ACTION SPECTRUM (620–640 nm) FOR HEMATOPORPHYRIN DERIVATIVE INDUCED CELL KILLING

Abstract— Monochromatic red light generated by a tunable dye laser is currently being utilized for the treatment of solid tumors with hematoporphyrin derivative (HpD) photoradiation therapy (PRT). Experiments were performed using mammalian cells to determine the most efficient wavelength of red light (620 to 640 nm range) for HpD induced cellular photoinactivation. Decrease in the clonogenic potential of Chinese hamster ovary (CHO) cells was examined following both short (I h) and extended (12 h) HpD incubation times. Maximal photosensitization was observed with wavelengths ranging from 630 to 632.5 nm and the action spectra for cell killing matched the absorption spectra for HpD bound to cells. Similar observations were obtained following both short and extended HpD-cell incubation times. The potential relevance of these results as they relate to clinical HpD PRT are discussed.     

MEMBRANE LYSIS IN CHINESE HAMSTER OVARY CELLS TREATED WITH HEMATOPORPHYRIN DERIVATIVE PLUS LIGHT

Abstract Chinese hamster ovary cells in exponential growth were incubated with various concentrations of hematoporphyrin derivative (HpD). Cellular porphyrin content was determined after 2 h incubation at 37°C using [³H]-hematoporphyrin derivative. Photoactivation of cell-bound HpD by red light resulted in a family of survival curves with terminal slopes proportional to cellular HpD concentration. The degree of cellular lysis, assayed 1 h after illumination using a chromium-51 labeling technique, was also found to be related to cellular HpD concentration. The amount of 51Cr released increased with post-irradiation incubation to a level parallel to cell lethality as measured by colony formation. These data suggest that lysis of the cell membrane may be largely responsible for cellular inactivation following HpD photoirradiation.     

TARGETING ACTIVATED LYMPHOCYTES WITH PHOTODYNAMIC THERAPY: SUSCEPTIBILITY OF MITOGEN-STIMULATED SPLENIC LYMPHOCYTES TO BENZOPORPHYRIN DERIVATIVE (BPD) PHOTOSENSITIZATION

Abstract— Benzoporphyrin derivative monoacid ring A (BPD), a hydrophobic chlorin-like porphyrin derivative, which fluoresces strongly at 690 nm, may have potential for both oncologic and nononcologic applications in photodynamic therapy (PDT). To study the influence of cellular characteristics on the uptake of BPD, the murine tumor cell line (P815), and in vitro and in vivo concanavalin A (Con A)-stimulated and unstimulated murine splenic lymphocytes were incubated with 2 µg/mL BPD at 37°C for 0–60 min. At various times, cells were lysed and the amount of BPD taken up by the cells was quantified by fluorescence measurements. The subsets of cells taking up BPD were analyzed using a panel of monoclonal antibodies and the Coulter XL* fluorescence-activated cell sorter. Furthermore, Con A-stimulated and unstimulated spleen cells were incubated with 0–50 ng/mL of BPD for 1 h prior to exposure to red light (7.2 J/cm²). Cell survival 24 h post-PDT was measured by the MTT assay. We found that the rapidly dividing tumor cell line and mitogen-stimulated murine T cells (mainly CD4V IL-2R⁺) took up significantly more BPD (5–10-fold) than do unstimulated splenic lymphocytes. Increased BPD uptake correlated with greater photoinactivation when these cells were exposed to light at a wavelength of 690 nm. These findings suggest that activated cells of the immune system may be a target for photoinactivation by BPD.     

