Nucleolar asynchrony observed in HL-60 leukemic granulocytic precursors resistant to 5-aminolaevulinic acid-based photodynamic treatment

To provide more information on the 5-aminolaevulinic acid (ALA)-induced photodynamic effect on nucleoli, morphologically expressed nucleolar asynchrony (the presence of 'active' large nucleoli with an uniform distribution of RNA and 'resting' ring-shaped nucleoli in one and the same nucleus) was studied in cultured HL-60 leukemic granulocytic precursors using a simple cytochemical procedure for the demonstration of RNA. Nucleolar asynchrony was mainly expressed in cells which were apparently resistant to ALA-based photodynamic treatment (PDT) since most of them (about 75%) exhibited this phenomenon.     

5-aminolevulinic acid-based photodynamic therapy in leukemia cell HL60

A study to explore the optimal experimental parameters and the photosensitization of 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) in promyelocytic leukemia cell HL60 has been conducted, in which HL60 cells and their control groups, peripheral blood mononuclear cell (PBMC), first are incubated with different concentrations of ALA in dark for different periods of time and then followed by irradiating with different wavebands for different fluences. Fluorescence microscope and spectrofluorometer have been used to detect the fluorescence of protoporphyrin IX (PpIX) endogenously produced by ALA. The response of the cells to ALA-PDT was evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2-5-diphenyl-2H-tetrazolium bromide (MTT) assay (interval between irradiation and the MTT assay is 24 h) and by flow cytometry (the length of time between irradiation and the flow assay is 30 min). MTT results will reflect the relative number of metabolically active mitochondria in the population. Propidium iodide uptake in flow cytometry will test for membrane damage. The results of parameter experiments were obtained: 1 x 10(5)/mL HL60 cell was first incubated with 1 mmol/L ALA in dark for 4 h and the maximum fluorescence of PpIX level appeared; then irradiated with 410 nm (4 mW/cm2) for 14.4 J/cm2 and maximum photodamage to membrane and mitochondrial function of HL60 cell resulted. With the normal granulocytes, such response was not detected. Therefore a hypothetical idea can be brought forward that ALA-based PDT can be used for inactivation of leukemia cell HL60 and these optimal parameters may be useful for clinical application.     

Early apoptotic features of K562 cell death induced by 5-aminolaevulinic acid-based photodynamic therapy

5-Aminolaevulinic acid-based photodynamic therapy (ALA-PDT) is used to eliminate cancerous cells through photoactivation of endogenously formed protoporphyrin IX (PPIX) following the administration of PPIX precursor, 5-aminolaevulinic acid (ALA). We report on the kinetics of PPIX accumulation and the mechanism of cytotoxic effects of ALA-PDT studied in the chronic myelogenous leukaemia derived cell line K562. The PPIX distribution and, consequently, cytotoxic effects were found to be heterogenous. A subpopulation of K562 cells accumulating PPIX to a lesser extent exhibits only transient cell cycle arrest. A fraction of cells, probably those with higher PPIX accumulation, are irreversibly damaged by ALA-PDT. We detected several signs of an early apoptosis: lowering of Bcl-xL expression, decrease of the mitochondrial and plasma membrane potential, the cytochrome c release into the cytoplasm, and the unmasking of the mitochondrial antigen 7A6. However, late apoptotic events were lacking: neither caspase-3 activation nor DNA fragmentation occurred. Instead, rapidly progressing cell necrosis resulting from plasma membrane damage was observed. We suggest that the high level of the antiapoptotic heat-shock proteins HSP70 and HSP27 found by us in the K562 cells is responsible for the inhibition of the apoptotic process upstream of caspases activation.     

