Photosensitization of human skin cell lines by ATMPn (9-acetoxy-2,7,12,17-tetrakis-(beta-methoxyethyl)-porphycene) in vitro: mechanism of action

9-Acetoxy-2,7,12,17-tetrakis-(beta-methoxyethyl)-porphycene (ATMPn) is a promising new photosensitizer characterized by high absorption around 640 nm and high singlet oxygen yield. To study the mechanism of action in vitro we have investigated uptake, intracellular localization, cell survival and ultrastructural changes following photodynamic treatment in human cell lines derived from the skin (SCL1 and SCL2, squamous cell carcinoma; HaCaT keratinocytes; N1 fibroblasts). Using flow cytometry we have determined the cellular fluorescence as a marker for the uptake of ATMPn after incubation for 60 min. Co-staining with ATMPn and fluorescent dyes specific for cell organelles reveals an intracellular localization of ATMPn in lysosomes. Following irradiation using an incoherent light source (580-740 nm) and a light fluence of 24 J cm-2, phototoxicity is determined by means of the 3-4.5 dimethylthiazol-2,5 diphenyl tetrazolium bromide (MTT) assay. For all cell lines ATMPn concentrations above 15 nM yield a significant phototoxic effect. The 50% effective concentration, EC50, for SCL1 cells is 11.2 +/- 2.9 nM ATMPn. ATMPn uptake and phototoxicity are more effective for HaCaT and SCL1 as compared to SCL2 and N1 cells. Growth curves confirmed the results of the MTT assay. Because of the high lysosomal accumulation of ATMPn, already low photosensitizer concentrations without dark toxicity yield a high photodynamic effect. Immunofluorescence and electron microscopy reveal damage to tonofilaments, plasma membrane and mitochondria, indicating a mechanism unrelated to apoptosis. A dose yielding complete cell killing, as needed for oncological indications, might lead to necrosis, whereas lower sub-lethal doses result in induction of apoptosis.     

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.     

DNA ligand Hoechst-33342 enhances UV induced cytotoxicity in human glioma cell lines

The effects of minor groove binding ligand bisbenzimidazole derivative Hoechst-33342 on the cellular response to UV damage have been studied in two human glioma cell lines BMG-1 and U-87 grown as monolayer cultures. Treatment induced cell death (macro-colony assay) and growth inhibition, potential lethal damage recovery, cytogenetic damage (micronuclei formation) and proliferation kinetics were studied as parameters for cellular response. Pre and post-irradiation treatment with Hoechst-33342 (1-20 microM) enhanced the UV-induced growth inhibition and cell death in a concentration dependent manner in both cell lines. At higher Hoechst-33342 concentrations (>5 microM), the cytotoxic effects of the combination (Hoechst-33342+UV) were highly synergistic and mainly mediated through apoptosis implying the possible interactions of lesions caused by both the agents. The enhanced cell death due to Hoechst-33342 was accompanied by a significant increase (2-3 folds at 5 microM) in UV-induced micronuclei formation in BMG-1 cells. Under these conditions, Hoechst-33342 also enhanced the UV-induced cell cycle delay, mainly due to S and G(2) blocks. The increase in UV-induced micronuclei formation observed after treatment with Hoechst-33342 indicates that the DNA bound Hoechst-33342 may interfere with the rejoining of DNA strand breaks. Since the treatment of cells with the replication inhibitor aphidicolin reduced the enhancement of UV induced cytotoxicity by Hoechst-33342, ongoing DNA replication appears to stimulate Hoechst-33342 and UV-induced cytotoxicity.     

p53‐Dependent and p53‐Independent Responses of Cells Challenged by Photosensitization

