Successful cutaneous delivery of the photosensitizer silicon phthalocyanine 4 for photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) has been shown to be effective in the treatment of malignancies of a variety of organ systems, including the lungs, bladder, gastrointestinal tract and skin. Cutaneous lesions serve as ideal targets of PDT because of the accessibility of the skin to light. To achieve optimum results, the photosensitizer must be delivered effectively into the target layers of the skin within a practical timeframe, via noninvasive methods. AIM: To determine whether topical application of a second-generation photosensitizer, silicon phthalocyanine (Pc) 4 [SiPc(OSi(CH3)2 (CH2)3 N(CH3)2)(OH)], results in effective penetration of the skin barrier. METHODS: Penetration of Pc 4 was evaluated using standard Franz-type vertical diffusion cell experiments on surrogate materials (silicone membranes) and laser-scanning confocal microscopy of normal skin biopsy samples from human volunteers. RESULTS: The Franz diffusion data indicate that Pc 4 formulated in an ethanol/propylene glycol solution (70/30%, v/v) can penetrate the membrane at a flux that is appreciable and relatively invariant. Using the same formulation, Pc 4 uptake could be detected in human skin via laser-scanning confocal microscopy. CONCLUSION: After topical application, Pc 4 is absorbed into the epidermis in as little as 1 h, and the absorption increased with increasing time and dose. Pc 4 can be effectively delivered into human skin via topical application. The data also suggest that the degree of penetration is time- and dose-dependent.

Photodynamic therapy-induced death of HCT 116 cells: Apoptosis with or without Bax expression.

Cell death following photodynamic therapy (PDT) with the photosensitizer Pc 4 involves the intrinsic pathway of apoptosis. To evaluate the importance of Bax in apoptosis after PDT, we compared the PDT responses of Bax-proficient (Bax(+/-)) and Bax knock-out (BaxKO) HCT116 human colon cancer cells. PDT induced a slow apoptotic process in HCT Bax(+/-) cells following a long delay in the activation of Bax and release of cytochrome c from mitochondria. Although cytochrome c was not released from mitochondria following PDT in BaxKO cells, an alternative mechanism of caspase-dependent apoptosis with extensive chromatin and DNA degradation was found in these cells. This alternative process was less efficient and slower than the normal apoptotic process observed in Bax(+/-) cells. Early events upon PDT, such as the loss of mitochondrial membrane potential, photodamage to Bcl-2, and activation of p38 MAP kinase, were observed in both HCT116 cell lines. In spite of differences in the efficiency and mode of apoptosis induced by PDT in the Bax(+/-) and BaxKO cells, they were found to be equally sensitive to killing by PDT, as determined by loss of clonogenicity. Thus, for Pc 4-PDT, the commitment to cell death occurs prior to and independent of Bax activation, but the process of cellular disassembly differs in Bax-expressing vs. non-expressing cells.

Bax is essential for mitochondrion-mediated apoptosis but not for cell death caused by photodynamic therapy.

The role of Bax in the release of cytochrome c from mitochondria and the induction of apoptosis has been demonstrated in many systems. Using immunocytochemical staining, we observed that photodynamic therapy (PDT) with the photosensitiser Pc 4 induced Bax translocation from the cytosol to mitochondria, and the release of cytochrome c from mitochondria as early signalling for the intrinsic pathway of apoptosis in human breast cancer MCF-7c3 cells. To test the role of Bax in apoptosis, MCF-7c3 cells were treated with Bax antisense oligonucleotides, which resulted in as much as a 50% inhibition of PDT-induced apoptosis. In the second approach, Bax-negative human prostate cancer DU-145 cells were studied. Following PDT, the hallmarks of apoptosis, including the release of cytochrome c from mitochondria, loss of mitochondrial membrane potential, caspase activation, and chromatin condensation and fragmentation, were completely blocked in these cells. Restoration of Bax expression in DU-145 cells restored apoptosis, indicating that the resistance of DU-145 cells to PDT-induced apoptosis is due to the lack of Bax rather than to another defect in the apoptotic machinery. However, despite the inhibition of apoptosis, the Bax-negative DU-145 cells were as photosensitive as Bax-replete MCF-7c3 cells, as determined by clonogenic assay. Thus, for Pc 4-PDT, the commitment to cell death occurs prior to Bax activation.