Protein changes in HL60 leukemia cells associated with 5-aminolevulinic acid-based photodynamic therapy. Early effects on endoplasmic reticulum chaperones

Using two-dimensional electrophoresis we investigated the effect of 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT; induction with 1 mM ALA for 4 h followed by blue light dose of 18 J/cm2) on the protein expression in HL60 leukemia cells. ALA-PDT resulted in extensive qualitative and quantitative changes in the protein pattern of HL60 cell lysates. Of more than 1350 protein spots recognized on the protein maps of ALA-induced cells, seven proteins were enhanced and 17 suppressed following irradiation. Three of these, calreticulin precursor, p58 microsomal protein (ERp57) and protein disulfide isomerase (p55) have been identified by matrix-assisted laser desorption and ionization-mass spectrometry and the pI/molecular weight parameters of the affected proteins were estimated by computer analysis. The findings suggest participation of endoplasmic reticulum Ca(2+)-binding chaperones and/or Ca2+ signaling in ALA-PDT mediated cytotoxicity.     

Effects of static magnetic field on human leukemic cell line HL-60

A number of structures with magnetic moments exists in living organisms that may be oriented by magnetic field. While most experimental efforts belong to the area of effects induced by weak and extremely low-frequency electromagnetic fields, we attempt to give an attention to the biological effects of strong static magnetic fields. The influence of static magnetic field (SMF) on metabolic activity of cells was examined. The metabolic activity retardation is observed in human leukemic cell line HL-60 exposed to 1-T SMF for 72 h. The retardation effect was observed as well as in the presence of the mixture of the antineoplastic drugs 5 fluorouracil, cisplatin, doxorubicin and vincristine.     

Mitochondrial and endoplasmic reticulum stress-induced apoptotic pathways are activated by 5-aminolevulinic acid-based photodynamic therapy in HL60 leukemia cells

We studied the mechanism of the cytotoxic effects of 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT; induction with 1 mM ALA for 4 h followed by a blue light dose of 18 J/cm(2)) on the human promyelocytic leukemia cell line HL60 using biochemical and electron microscopy methods. The disruption of mitochondrial membrane potential, deltapsi(m), was paralleled by a decrease in ATP level, unmasking of the mitochondrial antigen 7A6, release of cytochrome c into the cytoplasm, activation of caspases 9 and 3 and cleavage of poly(ADP-ribose) polymerase (PARP). This was followed by DNA fragmentation. These data suggest that ALA-PDT activates the mitochondrial apoptotic pathway. The level of endoplasmic reticulum Ca(2+)-binding chaperones ERp57 and ERp72 and of anti-apoptotic proteins Bcl-2 and Bcl-x(L) was decreased whereas that of Ca(2+)-binding protein calmodulin and the stress protein HSP60 was elevated following ALA-PDT. Inhibition of the initiator caspase 9, execution caspase 3 and Ca(2+)-dependent protease m-calpain, did not prevent DNA fragmentation. We conclude that, in our in vitro model, ALA-based photodynamic treatment initiates several signaling processes in HL60 cells that lead to rapidly progressing apoptosis, which is followed by slow necrosis. Two apoptotic processes proceed in parallel, one representing the mitochondrial pathway, the other involving disruption of calcium homeostasis and activation of the endoplasmic reticulum stress-mediated pathway.     

The toxic effects of organometals on the lands cycle in HL-60 cells

The concentration of free fatty acids within cells is mainly dependent upon the following enzyme activities: liberation by phospholipase A₂ (PLA₂), activation of free acids by acyl-CoA-synthetase and re-esterification by lysophospholipid acyltransferase (LAT). In many cell types, especially those of the haematopeotic system, this deacylation-reacylation cycle (‘Lands cycle’) plays an important role in the regulation of free fatty acid concentration, above all that of arachidonic acid. We have shown here that heavy-metal compounds affect this cycle mainly at two points and thereby lead to an increase of free fatty acids. On the one hand, organometals cause an inhibition of the reacylation of lysophospholipids; and on the other, the induction of PLA₂ activity produces the same result. All compounds investigated such as methylmercury chloride (MeHgCl), diethyltriethyl-, and trimethyl-lead chloride (Et₂PbCl₂, Et₃PbCl, Me₃PbCl) as well as trimethyltin chloride (Et₃SnCl) and di-t-butyltin dichloride (t-Bu₂SnCl₂) show at least one of these effects. In the case of Et₃PbCl, the use of PLA₂-inhibitors or pertussis toxin causes a drastic decrease in the amount of arachidonic acid liberated. These experiments demonstrate that the organometallic compounds inhibit the reacylation and/or stimulate the deacylation of fatty acids that are involved in many important biological or pathological mechanisms. The results suggest that in differentiated HL-60 cells the organometal compounds stimulate the Lands cycle by increasing the activity of the PLA₂, possibly via a signal-transduction mechanism, and this effect is intensified via an inhibition of reesterification.     

