Cellular uptake, subcellular localization and photodamaging effect of temoporfin (mTHPC) in nasopharyngeal carcinoma cells: comparison with hematoporphyrin derivative.

Temoporfin (meta-tetra (hydroxyphenyl)chlorin; mTHPC) potentiated a 100-fold higher cytotoxic effect than hematoporphyrin derivative (HPD) on two nasopharyngeal carcinoma cell lines (HK1 and CNE2) in terms of the overall photodynamic therapy (PDT) dose. The cellular uptake, evaluated by flow cytometry and spectrophotometry demonstrated that mTHPC exhibited higher uptake ability than HPD. Confocal laser scanning microscopy detection for both the sensitizer and mitochondria probe on the same cell images revealed that both drugs accumulated diffusely in the cytoplasm and that mitochrondria is a target organelle. Photo-activation ruptured the mitochrondria, with more pronounced mitochondrial damage being observed in mTHPC-PDT course. This correlated well with the cell photokilling efficiency of mTHPC.

A comparison of the photodynamic effects of temoporfin (mTHPC) and MC540 on leukemia cells: efficacy and apoptosis.

The photodynamic effects of temoporfin (meso-tetrahydroxyphenylchlorin, mTHPC) and merocyanine 540 (MC540) in murine myeloid leukemia M1 and WEHI 3B (JCS) cells were compared. The mTHPC was found to be more potent and selective. At a lethal dosage of 90% killing (LD90), only 1.3 microM of mTHPC and 4.2 kJ/m2 of light irradiation was required, which was a 20-fold lower drug concentration and 11-fold smaller light dose than that required when using MC540. Meanwhile, three times less, or 15%, of the coincubated erythrocytes were destroyed by mTHPC than by MC540. Confocal micrographs showed that both drugs accumulated diffusely inside the cytoplasm in a very similar fashion, but mTHPC induced a more extensive apoptosis in photosensitized JCS cells. For example, at LD90, mTHPC practically killed all JCS cells via apoptosis and cleaved the DNA to extremely small 150 base-pair fragments. In contrast, among the JCS cells killed by MC540, about 88% died via apoptosis and large DNA fragments were abundant. Relative to MC540, the ability of mTHPC to trigger large-scale and thorough apoptosis in leukemia cells may help explain its potency and selectivity.

The binding characteristics and intracellular localization of temoporfin (mTHPC) in myeloid leukemia cells: phototoxicity and mitochondrial damage .

The state of aggregation of the photosensitizer meso-tetrahydroxyphenylchlorin (mTHPC) in both cell free and intracellular environment was elucidated by comparing its absorption and excitation spectra. In methanol, mTHPC existed as monomers and strongly fluoresced. In aqueous solutions such as phosphate-buffered saline (PBS), mTHPC formed nonfluorescent aggregates. Some portion of mTHPC monomerized in the presence of 10% fetal calf serum PBS. In murine myeloid leukemia M1 and WEHI-3B (JCS) cells, cytoplasmic mTHPC were monomeric. By using organelle-specific fluorescent probes, it was found that mTHPC localized preferentially at the mitochondria and the perinuclear region. Photodynamic treatment of mTHPC-sensitized leukemia cells caused rapid appearance of the apoptogenic protein cytochrome c in the cytosol. Results from flow cytometric analysis showed that the release of cytochrome c was especially pronounced in JCS cells, and well correlated with the extent of apoptotic cell death as reported earlier. Electron microscopy revealed the loss of integrity of the mitochondrial membrane and the appearance of chromatin condensation as early as 1 h after light irradiation. We conclude that rapid release of cytochrome c from photodamaged mitochondria is responsible for the mTHPC-induced apoptosis in the myeloid leukemia JCS and M1 cells.

Subcellular localization of merocyanine 540 (MC540) and induction of apoptosis in murine myeloid leukemia cells.

Subcellular localization of photosensitizers is thought to play a critical role in determining the mode of cell death after photodynamic treatment (PDT) of leukemia cells. Using confocal laser scanning microscopy and fluorescent organelle probes, we examined the subcellular localization of merocyanine 540 (MC540) in the murine myeloid leukemia M1 and WEHI 3B (JCS) cells. Two patterns of localization were observed: in JCS cells, MC540 was found to localize on the plasma membrane and mitochondria; and in M1 leukemia cells, MC540 was found to localize on lysosomes. The relationship between subcellular localization of MC540 and PDT-induced apoptosis was investigated. Apoptotic cell death, as judged by the formation of apoptotic nuclei, was observed 4 h after irradiation in both leukemia cell lines. Typical ladders of apoptotic DNA fragments were also detected by DNA gel electrophoresis in PDT-treated JCS and M1 cells. At the irradiation dose of 46 kJ/m2 (LD90 for JCS and LD86 for M1 cells), the percentage of apoptotic JCS and M1 cells was 78 and 38%, respectively. This study provided substantial evidence that MC540 localized differentially in the mitochondria, and the subsequent photodamage of the organelle played an important role in PDT-mediated apoptosis in myeloid leukemia cells.

