Uptake Kinetics and Intracellular Localization of Hypocrellin Photosensitizers for Photodynamic Therapy: A Confocal Microscopy Study

Miller, Gerald G.; Brown, Kevin M.; Moore, Ronald B.; McPhee, Malcolm S.; Diwu, Zhenjun; Liu, Jixiang; Huang, Liren; Lown, J. William; Begg, David A.; Chlumecky, Vera; Tulip, J.

doi:10.1111/j.1751-1097.1995.tb09880.x

Cited by 53 publications

(32 citation statements)

References 24 publications

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“…Others have found that photosensitizers localize to a variety of cellular sites including the outer membrane, lysosomes, mitochondria and the nucleus resulting in damage to these structures upon light exposure (Morgan et al, 2000;Oleinick and Evans, 1998). Although mitochondria are primary targets for some photosensitizers, other organelles were also considered as sites of action (Granville and Hunt, 2000;Lee et al, 1995;Miller et al, 1995). The present data provides evidence that internal membranes, particularly, the ER/Golgi apparatus is a distinct site from which an apoptotic signal can emerge.…”

Section: Discussionsupporting

confidence: 49%

Mechanism of colon cancer cell apoptosis mediated by pyropheophorbide-a methylester photosensitization

et al. 2001

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Pyropheophorbide-a methylester (PPME) is a second generation of photosensitizers used in photodynamic therapy (PDT). We demonstrated that PPME photosensitization triggered apoptosis of colon cancer cells as measured by using several classical parameters such as DNA laddering, PARP cleavage, caspase activation and mitochondrial release of cytochrome c. Preincubation of cells with N-acetyl cysteine (NAC) or pyrolidine dithiocarbamate (PDTC) protected against apoptosis mediated by PPME photosensitization showing that reactive oxygen species (ROS) are involved as second messengers. On the other hand, photosensitization carried out in the presence of deuterium oxide (D 2 O) which enhances singlet oxygen ( 1 O 2 ) lifetime only increases necrosis without aecting apoptosis. Since PPME was localized in the endoplasmic reticulum (ER)/Golgi system and lysosomes, other messengers than ROS were tested such as calcium, Bid, Bap31, phosphorylated Bcl-2 and caspase-12 but none was clearly identi®ed as being involved in triggering cytochrome c release from mitochondria. On the other hand, we demonstrated that the transduction pathways leading to NF-kB activation and apoptosis were clearly independent although NF-kB was shown to counteract apoptosis mediated by PPME photosensitization. Oncogene (2001) 20, 4070 ± 4084.

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Section: Discussionsupporting

confidence: 49%

Mechanism of colon cancer cell apoptosis mediated by pyropheophorbide-a methylester photosensitization

et al. 2001

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“…18 The cells were viewed directly in 35-mm petri dishes to which a small quantity of mounting medium (50:50, PBS:glycerol) and a coverslip were added. For uniformity, all physical parameters pertaining to fluorescence illumination and detection were held constant as follows: ×63 or ×100 oilimmersion objective lenses; KP 590 short-pass excitation filter; dual dichroic beam-splitter 488/568; BP535 (fluorescein) barrier filter; LP 590 (Texas red) barrier filter; photomultiplier gain, 800 V; pinhole, 60; offset, −45.…”

Section: Clsmmentioning

confidence: 99%

Cytoplasmic delivery of a macromolecular fluorescent probe by poly(d,l-lactic-co-glycolic acid) microspheres

Newman

Kwon

Miller

et al. 2000

J. Biomed. Mater. Res.

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A macromolecular fluorescent probe encapsulated in poly(d, l-lactic-co-glycolic acid) (PLGA) microspheres was used as a model for studying cytoplasmic delivery of antigens. We hypothesized that Texas red dextran loaded in PLGA microspheres would be delivered to the cytoplasm and that cytoplasmic delivery would be affected by polymer molecular weight. Cellular localization of the Texas red dextran was investigated at two different molecular weights of PLGA: 6000 and 60,000 g/mol. Intracellular degradation and processing of Texas red dextran-loaded PLGA microspheres by mouse peritoneal macrophages was monitored both in vitro and in vivo for a 7-day period using confocal laser scanning microscopy (CLSM). The results revealed cytoplasmic delivery of the fluorescent probe at both molecular weights of PLGA. Furthermore, the CLSM images showed that both in vitro and in vivo, the kinetics of microsphere degradation and cytoplasmic delivery were more rapid for the 6000 g/mol PLGA microspheres than the 60,000 g/mol PLGA microspheres. Hence, this study provides physical evidence that PLGA microspheres are capable of cytoplasmic delivery and that delivery to the cytosol can be controlled by modifying formulation parameters such as polymer molecular weight.

