PRELIMINARY
NOTES
An immunological approach to the isolation of factors with mitotic activity from the plasmodial stage of the myxomycete
earlier induction of mitosis in test plasmodia. It is based on the idea, that factors which are indispensable for the initiation of Physarum polycephalum mitosis do exist in greater amounts in cells just prior to mitosis. However, these factors J. BLESSING’ and H. LEMPP, Abteilung fir klinische Morphologic, Zentrum fiir klinische Grundare exhausted during mitosis, becoming lagenforschung, Universittit Urn, D-79 Urn, BRD nearly non-existent in cells of early S phase. The experimental procedure deals with Summary. Nuclear divisions in plasmodia ofPhysarum polycephalum were advanced by applying immunothe purification of cell-free extracts from logically purified plasmodial extracts of late G2 phase on the surface of plasmodia which were 1.5 h before late G2 plasmodia with antisera prepared the third mitosis. The purification of G2 extracts was achieved by interaction of antibodies prepared against against the antigens of early S phase plasthe antigens of early S phase plasmodia with the anti- modia. gens of late G2 plasmodia. There was no advancement of mitosis by extracts prepared from early S phase plasmodia. Untreated G2 extracts did not accelerate mitosis with the same effectiveness as did antibody purified G2 extracts.
Further progress in the understanding of mitotic regulation at the molecular level depends on the isolation of the factors which are responsible for the initiation and completion of mitosis. The acellular slime mould Physarum polycephalum is a suitable experimental organism for studies of molecular processes which participate in the mitotic events. Its usefulness is greatly enhanced by the nearly absolute natural synchrony of nuclear division. Rusch et al. [l] have shown that the timing of mitosis in Physarum polycephalum can be altered by fusing two plasmodia at different stages of the mitotic cycle. Further experiments with this organism [2], as well as studies done on mammalian cells [3] and the protozoan Tetrahymena [4], are the basis for concluding that the factors which are required for mitosis are formed in the cytoplasm and increase in amount during the cell cycle, reaching a maximum at late G2 phase just prior to the beginning of prophase. In this paper a new method is described, which allows to engender an
Materials
and Methods
Culture of fhe organism and preparation of plasmodial extracts. Mitotically synchronized surface plasmodia
were prepared from agitated suspension cultures as described by Guttes et al. [5] and grown at 26°C in the dark. The mitotic cycle took about 9 h. Early S phase plasmodia were harvested 30 min after metaphase, and late G2 phase plasmodia 45 min before metaphase of the third mitotic division. The harvested plasmodia were washed, sonicated and centrifuged (30 min at 8 000 g) and the supematants were further investigated. The extracts were freshly prepared for every assay from four cultures each. The plasmodia were sonicated in an equal volume of sterile aqua dest., pH 4.6, because extracts, which are prepared by this method and placed on the surface of plasmodia do not destroy the cultures. Oppenheim & Katzir have reported that plasmodial extracts need to be dialysed against Tris buffer to avoid the destruction of recipient plasmodia [2]. This negative phenomenon, however, does not occur when plasmodial extracts are prepared according to our above-mentioned method (Blessing & Lempp, unpublished data). Antibody-forming
procedure
and purification
of G2
extracfs. Samples of supematants from sonicated early S phase plasmodia were injected into rabbits (New Zealand white). Each animal was injected with 2 ml of the extract (standardized on 5 mg protein/ml extract) by subcutaneous administration. Several booster injections were given during the following 6-7 months. Antisera were harvested from the animals 7 days after the last booster injection. The sera were inactivated at 56°C for 30 min, then lyophilized and stored. Immune sera from 12rabbits, which were good producers of anti-S antibodies, were pooled and samples for the purification of G2 extracts were taken from this stock. Surface plasmodia were grown until 45 min before
’ Present address: Max Planck Institut fiir Virusforschung Molekularbiologische Abteilung, Spemannstr. i.5, 74 Tiibingen, Germany. Exp Cell Rrs 113 (1978)
436
Preliminary
notes
Table 1. Effect of antibody-treated
cell-free extracts of Physarum polycephalum on the advancement of mitosis Treatment of test plasmodia Medium control Antibody control Untreated early S-extract Antibody-treated early S-extract Untreated late G2 extract Antibody-treated late G2 extract
10 f
l-l
Acceleration of metaphase (mitt)= in recipient plasmodia 0+2.5* 0.2k2.6 0+2.9 Ok2.4 854.2 35+6.3
(2 Mean values of 20 exoeriments each. * S.D. of the individual values. Plasmodia from the same stage of the cell cycle (1.5 h before the third metaphase) were removed from ‘their nutrient medium and transferred into dry Petri dishes. Either 0.12 ml of the antibody-treated late G2 phase extract or 0.12 ml of the control fluids as indicated above were carefully distributed over the surface of each plasmodium. After 25 min of starvation to facilitate complete absorption, the medium was added and the samples were removed for light microscopic observation. The advancement of mitosis was greatest in plasmodia which were incubated with anti-S antibody-treated late G2 extract.
