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The phenomenon of rural depopulation is seen in many places around the world as young adults move to urban areas where there is greater access to employment, government services and social activities. In Cyprus rural villages exemplify this pattern of demographic decline. Depopulation creates a cycle of loss that influences community identity and feelings of belonging. In this paper I argue that heritage may play a role in building community resilience in socially and economically marginalised rural areas. I focus on the heritage work of a Cypriot regional primary school – how its teachers and pupils created a new common sense of identity for the school, its pupils and the rural villages that the school serves. This case illustrates how even small heritage initiatives may enliven, strengthen and create new social networks – resources necessary to maintain a sense of place, build and sustain community resilience in rural areas.

Arylazopyrazole derivatives based on four core structures (4pzMe, 3pzH, 4pzH, and 4pzH-F2) and functionalized with a dodecanoate group were demonstrated to store thermal energy in their metastable Z isomer liquid phase and release the energy by optically triggered crystallization at -30 °C for the first time. Three heat storage-release schemes were discovered involving different activation methods (optical, thermal, or combined) for generating liquid-state Z isomers capable of storing thermal energy. Visible light irradiation induced the selective crystallization of the liquid phase via Z-to-E isomerization, and the latent heat stored in the liquid Z isomers was preserved for longer than 2 weeks unless optically triggered. Up to 92 kJ/mol of thermal energy was stored in the compounds, demonstrating remarkable thermal stability of Z isomers at high temperatures and liquid-phase stability at temperatures below 0 °C.

There are a subset of short gamma-ray bursts (SGRBs) which exhibit a rebrightening in their high-energy light curves known as extended emission. These bursts have the potential to discern between various models proposed to describe SGRBs as any model needs to account for extended emission. In this paper, we combine fallback accretion into the magnetar propeller model and investigate the morphological changes fallback accretion has on model light curves and fit to the afterglows of 15 SGRBs exhibiting extended emission from the Swift archive. We have parameterised the fallback in terms of existing parameters within the propeller model and solved for the disc mass and angular frequency of the magnetar over time. We then apply a Markov chain Monte Carlo routine to produce fits to the data. We present fits to our extended emission SGRB sample that are morphologically and energetically consistent with the data provided by Swift BAT and XRT telescopes. The parameters derived from these fits are consistent with predictions for magnetar properties and fallback accretion models. Fallback accretion provides a noticeable improvement to the fits of the light curves of SGRBs with extended emission when compared to previous work and could play an important role in explaining features such as variability, flares and long dipole plateaux.

G‐quadruplex nucleic acid structures have long been studied as anticancer targets whilst their potential in antiparasitic therapy has only recently been recognized and barely explored. Herein, we report the synthesis, biophysical characterization, and in vitro screening of a series of stiff‐stilbene G4 binding ligands featuring different electronics, side‐chain chemistries, and molecular geometries. The ligands display selectivity for G4 DNA over duplex DNA and exhibit nanomolar toxicity against Trypasanoma brucei and HeLa cancer cells whilst remaining up to two orders of magnitude less toxic to non‐tumoral mammalian cell line MRC‐5. Our study demonstrates that stiff‐stilbenes show exciting potential as the basis of selective anticancer and antiparasitic therapies. To achieve the most efficient G4 recognition the scaffold must possess the optimal electronics, substitution pattern and correct molecular configuration., Stiff scaffolds: Stiff‐stilbene has very recently emerged as a promising scaffold for the design of selective G‐quadruplex DNA ligands. We demonstrate the importance of substituent chemistry and molecular geometry on the activity of this novel ligand series. Compounds exhibit up to 700‐fold selective toxicity to parasitic organisms and 30‐fold selective toxicity to cervical cancer cells over a non‐tumoral mammalian cell model.

G-quadruplex nucleic acid structures have long been studied as potential anticancer targets while their potential in antiparasitic therapy has only recently been recognized but barely explored. Herein we report the synthesis, biophysical characterization and in vitro screening of a series of stiff-stilbene G4 binding ligands featuring differing electronics, side-chain chemistries and molecular geometries. The ligands display selectivity for G4 DNA over duplex DNA and exhibit nanomolar toxicity against Trypasanoma brucei and HeLa cancer cells whist remaining up to two orders of magnitude less toxic to non-tumoral mammalian cell line MRC5. Our study demonstrates that stiff-stilbenes show exciting potential as the basis of selective anticancer and antiparasitic therapies. In order to achieve the most efficient G4 recognition the scaffold must possess the optimal electronics and substitution pattern and correct molecular geometry.