Photodynamic therapy-induced apoptosis in epidermoid carcinoma cells. Reactive oxygen species and mitochondrial inner membrane permeabilization.

Photodynamic therapy (PDT), a novel and promising cancer treatment that employs a combination of a photosensitizing chemical and visible light, induces apoptosis in human epidermoid carcinoma A431 cells. However, the precise mechanism of PDT-induced apoptosis is not well characterized. To dissect the pathways of PDT-induced apoptosis, we investigated the involvement of mitochondrial damage by examining a second generation photosensitizer, the silicon phthalocyanine 4 (Pc 4). By using laser-scanning confocal microscopy, we found that Pc 4 localized to cytosolic membranes primarily, but not exclusively, in mitochondria. Formation of mitochondrial reactive oxygen species (ROS) was detected within minutes when cells were exposed to Pc 4 and 670-675 nm light. This was followed by mitochondrial inner membrane permeabilization, depolarization and swelling, cytochrome c release, and apoptotic death. Desferrioxamine prevented mitochondrial ROS production and the events thereafter. Cyclosporin A plus trifluoperazine, blockers of the mitochondrial permeability transition, inhibited mitochondrial inner membrane permeabilization and depolarization without affecting mitochondrial ROS generation. These data indicate that the mitochondrial ROS are critical in initiating mitochondrial inner membrane permeabilization, which leads to mitochondrial swelling, cytochrome c release to the cytosol, and apoptotic death during PDT with Pc 4.

Photodynamic therapy in oncology.

Photodynamic therapy (PDT) is a cancer treatment modality that is based on the administration of a photosensitiser, which is retained in tumour tissues more than in normal tissues, followed by illumination of the tumour with visible light in a wavelength range matching the absorption spectrum of the photosensitiser. The photosensitiser absorbs light energy and induces the production of reactive oxygen species in the tumour environment, generating a cascade of events that kills the tumour cells. The first generation photosensitiser, Photofrin (porfirmer sodium), has been approved for oesophageal and lung cancer in the US and has been under investigation for other malignant and non-malignant diseases. Sub-optimal light penetration at the treatment absorption peak of Photofrin and prolonged skin photosensitivity in patients are limiting factors for this preparation. Several new photosensitisers have improved properties, especially absorption of longer wavelength light which penetrates deeper into tissue and faster clearance from normal tissue. This paper reviews the current use of first- and second-generation photosensitisers in oncology. The use of PDT in oncology has been restricted to certain cancer indications and has not yet become an integral part of cancer treatment in general. The main advantage of PDT is that the treatment can be repeated multiple times safely, without producing immunosuppressive and myelosuppressive effects and can be administered even after surgery, chemotherapy or radiotherapy. The current work on new photosensitisers and light delivery equipment will address some of the present shortcomings of PDT. Much has been learned in recent years about the mechanisms of cellular and tissue responses to PDT and protocols designed to capitalise on this knowledge showed lead to additional improvements.

Photochemical destruction of the Bcl-2 oncoprotein during photodynamic therapy with the phthalocyanine photosensitizer Pc 4.