the third metaphase and then harvested. Extracts from these late G2 plasmodia (prepared by sonication and centrifugation) were incubated with antiserum against the antigens of S phase plasmodia at 37°C for 1 h. The volume ratio of G2 extract to antiserum was 2.4 : 1. It was determined bv dilution series. After incubation the antibody-antigen complexes were spun down at 30000 mm for 30 min (SW 30 rotor. Sninco ultracentrifuge) and the super&ant tested ‘as’ to its ability of mitotic induction in test plasmodia. Non-immune serum of untreated rabbits was incubated in the same way with G2 extracts which did not result in any precioitation with Phvsnrum antigens. Tests on mitdtic acceleraiion. Recipient plasmodia from the same stage of the cell cycle (1.5 h before the third metaphase) were cut aseptically into six equal pieces (the size of each was 3.6 cm? and placed in small separate Petri dishes. 0.12 ml of either G2 antibody-treated cell-free extract or control fluids were placed at the same time on the recipient plasmodia and spread evenly on their surface. To facilitate complete absorption of the fluids, the treated plasmodia were starved for 25 min at 26°C in the dark. After this interval, medium was added and samples for smear preparations were taken during the continuing phase of the cell cycle in order to observe microscopically the onset of mitosis. This experiment was repeated twenty times. The time difference in mitotic onset between Exp Cd Res 113 (1978)
12
0...1..1.1 0
6
16
24
32
40
48
Fig. 1. Abscissa: advance of metaphase (min); ordinate: no. of observations. Histograms of the difference in metaphase times for plasmodia which were either incubated with (a) untreated G2 extracts, or (b) anti-S antibody-treated G2 extracts. starved and non-starved cultures was 30 min. Control studies done with plasmodia from the same stage of the cell cycle (1.5 h before third metaphase) which were cut into six equal pieces showed that mitotic division between the separated parts of the same donor plasmodium occurs within 5 min.