Arylazopyrazoles show significant potential as next‐generation photoswitches, in particular because of the high thermal stability of their Z‐isomers. Herein we investigate the potential to perform Z−E isomer chemical switching of arylazopyrazoles using acid. We show that acid‐accelerated isomerisation allows for robust Z−E switching using mild acids and opens up the possibility to use light‐acid cycles to near quantitatively and reversibly switch the arylazopyrazoles at room temperature. We attribute the chemical switching to azonium formation, facilitated by mesomeric stabilisation by the pyrazole ring. Coupled with their exceptionally long Z‐isomer half‐lives, we believe our study may open up a wider array of functional opportunities for the arylazopyrazoles.

Understanding the regulation and control of heme/porphyrin biosynthesis is critical for the optimization of the delta-aminolevulinic-acid (ALA)-mediated photodynamic therapy of cancer, in which endogenously produced protoporphyrin IX (PPIX) is the photosensitizer. The human breast cancer cell line MCF-7, the rat mammary adenocarcinoma cell line R3230AC, the mouse mammary tumor cell line EMT-6 and the human mesothelioma cell line H-MESO-1 were used to study ALA-induced PPIX levels and their relationship to delta-aminolevulinic acid dehydratase (ALA-D) activity in vitro. Incubation of these cell lines with 0.5 mM ALA for 3 h resulted in a significant increase in PPIX accumulation, compared with control cells, but there was no significant change in ALA-D activity. Exposure of cells incubated with ALA to 30 mJ/cm2 of fluorescent light, a dose that would cause a 50% reduction in cell proliferation, did not significantly alter the activity of ALA-D. Increasing the activity of porphobilinogen deaminase (PBGD), the enzyme immediately subsequent to ALA-D, by four- to seven-fold via transfection of cells with PBGD complementary DNA did not alter the activity of ALA-D. However, incubation of cells with various concentrations of succinyl acetone, a potent inhibitor of ALA-D, caused a concomitant decline in both PPIX accumulation and ALA-D activity. These data imply that when cells are exposed to exogenous ALA, ALA-D is an important early-control step in heme/porphyrin biosynthesis and that regulation of PPIX synthesis by this dehydratase may impact the effectiveness of ALA-mediated photosensitization.

Recently, we reported that the delta-aminolevulinic acid (delta-ALA)-induced increase in porphobilinogen deaminase (PBGD) activity was closely correlated with an increase in the accumulation of protoporphyrin IX (PPIX), resulting in augmented phototoxicity. In this report, we asked whether increasing the cellular expression of PBGD by use of gene transfection techniques in vitro would further enhance delta-ALA-induced PPIX accumulation and hence, phototoxicity. For these experiments we constructed plasmid vectors containing the PBGD-DNA, using a reverse transcription-polymerase chain reaction-generated cDNA fragment encoded from its published sequence. Subsequently, transfection of the human mammary tumor cell line, MCF-7, and the human mesothelioma cell line, H-MESO-1, with the PBGD-DNA-containing plasmids was shown to produce a 2.5-2.7-fold increase in enzyme activity. Twenty-four hours after completion of the transfection procedure, transfectants were exposed for 3 h to 0.5 mM delta-ALA. Exposure of either wild type or transfectants to delta-ALA led to measurable levels of PPIX. Although this produced a modest but significant increase in intracellular PPIX content in H-MESO-1 cells compared to wild-type cells incubated with delta-ALA alone, the increase above the transfection control did not reach statistical significance. Likewise, a significant increase in PPIX was not observed in transfected MCF-7 cells subsequently exposed to delta-ALA. These data demonstrate that transient transfection of cells with the cDNA of PBGD was successful in elevating enzyme activity in both tumor cell lines, but this did not result in a comparable difference in the levels of PPIX. Such an approach for the study of other enzymes in the heme pathway should provide a model to better define rate-limiting steps in the delta-ALA induction of PPIX, and ultimately, to enhance the effectiveness of photodynamic therapy.

