Cytotoxic, nuclear, and growth inhibitory effects of photodynamic drugs on pancreatic carcinoma cells

ELSEVIER

CANCER LETTERS Cancer Letters 102 (1996) 3947

Cytotoxic, nuclear, and growth inhibitory effects of photodynamic drugs on pancreatic carcinoma cells Amanda J. Ward, E. Keith Matthews* Department of Pharmacology, University of Cambridge, Tennis Court Road,

CambridgeCB2lQ.l, fJK

Received4 January1996;accepted18January1996

Abstract

The light-activated drugs AlPcS4 and T4MPyP were studied in a pancreatic carcinoma cell line for their effects on DNA integrity, cell division, proliferation, and survival. The micronucleus assaymeasurednuclear changesand also the number of actively dividing cells while, under similar conditions, the M7T assaymeasuredcell survival. When tumour cells were exposed to light, pre-treatmentwith AlPcS4 induced more micronuclei than did FMPyP at the samelevels of cell division and survival. Both drugs showed a correlation between phototoxicity and changesto DNA integrity so establishing micronuclei formation as an important indicator of photodynamic drug action on tumour cells. Keywords:

Photodynamic drug action; Pancreaticcarcinoma cells; Micronucleus; M’IT assay;Phototoxicity; Growth inhibi-

tion

1. Introduction Photosensitizers are molecules which when activated by light have toxic effects on cells in vivo and in vitro [I]. Light-activated photosensitizers provide the basis for photodynamic therapy (PDT), and are used in the treatment of various forms of cancer. More recently the potential benefits of PDT in diseases such as psoriasis and rheumatoid arthritis have been recognized [2].

Abbreviations:MN, micronucleus;MTT, 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide; AlPcS4, aluminium phthalocyaninetetrasulphonate;T4MPyP,mesotetra(4N-methylpyridyl)porphine;FCS,fetal calf serum;PBS,phosphatebuffed saline. * Corresponding author.Fax: +44 1223334040

The activation of a photosensitizer by light results in the localised production of highly reactive singlet oxygen [3]. Although the mechanisms of cytotoxicity in PDT are still not clear, reactive oxygen species are known to be capable of affecting DNA integrity [4,5], and tumour tissue shows greater DNA sensitivity than does normal tissue [6,7]. Tumour cells also show greater sensitivity, above that of normal cells, to PDT [8,9]. The increased sensitivity of tumour cells to reactive oxygen species may be due to aberrations of chromosome 11, since insertion of normal chromosome 11 into tumour cells protects them against the DNA-damaging effects of activated neutrophils and xanthinetxanthine oxidase [6], as well as X-ray treatment [7]. Chromosome 11 alteration is a common event in tumorigenesis [ 10,l l] and loss of heterozygosity occurs in both exocrine and

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endocrine carcinoma of the pancreas[ 121.Carcinoma of the exocrine pancreasis a leading cause of cancer mortality with poor prognosis for survival. The most effective multimodal palliative treatmentsresult in an average survival of 8-15 months [ 131.It is therefore important to note that recent studies on rodent and human cell lines have revealed PDT to be a potentially effective treatment of pancreatic exocrine tumours [14,15]. In the present investigation, a ductal pancreatic carcinoma cell line, H2T, was selected to test the effects of PDT on pancreatic cells in vitro. The study was performed to obtain insight into changesin DNA integrity induced by two light-activated drugs, and to relate this effect to decreased proliferative cell growth, or to direct cytolytic action. Little is yet known of a link between any of these parametersin PDT. The drugs chosen were AIPcS4 which, since it is hydrophilic, does not bind readily to DNA [16], and FMPyP which is a lipophilic DNA intercalator [ 171 and therefore more nuclear-selective [18]. To assess simultaneously the cytotoxic, nuclear and growth inhibitory effects of a given drug we have combined two techniques. Both are simple, rapid methods. The micronucleus (MN) assay is a microscopic technique which quantitates both genetic alterations, in the form of micronuclei, and also changes in the number of cells actively dividing. Micronuclei arise from acentric chromosomal fragments or entire chromosomes excluded from the main nucleus when the cell divides. The MIT assay measurescell survival spectroscopically, the number of viable cells being related to the mitochondrial dehydrogenaseactivity of intact cells.