Timing of 5-aminolaevulinic acid-induced photodynamic therapy for the treatment of patients with Barrett's oesophagus

5-Aminolaevulinic acid-induced photodynamic therapy (ALA-PDT) is being used as an experimental treatment of Barrett's oesophagus (BE), a pre-malignant disorder in the distal oesophagus. The present study aims to acquire detailed knowledge on the pharmacokinetics of ALA and the photosensitizer protoporphyin IX (PPIX) in tissues and plasma of patients with BE to provide a rationale for the conditions used in ALA-PDT. A total of 26 patients with BE were randomized to varying time intervals between ingesting 60 mg/kg ALA and undergoing an endoscopy with biopsies of BE, normal oesophageal and gastric mucosa. At 1, 2, 7, 8 and 24 h, two patients at each time, and at 3, 4, 5 and 6 h, four patients at each time after ALA ingestion were included. ALA, porphyrin intermediates and PPIX were determined in all biopsy and plasma samples. The maximum concentration of PPIX was found earlier in BE (4.6+/-0.5 h) than in squamous epithelium (SQ) (6.6+/-2.2 h) (P<0.05). PPIX concentrations were higher in SQ than in BE especially at longer time intervals. In addition, tissue ALA concentrations were found to be 20-fold higher than the plasma concentrations at 1 h after ALA ingestion, suggesting uptake from the oesophageal lumen. Skin photosensitivity was short-lasting but often debilitating. Our results provide a rationale for the use of ALA-PDT for the treatment of BE at 4-5 h after ALA ingestion and for local application of ALA in the oesophagus. Patients undergoing ALA-PDT must be strongly advised to avoid sunlight for at least 24-36 h.     

A quantitative comparison of 5-aminolaevulinic acid- and methyl aminolevulinate-induced fluorescence, photobleaching and pain during photodynamic therapy

The characteristics of protoporphyrin IX (PPIX) fluorescence in superficial basal cell carcinoma (sBCC) and carcinoma in situ (Bowen's Disease, BD) following application of 5-aminolaevulinic acid (5-ALA) and its methyl ester (methyl aminolevulinate [MAL]) before, during and after photodynamic therapy (PDT) were investigated in 40 patients. Photosensitizer prodrug penetration can limit PDT efficacy and understanding the characteristics of PPIX fluorescence through fluorescence spectroscopy, may improve knowledge of photosensitizer delivery. Fluorescence intensity was assessed quantitatively, and the rate of photobleaching was determined by fitting an exponential decay. As a secondary end-point, PDT-induced pain was also measured continuously during treatment using a novel hand-held device, known as a pain logger. In vivo PPIX fluorescence was shown to decrease during irradiation, allowing the in vivo photobleaching of PPIX to be monitored. No significant difference was found between ALA- or MAL-induced PPIX fluorescence in lesions of sBCC and BD (P>0.05), indicating no detectable difference in PPIX kinetics for the two prodrugs as assessed by these measures. Pain, as assessed by the logger device, showed high interindividual variability and pain levels tended to be higher initially, decreasing during treatment. No difference was seen in pain experienced during ALA-or MAL-PDT (P>0.05).     