Photodynamic therapy (PDT), a novel treatment for a variety of human malignancies, usually consists of visible light irradiation of lesions following the systemic administration of a photosensitizer. Induction of the endogenous photosensitizer protoporphyrin IX by the systemic or topical administration of delta-aminolevulinic acid (delta-ALA) is being investigated for use in PDT. We have determined that the incubation of two human and two rodent tumor cell lines in culture with delta-ALA over a 24 h period results in an increase in the accumulation of fluorescent porphyrins in all of these cell lines. However, the two human cell lines produce fluorescent porphyrin at different rates from those seen in the rodent cell lines. The uptake of 14C-delta-ALA was concentration dependent, similar for all the cell lines studied and rapidly reached an intra/extracellular equilibrium after delta-ALA was added to the culture medium. The increase in intracellular fluorescent porphyrin was dependent on the level of delta-ALA in the medium and the incubation time and was directly related to the phototoxicity observed upon exposure of cultured monolayers to light. The data demonstrate that equivalent levels of phototoxicity can be attained by exposing cells to 0.04 mM delta-ALA for 24 h or to 0.5 mM delta-ALA for 2 h. These findings may have implications for optimization of PDT treatment regimens that use delta-ALA.

We examined the effectiveness of systemic administration of delta-aminolevulinic acid (delta-ALA) to induce endogenous protoporphyrin as a regimen for use in photodynamic therapy (PDT) of transplanted R3230AC rat mammary adenocarcinomas in vivo. Levels of porphyrins synthesized in various tissues after systemic administration of delta-ALA differed, with their accumulation in tumor tissue being dependent on both the dose and the time after delta-ALA administration. Tumor, liver, and intestine contained greater than 3.0 micrograms porphyrin/g tissue at 3 h after delta-ALA injection, whereas porphyrin levels in rat skin and muscle at that time were an order of magnitude lower. Analysis of tissue by HPLC revealed that the predominant porphyrin synthesized in tumors was protoporphyrin IX, whereas in liver, 18% of the total porphyrin detected was protoporphyrin IX, and in muscle, it was undetectable. Time-dependent studies of the uptake of 14C label from delta-ALA into the various tissues were not predictive of either the total amount of porphyrin or which porphyrin species would be present at 3 h after delta-ALA injection. Additionally, no simple relationship was apparent between the activities of certain selected enzymes involved in heme biosynthesis and the concentrations of porphyrins in the different tissues. High levels of tumor protoporphyrin IX were sustained by administration of two sequential doses of delta-ALA, at 3.0 and 1.5 h prior to irradiation. Using these treatment conditions, we inhibited R3230AC growth to an extent that was comparable to that obtained for Photofrin-induced PDT. High energy phosphate metabolism, measured by nuclear magnetic resonance spectroscopy in vivo, was dramatically impaired after delta-ALA-based PDT, with tumor ATP levels reduced to near zero by 4 h after irradiation. Our results demonstrated that delta-ALA-based PDT may be an alternative to current treatment protocols that use exogenously administered photosensitizers.

Cytochrome c oxidase activity in isolated tumor mitochondria was inhibited by exposure to 570-800 nm of light after addition of cationic dye 1. The alteration in enzyme activity is a consequence of the formation of the highly reactive oxygen species, singlet oxygen, a photochemical product resulting from the irradiation (770 nm of light) of dye 1 in the presence of oxygen. The oxidation of dye 1 by singlet oxygen produces dye 2. Irradiation of mitochondrial suspensions treated with 10(-5) M solutions of dye 2 with 489 +/- 5 nm of light also caused inhibition of cytochrome c oxidase activity, but the addition of scavengers of singlet oxygen, hydrogen peroxide, superoxide, and hydroxyl radical did not prevent the observed inhibition. Similarly, lowering the atmospheric oxygen concentration to 3% had no effect on mitochondrial inhibition. In solution, photoreduction of dye 2 occurred at an increased rate in the presence of 0.01 M tryptophan. ESR studies of dye 2 with 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) (0.1 M) and added tryptophan (0.01 M) suggested the photoproduction of hydroxyl radicals as a possible mechanism for its photodynamic action. We interpret the data as indicating the possibility that after the photooxidative transformation of dye 1 to dye 2, a second mechanism becomes operative for further photodynamic inhibition of enzyme activity. The in vivo or in vitro conversion of dye 1 to dye 2 would allow dye 1 to function as a novel, dual-action photosensitizer.

