P53 Family Proteins Research Articles

Oncogenic Viral Protein Interactions with p53 Family Proteins

Oncogenic Viral Protein Interactions with p53 Family Proteins

Regulation of apoptosis in the ischemic penumbra in the first day post-stroke.

Regulation of apoptosis in the ischemic penumbra in the first day post-stroke.

DMPK is a New Candidate Mediator of Tumor Suppressor p53-Dependent Cell Death

Tumor suppressor p53 plays an integral role in DNA-damage induced apoptosis, a biological process that protects against tumor progression. Cell shape dramatically changes when cells undergo apoptosis, which is associated with actomyosin contraction; however, it remains entirely elusive how p53 regulates actomyosin contraction in response to DNA-damaging agents. To identify a novel p53 regulating gene encoding the modulator of myosin, we conducted DNA microarray analysis. We found that, in response to DNA-damaging agent doxorubicin, expression of myotonic dystrophy protein kinase (DMPK), which is known to upregulate actomyosin contraction, was increased in a p53-dependent manner. The promoter region of DMPK gene contained potential p53-binding sequences and its promoter activity was increased by overexpression of the p53 family protein p73, but, unexpectedly, not of p53. Furthermore, we found that doxorubicin treatment induced p73 expression, which was significantly attenuated by downregulation of p53. These data suggest that p53 induces expression of DMPK through upregulating p73 expression. Overexpression of DMPK promotes contraction of the actomyosin cortex, which leads to formation of membrane blebs, loss of cell adhesion, and concomitant caspase activation. Taken together, our results suggest the existence of p53-p73-DMPK axis which mediates DNA-damage induced actomyosin contraction at the cortex and concomitant cell death.

Reciprocal Crosstalk Between YAP1/Hippo Pathway and the p53 Family Proteins: Mechanisms and Outcomes in Cancer.

The YAP1/Hippo and p53 pathways are critical protectors of genome integrity in response to DNA damage. Together, these pathways secure cellular adaptation and maintain overall tissue integrity through transcriptional re-programing downstream of various environmental and biological cues generated during normal tissue growth, cell proliferation, and apoptosis. Genetic perturbations in YAP1/Hippo and p53 pathways are known to contribute to the cells’ ability to turn rogue and initiate tumorigenesis. The Hippo and p53 pathways cooperate on many levels and are closely coordinated through multiple molecular components of their signaling pathways. Several functional and physical interactions have been reported to occur between YAP1/Hippo pathway components and the three p53 family members, p53, p63, and p73. Primarily, functional status of p53 family proteins dictates the subcellular localization, protein stability and transcriptional activity of the core component of the Hippo pathway, Yes-associated protein 1 (YAP1). In this review, we dissect the critical points of crosstalk between the YAP1/Hippo pathway components, with a focus on YAP1, and the p53 tumor suppressor protein family. For each p53 family member, we discuss the biological implications of their interaction with Hippo pathway components in determining cell fate under the conditions of tissue homeostasis and cancer pathogenesis.

Gliotoxin Enhances Autophagic Cell Death via the DAPK1-TAp63 Signaling Pathway in Paclitaxel-Resistant Ovarian Cancer Cells.

Death-associated protein kinase 1 (DAPK1) expression induced by diverse death stimuli mediates apoptotic activity in various cancers, including ovarian cancer. In addition, mutual interaction between the tumor suppressor p53 and DAPK1 influences survival and death in several cancer cell lines. However, the exact role and connection of DAPK1 and p53 family proteins (p53, p63, and p73) in drug-resistant ovarian cancer cells have not been studied previously. In this study, we investigated whether DAPK1 induction by gliotoxin derived from marine fungus regulates the level of transcriptionally active p63 (TAp63) to promote apoptosis in an autophagy-dependent manner. Pre-exposure of paclitaxel-resistant ovarian cancer cells to gliotoxin inhibited the expression of multidrug resistant-associated proteins (MDR1 and MRP1-3), disrupted the mitochondrial membrane potential, and induced caspase-dependent apoptosis through autophagy induction after subsequent treatment with paclitaxel. Gene silencing of DAPK1 prevented TAp63-mediated downregulation of MDR1 and MRP1-3 and autophagic cell death after sequential treatment with gliotoxin and then paclitaxel. However, pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, had no effect on the levels of DAPK1 and TAp63 or on the inhibition of MDR1 and MRP1-3. These results suggest that DAPK1-mediated TAp63 upregulation is one of the critical pathways that induce apoptosis in chemoresistant cancer cells.