THE EFFECT OF PORPHYRIN PHOTOSENSITIZERS ON THE TOXICITY OF l,3-BIS(2-CHLOROETHYL)-1- NITROSOUREA IN C57BL/6J MICE

Abstract The most widely used agents for photodynamic therapy are the porphyrin photosensitizers. It has been shown that hematoporphyrin derivative (HpD) can cause murine marrow hypercellularity and splenic hypertrophy. We have examined the effect on survival and marrow cellularity of high dose l,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) after HpD or dihematoporphyrin ether (DHE) pretreatment in C57BL/6J mice.
The lethal toxicity of the LD_S0+ 10% dose of BCNU (60 mg kg⁻¹) was significantly reduced by pretreatment with HpD when the HpD was administered at least 3 days prior to the BCNU. HpD administered 1 or 2 days prior to BCNU or after BCNU had no effect. The percent death rate was reduced from 80 to 0% when HpD was administered 7 and 5 days prior to BCNU.
No alteration of the lethal toxicity rate of BCNU at doses of 80 mg kg⁻¹ were identified with DHE pretreatment although some increase in median survival was noted in two groups. Some reduction in lethal toxicity was noted when 60 mg kg⁻¹ BCNU was used and the pretreatment dose of DHE was 10 or 25 mg kg⁻¹ given twice 3 days apart. Furthermore, a significant reduction of BCNU induced marrow cell depletion was found when low doses of DHE were used as pretreatment. High doses of DHE resulted in marrow depletion. Both HpD and DHE altered the toxicity of BCNU.
Should porphyrin photosensitizers, which alone have little toxicity, prove to protect against nitrosourea toxicity then an important dose limiting factor (myelotoxicity) could be altered if not reduction in the tumouricidal activity occurs.     

PHOTOSENSITIZED INACTIVATION OF CHINESE HAMSTER CELLS BY PHTHALOCYANINES

Chloroaluminum phthalocyanine was found to sensitize cultured Chinese hamster cells upon exposure to white fluorescent light. Elimination of wavelengths below 370 nm did not reduce the effect significantly, indicating that the effective wavelengths were those absorbed by the Q band (600–700 nm) of phthalocyanine. The magnitude of the photosensitizing effect increased with the dye concentration and the time of its contact with the cells prior to light exposure. Although photosensitization was drastically reduced in the absence of oxygen, the lack of effect of glycerol and D₂0 during exposure suggests that neither hydroxyl radicals nor ¹O₂ are responsible for the cytotoxic response. The efficiency of the photosensitized induced cell killing did not vary with the position of the cells in the cell cycle, in contrast to exposure to X-rays. The improved spectral properties, the reported low toxicity and the selective retention by neoplasms, make phthalocyanines promising candidates for use in photodynamic therapy of cancer.     

PHOTODYNAMIC EFFECTS ON CELLS IN VITRO EXPOSED TO HEMATOPORPHYRIN DERIVATIVE and LIGHT

Abstract Several effects of hematoporphyrin derivative (HpD) and light on NHIK 3025 cells in vitro were studied. The treatment resulted in a partly repairable reduction of the rate of thymidine incorporation into DNA, a division delay, a reduced rate of protein synthesis, a reduced rate of active cellular uptake of α-aminoisobutyrate, a reduction in the colony-forming ability and an increased permeability of the cell membrane to chromate. Thymidine incorporation was by far the most sensitive parameter studied. However, comparison of the photodynamic effects after 1 and 18 h incubation with HpD prior to irradiation indicated that neither the reduced rate of DNA synthesis nor any of the other observed effects was the main primary cause of cell inactivation under all conditions. Several of the effects, such as increased permeability of the cell membrane to chromate, reduction in the rate of protein synthesis and reduction in the rate of repair of the damage to the mechanism of DNA synthesis, were clearly of secondary nature. When seen in relation to cellular survival, membrane damage was more important after short incubation times with HpD than after long incubation times.     

