Heyke Diddens

Photodynamic targeting of human retinoblastoma cells using covalent low-density lipoprotein conjugates.

Combination of photosensitizers with carrier molecules has been shown to enhance the efficiency of photodynamic therapy (PDT). Owing to an increased expression of their receptors on some malignant and proliferating cells, low-density lipoproteins (LDLs) are potential endogenous carriers. A photosensitizer, chlorin e6 (Ce6), was covalently bound to LDL via carbodiimide activation. The Ce6-LDL conjugate was evaluated on a fibroblast cell line with defined LDL receptor expression and a retinoblastoma cell line (Y79). Uptake of free Ce6 and Ce6 either covalently bound to or complexed with LDL was measured by spectrofluorimetry. Phototoxicity after irradiation at 660 nm was determined by a mitochondrial activity assay (MTT). Covalent binding to LDL significantly increased the uptake of Ce6 for both cell lines by a factor of 4-5. A Ce6: LDL binding ratio of 50:1 was optimal. A receptor-mediated uptake was demonstrated by saturability and competitive inhibition by free LDL. Binding also occurred at 2 degrees C and was attributed to non-specific associations. Irradiation with 10 J cm-2 of 660 nm light after treatment of cells with Ce6-LDL conjugate reduced the MTT activity by 80%, while free or mixed Ce6 induced a maximum of 10% reduction in the MTT activity following identical treatment conditions. These data suggest that targeting of LDL receptor-bearing cells using covalently bound carriers, such as LDL, might increase the efficiency and selectivity of PDT. Intraocular tumours such as retinoblastomas could be appropriate targets for such an approach owing to the ease of access of light sources and the need for non-invasive approaches in sensitive ocular sites.

Laparoscopic Fluorescence Detection of Ovarian Carcinoma Metastases Using 5-Aminolevulinic Acid-Induced Protoporphyrin IX

The aim of the current clinical study was to evaluate the in vivo fluorescence detection of ovarian carcinoma metastases in a second‐look laparoscopic procedure after intraperitoneally applied 5‐aminolevulinic acid (ALA).

Mdr1/P-glycoprotein, topoisomerase, and glutathione-S-transferase pi gene expression in primary and relapsed state adult and childhood leukaemias.

In a variety of adult and childhood leukaemia cell samples collected at different states of the disease, we analysed in a series of sequentially performed slot-blot or Northern-blot hybridisation experiments the expression of genes possibly involved in multiple drug resistance (MDR) (mdr1/P-glycoprotein, DNA topoisomerase II, glutathione-S-transferase pi), and the expression of the DNA topoisomerase I and histone 3.1 genes. Occasionally, P-glycoprotein gene expression was additionally examined by indirect immunocytofluorescence using the monoclonal antibody C219. No significant difference in mdr1/P-glycoprotein mRNA levels between primary and relapsed state acute lymphocytic leukaemias (ALL) was seen on average. Second or third relapses, however, showed a distinct tendency to an elevated expression of this multidrug transporter gene (up to 10-fold) in part well beyond the value seen in the moderately cross-resistant T-lymphoblastoid CCRF-CEM subline CCRF VCR 100. Increased mdr1/P-glycoprotein mRNA levels were also found in relapsed state acute myelogenous leukaemias (AML), and in chronic lymphocytic leukaemias (CLL) treated with chlorambucil and/or prednisone for several years. Topoisomerase I and topoisomerase II mRNA levels were found to be very variable. Whereas in all but one case of CLL topoisomerase II mRNA was not detected by slot-blot hybridizations, strong topoisomerase I and topoisomerase II gene expression levels, frequently exceeding the levels monitored in the CCRF-CEM cell line, were seen in many cell samples of acute leukaemia. If topoisomerase II mRNA was undetectable, expression of topoisomerase I was clearly visible throughout. These observations might be valuable considering the possible treatment with specific topoisomerase I or topoisomerase II inhibitors. Significant positive correlations were found (i) for topoisomerase I and histone 3.1 gene expression levels in general (P less than 0.001), and (ii) in the CLL samples additionally for the expression levels of the mdr1 gene, and the histone 3.1, topoisomerase I, and glutathione-S-transferase pi genes, respectively.