Photocytotoxic and DNA damaging effect of temoporfin (mTHPC) and merocyanine 540 (MC540) on nasopharyngeal carcinoma cell.

Photodynamic therapy (PDT) is a new approach to cancer treatment for a variety of malignant tumors. In this study, two clinical photosensitizers, Temoporfin (meta-tetra-hydroxyl-phenyl-chlorin; mTHPC) and merocyanine 540 (MC540), were selected to explore for their photocytotoxic and genotoxic effects on nasopharyngeal carcinoma cells (NPC/HK1 and CNE2). Results of tetrazolium reduction assay showed that 80% cell killing were achieved for both cell lines at 0.4 microg/ml mTHPC for 24 h incubation and then with 40 kJ/m2 light irradiation, whereas 40 microg/ml MC540 with 50 kJ/m2 light dosage was required to attain the same level of phototoxicity for NPC/HK1. On the contrary, NPC/CNE2 was quite resistant to MC540. Hence, mTHPC-mediated PDT exerted a more potent effect than MC540-mediated PDT, even though the molar extinction coefficient of the main absorption peak for MC540 is much higher than that of mTHPC. Dark cytotoxicity remained negligible for both sensitizers. Comet assay was used to evaluate the DNA strand break and potential genotoxic effect induced by mTHPC and MC540 on the NPC cells. No DNA strand break was detected in the absence of light, and under sublethal treatment (LD25) for either sensitizer-loaded cells. Confocal laser scanning microscopy showed that mTHPC and MC540 localized in the cytoplasm but not in the nucleus of the tumor cells, which provided evidence for undetectable DNA damage under dark and low photodynamic dose.

A study of the binding of merocyanine 540 to myeloid leukemia M1 cells using an intensified charge-coupled device for fluorescence imaging microscopy.

The binding of merocyanine 540 (MC540) to murine myeloid leukemia (M1) cells and normal erythrocytes was measured by fluorescence digital imaging microscopy using an intensified charge-coupled device. It was found that, on average, about three times more MC540 were bound to a unit membrane area of M1 cells than erythrocytes, a result consistent with previous studies. However, it was shown for the first time that MC540 binding varied significantly from one M1 cell to the next, and about 15% of the sensitized M1 cells were as MC540-negative as normal erythrocytes. Using the leukemic inhibitory factor as a differentiation inducer, M1 cells were induced to differentiate into mature macrophage-like cells in vitro. Such treatment lowered the average MC540 binding by about one-third but did not affect the cell-to-cell variation significantly.

Attitudes of Victorian dentists to removable partial denture prosthodontics: treatment planning.

A survey of the attitudes of dentists in Victoria to various aspects of the treatment planning phase of removable partial denture prosthodontics was undertaken. Data were analysed and compared with those obtained from an identical study in South Australia. Those Victorian dentists who graduated in the 1960s, those who worked in multiple-practitioner practices, or non-rural areas, and those who had an added interest in removable prosthodontics were more likely to stress the importance of a greater number of attributes than those who graduated in the 1950s or 1970s, or those who worked alone, in rural areas, or had no particular interest in the discipline. Results indicated also that Victorian dentists tended to place greater emphasis on many of the concepts embraced by the questions asked than their interstate colleagues. It is suggested that, in addition to formal education, intraprofessional relationships and interest in a discipline are factors which might influence the concepts and attitudes of general practitioners.

Adsorption of bovine serum albumin on fused silica: Elucidation of protein-protein interactions by single-molecule fluorescence microscopy.

The adsorption of bovine serum albumin (BSA) on fused silica at pH 4.7 was studied at the single molecules level by total-internal-reflection fluorescence microscopy. This pH value was the isoelectric point of BSA. At low [BSA] of 20pM, protein molecules adsorbed as monomers. At intermediate [BSA] of 500pM, protein molecules adsorbed as clusters of about five monomers on average. Both monomers and clusters had adsorption rate coefficients of the order 10(-7)ms(-1) and desorption rate coefficients of about 2x10(-2)s(-1). The respective steady-state coverage was about 10x higher than that at neutral pH, presumably because of the more favorable BSA-silica electrostatics. At pH 4.7 and with [BSA] higher than 100nM, adsorption begot further adsorption to produce nonlinear isotherms. The coverage at 1microM BSA was 2.5x that of the linearly extrapolated coverage. This suggests that at pH 4.7, solute-adsorbate affinity was the dominant factor that explains the enhanced adsorption observed in ensemble measurements.

Adsorption kinetics of bovine serum albumin on fused silica: population heterogeneities revealed by single-molecule fluorescence microscopy.