THE SUSCEPTIBILITY OF MUNG BEAN CHLOROPLASTS TO PHOTOINHIBITION IS INCREASED BY AN EXCESS SUPPLY OF IRON TO PLANTS: A PHOTOBIOLOGICAL ASPECT OF IRON TOXICITY IN PLANT LEAVES

In an attempt to elucidate the underlying mechanisms for iron toxicity in plants, the combined effects of iron overload and light intensities on the photosynthetic capacity of leaves were particularly focussed upon in this study, using mung bean seedlings grown under varied conditions regarding the supply of light and iron. The seedlings, when supplied with excess iron (up to 1.0 m M ) and low light (40 W/m²), did not suffer any loss of photosynthesis; further, the typical symptoms of iron toxicity, as shown in the leaves grown in sunlight at ca 450 W/m² on an average, were not seen in those. Nonetheless, excess iron supply resulted in a marked increase in photosensitivity of the low light-adapted seedlings. A large portion of iron accumulated in chloroplasts by the supply of excess iron was found to be incorporated into thylakoids as nonheme iron (NHI), which acts as a potent sensitizer, photogenerating singlet oxygen (¹O₂). The generation rate of ¹O₂ from thylakoids linearly increased with increasing content of NHI; this was in parallel with the NHI content dependence of photoinactivation rates of photosynthetic electron transport and key enzymes of the Calvin cycle in chloroplasts. The results suggest that Fe-dependent photosensitization reactions, occurring via the ¹O₂ mechanism, may be deeply involved in cellular processes leading to developing iron toxicity symptoms in plants.     

CYTOPROTECTION AGAINST MEROCYANINE 540-SENSITIZED PHOTOINACTIVATION OF THE Na+,K+-ADENOSINE TRIPHOSPHATASE IN LEUKEMIA CELLS: GLUTATHIONE AND SELENOPEROXIDASE INVOLVEMENT

When irradiated with broad-band visible light in the presence of merocyanine 540 (MC540), murine leukemia L1210 cells grown under selenium-deficient conditions (Se(-) cells) accumulated lipid hydroperoxides and lost viability more rapidly than selenium-satisfied controls (Se(+) cells). These findings suggest that cytoprotection against photoperoxidation and photokilling is mediated at least in part by selenoperoxidase (SePX) action. Similar protection against photoinactivation of an intrinsic membrane enzyme, the Na⁺,K⁺-ATPase, has been observed. Thus, irradiation of MC540-sensitized Se(-) cells resulted in an immediate and progressive inactivation of ouabain-sensitive Na⁺, K⁺-ATPase; by contrast, activity loss in Se(+) cells was preceded by a prominent lag. Enzyme photoinactivation in Se(-) cells was inhibited by ebselen, an SePX mimetic, confirming that SePX(s) is (are) involved in natural protection. Desferrioxamine treatment (iron sequestration/inactivation) resulted in higher hydroperoxide levels and slower Na⁺,K⁺-ATPase inactivation during MC540/light exposure, whereas ferric-8-hydroxyquinoline treatment (iron supplementation) had the opposite effect. Thus, iron appears to play an important role in both of these processes. In contrast, photoinactivation of another intrinsic enzyme in L1210 cells, acetylcholinesterase (AChE), was unaffected by selenium or iron manipulation. On the basis of these findings, we propose that lipid peroxidation plays an important role in the photoinactivation of Na⁺,K⁺-ATPase, but not AChE. This is consistent with the fact that Na⁺, K⁺-ATPase's active site lies within the membrane bilayer, whereas AChE's active site lies outside the bilayer.     

CELLULAR BINDING OF HEMATOPORPHYRIN DERIVATIVE IN HUMAN BLADDER CANCER CELL LINES: KK-47

Abstract— The fluorescence lifetime and degree of fluorescence polarization of hematoporphyrin derivative (HpD) have been investigated using different solutions: organic and micellar solutions. Ham's F12 medium, and KK-47 cell suspension. The lifetime and polarization degree in organic and micellar solutions did not change with increasing incubation time, but the polarization degree in the cell suspensions temporarily increased at the initial incubation time and then decreased 4 h after incubation. The lifetime in the cell suspensions exhibited a bi-phasic exponential decay. The results obtained suggested that mainly dimeric HpD may bind weakly to the cell membrane, and then slowly be distributed throughout the cytoplasm. The polarity and viscosity of the intracellular loci containing HpD were evaluated from the fluorescence polarizations of HpD in MeOH-H₂O mixtures and ethylene glycol(EG)-MeOH mixtures. The dielectric constant and viscosity of the loci containing HpD were 35 and 11 cp, respectively. Accordingly, the intracellular location of HpD were considered relatively hydrophilic loci of the cells.     