Results and Discussion polycephalum synthesizes initiators of mitosis and there is some evidence that these substances are of proteinaceous nature [2,6-121. It is probable that the mitotically active factors are only present in premitotic cells, that they are consumed during mitosis and that they are thus essentially absent in post-mitotic cells. The antigenic pattern of early S phase and late G2 phase plasmodia thus differs essentially only with regard to ‘their unequal quantity of mitotic active substances. It is therefore hypothesized that treatment of late G2 extracts with anti-S antibodies should result in an enrichment of the latter with respect to factors which stimulate mitosis. If this hypoth-
Physarum
Preliminary
esis is correct, an acceleration of mitosis must follow, provided that a suitable biological system is incubated with a G2 extract which has been treated in the above described manner. As such a test system plasmodia from late G2 phase (1.5 h prior to the third mitosis) were incubated with antibody-purified late G2 extract. The result is shown in table 1. It is evident that anti-S antibody-treated late G2 extract effects the strongest acceleration in the test plasmodia. Antibody-containing serum as well as nonimmune serum by itself has no effect on the initiation of mitosis and on the integrity of the plasmodial tissue. Neither did untreated or anti-S antibody-treated extracts of early S phase plasmodia, which were harvested 30 min after metaphase, accelerate mitosis. In plasmodial segments, which were incubated with anti-S antibody-treated late G2 extract, mitosis occurred 35 min earlier with a single standard deviation of +6 min for the individual values (spreads from 18 to 35 min) than in control parts which were incubated with culture medium. In comparison with untreated late G2 extract (acceleration 854 min) the advancement of metaphase caused by anti-S antibodytreated late G2 extract was 27 min. A detailed presentation of timely occurrence of mitosis in the 20 cases which have been tested so far with antibody-treated or untreated late G2 extracts is given in fig. 1. The results of the experiment can be interpreted in the following manner: The earlier initiation of mitosis by antibody-treated late G2 extract indicates that anti-S antibodies are able to change the usual composition of late G2 extracts. The alteration can either be based on removal of mitosis-inhibiting factors, removal of inert substances, or on both modification processes. A further indication can be derived from our experiment. The effect of histone ki-
notes
437
nase on the induction of mitosis which was studied recently [14, 151 indicates that about 2 h prior to metaphase (Matthews, personal communication) this enzyme seems to play an active role in the sequence of events which finally induce the nuclear and cellular cleavage [ 131.In earlier experiments Cummins has shown that the inhibition of protein synthesis just before the beginning of prophase prevents the onset of mitosis. Our experiment shows that antibody-treated extracts from plasmodia 45 min before metaphase are able to induce mitosis earlier in plasmodia which are 1.5 h before metaphase. When extracts from plasmodia 45 min before metaphase are capable to induce earlier initiation of mitosis in plasmodia that have already passed the cell cycle point of histone-kinase activity, it follows that these extracts must contain still other mitotic stimulating factors besides histone kinase. This means that in the interval of the cell cycle, between 2 h prior to metaphase up to the beginning of prophase, additional factors with mitosis-stimulating activity besides histone kinase must be generated, probably of proteinaceous nature. At present we are engaged in developing specific antisera against purified proteins from the afore-mentioned interval and, with the aid of these antisera, to establish which components of late G2 extracts (-45 min extracts) give evidence of a mitosis-stimulating activity. References 1. Rusch, H P, Sachsenmaier, W, Behrens, K & Gruyter, V, .I cell biol3 1 (1966) 204. 2. Oppenheim, A & Katzir, N, Exp cell res 68 (1971) 224. 3. Rao, P N & Johnson, R T, Nature 224 (1970) 159. 4. Rasmussen, L & Zeuthen, E, Compt rend trav lab Carlsberg 32 (1962) 333. 5. Guttes, E & Guttes, S, Methods in cell physiology (ed D M Prescott) vol. 1, p. 43. Academic Press, New York and London (1964). Exp Cd RCF 113 (19781
438
Preliminary
notes
6. Blessing, J, Molecular base of malignancy (ed E Deutsch, K Moser, H Rainer & A Stacher) p. 139. Thieme, Stuttgart (1976). 7. Cummins, J E, Brewer, E N & Rusch, H P, J cell biol27 (1965) 337. 8. Sisken, J E &Wilkes, E, J cell biol34 (1967) 97. 9. Sisken, J E & Iwasaki, T, Exp cell res 55 (1969) 161. 10. Mueller, G C & Kajiwara, K, Symp fundamental cancer res 19th, p. 452. Houston (1965). 11. Mueller, G C & Kajiwara, K, Biochim biophys acta 119(1966) 557. 12. Melvin, J B, Exp cell res 45 (1967) 559. 13. Jockusch, B M,‘Naturwiss 62 (1975) 283. 14. Inglis, R J, Langan, T A, Matthews, H R, Hardie, D G & Bradbury, E M, Exp cell res 97 (1976) 418. 15. Hardie, D G, Matthews, H R & Bradbury, E M, Eur j biochem 66 (1976) 37. Received October 29, 1976 Revised version received January 4, 1978 Accepted January 23, 1978
Normal level of unscheduled DNA synthesis in Werner’s syndrome fibroblasts in culture TOSHIAKI HIGASHIKAWA and YOSHISADA FUJIWARA,’ Department of Radiation Biophysics,
Kobe University School of Medicine, 7-12, Ikuta-ku, Kobe 650, Japan
Kusunoki-cho
We investigated UV-induced unscheduled DNA synthesis (UDS) in skin Bbroblasts from seven unrelated patients with clinically apparent Werner’s syndrome (VVS). WS cells exhibited greatly abbreviated in vitro lifespans, the extents of which ranged from about 20 to 50% of the normal. However, WS cells in early and senescent phases of growth showed the same quantity of DNA repair following UV exposure as did normal Iibroblasts.
