Psoralen-photosensitized disaggregation of

HeLa Cx43 spheroids

Eugene P. Lysenko ^a, Fritz Pliquett ^b, Siegfried Wunderlich ^b,

Alexander Ya. Potapenko ^a

^a Department of Medical and Biological Physics, Russian State Medical University, Moscow 117869, Russia;

e-mail: elysenko@hotmail.com

^b Institute of Medical Physics and Biophysics, Leipzig University, Leipzig 04103, Germany

Abstract

Disaggregation of spheroids grown from HeLa cells with connexin Cx43 induced by PUVA-treatment (psoralen + UVA irradiation) or by photooxidized psoralen (POP) was studied. It was found that both PUVA and POP induced disaggregation of spheroids. The rate of disaggregation was estimated as inverse time of disaggregation of 50% or 100% spheroids in suspension (1/t₅₀ or 1/t₁₀₀, respectively). It was found that the rate of PUVA-induced disaggregation increased with the increase of UVA-fluence up to 85 kJ/m². Photosensitization coefficient was the highest at low UVA-fluences and significantly decreased with an increase in UVA-fluence. Simultaneously with a registration of disaggregation, the viability of cells in spheroids was estimated with the use of trypan blue stain. It was shown that at small fluences of PUVA-treatment the process of disaggregation occurred without the formation of trypan positive cells in spheroids. POP-induced disaggregation of HeLa Cx43 spheroids implies that PUVA-disaggregation, at least partially, may occur through the action of POP-products.

Introduction

          Psoralens [furocoumarins (FC)] combined with UVA-irradiation (320-400 nm) of skin (PUVA-therapy) or blood leukocytes (photopheresis) are used for the treatment of cutaneous and autoimmune diseases (psoriasis, T-cell lymphoma, neurodermit, etc.) [1-3]. PUVA-treatment is accompanied by side toxic effects (erythema, oedema, etc.). Photochemical mechanisms of both beneficial and harmful effects have not been established in detail and are still a matter of discussions. Four types of psoralen photochemical reactions proceed in skin: generation of free radicals, (Type I), generation of singlet oxygen (Type II), photoaddition to DNA (Type III) and photooxidation of psoralen (Type IV). These reactions are the basis of therapeutic and side effects. It is assumed that therapeutic effects of PUVA and photopheresis are connected with their immunomodulative activitiy [1-3], and side effects of PUVA may be caused by photooxidative psoralen reactions with cell membrane components [4].

One of the mechanisms of PUVA-treatment may be realized through the formation of psoralen photooxidation products (POP), which have been shown to possess a significant biological activity. POP-products wasere found to induce the oxidation of unsaturated lipids and 3,4-dihydroxyphenylalanine [4], bind covalently bind to proteins [5], inactivate enzymes [6], modulate superoxide radical production by phagocytes [7], induce mutagenic and lethal effects in microorganisms [8, 9], inhibit hemotactic activity of polymorphonuclear neutrophils [10], modulate T-cell immune system in mice [11, 12], and induce therapeutic effects in patients with skin diseases [13]. Both PUVA and POP was shown to damage plasma membranes of erythrocytes [4, 14] and leukocytes [7] in cell suspensions. An action of PUVA and POP on cell aggregates was not studied. Thus, in this work we have investigated the action of PUVA and POP on multicell spheroids, which were grown from HeLa cells with connexin Cx43.

Materials and methods

Epithelioid HeLa cell lines of a human cervix carcinoma were grown at pH 7.4 and 37^o C in humidified incubator with 5% CO₂ in Dulbecco's modified Eagle's medium as described by D. F. Huelser [15]. Spheroids were initiated by seeding single-cell suspensions (1 to 2 x 10⁶ cells per dish) in 94 mm diameter plastic Petri dishes with nonadherent surfaces. Within 3 to 7 days, the aggregated cells were transferred into spinner flasks filled with 60 ml medium and cultured at 120 rpm [15].

Disaggregation of spheroids was observed using a microscope “Leica MS5” (Germany) (ocular 10^x/21B; lens 1.6^x, 2.5^x and 4^x) supplied with a camera “Minolta X-700”.