The p53 protein exerts fundamental roles in cell responses to a variety of stress stimuli. It has clear roles in controlling cell cycle, triggering apoptosis, activating autophagy and modulating DNA damage response. Little is known about the role of p53 in autophagy‐associated cell death, which can be induced by photoactivation of photosensitizers within cells. The photosensitizer 1,9‐dimethyl methylene blue (DMMB) within nanomolar concentration regimes has specific intracellular targets (mitochondria and lysosomes), photoinducing a typical scenario of cell death with autophagy. Importantly, in consequence of its subcellular localization, photoactive DMMB induces selective damage to mitochondrial DNA, saving nuclear DNA. By challenging cells having different p53 protein levels, we investigated whether p53 modulates DMMB/light‐induced phototoxicity and cell cycle dynamics. Cells lacking p53 activity were slightly more resistant to photoactivated DMMB, which was correlated with a smaller sub‐G1 population, indicative of a lower level of apoptosis. DMMB photosensitization seems to induce mostly autophagy‐associated cell death and S‐phase cell cycle arrest with replication stress. Remarkably, these responses were independent on the p53 status, indicating that p53 is not involved in either process. Despite describing some p53‐related responses in cells challenged by photosensitization, our results also provide novel information on the consequences of DMMB phototoxicity.     

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.     

Involvement of Heat-Shock Protein 70 and P53 Proteins in Attenuation of UVC-lnduced Apoptosis by Thermal Stress in Hepatocellular Carcinoma Cells

Induction of apoptosis is a function of external stimuli and cellular gene expression. Many cells respond to DNA damage by the induction of apoptosis, which depends on a functional p53 protein and is signaled by elevation of p53 levels. In this study, we found that a prior exposure to mild stress (42 degrees C) can protect HepG2 (p53+/+) cells from a subsequent UVC-induced apoptosis determined by DNA fragmentation and ratio of sub-G1 peak, but no heat-enhanced protection was found in Hep3B (p53-/-) cells. Although a similar inductive pattern of HSP70 protein and mRNA was detected in the two cell lines under thermal stress, the effect of thermal stress on UVC-induced apoptosis in HepG2 and Hep3B cells was obviously different. Overexpression of HSP70 by transient transfection of HSP70 expression vector in HepG2 cells significantly inhibited UVC-induced cell death; however, this inhibitory effect did not occur in transfected-Hep3B cells. Treatment of HepG2 cells with p53-specific antisense oligonucleotide could effectively block the antiapoptotic effect of thermal stress on UVC-induced apoptosis and increase of intracellular wild-type p53 protein by transfecting wtp53 expression plasmid into Hep3B cells yielded more resistance to UVC irradiation after prior thermal stress exposure. The results reveal an involvement of p53 in the antiapoptotic effect of thermal stress on UVC irradiation. Finally, a p53 protein increase was detected in UVC-treated HepG2 cells and could be coimmunoprecipitated with HSP70 after a thermal stress treatment. Prolonged p53 binding activity and enhanced expression of p53-controlled genes such as G1 arrest and DNA damage 45 and wild-type p53 activation factor 1/Cdk-interacting protein 1 by thermal stress are also observed in UVC-irradiated HepG2 cells. Based on these results, we propose that the antiapoptotic effect of thermal stress is mediated by increasing HSP70 and modulating intracellular p53 function.     

Intracellular localization is a cofactor for the phototoxicity of protoporphyrin IX in the gastrointestinal tract: in vitro study

Photodynamic therapy (PDT) is a new treatment modality for solid tumors as well as for flat lesions of the gastrointestinal tract. Although the use of 5-aminolevulinic acid-induced protoporphyrin IX (PPIX) shows important advantages over other photosensitizers, the main mechanisms of phototoxicity induced are still poorly understood. Three human colon carcinoma cell lines with variable degrees of differentiation and a normal colon fibroblast cell line were used to generate a suitable in vitro model for investigation of photosensitizer concentration as well as the applied light dose. Also, the effects of intracellular photosensitizer localization on efficiency of PDT were examined, and cellular parameters after PDT (morphology, mitochondrial transmembrane potential, membrane integrity and DNA fragmentation) were analyzed to distinguish between PDT-induced apoptosis from necrosis. The fibroblast cell line was less affected by phototoxicity than the tumor cells to a variable degree. Well-differentiated tumor cells showed higher toxicity than less-differentiated cells. After irradiation, cell lines with cytosolic or mitochondrial PPIX localization indicate a loss of mitochondrial transmembrane potential resulting in growth arrest, whereas membrane-bound PPIX induces a loss of membrane integrity and consequent necrosis. Although the absolute amount of intracellular photosensitizer concentration plays the main determining role for PDT efficiency, data indicate that intracellular localization has additional effects on the mode of cell damage.     

