Poly-D-lysine has been reported to induce a triggering of mitosis in plant cells due to a selective stimulatory effect on cells arrested in G 2. Root-tip cells of Allium cepa L. were first exposed to maleic hydrazide (MH) early in the cell cycle and posttreated with different concentrations of the polycationic agent while in G 2. The result was a dose-dependent potentiation of chromosome damage observed at metaphase without any apparent effect induced by poly-D-lysine itself. The enhancement of the yield of chromosomal aberrations was concomitant with an increase in the frequency of mitosis.
In order to test further the stimulatory effect of poly-D-lysine on mitosis, as well as the consequences of a shortening of the time available for repair, cells synchronized by protracted treatment with 5-aminouracil (5-AU), which also induces chromosome damage, were allowed to recover in the presence of the polycationic compound. Our data show that a premature arrival at mitosis resulted in an increase in the frequency of damaged cells observed.
Inhibitors of DNA repair have been reported to be capable of increasing the frequency of chro- mosomal aberrations (CA) induced by physical and chemical mutagens in both plant and animal cells (Kihlman and Natarajan, 1984). Experimen- tal evidence suggests that total or partial removal of induced lesions takes place in S or G 2, and that these repair processes seem to require DNA synthesis (Taylor et al., 1962; Kihlman et al., 1982; Natarajan et al., 1982). It has been proposed
Correspondence: Dr. F. Cortrs, Departamento de Biologia Celular, Facultad de Biologia, Avda. Reina Mercedes, s /n , E-41012 Sevilla (Spain).
that CA would arise in connection with chro- mosome condensation in G 2 when this repair synthesis fails or makes mistakes in the presence of inhibitors that disturb the DNA precursor pool, such as hydroxyurea (HU), 5-fluorodeoxyuridine (FdUrd) or 2'-deoxyadenosine (dAdo) (Kihlman et al., 1982), or inhibitors of DNA polymerase, such as aphidicolin or cytosine arabinoside (araC) (Natarajan et al., 1982).
The exact molecular mechanisms by which caf- feine exerts its potentiating action on CA, on the other hand, are still largely unknown. Mutagen- treated plant cells are most sensitive to caffeine posttreatment during S phase (Andersson and Kihlman, 1987; Cort4s et al., 1987; Mateos et al.,
148
1989) while human lymphocytes appear to be most sensitive during G 2 (Kihlman et al., 1982). The current view is that there are different repair mechanisms operating in S (Lehmann, 1974; Rob- erts, 1978; Painter, 1980) and in G 2 (Kihlman et al., 1982; Kihlman and Natarajan, 1984).
Focusing on G 2 repair, it has been proposed that caffeine, by reducing the G 2 delay induced by physical and chemical mutagens, would shorten the time available for repair and cells would arrive prematurely at mitosis with an enhanced frequency of CA (Kihlman et al., 1982; Kihlman and Natarajan, 1984; Schlegel and Pardee, 1986). As mentioned above, contrasting with the strong potentiating effect of caffeine in G 2 in human lymphocytes, plant cells appear to be rather insen- sitive (Hartley-Asp et al., 1980; Andersson and Kihlman, 1987; Cort6s et al., 1987; Mateos et al., 1989), and caffeine only seems to be able to en- hance chromosome damage in G 2 at fairly high concentrations, that per se induce CA (Gonzfilez- Fernfindez and Lbpez-Sfiez, 1982; Gonzfilez- Fernfindez et al., 1985).
Poly-D-lysine has been reported to trigger the onset of mitosis in All ium sativum L. primary root meristems, and this effect has been attributed to a selectively stimulating effect on G 2 cells (Brulfert and Benbadis, 1985; Benbadis et al., 1985).
We report here that All ium cepa L. root-tip cells exposed to maleic hydrazide (MH) early in the cell cycle and posttreated with poly-D-lysine while in G 2 show a dose-dependent enhancement of the yield of CA observed at metaphase. To test further the hypothesis of a triggering of mitosis in damaged cells delayed in G2, cells were treated with the DNA-synthesis inhibitor 5-aminouracil (5-AU) and allowed to recover in the absence or presence of poly-D-lysine. Our results show that poly-D-lysine is able to induce a premature arrival at mitosis of cells synchronized by 5-AU.
Materials and methods
Culture conditions Roots of All ium cepa L. bulbs (2n = 16), 15-30
g in weight, were grown in the dark in cylindrical glass receptacles with about 70 ml of tap water at 25 _+ 0.5°C. Air was bubbled continuously at a
rate of 10-20 m l / m i n and the tap water was renewed at 24-h intervals. The treatments began when the roots reached a length of 15-20 mm. All the experiments described in this paper were done at least twice, using 2 bulbs per treatment.
Chemicals
MH (Sigma) was dissolved in tap water at 37 ° C, and the pH of the solution was adjusted to 6.0 at room temperature. The final concentration of MH (0.3 mM) was chosen to induce frequencies of chromatid-type aberrations low enough to de- tect any potentiation resulting from treatment with poly-D-lysine in G 2. Poly-D-lysine (Sigma) was prepared as a frozen stock solution at a concentra- tion of 1 m g / m l in distilled water. Three con- centrations were tested (10, 15, and 20 /~g/ml) simultaneously with 0.05% colchichine. 5-AU (Sigma) was also dissolved in tap water at a con- centration of 0.5 mM.