Summary.
WS is an autosomal recessive hereditary disease [4], and patients who suffer from it manifest strikingly diminished lifespans together with other clinically apparent symptoms of premature senility [4, 141.Successful culture of skin fibroblasts from WS patients has been notoriously difficult, and in vitro lifespans of WS cells, like progeria cells [3, 51, have been suggested to be shorter than those of tibroblasts from normal donors at the same or similar age [4, 7, 141. Regarding the relationship between Exp Cd Res 113 (1978)
DNA repair and human cell aging in vitro, the results obtained so far have not been unequivocal [l, 2, 7, 16, 19, 201. In some reports, progeria cells have been shown to be defective in the repair of single-strand DNA breaks (SSBs) induced by ionizing radiation [5, 61. We have previously indicated normal repair of X-ray-induced SSBs in WS cells, and suggested a probably normal level of UDS in a few WS strains from preliminary results after a single dose of 20 J/m2 at 254 nm [7]. Here we describe the success in difficult cultures of seven unrelated WS strains and their UDS kinetics as functions of UV dose and post-irradiation time. This is probably the first detail to show that WS cells with a limited in vitro lifespan can perform quantitatively normal UDS even in the terminally senescent phase of growth. Materials
and Methods
The diploid tibroblast strains used were NHSF6, 7a [9-111 and 46, from 6-, 7- and 46-year-old normals, respectivelv, seven WS strains from unrelated patients [7, table 11, and a xeroderma pigmentosum strain, XP4KO r91, from an XP patient with neurological complication (De San&s-Cacchione syndrome). The cells were derived from unoer arm skins of the’donors, and cultured in Eagle’s=MEM (Gibco, New York) ulus 15% fetal calf serum (Flow Lab., Md) in a water-saturated atmosphere of 5 % CO* in air using Falcon plastic Petri dishes (Div. of BioQuest, Calif.), as described previouslv [9-l 11.Cell cultures were passaged by the‘1 : 2 and -1-4 split regimens for WSs-and NHSFs, respectively, and the population doubling level (PDL) was calculated as described in the legend to table 1. The cells attached on coverslips were exposed to oredominantlv 254 nm UV from a 15 W low-pressure mercury lamp (Toshiba Electric Co., Tokyo) at an incident intensitv of 1 J/m’. set r9-111. For the doseUDS relation,-early and late passage WS cells and Hayflick phase II NHSF6 cells were first incubated in 5 &i/ml of [methyl-3H]thymidine (5 Ci/mmole, Radiochemical Centre, Amersham, UK) for 1 h at 37°C to label S phase cells densely, UV-irradiated (0 to 40 J/m’), and reincubated for an additional 4 h in 5 @/ml of [3H]thymidine plus 2 mM hydroxyurea (K & K Lab., Calif.) to detect UDS, as described previously [ 111.In experiments of the UDS kinetics as a function of postUV time (see fig. 3), prelabeled (1.7 &i/ml, I h) ’ To whom requests for reprints should be addressed.