Comparison of phototoxicity mechanism between pulsed and continuous wave irradiation in photodynamic therapy

A study has been conducted in which HeLa cells are incubated with hematoporphyrin derivative (HpD) for 1 h (1 microgram/ml of HpD in PBS) to compare the use of continuous wave (CW) and pulsed laser (10 Hz repetition rate and 7-9 ns pulse width) light for photodynamic therapy. Cytotoxic effects on the cells are evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2-5-diphenyl-2H-tetrazolium bromide (MTT) assay and the fluorescein diacetate (FDA)/propidium iodide (PI) stain method using a flow cytometer. The type of cell death is estimated by analysis of the DNA content and observation of the nuclear morphology. The cytotoxicity ratio of cells irradiated by pulsed laser light is estimated to be lower than that for CW laser light. The viability of cells that received pulsed laser light gradually decreases, whereas no significant changes are found in the cells irradiated with CW laser light with the elapse of post-irradiation time. The type of cell death differs between the pulsed and CW laser light irradiations. These findings suggest that the cytotoxic efficacy of the excitation light source is displayed by the difference in the type of cell death, namely apoptosis or necrosis.     

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.     

Review of photodynamic therapy for gastrointestinal tumours in the past 6 years in China

Patients (142) with advanced gastrointestinal (GI) cancers were treated by photodynamic therapy (PDT) from September 1982 to December 1988. Haematoporphyrin derivative (HpD) (5 mg kg-1) was intravenously given 48-72 h prior to PDT. The light source was an argon dye laser emitting at 630 nm. The entire tumour was irradiated with a light dose of 100-250 J cm-2. Fifteen patients (10.6%) showed complete response (CR), 53 (37.3%) showed partial response (PR) and 32 (22.5%) showed mild response (MR). The clinical results show that the estimated energy dose (EED) of 200-250 J cm-2 is appropriate. All patients were treated by PDT and adjuvant chemotherapy showing good results with a follow-up of 1-5 years; 13 out of 15 patients in the CR group are alive (86.7%); 12 patients survived more than 2 years (8.8%).     

PHOTOINACTIVATION OF CHINESE HAMSTER CELLS BY HEMATOPORPHYRIN DERIVATIVE AND RED LIGHT

Abstract— The use of hematoporphyrin derivative (HpD) has previously been demonstrated to be beneficial in clinical cancer therapy. This paper describes cell culture studies used to examine HpD phototherapy in Chinese hamster ovary cells (line CHO). Survival curves have been obtained for both direct HpD toxicity and HpD induced photoinactivation. Examination of HpD induced photoinactivation as a function of stage in the cell growth cycle has also been performed, as has the quantitative measurement of HpD uptake in cells (using ³H-HpD) as a function of cellular incubation time, serum concentration in the incubation medium, and cell cycle position. In the absence of light, no toxicity was observed for HpD incubation levels of up to 400 μg/m/ when incubations times were 3 h or less. Exposure of cells to light alone (> 590 nm, 4.0 mW/cm²) for 9 min was also found to be completely nontoxic. Survival curves obtained for exponentially growing cells labeled with various concentrations of HpD and subsequently illuminated with red light exhibited a threshold or shoulder region at short exposure times followed by exponential killing at longer exposure times. The cell cycle response curves for HpD induced photoinactivation of synchronized CHO cells was nearly flat, indicating no variation in sensitivity for cells treated at time periods from 6 to 15 h after mitosis. Additon of serum to the incubation medium resulted in improved plating efficiency and reproducible survival curves but decreased cellular uptake of HpD.     

