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Response to X-ray- and cis Pt-induced DNA damage in Stylonychia lemnae (Ciliata, Protozoa)

Posted on: Sunday, 28 September 2003, 06:00 CDT

The macronuclei of the ciliated protozoan Stylonychia lemnae are highly resistant to several DNA-damaging agents, but the micronuclei are sensitive. Possible reasons for these differences were investigated. Although both kinds of nuclei continue to synthesize DNA and repair damage after application of Cisplatin, the micronuclear division rate slows down, and its DNA is degraded. Apparently only the macronuclei repair damage successfully. After treatment with X-rays, the micronuclei are lost because they are unable to divide. In the macronucleus no recombination repair was found. Our hypothesis is that the macronucleus can survive because it has enough copies of each gene to cope with the loss of some of them.

Key words: Ciliates, DNA repair, cisdiamminedichloroplatinum (cis Pt), X-rays.

Introduction

Ciliates contain two nuclei with a different organization of their genetic material and with different functions. The macronuclei (Ma) are responsible for nearly all transcription. Their DNA is organized, at least in Stylonychia lemnae, in minichromosomes generally carrying only one protein-coding gene. Their copy number is high (several thousand). The diploid micronuclei (Mi) are typical eucaryotic nuclei. Their role is to go through meiosis during sexual reproduction and later (after syncaryon formation) to develop a new Ma. (See reviews by: Ammermann 1990 and Prescott 1994).

In a previous paper it was shown that after treatment of S. lemnae cells with DNA damaging agents (cisdichlorodiammineplatinum II (= cis Pt) or irradiation with X-rays or UV) the Mi are damaged and finally lost, while the Ma seem to be unaffected. While DNA repair was observed after cis Pt treatment but not after X-ray irradiation, the result was always the loss of the Mi (Ammermann 1988).

In this study we tried to understand the different effects of cis Pt and X-rays on the Ma and Mi. The drug cis Pt has become one of the most widely used anticancer drugs. Its cytotoxic effects derive from its covalent interactions with DNA. It forms primarily 1,2-d (GpG) - and 1,2-d (ApG) - intrastrand crosslinks. How these adducts lead to cell death, however, is not clear (Chu 1994; Mello et al. 1998). An inhibition of DNA synthesis, once thought to be the main factor, does not explain the effects (Chu 1994). Often the cells are driven into apoptosis (Chu 1994). In S. mytilus treatment with cis Pt leads to the loss of the Mi (Dutta et al. 1982).

Ionizing radiation causes single and double strand DNA breaks. Depending on the extent of damage the cells either die or they repair both forms of damage efficiently (Jackson and Jeggo 1995). Like cis Pt, X rays cause the loss of the Mi in S. lemnae (Ammermann 1970).

Material and methods

Cells: S. lemnae were cultivated in a modified Pringsheim's solution with the phytoflagellate Chlorogonium elongatum as food (Ammermann et al. 1974).

Chemicals: Cis Pt (Sigma, D-82939 Deisenhofen) was prepared as stock solution (15 mg/ml DMSO), and the desired amount was added to the culture medium (S. lemnae survives up to 2% DMSO for several hours without damage). A dose of 150 pg cis Pt/ml culture medium (= 500 [mu]M) for 4 hours is sufficient for Mi removal and was used if not described otherwise. Cells treated with DMSO (but without cis Pt) were always used as controls.

X-ray irradiation: The irradiation was done in a sterilization chamber (for medical equipment) with Co-60 as the source for the X- rays. The usual dose was 1350 Gy within 15 minutes (dose rate of 90 Gy/min), if not otherwise stated.

Vectors and Microinjection: The details of the vectors used and of the microinjection are described (see Fig. 3 and Skovorodkin et al. 1999; 2001).

Light microscopy and Autoradiography: Details are described in Ammermann et al. (1974) and Ammermann (1988).

DNA analysis: The methods for PCR reactions and Southern blot analysis are also as described previously (Skovorodkin et al. 2001).

Results

Effects caused by cis Pt

The basis for the reported different sensitivities of Ma and Mi to cis Pt could be one or more of the following: Different DNA repair capacity of the nuclei, their different mode of division, the inhibition of Mi DNA synthesis, or a different reaction of the Ma and Mi DNA to the bound cis Pt.