NOTES
An immunological approach to the isolation of factors with mitotic activity from the plasmodial stage of the myxomycete
earlier induction of mitosis in test plasmodia. It is based on the idea, that factors which are indispensable for the initiation of Physarum polycephalum mitosis do exist in greater amounts in cells just prior to mitosis. However, these factors J. BLESSING’ and H. LEMPP, Abteilung fir klinische Morphologic, Zentrum fiir klinische Grundare exhausted during mitosis, becoming lagenforschung, Universittit Urn, D-79 Urn, BRD nearly non-existent in cells of early S phase. The experimental procedure deals with Summary. Nuclear divisions in plasmodia ofPhysarum polycephalum were advanced by applying immunothe purification of cell-free extracts from logically purified plasmodial extracts of late G2 phase on the surface of plasmodia which were 1.5 h before late G2 plasmodia with antisera prepared the third mitosis. The purification of G2 extracts was achieved by interaction of antibodies prepared against against the antigens of early S phase plasthe antigens of early S phase plasmodia with the anti- modia. gens of late G2 plasmodia. There was no advancement of mitosis by extracts prepared from early S phase plasmodia. Untreated G2 extracts did not accelerate mitosis with the same effectiveness as did antibody purified G2 extracts.
Further progress in the understanding of mitotic regulation at the molecular level depends on the isolation of the factors which are responsible for the initiation and completion of mitosis. The acellular slime mould Physarum polycephalum is a suitable experimental organism for studies of molecular processes which participate in the mitotic events. Its usefulness is greatly enhanced by the nearly absolute natural synchrony of nuclear division. Rusch et al. [l] have shown that the timing of mitosis in Physarum polycephalum can be altered by fusing two plasmodia at different stages of the mitotic cycle. Further experiments with this organism [2], as well as studies done on mammalian cells [3] and the protozoan Tetrahymena [4], are the basis for concluding that the factors which are required for mitosis are formed in the cytoplasm and increase in amount during the cell cycle, reaching a maximum at late G2 phase just prior to the beginning of prophase. In this paper a new method is described, which allows to engender an
Materials
and Methods
Culture of fhe organism and preparation of plasmodial extracts. Mitotically synchronized surface plasmodia
were prepared from agitated suspension cultures as described by Guttes et al. [5] and grown at 26°C in the dark. The mitotic cycle took about 9 h. Early S phase plasmodia were harvested 30 min after metaphase, and late G2 phase plasmodia 45 min before metaphase of the third mitotic division. The harvested plasmodia were washed, sonicated and centrifuged (30 min at 8 000 g) and the supematants were further investigated. The extracts were freshly prepared for every assay from four cultures each. The plasmodia were sonicated in an equal volume of sterile aqua dest., pH 4.6, because extracts, which are prepared by this method and placed on the surface of plasmodia do not destroy the cultures. Oppenheim & Katzir have reported that plasmodial extracts need to be dialysed against Tris buffer to avoid the destruction of recipient plasmodia [2]. This negative phenomenon, however, does not occur when plasmodial extracts are prepared according to our above-mentioned method (Blessing & Lempp, unpublished data). Antibody-forming
procedure
and purification
of G2
extracfs. Samples of supematants from sonicated early S phase plasmodia were injected into rabbits (New Zealand white). Each animal was injected with 2 ml of the extract (standardized on 5 mg protein/ml extract) by subcutaneous administration. Several booster injections were given during the following 6-7 months. Antisera were harvested from the animals 7 days after the last booster injection. The sera were inactivated at 56°C for 30 min, then lyophilized and stored. Immune sera from 12rabbits, which were good producers of anti-S antibodies, were pooled and samples for the purification of G2 extracts were taken from this stock. Surface plasmodia were grown until 45 min before
’ Present address: Max Planck Institut fiir Virusforschung Molekularbiologische Abteilung, Spemannstr. i.5, 74 Tiibingen, Germany. Exp Cell Rrs 113 (1978)
436
Preliminary
notes
Table 1. Effect of antibody-treated
cell-free extracts of Physarum polycephalum on the advancement of mitosis Treatment of test plasmodia Medium control Antibody control Untreated early S-extract Antibody-treated early S-extract Untreated late G2 extract Antibody-treated late G2 extract
10 f
l-l
Acceleration of metaphase (mitt)= in recipient plasmodia 0+2.5* 0.2k2.6 0+2.9 Ok2.4 854.2 35+6.3
(2 Mean values of 20 exoeriments each. * S.D. of the individual values. Plasmodia from the same stage of the cell cycle (1.5 h before the third metaphase) were removed from ‘their nutrient medium and transferred into dry Petri dishes. Either 0.12 ml of the antibody-treated late G2 phase extract or 0.12 ml of the control fluids as indicated above were carefully distributed over the surface of each plasmodium. After 25 min of starvation to facilitate complete absorption, the medium was added and the samples were removed for light microscopic observation. The advancement of mitosis was greatest in plasmodia which were incubated with anti-S antibody-treated late G2 extract.