          In experiments with PUVA-induced disaggregation, about 200 spheroids of approximately equal size (200 - 250 mm in diameter) were twice washed in fresh Dulbecco's medium and placed in 35 mm diameter plastic tissue culture dishes with psoralen dissolved in Dulbecco's medium

(10^-4 M, 1% of ethanol). After dark incubation during 30 min, spheroids were UVA-irradiated (365 nm) in dishes by incident light of “HQV-125” lamp (Germany) at 37^o C at various fluences. The fluence rate of UVA-irradiation (365 nm) measured by “Waldman UV-meter” (Germany) was equal to 25 W/m² on the surface of the solution. Since the spheroids were on the bottom of dishes during irradiation, we took into account a decrease in the fluence rate due to light absorption in 4 mm layer of red Dulbecco's medium above the spheroids. The absorption coefficient (1-T) measured at 365 nm was equal to 0.3, where T is transmittance of the layer. Three aliquots of PUVA-treated spheroids (about 50 spheroids with 100 ml of irradiated solution in each one) were placed in 6 mm diameter holes of tissue culture testplate and incubated at 37^oC. During incubation, cells and small aggregates moved away from the spheroids. We considered spheroids to be disaggregated, if their diameter decreased from 200-250 mm to 30-40 mm. The shares of not disaggregated spheroids were estimated in each hole at intervals, and mean values + SEM were calculated.

In control, spheroids were UVA-irradiated in Dulbecco's medium without psoralen (with 1% of ethanol), or not irradiated, but incubated in darkness with psoralen (10^-4 M, 1% of ethanol), or incubated without psoralen (only with 1% of ethanol).

The rate of disaggregation of spheroids may be estimated as the value inverse to the time of some standard share of spheroid disaggregation. We estimated the value inverse to the time of 50% or 100% disaggregation of spheroids in the sample (1/t₅₀ or 1/t₁₀₀, respectively) similarly to the rate of PUVA- or POP-induced hemolysis [16, 4, 14]. In the same fashion we estimated the rate of trypan positive cell formation: as inverse time of formation of 50% trypan positive cells in not deaggregated spheroids or the inverse time of formation of 50% trypan positive cells in the suspension after a complete disaggregation of spheroids.

          Photosensitization coefficient was determined at various fluences from the fluence dependence of the rates of PUVA- and UVA-induced cell disaggregation (curves 1 and 2, respectively, in Fig. 3):

       [(1/t₅₀)_PUVA - (1/t₅₀)_Ps] - [(1/t₅₀)_UVA - (1/t₅₀)₀]

k_s = ----- -------------------------------------------------

(1/t₅₀)_UVA - (1/t₅₀) ₀

where (1/t₅₀)_PUVA and (1/t₅₀)_UVA are the rates of PUVA-and UVA-induced disaggregation, respectively; (1/t₅₀)_Ps and (1/t₅₀)₀ are the rates of dark psoralen effect and spontaneous disaggregation (without any influences), respectively.

          In the experiments with PUVA-induced formation of trypan positive cells in spheroids, about 300 PUVA-treated sphreoids were divided into 15 aliquots with 100 mm of irradiated psoralen solution in each one, and were placed in 6 mm diameter holes of tissue culture testplate for incubation at 37^oC. At intervals, the contents of holes were mixed with 0.2 % solution of trypan blue in relation 1:1, and the share of trypan positive cells in each spheroid was estimated. If spheroids were disaggregated, the share of trypan positive cells in suspension of disaggregated cells was estimated. The mean values + SEM were calculated.

          In the experiments with POP-induced disaggregation, irradiation conditions were analogous to described above for the experiments with PUVA-induced disaggregation. Psoralen dissolved in fresh Dulbecco’s medium (10^-4 M, 1% of ethanol) was UVA-irradiated at various fluences in 35 mm diameter plastic tissue culture dishes in the air at 37^oC. Just after irradiation, about 200 spheroids were placed in the photooxidized solution. Then three portions by 100 ml of POP with about 50 spheroids in each one were placed in 6 mm holes of tissue cultural testplate and incubated at 37^oC. The shares of not disaggregated spheroids were determined in each hole at intervals, and mean values + SEM were calculated.

Results

          Fig. 1 illustrates the disaggregation (1, k₁, k₂) and trypan positive cell formation (1’, k₁’, k₂’) in HeLa Cx43 spheroids after PUVA-treatment (1, 1’) and in the process of dark incubation (in control) of spheroids with psoralen (k₂, k₂’) or without psoralen (k₁, k₁’).

Fig. 1. Spheroids twice washed in Dulbecco’s medium were incubated with psoralen (10^-4 M, 1% of ethanol), then UVA-irradiated (365 nm, 25 W/m²) with the fluence of 19 kJ/m² (1, 1’) and incubated at 37^oC. X axis indicates incubation time (hours) after UVA irradiation; Y axis indicates the share of not disaggregated spheroids during incubation after PUVA treatment (left) or the share of trypan positive cells in spheroids (or in cell suspension after complete their disaggregation) (right). In control, spheroids were incubated in Dulbecco’s medium without psoralen (1% of ethanol) (k₁, k'₁) or with psoralen (10^-4 M, 1% of ethanol) (k₂, k'₂).