1 mg/ml in dimethylsulphoxide, at -20°C. The 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MIT) solution, 3.75 mg/ml in phosphatebuffered saline (PBS), was stored at 4°C and sterilized by filtration before use. Solubilizing solution (20% w/v sodium dodecyl sulphate in 50% v/v N,Ndimethylformamide/distilled water (pH 4.7) with 2.5% glacial acetic acid/l N HCl) was stored at room temperature. 2.2. Illumination

Cell cultures to be exposedto light were placed on a Sigma T2203 illuminator, at 0.47 mW/cm2 without a filter, or 0.2 mW/cm* with a >570 nm filter. The fluorescent tubes of the illuminator were repositioned to ensure an even illumination intensity. Light power was measured with a Melles Griot Power Meter. Light emitted, without a filter, spanned the visible wavelength range from 400 to 750 nm. 2.3. Cell culture

The pancreatic carcinoma cell line H2T was a gift from ProfessorC.M. Townsend, University of Texas Medical Branch at Galveston, TX, USA. The culture was derived from a transplantable adenocarcinoma induced in the ductal pancreasof the Syrian golden hamster by N-nitrosobis(2-oxopropyl)amine. Cells were grown in D-MEM, supplemented with 10% FCS and 1% L-glutamine. The stock culture was maintained in 75-cm* culture flasks at 37°C in a humidified incubator with 5% CO*. 2.4. Micronucleus assay

2. Materials and methods 2. I. Drugs and chemicals

FMPyP was obtained from Sigma Chemical Co., Poole, UK and AlPcS4 from Porphyrin Products Inc., Logan, UT, USA. Stock solutions of both agents were made in distilled water and stored at 4°C. Before use the solutions were filter-sterilized and diluted to required concentrations using Dulbecco’s modified Eagle’s media, without phenol red (DMEM), and without fetal calf serum (FCS). Cytochalasin B was stored as a sterile stock solution of

The MN assay was performed on cell cultures growing on sterile 22-mm* glass coverslips placed in 10 X 30 -mm* tissue-culture dishes. These coverslip cultures were prepared by seeding pancreatic carcinoma cells (20 x lo4 cells/dish) in D-MEM medium containing the photodynamic drug (total dish volume 1 ml). The cultures were incubated with the drug for 4 h in the dark at 37°C. After the incubation period, all dishes of cell cultures were washed as follows. The medium containing drug was removed by gentle suction from the dishes and the cells were washed once with PBS. To

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the washed dishes fresh medium containing FCS was added. Dishes to be exposed to the light were placed on the illuminator for 15 min. Cytochalasin B was then added at a concentration of 2 ,ugldish to identify cells that had passed through one division cycle after treatment. The drug prevents cytokinesis and thus cells undergoing karyokinesis after treatment will appear binucleated [19]. Cytochalasin B at 2,ug/dish was the optimum concentration at which to capture all dividing cells (data not shown). The cultures were incubated for 20 h prior to harvest. At that time, the coverslips were removed from the dishes, dipped in PBS, air-dried and fixed for 20 min with carnoys (methanol/glacial acetic acid in a ratio of 3:l). The cellular DNA was stained with the Feulgen reaction as described by Yi et al. [20]. To delineate the cell cytoplasm, cells were counterstained with a 0.5% solution of fast green, prepared in 95% ethanol. The coverslip cultures were then dehydrated through an alcohol series and mounted in Eukitt mountant on glass slides. The slides were blind-coded and a total of 9001500 binucleated cells were analyzed from three independent experiments. From the total number of cells a percentage of binucleated cells was calculated. The binucleated cells were scored for the presence of micronuclei using criteria described by Rosin [21]. 2.5. Tetrazolium assay The cell seeding, drug treatment, and light exposure procedures were identical to those of the MN assay but for the MTT assay it was not necessary to add Cytochalasin B to the dishes. After the 20 h of incubation at 37°C 200~1 of MTT was added to each dish and the cultures incubated for a further 4 h. The yellow tetrazolium MIT salt is converted, via intact mitochondrial dehydrogenase activity in living cells, to a blue-purple coloured formazan product [22-241. At the end of the 4 h incubation period, 1 ml of solubilizing solution was added. The dishes were kept in a humidified and darkened box for a further 24 h to allow for complete solubilization. The solubilized product of each dish was transferred to a well plate for reading in a TiterTech Multiskanner (MCC 340). The optical density of the formazan product was measured spectrophotometrically at 540 nm and the optical density of the