the third metaphase and then harvested. Extracts from these late G2 plasmodia (prepared by sonication and centrifugation) were incubated with antiserum against the antigens of S phase plasmodia at 37°C for 1 h. The volume ratio of G2 extract to antiserum was 2.4 : 1. It was determined bv dilution series. After incubation the antibody-antigen complexes were spun down at 30000 mm for 30 min (SW 30 rotor. Sninco ultracentrifuge) and the super&ant tested ‘as’ to its ability of mitotic induction in test plasmodia. Non-immune serum of untreated rabbits was incubated in the same way with G2 extracts which did not result in any precioitation with Phvsnrum antigens. Tests on mitdtic acceleraiion. Recipient plasmodia from the same stage of the cell cycle (1.5 h before the third metaphase) were cut aseptically into six equal pieces (the size of each was 3.6 cm? and placed in small separate Petri dishes. 0.12 ml of either G2 antibody-treated cell-free extract or control fluids were placed at the same time on the recipient plasmodia and spread evenly on their surface. To facilitate complete absorption of the fluids, the treated plasmodia were starved for 25 min at 26°C in the dark. After this interval, medium was added and samples for smear preparations were taken during the continuing phase of the cell cycle in order to observe microscopically the onset of mitosis. This experiment was repeated twenty times. The time difference in mitotic onset between Exp Cd Res 113 (1978)
12
0...1..1.1 0
6
16
24
32
40
48
Fig. 1. Abscissa: advance of metaphase (min); ordinate: no. of observations. Histograms of the difference in metaphase times for plasmodia which were either incubated with (a) untreated G2 extracts, or (b) anti-S antibody-treated G2 extracts. starved and non-starved cultures was 30 min. Control studies done with plasmodia from the same stage of the cell cycle (1.5 h before third metaphase) which were cut into six equal pieces showed that mitotic division between the separated parts of the same donor plasmodium occurs within 5 min.