A set of structurally distinct o-substituted tetraphenylporphyrins, the picket fence porphyrins, were evaluated for their ability to photosensitize tumor mitochondria in vitro, in vivo-in vitro, and tumor implants in situ. Differential photosensitized inactivation efficiencies toward mitochondrial enzymes in vitro are reported for the 12 compounds studied as a function of side chain length and isomer structure. Fluorescence studies in aqueous solution coupled with mitochondrial uptake studies indicate that the observed range of inactivation efficiencies are due to different inherent solubilization properties for the picket fence porphyrins. Studies with the most soluble compound, 3,1-meso-tetrakis(o-propionamidophenyl)porphyrin, using an in vivo-in vitro protocol indicate that a more effective photosensitization can be obtained by using an interval of 4 h between photosensitizer administration and irradiation as compared to 24 h for Photofrin II. Irradiation of tumors in vivo 4 h following administration of 3,1-meso-tetrakis(o-propionamidophenyl)porphyrin, resulted in a mean tumor doubling time more than eight times longer than that observed for untreated tumors. 31P NMR spectroscopy in situ indicated that photodynamic therapy using 3,1-meso-tetrakis(o-propionamidophenyl)porphyrin induced a rapid and significant reduction in high energy phosphate metabolites.

Clinical photodynamic therapy consists of the systemic administration of a derivative of hematoporphyrin (Photofrin II) followed by exposure of malignant lesions to continuous visible laser irradiation. We investigated the effects of various modifications of laser light delivery on the efficacy of photodynamic therapy in controlling R3230AC mammary tumor growth. We observed a significant delay in growth (from initial to 2 times initial volume) of tumors exposed to periodic irradiation (100 mW/cm2/0.25 h, 1-h dark interval, 100 mW/cm2/0.25 h; total fluence, 180 J/cm2), compared to untreated controls or to tumors receiving continuous irradiation at the same total fluence. Other periodic light treatment regimens, consisting of 3-, 6-, or 24-hr dark intervals, delayed tumor growth but not significantly more than continuous irradiation at the same fluence. A biochemical basis was sought by comparing continuous versus periodic irradiation for effects on mitochondrial or cytosolic enzymes in vivo. Although both cytochrome c oxidase and pyruvate kinase activities were reduced dramatically during the first 24 h by continuous or periodic irradiation schemes, recovery of enzyme activity to initial levels took longer after the periodic irradiation protocol (168 h), compared to the continuous irradiation regimen (72 h). We observed a significantly greater delay in the growth of tumors exposed to 50 mW/cm2/2 h continuously, compared to controls or to tumors exposed to the same total fluence but with light delivered at 100 or 200 mW/cm2. The data presented here indicate that the efficacy of photodynamic therapy could be significantly increased by modifications in the delivery of photoradiation.

Flares in the X-ray afterglow of gamma-ray bursts (GRBs) share more characteristics with the prompt emission than the afterglow, such as pulse profile and contained fluence. As a result, they are believed to originate from late-time activity of the central engine and can be used to constrain the overall energy budget. In this paper, we collect a sample of $19$ long GRBs observed by \emph{Swift}-XRT that contain giant flares in their X-ray afterglows. We fit this sample with a version of the magnetar propeller model, modified to include fallback accretion. This model has already successfully reproduced extended emission in short GRBs. Our best fits provide a reasonable morphological match to the light curves. However, $16$ out of $19$ of the fits require efficiencies for the propeller mechanism that approach $100\%$. The high efficiency parameters are a direct result of the high energy contained in the flares and the extreme duration of the dipole component, which forces either slow spin periods or low magnetic fields. We find that even with the inclusion of significant fallback accretion, in all but a few cases it is energetically challenging to produce prompt emission, afterglow and giant flares within the constraints of the rotational energy budget of a magnetar., Comment: 8 pages, 2 figures

The value of estrogen receptor (ER) analysis in primary breast cancer samples as a potential prognostic factor was examined in three clinical situations: time to recurrence in patients with no therapy after mastectomy, failure of patients receiving adjuvant therapy, and response of advanced disease patients to cytotoxic chemotherapy. Other prognostic factors analyzed were menopausal and nodal status. In none of these clinical settings were we able to demonstrate the usefulness of ER status as a prognosticator of the disease course or its response to therapy.

