TOXICOLOGY: Subcellular Targets of Anticancer Therapy
March 22-24 , 2006

Place: Room Prof. Dr. Fernando Manuel Oliveira Sá, National Institute of Legal Medicine, School of Medicine (Sala 27 do Instituto de Medicina Legal, Faculdade de Medicina)
Lecturers:  
  Sten Orrenius
  Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Box 210, SE-171 77 Stockholm, Sweden
  Catherine Brenner
  Centre National de la Recherche Scientifique, Formation de Recherche en Evolution 2445, Universite de Versailles/St. Quentin,Versailles, France
  Ulf Rapp
  Institut fur Medizinische, Strahlenkunde und Zellforschung (MSZ), Bayerische Julius-Maximilians-Universitat, Universitat Wurzburg, Versbacher-Strasse 5, Wurzburg, Germany
  Jon Holy
  Department of Anatomy and Cell Biology, University of Minnesota School of Medicine, Duluth, MN, USA
Coordinators:



  Paulo J. Oliveira
  Leonor M. Almeida
  Carlos M. Palmeira
  Joao Laranjinha
 

Center for Neurosciences and Cellular Biology of Coimbra, Department of Zoology and School of Pharmacy, University of Coimbra
 
Informations:  
  Paulo J. Oliveira

Area: CELL AND MOLECULAR TOXICOLOGY

Objectives:

The objective of this course is to describe some mechanisms by which anti-cancer drugs interact with intracellular targets. The nuclei, mitochondria, cytoskeleton and signaling pathways will be specially focused in this course. Due to the importance of mitochondria in many cellular processes of life and death, several lectures will focus on the role of mitochondria and especially of the permeability transition pore as a target for anti-cancer therapy.
The lecturers will also spend some time providing theoretical background on biological systems and techniques that can be used by the researcher as a tool to study the anti-neoplasic potential of some novel drugs. The program will also count with some presentations of Ph.D. students from the University of Coimbra and from other Universities who presently work in the topic of the course. The objective of the presentations (three/four each day) is to stimulate discussion between the students presenting their work and the audience. The discussions will be moderated by the lecturers.

The course is the 2006 edition of the “International Courses on Toxicology”, organized by the Cell and Molecular Toxicology Group of the CNC on a yearly basis.

TASKS FOR DOCTORAL STUDENTS
A different type of assignment will be proposed for this course. Students will be divided into 6 pre-defined groups of 2 students each. Each group will be in charge of writing a short review manuscript about a specified topic related with the theme of the course. The text should be written in English, with 1 figure and appropriate references. It must be written in font size 12 and double-spaced (maximum 6 pages, including references).
Groups and themes:
 
1-
 The mitochondrial permeability transition pore as a target for anti-cancer therapy
Ana Cristovão/Mariana Bexiga
2-
Confocal microscopy as a tool to study morphological alterations induced by anti-neoplastic agents - examples and advantages vs. normal epifluorescence microscopy
Ana Teles/Ricardo Marques
3-
 Vital imaging of multi-cellular spheroids to study anti-cancer drug accumulation and cellular effects

Ricardo Soares/ Catarina Pimentel
4–
 Multi-drug resistance proteins and failure of anti-neoplastic treatments
Helena Domingues/Joana Lourenço
5–
 Cell surface NADH oxidases as a target in anti-cancer therapy
Gisela Silva/Carina Santos
6–

Endoplasmic reticulum-mediated cell death and anti-cancer therapy
Eduardo Ferreira/Lígia Gomes
The manuscripts should be delivered March 24 (the last day of the course) or earlier. I will be available for further details (pauloliv@ci.uc.pt, or at the Zoology Department). If needed, during the course the students can interact with the lecturers in order to obtain further insights on the topics.
Several papers on the subjects can be easily found on Pubmed or Current Contents.
COURSE SCHEDULE:
Wednesday, March 22
9:00 – 10:00
 Course opening
10:00 – 11:00
 Mitochondrial regulation of cell death
Sten Orrenius
11:00-11:30
 Coffee break
11:30-12:30
 Nuclear and Cytoskeletal Targets of Novel Phytochemicals with Chemotherapeutic Potential
Jon Holy
   
14:00-16:30
 Round Table 1 (includes four short presentations with general discussion).
Head of discussion: Catherine Brenner, Ulf Rapp
Thursday, March 23
9:00 – 10:00
 Mitochondrial membrane permeabilization involving the permeability transition pore
Catherine Brenner
10:00 – 11:00
 Mitogenic signaling
Ulf Rapp
11:00-11:30
 Coffee break
11:30-12:30
 Role of caspase-2 in DNA damage-induced apoptosis
Sten Orrenius
   
14:00-16:30

Round Table 2 (includes four short presentations with general discussion).