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control cultures was set at 100% survival after appropriate background subtraction. No loss of viability occurred in control cultures, after 24 h incubation, as measured by trypan blue staining. 2.4. Statistical analysis Data were obtained from at least three separate experiments and the results are expressed as mean values and standard error of the mean (SE) for each data point. Comparison within or between light and dark treatments was made with an unpaired t-test. The P value for significance was 0.05.

3. Results 3.1. Micronuclei formation and reduction in proliferation There was no significant difference between either micronuclei formation, or division rate, in control cultures of pancreatic tumour cells when exposed to drug in the dark, or when exposed to light treatment in the absence of drug. There was, however, a significant concentrationdependent induction of micronuclei when the tumour cells were pre-treated with AlPcS4 or FMPyP and then exposed to light (P < 0.05) (Fig. 1). Illumination after pre-treatment with either drug also resulted in a concentration-dependent increase in the number of cells that had not divided (P < 0.05) (Fig. 2). Drug treatment in the absence of illumination had no such effect on micronuclei induction and no reduction of cell division occurred with increasing drug concentration. By comparing the effects on tumour cells exposed to light at the maximum concentration of drug employed (Fig. l), i.e. when -85% of the cells are not dividing (Fig. 2), it can be seen that the frequency of micronuclei formation tripled with AlPcS4 and doubled with T4MPyP pre-treatment. 3.2. Cell survival and cytotoxicity There was no significant difference in the survival rate of cells in control cultures exposed to drug and kept in the dark, or when the cells were exposed to light but in the absence of drug. A significant in-

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Fig. 1. Micronuclei formation: concentration-dependenteffects of (A) AIPcS4 and (B) 9MPyP on the % micronuclei in illuminated (Cl) or non-illuminated (m) H2T cells.

crease in toxicity was observed however with increasing concentration of AlPcS4 or T4MPyP treatment followed by illumination (P < 0.05) (Fig. 3). AlPcS4 showed some degree of dark toxicity, but no more than 16 + 5%, and the toxicity in the light was significantly greater (P c 0.05) at concentrations >lOpM. 3.3. Growth inhibition When the tumour cells were treated with the lightactivated drugs there was a significant decreasein the number of dividing cells as observed with the MN assay(Fig. 2). By combining the results from the MN and M’lT assaysit was possible to establish the relative contributions of toxicity and growth inhibition to

0

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the number of cells not dividing. Thus MlT values for toxicity (Fig. 3) were subtracted from the values for cells that were not dividing (Fig. 2) to give the percentageof cells that were growth inhibited (Fig. 4). For both drugs there was an initial increase in growth inhibition with increasing concentration (P c 0.05). However, as the percentage of cells that were not dividing increasedbeyond 70% (seeFig. 2), the balance between growth inhibition and toxicity becameoutweighed by cytotoxicity. 3.4. Relationship betweenmicronuclei formation and cytotoxicity A further advantageof combining the results from both assays for light-activated drug treatment was

04 0

1 0.05

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Fig. 2. Cell division: concentration-dependenteffects of (A) AlPcS4 and (B) ?4MPyP on the division rates in H2T cells, illuminated (0) or non-illuminated (m).