Jorunnamycin A from Xestospongia sp. Suppresses Epithelial to Mesenchymal Transition and Sensitizes Anoikis in Human Lung Cancer Cells.

Metastasis is a key driving force behind the high mortality rate associated with lung cancer. Herein, we report the first study revealing the antimetastasis activity of jorunnamycin A, a bistetrahydroisoquinolinequinone isolated from a Thai blue sponge Xestospongia sp. evidenced by its inhibition of epithelial to mesenchymal transition (EMT), sensitization of anoikis, and suppression of anchorage-independent survival in human lung cancer cells. Treatment with jorunnamycin A (0.05-0.5 μM) altered the expression of p53 and Bcl-2 family proteins, particularly causing the down-regulation of antiapoptosis Bcl-2 and Mcl-1 proteins. Under detachment conditions for 12 h, jorunnamycin A-treated cells exhibited diminution of pro-survival proteins p-Akt and p-Erk as well as the survival-promoting factor caveolin-1. Corresponding with the inhibition on the Akt and Erk pathway as well as activation of p53, there was an increase in the epithelial marker E-cadherin and a remarkable decrease of EMT markers and associated proteins including vimentin, snail, and claudin-1. As the loss of anchorage dependence is an important barrier to metastasis, the observed inhibitory effects of jorunnamycin A on the coordinating networks of EMT and anchorage-independent growth emphasize the potential development of jorunnamycin A as an effective agent against lung cancer metastasis.

Abstract 5308: BRMS1L, a metastatic suppressor gene, is transcriptionally regulated by p53

Abstract The tumor suppressor p53 plays a pivotal role in the cell fate determination in response to diverse upstream signals. p53 regulates a number of genes that are involved in cell cycle arrest, apoptosis, senescence, and maintenance of genomic stability. Recent studies revealed that p53 is also important for the regulation of cell invasion and migration. Our group has previously revealed that the p53 transactivates anti-invasion genes such as CLCA2, ICAM2, and LIMA1. However, the role of the p53 downstream pathway in cancer invasion, migration, and metastasis remains unclear. In this study, microarray analysis showed that breast cancer metastasis-suppressor 1- like (BRMS1L) is upregulated by p53. We identified two responsive elements of p53 family proteins in the first intron and upstream of BRMS1L. These response elements are well conserved among mammals. Functional analysis showed that ectopic expression of BRMS1L inhibited cancer cell invasion and migration; knockdown of BRMS1L by siRNA induced the opposite effect. Importantly, clinical databases revealed that reduced BRMS1L expression correlated with poor prognosis in patients with breast and brain cancer. Together, these results strongly demonstrate that BRMS1L is one of the mediators downstream of the p53 pathway, and that BRMS1L inhibits cancer cell invasion and migration, which are essential steps in cancer metastasis. Collectively, our results suggest that BRMS1L is involved in cancer cell invasion and migration, and could be a therapeutic target for cancer. Citation Format: Takashi Tokino, Ryota Koyama, Masashi Idogawa, Yasushi Sasaki. BRMS1L, a metastatic suppressor gene, is transcriptionally regulated by p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5308.

C-terminal α Domain of p63 Binds to p300 to Coactivate β-Catenin

TP63 (p63), a member of the tumor suppressor TP53 (p53) gene family, is essential for ectodermal tissue development and suppresses malignant progression of carcinomas. The most abundant isoform, ΔNp63α (referred to as p63), lacks the N-terminal transactivation (TA) domain, and was originally characterized as a dominant-negative type suppressor against p53 family proteins. It also binds to TCF/LEF to inhibit β-catenin. Nevertheless, transcriptional activation by p63 has also been observed in varied systems. To understand the puzzling results, we analyzed the structure–function relationship of p63 in the control of β-catenin-dependent transcription. p63 acted as a suppressor of moderately induced β-catenin. However, when nuclear targeted S33Y β-catenin was applied to cause the maximum enhancer activation, p63 displayed a β-catenin-coactivating function. The DNA-binding domain of p63 and the target sequence facilitated it. Importantly, we newly found that, despite the absence of TA domain, p63 was associated with p300, a general adaptor protein and chromatin modifier causing transcriptional activation. C-terminal α domain of p63 was essential for p300-binding and for the coactivator function. These results were related to endogenous p63-p300 complex formation and Wnt/β-catenin-responsive gene regulation by p63 in squamous cell carcinoma lines. The novel p63-p300 interaction may be involved in positive regulation of gene expression in tissue development and carcinogenesis.

