4,6,4′-Trimethylangelicin induces interstrand cross-links in mammalian cell DNA

Journal of Photochemistry and Photobiology B: Biology 26 (1994) 197-201

ELSEVIER

Rapid

Communication

4,6,4’-Trimethylangelicin induces interstrand mammalian cell DNA F. Bordin”,

C. Marzano,

C. Gatto,

F. Carlassare,

cross-links in

P. Rodighiero,

F. Baccichetti

Dipmtimento di Science Farmaceutiche,Universitddi Padova, Vii Manolo 5, 35131 Padua, Italy

Received 30 May 1994; accepted 17 June 1994

Abstract 4,6,4’-Trimethylangelicin,

cross-links; Alkaline elution; Two-step irradiation

1. Introduction

Angelicin derivatives have been prepared and studied in an attempt to obtain new monofunctional drugs for photochemotherapy capable of photobinding to DNA on UVA irradiation without forming interstrand crosslinks (ISCs) [l]. They are potentially bifimctional compounds, having two photoreactive sites at the 3,4 and 4’,5’ positions, but in general they behave as monofunctional compounds because their angular molecular structure restrains ISC formation. In fact, both pyrone

side and furan side monoadducts have been isolated from the photoreactions of 4’-methylangelicin (MA) [2] and 4,6,4’-trimethylangelicin (TMA) with DNA [31Of the angelicin derivatives, the most interesting compound is certainly TMA [4,5]. Various studies have described it as a very active photosensitizing drug, capable of inducing strong antiproliferative [5] and genotoxic [6,7] effects; in some cases the effects are *Corresponding

author.

loll-1344/94/$07.00 0 1994 Elsevier Science S.A. AlI rights reserved SSDI 1011-1344(94)07040-U

difficult to explain considering only its capacity to link monofunctionally to DNA. The investigation of the photochemical reactions of TMA with the Kpn linker (an oligonucleotide having the structure 5’-d(CGGTACCG)3’) showed the formation of small amounts of cross-linked nucleotide [8]. Very recently, Chen and coworkers [9] reported the formation of ISCs in pBR322 and Ml3 DNA in vitro by TMA sensitization. In this paper, using alkaline elution, we show that TMA is capable of inducing ISCs in mammalian cell DNA in vivo. Moreover, we confirm these data by studying the clonal growth capacity of TMA-sensitized cells using the two-step irradiation method. This procedure has been employed for several years to demonstrate the increased antiproliferative or genotoxic effects of the conversion of monoadducts into crosslinks [lo]. As reference compounds, we used 8-methoxypsoralen and 3-carbethoxypsoralen as known positive [l] and negative [9,11] cross-linking agents respectively; for comparison we also performed some experiments with two compounds related to TMA, MA and 4,4’dimethylangelicin (DMA). Fig. 1 shows the molecular structure of the tested compounds.

F. Bordin et al. 11. Photochem.

198

Fi

R=H;

R,

R,

= H

=COOCH

H

8-Methoxypsoralen

@-MOP)

3-Carbethoxypsoralen (3-CPs)

25

R

I1

R

2/

\

I 0’00

p R=CH 3’I R, =R R=

R, =CH3;

R=

R1

4’-methylangelicin (MA)

=H

4.4’dimethylangelicin

R2=H

= R2=CH

Fig. 1. Molecular

I+

2

structure

3

@MA)

4.6.4’-trtmethylangelicin

of the compounds

B: Biol. 26 (1994) 197-201

2.3. WA irradiation

WoR’ R = OCH3;

Photobiol.

(TMA)

tested.

2. Materials and methods

2.1. Chemicals MA, DMA and TMA were prepared as described previously [12,13]; 8methoxypsoralen (8-MOP) was purchased from Chinoin, Milano, Italy. 3-Carbethoxypsoralen (3-CPs) was a gift from Professor D. Averbeck, Institute Curie, Paris. The compounds were dissolved in dimethyl sulphoxide (DMSO, 1 mg ml-‘) and the solutions were kept in the dark. Just before the experiments, a calculated amount of drug solution was added in the dark to the growth medium containing the cells to be sensitized, to a final DMSO concentration of 0.5%. 3H-Thymidine (4.77 TBq mM_‘) and 14Cthymidine (2.2 GBq mM_l) were obtained from Amersham International Inc., UK. Proteinase K was purchased from Boehringer Mannheim GmbH, Germany. Tetrapropylammonium hydroxide, 1 M aqueous solution, was obtained from Sigma Chimica, Milano, Italy. 2.2. Mammalian cells Pseudo-diploid Chinese hamster ovary (CHO) cells and human HeLa cells were a gift from Professor F. Majone, Department of Biology of Padua University. CHO cells were used for ISC detection by alkaline elution; they were grown in Nutrient Mixture F-12 Ham medium (Sigma Chimica, Milano, Italy) containing 5% calf serum by incubation at 37 “C in a 5% carbon dioxide atmosphere. HeLa cells were used for studies on clonal growth ability, using the same medium supplemented with 10% calf serum; the plating efficiency was about 90%. Cell labelling and clonal growth studies were carried out as described in Ref. [14].