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Fig. 3. Cytotoxicity: concentration-dependenteffects of (A) AIPcS4 and (B) tiMPyP on the % toxicity in illuminated (0) or nonilluminated (a) H2T cells.

that it allowed any DNA-damaging effects (MN assay) to be related to increasing toxicity (MTT assay). Although there appeared to be little correlation between increasing concentrations of either AlPcS4 or T4MPyP (Fig. 5) and the magnitude of toxicity or of micronuclei formation, linear regression analysis of the data revealed a highly positive correlation between micronuclei formation and increasing cytotoxicity (AlPcS4, r = 0.9975; T4MPyP, r = 0.9965) (Fig. 6). 4. Discussion Treatment of pancreatic tumour cells with the photodynamic drugs AlPcS4 or VMPyP, followed by

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exposure to light was found to result in a concentration-dependent increase in micronuclei formation. It also resulted in fewer dividing cells, an effect due in part to a decrease in cell survival with increasing drug concentration. There appeared to be no close relationship between the increase in toxicity and the induction of micronuclei, with respect to the concentration of either drug. However, a correlation was observed when the extent of cytotoxicity was compared directly with the frequency of micronuclei formation. Illumination of cells in the absence of either drug had no significant effect on the cells. Likewise, no significant changes occurred in the measuredresponseswhen cells were incubated with increasing concentrationsof eachdrug in the dark.

II0

0.05

0.10

0.15

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Fig. 4. Growth inhibition: concentration-dependenteffects of (A) AIPcS4 and (B) +MPyP on the % growth inhibition in H2T cells, ilbm$ nated (Cl) or non-illuminated (W).

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=I,, 0

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Fig. 5. Incidence of micronuclei (96) (0) and toxicity (8) (0) in H2T cells exposed to light with increasing concentrations of (A) AIPcS4 and (B) +MPyP.

The advantage of combining the MN assay with the MTI assay is that it allows the proliferative activity, cytotoxicity and sensitivity to DNA damageto be evaluated simultaneously. When clonogenic assaysare used for cell survival, results are obtained 67 generations post-treatment and thus do not readily correlate with acute, or immediate effects. All results presented in this paper represent the responses to photodynamic drug treatment during the first 20 h; other time-frames could easily be selected. 70 60 50 T 6 >r C .-0 :: l-

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Fig. 6. Correlation between micronuclei (%) and toxicity (8) for AlPcS4 (0) and ?MPyP (0). Linear regression analysis with biaxial error bars.

This is the first study in which the induction of micronuclei, together with a decreasein cell division and survival, has been compared for a dose range of photodynamic drugs. T4MPyP has previously been shown to induce micronuclei (but not using the cytokinesis-block method) in HeLa cells at a single dose, and with minimal toxicity as measured by trypan blue staining [ 18). Light-activated drugs do not need to be bound directly to DNA in order to induce photodynamic effects in tumour cells [ 17,251.In the present experiments A1PcS4,when exposed to light, induced increasingly more DNA damage than T4MPyP, at the same levels of division and survival. Furthermore, Kvam and Moan found that AlPcS4 was capable of inducing more double-strand breaks than Photofrin II and tetra(3-hydroxyphenyl)porphyrin (3THPP) when compared at the samelevel of cell survival in NHIK 3025 cells [26]. Photosensitizers when activated by light act primarily via the generation of singlet oxygen [3]. The localization patterns of AIPcS4 and T4MPyP within the cell would suggest that the sites of initiation of cytotoxicity are different, AlPcS4 localizing to the cytoplasm and particularly lysosomes [16] while T4MPyP shows preference for nuclear localization [18]. By its close proximity to the DNA, T4MPyP may be able to induce direct, lethal damage via singlet oxygen. Singlet oxygen has a life span of ~3 ,us and a diffusion radius of 0.1 ,um [27]. A1PcS4 may require the production of more stable, secondary DNA-targeting agents, such as those generated by