The adsorption of bovine serum albumin (BSA) on fused silica at neutral pH was investigated at the single-molecule level by total internal reflection fluorescence microscopy. Dye-labeled BSA molecules that adsorbed on the quartz surface lit up as discrete, fluorescent dots which eventually disappeared upon desorption. Movies of these events offered unprecedented details for kinetics modeling. The results suggested that 99.3% of the BSA was not sticky, and even if adsorbed, it would desorb in minutes. In contrast, the remaining 0.7% was not only sticky, but would anchor in due course. Such population heterogeneity, otherwise masked in ensemble measurements, sheds new light on our understanding of protein adsorption. The methodology is also generally applicable to the studies of macromolecules at interfaces.

Minimally destructive analysis of aluminum alloys by resonance-enhanced laser-induced plasma spectroscopy.

Aluminum alloys were analyzed for minor components and trace impurities using double pulse resonance-enhanced laser-induced plasma spectroscopy. The first laser pulse at 532 nm ablated the sample. The second laser pulse at 396.15 nm resonantly excited the Al atoms to rekindle the plasma plume. Emissions from Mg, Cu, Si, and Na were observed. At laser energies below the damage threshold, the analyte emissions were already orders of magnitude above the background noise. Nonresonant probes of comparable sensitivity would melt and deform the sample surface. Because of the lower etch rate of resonant probes, depth profiling at nanometer resolution was possible. Using this method, the variation of [Na] with depth was measured for high-purity samples. In contrast, nonresonant probes required 5 times the fluence and proportionally poorer resolution. Worse yet, the associated heating and laser remelting modified the [Na] profile.

Sub-part-per-billion analysis of aqueous lead colloids by ArF laser induced atomic fluorescence.

Highly sensitive analysis of aqueous lead carbonate colloids was demonstrated by two-pulse laser-induced atomic fluorescence. The first laser pulse at 1064 nm ablated the sample solution to create an expanding plume. The colloids, being heavier, trailed behind and became concentrated. They were then intercepted by an ArF laser pulse that induced prompt atomic fluorescence at 405.8 nm from the lead atoms. The detection limit for lead was 0.24 ppb. Tap water was analyzed, and lead emissions were clearly observed. Time-resolved spectroscopy revealed that the efficient 193-nm excitation of the analytes was more universal than expected. That was confirmed by the successful application of the technique to colloids and alloys other than lead.

Distribution of sodium and potassium within individual human erythrocytes by pulsed-laser vaporization in a sheath flow.

Simultaneous determination of the amounts of Na and K inside single human erythrocytes was accomplished by laser vaporization and monitoring the atomic emission produced. By using a modified sheath-flow arrangement, detection of 8 fg (0.35 fmol) of Na is possible with one laser pulse. By using Poisson statistics, one can obtain single-cell information even when multiple cells are vaporized per laser pulse. The intracellular contents for a given individual were found to vary significantly. The +/- 55% and +/- 155% variations for Na and K, respectively, cannot be explained by changes in cell volume. There is only a weak correlation between the Na and K contents in single cells. The results reflect the age distribution of erythrocytes in the sample. Presumably, the enzymes regulating ion transport lose their activities in the older cells.

Detection of sodium and potassium in single human red blood cells by 193-nm laser ablative sampling: a feasibility demonstration.

The feasibility of quantifying sodium and potassium in single human erythrocytes was demonstrated by spectrochemical analysis of emissions from plasmas produced by 193-nm laser ablation of blood cells confined in a sheath flow. In one scheme, single blood cells that happened to be in the ablation volume were sampled. In another scheme, individual blood cells were first sighted and then synchronously ablated downstream. Plasma emission spectra of single ablated cells were captured, and the ratios of the analyte line intensity to the root-mean-square fluctuation of the continuum background were measured to be about 18 for sodium and 30 for potassium.

Endogenous production of protoporphyrin IX induced by 5-aminolevulinic acid in leukemia cells.

AIM: To explore the photosensitization of 5-aminolevulinic acid (ALA) in myeloid leukemia cell line. METHODS: Using the technique of fluorescence spectra, the A LA induced protoporphyrin IX (Pp IX) was measured in myeloid leukemia JCS cells. Cofocal laser scanning microscopy (CLSM) combined with fluorescence organelle probe was used to detect the localization of Pp IX in JCS cells at the subcellular levels. MTT assay was used to measure the cell survival after light irradiation. RESULTS: ALA successfully produced endogenous PpI X in leukemia JCS cells. PpIX was observed to be distributed in the cytoplasm and mitochondria was exhibited as the one of binding sites of PpIX. As a photosensitizer, PpIX initiated photodynamic reaction after light irradiation and effectively photodamaged leukemia cells. CONCLUSION: ALA-based photosensitization could be used for inactivation of leukemia cells.