Photodynamic therapy (PDT), utilizing a photosensitizer and visible light, causes localized oxidative damage. With the mitochondrial photosensitizer Pc 4, PDT induces apoptosis, yet its molecular targets are not known. Here, the anti-apoptotic protein Bcl-2 is shown to be highly sensitive to PDT, as judged on Western blots by the disappearance of anti-Bcl-2-reactive material from the position of the native 26 kDa protein. The loss of Bcl-2 was PDT dose dependent and was observed for both endogenous and overexpressed Bcl-2 in several cell lines, immediately after PDT, and with chilled cells. It was accompanied by a trace of a 23-kDa cleavage product as well as high-molecular weight products that may result from photochemical crosslinking. PDT-induced Bcl-2 loss occurred in MCF-7 cells that do not express caspase-3 or in the presence of protease inhibitors, but was prevented, along with the induction of apoptosis, by the singlet oxygen scavenger L-histidine. Loss of FLAG-Bcl-2 was observed with both anti-FLAG and anti-Bcl-2 antibodies, indicating loss of native protein rather than simple BCL-2-epitope destruction. Photochemical damage was not observed in Bcl-x(L), Bax, Bad, the voltage-dependent anion channel, or the adenine nucleotide translocator. Therefore, Bcl-2 is one target of PDT with Pc 4, and PDT damage to Bcl-2 contributes to its efficient induction of apoptosis.

Dissociation of mitochondrial depolarization from cytochrome c release during apoptosis induced by photodynamic therapy.

Photodynamic therapy (PDT) with the phthalocyanine photosensitizer Pc 4 induces rapid apoptosis in mouse lymphoma (LY-R) cells, initiating with the release of cytochrome c from mitochondria. It has been proposed that the opening of the mitochondrial membrane permeability transition pores, which results in the dissipation of the mitochondrial membrane potential (Deltapsi(m)), is essential for the escape of cytochrome c from mitochondria into the cytosol as well as for apoptotic cell death. Therefore, we have assessed the correlation between the loss of Deltapsi(m)and the release of cytochrome c following PDT. Treatment of LY-R cells with 300 nM Pc 4 and 60, 90 or 120 mJ/cm(2)of red light resulted in apoptosis of 80-90% of the cells, accompanied by >20-fold elevation in caspase-3-like activity within one h. At all 3 doses of PDT employed here, the majority of the cytochrome c was released from mitochondria at 15 min after irradiation, as determined by an immunohistochemical method. In contrast, the loss of Deltapsi(m)following PDT, as monitored by the uptake of JC-1 or Rh-123, depended on the PDT dose and the post-treatment time. In spite of the release of cytochrome c at 15 min after each of the 3 doses, a corresponding loss of Deltapsi(m)was observed only for those cells that received the highest dose of PDT. Virtually all cells that received one of the lower doses of PDT (300 nM Pc 4 plus 60 or 90 mJ/cm(2)) maintained normal Deltapsi(m). Hence, our results support the conclusion that the release of cytochrome c from mitochondria resulting from Pc 4-PDT-induced photodamage is independent of the loss of Deltapsi(m). Therefore, it is important to consider a range of doses of this or other apoptotic stimuli in deciphering the relationship of metabolic responses that contribute to apoptosis.

Phthalocyanine 4 photodynamic therapy-induced apoptosis of mouse L5178Y-R cells results from a delayed but extensive release of cytochrome c from mitochondria.

Photodynamic therapy (PDT) activates the mitochondrial pathway of apoptosis, for which the release of cytochrome c into the cytosol is considered critical. To further elucidate the role of cytochrome c release in PDT-induced apoptosis, we monitored cytochrome c localization immunocytochemically and related it to nuclear apoptosis of the same cells. When mouse L5178Y-R cells were treated with 300 nM phthalocyanine (Pc) 4 and 0-75 mJ/cm(2) red light, cytochrome c release had a dose response similar to that of clonogenic cell killing, with nearly identical threshold doses. Within individual cells, the release of cytochrome c appeared to be an all-or-none phenomenon. Moreover, it was tightly associated with activation of a caspase-3-like protease and changes in nuclear morphology. Thus, in response to Pc 4-PDT, the release of cytochrome c from mitochondria is a key determinant of apoptotic cell death.