Results and Discussion polycephalum synthesizes initiators of mitosis and there is some evidence that these substances are of proteinaceous nature [2,6-121. It is probable that the mitotically active factors are only present in premitotic cells, that they are consumed during mitosis and that they are thus essentially absent in post-mitotic cells. The antigenic pattern of early S phase and late G2 phase plasmodia thus differs essentially only with regard to ‘their unequal quantity of mitotic active substances. It is therefore hypothesized that treatment of late G2 extracts with anti-S antibodies should result in an enrichment of the latter with respect to factors which stimulate mitosis. If this hypoth-
Physarum
Preliminary
esis is correct, an acceleration of mitosis must follow, provided that a suitable biological system is incubated with a G2 extract which has been treated in the above described manner. As such a test system plasmodia from late G2 phase (1.5 h prior to the third mitosis) were incubated with antibody-purified late G2 extract. The result is shown in table 1. It is evident that anti-S antibody-treated late G2 extract effects the strongest acceleration in the test plasmodia. Antibody-containing serum as well as nonimmune serum by itself has no effect on the initiation of mitosis and on the integrity of the plasmodial tissue. Neither did untreated or anti-S antibody-treated extracts of early S phase plasmodia, which were harvested 30 min after metaphase, accelerate mitosis. In plasmodial segments, which were incubated with anti-S antibody-treated late G2 extract, mitosis occurred 35 min earlier with a single standard deviation of +6 min for the individual values (spreads from 18 to 35 min) than in control parts which were incubated with culture medium. In comparison with untreated late G2 extract (acceleration 854 min) the advancement of metaphase caused by anti-S antibodytreated late G2 extract was 27 min. A detailed presentation of timely occurrence of mitosis in the 20 cases which have been tested so far with antibody-treated or untreated late G2 extracts is given in fig. 1. The results of the experiment can be interpreted in the following manner: The earlier initiation of mitosis by antibody-treated late G2 extract indicates that anti-S antibodies are able to change the usual composition of late G2 extracts. The alteration can either be based on removal of mitosis-inhibiting factors, removal of inert substances, or on both modification processes. A further indication can be derived from our experiment. The effect of histone ki-
notes
437
nase on the induction of mitosis which was studied recently [14, 151 indicates that about 2 h prior to metaphase (Matthews, personal communication) this enzyme seems to play an active role in the sequence of events which finally induce the nuclear and cellular cleavage [ 131.In earlier experiments Cummins has shown that the inhibition of protein synthesis just before the beginning of prophase prevents the onset of mitosis. Our experiment shows that antibody-treated extracts from plasmodia 45 min before metaphase are able to induce mitosis earlier in plasmodia which are 1.5 h before metaphase. When extracts from plasmodia 45 min before metaphase are capable to induce earlier initiation of mitosis in plasmodia that have already passed the cell cycle point of histone-kinase activity, it follows that these extracts must contain still other mitotic stimulating factors besides histone kinase. This means that in the interval of the cell cycle, between 2 h prior to metaphase up to the beginning of prophase, additional factors with mitosis-stimulating activity besides histone kinase must be generated, probably of proteinaceous nature. At present we are engaged in developing specific antisera against purified proteins from the afore-mentioned interval and, with the aid of these antisera, to establish which components of late G2 extracts (-45 min extracts) give evidence of a mitosis-stimulating activity. References 1. Rusch, H P, Sachsenmaier, W, Behrens, K & Gruyter, V, .I cell biol3 1 (1966) 204. 2. Oppenheim, A & Katzir, N, Exp cell res 68 (1971) 224. 3. Rao, P N & Johnson, R T, Nature 224 (1970) 159. 4. Rasmussen, L & Zeuthen, E, Compt rend trav lab Carlsberg 32 (1962) 333. 5. Guttes, E & Guttes, S, Methods in cell physiology (ed D M Prescott) vol. 1, p. 43. Academic Press, New York and London (1964). Exp Cd RCF 113 (19781
438
Preliminary
notes
6. Blessing, J, Molecular base of malignancy (ed E Deutsch, K Moser, H Rainer & A Stacher) p. 139. Thieme, Stuttgart (1976). 7. Cummins, J E, Brewer, E N & Rusch, H P, J cell biol27 (1965) 337. 8. Sisken, J E &Wilkes, E, J cell biol34 (1967) 97. 9. Sisken, J E & Iwasaki, T, Exp cell res 55 (1969) 161. 10. Mueller, G C & Kajiwara, K, Symp fundamental cancer res 19th, p. 452. Houston (1965). 11. Mueller, G C & Kajiwara, K, Biochim biophys acta 119(1966) 557. 12. Melvin, J B, Exp cell res 45 (1967) 559. 13. Jockusch, B M,‘Naturwiss 62 (1975) 283. 14. Inglis, R J, Langan, T A, Matthews, H R, Hardie, D G & Bradbury, E M, Exp cell res 97 (1976) 418. 15. Hardie, D G, Matthews, H R & Bradbury, E M, Eur j biochem 66 (1976) 37. Received October 29, 1976 Revised version received January 4, 1978 Accepted January 23, 1978
Normal level of unscheduled DNA synthesis in Werner’s syndrome fibroblasts in culture TOSHIAKI HIGASHIKAWA and YOSHISADA FUJIWARA,’ Department of Radiation Biophysics,
Kobe University School of Medicine, 7-12, Ikuta-ku, Kobe 650, Japan
Kusunoki-cho
We investigated UV-induced unscheduled DNA synthesis (UDS) in skin Bbroblasts from seven unrelated patients with clinically apparent Werner’s syndrome (VVS). WS cells exhibited greatly abbreviated in vitro lifespans, the extents of which ranged from about 20 to 50% of the normal. However, WS cells in early and senescent phases of growth showed the same quantity of DNA repair following UV exposure as did normal Iibroblasts.