TP53BP2 decreases cell proliferation and induces autophagy in neuroblastoma cell lines

Tumor protein p53-binding protein 2 (TP53BP2), a member of the apoptosis-stimulating protein of p53 (ASPP) family, has previously been reported to be associated with tumor development. However, to the best of our knowledge, the role of TP53BP2 in neuroblastoma has not been elucidated. The aim of the present study was to investigate the function of TP53BP2 in the proliferation and autophagy of neuroblastoma. An expression vector that expresses TP53BP2-specific short hairpin RNA (shTP53BP2) was used for the experimental group and green fluorescent protein short hairpin RNA was used as a control. Cell proliferation was measured using MTT assays, self-renewal was evaluated using soft agar assays, light chain 3 (LC3) II expression level was examined by western blot and immunofluorescence analysis, and the autophagy-related 3 homolog (ATG3), autophagy-related 5 homolog (ATG5) and autophagy-related 9 homolog (ATG7) expression levels were examined using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). A genomics analysis revealed that TP53BP2 expression was associated with the survival of patients with neuroblastoma. Western blot and RT-qPCR assays indicated that TP53BP2 could be implicated in neuroblastoma, as the proliferative ability of the experimental group decreased compared with that of the control group (P<0.001) and the expression levels of genes associated with autophagy, including LC3 II. ATG3, ATG5 and ATG7, increased in the experimental group. In conclusion, an increased expression of TP53BP2 in patients with neuroblastoma may be associated with poor survival and shTP53BP2 may decrease the proliferative abilities of neuroblastoma cells, including BE(2)C and SK-N-DZ cell lines. In addition, the LC3 II, ATG3, ATG5 and ATG7 expression levels increased and were associated with increased rates of autophagy following upregulation of TP53BP2.

Serine 195 phosphorylation in the RNA-binding protein Rbm38 increases p63 expression by modulating Rbm38’s interaction with the Ago2–miR203 complex

The p63 transcription factor, a p53 family protein, regulates genes involved in various cellular processes, including cell growth and differentiation. We previously showed that RNA-binding motif protein (Rbm38) is a p63 target and, in turn, regulates p63α mRNA stability by binding to the AU/U-rich element in its 3'UTR. Interestingly, Rbm38 can be phosphorylated at serine 195, altering its ability to regulate mRNA translation. However, whether the Ser-195 phosphorylation affects Rbm38's ability to destabilize p63 mRNA remains unclear. Here, using MCF7 and HaCaT cells, we showed that ectopic expression of phosphomimetic Rbm38-S195D increases, whereas WT Rbm38 and nonphosphorylatable Rbm38-S195A decrease p63α protein and transcript levels. We also found that upon activation of glycogen synthase kinase 3β (GSK3β), phosphorylation of Rbm38 at Ser-195 is increased, enhancing p63α expression in an Rbm38-dependent manner. To confirm this, we generated mouse embryo fibroblasts (MEFs) in which Ser-193 in mouse Rbm38 (equivalent to Ser-195 in human Rbm38) was substituted with aspartic acid (Rbm38S193D/S193D ) or alanine (Rbm38S193A/S193A ). We observed that the p63 transcript level was increased in Rbm38S193D/S193D MEFs, but decreased in Rbm38S193A/S193A MEFs. Mechanistically, we found that WT Rbm38, but not Rbm38-S195D, is required for p63 mRNA degradation mediated by microRNA 203 (miR203). Furthermore, we noted that Argonaute 2 (Ago2), a key regulator in microRNA-mediated mRNA decay, associates with WT Rbm38, and this association was reduced by Ser-195 phosphorylation. Together, our results reveal a critical mechanism by which Ser-195 phosphorylation in Rbm38 increases p63 expression by attenuating the association of Rbm38 with the Ago2-miR203 complex.

Role of p53 Family Proteins in Metformin Anti-Cancer Activities.

Metformin has been used as therapy for type 2 diabetes for many years. Clinical and basic evidence as indicated that metformin has anti-cancer activities. It has been well-established that metformin activates AMP-activated protein kinase (AMPK), which in turn regulates energy homeostasis. However, the mechanistic aspects of metformin anti-cancer activity remain elusive. p53 family proteins, including p53, p63 and p73, have diverse biological functions, including regulation of cell growth, survival, development, senescence and aging. In this review, we highlight the evidence and mechanisms by which metformin inhibits cancer cell survival and tumor growth. We also aimed to discuss the role of p53 family proteins in metformin-mediated suppression of cancer growth and survival.

The long noncoding RNA HIF1A-AS2 facilitates cisplatin resistance in bladder cancer.