Photodynamic therapy-induced death of MCF-7 human breast cancer cells: a role for caspase-3 in the late steps of apoptosis but not for the critical lethal event.

Photodynamic therapy (PDT) causes mitochondrial damage and induces apoptosis through release of cytochrome c and activation of caspase-3. To test whether caspase 3 is the sole executioner of apoptosis and its role in overall cell lethality, we compared the response of MCF-7c3 cells that express a stably transfected CASP-3 gene to that of parental MCF-7:SW8 cells transfected with vector alone (MCF-7v). Following photosensitization with the phthalocyanine Pc 4 and red light, cytochrome c was released from the mitochondria to equivalent extents in the two cell lines. However, the appearance of apoptotic indicators, such as active caspase-3 (DEVDase), cleavage of poly(ADP-ribose) polymerase, and oligonucleosomal DNA fragmentation, was observed only in MCF-7c3 cells during the first 6 h after photosensitization. Although production of 50-kb DNA fragments and chromatin condensation were found in PDT-treated MCF-7v cells by 20-24 h posttreatment, the rate and extent of apoptosis were much less than in MCF-7c3 cells. MCF-7c3 cells were more sensitive to photosensitization than were MCF-7v cells when assayed for loss of viability by reduction of a tetrazolium dye. However, the two cell lines were equally sensitive to photodynamic killing when evaluated by a clonogenic assay. These results show (a) the importance of assessing overall cell death by clonogenic assay; (b) that the critical lethal event is independent of caspase-3, perhaps at or near the release of cytochrome c from mitochondria; and (c) that the caspase-3-mediated events appear to be irrelevant in determining overall killing of cells.

Photodynamic therapy: a new concept in medical treatment.

A new concept in the therapy of both neoplastic and non-neoplastic diseases is discussed in this article. Photodynamic therapy (PDT) involves light activation, in the presence of molecular oxygen, of certain dyes that are taken up by the target tissue. These dyes are termed photosensitizers. The mechanism of interaction of the photosensitizers and light is discussed, along with the effects produced in the target tissue. The present status of clinical PDT is discussed along with the newer photosensitizers being used and their clinical roles. Despite the promising results from earlier clinical trials of PDT, considerable additional work is needed to bring this new modality of treatment into modern clinical practice. Improvements in the area of light source delivery, light dosimetry and the computation of models of treatment are necessary to standardize treatments and ensure proper treatment delivery. Finally, quality assurance issues in the treatment process should be introduced.

Quantitative analysis of Pc 4 localization in mouse lymphoma (LY-R) cells via double-label confocal fluorescence microscopy.

Photodynamic therapy (PDT) is a novel cancer therapy that uses light-activated drugs (photosensitizers) to destroy tumor tissue. Reactive oxygen species produced during PDT are thought to cause the destruction of tumor tissue. However, the precise mechanism of PDT is not completely understood. To provide insight into the in vitro mechanisms of PDT, we studied the subcellular localization of the photosensitizer HOSiPcOSi(CH3)2-(CH2)3N(CH3)2 (Pc 4) in mouse lymphoma (LY-R) cells using double-label confocal fluorescence microscopy. This technique allowed us to observe the relative distributions of Pc 4 and an organelle-specific dye within the same cell via two, spectrally distinct, fluorescence images. To quantify the localization of Pc 4 within different organelles, linear correlation coefficients from the fluorescence data of Pc 4 and the organelle-specific dyes were calculated. Using this measurement, the subcellular spatial distributions of Pc 4 could be successfully monitored over an 18 h period. At early times (0-1 h) after introduction of Pc 4 to LY-R cells, the dye was found in the mitochondria, lysosomes and Golgi apparatus, as well as other cytoplasmic membranes, but not in the plasma membrane or the nucleus. Over the next 2 h, there was some loss of Pc 4 from the lysosomes as shown by the correlation coefficients. After an additional incubation period of 2 h Pc 4 slowly increased its accumulation in the lysosomes. The highest correlation coefficient (0.65) was for Pc 4 and BODIPY-FL C5 ceramide, which targets the Golgi apparatus, and also binds to other cytoplasmic membranes. The correlation coefficient was also high (0.60) for Pc 4 and a mitochondria-targeting dye (Mitotracker Green FM). Both of these correlation coefficients were higher than that for Pc 4 with the lysosome-targeting dye (Lysotracker Green DND-26). The results suggest that Pc 4 binds preferentially and strongly to mitochondria and Golgi complexes.

