Protein Expression Analysis in Uterine Cervical Cancer for Potential Targets in Treatment.

Specific markers in lesions of the human uterine cervix cancer (UCC) are still needed for prognostic, diagnostic and/or therapeutic purposes. In this study we evaluated key molecules at protein level between normal epithelium, cervical intraepithelial neoplasia (CIN1-3) and invasive cancer of a group of molecules previously reported at mRNA level. For that purpose, human formalin-fixed paraffin embedded tissue microarrays (TMAs) were constructed containing 205 Mexican tissue core specimens. Immunohistochemistry and quantitative analysis of histological staining was performed against twenty-two distinct proteins for each core and the processing platform ImageJ. In the progression of the disease we found key statistical differences for the proteins SEL1, Notch3 and SOCS3. High expressions of SEL1L, Notch3 and SOCS3 have potential value to increase the prognostic of UCC in combination with markers such as p16INK4a. This study identified key drivers in cervical carcinogenesis that should be evaluated for the development of UCC therapies.

Endogenous XIAP, but not other members of the inhibitory apoptosis protein family modulates cerebellar granule neurons survival.

Programmed cell death plays a critical role during cerebellar development. In particular, it has been shown in vivo and in vitro that developing cerebellar granule neurons (CGN) die apoptotically. Apoptosis involves a series of morphological changes and the activation of caspases. Inhibitor of apoptosis proteins (IAPs) is implicated in negative regulation of caspase activation and apoptotic cell death. Although apoptotic death of CGN has been extensively studied, there is no information about the role of IAPs in the developing cerebellum. Here, we studied the participation of some members of IAPs in the survival of the developing rat CGN in culture and under physiological conditions. Under these conditions, we found a differential expression pattern of cIAP-1, cIAP-2, XIAP and survivin during cerebellar development in an age-dependent manner, highlighting the significant increase of XIAP levels. We also detected an interaction between XIAP and caspase 3 at postnatal day (P) 12 and 16. On the other hand, we found a significant decrease of XIAP levels in cultured CGN maintained in chronic potassium deprivation, an apoptotic condition, suggesting a possible relationship between XIAP levels and neuronal viability. Under these conditions, we also detected the interaction of XIAP with active caspase-3. The down-regulation of XIAP in CGN cultured under survival conditions (chronic potassium depolarization) induced a reduction of cell viability and an increment of apoptotic cells. These findings support the idea that IAPs could be involved in the survival of CGN and that XIAP might be critical for neuronal survival in cerebellar development and during chronic depolarization in cultured CGN through a mechanism involving caspase inhibition.

Role for apoptosis-inducing factor in the physiological death of cerebellar neurons.

Apoptosis-inducing factor (AIF) is implicated in caspase-independent apoptotic-like death. AIF released from mitochondria translocates to the nucleus, where it mediates some apoptotic events such as chromatin condensation and DNA degradation. Here, the role of AIF in the neuronal death was studied under physiological conditions. When we analyzed the cellular localization of AIF during cerebellar development, we found a significant increase in the number of neurons with nuclear AIF localization in an age-dependent manner. On the other hand, cerebellar granule neurons (CGN) chronically cultured in low concentration of potassium (5 mM; K5) die with apoptotic-like characteristics after five days. In the present study we found that K5 induces a caspase-dependent apoptotic-like death of CGN as well as a late nuclear translocation of AIF. When CGN death induced by K5 was carried out in the presence of a general inhibitor of caspases, there was a slight decrement of cell death, but neurons eventually died by showing apoptotic-like features such as phosphatidylserine translocation and nuclear condensation. Besides, there was a significant increment of nuclear AIF translocation. These findings support the idea that AIF could be involved in apoptotic-like death of CGN and that it could be an alternative mechanism of neuronal death during cerebellar development.

Oxidation of biomolecules in the apoptotic death of cerebellar granule neurons induced by potassium deprivation.