Photosensitizers in dermatology

Systemic injection of hematoporphyrin derivative (HpD) in contribution with visible light (red or blue-green) delivered by laser was used to treat a patient with psoriasis. The psoriatic lesions responded vigorously to laser treatments, forming eschars by 1 week post irradiation. In contrast, only minimal erythema was observed in the noninvolved, clinically normal appearing skin. Two approaches for localized HpD administration were investigated in the guinea-pig and minipig models as a means of achieving local photodynamic effects. Intracutaneous injection of HpD produced localized cutaneous photosensitization with either UVA or red light. Azone increased percutaneous penetration of HpD in human skin in vitro. Topical application of HpD and irradiation with UVA produced localized cutaneous photosensitivity and inhibition of epidermal DNA synthesis.     

Fluorescence of hematoporphyrin in living cells and in solution

The fluorescence properties of hematoporphyrin (Hp) and its derivative (HpD) were investigated in leukemia cells and in normal lymphocytes under a microscope, and the results were compared with those in solution. The spectra and the time behaviour of Hp (or HpD) fluorescence in living cells were found to be almost the same as those in Hp solution of very high concentration. This implies that Hp is much more concentrated in the cells than in the medium. It was also found that irradiation with intense light easily gives rise to a photoproduct which gives an additional peak in the fluorescence spectrum. Possible methods to increase the sensitivity of cancer detection and localization are discussed.     

Comparison of photodynamic targets in a carcinoma cell line and its mitochondrial DNA-deficient derivative

The relative contribution, to cell death, of photodynamic damage to respiratory proteins (known targets of photodynamic therapy with many photosensitizers) and other cellular sites was examined. The models were a human ovarian carcinoma cell line 2008, and its mitochondrial DNA-deficient derivative ET3, which lacks several key respiratory protein subunits. Phototoxicity was compared in the two cell lines with photosensitizers that localized to different cellular compartments. Photosensitizers included Victoria Blue BO (VBBO; mitochondria); Photofrin with a short incubation, (plasma membrane) or a long incubation (intracellular membranes including mitochondria); and Nile Blue A (NBA; lysosomes). Photosensitizer content and localization did not differ between the 2008 and ET3 cells. For sensitizers without a primary mitochondrial localization (NBA and Photofrin with a short incubation), there was no significant difference between 2008 and ET3 toxicity. Consistent with a mitochondrial localization of VBBO and independence from respiratory-chain damage, ET3 cells were less susceptible than 2008 to both dark- and light-activated VBBO-mediated damage. Statistical analysis of the data demonstrated minimal photobleaching of VBBO and a significant difference between the phototoxicity curves of ET3 and 2008. For Photofrin with a long incubation, dark- and phototoxicity effects were similar for both cell lines. Inhibition of respiratory enzymes is thus only a minor component of Photofrin-mediated (long incubation) phototoxicity in these cell lines and is overwhelmed by more significant damage elsewhere, whereas it is a major but not the exclusive element of death mediated by VBBO.     

BIOLOGICAL ACTIVITIES OF PHTHALOCYANINES. XIII. THE EFFECTS OF HUMAN SERUM COMPONENTS ON THE in vitro UPTAKE AND PHOTODYNAMIC ACTIVITY OF ZINC PHTHALOCYANINE

Abstract— The effect of human serum components on the photodynamic activity of zinc phthalocyanine (ZnPc) toward Chinese hamster fibroblasts (lineV–79) was studied. Photodynamic activities were correlated with cellular uptake of radiolabeled [⁶⁵Zn]ZnPc, which allowed corrections to be made for the amount of sensitizer present in the cells at the time of irradiation and to express photodynamic efficiences on a cellular dye concentration basis. All serum components, with the exception of high-density lipoproteins, inhibit uptake of ZnPc byV–79 cells, when compared to incubation of ZnPc with the same cells in serum-free medium. High-density lipoproteins increased ZnPc uptake by 23%, but the photodynamic efficiency corrected for the cellular ZnPc concentration was unaffected. Very low-density lipoprotein and globulins decreased ZnPc cell uptake but likewise did not affect the cellular photodynamic efficiency of the dye. In contrast low-density lipoprotein and albumin, while inhibiting ZnPc cell uptake, increased the cellular photodynamic efficiency of ZnPc, suggesting that these proteins facilitate localization of the dye at cellular targers sensitive to photodynamic damage and vital to cell survival. We conclude from these results that association of ZnPc with serum components can have important, and widely differing, effects on both degree of uptake and cellular distribution of the photosensitizer.     