Photodynamic therapy with the phthalocyanine photosensitizer Pc 4 of SW480 human colon cancer xenografts in athymic mice.

Photodynamic therapy (PDT) using the silicon phthalocyanine photosensitizer Pc 4 [HOSiPcOSi(CH3)2(CH2)3N-(CH3)2] is an oxidative stress associated with induction of apoptosis in various cell types. We assessed the effectiveness of Pc 4-PDT on SW480 colon cancer xenografts grown in athymic nude mice. Animals bearing xenografts were treated with 1 mg/kg body weight Pc 4 and 48 h later were irradiated with 150 J/cm2 672-nm light from a diode laser delivered at 150 mW/cm2. Biochemical studies were performed in xenografts resected at various time points up to 26 h after Pc 4-PDT treatment, whereas tumor size was evaluated over a 4-week period in parallel experiments. In the tumors resected for biochemical studies, apoptosis was visualized by activation of caspase-9 and caspase-3 and a gradual increase in the cleavage of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) to a maximum of approximately 60% of the total PARP present at approximately 26 h. At that time all Pc 4-PDT-treated tumors had regressed significantly. Two signaling responses that have previously been shown to be associated with Pc 4-PDT-induced apoptosis in cultured cells, p38 mitogen-activated protein kinase and p21/WAF1/Cip1, were examined. A marked increase in phosphorylation of p38 was observed within 1 h after Pc 4-PDT without changes in levels of the p38 protein. Levels of p21 were not altered in the xenografts in correspondence with the presence of mutant p53 in SW480 cells. Evaluation of tumor size showed that tumor growth resumed after a delay of 9-15 days. Our results suggest that: (a) Pc 4-PDT is effective in the treatment of SW480 human colon cancer xenografts independent of p53 status; (b) PARP cleavage may be mediated by caspase-9 and caspase-3 activation in the Pc 4-PDT-treated tumors; and (c) p38 phosphorylation may be a trigger of apoptosis in response to PDT in vivo in this tumor model.

Promotion of photodynamic therapy-induced apoptosis by stress kinases.

Photodynamic therapy (PDT), a cancer treatment that employs a photosensitizer and visible light, induces apoptosis in murine LY-R leukemic lymphoblasts and in CHO cells, but the rate and extent of apoptosis are much greater in LY-R cells. Three MAPK family members, ERK1/ERK2, SAPK/JNK, and p38/HOG, are important intermediates in signal transduction pathways. To ascertain whether activation of one or more MAPKs could mediate PDT-induced apoptosis, Western blot analysis has been performed on the proteins of LY-R and CHO cells at various times following lethal (90 - 99% cell kill) doses of PDT photosensitized by the phthalocyanine Pc 4. The blots were probed with antibodies to each of the proteins as well as antibodies specific for the activated (phosphorylated) forms of each kinase. Of the three MAPK types, only the p46 and p54 SAPK/JNKs were found to be activated by PDT in LY-R cells, with a maximum approximately threefold increase in the content of the phosphorylated forms reached in 30 - 60 min. An even larger relative activation was observed in CHO cells. PDT did not affect ERK and p38/HOG activation in LY-R cells. In the case of CHO cells, however, ERK2 was slightly activated at 5 min post-PDT, then declined, and p38/HOG was strongly activated from 5 to 60 min post-PDT. A specific inhibitor (PD98059) of MEK1, the kinase that activates ERK, had little or no effect on PDT-induced apoptosis in either LY-R or CHO cells. In contrast, a specific inhibitor of p38/HOG (SB202190) blocked PDT-induced apoptosis in LY-R cells with a lesser effect in CHO cells. The results suggest that both the SAPK and p38/HOG cascades can be stimulated by PDT and that the latter participates in both rapid and slow PDT-induced apoptosis. Furthermore, the high level of constitutively active p38/HOG in LY-R cells may poise those cells for rapid activation of apoptosis following PDT.