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lipid peroxidation and arachidonic acid metabolism [28,29]. Just as the basis of DNA damageis unlikely to be identical for the two drugs, so the extent of the damage may differ qualitatively and quantitatively, but ultimately yield the same effect of eliminating tumour cells. The inhibition of DNA repair has been proposed as a cause of cell killing during PDT [3032]. Penning et al. observed that the ability to repair X-ray-induced DNA strand breaks in L929 fibroblasts was completely abolished by preceding it with light-activated drug treatment [31]. If AlPcS4 and T4MPyP both induce DNA damagethat is lethal and cannot be repaired then cell death will occur in both cases as a direct result of persistent DNA damage. Micronucleus formation represents a good indicator of DNA damagethat is evidently not repairable. Both AlPcS4 [33] and TQMPyPwhen light-activated can disrupt microtubule organization [34]. Dysfunction of microtubule dynamics could be responsible for a decreasein the number of dividing cells observed as a result of light-activated drug treatment, and/or for failure of chromosomesto segregatecorrectly, which would increase micronucleus formation. When Juarranz et al. treated HeLa cells with T4MPyP they found the effect on the microtubules to be transient when cell survival was between 90 and 60%. With less than 10% survival there was irreversible alteration of microtubule function [34]. Comparing AlPcS4 and tetrabydroxy- and monosulphonated meso-tetraphenylporphines (3-THPP and TPPSl), Berg and Moan found that mitotic inhibition was more extensive for AlPcS4, which may relate to the fact that A1PcS4is highly water soluble while the porphines are lipophilic sensitizers [33]. To date, there has been no study using the cytokinesis-blocked MN assaythat has revealed the relative contributions of toxicity and growth inhibition to the number of cells that are not dividing. By combining the MTT assaywith the MN assay,it is now possible to determine the growth inhibitory effects. For both drugs, growth inhibition increased to a point where approximately 70% of the cells were not dividing, 20% being non-viable and 50% in a state of growth inhibition. With further reduction in division, lack of viability gradually becamethe major reason for cells not dividing. As the MN assaynow allows a view of the stages before complete toxicity intervenes, an important question can be addressed,that is: how

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many growth inhibited cells will eventually recover and how many will become non-viable through necrotic or apoptotic mechanisms? Although it is helpful to observe a correlation when comparing a particular type of damage to the toxic effect of PDT, it should be realized that what appearsto be relatively little DNA disruption may in some casesbe fatal for the cell, whereas, some enzyme activities may be inhibited considerably but without severe consequences[30,32]. Nonetheless, some form of relationship, either causal or consequential, is implied by the correlation between the induction of micronuclei and toxicity observed for both drugs in this study. It may for example involve Ca2+-dependentendonucleaseactivation and necrosis or apoptosis [35] following an increase in intracellular Ca2+by photodynamic drug action [36]. Whatever the precise sequenceof events in PDT culminating in the elimination of tumour cells the appearance of micronuclei is a good indicator of lethal DNA damage to tumour cells whether produced directly or indirectly. In summary, the MN assay allows for expansion of the parametersthat can be measuredin assessing the effects of PDT. In addition to a knowledge of lethal chromosomal changes,the point at which toxicity begins to predominate over growth inhibition and eventual cell recovery may be valuable in terms of determining lower levels of drug delivery, or light intensity, for PDT. To maximize the use of PDT it is important to identify factors that increase the sensitivity of tumour cells. Future studies will focus on the fact that many tumours show loss of heterozygosity on chromosome 11, and defects on this chromosome may account for the increased sensitivity of tumour cells to the oxidative stressof PDT when compared to normal cells. References [I] Penning, L.C. and Dubbelman, T.M.A.R. (1994) Fundamentals of photodynamic therapy: cellular and biochemical aspects. Anti-Cancer Drugs, 5, 139-146. [2] Levy, J.G. (1994) Photosensitizersin photodynamic therapy. Semin. Oncol., 21 (Suppl. 15). 4-10. [3] Weishaupt, K.R., Gomer, C.J. and Dougherty, T.J. (1976) Identification of singlet oxygen as the cytotoxic agent in photo-activation of a murine tumour. Cancer Res., 36.2326. [4] Cerutti, P. (1985) Prooxidant states and tumour promotion. Science,227, 375-38 1.