Fixation and squashing, o f roots The roots were either fixed directly in a 3 :1
methanol-acet ic acid mixture for the determina- tion of mitotic index and the scoring of anomalous ana-telophases, or after a 3-h treatment with 0.05% colchicine (Sigma) for mitotic arrest and subse- quent scoring of aberrations at metaphase. The meristems were prepared in all cases as Feulgen squashes and made permanent by the dry-ice method (Conger and Fairchild, 1953). 2000 cells per point were scored for mitotic index (MI) val- ues, while for analysis of chromosome damage 1000 ana-telophases or 200 metaphases were studied.
Results and discussion
G 2 effect ofpoly-D-lysine on MH-induced chromatid aberrations
Autoradiographic experiments indicate that more than 99% of A. cepa L. root-tip cells com- plete their D N A synthesis 3 h before metaphase (Mateos et al., 1989). Table 1 shows that exposure to poly-D-lysine for 2.5 h, simultaneously with colchicine, did not lead to any increase in the yield of chromatid aberrations (CA). Nevertheless, when the cells were first exposed to 0.3 mM M H for 2 h and, after a recovery of 19 h, to poly-D-lysine in
149
TABLE 1
G 2 EFFECTS OF POLY-D-LYSINE ON THE FREQUENCIES OF C H R O M A T I D - T Y P E ABERRATIONS IN A. cepa ROOT-TIP CELLS PRE-TREATED WITH 0.3 m M MALEIC H Y D R A Z I D E (MH)
Mutagen Poly-D-lysine Abnormal Aberrations per 100 cells a Mitotic
treatment (/~g/ml) metaphases Chromatid Isochromatid Chromatid Total index b
a 200 cells were scored in each case. b At least 1000 cells scored.
G 2, a dose-dependent enhancement in the fre- quency of CA induced by MH was observed. Regarding the different types of CA, chromatid breaks were drastically potentiated while exchange aberrations remained unaffected. This result seems to indicate that poly-D-lysine is able to interfere with a putative DNA-repair process that takes place in G 2.
As can be seen in Table 1 (MI) the enhance- ment of the frequency of CA was concomitant with an increase in the frequency of mitosis when the MH-treated cells were exposed to poly-D-lysine in the G 2 phase.
Triggering of mitosis in 5-A U-synchronized cells When A. cepa L. root-tip cells are treated with
0.5 mM 5-AU for 18 h at 25°C, the mitotic index (MI) drops from the beginning of the treatment, reaching very low values, but when the drug is removed the accumulated cells end their inter- phase roughly synchronously and the MI rises until it reaches a maximum after 8-10 h of re- covery (Diez et al., 1976; Gonzfilez-Fern~mdez and L6pez-Sfiez, 1982; Cort6s et al., 1983). Taking advantage of the high resolving power provided by the FPG technique for the cytological detection of DNA replication we have demonstrated a prefer- ential DNA-synthesis inhibition by 5-AU at late S (Cort6s et al., 1983), in agreement with earlier reports (Scheuermann and Klaffke-Lobsien, 1973; Diez et al., 1976).
As can be seen in Fig. 1, in 5-AU-synchronized cells recovered in the presence of 20 t tg /ml poly- o-lysine the mitotic wave was observed 2 h earlier than in the cells recovering in the absence of poly-D-lysine, with a concomitant potentiation of the frequency of anomalous ana-telophases (Figs. 1 and 2). Since there is a preferential inhibition at late S by 5-AU (Cort6s et al., 1983), our present results seem to demonstrate that poly-D-lysine is
5° t 4C
252
38 2 9 8 , lm73 15.9
v 3C
c - 171 L, 2C
1(
T i m e ( h o u r s )
Fig. 1. Evolution of mitotic index (MI) in root-tip cells syn- chronized by protracted treatment with 5-AU, which also results in chromosome damage, and allowed to recover in the presence (e) or absence (v) of 20 •g /ml poly-D-lysine. Where the MI was high enough for an accurate scoring of anomalous
ana-telophases, the percent of damaged cells is given.
150
I
Q
it
Q
e
Fig. 2. A. cepa root-tip cells treated with 5-AU for 18 h and fixed after a recovery period of 4 h in the presence of 20 /Lg/ml poly-D-lysine. Note the high number of mitotic cells as well as the heavily damaged ana-telophases (arrows) and micronuclei
(arrowheads).
able to trigger mitosis in cells arrested in 6 2
recovering from 5-AU treatment, and that this cancellation of G 2 delay in damaged cells results in a potentiation of chromosome damage.
At present, an explanation for the premature onset of mitosis induced by poly-D-lysine at the molecular level is not available. It has been sug- gested that this polycationic compound, due to its strong affinity in vitro for different cell compo- nents (DNA, RNA, proteins and elements of the cytoskeleton), could mimic some effects of poly- amines which have been shown to play a role in many cell processes affecting proliferation (Brul- fert and Benbadis, 1985; Benbadis et al., 1985; Tabor and Tabor, 1984).
In our opinion, the induction of mitosis by poly-D-lysine without any concomitant induction of chromosome damage offers a unique opportu-
nity to study G 2 repair in plant cells pretreated with mutagens.
Acknowledgement
This study was partly supported by the Direc- ci6n General de Universidades e Investigaci6n de la Junta de Andalucia (Spain).
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