Induction of chromosomal aberrations by restriction endonucleases encapsulated in liposomes

Mutation Research, 262 (1991) 177-181

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© 1991 ElsevierSciencePublishers B.V. (BiomedicalDivision)0165-7992/91/$ 03.50 ADONIS 0165799291000188 MUTLET0466

Induction of chromosomal aberrations by restriction endonucleases encapsulated in liposomes Francisc Mixich Department of Cell Biology, Faculty of Medicine Craiova, 1100 Craiova (Romania)

(Received3 April 1990) (Revisionreceived23 October 1990) (Accepted29 October 1990)

Keywords:Chromosomalabberations;Restrictionendonucleases;Liposomes

Summary We used liposomes to deliver the restriction endonucleases B a m H I and Sinai into human heteroploid HEp-2 cells. With this method very low concentrations of enzymes (2 units/ml) were active in the production of chromosomal aberrations. Sinai, which produces blunt-ended double-strand breaks in the DNA molecule, induces chromosomal aberrations more effectively than B a m H I , which produces cohesive ends. Our results indicate that liposomes are suitable vectors for introducing restriction endonucleases into cultured human cells.

Classical mutagens such as radiations, chemicals and viruses can produce chromosomal aberrations either directly or indirectly. Recently it has been shown that chromosomal aberrations can be induced by endonucleotic enzymes (for reviews see Obe et al., 1987; Zajac-Kaye, 1987). Indirect evidence for the induction of chromosomal aberrations with endonucleases was observed in human embryonic fibroblasts infected with herpes simplex virus coding for DNase (Sablina, 1978). Exogenous administration of endonucleases to living mammalian cells can produce chromo-

Correspondence: Dr. F. Mixich,Department of Call Biology, Facultyof MedicineCraiova, 1100Craiova (Romania).

somal aberrations as demonstrated with Neurospora endonuclease (Natarajan and Obe, 1978; Obe and Natarajan, 1980; Nowak and Obe, 1984; Nowak et al., 1984; Sen and Hittelman, 1984) and restriction endonucleases (Obe et al., 1987). Treatment of heteroploid EUE cells, human lymphocytes or Syrian hamster embryo cells (SHE) with pancreatic deoxyribonuclease (DNas¢ I) gave negative results (Zajac-Kaye and Ts'o, 1984; Nuzzo et al., 1987). Treatment of cells with DNase I in liposomes led to chromosomal aberrations, mutations at the HPRT locus and neoplastic transformation (Zajac-Kaye and Ts'o, 1984; Nuzzo et al., 1987). Restriction endonucleases (RE) produce only one type of alteration in DNA, namely DNA

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double-strand breaks. Various treatment protocols, namely permeabilization of cells with inactivated Sendai virus (Bryant, 1984; Natarajan and Obe, 1984; Bryant and Christie, 1989), treatment of cell pellets with high concentrations of RE (Obe et al., 1985), and osmotic shock (Winegar and Preston, 1988; Bryant and Christie, 1989), gave similar results. In order to facilitate the uptake of REs, we encapsulated BamHI, which produces cohesive ends, and SmaI, which produces blunt ends, in liposomes, and treated the cells with the liposomes. Using this method we obtained chromosomal aberrations with doses of REs that were much lower than those used by other authors. Materials and methods

Reencapsulation in liposomes Liposomes were prepared according to a reversephase evaporation procedure (Szoka and Papahadjopoulos, 1978), modified to avoid enzymatic degradation. In brief, 26.4 /~mole of egg yolk phosphatidylcholine (Sigma, U.S.A.), 12.8/zmole of cholesterol (Sigma, U.S.A.), and 4 /~mole of diacetylphosphate (Fluka, Switzerland) were dissolved in 5 ml diethylether. To the organic solution, 1.5 ml of assay buffers were added as given by Sigma (SmaI: buffer A; BamHI: buffer B). Buffer A was composed of Tris 15 mmole, MgC12 6 mmole, KC1 15 mmole, 2-mercaptoethanol 6 mmole, and bovine serum albumin (BSA) 100 /zg/ml; the pH was adjusted to 8.0 with HCI 0.2 mole. Buffer B was composed of Tris 20 mmole, MgCI2 7 mmole, NaC1 100 mmole, 2-mercaptoethanol 2 mmole, and BSA 100/~g/ml; the pH was adjusted to 7.4 with HCI 0.2 mole. Biphasic mixtures composed of lipid-diethylether solution and aqueous buffer were ultrasonicated for 30 s, and RE was added to the obtained emulsion resulting in a final concentration of enzyme of about 2 units/ml of final suspension of liposomes. After additional vortexing of the emulsion for 5 min, diethylether was evaporated in vacuo using a rotary evaporator at 20°C. To the resulting gel 1.5

ml of buffer A or buffer B was added. The final suspension of liposomes was prepared by vortexing for another 10 min and ultrafiltration using a Millipore membrane (0.22/zm). To remove free RE, 2 ml of liposomes was passed through a Sephadex G-200 column (1.5 cm x 30 cm) which was eluted with phosphate-buffered saline (PBS) at pH 7.4. Using the same technique liposomes with buffer A and buffer B but without RE (empty liposomes) were prepared.

Cell-culture treatment Human HEp-2 cells (Cantacuzino Institute, Bucharest), growing in MEM medium supplemented with 10% calf serum, were trypsinized 2 days after the last passage, and suspended in 10 ml of fresh medium. The cells were pelleted (100 x g for 10 min) and suspended in PBS at pH 7.4. To 10 ml suspension with 5 x 10 6 cells, 1 ml liposomes with or without RE was added and incubated at 37°C for 20 min. The cells were centrifuged, washed once with PBS and resuspended in culture medium. Chromosome preparations were made 5 or 18 h after culture initiation.