Summary.
WS is an autosomal recessive hereditary disease [4], and patients who suffer from it manifest strikingly diminished lifespans together with other clinically apparent symptoms of premature senility [4, 141.Successful culture of skin fibroblasts from WS patients has been notoriously difficult, and in vitro lifespans of WS cells, like progeria cells [3, 51, have been suggested to be shorter than those of tibroblasts from normal donors at the same or similar age [4, 7, 141. Regarding the relationship between Exp Cd Res 113 (1978)
DNA repair and human cell aging in vitro, the results obtained so far have not been unequivocal [l, 2, 7, 16, 19, 201. In some reports, progeria cells have been shown to be defective in the repair of single-strand DNA breaks (SSBs) induced by ionizing radiation [5, 61. We have previously indicated normal repair of X-ray-induced SSBs in WS cells, and suggested a probably normal level of UDS in a few WS strains from preliminary results after a single dose of 20 J/m2 at 254 nm [7]. Here we describe the success in difficult cultures of seven unrelated WS strains and their UDS kinetics as functions of UV dose and post-irradiation time. This is probably the first detail to show that WS cells with a limited in vitro lifespan can perform quantitatively normal UDS even in the terminally senescent phase of growth. Materials
and Methods
The diploid tibroblast strains used were NHSF6, 7a [9-111 and 46, from 6-, 7- and 46-year-old normals, respectivelv, seven WS strains from unrelated patients [7, table 11, and a xeroderma pigmentosum strain, XP4KO r91, from an XP patient with neurological complication (De San&s-Cacchione syndrome). The cells were derived from unoer arm skins of the’donors, and cultured in Eagle’s=MEM (Gibco, New York) ulus 15% fetal calf serum (Flow Lab., Md) in a water-saturated atmosphere of 5 % CO* in air using Falcon plastic Petri dishes (Div. of BioQuest, Calif.), as described previouslv [9-l 11.Cell cultures were passaged by the‘1 : 2 and -1-4 split regimens for WSs-and NHSFs, respectively, and the population doubling level (PDL) was calculated as described in the legend to table 1. The cells attached on coverslips were exposed to oredominantlv 254 nm UV from a 15 W low-pressure mercury lamp (Toshiba Electric Co., Tokyo) at an incident intensitv of 1 J/m’. set r9-111. For the doseUDS relation,-early and late passage WS cells and Hayflick phase II NHSF6 cells were first incubated in 5 &i/ml of [methyl-3H]thymidine (5 Ci/mmole, Radiochemical Centre, Amersham, UK) for 1 h at 37°C to label S phase cells densely, UV-irradiated (0 to 40 J/m’), and reincubated for an additional 4 h in 5 @/ml of [3H]thymidine plus 2 mM hydroxyurea (K & K Lab., Calif.) to detect UDS, as described previously [ 111.In experiments of the UDS kinetics as a function of postUV time (see fig. 3), prelabeled (1.7 &i/ml, I h) ’ To whom requests for reprints should be addressed.