New 5-aminolevulinic acid esters--efficient protoporphyrin precursors for photodetection and photodynamic therapy

Photodetection (PD) and photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PPIX) accumulation are approaches to detect and treat dysplasia and early cancer in the gastrointestinal tract and in the urinary bladder. Because ALA-induced PPIX production is limited, we synthesized ALA ester hydrochlorides 3-22 and tested them in two different in vitro models (gastrointestinal tract: HT29-CCD18; urinary bladder: J82-UROTSA). PPIX accumulation after incubation with 0.12 mmol/L for 3 h and PPIX accumulation as a function of different incubation times were measured using flow cytometry. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were performed to check cellular dark toxicity. Phototoxicity after irradiation was tested. ALA nonafluorohexylester hydrochloride 11, ALA thiohexylester hydrochloride 13 and ALA dibenzyldiester dihydrochloride 19 induced appreciably increased PPIX levels and showed improved phototoxicity compared with the references ALA hydrochloride 1, ALA hexylester hydrochloride 3 and ALA benzylester hydrochloride 4. Thus, the new compounds 11, 13 and 19 are promising compounds for PD and PDT.     

An efficient synthesis of 5-aminolaevulinic acid (ALA)-containing peptides for use in photodynamic therapy

An efficient and cost-effective procedure has been devised for the preparation of urethane-protected 5-aminolaevulinic acid (5-ALA) dipeptide ester derivatives which avoids problems associated with the instability of 5-ALA under basic conditions. The procedure is also applicable to the direct synthesis of N-(α)-acetyl amino acid-ALA dipeptides in high enantiomeric purity as potential novel prodrugs for photodynamic therapy (PDT).     

Oxygen monitoring during 5-aminolaevulinic acid induced photodynamic therapy in normal rat colon. Comparison of continuous and fractionated light regimes

Currently, the clinical use of 5-aminolaevulinic acid (ALA) induced protoporphyrin IX (PPIX) for photodynamic therapy (PDT) is limited by the maximum tolerated oral ALA dose (60 mg/kg). Attempts have been made to enhance this treatment modality without increasing the administered dose of ALA. One way to do this is through light dose fractionation, where the irradiation is interrupted at a particular point for a short period of time. This can produce up to three times more necrosis than with the same light dose delivered without a break. An oxygen microelectrode was employed to study the effect of continuous and fractionated light regimes on the level of oxygen in the colon of normal Wistar rats during ALA PDT. A rapid decline in pO2 occurred close to the irradiation fibre as soon as the light dose commenced. With the fractionated regime, a partial recovery in pO2 was observed during the dark interval which was reversed soon after the second light fraction commenced. We have shown that the level of tissue oxygen at the treatment site is affected differently when the light dose is fractionated, than when continuous illumination is employed. This factor may at least partially explain the difference in outcome of these two treatment regimes. Further, oxygen measurements might prove to be a useful way of monitoring PDT treatments if they can predict whether tissue is likely to be viable following treatment.     

Simulations on the selectivity of 5-aminolaevulinic acid-induced fluorescence in vivo

