René Santus

Pyropheophorbide-a Methyl Ester-mediated Photosensitization Activates Transcription Factor NF-κB through the Interleukin-1 Receptor-dependent Signaling Pathway

Pyropheophorbide-a methyl ester (PPME) is a second generation of photosensitizers used in photodynamic therapy. We demonstrated that PPME photosensitization activated NF-κB transcription factor in colon cancer cells. Unexpectedly, this activation occurred in two separate waves, i.e. a rapid and transient one and a second slower but sustained phase. The former was due to photosensitization by PPME localized in the cytoplasmic membrane which triggered interleukin-1 receptor internalization and the transduction pathways controlled by the interleukin-1 type I receptor. Indeed, TRAF6 dominant negative mutant abolished NF-κB activation by PPME photosensitization, and TRAF2 dominant negative mutant was without any effect, and overexpression of IκB kinases increased gene transcription controlled by NF-κB. Oxidative stress was not likely involved in the activation. On the other hand, the slower and sustained wave could be the product of the release of ceramide through activation of the acidic sphingomyelinase. PPME localization within the lysosomal membrane could explain why ceramide acted as second messenger in NF-κB activation by PPME photosensitization. These data will allow a better understanding of the molecular basis of tumor eradication by photodynamic therapy, in particular the importance of the host cell response in the treatment.

Optical Properties of the Skin

Skin consists of three main layers—the epidermis, separated from the underlying dermis by a basement membrane; the dermis, with collagen and elastic fibers produced by fibroblasts, blood and lymph vessels, hair follicles, sweat and sebaceous glands, erector pilae smooth muscles, and nerves; and the sub epidermal tissue, consisting of a fat layer. The epidermis of the skin, further consists of four layers—the stratum germinativum, the stratum spinosum, the stratum granulosum, and the stratum corneum. When radiation strikes the skin, part is absorbed, part is remitted, and part is transmitted through the successive layers of skin, until the energy of the incident beam is dissipated. The transmittance of a planar sample is defined as that fraction of the radiation incident on one side of the sample that passes through and emerges from the other side of the sample. Remittance is defined as that fraction of the radiation incident on one side of a sample that returns from or through the same side. The term diffuse reflectance is spatially isotropic remitted radiation. The regular reflectance of an incident beam normal to skin is 4–7% over the spectrum from 250 to 3000 nm. The action spectrum wavelengths of photobiologic responses are those absorbed by specific chromophores that initiate the photochemical reactions. The remittance of incident radiation with respect to skin color and the Kubelka-Munk model for radiation transfer in a scattering, absorbing medium is also described in the chapter.

Fluorescence Probes in Oncology

The Nature of Light Introduction to Photophysics Basic Approaches to the Experimental Study of Chemical and Biological Luminescence Methods and Instrumentation New Methods. Theoretical Basis and Potential Applications to Biology Fluorescent Probes Applications of Fluorescence Techniques to Study Biological Processes in Normal and Pathological Cells Cell Physiopathology.

Photochemistry of Biological Molecules

The excitation of chromophores determines the sequence of secondary dark reactions and structural rearrangements, giving rise to stable products whose chemical structure can be established with the technical armamentarium of the chemist. This chapter gives a detailed account of some of the important photoproducts of the biological molecules (amino acids, nucleic acid bases, and lipids) constituting the basic units of living cells. As far as direct photochemistry is concerned, the discussion is limited to the photochemical changes of these molecules on excitation in their first excited singlet state. It must be kept in mind that the bandwidth of the optical absorption corresponding to this excited state must somewhat overlap the solar radiation spectrum reaching the ground (wavelengths >290 nm), to be of biological significance. Because most of the photoreactions essential to life occur in the presence of oxygen, the chapter only considers photoproducts that can be formed by direct photolysis under aerobic conditions or during photosensitized reactions.

From Photophysics to Photochemistry: Determination of Primary Processes in Direct or Sensitized Photoreactions

The involvement of sunlight in promoting chemical reactions, such as the production of chlorophyll by plants, is common knowledge. Indeed, plants use sunlight to perform photochemical reactions that ultimately provide all the food supplies of the animal world. The goal of photobiologists is to understand the quantitative aspects of this holistic relationship between sunlight (or in a broader sense, electromagnetic radiations) and man. In this chapter it is emphasized that laser flash spectroscopy is the essential tool of molecular photobiologists for characterizing and studying the singlet- and triplet-state properties of important biological molecules and photosensitizers, and for investigating the primary photochemical processes of biological chromophores. It then describes how the mechanistic aspects and product analyses of direct or sensitized photochemical reactions with biological relevance can be studied by much less expensive steady state irradiation techniques. The therapeutic benefit of such a substitution reaction is also examined in this chapter.