CELLULAR UPTAKE OF HYDROXYETHYLVINYLDEUTEROPORPHYRIN(HVD) AND PHOTOINACTIVATION OF CULTIVATED HUMAN LEUKEMIA (REH6) CELLS

Abstract— The in vitro incorporation of purified hydroxyethylvinyldeuteroporphyrin (HVD) into cells (Reh6) derived from an acute lymphocytic human leukemia is investigated using quantitative extraction techniques and fluorescence spectroscopy. A fast incorporation step (< 2 min) is characterized by its dependence on the porphyrin concentration in the incubation medium which suggests a saturation process. It is followed by a slower uptake, the rate of which linearly depends on the porphyrin concentration. No preferential uptake of aggregated form of HVD, which is shown to dimerize with an equilibrium constant of 9.7 × 10⁵ M ⁻¹, can be evidenced. As inferred from fluorescence spectra of cell suspensions and those of HVD dissolved in aqueous and micellar solutions as references, the porphyrin is mainly located in membrane structures and to a lower extent in cytoplasm. Cell photoinactivation does not depend on the incubation time but is only related to the intracellular porphyrin concentration.     

In vitro survival of nonsmall cell lung cancer cells following combined treatment with ionizing radiation and photofrin-mediated photodynamic therapy

It has been suggested that combination high dose rate (HDR) intraluminal brachytherapy and photodynamic therapy (PDT) in nonsmall cell lung cancer (NSCLC) may improve efficacy of treatment, reduce toxicity and enhance quality of life for patients. To provide a cellular basis for this we examined the in vitro sensitivity of MRC5 normal lung fibroblasts and four NSCLC cell lines following HDR radiation, PDT and combined HDR radiation and PDT. HDR radiation was cobalt-60 gamma rays (1.5–1.9 Gy min⁻¹). For PDT treatment, cells were exposed to 2.5 μg mL⁻¹ Photofrin for 18–24 h followed by light exposure (20 mW cm⁻²). For combined treatment cells were exposed to Photofrin and then either exposed to light and 15–30 min later exposed to HDR radiation or exposed to HDR radiation and 15–30 min later exposed to light. D₃₇ values calculated from clonogenic survival curves indicated a six-fold difference in HDR radiation sensitivity and an eight-fold difference in PDT sensitivity. The effect of combined treatment was not significantly different from an additive effect of the individual treatment modalities for the NSCLC cells, but was significantly less than additive for the MRC5 cells. These results suggest an equivalent tumor cell kill may be possible at reduced systemic effects to patients.     

Ca2+ INFLUX INDUCED BY PHOTODYNAMIC ACTION IN HUMAN CEREBRAL GLIOMA (U-87 MG) CELLS: POSSIBLE INVOLVEMENT OF A CALCIUM CHANNEL

Abstract The plasma membrane has been implicated as a critical target of photodynamic action on cells. We have observed that the photosensitization of human cerebral glioma (U-87 MG) cells by hematoporphyrin derivative (HpD) causes a large increase in intracellular calcium [Ca²⁺]. This increase in [Ca²⁺]_i was solely due to the influx of extracellular Ca²⁺ through the plasma membrane and showed a dependence on HpD concentration, light dose and concentration of calcium in the extracellular medium. The magnitude of the Ca²⁺ influx decreased with increasing postirradiation time, which suggests that the cell membrane partially recovers from the photodynamic injury. The photoinduced Ca²⁺ influx was inhibited by the Ca²⁺ channel blocker diltiazem and the reducing agent dithioerythritol. These findings are discussed in terms of possible activation of a Ca²⁺ channel as a result of photosensitization.     