5-Aminolevulinic acid-induced protoporphyrin-IX accumulation and associated phototoxicity in macrophages and oral cancer cell lines

Studies were carried out on 5-aminolevulinic acid (ALA)-induced protoporphyrin (PpIX) synthesis in mice peritoneal macrophages and two human oral squamous cell carcinoma (OSCC) cell lines NT8e and 4451. Cells were treated with 200 microg/ml ALA for 15 h and PpIX accumulation was monitored by spectrofluorometry and phototoxicity to red light (630+/-20 nm) was measured by MTT assay. PpIX accumulation was higher in macrophages as compared to OSCC cells under both normal serum concentration (10%) and conditions of serum depletion. The results on phototoxicity measurements correlated well with the levels of PpIX accumulation in both macrophages and cancer cells. While red light caused 20% phototoxicity in macrophages, no phototoxicity was seen in 4451 cells at 10% serum. Decrease in serum concentration to 5% and 1% led to higher phototoxicity corresponding to 40% and 70% in macrophages and 10% and 15% in 4451 cells. Similar results were obtained in NT8e cell line. Propidium iodide staining followed by fluorescence microscopic observations on photodynamically treated co-culture of murine or human macrophages and cancer cells showed selective damage to macrophages. These results suggest that in OSCC, macrophages would contribute more to tumor PpIX level than tumor cells themselves and PDT may lead to selective killing of macrophages at the site of treatment. Since macrophages are responsible for production and secretion of various tumor growth mediators, the effect of selective macrophage killing on the outcome of PDT would be significant.     

SUBCELLULAR LOCALIZATION OF HEMATOPORPHYRIN DERIVATIVE IN BLADDER TUMOR CELLS IN CULTURE

Mitochondria have been implicated as a primary subcellular site of porphyrin localization and photodestruction. However, other organelles including the cell membrane, lysosomes and nucleus have been shown to be damaged by hematoporphyrin derivative (HpD) photosensitized destruction as well. In this study we attempted to follow the translocation of the fluorescent components of HpD in human bladder tumor cells (MGH-U1) in culture to determine whether specific subcellular localization occurs over time. Following a 30 min exposure to HpD the cellular fluorescence was examined immediately and 1, 2, 4, and 24 h after HpD removal using fluorescence microscopy and an interactive laser cytometer. The in vitro translocation of dye appeared to be fairly rapid with fluorescence present at the cell membrane and later (1-2 h) within a perinuclear area of the cytoplasm. To determine whether HpD had become concentrated into a specific subcellular organelle, these fluorescence distribution patterns were compared with fluorescent marker dyes specific for mitochondria, endoplasmic reticulum and other membranous organelles. The HpD fluorescence did not appear to be as discrete as the dyes specific for mitochondria or endoplasmic reticulum but appeared similar to the diffuse cytomembrane stain. Finally, the interaction between the fluorescent components of HpD and the cellular constituents was evaluated using a "fluorescence redistribution after photobleaching" technique. The results indicated that the mean lateral diffusion for HpD in MGH-U1 cells was 1.05 x 10(-8) cm2/s, a rate closer to that of lipid diffusion (10(-8)) than that of protein diffusion (10(-10)).(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypericum perforatum L. extract - novel photosensitizer against human bladder cancer cells