Etk/Bmx, a PH-domain containing tyrosine kinase, protects prostate cancer cells from apoptosis induced by photodynamic therapy or thapsigargin.

Prostate carcinoma (PCA) is the most frequently diagnosed malignancy in American men. PCA at advanced stages can both proliferate abnormally and resist apoptosis. Among the many known signal transduction pathways, phosphatidylinositide-3'OH kinase (PI3-kinase) has been shown to play an important role in cell survival and resistance to apoptosis. In this study, we investigate the involvement of Etk/Bmx, a newly discovered tyrosine kinase that is a substrate of PI3-kinase, in protection of prostate cancer cells from apoptosis. Parental LNCaP cells and two derivative cell lines, one overexpressing wild type Etk (Etkwt) and the other expressing a dominant negative Etk (EtkDN), were used to study the function of Etk. The cells were treated with photodynamic therapy (PDT), a newly approved cancer treatment which employs a photosensitizer and visible light to produce an oxidative stress in cells, often leading to apoptosis. Our results indicate that PDT induces apoptosis in LNCaP cells, as measured by DNA fragmentation and by cleavage of poly(ADP-ribose) polymerase (PARP), and moreover, the extent of apoptosis was much reduced in Etkwt cells as compared to LNCaP or EtkDN cells. Assay of overall cell viability confirmed that Etkwt cells were considerably less sensitive to PDT than were the parental LNCaP or EtkDN cells. Similar results were found in response to thapsigargin (TG). A specific inhibitor of PI3-kinase, LY294002, abolished Etk activity and markedly increased TG-induced PARP cleavage. The results suggest that Etk/Bmx is an efficient effector of PI3-kinase and that the newly described PI3-kinase/Etk pathway is involved in the protection of prostate carcinoma cells from apoptosis in response to PDT or TG.

Photodynamic therapy-induced apoptosis in lymphoma cells: translocation of cytochrome c causes inhibition of respiration as well as caspase activation.

L5178Y-R mouse lymphoma (LY-R) cells undergo rapid apoptosis when treated with photodynamic therapy (PDT) sensitized with the silicon phthalocyanine Pc 4. In this study we show that cytochrome c is released into the cytosol within 10 min of an LD99.9 dose of PDT. Cellular respiration is inhibited by 42% at 15 min, and 60% at 30 min after PDT treatment, and caspase 3-like protease activity is elevated by 15 min post-PDT. In digitonin-permeabilized cells addition of cytochrome c to the respiration buffer reverses PDT-induced inhibition of state 3 respiration via Complex I by 40-60%, and via Complex III by 50-90%. In contrast, extramitochondrial cytochrome c does not stimulate respiration in permeabilized control cells, and catalyzes only a low rate of oxygen consumption via electron transfer to cytochrome b5 on the outer mitochondrial membrane. These results demonstrate that PDT-induced inhibition of respiration is primarily due to leakage of cytochrome c into the cytosol rather than to damage to the major enzyme complexes of the electron transport chain. Whether or not inhibition of respiration influences the time course or extent of Pc 4-PDT-induced apoptosis in LY-R cells is not clear at the present time.

Phthalocyanine 4 (Pc 4) photodynamic therapy of human OVCAR-3 tumor xenografts.