The Effect of Charge on Cellular Uptake and Phototoxicity of Polylysine Chlorine6Conjugates

Abstract— The effect of charge on the cellular uptake, localization and phototoxicity of conjugates between chlorin_e6 ( c _e6) and poly-L-lysine was studied in vitro. These conjugates (average MW35–55 kDa) were synthesized to have polycationic, polyanionic or neutral charges. Two human cell lines (A431 epidermoid carcinoma cells and EA.hy926 hybrid endothelial cells) were studied and the cellular uptake of c _e6 delivered by the conjugates of varying charge and free c _e6 was measured at conjugate c _e6 equivalent concentrations up to 0.4 μM. Uptake was time and concentration dependent and temperature dependent in the case of neutral and anionic conjugates. Relative uptake at 6 h for A431 cells was 73:15:4:1 and for EA. hy926 cells was 63:11:3:1 for cationic, anionic, neutral and free c _e6, respectively, but EA. hy926 cells took up 1.5-2 times as much c _e6 from all the conjugates as A431 cells. Localization as studied by fluorescence microscopy indicated that the cationic conjugate was in aggregates bound to the plasma membrane, while the other forms were internalized in organelles and membranes. Phototoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-diphenyltetra-zolium bromide (MTT) assay after irradiation with5–20 J cm⁻²of 666 nm light. In contrast to the uptake, the order of phototoxicity for both cell types per mole of c _e6 uptake per cell was neutral ≫ anionic > cationic > free c _e6. Polymeric c _e6 conjugates bearing positive, negative and neutral charges may have different tissue-localizing properties and could play a role in photodynamic therapy.     

HepG2 human hepatocarcinoma cells: an experimental model for photosensitization by endogenous porphyrins

Endogenous protoporphyurin IX (PpIX) synthesis after δ-aminolaevulinic acid (ALA) administration occurs in cancer cells in vivo; PpIX, which has a short half-life, may thus constitute a good alternative to haematoporphyrin derivative (HPD) (or Photofrin). This study assesses the ability of the human hepatocarcinoma cell line HepG2 to synthesize PpIX in vitro from exogenous ALA, and compares ALA-induced toxicity and phototoxicity with the photodynamic therapy (PDT) effects of HPD on this cell line.

ALA induced a dose-dependent dark toxicity, with 79% and 66% cell survival for 50 and 100 μg ml⁻¹ ALA respectively after 3 h incubation; the same treatment, followed by laser irradiation (λ = 632 nm, 25 J cm⁻²), induced a dose-dependent phototoxicity, with 54% and 19% cell survival 24 h after PDT. Whatever the incubation time with ALA, a 3 h delay before light exposure was found to be optimal to reach a maximum phototoxicity.

HPD induced a slight dose-dependent toxicity in HepG2 cells and a dose- and time-dependent phototoxicity ten times greater than that of ALA-PpIX PDT. After 3 h incubation of 2.5 and 5 μg ml⁻¹ HPD, followed by laser irradiation (λ = 632 nm, 25 J cm⁻²), cell survival was 59% and 24% respectively at 24 h.

Photoproducts induced by light irradiation of porphyrins absorb light in the red spectral region at longer wavelengths than the original porphyrins. The possible enhancement of PDT effects after HepG2 cell incubation with ALA or HPD was investigated by irradiating cells successively with red light (λ = 632 nm) and light (λ = 650 nm). The total fluence was kept constant at 25 J cm⁻². For both HPD and ALA-PpIX PDT, phototoxicity was lower when cells were irradiated for increased periods with λ = 650 nm light than with λ = 632 nm light alone. This suggests that any photoproducts involved either have a short life or are poorly photoreactive.

Not all cell lines can synthesize PpIX after ALA incubation. HepG2 cells, which can synthesize enzymes and precursors of endogenous porphyrin synthesis, represent a good in vitro model for experiments using ALA-PpIX PDT. In addition, ALA-PpIX PDT may represent a new, specific treatment for hepatocarcinomas.     

