Christoffer Tamm

DNA damage induces two distinct modes of cell death in ovarian carcinomas.

Activation of p53 by cellular stress may lead to either cell cycle arrest or apoptotic cell death. Restrictions in a cells ability to halt the cell cycle might, in turn, cause mitotic catastrophe, a delayed type of cell death with distinct morphological features. Here, we have investigated the contribution of p53 and caspase-2 to apoptotic cell death and mitotic catastrophe in cisplatin-treated ovarian carcinoma cell lines. We report that both functional p53 and caspase-2 were required for the apoptotic response, which was preceded by translocation of nuclear caspase-2 to the cytoplasm. In the absence of functional p53, cisplatin treatment resulted in caspase-2-independent mitotic catastrophe followed by necrosis. In these cells, apoptotic functions could be restored by transient expression of wt p53. Hence, p53 appeared to act as a switch between apoptosis and mitotic catastrophe followed by necrosis-like lysis in this experimental model. Further, we show that inhibition of Chk2, and/or 14-3-3σ deficiency, sensitized cells to undergo mitotic catastrophe upon treatment with DNA-damaging agents. However, apoptotic cell death seemed to be the final outcome of this process. Thus, we hypothesize that the final mode of cell death triggered by DNA damage in ovarian carcinoma cells is determined by the profile of proteins involved in the regulation of the cell cycle, such as p53- and Chk2-related proteins.

Differential regulation of the mitochondrial and death receptor pathways in neural stem cells

Despite an increasing interest in neural stem cell (NSC) research, relatively little is known about the biochemical regulation of cell death pathways in these cells. We demonstrate here, using murine‐derived multipotent C17.2 NSCs, that cells undergo mitochondria‐mediated cell death in response to apoptotic stimuli such as oxidative stress induced by 2,3‐dimethoxy‐1,4‐naphthoquinone (DMNQ). In particular, treated cells exhibited apoptotic features, including Bax translocation, cytochrome c release, activation of caspase‐9 and ‐3, chromatin condensation and DNA fragmentation. Although C17.2 cells possess the Fas receptor and express procaspase‐8, agonistic Fas mAb treatment failed to induce apoptosis. Fas treatment activated the extracellular signal‐regulated protein kinase (ERK) pathway, which may have an antiapoptotic as well as a growth stimulating role. Combined, our findings indicate that while NSCs are sensitive to cytotoxic stimuli that involve an engagement of mitochondria, Fas treatment does not induce death and may have an alternative role.

Voltage-dependent anion channels (VDAC) in the plasma membrane play a critical role in apoptosis in differentiated hippocampal neurons but not in neural stem cells

microRNAs (miRNAs) are small non-coding RNAs that regulate a large variety of cellular processes including differentiation, apoptosis and proliferation. Several miRNAs display defective expression patterns in human tumors with the consequent alteration of target oncogene or tumor suppressor genes. Many of these miRNAs modulate the major proliferation pathways through direct interaction with critical regulators such as RAS, PI3K/PTEN or ABL, as well as members of the retinoblastoma pathway, Cyclin-CDK complexes or cell cycle inhibitors of the INK4 or Cip/Kip families. A complex interplay between miRNAs and MYC or E2F family members also exists to modulate cell cycle-dependent transcription during normal or tumoral proliferation. The ability of miRNAs to modulate these proliferation pathways may have relevant implications not only in physiological or developmental processes but also in tumor progression or cancer therapy.

Caspase-2 activation in neural stem cells undergoing oxidative stress-induced apoptosis