The polar methanolic fraction (PMF) of the Hypericum perforatum L. extract has recently been developed and tested as a novel, natural photosensitizer for use in the photodynamic therapy (PDT), and photodynamic diagnosis (PDD). PMF has been tested on HL-60 leukemic cells and cord blood hemopoietic progenitors. In the present study, the efficacy of PMF as a phototoxic agent against urinary bladder carcinoma has been studied using the T24 (high grade metastatic cancer), and RT4 (primary low grade papillary transitional cell carcinoma) human bladder cancer cells. Following cell culture incubation, PMF was excited using 630 nm laser light. The photosensitizer exhibited significant photocytotoxicity in both cell lines at a concentration of 60microg/ml, with 4-8 J/cm(2) light dose, resulting in cell destruction from 80% to 86%. At the concentration of 20microg/ml PMF was not active in either cell line. These results were compared with the results obtained in the same cell lines, under the same conditions with a clinically approved photosensitizer, Photofrin. Photofrin was used in the maximum clinically tolerable dose of 4microg/ml, and it was also excited with 630 nm laser light. In the T24 cell Photofrin exhibited slightly less photocytotocixity, compared with PMF, resulting in 77% cell death with 8J/cm(2) light dose. However, against the RT4 cells Photofrin resulted in minimal cell death (9%) with even 8J/cm(2) light dose. Finally, the type of cell death induced by PMF photoactivation was studied using flow cytometry and DNA laddering. Cell death by PMF photodynamic action in these two bladder cell lines is caused predominently by apoptosis. The reported significant photocytotoxicity, selective localization, natural abundance, easy, and inexpensive preparation, underscore that the PMF extract hold the promise of being a novel, effective PDT photosensitizer.     

PHOTOBLEACHING OF PORPHYRINS USED IN PHOTODYNAMIC THERAPY AND IMPLICATIONS FOR THERAPY

Abstract— The development of an extraction procedure to quantitate dihematoporphyrin ether (DHE) concentration in tissues correlated to fluorescence measurements from instrumentation developed for in vivo fluorimetry was examined. In vivo fluorometric results from mouse mammary carcinoma (SMT-F) were calibrated against results of the chemical extraction assay quantitated spectrophotometrically. Fluorescence and drug extractable levels increase in a linear fashion with injected dose. Loss of porphyrin fluorescence (photobleaching) and intra-tumoral porphyrin level has been demonstrated both in vitro (NHIK cells) and in vivo (SMT-F tumor) during illumination with light following exposure to Hpd or DHE. This process is essentially independent of porphyrin tumor level in vivo and could lead to tumor protection at very low porphyrin levels. On the other hand, this photobleaching process which occurs concurrent with cellular inactivation and tissue damage due to the photodynamic process can be exploited to protect normal tissue during photodynamic therapy (PDT) and thus greatly enhance the therapeutic ratio. This has been demonstrated in patients undergoing PDT.     

The role of the p53 tumor suppressor in the response of human cells to photofrin-mediated photodynamic therapy

Although there is evidence that the p53 tumor suppressor plays a role in the response of some human cells to chemotherapy and radiation therapy, its role in the response of human cells to photodynamic therapy (PDT) is less clear. In order to examine the role of p53 in cellular sensitivity to PDT, we have examined the clonogenic survival of normal human fibroblasts that express wild-type p53 and immortalized Li-Fraumeni syndrome (LFS) cells that express only mutant p53, following Photofrin-mediated PDT. The LFS cells were found to be more resistant to PDT compared to normal human fibroblasts. The D37 (LFS cells)/D37 (normal human fibroblasts) was 2.8 +/- 0.3 for seven independent experiments. Although the uptake of Photofrin per cell was 1.6 +/- 0.1-fold greater in normal human fibroblast cells compared to that in LFS cells over the range of Photofrin concentrations employed, PDT treatment at equivalent cellular Photofrin levels also demonstrated an increased resistance for LFS cells compared to normal human fibroblasts. Furthermore, adenovirus-mediated transfer and expression of wild-type p53 in LFS cells resulted in an increased sensitivity to PDT but no change in the uptake of Photofrin per cell. These results suggest a role for p53 in the response of human cells to PDT. Although normal human fibroblasts displayed increased levels of p53 following PDT, we did not detect apoptosis or any marked alteration in the cell cycle of GM38 cells, despite a marked loss of cell viability. In contrast, LFS cells exhibited a prolonged accumulation of cells in G2 phase and underwent apoptosis following PDT at equivalent Photofrin levels. The number of apoptotic LFS cells increased with time after PDT and correlated with the loss of cell viability. A p53-independent induction of apoptosis appears to be an important mechanism contributing to loss of clonogenic survival after PDT in LFS cells, whereas the induction of apoptosis does not appear to be an important mechanism leading to loss of cell survival in the more sensitive normal human fibroblasts following PDT at equivalent cellular Photofrin levels.