Chemical instability of 5-aminolevulinic acid used in the fluorescence diagnosis of bladder tumours

Aqueous solutions of 5-aminolevulinic acid (ALA) prepared for intravesical instillation in the framework of a clinical study on the fluorescence diagnosis of urothelial bladder cancer were found to be unstable. This chemical instability of ALA was studied in aqueous solution of 37 degrees C as a function of concentration, pH and reaction time. Our investigations showed that the reaction of ALA is an irreversible process, which yields at least two reaction products in the pH range studied (pH lower than 8): the 2,5-(beta-carboxyethyl)dihydropyrazine and the 2,5-(beta-carboxyethyl)pyrazine. As a result of these studies, the conditions for the preparation of ALA solutions to be used for intravesical instillation were optimized: solution of ALA in phosphate-buffered saline at a concentration of 0.18 M (3 g of ALA in 100 ml) neutralized to pH 5, were prepared and stored on ice until use. Solutions prepared under these conditions were stable and were used for fluorescence diagnosis of bladder tumours with successful results. The effect of the pH and the composition of the urine on the extent of the reaction of ALA and on the nature of its reaction products formed during instillation was investigated by comparing the urine of patients before and immediately after instillation of ALA.

Expression of a P-glycoprotein gene is inducible in a multidrug resistant human leukemia cell line

A human T lymphoblastoid CCRF-CEM cell line exhibiting cross resistance to a variety of drugs was selected with increasing doses of actinomycin D. A subline, designated CCRF ACTD400+, was permanently cultured in the presence of 400 ng/ml Actinomycin D for several months. Using a fragment of the human mdr1 cDNA we found high expression of a 5 kb mRNA species which was not detectable in the sensitive parental CCRF-CEM cell line. The extent of the mdr-mRNA expression in resistant cells, however, depended on the presence or absence of actinomycin D in the culture medium: when the inhibitor was omitted, the expression decreased to about 60% after one month. In reverse, the steady state level of the P-glycoprotein mRNA increased about 2.5-fold within 72 h after the original dose of the drug was added again. In further experiments we recorded the actinomycin D or adriamycin dose response curves of the variously treated sublines by evaluation of [3H]uridine or [3H]thymidine incorporation, respectively, into acid insoluble material. Consistently, the drug sensitivity of the respective macromolecular synthesis was found to decrease with increasing mdr-mRNA levels.

Lipid metabolism in retinal pigment epithelium (RPE): a possible role of LDL receptors

SummaryPurpose: The retinal pigment epithelium (RPE) regulates the lipid metabolism of the photoreceptors by catalysis of membrane outer segments and via choriocapillary perfusion is also exposed to the regulation of blood lipid levels. Since the uptake and metabolism of cholesterol are mediated by specific low-density lipoprotein (LDL) receptors, expression and regulation of this receptortype were studied in RPE cultures. Methods: In vitro experiments were carried out in transformed (SV40) human RPE cells. Human fibroblasts were used as a comparative cell line with known receptor expression. LDL was coupled to a fluorescent marker (DiI); receptor binding was quantified by flow cytometry. Expression and saturation characteristics were determined. LDL metabolism was examined by variation of the temperature (4 and 37 °C). LDL and DiI-LDL showed competition at the receptor. Results: RPE cells demonstrated a higher uptake of DiI-LDL than fibroblasts. Expression could be further stimulated by culture conditions. Uptake kinetics were saturable with complete saturation at 50 μg/ml DiI-LDL. LDL uptake was shown to be temperature-dependent, indicating an energy-dependent pathway. Conclusions: RPE cells exhibit significant expression of receptors for native LDL, possibly mediating the lipid metabolism of the RPE-photoreceptor complex, as well as the uptake of blood lipids. Lack of regulation of the receptor for LDL may lead to intracellular accumulation of lipids, which may play a role in the pathogenesis of AMD.ZusammenfassungHintergrund: Das retinale Pigmentepithel (RPE) reguliert den Lipidmetabolismus der Photorezeptoren durch Katalyse von Außensegmenten und ist über die Aderhautperfusion der Regulation der Blutlipide ausgesetzt. Da die Aufnahme von Cholesterin und Phospholipiden über spezifische Low-Density-Lipoprotein-(LDL)-Rezeptoren erfolgt, wurden Expression und Regulation diese Rezeptortypes im RPE untersucht. Material und Methode: In vitro-Experimente wurden an transformierten (SV40-) RPE-Kulturen mit humanen Fibroblasten als Vergleichskultur durchgeführt. Die Rezeptorkinetik wurde mit fluoreszenzmarkiertem LDL mittels Flowzytometrie untersucht. Ergebnisse: RPE-Zellen zeigten eine höhere LDL-Rezeptor-Aktivität als Fibroblasten. Die Bindungskinetik war saturabel und temperaturabhängig, vereinbar mit einem rezeptorvermittelten Aufnahmesystem. Die LDL-Aufnahme war induzierbar und durch Zugabe von unmarkiertem LDL kompetitiv hemmbar. Schlußfolgerung: RPE-Zellen besitzen eine hohe Dichte von Rezeptoren für natives LDL, über die möglicherweise der Lipidmetabolismus sowohl des RPE-Photorezeptorkomplexes als auch der Blutfette gesteuert wird. Ein Verlust der Regulierbarkeit des Rezeptors für LDL kann zu einer intrazellulären Akkumulation von Lipidstoffwechselprodukten führen, die eine Rolle in der Pathogenese der AMD spielen könnte.