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[5] Ward, A.J., Olive, P.L., Burr, A.H. and Rosin, M.P. (1994) Response of tibroblast cultures from ataxia-telangiectasia patients to reactive oxygen species generated during inflammatory reactions. Environ. Mol. Mutagen., 24, 103111. [6] Ward, A.J., Olive, P.L., Burr, A.H. and Rosin, M.P. (1993) A sensitivity to oxidative stress is linked to chromosome 11 but is not due to a difference in single strand DNA breakage or repair. Mutat. Res., 294.299-308. (71 Parshad, R., Price, F.M., Oshimura, M., Barrett, J.C., Satoh, H., Wiessman, B.E., Stanbridge, E.J. and Sanford, K.K. (1992) Complementation of a DNA-repair deficiency in six human tumour cell lines by chromosome 11. Hum. C&et., 88,524-528. [8] Mang, T.S. and Wieman, T.J. (1987) An investigation of photodynamic therapy in the treatment of pancreatic carcinoma: dihematophyrin ether uptake and photobleaching kinetics. SPIE, 847, 116-121. [9] Barrett, A.J., Kennedy, J.C., Jones, R.A., Nadeau, P. and Pottier, R. (1990) The effect of tissue and cellular pH on the selective biodistribution of porphyrin-type photochemotherapeutical agents: a volumetric study. J. Photochem. Photobiol., 6, 309-323, [IO] Hopman, A.H.N., Moesker, 0.. Smeets, A.W.G.B., Pauwels, R.P.E., Vooijs, G.P. and Ramaekers, F.C.S. (1991) Numerical chromosome 1,7,9, and 11 aberrations in bladder cancer detected by in situ hybridization. Cancer Res., 51, 644-651. [11] Takita, K., Sato, T., Miyagi, M., Wataatani, M., Akiyama, F., Sakamoto, G., Kasumi, F., Abe, R. and Nakamua, Y. (1992) Correlation of loss of alleles on the short arms of chromosome 11 and 17 with metastasis of primary breast cancer to lymph nodes. Cancer Res., 52,3914-3917. [12] Ding, S.-F., Habib, N.A., Delhanty, J.D.A., Bowles, L., Greco, L., Wood, C., Williamson, R.C.N. and Dooley, J.S. (1992) Loss of herterozygosity on chromosome 1 and 11 in carcinoma of the pancreas. Br. 1. Cancer, 65.809-812. [I 31 Huibregtse, K. (1990) Non-operative palliation of pancreatic cancer. In: Clinical Gastroenterology: Cancer of the Pancreas, pp. 995-1004. Editor: J.P. Neoptolemos. Bailliere Tindall, London. [14] Schroder, T., Chen, I.W., Sperling, M., Bell, R., Bracket& K. and Joffe, S.N. (1988) Hematoporphyrin derivative uptake and photodynamic therapy in pancreatic carcinoma. J. Surg. Oncol., 38, 4-9. [15] Moesta, K.T., Dmytrijuk, A., Schlag, P. and Mang, T.S. (1992) Individual in vitro sensitivities of human pancreatic carcinoma cell lines to photodynamic therapy. SPIE, 1645, 43-5 1. [16] Moan, J., Berg, K., Bommer, J.C. and Western, A (1992) Action spectra of phthalocyanines with respect to photosensitization of cells. Photochem. Photobiol., 56, 171-175. [17] Fiel, R.J., Datta-Gupta, N., Mark, E.H. and Howard, J.C. (1981) Induction of DNA damage by porphyrin photosensitizers. Cancer Res., 41,3543-3545. [18] Villanueva, A., Juarranz, A., Diaz, V., Gomez, J. and Canete, M. (1992) Photodynamic effects of a cationic mesosubstituted porphyrin in cell cultures. Anti-Cancer Drug Design, 7, 297-303.