EFFICIENT PHOTODYNAMIC ACTION OF VICTORIA BLUE BO AGAINST THE HUMAN LEUKEMIC CELL LINES K-562 AND TF-1

Abstract— Photodynamic induced cytotoxicity by Victoria blue BO (VB-BO), merocyanine 540 (MC540), Nile blue A (NB) and 4-tetrasulfonatophenyl-porphyrin (4-TSPP) has been studied on two human leukemic cell lines: K-562 and TF-1. Cells were incubated with dyes and irradiated with different doses of white light. Cell survival was assessed by propidium iodide (PI) staining using flow cytometry analysis. Concentrations of 5 x 10 ⁸ M VB-BO were found to kill 75% of cells, and a concentration of 1 × 10⁻⁷ M induced more than 99% of cell killing. To obtain the same cytotoxic level, the presence of 2.6 × 10⁻⁵ M of MCS40 during irradiation was needed. Under the conditions used, NB was ineflective as a photosensitizer, although uptake studies showed that this dye was taken by the cells in much greater amounts than any other studied dye. Cell cycle distribution of TF-1 cells, surviving MC540 or VB-BO photoscnsitization has bccn studied by flow cytometry analysis after staining with Hoechst 33342 and PI. It was found that cells in G₁ phase were slightly more resistant toward MCS40– and VB-BO-mediated photosensitization than cells in other phascs of the ccll cycle     

GROWTH DELAY STUDIES OF THE RESPONSE OF V-79 MULTICELL SPHEROIDS EXPOSED TO DHE and RED LIGHT

Abstract Multicell tumour spheroids (MTS) of V-79 Chinese hamster cells have been used to study the role of a number of treatment and microenvironmental parameters in the modification of tumour response to Photodynamic Therapy (PDT) using visible light in combination with the photosensitizing compound dihematoporphyrin ether (DHE). The kinetics of DHE uptake into MTS, determined by fluorimetry of extracted porphyrins, indicate that after extended incubation (i.e. 24 h) the mean cellular DHE content in larger (˜300 μ.m and 400 u.m) MTS is significantly less than that for smaller (˜200 μm) MTS, consistent with a hypothesis that DHE uptake into the internal regions of spheroids is diffusion-limited. The response of spheroids to PDT, as assessed by the endpoint of growth delay, indicates that the kinetics of spheroid volume alteration and cell loss, as well as the potential for regfrrwth, are markedly dependent on both the drug and light exposure levels used. The oxygen dependence of this response has been investigated after light irradiation of spheroid cultures equilibrated with either 21% O₂ (i.e. air) or 0% 0₂ (i.e. N₂). While treatment in air results in significant growth delay, the growth kinetics of DHE-treated spheroids irradiated under N₂ were essentially unchanged from those of untreated spheroids. These observations clearly demonstrate an important role for oxygen, at the time of irradiation, in determining the response of spheroids to PDT.     

RECOVERY OF SUBCHROMOSOMAL DNA SYNTHESIS IN SYNCHRONOUS V-79 CHINESE HAMSTER CELLS AFTER ULTRAVIOLET LIGHT EXPOSURE

Abstract— Previous work obtained from Chinese hamster V-79 cells indicated that, immediately following exposure, UV-induced lesions acted as blocks to elongation of nascent strands, but gradually lost that ability over a 10 h period after exposure to 10 J/m². The work reported herein attempted to examine possible cell cycle mediated alterations in the recovery of DNA synthesis. Kinetic incorporation of radiolabeled thymidine studies indicated that there may have been a more rapid recovery of DNA synthesis in cells irradiated in G₁ or G₂ vs cells irradiated in S phase. DNA fiber autoradiograms prepared from synchronous cells indicated that after irradiation in any phase of the cell cycle, the length of newly synthesized DNA was equal to control lengths 1 h after exposure to 5.0 J/m² (or 1 h after entering S phase for cells irradiated in G₁ or G₂). This observed recovery was not solely due to an excision process. No cell cycle mediated difference in the number of dimers induced or removed as a function of cell cycle position was observed. These results appear to be consistent with a continuum of effects, with initiation effects dominating the response at low fluences, gapped synthesis at intermediate fluences and elongation inhibition at high fluences. The fluences at which each event dominates may be cell-line specific.     