The role of insulin as a regulator of estrogen receptors (ER's) was investigated in 7,12-dimethylbenz(a)anthracene-induced mammary tumors in diabetic and insulin-treated rats. Induction of diabetes with streptozotocin produced regression of lesions classified as insulin dependent; lesions that continued to grow in diabetic rats were classified as insulin independent. Compared to tumors in intact hosts [ER, 39 +/- 4 (S.E.) fmol/mg cytosol protein], regressing insulin-dependent lesions had ER levels of 8.5 +/- 0.7 fmol, and insulin-independent tumors had ER levels of 24 +/- 3 fmol. Treatment of diabetic rats with insulin 8 IU/day, caused insulin-dependent regressing tumors to resume growth; these lesions had ER levels of 53 +/- 10 fmol. Insulin-independent lesions in diabetic rats demonstrated two patterns after treatment with insulin; continued growth resulted in tumors having ER levels of 28 +/- 11 fmol, whereas insulin-induced tumor regression resulted in tumors that demonstrated ER levels of 40 +/- 6 fmol/mg cytosol protein, a value equal to the level of ER in tumors growing in intact rats. Scatchard analysis of the saturation-binding data gave linear representations, and the estimated Kd for the ER was comparable for all groups, ranging from 0.44 to 1.90 x 10(-9) M. Several additional tumors were classified as demonstrating static growth. When this behavior represented a response due to insulin treatment, ER levels were elevated. Static tumors remaining static after insulin treatment demonstrated low ER levels. We conclude that (a) cessation of tumor growth after induction of diabetes resulted in reduction of ER levels, (b) treatment with insulin that resulted in an altered tumor growth was accompanied by elevated ER levels, and (c) insulin may play a role in regulation of ER independent of tumor growth.

The photosensitizing activity of hematoporphyrin derivative (HPD) was investigated by studying selected enzymes localized to mitochondria and cytosol of R3230AC mammary adenocarcinomas. Experiments in vitro demonstrated that mitochondrial succinate dehydrogenase was inhibited in a drug dose- and light exposure time-related manner; at 7.0 micrograms of HPD per ml or higher, enzyme activity was inhibited greater than 50% after 15 min of photoradiation. The three cytosol enzymes studied under the same conditions in vitro demonstrated different photosensitivities. Pyruvate kinase activity was significantly inhibited in a dose- and time-related fashion, whereas lactate dehydrogenase was inhibited to a lesser extent, and glucose phosphate isomerase activity was inhibited only at the highest dose (70 micrograms of HPD per ml) used. The time-course of these responses was examined with an in vivo-in vitro protocol, consisting of photoradiation of mitochondria and cytosol prepared from tumors obtained at various times (up to 1 week) after a single injection of HPD (80 mg/kg). Pyruvate kinase activity was markedly inhibited at early times returning to initial levels by 48 hr; neither lactate dehydrogenase nor glucose phosphate isomerase was inhibited by this treatment. Mitochondrial succinate dehydrogenase and cytochrome c oxidase activities displayed significant photoradiation-induced inhibitions, with greatest inhibition occurring between 24 and 96 hr after injection of HPD; at 1 week, succinate dehydrogenase activity had returned to its initial level, but cytochrome c oxidase activity remained significantly inhibited. These data suggest that HPD-induced photosensitization of mitochondria may be an important site of action contributing to tumor cell cytotoxicity and regression as a result of photoradiation therapy.

Primary breast cancers from 54 patients undergoing postsurgical adjuvant chemotherapy were analyzed for several enzymes and estrogen receptors to determine if biochemical parameters might offer a prognostic index for determining outcome of therapy. Recurrence of disease during therapy or within 12 months after cessation of therapy was the criterion for classifying the patient as a treatment failure. Recurrence rates were similar in ER-positive vs. ER-negative patients, indicating that estrogen receptor status offered no predictive value in this series. However, patients who failed therapy had tumors that displayed significantly lower activities of lactate dehydrogenase, glucosephosphate isomerase, and glucose 6-phosphate dehydrogenase vs. patients who showed no evidence of recurrence for this observation period. When either premenopausal or ER-negative patients were compared, a similar observation was made; low enzyme activities occurred in patients failing to benefit from adjuvant chemotherapy. Based on this series of patients, it is suggested that a selected enzyme activity profile may offer a predictive index for recurrence in the adjuvant chemotherapy setting.