Chemotherapy drug resistance frequently happens in more than 50% of bladder cancer patients and is the major obstacle for the bladder cancer therapy. Recent studies have shown that long noncoding RNA (lncRNA) is involved in the development of chemoresistance. In this study, we reported hypoxia inducible factor 1α-antisense RNA 2 (HIF1A-AS2), as a subtype-specific hypoxia inducible lncRNA, is upregulated in bladder cancer cells and tissue after cisplatin (Cis) treatment. The induction of HIF1A-AS2 in bladder cancer cells rendered resistance to Cis-induced apoptosis. Silencing HIF1A-AS2 in Cis-resistant bladder cancer cells was re-sensitized to Cis-induced apoptosis. Mechanically, we found that HIF1A-AS2 suppressed the transcription activity of p53 family proteins by promoting the expression of high-mobility group A1(HMGA1). The induction of HMGA1 physically interacts with p53, p63, and p73, and therefore constrains their transcriptional activity on Bax. Knockdown of HIF1A-AS2 or HMGA1 rescued the expression of Bax, which therefore enhanced the killing effect of Cis. Furthermore, we also found that the expression of HIF1A-AS2 was higher in the human bladder tumor tissues after Cis treatment, and was positive correlated to the expression of HIF1α and HMGA1. This study suggests that upregulated HIF1A-AS2 hampers the p53 family proteins dependent apoptotic pathway to promote Cis resistance in bladder cancer. Our data suggested that HIF1A-AS2 plays oncogenic roles and can be used as a therapeutic target for treating human bladder cancer.

PO-148 Activin a upregulation mediated by P63 promotes migration and invasion of oral cancer cells

IntroductionTGF-β family proteins, Activins are homo/heterodimers of β subunits βA, βB, βC and βE, while Inhibins are αβ heterodimers. Relative abundance of each monomer dictates which dimer is predominantly formed. Activins bind to typeII receptor on cell membrane and trigger their tetramerization with typeI receptor, ALK4 phosphorylating SMAD 2/3 which translocate to nucleus and regulate transcription. Activins are reported to be up regulated in various cancers including HNSCC; however, these are transcriptome-scale studies that have indicated altered βA expression, without considering other subunits’ expression or their dimerization. An oncogenic role for Activins has been demonstrated in cancer; albeit, the precise mechanisms downstream of Activin or molecules regulating its expression/function are vaguely understood. This study evaluated the expression of Activins and phenotypes that are affected by its altered expression in oral cancer; with emphasis on Activin-SMAD 2/3 signalling as the downstream mechanism. We also tested the possible role of p53 family proteins in regulating Activin gene expression and functions.Material and methodsActivin subunits’ expression was assessed in oral cancer celllines and tissues by qRT-PCR, IHC and Western blot. Wound healing, Matrigel Invasion, and MTT assays were performed post knockdown of Activin βA and SMAD 2/3 using siRNA and post treatment with recombinant Activin A. We tested the regulation of Activin βA expression by p53 and p63 using ChIP assay and analysed the effect of p63 knockdown in context of Activin expression on oral cancer cells.Results and discussionsActivin βA was significantly overexpressed in oral cancer cells and tissues compared to normal counterparts at transcript and protein level. Knockdown of βA resulted in decreased, while treatment with recombinant Activin A resulted in increased migration and invasion of oral cancer cells. Knockdown of SMAD 2/3 also led to similar effects, indicating that Activin A contributes to these phenotypes through SMAD2/3 signalling pathway. The ChIP experiments demonstrated that βA promoter could be bound by p63, a p53 family member with established oncogenic functions. Recombinant Activin A treatment could rescue the loss of migration upon p63 knockdown suggesting that p63 could be an upstream regulator of Activin expression and function in oral cancer.ConclusionThe study revealed a novel role of p63 in regulating Activin A expression. We also deciphered the mechanism downstream of p63-Activin A axis that contributes to migration and invasion of oral cancer.

IASPP\u2013PP1 complex is required for cytokinetic abscission by controlling CEP55 dephosphorylation

Cytokinesis is the last step of cell division and is concluded by the abscission of the intercellular bridge that connects two daughter cells. The tight regulation of cytokinesis completion is essential because cytokinesis failure is associated with various human diseases. Here, we report that iASPP, a member of the apoptosis-stimulating proteins of p53 (ASPP) family, is required for proper cell division. iASPP depletion results in abnormal midbody structure and failed cytokinesis. We used protein affinity purification methods to identify the functional partners of iASPP. We found that iASPP associates with centrosomal protein of 55 kDa (CEP55), an important cytokinetic abscission regulator. Mechanically, iASPP acts as a PP1-targeting subunit to facilitate the interaction between PP1 and CEP55 and to remove PLK1-mediated Ser436 phosphorylation in CEP55 during late mitosis. The latter step is critical for the timely recruitment of CEP55 to the midbody. The present observations revealed a previously unrecognized function of iASPP in cytokinesis. This function, in turn, likely contributes to the roles of iASPP in tumor development and genetic diseases.