Cerebellar granule neurons (CGN) cultured in a 25 mM KCl medium (K25) die apoptotically when they are transferred to a medium containing 5 mM KCl (K5). It has been previously shown that apoptotic death of CGN induced by K5 is mediated by an increase in the levels of reactive oxygen species (ROS). ROS may participate in the apoptotic program either as signaling molecules or as effectors by causing oxidative damage to lipids, DNA and proteins. In this study we evaluated ROS production in CGN treated with K5 for different periods of time and evaluated a possible correlation between ROS production and oxidation of DNA proteins and two lipid peroxidation products, conjugated dienes and malondialdehyde. Under these conditions, we found two episodes of ROS generation, one at an early time (4 h) and another at a later time point (18-24 h). We also identified two peaks in the formation of conjugated dienes, the initial and transient by-products of lipoperoxidation. The first one occurred after 4 h of K5 treatment and the other was observed after 18 h, both of them correlated with the formation of ROS. In contrast, we found significant levels of the late product of lipidperoxidation, malondialdehyde only after 18 h of treatment. Besides, we did not find significant levels of DNA and protein oxidation products that could be correlated with the observed ROS production. These results support the idea that ROS produced early by K5 treatment could act primarily as a signal of the apoptotic cell death and that ROS produced later could be mainly a product of the cell death that could contribute directly to this process.

Effect of N-methyl-D-aspartate receptor blockade on caspase activation and neuronal death in the developing rat cerebellum.

In vitro studies have demonstrated that N-methyl-D-aspartate (NMDA) receptor activation rescue cerebellar granule neurons (CGN) from apoptotic death. It has been suggested that this effect mimics the transient glutamate receptors activation by mossy fibers during cerebellar development. We reported previously that CGN from postnatal days 2-4 (P2-P4) rats increased cell survival in response to NMDA treatment. In this study, we evaluated the effect of dizocilpine (MK-801) administrated for three consecutive days on the apoptotic death of CGN during development. MK-801 treatment decreased the large number of CGN condensed nuclei found at P8, but this drug increased the proportion of condensed nuclei at P16. We also found a high activity of caspases during the first postnatal week that decreased during development. MK-801 treatment did not modify the activity of caspase-8 at any age, but decreased caspase-9 activity at P8 and increased the activity of caspase-1 (76%) at P8, caspase-3 (160%) at P16 and caspase-9 (50%) at P12. These results suggest that NMDA receptor stimulation regulates the activity of caspases in a differential way and plays an important role in the in vivo CGN death during postnatal development.

Effect of NMDA antagonists on the death of cerebellar granule neurons at different ages.

Cerebellar granule neurons (CGN) are the most abundant neuronal type in the cerebellum. During development, these cells migrate from the external to the internal granule layer (IGL), where they receive excitatory glutamatergic and cholinergic contacts from mossy fibers. During this period of development a large proportion of CGN are eliminated via apoptosis. In vitro studies have demonstrated that when CGN are obtained from rats at postnatal day 8 (P8), the sustained activation of N-methyl-D-aspartate (NMDA) receptor at 2-4 days in vitro rescues neurons from cell death. The NMDA action on cultured CGN could mimic the in vivo actions of the transient activation of the glutamate receptors by the transmitter released by mossy fibers by P12. However, some results suggest that glutamate stimulation could be relevant for CGN at earlier stages of development. In this study we evaluated the effect of NMDA receptor stimulation or blockade on the cell death of both in vivo and cultured CGN obtained from P2 to P8 rats. Our results showed that the blockade of NMDA receptors with the antagonists D,L-2-amino-5-phosphonovaleric acid or dizocilpine (MK-801) reduces cell survival to 20-40%, whereas NMDA treatment increases neuronal survival by approximately 50-60%. In vivo, the treatment with MK-801 reduced the number of apoptotic CGN in the molecular layer (ML) from P5 to P8. These results suggest that NMDA receptor stimulation plays a critical role in the regulation of CGN death during the first week of rat cerebellar development.