Head of discussion: Jon Holy, Sten Orrenius
Friday, March 24
9:00 – 10:00
 Enzymatic Assays for Toxicological Studies in Isolated Mitochondria and in Proteoliposomes
Catherine Brenner
10:00-10:30
 Coffee break
10:30 – 11:30
 Morphological Approaches Toward Assessing Cellular Responses to Novel Anti-cancer Chemotherapeutics
Jon Holy
11:30 – 13:00
 Cancer stem cells and reversibility of differentation
Ulf Rapp
   
16:00 – 17:00
 Closing seminar:
Mitochondria, oxidative stress and apoptosis
Sten Orrenius

Lecturers, Affiliations and Selected publications
Sten Orrenius - Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Box 210, SE-171 77 Stockholm, Sweden
Zhivotovsky B, Orrenius S: Caspase-2 function in response to DNA damage. Biochem Biophys Res Commun 2005, 331:859-867.
Elinder F, Akanda N, Tofighi R, Shimizu S, Tsujimoto Y, Orrenius S, Ceccatelli S: Opening of plasma membrane voltage-dependent anion channels (VDAC) precedes caspase activation in neuronal apoptosis induced by toxic stimuli. Cell Death Differ 2005, 12:1134-1140.
Orrenius S: Mitochondrial regulation of apoptotic cell death. Toxicol Lett 2004, 149:19-23.
Gogvadze V, Robertson JD, Enoksson M, Zhivotovsky B, Orrenius S: Mitochondrial cytochrome c release may occur by volume-dependent mechanisms not involving permeability transition. Biochem J 2004, 378:213-217.

Enoksson M, Robertson JD, Gogvadze V, Bu P, Kropotov A, Zhivotovsky B, Orrenius S: Caspase-2 permeabilizes the outer mitochondrial membrane and disrupts the binding of cytochrome c to anionic phospholipids. J Biol Chem 2004, 279:49575-49578.
Catherine Brenner - Centre National de la Recherche Scientifique, Formation de Recherche en Evolution 2445, Universite de Versailles/St. Quentin,Versailles, France.
Weaver JG, Tarze A, Moffat TC, Lebras M, Deniaud A, Brenner C, Bren GD, Morin MY, Phenix BN, Dong L, et al.: Inhibition of adenine nucleotide translocator pore function and protection against apoptosis in vivo by an HIV protease inhibitor. J Clin Invest 2005, 115:1828-1838.
Deniaud A, Brenner C, Kroemer G: Mitochondrial membrane permeabilization by HIV-1 Vpr. Mitochondrion 2004, 4:223-233.
Brenner C, Le Bras M, Kroemer G: Insights into the mitochondrial signaling pathway: what lessons for chemotherapy? J Clin Immunol 2003, 23:73-80.
Belzacq AS, Brenner C: The adenine nucleotide translocator: a new potential chemotherapeutic target. Curr Drug Targets 2003, 4:517-524.
Brenner C, Kroemer G: Apoptosis. Mitochondria--the death signal integrators. Science 2000, 289:1150-1151.
Ulf Rapp - Institut fur Medizinische, Strahlenkunde und Zellforschung (MSZ), Bayerische Julius-Maximilians-Universitat, Universitat Wurzburg, Versbacher-Strasse 5, Wurzburg, Germany.

Hekman M, Fischer A, Wennogle LP, Wang YK, Campbell SL, Rapp UR: Novel C-Raf phosphorylation sites: serine 296 and 301 participate in Raf regulation. FEBS Lett. 2005, 579(2): 464-468.
Gentschev I, Fensterle J, Schmidt A, Potapenko T, Troppmair J, Goebel W, Rapp UR: Use of a recombinant Salmonella enterica serovar Typhimurium strain expressing C-Raf for protection against C-Raf induced lung adenoma in mice. BMC Cancer. 2005,
9;5(1):15. 
Fensterle J, Becker JC, Potapenko T, Heimbach V, Vetter CS, Brocker EB, Rapp UR: B-Raf specific antibody responses in melanoma patients. BMC Cancer 2004, 4:62.
Rapp UR, Fensterle J, Albert S, Gotz R: Raf kinases in lung tumor development. Adv Enzyme Regul 2003, 43:183-195.
Rennefahrt U, Illert B, Greiner A, Rapp UR, Troppmair J: Tumor induction by activated JNK occurs through deregulation of cellular growth. Cancer Lett 2004, 215:113-124.
Jon Holy - Department of Anatomy and Cell Biology, University of Minnesota School of Medicine, Duluth, MN, USA