The knowledge of the exact time course of a photosensitizer in tumour and surrounding host tissue is fundamental for effective photodynamic therapy (PDT) and fluorescence-based diagnosis. In this study the time course of porphyrin fluorescence following topical application of 5-aminolaevulinic acid (ALA) using different formulations, concentrations and incubation times has been measured in amelanotic melanomas (A-Mel-3) (n = 54) grown in transparent dorsal skinfold chambers of Syrian golden hamsters and in human basal cell carcinomas (BCCs) (n = 40) in vivo. To simulate the accumulation of ALA-induced protoporphyrin IX (Pp IX), a three-compartment model has been developed and rate constants have been determined. The kinetics of both the A-Mel-3 tumours and the BCCs show a significantly higher fluorescence intensity in tumour as compared to normal surrounding host tissue. Maximal fluorescence intensity in A-Mel-3 tumours as a percentage of the reference standard used occurs 150 min post incubation (p.i.) using a 1, 3 or 10% (vol.) ALA solution buffered to pH 7.4 and 1 h incubation time. After a 4 h incubation time maximal fluorescence intensity in tumour is measured shortly p.i. A concentration of 10% ALA does not increase the fluorescence intensity as compared to 3% ALA following 4 h incubation, but either 3 or 10% ALA yields a significantly higher fluorescence after 4 h incubation time as compared to 1 h. The fluorescence intensity following an 8 h incubation reaches its maximum directly p.i. for all concentrations and then decreases exponentially. The fluorescence intensity in the surrounding host tissue shows no statistically significant difference regarding concentration or incubation time. At least during the first hour p.i., the fluorescence intensity measured in the surrounding tissue is lower as compared to that in the tumour in all groups. 24 h after topical application hardly any fluorescence is detectable in tumour or surrounding host tissue in all experimental groups. Incubating human BCCs with a 20% ALA cream (water-in-oil emulsion) or a 20% ALA gel (containing 40% dimethyl sulfoxide) for approximately 2 h yields a similar fluorescence intensity directly after incubation for either cream or gel. However, while yielding a maximum 120 min p.i. with cream, the fluorescence intensity increases for a longer time (about 2-3 h p.i.) and up to higher values using the gel formulation. In surrounding normal skin, cream as well as gel formulation yields a similar fluorescence intensity directly after incubation. Afterwards the fluorescence intensity decreases slowly using the cream whereas a further increase of the fluorescence intensity is measured in the normal skin with a maximum 240 min p.i. using the gel formulation. The results of the proposed three-compartment model indicate that the observed selectivity of accumulated porphyrins following topical application of ALA is mainly governed by an increased ALA penetration of the stratum corneum of the skin, an accelerated ALA uptake into the cell and a higher porphyrin formation in tumour as compared to normal skin tissue, but not by a reduced ferrocheletase activity.     

Purification and Characterization of DNA Methylase From HL-60 Cells

A solid leukemia sarcoma has been successfully developed after subcutaneous inoculation of the cultured human promyelocytic leukemia cells (HL-60 cells) into unde mice. The solid leukemia sarcoma is a more plantiful source than the cultured cells for enzymatic study and its growing environment is closer to that of the human body than the cultured cells.We establish an efficient procedure of purifying HL-60 cells DNA methylase which includes: disruption of HL-60 cells by homogenization and sonication, removing the cell fragments and cellular particles by centrifuge and ultracentrifuge (105.000 g); removing endogenous DNA by streptomycin sulfate, salting out by (NH4)2SO4, ion exchange chromatography on DEAE-cellulose (DE-52), gel filtration over Sephadex G-100 column.The DNA methylase from HL-60 cells has been purified 204 fold by this procedure. The purified enzyme shows a single-band on PG-PAGE. A 479-kD molecular weight of this enzyme is measured by PG-PAGE. The enzyme properties of HL-60 DNA methylase     

Apoptosis and membrane permeabilisation induced by macranthoside B on HL-60 cells

Triterpene saponins are throught to be potential anti-tumour agents in many cell types. This study aims to evaluate the cytotoxic activity and mechanism of a triterpene saponin, macranthoside B (MB), isolated from Lonicera macranthoides Hand.-Mazz. (Caprifoliaceae). A cell viability assay showed that MB inhibited cell growth of a panel of six cancer cell lines, especially in human acute promyelocytic leukaemia HL-60 cells, with an IC50 value of 3.8 μmol. A hypodiploid cells assay and an annexin-V-FITC/PI double staining assay showed a significant increase of apoptosis in a dose-dependent manner on HL-60 cells both 24 and 48 h after MB treatment. MB-induced apoptosis was through the caspase-mediated pathway, by activation of caspase-3. Furthermore, a lactate dehydrogenase (LDH) release test suggested that an MB-cholesterol interaction led to the rearrangement of the lipid bilayer and to subsequent cell membrane impairment. Taken together, these findings demonstrate that MB may exhibit cytotoxic activity against HL-60 cells by inducing apoptosis via caspase-dependent pathways and also membrane permeabilisation.     