Resistance to methotrexate and multidrug resistance in childhood malignancies

Resistance to drugs, either primary or acquired, is a main problem in cancer chemotherapy. The paper summarizes our results in regard to resistance to methotrexate and multiple drug resistance in human cell lines of pediatric malignancies and in children with resistant cancer. In cell lines as well as in children we could demonstrate amplification of the gene coding for dihydrofolate reductase as a cause for resistance to MTX. Procedures to overcome drug resistance such as treatment with high dose MTX and leucovorin rescue are discussed. The increased expression of the mdrl gene coding for the P-glycoprotein is related to multidrug resistance. This could be shown in cell lines and in children. The expression decreased when the drug, used for induction of resistance, was omitted for a few weeks from the cell culture medium. Readdition of the drug caused a rapid increase of expression. For the first time data in children are presented which demonstrate the amplification of the gene coding for dihydrofolate reductase or increased expression of the mdrl gene as cause of drug resistance. The clinical implications of these findings are discussed.

Photodynamic laser therapy for rheumatoid arthritis Cell culture studies and animal experiments

Abstract The introduction of arthroscopic techniques has improved the surgical therapy of rheumatoid arthritis. The additional application of the holmium:yttrium aluminum garnet (Ho:YAG) laser likewise holds great promise by providing complete hemorrhagic control. Unfortunately, a minimally invasive solution for use in smaller joints has not yet emerged. The present study describes the possible treatment of these joints by means of photodynamic laser therapy. Cell culture studies with human synovial fibroblasts obtained from patients with rheumatoid arthritis have demonstrated a cytotoxic effect after administration of Photosan-3 as a photosensitizer and subsequent laser irradiation at 630 nm. For the in vivo studies, IgG-induced arthritis in rabbits, which is histologically consistent with the proliferative phase of rheumatoid arthritis, was used as the animal model. The histologic picture following photodynamic laser therapy with Photosan-3 revealed complete synovial destruction which also extended to the border of the subjacent joint capsule. In contrast, bradytrophic structures, e.g. cartilage, menisci, and ligaments, remained unchanged at both the macroscopic and microscopic levels. Therefore, photodynamic laser therapy can be considered a new method in the surgical treatment of inflammatory disease of the synovial membrane. It has the advantage of being minimally invasive, while offering a high degree of efficacy and selectivity.

Spectral characterization of the benzoporphyrin derivative monoacid ring— A photoproduct formed in fetal calf solutions during irradiation with 694 nm continuous-wave radiation

Benzoporphyrin derivative monoacid ring A (BPD-MA) is a second-generation photosensitizer for photodynamic therapy (PDT) that has shown good results in phase I clinical trials. Similar to other porphyrin derivatives, BPD-MA readily photobleaches during in-vivo PDT treatment. This study investigated the photodegradation of BPD-MA in fetal calf serum (FCS) solutions in vitro. Absorption and fluorescence spectra from dilute solutions of BPD-MA in 10% FCS were recorded before and immediately after irradiation with light at 694 nm. After irradiation, the appearance of a new fluorescence emission band at 650 nm and changes in the fluorescence excitation spectra indicate the formation of a photoproduct. Photoproduct formation was observed only when BPD-MA was bound to FCS and in oxygenated solutions. The spectroscopy of the photoproduct is consistent with the reaction of an oxygen species with the ring B vinyl group, forming a hydroxyaldehyde photoproduct. Monitoring the increase in photoproduct fluorescence during treatment may provide an in-vivo dosimeter to measure PDT efficacy.

Chemical instability of 5-aminolevulinic acid (ALA) in aqueous solution

Five-aminolevulinic acid (ALA) is being used to induce formation and accumulation of endogenous protoporphyrin IX both in preclinical and clinical studies on photodynamic therapy and diagnosis. At the high concentrations needed for clinical application, ALA is not stable in aqueous solutions in the neutral to basic pH range. This could be of critical importance for the clinical use of ALA. The chemical instability of ALA was studied by using UV-Vis absorption spectroscopy. Our results show that ALA undergoes a chemical reaction, which is a nonreversible condensation process yielding two different products -- a hydropyrazine and a pyrazine, the latter formed by oxidation of the hydropyrazine. Preliminary kinetic studies of this reaction showed a complex pH-, concentration and temperature-dependency of the reaction rate. On the basis of these studies we optimized conditions for the preparation of ALA solutions to be used in clinical trials, leading to the expected accumulation of protoporphyrin IX in tumor cells.