In a previous paper it was shown that both Ma and Mi are able to repair DNA to the same extent, evident as unscheduled DNA synthesis after cis Pt treatment. Nevertheless the Mi disappear while the Ma remain (Ammermann 1988). Therefore, the following other characteristics were tested:

Mi division: Cells were treated with cis Pt, then 60 clones were started, and the decrease of their Mi number was followed during the next 7 divisions. After each division all cells of 15% of the clones were fixed and stained (Feulgen). The cells before treatment contained on average 3.4 Mi/cell. Two divisions after the treatment this number was 2.7 Mi/cell, five divisions after treatment 1.2 Mi/ cell, seven divisions after irradiation 0.9 Mi/cell. Without any Mi division one would expect after 3 cell divisions 0.4 Mi/cell (3.4 [arrow right] 1.7 [arrow right] 0.85 [arrow right] 0.43). The slow decrease shows that Mi divisions occur, but not enough to cope with the cell division rate. A light microscopic inspection of dividing cells confirmed this result. In some treated cells individual (never all) mitotic Mi were found; however, often they possessed abnormalities (comet-shaped dividing Mi, unequal division of Mi). This inspection showed additionally, that the interphase Mi, which are very regular in appearance in untreated cells (spherical, 6 [mu]m diameter, homogeneous content), also show many abnormalities (malformed, clumped chromatin in a part of the enlarged, but otherwise "empty" Mi, see Fig. 1).

Fig. 1. Micronuclei of cis Pt- treated S. lemnae (B-F) and of an untreated cell (A) stained by Feulgen reaction.

DNA synthesis: Cells were treated with cis Pt, then clones were started. The clones were divided into seven groups and one group of clones were given ^sup 3^H-thymidine before the first division, a second group before the second division, etc. to the seventh group before the seventh division. Each group was fixed after the subsequent division. Autoradiographs showed clearly that both Ma and Mi synthesize DNA before all 7 divisions. The Ma DNA synthesis occurred (as usual) in replication bands, and the Mi DNA synthesis occurred (also as in untreated cells) at the end of the S phase of the Ma. The unscheduled DNA synthesis, which was earlier described to occur after cis Pt treatment (Ammermann 1988), was demonstrated in G1 phase nuclei. Although both nuclei synthesize DNA, it is possible that the extent of synthesis is not the same (incomplete DNA synthesis may occur, e. g. in the Mi?).

DNA damage: To detect damage caused by cis Pt the low MW Ma DNA and the high MW Mi DNA were analyzed by agarose gel electrophoresis during several days after treatment with the drug. The result (Fig. 2) shows that the Ma DNA was not detectably affected by the treatment. Even the fraction of the rather higher molecular weight DNA of the Ma, the rDNA genes (arrow in Fig. 2) was unaffected. The Mi DNA showed, however, a different picture: Its high molecular weight DNA has been broken down, beginning (recognizably) 2-3 days after treatment.

Fig. 2. Macronuclear and micronuclear DNA isolated immediately (0) and 1-5 days after cis Pt treatment. The DNA was separated on a 1% agarose gel. The arrow points to the rDNA genes of the macronuclei. Lanes 1 and 14 show marker DNA ([lambda]-DNA, digested with Hind III/Eco RI).

Effects caused by X-rays

In contrast to the observations after cis Pt treatment, no unscheduled synthesis of DNA, which could be interpreted as DNA repair, was found after X-ray treatment (Ammermann 1988). Therefore, we investigated whether the different division mode of Ma and Mi could explain their different sensitivity. We tested also whether DNA double strand breaks are observable, and we turned then again to other possible repair pathways:

Mi division: It was supposed in the past that the Mi are unable to divide after irradiation and therefore are lost during subsequent divisions (Frick 1967). To test this assumption, clones were isolated after irradiation and fed. The average number of Mi in the clones several divisions later was counted (as described above). The cells contained before irradiation on the average 3.65 Mi/cell. One division after irradiation this number was 2.8 Mi/cell, after two divisions 0.8 Mi/cell. It is apparent that the decrease of the Mi number is much faster than after cis Pt treatment. The data support the hypothesis that the Mi are lost because they are unable to divide.

DNA damage: It was tested whether DNA damage was recognizable (as described above and in the legend of Fig. 2). Neither the Ma nor the Mi DNA showed any decrease of molecular weight in an agarose gel (not shown). It was, however, not possible to follow the fate of the Mi DNA after the third cell division, because these nuclei disappeared so fast (see above).

