 20. Ikai, K., Danno, K., Imamura, S. & Ueda, K. (1982) J. Derma- low- tol. 9, 125-129. ered level of poly(ADP-ribose) synthesis may induce cellular 21. Ikai, K., Ueda, K. & Hayaishi, 0. (1980) J. Histochem. Cyto- differentiation of teratocarcinoma cells. However, apparent- chem. 28, 670-676. ly contradictory observations were also reported; Farzaneh 22. Niwa, O., Yokota, Y., Ishida, H. & Sugahara, T. (1983) Cell et al. (42) observed an increase, rather than a decrease, in 32, 1105-1113. poly(ADP-ribose) synthesizing activity during differentiation 23. Jakob, H., Boon, T., Gillard, J., Nicholas, J. F. & Jacob, F. of cultured chicken embryonal myoblasts due, probably, to (1973) Ann. Microbiol. (Paris) 124B, 269-282. an increase in DNA strand breaks, and they further showed 24. Gautsh, J. W. (1980) Nature (London) 285, 110-112. that inhibitors of poly(ADP-ribose) synthetase inhibited dif- 25. Berger, N. A., Weber, G. & Kaichi, A. S. (1978) Biochim. ferentiation of these cells. Johnstone and Williams (43) also Biophys. Acta 519, 87-104. 26. Okayama, H., Edson, C. M., Fukushima, M., Ueda, K. & demonstrated that various inhibitors of poly(ADP-ribose) Hayaishi, 0. (1977) J. Biol. Chem. 252, 7000-7005, synthetase diminished differentiation of mitogen-stimulated 27. Jones, P., Benedict, W., Strickland, S. & Reich, E. (1975) Cell human peripheral lymphocytes, probably through inhibition 5, 323-329. of rejoining of single-strand breaks present in the lympho- 28. Purnell, M. R. & Whish, W. J. D. (1980) Biochem. J. 185, 775- cyte DNA. Although the reason for the discrepancy remains 777. to be investigated, as does a contribution of DNA strand 29. Liau, G., Ong, D. E. & Chytil, F. (1981) J. Cell Biol. 91, 63- breakage to differentiation of myoblasts and other cells, it 68. seems plausible that a marked change, decrease or increase, 30. Takase, S., Ong, D. E. & Chytil, F. (1979) Proc. Natl. Acad. in the cellular level of poly(ADP-ribose) might have pro- Sci. USA 76, 2204-2208. found effects 31. Fuchs, E. & Green, H. J. (1981) Cell 25, 617-625. on the structure of chromatin (44, 45), thereby 32. Strickland, S., Smith, K. K. & Marotti, K. R. (1980) Cell 21, activating a set of genes involved in cellular differentiation. 347-355. 33. Kraft, A. S. & Anderson, W. B. (1983) J. Biol. Chem. 258, We thank Drs. 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