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“…The unbound fluorescent antibody was removed by extensive washing in PBS, and the slides were covered with coverslips for confocal microscopy using PBS/glycerol, 1:1 as a mounting medium. The instrumentation and the procedures for the confocal laser scanning microscopy have been described previously (38).…”

Section: Fig 4 Activities Of Recombinant Pnk a Comparison By Sds-mentioning

confidence: 99%

Molecular Characterization of a Human DNA Kinase

Karimi‐Busheri¹,

Daly²,

Robins³

et al. 1999

Journal of Biological Chemistry

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229

172

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Human polydeoxyribonucleotide kinase is an enzyme that has the capacity to phosphorylate DNA at 5-hydroxyl termini and dephosphorylate 3-phosphate termini and, therefore, can be considered a putative DNA repair enzyme. The enzyme was purified from HeLa cells. Amino acid sequence was obtained for several tryptic fragments by mass spectrometry. The sequences were matched through the dbEST data base with an incomplete human cDNA clone, which was used as a probe to retrieve the 5-end of the cDNA sequence from a separate cDNA library. The complete cDNA, which codes for a 521-amino acid protein (57.1 kDa), was expressed in Escherichia coli, and the recombinant protein was shown to possess the kinase and phosphatase activities. Comparison with other sequenced proteins identified a P-loop motif, indicative of an ATP-binding domain, and a second motif associated with several different phosphatases. There is reasonable sequence similarity to putative open reading frames in the genomes of Caenorhabditis elegans and Schizosaccharomyces pombe, but similarity to bacteriophage T4 polynucleotide kinase is limited to the kinase and phosphatase domains noted above. Northern hybridization revealed a major transcript of approximately 2.3 kilobases and a minor transcript of approximately 7 kilobases. Pancreas, heart, and kidney appear to have higher levels of mRNA than brain, lung, or liver. Confocal microscopy of human A549 cells indicated that the kinase resides predominantly in the nucleus. The gene encoding the enzyme was mapped to chromosome band 19q13.4.Transient DNA strand breaks and short gaps are frequently observed in cellular DNA. Many arise during regular cellular activity such as DNA replication, recombination, or differentiation. Others occur as a consequence of exposure to endogenous or exogenous DNA damaging agents. Repair of these strand interruptions is usually mediated by DNA ligases and polymerases. Both of these classes of enzymes require 3Ј-hydroxyl DNA termini, and the DNA ligases also require 5Ј-phosphate termini. However, the termini generated by nucleases, such as DNase II, and many produced by ionizing radiation bear 3Ј-phosphate and 5Ј-hydroxyl groups (1-4), and therefore must be processed before they can be acted upon by DNA ligases or polymerases.One enzyme that possesses the capacity to both phosphorylate 5Ј-hydroxyl termini and dephosphorylate 3Ј-phosphate termini is polynucleotide kinase (PNK).1 The PNK from T4 phage has found widespread application in molecular biology, especially for radiolabeling DNA and oligonucleotides (5). It can act on DNA and RNA and even phosphorylate nucleoside 3Ј-monophosphates. However, the main cellular function of the T4 enzyme is not to repair DNA, but rather to counter the action of a phage endoribonuclease that cleaves tRNA (6). Eukaryotic PNKs fall into two categories depending on whether their preferred substrate is DNA or RNA (7). While both can phosphorylate 5Ј-termini, only the former have an associated 3Ј-phosphatase activity (8 -12).Mammalian DNA kinases have ...

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Uptake Kinetics and Intracellular Localization of Hypocrellin Photosensitizers for Photodynamic Therapy: A Confocal Microscopy Study

Cited by 53 publications

References 24 publications

Mechanism of colon cancer cell apoptosis mediated by pyropheophorbide-a methylester photosensitization

Mechanism of colon cancer cell apoptosis mediated by pyropheophorbide-a methylester photosensitization

Cytoplasmic delivery of a macromolecular fluorescent probe by poly(d,l-lactic-co-glycolic acid) microspheres

Molecular Characterization of a Human DNA Kinase

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