The fluence of UVA-irradiation was 19 kJ/m². It is seen that PUVA-induced disaggregation of spheroids goes faster than the formation of trypan positive cells (curves 1 and 1’, respectively). In control both processes developed essentially slower.

             Fig. 2 presents bar diagrams of the rates 1/t₅₀ (A) and 1/t₁₀₀ (B) of PUVA-induced spheroid disaggregation (UVA-fluence was equal to 28 kJ/m²). Obviously, both 1/t₅₀ and 1/t₁₀₀ values may be used for estimation of the rate of spheroid disaggregation. It is seen from these diagrams that the rate of PUVA-induced disaggregation is about 4 - 8 times higher than the rates in controls.

Fig.2. The rates of PUVA-induced (A, B) and POP-induced (C) disaggregation of HeLa Cx43 spheroids. (A, B) Spheroids twice washed in Dulbecco’s medium were incubated with psoralen (10^-4 M, 1% of ethanol), then were UVA irradiated (365 nm, 25 W/m²) with the fluence of 28 kJ/m² and incubated at 37^oC. In control, spheroids were incubated in Dulbecco’s medium with 1% of ethanol or with psoralen (10^-4 M, 1% of ethanol) without UVA-irradiation, or were UVA-irradiated without psoralen (in the presence of 1% of ethanol). (C) Psoralen dissolved in Dulbecco’s medium (10^-4 M, 1% of ethanol) was UVA-irraddiated (365 nm, 40 W/m²) with the fluence of 136 kJ/m² in the air. Just after irradiation, HeLa Cx43 spheroids were placed in POP and were incubated at 37^oC. In control, spheroids were incubated in Dulbecco’s medium with non-irradiated psoralen (10^-4 M, 1% of ethanol), or without psoralen (in the presence of 1% of ethanol). The rate of disaggregation was estimated as the inverse time for 50 % (A) or 100 % (B, C) of spheroid disaggregation in the suspension.

Bar diagram (C) in Fig. 2 presents the rate 1/t₁₀₀ of POP-induced disaggregation of HeLa Cx43 spheroids in compared with the rates in controls (after incubation of spheroids with non-irradiated psoralen or with 1% of ethanol). The fluence of UVA-irradiation of psoralen solution was equal to 136 kJ/m². It is seen that the rate of POP-induced disaggregation is about 5 times higher than the rates in controls.

Fig. 3 presents the fluence dependences of the rates (1/t₅₀) of PUVA-induced processes of disaggregation (curve 1) and trypan positive cell formation (curve 3) in HeLa Cx43 spheroids. Control curve 2 describes UVA-induced disaggregation of spheroids. It is seen that at low UVA-fluences the rate of PUVA-induced disaggregation is essentially higher than the rate of PUVA-induced trypan positive cell formation. With the increase in the fluences, this difference became less, and in the fluence equal to about 80 kJ/m² they were close.

The dependence of photosensitisation coefficient on the UVA-fluence is presented in Fig. 3 (B). It is seen that this coefficient has the highest values at low UVA-fluences (5-10 kJ/m²) and significantly decreases with an increase in UVA-fluence.

Fig. 3. (A) Fluence dependences of the rates (1/t₅₀) of PUVA-induced (1) or UVA-induced (2) cell disaggregation and PUVA-induced trypan positive cell formation (3) in HeLa Cx43 spheroids. (B) Fluence dependence of photosensitization coefficient Spheroids twice washed in Dulbecco’s medium were incubated with psoralen (10^-4 M, 1% of ethanol), then UVA-irradiated (365 nm, 25 W/m²) with various fluences and incubated at 37^oC (1, 3). In control (2), spheroids were preincubated with only 1% of ethanol (without psoralen) before UVA-irradiation. A rate of disaggregation was estimated as the inverse time of disaggregation of 50 % of spheroids. A rate of trypan positive cell formation was estimated as the inverse time of the appearance of 50 % of trypan positive cells in spheroids or in cell suspension after their complete disaggregation. Photosensitization coefficient (k_s) was calculated as described in Materials and Methods section.

Obtained results evidence that both PUVA and POP-treatments induce disaggregation of HeLa Cx43 spheroids with the highest value of photosensitization coefficient at low fluences. One can suggest that PUVA-induced disaggregation, at least partially, may occur through the formation of psoralen photooxidation products.

Acknowledgements.

This research work was made possible by the Grant No RUS-114-96 from BMBF (Germany) and partlially by the Grant No 98-04-49054 from Russian Foundation for Basic Research (Russia).

We thank Mrs. I. Schaedlich (Institute of Medical Physics and Biophysics of Leipzig University) for assistance in preparing HeLa Cx43 spheroids.

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