Cell cultures containing the drug to be studied were incubated at room temperature for 15 min in the dark to allow drug penetration and then placed into Petri dishes (5 cm in diameter; 3 ml) in an ice bath. After cooling for 15 min, the samples were exposed to UVA light; samples for alkaline elution were also kept in ice during successive treatments (e.g. y irradiation), until the lysis step. UVA exposures were performed using Philips HPW 125 lamps, provided with a builtin Philips filter; their emission was in the range 320-400 nm, with a maximum, over 90% of the total, at 365 nm; the irradiation intensity, determined by a UV-X radiometer (Ultraviolet Products Inc., Cambridge, UK), was 5.5 X 10Y4 kJ m-‘. The two-step irradiations were accomplished by exposing cells to a small UVA dose in the presence of the compound examined; according to the different photosensitizing activities of the compounds, appropriate drug concentrations and UVA doses were chosen to obtain approximately the same surviving fraction. The furocoumarin-containing medium was removed, the cells were washed twice with drug-free medium and were further irradiated with increasing UVA doses in the absence of the drug [lo]. The clonal growth ability of the treated cells was assayed. 2.4. IX

detection

Alkaline elution was carried out according to Kohn [15]; the experiments were performed using untreated cells labelled by incubation with 3H-thymidine as internal standard and treated cells labelled with 14C-thymidine. Cells were kept in ice during irradiation and before lysis. Elutions were performed using polycarbonate filters (pores 2 pm in diameter); a Gilson Minipuls peristaltic pump and a Gilson fraction collector were used. y-Ray irradiations (about 6 Gy) were accomplished at O-5 “C using a 6oCo source (working at the Reparto Applicazioni, Legnaro, Padova, Istituto di Fotochimica e Radiazioni d’Alta Energia, FRAE, CNR) with a dose rate of 270 rad min-‘, determined by Fricke solution. Radioactivity measurements were performed using Instage1 (Packard Instruments, Meriden, CT, USA) as scintillating fluid and a Packard A 300 CD spectrometer. Double-isotope counting was accomplished automatically on the basis of quenching curves obtained using 3H and 14C radioactivity standards. The data were expressed as the cross-linking coefficient (CC), according to the following equation cc=

l/z

( ) 2

-1

1

where R, is the DNA retention observed with control cells and R, is the retention of DNA from photosensitized cells, both treated with 6 Gy of y rays [15].

F. Bordin et al. / J. Photochem.

Photobiol.

B: Biol. 26 (1994) 197-201

199

2.5. Clonal growth HeLa cells ((1.5-2) X 105) were grown for 24 h; the medium was then replaced by a fresh solution containing the drug studied; after UVA irradiation, 200 treated cells were seeded again and incubated as above for 7 days in growth medium containing 10% calf serum. The colonies were stained and counted, eliminating those formed by less than 50 cells; the clonal growth efficiency is the ratio between the number of scored colonies and the number of seeded cells. The plating efficiency was about 90%. UVA

3. Results and discussion Fig. 2 shows the detection of ISCs in CHO cells by alkaline elution; &MOP and TMA were studied at 20 PM concentration and 3-CPs at 39 PM; the UVA dose was 5 kJ mp2. As expected, S-MOP and 3-CPs behave clearly as positive and negative DNA cross-linking references respectively. In fact, the bulk of DNA from cells sensitized by 8-MOP is retained on the filter, whereas with 3-CPs this amount is very small and similar to that obtained with standard cells exposed to y rays only. TMA sensitization generates an elution profile intermediate between those of &MOP and y rays; this result is consistent with the induction of a moderate amount of ISCs by TMA sensitization. Fig. 3 shows the quantitative data obtained in various experiments; alkaline elution does not give absolute data, but only a parameter proportional to the number of ISCs (CC). We have also tested two other methyl-

1

0

2

4 8 6 FRACTION NUMBER

10

12

Fig. 2. KC formation in CHO cell DNA; cells were exposed to 5 kJ m-* in the presence of the compound studied, irradiated with 6 Gy of -y rays and then submitted to alkaline elution. The drug concentrations and symbols are as follows: untreated control (W); control irradiated with 6 Gy of y rays (0); sample treated with 6 Gy of y rays and UVA irradiated in the presence of S-MOP, 20 PM (x ), TMA, 20 PM (A) and 3-CPs, 39 PM ( q).