[19] Fenech, M. and Morley, A.A. (1985) Measurement of micronuclei in lymphocytes. Mutat. Res., 147, 29-36. [20] Yi, M., Rosin, M.P. and Anderson, C.K. (1990) Response of fibroblast cultures from ataxia-telangiectasia patients to oxidative stress. Cancer Lctt., 54.43-50. [21] Rosin, M.P. (1992) The use of the micronucleus test on exfoliated cells to identify anti-clastogenic action in humans: a biological marker for the efficacy of chemopreventive agents. Mutat. Res., 267.265-276. [22] Mossman, T. (1983) Rapid colorimettic assay for cellular growth and survival: application to proliferation and cytotoxic assays. J. Immunol. Methods, 65.5563. [23] Hansen, M.B., Nielsen, SE. and Berg, K. (1989) Reexamination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J. Immunol. Methods, 119, 203-210. [24] Ratcliffe, S.L. and Matthews, E.K. (1995) Modification of the photodynamic action of d-aminolaevulinic acid (ALA) on rat pancreatoma cells by mitochondrial benzodiazepine receptor ligands. Br. J. Cancer, 71.300-305. [25] Boye, E. and Moan, J. (1980) The photodynamic effects of hematoporphyrin on DNA. Photochem. Photobiol., 3 1, 223228. [26] Kvam, E. and Moan, J. (1990) A comparison of three photosensitizers with respect to efficiency of cell inactivation, fluorescence quantum yield and DNA strand breaks. Photothem. Photobiol., 52, 769-773. [27] Jori, Cl. and Spikes, J.D. (1981) Photosensitized oxidations in complex biological structures. In: Oxygen and Oxyradicals in Chemistry and Biology, pp. 441-457. Editors: M.A.J. Rodgers, and E.L. Power. Academic Press, New York. [28] Vaca, C.E., Wilhelm, J. and Harms-Ringdahl, M. (1988) Interaction of lipid pcroxidation products with DNA. A review. Mutat. Res., 195, 137-149. [29] Al-La&h, M., Matthews, E.K. and Cui, Z.J. (1993) Photodynamic drug action on isolated rat pancreatic acini. Biochem. Pharmacol., 46,567-573. [30] Boegheim, J.P.J. Dubbelman, T.M.A.R., Mullenders, L.H.F. and VanSteveninck, J. (1987) Photodynamic effects of hematoporphyrin derivative on DNA repair in murine L929 Iibroblasts. Biochem. J., 244.711-715. [31] Penning, L.C., Lagerberg, J.W.M., VanDierendonck, J.H., Comelisse, C.J., Dubbelman, T.M.A.R. and VanSteveninck. J. (1994) The role of DNA damage and inhibition of poly(ADP-ribosyl)ation in loss of clonogenicity of murine L929 tibroblasts, caused by photodynamically induced oxidative stress. Cancer Res., 54, 5561-5567. [32] Penning, L.C., Tijssen, K., Boegheim, J.P.J., van Steveninck, J. and Dubbelman T.M.A.R. (1994) Relationship between photodynamically induced damage to various cellular parameters and loss of clonogenicity in different cell types with hematopotphyrin derivative as sensitizer. Biochim. Biophys. Acta, 1221.250-258. [33] Berg, K. and Moan, J. (1992) Mitotic inhibition by phenylporphines and tetrasulfonated aluminium phthalocyanine in combination with light. Photochem. Photobiol., 56, 333339. [34] Juarranz, A., Villanueva, A., Diaz, V. and Canete, M. (1995)

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Photodynamic effects of the cationic porphyrin, mesotetra(4Nmethylpyridyl)porphine, on microtubules of HeLa cells. J. Photochem.Photobiol. B, Biol., 27,47-53. [35] He, X.-Y., Sikes, R.A., Thomsen, S., Chung, L.W.K. and Jacques,S.L. (1994) Photodynamic therapy with photofrin II induces programmed cell death in carcinoma cell lines. Photochem.Photobiol., 59.4681173.

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[36] Al-La& M. and Matthews, E.K. (1994) Calcium-dependent photodynamic action of di-and tetrasulphonated aluminium phthalocyanine on normal and tumour-derived rat pancreatic exocrine cells. Br. J. Cancer, 70, 893-899.