1. Holy J, Lamant G, Perkins E: Disruption of nucleocytoplasmic trafficking of cyclin D1 and topoisomerase II by sanguinarine. BMC Cell Biology 2005 (submitted).
Rolo AP, Palmeira CM, Holy J, Wallace KB: Role of mitochondrial dysfunction in combined bile acid-induced cytotoxicity: the switch between apoptosis and necrosis. Toxicol Sci 2004, 79:196-204.
Holy J: Curcumin inhibits cell motility and alters microfilament organization and function in prostate cancer cells. Cell Motil Cytoskeleton 2004, 58:253-268.
Holy J: Curcumin disrupts mitotic spindle structure and induces micronucleation in MCF-7 breast cancer cells. Mutat Res 2002, 518:71-84.
Pierce T, Worman HJ, Holy J. Neuronal differentiation of NT2/D1 teratocarcinoma cells is accompanied by a loss of lamin A/C expression and an increase in lamin B1 expression. Exp Neurol. 1999,157(2), 241-50. 
Coordinators

Paulo J. Oliveira, Ph.D.
Ascensao A, Magalhaes J, Soares JM, Ferreira R, Neuparth MJ, Marques F, Oliveira PJ, Duarte JA: Moderate endurance training prevents doxorubicin-induced in vivo mitochondriopathy and reduces the development of cardiac apoptosis. Am J Physiol Heart Circ Physiol 2005, 289:H722-731.
Oliveira PJ, Bjork JA, Santos MS, Leino RL, Froberg MK, Moreno AJ, Wallace KB: Carvedilol-mediated antioxidant protection against doxorubicin-induced cardiac mitochondrial toxicity. Toxicol Appl Pharmacol 2004, 200:159-168.
Oliveira PJ, Seica R, Coxito PM, Rolo AP, Palmeira CM, Santos MS, Moreno AJ: Enhanced permeability transition explains the reduced calcium uptake in cardiac mitochondria from streptozotocin-induced diabetic rats. FEBS Lett 2003, 554:511-514.

Leonor Almeida, Ph.D.
Custodio JB, Cardoso CM, Almeida LM: Thiol protecting agents and antioxidants inhibit the mitochondrial permeability transition promoted by etoposide: implications in the prevention of etoposide-induced apoptosis. Chem Biol Interact 2002, 140:169-184.
Cardoso CM, Almeida LM, Custodio JB: 4-Hydroxytamoxifen is a potent inhibitor of the mitochondrial permeability transition. Mitochondrion 2002, 1:485-495.
Brito P, Almeida LM, Dinis TC: The interaction of resveratrol with ferrylmyoglobin and peroxynitrite; protection against LDL oxidation. Free Radic Res 2002, 36:621-631.

Joao Laranjinha, Ph.D.
Ledo A, Frade J, Barbosa RM, Laranjinha J: Nitric oxide in brain: diffusion, targets and concentration dynamics in hippocampal subregions. Mol Aspects Med 2004, 25:75-89.
Antunes F, Nunes C, Laranjinha J, Cadenas E: Redox interactions of nitric oxide with dopamine and its derivatives. Toxicology 2005, 208:207-212.
Nunes C, Almeida L, Laranjinha J: Synergistic inhibition of respiration in brain mitochondria by nitric oxide and dihydroxyphenylacetic acid (DOPAC). Implications for Parkinson's disease. Neurochem Int 2005, 47:173-182.

Carlos Palmeira, Ph.D.
Palmeira CM, Rolo AP: Mitochondrially-mediated toxicity of bile acids. Toxicology 2004, 203:1-15.
Rolo AP, Palmeira CM, Holy JM, Wallace KB: Role of mitochondrial dysfunction in combined bile acid-induced cytotoxicity: the switch between apoptosis and necrosis. Toxicol Sci 2004, 79:196-204.
Rolo AP, Palmeira CM, Wallace KB: Mitochondrially mediated synergistic cell killing by bile acids. Biochim Biophys Acta 2003, 1637:127-132.