Juan C. Stockert

MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets

Although MTT is widely used to assess cytotoxicity and cell viability, the precise localization of its reduced formazan product is still unclear. In the present study the localization of MTT formazan was studied by direct microscopic observation of living HeLa cells and by colocalization analysis with organelle-selective fluorescent probes. MTT formazan granules did not colocalize with mitochondria as revealed by rhodamine 123 labeling or autofluorescence. Likewise, no colocalization was observed between MTT formazan granules and lysosomes labeled by neutral red. Taking into account the lipophilic character and lipid solubility of MTT formazan, an evaluation of the MTT reaction was performed after treatment of cells with sunflower oil emulsions to induce a massive occurrence of lipid droplets. Under this condition, lipid droplets revealed a large amount of MTT formazan deposits. Kinetic studies on the viability of MTT-treated cells showed no harmful effects at short times. Quantitative structure-activity relations (QSAR) models were used to predict and explain the localization of both the MTT tetrazolium salt and its formazan product. These predictions were in agreement with experimental observations on the accumulation of MTT formazan product in lipid droplets.

Morphological criteria to distinguish cell death induced by apoptotic and necrotic treatments.

We present a comparative study of apoptotic and necrotic morphology (light and scanning electron microscopy), induced by well known experimental conditions (photodynamic treatments, etoposide, hydrogen peroxide, freezing-thawing and serum deprivation) on cell cultures. Our results indicate that morphological criteria (apoptotic cell rounding and shrinkage, and appearance of membrane bubbles in early necrosis) allow to distinguish these cell death mechanisms, and also show that, independently of the damaging agents, the necrotic process occurs in a characteristic sequence (coalescence of membrane bubbles in a single big one that detaches from cells remaining on the substrate).

Porphycenes: Facts and Prospects in Photodynamic Therapy of Cancer

The photodynamic process induces cell damage and death by the combined effect of a photosensitizer (PS), visible light, and molecular oxygen, which generate singlet oxygen ((1)O(2)) and other reactive oxygen species that are responsible for cytotoxicity. The most important application of this process with increasing biomedical interest is the photodynamic therapy (PDT) of cancer. In addition to hematoporphyrin-based drugs, 2nd generation PSs with better photochemical properties are now studied using cell cultures, experimental tumors and clinical trials. Porphycene is a structural isomer of porphyrin and constitutes an interesting new class of PS. Porphycene derivatives show higher absorption than porphyrins in the red spectral region (lambda > 600 nm, epsilon > 50000 M-(1)cm(-1)) owing to the lower molecular symmetry. Photophysical and photobiological properties of porphycenes make them excellent candidates as PSs, showing fast uptake and diverse subcellular localizations (mainly membranous organelles). Several tetraalkylporphycenes and the tetraphenyl derivative (TPPo) induce photodamage and cell death in vitro. Photodynamic treatments of cultured tumor cells with TPPo and its palladium(II) complex induce cytoskeletal changes, mitotic blockage, and dose-dependent apoptotic or necrotic cell death. Some pharmacokinetic and phototherapeutic studies on experimental tumors after intravenous or topical application of lipophilic alkyl-substituted porphycene derivatives are known. Taking into account all these features, porphycene PSs should be very useful for PDT of cancer and other biomedical applications.

Epigenetic disruption of ribosomal RNA genes and nucleolar architecture in DNA methyltransferase 1 (Dnmt1) deficient cells

The nucleolus is the site of ribosome synthesis in the nucleus, whose integrity is essential. Epigenetic mechanisms are thought to regulate the activity of the ribosomal RNA (rRNA) gene copies, which are part of the nucleolus. Here we show that human cells lacking DNA methyltransferase 1 (Dnmt1), but not Dnmt33b, have a loss of DNA methylation and an increase in the acetylation level of lysine 16 histone H4 at the rRNA genes. Interestingly, we observed that SirT1, a NAD+-dependent histone deacetylase with a preference for lysine 16 H4, interacts with Dnmt1; and SirT1 recruitment to the rRNA genes is abrogated in Dnmt1 knockout cells. The DNA methylation and chromatin changes at ribosomal DNA observed are associated with a structurally disorganized nucleolus, which is fragmented into small nuclear masses. Prominent nucleolar proteins, such as Fibrillarin and Ki-67, and the rRNA genes are scattered throughout the nucleus in Dnmt1 deficient cells. These findings suggest a role for Dnmt1 as an epigenetic caretaker for the maintenance of nucleolar structure.

Fluorescent cationic probes for nuclei of living cells: why are they selective? A quantitative structure–activity relations analysis

Selectivity of nuclear probes is controlled by competitive accumulation of the probe by cellular organelles as well as the high affinity for nucleic acids. Physicochemical features of probes which favor nucleic acid binding include cationic character and a planar aromatic system above a minimum size. Features of probes which permit entry into cells are low protein and lipid binding. Features which reduce accumulation in non-nuclear sites include high base strength and hydrophilicity of the cation. The overall quantitative structure–activity (QSAR) model specifying nuclear accumulation may be expressed as follows: CBN<40; 8>log Pneutral species>0; AI<8; Z>0; -510; LCF>17; LCF/CBN>0.70 (where CBN is the conjugated bond number, log Px the logarithm of the water–octanol partition coefficient of species x, AI the amphilicity index, Z the electric charge, pKa the negative logarithm of the equilibrium constant for the free base–protonated base reaction, and LCF the largest conjugated fragment). Preliminary applications of the QSAR model—to the selection of anticancer drugs, minimization of dye and drug toxicity and the designed synthesis of fluorescent probes—are outlined.

