The role of peroxisomes in oxidative stress

Peroxisomes are one of the main sites in the cell where oxygen free radicals are both generated and scavenged. The balance between these two processes is believed to be of great importance for proper functioning of cells and has been implicated in aging and carcinogenesis. To gain more insight in the functioning of the peroxisome, we developed a new method to measure the biophysical properties of these organelles in the living cell. To achieve this we made use of small fluorescently labelled polypeptides with a peroxisomal targeting signal of type I. Using such a probe, we determined the peroxisomal matrix is alkaline and that the peroxisomal membrane is impermeable to protons under physiological conditions. Oxidative stress deriving from peroxisomal b-oxidation is to be detoxified by a set of anti-oxidant enzymes. In this thesis I show that next to the well known enzymes catalase and MnSOD, the sterol carrier protein 2 (SCP2) plays a role in the protection against oxidative stress. We show that mice lacking this protein have higher basal levels of lipid-peroxidation in the liver and develop liver tumours. In a model I propose that this peroxisomal protein shields poly-unsaturated fatty acyl-CoA esters from free radicals that are present in the organellar matrix. The regulation of anti-oxidant activity in the cell has been a matter of study. We show that MnSOD is directly regulated by the daf-16 like Forkhead transcription factors. Forkhead transcription factors are negatively regulated by phosphatidyl-inositol-3-OH-kinase (PI-3-kinase) through phosphokinase B (PKB). Catalase and SCP2 are also increased upon activation of the Forkheads, but until now we do not know whether this reflects a direct regulation. A model is proposed for the regulation of anti-oxidant capacity of the cell in relation to metabolism

Возможность прогнозирования клеточного типа увеальных меланом без использования инвазивных методов диагностики

Резюме. С помощью дискриминантного анализа установлена возможность определения клеточного типа меланомы увеального тракта в процессе проведения комбинированного (фотокоагуляция + брахитерапия) лечения. Разработана высокозначимая (l = 0,08; р = 0,002) дискриминантная модель, включающая ряд клинических (степень пигментации, пол, скорость роста меланомы) и иммунологических (количество Т- и В-лимфоцитов, процент Т-хелперов и др.) показателей. Особое место в модели занимают признаки, в наибольшей степени отражающие биологические особенности увеальных меланом различного клеточного состава, а именно — скорость изменения размера опухоли в процессе лечения и изменение показателей клеточного иммунитета. Ключевые слова: увеальная меланома, клеточный тип, клинико-морфологические, иммунологические показатели, дискриминантный анализ.Summary. Application of the discriminant analysis shows that it is possible to define the cell type of melanoma of uveal tract of the eye in the process of combined (photocoagulation + brachytherapy) treatment. A highly reliable (l= 0,08; р = 0,002) discriminant model was elaborated, involving a number of both clinical (pigmentation level, gender, melanoma growth rate) and immunological (number of T and B lymphocytes, T helper rate, etc.) indicators. In this model, especially important are those traits that most pronouncedly reflect the biological peculiarities of uveal melanomas of various cellular compositions, namely — the pace of tumor size growth in the process of treatment and changes in cell immunity indicators. Key Words: uveal melanoma, cell type, clinical and morphological, immunological indicators, discriminant analysis

The role of peroxisomes in oxidative stress

Peroxisomes are one of the main sites in the cell where oxygen free radicals are both generated and scavenged. The balance between these two processes is believed to be of great importance for proper functioning of cells and has been implicated in aging and carcinogenesis. To gain more insight in the functioning of the peroxisome, we developed a new method to measure the biophysical properties of these organelles in the living cell. To achieve this we made use of small fluorescently labelled polypeptides with a peroxisomal targeting signal of type I. Using such a probe, we determined the peroxisomal matrix is alkaline and that the peroxisomal membrane is impermeable to protons under physiological conditions. Oxidative stress deriving from peroxisomal b-oxidation is to be detoxified by a set of anti-oxidant enzymes. In this thesis I show that next to the well known enzymes catalase and MnSOD, the sterol carrier protein 2 (SCP2) plays a role in the protection against oxidative stress. We show that mice lacking this protein have higher basal levels of lipid-peroxidation in the liver and develop liver tumours. In a model I propose that this peroxisomal protein shields poly-unsaturated fatty acyl-CoA esters from free radicals that are present in the organellar matrix. The regulation of anti-oxidant activity in the cell has been a matter of study. We show that MnSOD is directly regulated by the daf-16 like Forkhead transcription factors. Forkhead transcription factors are negatively regulated by phosphatidyl-inositol-3-OH-kinase (PI-3-kinase) through phosphokinase B (PKB). Catalase and SCP2 are also increased upon activation of the Forkheads, but until now we do not know whether this reflects a direct regulation. A model is proposed for the regulation of anti-oxidant capacity of the cell in relation to metabolism

FOXO Transcription Factors Both Suppress and Support Breast Cancer Progression

Cancer Signaling networks and Molecular Therapeutic

Activation of forkhead box O transcription factors by oncogenic BRAF promotes p21cip1-dependent senescence

Oncogene-induced senescence (OIS) is a potent tumor-suppressive mechanism that is thought to come at the cost of aging. The Forkhead box O (FOXO) transcription factors are regulators of life span and tumor suppression. However, whether and how FOXOs function in OIS have been unclear. Here, we show a role for FOXO4 in mediating senescence by the human BRAFV600E oncogene, which arises commonly in melanoma. BRAFV600E signaling through mitogen-activated protein kinase/extracellular signal-regulated kinase kinase resulted in increased reactive oxygen species levels and c-Jun NH 2 terminal kinase-mediated activation of FOXO4 via its phosphorylation on Thr223, Ser226, Thr447, and Thr451. BRAFV600E-induced FOXO4 phosphorylation resulted in p21cip1-mediated cell senescence independent of p16 ink4a or p27kip1. Importantly, melanocyte-specific activation of BRAFV600E in vivo resulted in the formation of skin nevi expressing Thr223/Ser226-phosphorylated FOXO4 and elevated p21cip1. Together, these findings support a model in which FOXOs mediate a trade-off between cancer and aging

Cysteine oxidation triggers amyloid fibril formation of the tumor suppressor p16(INK4A).

The tumor suppressor p16(INK4A) induces cell cycle arrest and senescence in response to oncogenic transformation and is therefore frequently lost in cancer. p16(INK4A) is also known to accumulate under conditions of oxidative stress. Thus, we hypothesized it could potentially be regulated by reversible oxidation of cysteines (redox signaling). Here we report that oxidation of the single cysteine in p16(INK4A) in human cells occurs under relatively mild oxidizing conditions and leads to disulfide-dependent dimerization. p16(INK4A) is an all a-helical protein, but we find that upon cysteine-dependent dimerization, p16(INK4A) undergoes a dramatic structural rearrangement and forms aggregates that have the typical features of amyloid fibrils, including binding of diagnostic dyes, presence of cross-beta sheet structure, and typical dimensions found in electron microscopy. p16(INK4A) amyloid formation abolishes its function as a Cyclin Dependent Kinase 4/6 inhibitor. Collectively, these observations mechanistically link the cellular redox state to the inactivation of p16(INK4A) through the formation of amyloid fibrils