Photodynamic therapy (PDT) is a cancer treatment modality utilizing a photosensitizer, light and oxygen. Photodynamic therapy with Photofrin has been approved by the U.S. Food and Drug Administration for treatment of advanced esophageal and early lung cancer. Because of certain drawbacks associated with the use of Photofrin, there is a need to identify new photosensitizers for human use. The photosensitizer Pc 4 (HOSiPc-OSi[CH3]2[CH2]3N[CH3]2) has yielded promising PDT effects in many in vitro and in vivo systems. The aim of this study was to assess the usefulness of Pc 4 as a PDT photosensitizer for a human tumor grown as a xenograft in athymic nude mice. The ovarian epithelial carcinoma (OVCAR-3) was heterotransplanted subcutaneously in athymic nude mice. Sixty mice bearing OVCAR-3 tumors (approximately 80-130 mm3) were divided into six groups of 10 animals each, three for controls and three for treatment. The Pc 4 was given by tail vein injection, and 48 h later a 1 cm area encompassing the tumor was irradiated with light from a diode laser coupled to a fiberoptic terminating in a microlens (lambda = 672 nm, 150 J/cm2, 150 mW/cm2). Tumors of control animals receiving no treatment, light alone or Pc 4 alone continued to grow. Of animals receiving 0.4 mg/kg Pc 4 and light, one (10%) had a complete response and was cured (no regrowth up to 90 days post-PDT), while all others (90%) had a partial response and were delayed in regrowth. Of animals receiving 0.6 mg/kg Pc 4 and light, eight (80%) had a complete response, and two of these were cured. Of animals receiving 1.0 mg/kg Pc 4 and light, six (60%) had a complete response, and two of these were cured. In additional experiments, tumors from animals treated with Pc 4 (1 mg/kg) and light were removed 15, 30, 60 and 180 min post-PDT, and from these tumors DNA and protein were extracted. Agarose gel electrophoresis revealed the presence of apoptotic DNA fragmentation as early as 15 min post-PDT. Western blotting showed the cleavage of the 116 kDa native poly(ADP-ribose) polymerase (PARP) into fragments of approximately 90 kDa, another indication of apoptosis, and the presence of p21/WAF1/CIP1 (p21) in all PDT-treated tumors. These changes did not occur in control tumors. Pc 4 appears to be an effective photosensitizer for PDT of human tumors grown as xenografts in nude mice. Early apoptosis, as revealed by PARP cleavage, DNA fragmentation and p21 overexpression, may be responsible for the excellent Pc 4-PDT response. Clinical trials of Pc 4-PDT are warranted.

The Impact of Biology on Risk Assessment--workshop of the National Research Council's Board on Radiation Effects Research. July 21-22, 1997, National Academy of Sciences, Washington, DC.

The linear no-threshold extrapolation from a dose-response relationship for ionizing radiation derived at higher doses to doses for which regulatory standards are proposed is being challenged by some scientists and defended by others. It appears that the risks associated with exposures to doses of interest are below the risks that can be measured with epidemiological studies. Therefore, many have looked to biology to provide information relevant to risk assessment. The workshop reported here, "The Impact of Biology on Risk Assessment", was planned to address the need for additional information by bringing together scientists who have been working in key fields of biology and others who have been contemplating the issues associated specifically with this question. The goals of the workshop were to summarize and review the status of the relevant biology, to determine how the reported biological data might influence risk assessment, and to identify subjects on which more data are needed.

The photobiology of photodynamic therapy: cellular targets and mechanisms.