The possible relationship between estrogen receptors and response of breast cancer patients to chemotherapy was examined in two situations: patients with advanced disease and patients receiving post-surgical adjuvant treatment. In 73 patients with disseminated disease, no relationship between ER status and response to cytotoxic chemotherapy was observed. At this time, 44 of these cases have undergone extramural review; in these 44 cases, we observed no significant relationship between ER status and response to chemotherapy. In a series of 52 patients receiving adjuvant therapy, 27 patients were classified as failures due to recurrence. ER status did not offer prognostic value for failure of patients treated with postsurgical adjuvant therapy. We conclude that the prognostic value of ER data in nonhormonal therapy settings remains to be proven

The response to Photofrin II-induced photosensitization on the activities of mitochondrial monoamine oxidase (MAO) and adenylate kinase (AK) were studied in order to gain further insight into site specific effects. Utilizing intact mitochondria in vitro, both MAO, located on the cytoplasmic side of the outer mitochondrial membrane, and AK, located in the intermembrane space, were inhibited by exposure to Photofrin II plus light; inhibition was drug-dose and light-dose dependent. However, MAO activity was inhibited to a greater extent than AK; at 35 micrograms/ml of Photofrin II and 160 J/cm2, MAO activity was decreased by 80% whereas AK activity was inhibited by 30%. Higher doses of Photofrin II had no further effect on AK activity. Studies of photosensitization of AK in different mitochondrial preparations demonstrated that inhibition of activity was evident only when mitochondrial membranes containing sequestered porphyrins were present in the reaction mixture. Using an in vivo-in vitro protocol and sampling at 2 to 72 h after administration of 25 mg/kg of Photofrin II, photosensitization of MAO (30% inhibition) was seen at 2 h after drug treatment but inhibition of activity was not observed at later times. AK activity was unchanged over the entire time course. Compared to cytochrome c oxidase, located in the inner mitochondrial membrane and which displayed a sustained inhibition of activity, we suggest that inhibition of MAO or AK activities probably does not contribute to the tumor cytotoxicity under the usual conditions used for photodynamic therapy.

Hematoporphyrin derivative (HPD) plus photoradiation caused the inactivation of DNA polymerases from calf thymus and R3230AC rat mammary tumor. Photosensitization of purified DNA polymerase-alpha as well as two forms of DNA polymerase-delta (I and II) from calf thymus were evaluated. Although all polymerase enzyme forms were inactivated at 70 micrograms HPD/ml, DNA polymerase-delta II was the most sensitive, displaying a 90% inactivation under conditions that did not cause significant inactivation of the other polymerase forms. Unlike DNA polymerase-alpha, the delta-forms have an associated 3'- to 5'-exonuclease activity. The exonuclease associated with DNA polymerase-delta II was uniquely sensitive to a low level of HPD and light exposure. DNA polymerase-delta II can be distinguished from other polymerase forms in cell extracts by its relative insensitivity to the polymerase inhibitor N2-(p-n-butylphenyl)deoxyadenosine 5'-triphosphate. In cytosols prepared from calf thymus and R3230AC rat mammary tumors, DNA polymerase-delta II was preferentially inhibited by HPD plus light. Furthermore, in experiments in which tumor-bearing rats were administered HPD prior to preparation of tumor cytosols, DNA polymerase-delta II was specifically inactivated by exposure to light. These results are discussed in view of their possible role in cancer therapy, and the potential use of HPD as a specific inhibitory agent of DNA polymerase-delta II is suggested.

The effects of photodynamic therapy using 632 nm photoradiation emitted from an ion pumped dye laser system on the phosphate metabolite levels of rat mammary tumors were monitored by 31P-NMR spectroscopy. A dramatic decline to almost undetectable levels, in the ratio of whole tumor beta-ATP (NTP) to Pi was observed after systemic administration of 5 mg/kg Photofrin II 24 h prior to exposure of R3230AC rat mammary tumor to laser irradiation at 180 and 360 J/cm2 total fluence. This decline in ATP was accompanied by a concomitant increase in the levels of Pi relative to the total observable phosphate signals. Whole tumor pH was calculated from the chemical shift in inorganic phosphate using the water proton signal as reference. Under the same treatment conditions used to monitor the phosphate metabolites following Photodynamic Therapy, the pH of the tumor as a whole decreased approximately 0.35 units at the time when the beta-ATP to Pi ratios were lowest. This maximal decrease in whole tumor ATP levels and pH, which occurred at 4-6 h post irradiation, was followed by a gradual return to pre-treatment levels over a 24 h period. These results demonstrate that Photodynamic Therapy employing porphyrin photosensitization and monochromatic laser irradiation is effective in reducing both tumor high energy phosphate levels and pH. Depending on sensitizer dose and light fluence, metabolic inhibition, represented by depleted nucleoside triphosphates and elevated Pi, may be reversible.