DNA repair by recombination?

The most severe DNA damage, e.g. after X-ray treatment, is double strand breaks (DSBS). There are two processes known to occur in pro- and eucaryotes which can repair DSBS:

Homologous recombination between a broken DNA helix and \an undamaged homologous strand restores two repaired strands (comparable with recombination during meiosis (Shinohara and Ogawa 1995)). Two broken ends of a double helix can be sealed by nonhomologous end-joining (Kanaar et al. 1998; Featherstone and Jackson 1999). It was shown by Ammermann (1988), using autoradiography, that after X-ray-irradiation of S. lemnae no DNA synthesis which could be interpreted as DNA repair synthesis was visible. But recombination and end joining processes could have escaped detection because of the insensitivity of the method used. Therefore, it was investigated whether there are hints for one of these repair processes in the Ma.

Homologous recombination: We used a vector which we described earlier (Skovorodkin et al. 1999, 2001). Briefly, it is an [alpha]1- tubulin minichromosome of S. lemnae (Fig. 3A). Its noncoding 5'- leader and 3'-trailer sequences were replaced with sequences of the procaryotic neomycin phosphotransferase gene and a 19 bp tag was inserted in the coding region (Fig. 3B). This vector was injected successfully (proven with the PCR reaction, see Skovorodkin et al. 1999) into the Ma of three cells. To exclude interferences which may be caused by the loss of the Mi after irradiation, the vectors were injected into Ma of established (= well growing) cells without Mi (see Ammermann 1970). After the three transfected cell clones contained several hundred cells each, they were irradiated and clones were started. Three weeks later DNA was isolated. Hybridization of the DNA with labeled neomycin resistance gene probes showed that the injected gene was still in the Ma, and that both 5'-leader and 3'-trailer sequences were still present on only one chromosome which had the same size as the injected one (as present in unirradiated control cells). After a recombination process one would expect to find some of the 5'-leader and 3'- trailer sequences on two different minichromosomes. This result (no figure shown here) does not point to recombination processes between the [alpha]1-tubulin coding region of the endogeneous minichromosome and the homologous coding region of the injected vector. It supports earlier reports (Jacob, pers. comm.; Skovorodkin et al. 1999), that in the Ma of S. lemnae no homologous recombination processes are observable.

Fig. 3. Schematic drawings of the vectors used for microinjection. A. The minichromosome with the [alpha] 1-tubulin gene of S. lemnae. Dense stippling: coding sequence; light stippling: the 5'-leader (L) and 3'-trailer (T) noncoding sequences. The lengths (in bp) are given. Black: The telomeres (20 bp + 16 b- single stranded overhang). B. The noncoding sequences were replaced by neomycin phosphotransferase sequences (wide striped), and a tag sequence (white) was inserted into the coding region. C. The coding sequence was replaced by the coding region of the neomycin phosphotransferase gene (narrow stripes). The vector was cut with NcoI. For identification of both pieces probes l and t were used (see Fig. 4). For details of the vector construction see Skovorodkin et al. (1999, 2001).

Non-homologous recombination: For the following experiment a different vector was used, described in Skovorodkin et al. (2001). Here the coding region of S. lemnae [alpha]1-tubulin minichromosomes was replaced by the coding regions of the neomycin transferase gene (Fig. 3C). The noncoding regions were unchanged and remained, therefore, as in the endogenous minichromosome. Artificial minichromosomes were generated by digestion of pTubal-neo gene construct (Skovorodkin et al., 2001) with Apal restriction enzyme with following purification on a 1 % agarose gel using the QIAquick Gel Extraction Kit (Qiagen). In order to mimic one DNA damage double strand break caused by X-rays this linear vector was cut into two pieces with the restriction enzyme Ncol followed by a blunt-end reaction using the Klenow enzyme. As a result two types of DNA molecules were created, which each had a telomere at one side and an unprotected blunt end at the other side (Fig. 3C). Both DNA pieces were injected in equimolar amounts into the Ma of two cells. The presence of these molecules was checked 9 weeks (approx. 120 divisions) after injection with DIG-labelled probes corresponding to the 3' and 5'-end of the coding region (t and l in Fig. 3C) of the vector. The result is shown in Fig. 4. Both injected parts of the vector are present in the Ma, but no longer in the injected size. The smaller part (with L, sec Fig. 3C and Fig. 4B, lane 1) is now present in three larger editions (Fig. 4B, lane 3, arrows). The larger part (with T, see Fig. 3C, and Fig. 4C, lane 2) is also present in 3 larger versions (arrows m lane 3, Fig. 4C). Only one rather weak band (asterisk in Fig. 4) hybridizes with both probes, but shows also longer DNA molecules than the original vector.