Capillary zone electrophoresis/mass spectrometry of 5-aminolaevulinic acid and porphobilinogen

A capillary zone electrophoresis/electrospray ionisation mass spectrometry (CZE/ESI-MS) method has been developed for the separation and detection of 5-aminolaevulinic acid (ALA) and porphobilinogen (PBG). Capillaries were 70 cm long with an inner diameter of 75 micrometer and outer diameter of 375 micrometer. The buffer used was aqueous ammonium acetate (50mM, pH 5.2) with a co-axial 'make-up' flow of methanol/aqueous 0.1% formic acid (1:1 v/v) at a flowrate of 6 microL/min. A voltage of 20 kV was used for CZE and an ESI voltage of 3.5 kV. Full scan data was acquired over the range m/z 100-500 in positive ion mode, from which selected ion electropherograms were extracted; at m/z 132 for the protonated molecular ion of ALA and m/z 210 for the methylenepyrrolenine fragment ion of PBG. The protonated molecular ion of PBG, m/z 227, was found to be too facile to monitor, easily losing ammonia in the electrospray source and better sensitivity was achieved by monitoring the resulting fragment ion. The detection limits were circa 100 attomoles of ALA and 10 attomoles of PBG at a signal-to-noise ratio (S/N) better than 10, providing sufficient sensitivity for clinical use and offering advantages over existing techniques.     

The structure of organometals determines cytotoxicity and alteration of calcium homeostasis in HL-60 cells

There is increasing concern about the degradation and metabolisation as well as the biochemical mechanisms of action of organometallic compounds. They are known to be immunotoxic and/or neurotoxic. Because of their different toxic capacities, the development of a reliable correlation between molecular parameters and biochemical effects, which could be helpful in risk assessment, was an aim of this study. The tested organolead and -tin compounds decrease the viability of human cells in culture in a time- and concentration-dependent manner. Parabolic QSAR¹¹ The abbreviations used are: TMT, trimethyltin chloride; TET, triethyltin bromide; TPT, tripropyltin chloride; TBT, tri-n-butyltin chloride; DBT, di-n-butyltin dichloride; TEL, triethyllead chloride; DEL, diethyllead dichloride; TML, trimethyllead chloride; TPhL, triphenyllead chloride; QSAR, quantitative structure-activity relationships; TSA, total surface area; MW_ion, ionic molecular weight; fMLP, N-formyl-L-methionyl-L-leucyl-L-phenylalanine; fluo-3, fluo-3 free acid; fluo-3 AM, fluo-3 acetoxymethyl ester; Me₂SO, dimethyl sulfoxide; PLA₂, phospholipase A₂ (EC 3.1.1.4); FCS, fetal calf serum; HEPES, 4-(2-hydroxy-ethyl)-1-piperazineethanesulfonic acid; EGTA, [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid; [Ca²⁺]_i, cytosolic free Ca²⁺ concentration models yield an adequate correlation between toxicity expressed as LC₅₀ and structural parameters like ionic molecular weight (MW_ion) or total surface area (TSA). Two main chemical attributes of the organometals are probably responsible for such a parabolic relationship: the hydrophobic side chain and the polar metal atom. Furthermore, all tested organometal compounds evoke a persistent increase of the cytosolic free calcium concentration [Ca²⁺]_i. This effect is mainly due to an influx from the extracellular space. Further results suggest that Ca²⁺ enters the cell via opened calcium channels. Based on the essential role of Ca²⁺ within cellular signalling, the perturbation of calcium homeostasis appears to be an important event in final cell killing by organometals and it is most likely that other biochemical mechanisms, e.g. activation of phospholipase A₂, are possibly mediated by an increase of [Ca²⁺]_i.     