Combined fluorescence and ultrastructural mapping of living cells

The topographic analysis of fluorescence distribution has been carried out pixel-by-pixel by one dimensional, two-dimensional microspectrofluorometry and three-dimensional confocal fluorescence microscopy. Fluorescence emission spectra of NAD(P)H and benzo(a)pyrene (or metabolites) were recorded at different excitation wavelengths. Cell bioenergetics are monitored in normal and malignant cells as well as cells with genetic defects by coenzyme responses to microinjections of substrates and modifiers from key metabolic pathways in presence and absence of inhibitors and drugs active on mitochondrial structure and function. Cooperative interactions between organelles involved in detoxification mechanisms are observed in cells treated with fluorescent cytotoxic agents. Such interactions can be directly mapped by the fluorescence of cytotoxic agents, their reaction products or vital probes such as NBD ceramide for the Golgi apparatus. To identify the organelles involved parallel electron microscopic studies are carried out in cells first treated with the cytotoxic agent and then incubated with an electron opaque material. A recently developed combined X-ray laser microscope (COXRALM) holds the promise of carrying out combined phase-fluorescence-and X-ray microscopic observations of fluorescence and ultrastructural correlations in live cell probing. As further versatility is gained in such methods it may become possible to obtain a very detailed structure and function mapping of living cells within the context of cytomatrix analysis, metabolic compartmentation and organelle interactions.

Atlas of cell organelles fluorescence

Contents Vital Fluorescence Probes of Cell Organelles Mitochondrial Probes Lysosomal Probes Nuclear Probes Metabolic Probes Cytotoxic Drugs Lysosomotropic Agents Mitochondria-Toxic Agents Mitochondrial Inhibitors Photosensitizers Carcinogens and Cancer Chemotherapeutic Drugs: Agents Stimulating the Proliferation of the Endoplasmic Reticulum and Golgi Together with the Loading of Lysosomes Genetic Diseases Cell Differentiation and Cell Pathology Cell-to-Cell Communication The Study of Microecosystems Biotechnology Instrumentation Novel Methods and Instrumental Designs Two-Photon Excitation Microscopy Fourier Interferometry for Excitation-Emission Fluorescence Spectral Imaging Excitation-Emission Fluorescence Imaging Combined with Photoacoustic Microscopy. A Combined Fluorescence Imaging and Photoacoustic Microscopy Design for Studies in Cancer Cells Combined Confocal Fluorescence and Acoustic Microscopy CCOFAM without Necessity for Scanning Is the Study of Nanocompartments in Living Cells Feasible? Conclusion Index

Molecular Mechanism of Visual Transduction

When a rod cell of the retina absorbs light as a primary photoevent, an isomerization of the photoreceptor chromophore 11-cis-retinal to 11-trans - retinal occurs within picoseconds. The photoreceptor cells that make it possible to form black and white images in dim light, i.e., the rod cells, are exquisitely sensitive detectors. When a photon strikes the retina, the rhodopsin molecule (the macromolecule holding the retinal) that is struck reports the event with high efficiency, whereas the millions of other rhodopsin molecules in the cell remain silent. The photoreceptor of rod cells, rhodopsin, located in the rod disks as a transmembrane protein, has two components: cis-retinal, an organic molecule derived from vitamin A, and opsin, a protein that has the capacity to act as an enzyme. Retinal is nested at the center of a complex and highly structured protein environment, responsible for “tuning” retinal by influencing the spectrum of radiation it can absorb. When a photon is absorbed by cis-retinal, the energy of light straightens the bend in the retinal carbon chain, a bend that is because of the presence of hydrogen atoms attached to the C-11 and C-12 on the same side of the chain. In the transduction process that follows, a cascade of reactions results in a nerve signal. The chapter also explains signal transduction between the disk membrane and the rod outer membrane.

Biological Effects of Solar Ultraviolet Radiation

The ultraviolet (UV) absorption spectra of major epidermal chromophores (tryptophan, tyrosine, DNA, and urocanic acid) provide a glimpse of the primary targets of UVB effects. In the case of urocanic acid, the action spectrum is almost superposable with the action spectrum of immuno suppression. The combined action spectra of UVB–UVA irradiation for lethality, mutagenesis, and pyrimidine dimer formation show an abrupt break at about 330 nm. This chapter discusses DNA repair processes. The post replication repair process, which results in DNA daughter strand gaps, requires a complete growth medium with nutrients, whereas nucleotide excision repair occurs in buffer devoid of nutrients. Avoidance of post replication repair by cells in the stationary phase, with nutrients in the growth medium exhausted, results in the higher survival. The damaged part of a DNA molecule can be restored in situ , without breaking its sugar phosphate backbone, by photo reactivation (PR). The enzymatic splitting of cyclobutane-type pyrimidine dimers is mediated by intense blue light. The PR enzyme is DNA photolyase. In the dark, the enzyme binds tightly to a cyclobutane-type dimer to form an enzyme substrate complex. The absorption of light between 300 and 450 nm activates this complex. The pyrimidine dimer is converted to monomeric pyrimidines, and the enzyme is released.

Pathways of Molecular Excitation and Deactivation

The possible electron distributions defining the molecular orbitals and the energy levels of molecules in their ground and excited states are given by the approximate solutions of a generalized Schroodinger equation. The latter takes into account the electrostatic attractions and repulsions of protons and electrons, inter-nuclear vibrations, and the rotational movement of molecules as well as magnetic interactions because of electron and nuclear spins and orbital motion. Molecular orbitals can contain no more than two electrons. A transition between the two states of a molecule corresponds to the movement of one electron from one orbital to another. This chapter explains the pathways of molecular deactivation. Because an electron has spin angular momentum, and because moving charges generate magnetic fields, an electron has a magnetic moment, which arises from its spin. Similarly, an electron with orbital angular momentum is a circulating current, also generating a magnetic moment. The strength of the coupling and its effect on the energy levels of the molecule, depend on the relative orientation of the two angular momenta.