Fig. 4. In these 3 gels the following DNA probes were run: M=a molecular marker. Lane 2 the complete vector (see Fig. 3C). Lane 1 the vector cut with Ncol (see Fig. 3C). Lane 3 the DNA of one cell clone approximately 30 generations (3 weeks) after injection. The following DIG labeled probes were hybridized to the gels: A full sized coding region of the vector C (see Fig. 3C); Gel B: Part l of the vector (see Fig. 3C); Gel C: Part t of the vector (see Fig 3C). The asterisk in lane 3 marks a band which apparently contains both parts of the vector, but also additional sequences, because it is longer than the vector in lane 2.

We conclude that the cells restored the separate injected pieces to the original vector in only rare cases (if at all). At the same time we have to recognize that rejoining of the DNA fragments happened, but it occurred non randomly (otherwise we would see a smear instead of the discrete bands which are visible in Fig. 4). It is also interesting that the gel pattern of both injected clones was the same. It would clearly be informative (and is planned for the future) to investigate which DNA sequences are joined with the injected ones.

Do the S. lemnae Ma replace damaged DNA molecules?

The Ma contains several thousand copies of each DNA molecule (minichromosome). The mechanism of their distribution during division is not clear, but it is not by mitosis. It could be simple random distribution of the 100-300 x 106 minichromosomes.

One hypothesis for the low sensitivity to DNA damaging agents could therefore be that the nuclei lose all damaged (= broken, without both telomeres) minichromosomes. Because enough undamaged copies are left, the Ma could upregulate the lost DNA by an extra replication. The prerequisites of this hypothesis were demonstrated earlier: 1) The Ma of S. lemnae propagate injected minichromosomes with both telomeres, but lose them if the telomeres are lacking (Skovorodkin et al. 2001). 2) The Ma maintain a certain DNA content. It is able to regenerate lost parts (Grell 1973; in Stylonychia: Frick 1967) by extra DNA replication.

It was therefore investigated whether experimental data after X- ray treatment support this hypothesis. Spectrophotometric measurements showed no DNA loss of the Ma after ionizing radiation (Dittmann, Ph-D Thesis, 1978 Univ. Tubingen and pers. comm.). However, this method may be not sensitive enough to detect a small loss. Therefore, it was tested whether there are signs of extra (= upregulating) replication bands. Hypotrichous ciliates replicate their Ma DNA in easily visible replication bands (Gall 1959) which traverse the Ma once before division. S. lemnae cells were synchronized by starving them for 16 hours. They are then nearly all in the G1 phase. Then half of the cells were irradiated with X-ray (1660 Gy, 69 Gy/min), the other half remained unirradiated (control). After this treatment the irradiated cells looked (as usual) slightly damaged for an hour, recognizable by their slow movement. After this time they behaved normally again. Seven hours after irradiation clones were started from the irradiated and control cells, and they were fed. The first and second divisions were recorded. Every hour after feeding 20 of the control and the irradiated cells were stained, and the occurrence and position of their replication band was recorded.

The experiment showed that the irradiated cells lagged behind the control cells by 1-2 hours in progress of replication bands and m division. There was no hint of the occurrence of an extra replication band. Previous observations showed that it takes 6 hours for a replication band to traverse the Ma (Ammermann 1970).

These results make it unlikely that a complete extra replication of the irradiated Ma occurs, but they cannot exclude that a small part of the Ma is restored by extra replication.

Discussion

Although the treatments with cis Pt and X-rays both result in emicronucleated cells, the underlying damage of the nuclei and their reactions are different. Therefore the two agents are discussed separately.

Cis Pt: After cis Pt treatment unscheduled DNA synthesis was a sign of repair in both nuclei (Ammermann 1988). The mode of repair was not investigated, but in other eucaryotic cells an excision repair (with new DNA synthesis) was observed after cis Pt treatment (Chu 1994; Mello et al. 1998).