Photodynamic therapy (PDT) is dependent on the uptake of a photosensitizing dye, often a porphyrin-related macrocycle, by the tumor or other abnormal tissue that is to be treated, the subsequent irradiation of the tumor with visible light of an appropriate wavelength matched to the absorption spectrum of the dye, and molecular oxygen to generate reactive oxygen intermediates. The initial oxidative reactions lead to damage to organelles in which the dye is bound, culminating in cell death and destruction of the tumor or abnormal tissue. Apoptosis is a common mechanism of cell death after PDT both in vitro and in vivo. PDT also triggers the activation of several signal transduction pathways in the treated cells; some of these are stress responses aimed at cell protection, while others are likely to contribute to the cell death process. The photosensitizers of greatest interest in PDT bind to various cytoplasmic membranes but are not found in the nucleus and do not bind to DNA. Nevertheless, some DNA damage is produced that can lead to mutagenesis, the extent of which is dependent on the photosensitizer, the cellular repair properties and the target gene. Thus, in spite of generating some responses common to ionizing radiation and other oxidative stresses, PDT is unique in the subcellular localization of damage, the combination of signaling pathways that are activated, and rapid kinetics of the induction of cell death processes.

Association of ceramide accumulation with photodynamic treatment-induced cell death.

Ceramide, a stress-induced second messenger, has been associated with apoptosis in several malignant and non-malignant cell lines. We have shown that photodynamic treatment (PDT), using the phthalocyanine photosensitizer Pc 4 (HOSiPcOSi[CH3]2[CH2]3N[CH3]2), causes increased ceramide generation and subsequent induction of apoptosis in L5178Y-R (LY-R) mouse lymphoma cells. To test further if ceramide generation accompanies photocytotoxicity, we treated various cell lines with a PDT dose producing a 99-99.9% loss of clonogenicity. Like LY-R cells, human leukemia (U937) cells underwent rapid DNA fragmentation initiating within 1 h after PDT. Similarly, Chinese hamster ovary (CHO) cells showed rapid DNA laddering, beginning 1 h following the treatment. In contrast, mouse radiation-induced fibrosarcoma (RIF-1) cells showed no apoptosis within 24 h post-PDT, as judged by the absence of 50 kbp and oligonucleosome size DNA fragments, as well as no annexin V binding to cells with preserved membrane integrity. Using the same doses of PDT, we observed a time-dependent ceramide accumulation in all three cell lines. While a significant increase in ceramide levels was reached within 1 and 10 min in U937 and CHO cells, respectively, elevated ceramide production was measured only after 30 min in RIF-1 cells. In addition, exogenous N-acetyl-sphingosine was able to mimic PDT-induced apoptosis in U937 and CHO cells. We suggest that ceramide accumulation is associated with PDT-induced apoptosis and photocytotoxicity.

Variation in photodynamic efficacy during the cellular uptake of two phthalocyanine photosensitizers.

A decrease in the efficacy of photodynamic therapy (PDT) with phthalocyanine photosensitizers was observed for lymphoblastic murine and human cell lines as the time between the addition of the photosensitizer, aluminum phthalocyanine (AIPc), to the culture medium and exposure to light was increased from 4 h to 18 h. The total intracellular concentration of photosensitizer did not decrease significantly during this 18 h interval. For the murine cell lines, the maximum cytotoxic and mutagenic effects were observed when the time between addition of the photosensitizer and irradiation was between 1 and 4 h. The time course of the variations in efficacy did not vary greatly from one murine cell line to another, even though the cell lines differ markedly in the extent of their cytotoxic and mutagenic response. The time course of the variation was similar for cytotoxicity and mutagenicity, as well as for the induction of DNA fragmentation. The human lymphoblastic cell line, WTK1, showed less variation in survival and mutability with time than did the murine cell lines. With Pc 4 (HOSiPcOSi[CH3]2[CH2]3N[CH3]2) as the photosensitizer, the photocytotoxicity for murine L5178Y (LY)-S1 cells did not change significantly as the time between addition of Pc 4 and irradiation was increased from 2 to 18 h. However, the mutagenicity decreased by a factor of three during this interval. The mutagenicity of PDT with Pc 4 was much less in LY-S1 cells than that with AlPc. The results suggest that the variation in the efficacy observed for AlPc-induced photocytotoxicity is caused by changes in the intracellular distribution and/or the aggregation of the photosensitizer with time after its addition.