Chromosome preparation 90 min before fixation Colcemid (1 #g/ml) was added to the culture. The mitoses were detached from the monolayer by agitation, centrifuged and suspended in hypotonic solution (KCI 0.075 mmole, 30 min at 37°C), fixed 3 times in methanol/glacial acetic acid, 3:1, and dropped on icecold slides. The dry preparations were stained with 3% Giemsa stain. Chromosome anomalies were analyzed according to conventional criteria as chromatid- (gaps, chromatid breaks, single fragments, chromatid rearrangements) and chromosome-type (chromosome breaks, double fragments, dicentrics, and other chromosomal rearrangements) aberrations. Results

Table 1 shows the frequencies of aberrations found. There is no significant difference between

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TABLE l FREQUENCIES OF C H R O M O S O M A L ABERRATIONS IN HEp-2 CELLS TREATED W I T H RE, W I T H EMPTY LIPOSOMES OR W I T H RE E N C A P S U L A T E D IN LIPOSOMES Treatment

Recovery

Number of

Aberrant

Types of aberrations b

time (h)

metaphases analyzed a

cells (070)

Chromatid A

Control

B

Aberrations Chromosome

C

D

E

(070) F

G

5 18

300 300

4.6 6.3

6 6

4 7

-

4 7

1 4

-

-

5.33 8.00

Exogenous 5 Sinai in 18 PBS (2 U / m l )

300 300

8.3 5.0

9 6

6 2

-

4 5

7 4

-

-

8.66 5.66

Exogenous

5 18

300 300

3.3 4.6

4 4

2 3

-

6 3

1 6

-

-

4.33 5.33

Buffer A in liposomes

5 18

300 300

3.6 5.0

3 4

3 4

-

2 5

3 4

-

-

3.33 5.66

Buffer B in liposomes

5 18

300 300

6.6 6.0

5 6

2 5

-

7 3

7 4

2

-

7.00 6.66

Sinai in

5 18

300 300

26.3 (2.0) 73.6 (2.8)

50 36

27 29

6 15

10 96

18 78

18

3 60

38.00 111.33

5 18

300 300

16.0 (1.8) 20.6 (2.4)

18 21

22 20

2 6

8 31

10 16

1 l

II

20.33 35.33

BamHI in

1

PBS (2 U / m l )

liposomes

BamHI in liposomes

a 100 metaphases were analyzed in 3 independent experiments. b Total number in 3 independent experiments. Buffer A, assay buffer for Sinai (according to Sigma); Buffer B, assay for BamHI (according to Sigma). A, chromatid breaks; B, single fragments; C, chromatid exchanges; D, isochromatid breaks; E, double fragments; F, rearrangements (rings, translocations); G, dicentrics. Figures in parentheses indicate standard deviations for 3 experiments.

controls (untreated cultures, cultures treated with RE not included in liposomes, and empty liposomes), indicating that RE in a concentration of 2 units/ml or empty liposomes do not induce chromosomal aberrations. Sinai in liposomes produced chromosomal aberrations when the cells were treated for 5 or 18 h before fixation. Under the same conditions BamHI was less effective. Sinai, but not BamHI, induced predominantly chromatid aberrations following a recovery time of 5 h and chromosome aberrations following a recovery time of 18 h.

Discussion Our experiments show that the uptake of RE by living cells seems to be very effective when the enzymes are encapsulated in liposomes. The amount of enzymes necessary to produce aberrations seems to be very low, and, according to our experimental protocol, cell viability is hardly affected. The mechanism by which liposomes can interact with cells via fusion and endocytosis has been extensively studied (for reviews see Pagano and Weinstein, 1978; Ryman and Tyrrell, 1979; Poste,

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1980; Nicolau and Sene, 1982). An endocytotic mechanism is supported by the finding that the fate of the encapsulated material depends on the action of lysosomal enzymes. The fusion of liposomes with the plasmalemma was demonstrated morphologically by freeze-fracturing techniques. As compared with other techniques, the production of chromosomal aberrations with RE encapsulated in liposomes offers 4 advantages: (1) the active concentration of RE is very low; (2) cell viability is not affected; (3) results are reproducible; and (4) possible effects of permeabilizing agents are eliminated. We used two RE, Sinai, which produces bluntended double-strand breaks (recognition site CCC/GGG), and BamHI, which produces stickyended breaks (recognition site G/GATCC). Although some authors maintained that the frequencies of RE-induced chromosome aberrations do not depend on the type of double-strand breaks (Obe et al., 1985; Gustavino et al., 1986; Winegar and Preston, 1988), our results clearly demonstrate that SmaI, generating blunt-ended breaks, is more effective than BamHI, generating cohesive-ended breaks. These results agree with those obtained by others (Bryant, 1984; Natarajan and Obe, 1984; Bryant and Christie, 1989). But it has to be taken into account that the frequencies of recognition sites of different REs vary in the mammalian genome and it is likely that the effectivity of different REs to induce chromosomal aberrations depends on this (Obe et al., 1987). Our results confirm that REs produce aberrations independent of the cell cycle (Obe et al., 1987). This is partially supported by the predominance of chromatid aberrations in cultures examined 5 h after treatment and of chromosome aberrations in cultures examined 18 h after treatment. However, to confirm these quantitative data, it will be necessary to treat cells with bromodeoxyuridine in order to exclude second post-treatment metaphases. References Bryant, P.E. (1984) Enzymatic restriction of mammalian cell

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