Nevertheless only the Ma survive, and we conclude that the reason is their successful repair. It is intriguing to suppose that only the damaged Mi (and not the repaired Ma) give signals to the cytoplasm which leads (after 3-5 days) to its destruction. The degradation of some of the cell nuclei (e.g.: diploid and haploid Mi and old Ma during conjugation) is in ciliates a common process and is often named "apoptosis like" (in Tetrahymena: Davies et al. 1992; Santos et al. 2000; in Paramecium: Hirano et al. 1998; in Stylonychia: Maercker et al. 1999).

X-rays: The results support the suggestion of Frick (1967) that the Mi are unable to undergo mitosis after damage by X-rays and are therefore lost. We do not ha\ve an explanation why we do not see damage to DNA in the agarose gels (see below).

The results do not point to a hypothesis which could explain why the Ma are so resistant. Neither DNA repair synthesis (UDS) nor recombination repair could be demonstrated.

It may, therefore, be helpful to try to calculate the number of DNA double strand breaks (DSBS). Lobrich et al. (1995) calculated 36 DSBS in primary human fibroblast cell nuclei (6 x 109 bp DNA) per 1 Gy. Daly and Minton (1995) calculated for the prokaryote cell Deinococcus radiodurans 500 DSBS in the genome (3 x 10^sup 6^ bp) after irradiation with 17500 Gy. From these results it can be estimated that 1 Gy irradiation causes 20-30 DSBS per 3 x 10^sup 9^Bp.

One S. lemnae Mi contains around 18 pg DNA (Ammermann and Schlegel 1983), distributed in 350 (= 2n) chromosomes (Ammermann 1987). One chromosome contains on the average 5 x 107 bp, which (irradiated with 1350 Gy) would produce about 500 DSBS. It seems difficult to imagine that such chromosomes can go through mitosis.

One S. lemnae Ma contains 788 pg DNA (Ammermann and Schlegel 1983), distributed in 100-300 x 106 minichromosomes, each with (average value) 2500 bp. (Values for other hypotrich ciliates, see Hoffman et al. 1995). Less than 5% of the minichromosomes would therefore contain DSBS which means the majority of the minichromosomes suffer no DSBS. If these "less than 5%" minichromosomes are lost (due to the lack of telomeres, Skovorodkin et al. 2001), this loss would not be observable with spectrophotometric measurements, and it probably would not be seen on agarose gels. Our hypothesis is that the presence of minichromosomes in high copy numbers allows a loss of part of the Ma DNA without lethal consequences. This may be the reason why we could not detect repair of DNA after ionizing radiation: the cells simply do not need it.

It is remarkable that many of the S. lemnae strains collected in the USA are without Mi or with small Mi, in contrast to Eurasia, where a strain without Mi or with "small" Mi never was found (Ammermann, et al. 1989). Because the US strains are mortal, they have to originate anew from time to time. Conjugation without Mi does not create survivors, so cells without Mi have to develop anew regularly after conjugation from cells with Mi. It would be interesting to know the factor(s) which are responsible for the regular loss of the Mi in the US strains.

Acknowledgement: This work was supported by a grant from the DFG (Am 26/31-1) and by grants from the Russian Foundation of Fundamental Research (03-04-48505-a). Irina Zassoukhina was supported by a Stipendium from the DAAD. We are very much indebted to the Willy Ruesch AG, D-71394 Kernen, who allowed us to use their facilities for the irradiation experiments. We are also indebted to valuable comments from (unknown) reviewers.

Europ. J. Protistol. 39, 223-230 (2003)

(C) Urban & Fischer Verlag

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Skovorodkin I. Zassoukhina I. B., Hojak S., Ammermann D. and Gunzl A. (2001): Minichromosomal DNA replication in the macronucleus of the hypotrichous ciliate Stylonychia lemnae does not depend on nontelomenc sequences. Chromosoma 110, 352-359.

Dieter Ammermann1'*, Karl-Heinz Hellmer1, Irina Zassoukhina2 and Ilya Skovorodkin2

1Zoologisches Institut der Universitat, 72076 Tubingen, Auf der Morgenstelle 28, Germany; E-mail: dieter.ammermann@uni-tuebingen.de

2Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Avenue, 19464 St. Petersburg, Russia

Received: 27 January 2003; 12 May 2003. Accepted: 15 May 2003

*corresponding author

Copyright Urban & Fischer Verlag Jul 2003

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