Photodynamic therapy, which consists of treatment with hematoporphyrin derivative (HPD) followed by photoradiation with visible light, is a promising approach to treatment of various cancers. To gain further understanding of the mechanisms whereby this therapy produces cytotoxicity, we undertook a study of the mitochondrial proton-translocating adenosine triphosphatase, an enzyme performing the critical role of coupling electrochemical proton gradients to the formation of adenosine triphosphate. Exposure of submitochondrial particles to HPD and photoradiation in vitro caused a marked inhibition of proton transport; inhibition displayed both drug-dose and light-dose relationships. Inhibition of proton transport was correlated with inhibition of adenosine triphosphate hydrolysis, both demonstrating an inhibition rate of 2% min-1. Investigation of effects of HPD plus light on membrane integrity, measured by [3H]sucrose leakage from submitochondrial particles and by K+ leakage from asolectin phospholipid vesicles, indicated that discrete membrane alterations were not the likely cause of initial loss of pH gradient formation. Likewise, photosensitization by HPD to inhibit adenosine triphosphate hydrolysis and proton transport was not coincident with cross-linking of the major subunits of the enzyme. We conclude that mitochondrial function is severely impaired by photodynamic therapy and offers a potentially important target site leading to cytotoxicity.

The effects of hematoporphyrin derivative-induced photosensitization on the levels of adenosine triphosphate (ATP) in R3230AC mammary adenocarcinomas were studied. Enzymatically dissociated tumor cells were exposed to various doses of hematoporphyrin derivative (HPD) in vitro plus photoradiation. A drug and light dose-dependent decrease in cellular ATP levels was observed; ATP levels were reduced by 60% after treatment with 7.0 micrograms HPD per ml plus 0.72 J total energy density per cm2. Cell viability, assessed by exclusion of trypan blue, displayed an apparently coordinate behavior to ATP levels. The effects of hematoporphyrin derivative plus photoradiation were examined in the presence of oligomycin, an inhibitor of mitochondrial oxidative phosphorylation, or iodoacetate, an inhibitor of glycolysis, experiments designed to elucidate the site of action leading to reduced ATP levels. The results indicated that HPD-induced photosensitization had little additive effects to oligomycin-sensitive ATP production, whereas significant further reduction in ATP levels was obtained by HPD-induced photosensitization in the presence of iodoacetate. Taken together, along with earlier studies of selected mitochondrial enzymes, we conclude that HPD plus photoradiation inhibits mitochondrial function leading to reduction in cellular ATP levels and loss of viability.

The estrogen-binding capacity of mammary tumors induced by 7,12-dimethylbenz(a)anthracene was measured in lesions from animals after the ovariectomy, deprival of insulin (diabetes), or treatment with lergotrile mesylate to inhibit prolactin secretion. The average estrogen-binding capacity was 30 fmoles/mg cytosol protein in growing or static carcinomas from intact (control) animals. A significant reduction in estrogen-binding capacity was observed in regressing but not static mammary tumors from ovariectomized animals. In regressing and static tumors from diabetic rats, estrogen-binding capacity was significantly lower than in lesions from intact animals; this effect was not seen in growing tumors from diabetic rats. Tumors that were growing or static in lergotrile-treated animals showed reduced capacity to bind labeled estradiol. The effects of duration of hormone treatment or time of tissue storage on estrogen-binding capacity were examined and did not appear to be correlated with the decreased binding in tumors from treated animals. The results suggest that hormones capable of producing altered neoplastic growth may influence the level of estrogen receptors.

A logistic regression model, utilizing the activities of certain selected glycolytic enzymes and ER status measured on primary or recurrent lesions, has been applied to predict for response to combination chemotherapy regimens administered to women with advanced breast cancer. The clinical outcome of response or no response was evaluated retrospectively using criteria employed by cooperative group protocols. In 93 cases, 58/61 patients classified as nonresponders and 22/32 patients demonstrating objective responses would have been correctly designated, based on the 50% estimated probability as the level for separation of responders from nonresponders. The overall predictive accuracy of this model was 86%, with apparently greater accuracy for prediction of lack of response. Addition of estrogen receptor status to the model imparted no gain in accuracy of prediction. Application of this model to a prospective study is warranted.