Oxidative stress occurs as a consequence of disturbance in the balance between the generation of reactive oxygen species (ROS) and the antioxidant defence mechanisms. The interaction of ROS with DNA can cause single-, or double-strand breaks that subsequently can lead to the activation of p53, which is central for the regulation of cellular response, e.g. apoptosis, to a range of environmental and intracellular stresses. Previous reports have suggested a regulatory role of p53 in the early activation of caspase-2, upstream of mitochondrial apoptotic signaling. Here we show that excessive ROS formation, induced by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) exposure, induces apoptosis in primary cultured neural stem cells (NSCs) from cortices of E15 rat embryos. Following DMNQ exposure cells exhibited apoptotic hallmarks such as Bax oligomerization and activation, cytochrome c release, caspase activation and chromatin condensation. Additionally, we could show early p53 accumulation and a subsequent activation of caspase-2. The attenuation of caspase-2 activity with selective inhibitors could antagonize the mitochondrial signaling pathway and cell death. Overall, our results strongly suggest that DMNQ-induced oxidative stress causes p53 accumulation and consequently caspase-2 activation, which in turn initiates apoptotic cell death via the mitochondria-mediated caspase-dependent pathway in NSCs.

Cell death in adult neural stem cells.

Neural stem cells (NSC) were discovered to exist in the adult brain. These cells have a capacity for self-renewal and can generate both neuronal and glial cells. Stem cells undergo apoptosis as an essential component of neural development. Previous studies suggest that embryonic and perinatal progenitor cells undergo apoptosis via activation of caspases and involvement of the Fas death receptor. Nothing, however, is known about the mechanisms by which adult NSC undergo cell death. Here we show that adult NSC undergo apoptosis via the mitochondrial pathway with caspase-3 serving as the executioner caspase in the apoptotic machinery. We utilized a primary culture method which promotes the growth of adult NSC. Primary tissue was harvested from the ependymal layer and underlying subventricular zone from the adult rat. Procedures used in animal experimentation comply with the Karolinska Institutes regulations for care and use of laboratory animals. Cells were cultured for 6 ± 8 days in a serumfree medium supplemented with epidermal growth factor in order to develop clonal spheres of cells, which were subsequently trypsinized and re-cultured for 4 days to generate secondary spheres before a final trypsinization procedure and cell attachment onto poly-L-lysine coated slides. After 45 minutes of cell attachment, cells were exposed for 18 h to staurosporine (STS) (0.25 mM), a known inducer of apoptosis. At the time of exposure, cells were found to express nestin (Figure 1a), an

Mechanisms and modulation of neural cell damage induced by oxidative stress

Oxidative stress has been linked to several neurodegenerative disorders characterized by neuronal death. Apoptosis and necrosis are the two major forms of cell death that have been described in the nervous system, and stimuli inducing oxidative stress can cause both types of death, depending on the intensity and the duration of the insult. In the present article, we report on a series of studies from our laboratory describing the intracellular pathways activated by oxidative stress in differentiated neurons, such as cerebellar granule cells, and neural stem cells. Using in vitro/ex vivo experimental models, we have investigated whether the susceptibility to injuries can be affected by the occurrence of potential insults taking place during development. We have found that prenatal exposure to high levels of glucocorticoids renders neural cells, including stem cells, more sensitive to oxidative stress damage. Similar effects were seen after in utero exposure to methylmercury. The analysis of behavior has proven to be a sensitive tool to detect mild alterations induced by early stimuli that increase susceptibility to oxidative stress. Our findings contribute to the understanding of how early events may have long-term consequences by modifying intracellular processes that predispose the affected cells to dysfunction, which can be unmasked or worsen by subsequent exposure to further injuries.

Mechanistic insight into neurotoxicity induced by developmental insults

Epidemiological and/or experimental studies have shown that unfavorable prenatal environmental factors, such as stress or exposure to certain neurotoxic environmental contaminants, may have adverse consequences for neurodevelopment. Alterations in neurogenesis can have harmful effects not only for the developing nervous system, but also for the adult brain where neurogenesis is believed to play a role in learning, memory, and even in depression. Many recent advances in the understanding of the complex process of nervous system development can be integrated into the field of neurotoxicology. In the past 15 years we have been using cultured neural stem or progenitor cells to investigate the effects of neurotoxic stimuli on cell survival, proliferation and differentiation, with special focus on heritable effects. This is an overview of the work performed by our group in the attempt to elucidate the mechanisms of developmental neurotoxicity and possibly provide relevant information for the understanding of the etiopathogenesis of complex brain disorders.