A TEST OF DIFFERENT PHOTOSENSITIZERS FOR PHOTODYNAMIC TREATMENT OF CANCER IN A MURINE TUMOR MODEL

Abstract The efficiency of different sensitizers for photodynamic therapy (PDT) was tested using a model system with a C3H mammary carcinoma growing subcutaneously on the dorsal side of mouse feet. Growth curves were constructed from which growth delay and doubling time in the regrowth phase were calculated. As PDT induced oedema in the mouse foot, this model system also allowed assessment of normal tissue response.
The following sensitizers were tested: hematoporphyrin derivative (HpD), Photofrin II (PII), tetraphenylporphinetetrasulfonate (TPPS₄), acridine orange (AO), phthalocyanine tetrasulfonate (PCTS), Al- and Zn-phthalocyanine tetrasulfonate (A1PCTS and ZnPCTS). For tumor control, the following sensitizer efficiencies were found: PII > HpD > AIPCTS > TPPS₄ >>> ZnPCTS, PCTS, AO. With regard to sensitizing normal-tissue damage: PII > AIPCTS, TPPS₄ > HpD, ZnPCTS, PCTS. The results suggest that AIPCTS should be further evaluated for use in PDT.     

HEMATOPORPHYRIN DERIVATIVE-INDUCED PHOTODYNAMIC INHIBITION OF Na+/K+-ATPase IN L929 FIBROBLASTS, CHINESE HAMSTER OVARY CELLS AND T24 HUMAN BLADDER TRANSITIONAL CARCINOMA CELLS

The photodynamically induced inhibition of Na⁺/K⁺-ATPase with hematoporphyrin derivative as photosensitizer was studied in murine L929 fibroblasts, Chinese hamster ovary (CHO) cells and T24 human bladder transitional carcinoma cells. In T24 cells the inhibition of the enzyme activity appeared to be caused by ATP depletion rather than by direct damage from the enzyme itself. In L929 and CHO cells, on the other hand, the inhibition was caused by photodynamic damage from the enzyme molecule. For all three cell lines it was shown that a causal relationship between photodynamically induced reduction in Na⁺/K⁺-ATPase activity and the loss of clonogenicity is highly unlikely.     

RESISTANCE TO PHOTODYNAMIC THERAPY IN RADIATION INDUCED FIBROSARCOMA-1 and CHINESE HAMSTER OVARY-MULTI-DRUG RESISTANT CELLS in vitro

A degree of resistance to photodynamic therapy (PDT) has been induced in radiation-induced fibrosarcoma-1 (RIF-1) tumor cells by repeated photodynamic treatment with Photofrin (4 or 18 h incubation) in vitro to the 0.1-1% survival level, followed by regrowth from single surviving colonies. The resistance is shown as increased cell survival in the strain designated RIF-8A, compared to the wild-type RIF-1 cells, when exposed to increasing Photofrin concentration for 18 h incubation and fixed light exposure. No difference was found between RIF-1 and RIF-8A in the uptake of Photofrin per unit cell volume at 18 h incubation. Resistance to PDT was also observed in Chinese hamster ovary-multi-drug resistant (CHO-MDR) cells compared to the wild-type CHO cells, possibly associated with decreased cellular concentration of Photofrin in the former. By contrast, the PDT-resistant RIF-8A cells did not show any cross-resistance to Adriamycin, nor was there any significant drug concentration difference between RIF-1 and RIF-8A. These findings suggest that different mechanisms are responsible for PDT-induced resistance and multi-drug resistance.