DOSE (KJ/m’)

Fig. 3. ISC formation in CHO cell DNA; cells were exposed to increasing UVA doses in the presence of 8-MOP or TMA, both at 20 PM concentration. Samples were irradiated with 6 Gy of y rays and submitted to alkaline elution. The cross-linking coefficient was calculated according to Kohn [15]. Symbols: TM.4 (B); 8-MOP (Cl).

Table 1 ISC formation in DNA of CHO cells Compound

S-MOP MA DMA TMA 3-CPS

Concentration

UVA dose (kJ m-‘)

ISC

(PM)

(CC)

Relative activity

20 25 24 20 39

5 10 10 5 10

0.420
1.0 < 0.02 < 0.02 0.15
angelicins related to TMA (MA and DMA) for DNA cross-linking. Table 1 shows the data thus obtained. In comparison with &MOP, TMA induces a lower but still significant amount of ISCs, while MA and DMA appear to be entirely incapable of cross-linking DNA. Since the conversion of monoadducts into ISCs should lead to a notable increase in lethality, we carried out some experiments using two-step irradiation on HeLa cells. With this method, a first short UVA exposure induces mainly monoadducts; the unbound furocou‘marin molecules are washed out and the cells are further irradiated with increasing UVA doses in drugfree medium. The clonal growth ability of the treated cells is then assayed. In these experimental conditions, with a DNA cross-linking furocoumarin, some furan side monoadducts are converted into ISCs, with an evident increase in lethality [lo]. As reported in Table 2, 8-MOP, the cross-linking reference, behaves as expected, inducing a pronounced reduction in the surviving fraction after the second irradiation step. In contrast, with 3-CPs, no significant modifications in survival are observed after the second irradiation step, a result consistent with its well-known inability to cross-link DNA [9,11]. In these experiments, TMA behaves sim-

200 Table 2 Inhibition

F. Bordin et al. / J. Photochem.

of clonal growth in HeLa cells by two-step

Controls TMA

B: Biol. 26 (1994) 197-201

irradiation

(cLM)

First step (kJ m-‘)

2.2

0.03

Concentration at first step

Compound

Photobiol.

Successive step (kJ m-a)

0.12 0.24 0.48 8-MOP

5.18

0.12 0.12 0.24 0.48

3-CPS

4.8

0.67 0.66 1.32

SD, standard

Surviving fraction (*SD) 1.0*0.015 0.77 f 0.02 0.48 f 0.04 0.35 f 0.03 0.32*0.05 0.72 * 0.04 0.63 f 0.005 0.50 f 0.008 0.42 i- 0.02 0.80 i- 0.035 0.78 * 0.04 0.75 + 0.06

deviation.

ilarly to 8-MOP, with a remarkable decrease in the survival fraction in the second step as a function of UVA dose. Therefore we have demonstrated that TMA can photoinduce ISCs in DNA inside mammalian cells in vivo, in accordance with recent observations made in vitro [9]. This conclusion can explain some anomalous data obtained previously [5-71. The reason why TMA can form ISCs while other angelicins, such as MA and DMA, cannot remains to be elucidated; indeed, the molecular structures of DMA and TMA are very similar. We believe that the answer lies in their interaction with DNA in the dark, in particular with DNA inside a cell; it is possible that the presence of the methyl group at the 6 position modifies the arrangement of the intercalated molecule, making two adjacent pyrimidine bases accessible, thus allowing the formation of a small number of ISCs. Another problem is the description of TMA as a monofunctional compound; we believe that this is mainly due to the sensitivity of the method and the features of the materials used. Indeed, we have observed that commercial calf thymus DNA contains many singlestrand breaks (SSBs) and that heat denaturation increases their number; thus the sensitivity of ISC detection, based on the evaluation of reversibly renaturing DNA, is strongly affected [16]. Moreover, we should consider that MA and DMA, in contrast with TMA, can form DNA-protein crosslinks (DPCs) in mammalian cells [17]. At present the nature of such lesions is not known; however, a bifunctional adduct between a furocoumarin and a protein has been prepared in vitro [18]. Therefore we believe that similar adducts may link together DNA and proteins in sensitized cells. These data suggest that we need to redefine the properties of some furocoumarins. Thus

only derivatives having one photoreactive site in their molecule can be defined as monofunctional (e.g. 3CPs); we could call them intrinsically monofunctional compounds. In general, angelicins are monofunctional and not ISC-forming compounds even when they are intrinsically bifunctional derivatives. Finally, we should distinguish between ISC- and DPC-forming furocoumarins; for instance, TMA forms ISCs but no DPCs, whereas MA and DMA form DPCs but no ISCs.

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