The structure of organometals determines cytotoxicity and alteration of calcium homeostasis in HL-60 cells

There is increasing concern about the degradation and metabolisation as well as the biochemical mechanisms of action of organometallic compounds. They are known to be immunotoxic and/or neurotoxic. Because of their different toxic capacities, the development of a reliable correlation between molecular parameters and biochemical effects, which could be helpful in risk assessment, was an aim of this study. The tested organolead and -tin compounds decrease the viability of human cells in culture in a time- and concentration-dependent manner. Parabolic QSAR(1)(1) The abbreviations used are: TMT, trimethyltin chloride; TET, triethyltin bromide; TPT, tripropyltin chloride; TBT, tri- n-butyltin chloride; DBT, di- n-butyltin dichloride; TEL, triethyllead chloride; DEL, diethyllead dichloride; TML, trimethyllead chloride; TPhL, triphenyllead chloride; QSAR, quantitative structure-activity relationships; TSA, total surface area; MW(ion), ionic molecular weight; fMLP, N-formyl-L-methionyl-L-leucyl-L-phenylalanine; fluo-3, fluo-3 free acid; fluo-3 AM, fluo-3 acetoxymethyl ester; Me(2)SO, dimethyl sulfoxide; PLA(2), phospholipase A(2) (EC 3.1.1.4); FCS, fetal calf serum; HEPES, 4-(2-hydroxy-ethyl)-1-piperazineethanesulfonic acid; EGTA, [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid; [Ca(2+)](i), cytosolic free Ca(2+) concentration models yield an adequate correlation between toxicity expressed as LC(50) and structural parameters like ionic molecular weight (MW(ion)) or total surface area (TSA). Two main chemical attributes of the organometals are probably responsible for such a parabolic relationship: the hydrophobic side chain and the polar metal atom. Furthermore, all tested organometal compounds evoke a persistent increase of the cytosolic free calcium concentration [Ca(2+)](i). This effect is mainly due to an influx from the extracellular space. Further results suggest that Ca(2+) enters the cell via opened calcium channels. Based on the essential role of Ca(2+) within cellular signalling, the perturbation of calcium homeostasis appears to be an important event in final cell killing by organometals and it is most likely that other biochemical mechanisms, e.g. activation of phospholipase A(2), are possibly mediated by an increase of [Ca(2+)](i).     

5-Aminolevulinic acid-based photochemical internalization of the immunotoxin MOC31-gelonin generates synergistic cytotoxic effects in vitro

Photochemical internalization (PCI) is a novel method for the endosomal or lysosomal release of membrane-impermeable molecules into the cytosol of target cells. This novel technology is based on the photodynamically induced rupture of endocytic vesicles preloaded with molecules of therapeutic interest. PCI of the ribosome-inactivating plant toxin gelonin and the immunotoxin monoclonal antibody 31 (MOC31) gelonin has been performed previously by the use of the endocytic vesicle-localizing photosensitizers TPPS2a and AIPcS2a and light, demonstrating synergistic toxicity against the more than 20 different cell lines tested, most of them of neoplastic origin. In this study we demonstrate that 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) is also capable of inducing PCI of MOC31-gelonin in the human colon adenocarcinoma cell line WiDr. The cells were incubated with 1 mM 5-ALA for up to 8 h in serum-free medium and from 24 to 96 h in serum-containing medium. Fluorescence microscopical studies indicate a partial plasma membrane localization of PpIX when 5-ALA was applied under serum-free conditions. This plasma membrane localization was not seen when 5-ALA was given in the presence of serum. There was a granular component of the PpIX localization in addition to a diffuse cytoplasmic localization. The granular component resembled the localization of the fluorescent dye conjugate Alexa-gelonin and the lysosomal localizing dye acridine orange. Our present results provide evidence for an endocytic vesicle-associated fraction of PpIX after 5-ALA incubation of the WiDr cells. We demonstrate that PCI, by combining 5-ALA, MOC31-gelonin and light, induces a synergistic cytotoxic effect against the WiDr cells.