Acridine-orange differential fluorescence of fast- and slow-reassociating chromosomal DNA after in situ DNA denaturation and reassociation

A cytological technique based on heat denaturation of in situ chromosomal DNA followed by differential reassociation and staining with acridine orange was developed. Mouse nuclei and chromosomes in fixed cytological preparations show a red-orange fluorescence after thermal DNA denaturation (2–4 minutes at 100° C), and fluoresce green if denaturation is followed by a total DNA reassociation (two minutes or more at 65–66°C). — A reassociation time between a few and 60–90 seconds demonstrates the centromeric heterochromatin of chromosomes (which sometimes aggregate in the form of clusters) and the interphase chromocenters in green, the chromosomal arms fluorescing red-orange. Under the same conditions, the Y chromosome presents a pale green or yellow-green fluorescence along its chromatids, but its centromeric region fluoresces weakly. — The interpretation is suggested that the fast-reassociating chromosomal DNA (as detected by AO in centromeric heterochromatin and interphase chromocenters), represents repetitive DNA.

Do folate-receptor targeted liposomal photosensitizers enhance photodynamic therapy selectivity?

One of the current goals in photodynamic therapy research is to enhance the selective targeting of tumor cells in order to minimize the risk and the extension of unwanted side-effects caused by normal cell damage. Special attention is given to receptor mediated delivery systems, in particular, to those targeted to folate receptor. Incorporation of a model photosensitizer (ZnTPP) into a folate-targeted liposomal formulation has been shown to lead an uptake by HeLa cells (folate receptor positive cells) 2-fold higher than the non-targeted formulation. As a result, the photocytotoxicity induced by folate-targeted liposomes was improved. This selectivity was completely inhibited with an excess of folic acid present in the cell culture media. Moreover, A549 cells (folate receptor deficient cells) have not shown variations in the liposomal incorporation. Nevertheless, the differences observed were slighter than expected. Both folate-targeted and non-targeted liposomes localize in acidic lysosomes, which confirms that the non-specific adsorptive pathway is also involved. These results are consistent with the singlet oxygen kinetics measured in living cells treated with both liposomal formulations.

Loss of e-cadherin mediated cell-cell adhesion as an early trigger of apoptosis induced by photodynamic treatment

Photodynamic treatment with different photosensitizers (PSs) can result in the specific induction of apoptosis in many cell types. It is commonly accepted that this apoptotic response depends on the mitochondrial accumulation of the PS. Accumulation in other cellular organelles, such as lysosomes or the Golgi complex, and subsequent photodamage resulting in an apoptotic process has been also described. However, the role played by cell adhesion in apoptosis induced in epithelial cells after photodynamic treatment is not well characterized. Here, we have used a murine keratinocyte line, showing a strong dependence on E‐cadherin for cell–cell adhesion and survival, to analyze the relevance of this adhesion complex in the context of zinc(II)‐phthalocyanine (ZnPc) photodynamic treatment. We report that under apoptotic conditions, ZnPc phototreatment induces a rapid disorganization of the E‐cadherin mediated cell–cell adhesion, which largely preceded both the detachment of cells from the substrate, via β‐1 integrins and the induction of apoptotic mitochondrial markers. Therefore, the alteration in E‐cadherin, α‐ and β‐catenins adhesion proteins preceded the release of cytochrome c (cyt c) from mitochondria to the cytosol and the activation of caspase 3. In addition, blocking E‐cadherin function with a specific antibody (Decma‐1) induced apoptosis in this cell system. These results strongly suggest that the E‐cadherin adhesion complex could be the primary target of ZnPc phototreatment, and that loss of E‐cadherin mediated cell adhesion after early photodamage triggers an apoptotic response.

Photodynamic damage to HeLa cell microtubules induced by thiazine dyes

Abstract The aim of this study was to analyze possible alterations of the microtubule cytoskeleton of cultured cells subjected to photodynamic treatments with the thiazine dyes methylene blue or toluidine blue. Indirect immunofluorescence labeling of α-tubulin was performed in HeLa cells after 1 or 18 h of incubation with thiazines followed by red-light irradiation for 15 min [leading to surviving fractions (SF) of about 65% (SF65) or 1% (SF1), respectively]. Untreated control cells showed the normal distribution of interphase microtubules, whereas considerable or severe disorganization of the microtubule network was observed after SF65 or SF1 photodynamic treatments, respectively. A great amount of blebs showing homogeneous fluorescence was also found on the cell surface after SF1 treatments. Possible mechanisms responsible for the photodamage to microtubules induced by thiazine dyes are briefly discussed.

Protoporphyrin IX-dependent photodynamic production of endogenous ROS stimulates cell proliferation.

Photodynamic therapy using methyl 5-aminolevulinate (MAL) as a precursor of the photosensitizing agent protoporphyrin IX is widely used in clinical practice for the treatment of different pathologies, including cancer. In this therapeutic modality, MAL treatment promotes the forced accumulation of the endogenous photoactive compound protoporphyrin IX in target malignant cells. Subsequent irradiation of treated tissues with an appropriate visible light source induces the production of reactive oxygen species (ROS) that, once accumulated above a critical level, promote cell death. Here we demonstrate that a photodynamic treatment with low MAL concentrations can be used to promote a moderate production of endogenous ROS, which efficiently stimulates cell growth in human immortalized keratinocytes (HaCaT). We also show that this proliferative response requires Src kinase activity and is associated to a transient induction of cyclin D1 expression. Taken together, these results demonstrate for the first time that a combination of light and a photoactive compound can be used to modulate cell cycle progression through Src kinase activation and that a moderate intracellular increase of photogenerated ROS efficiently stimulates cell proliferation.