P73, like its p53 homolog, shows preference for inverted repeats forming cruciforms.

p73 is a member of the p53 protein family and has essential functions in several signaling pathways involved in development, differentiation, DNA damage responses and cancer. As a transcription factor, p73 achieves these functions by binding to consensus DNA sequences and p73 shares at least partial target DNA binding sequence specificity with p53. Transcriptional activation by p73 has been demonstrated for more than fifty p53 targets in yeast and/or human cancer cell lines. It has also been shown previously that p53 binding to DNA is strongly dependent on DNA topology and the presence of inverted repeats that can form DNA cruciforms, but whether p73 transcriptional activity has similar dependence has not been investigated. Therefore, we evaluated p73 binding to a set of p53-response elements with identical theoretical binding affinity in their linear state, but different probabilities to form extra helical structures. We show by a yeast-based assay that transactivation in vivo correlated more with the relative propensity of a response element to form cruciforms than to its expected in vitro DNA binding affinity. Structural features of p73 target sites are therefore likely to be an important determinant of its transactivation function.

The family of apoptosis-stimulating proteins of p53 is dysregulated in colorectal cancer patients.

The apoptosis-stimulating protein of p53 (ASPP) family is a newly identified family protein including ASPP1, ASPP2 and inhibitor of ASPP (iASPP), by which the tumor protein 53 (TP53)-mediated apoptotic process is selectively regulated. Downregulation of ASPP1/ASPP2 and upregulation of iASPP were revealed to be associated with a poor prognosis and metastasis in several types of cancer. However, to the best of our knowledge, the expression of ASPP in colorectal cancer (CRC) has not previously been investigated. The present study analyzed ASPP expression in human CRC tissues with multiple clinical and pathological profiles. A total of 41 patients diagnosed with CRC were enrolled in the present study. The expression of ASPP was detected by immunohistochemistry, immunofluorescence and reverse transcription-quantitative polymerase chain reaction. In addition, the variation in ASPP expression was examined in a number of pathological groups. The associations among ASPP expression, and the expression of TP53, plasma carcinoembryonic antigen (CEA) levels and α-fetoprotein (AFP) levels were also investigated. ASPP1 and ASPP2 expression was significantly reduced, while iASPP expression was elevated in CRC samples compared with expression in adjacent non-cancerous tissues. Downregulation of ASPP1 was detected in the TP53-positive group compared with the TP53-negative group. The increase in iASPP expression was correlated with the grade of malignancy, but not with regional lymph node status or metastases. The expression of ASPP2 was negatively correlated with plasma CEA levels. The results of the present study, not only enrich CRC epidemic and pathological data, but also provide valuable indices for CRC clinical treatment and prognosis.

Overexpression of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) in boys with cryptorchidism.

BackgroundThe ubiquitin-proteasome system regulate p53, caspase and Bcl-2 family proteins, and is crucial for the degradation of the defective germ cells in testes. Purpose: to evaluate the concentration of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) in the blood plasma of boys with cryptorchidism and if there is any correlation with patient age.MethodsPatients—50 boys aged 1–4 years (median = 2,4y.) with unilateral cryptorchidism. Exclusion criteria were: previous human chorionic gonadotropin treatment, an abnormal karyotype, endocrine or immunological disorders or any long-term medication. The control group—50 healthy, age matched boys (aged 1–4 years, median = 2,1y.), admitted to the Pediatric Surgery Department for planned herniotomy. To investigate UCHL1 in blood plasma of boys with cryptorchidism, we used a novel technique Surface PLASMON RESONANCE Imaging (SPRI).ResultsThe median concentration of UCHL1 in the blood plasma of boys with cryptorchidism, was 5-folds higher than in boys with inguinal hernia, whose testicles were located in the scrotum. We also noticed statistically significant difference between UCHL1 levels in boys with cryptorchidism up to 2 years old, and above 2 years old. Older boys, whose testicles since birth were located in the inguinal pouch or in the abdominal cavity, had higher concentration of UCHL1 in their blood plasma, than boys from younger group. In the group of cryptorchid boys, we also found slightly lower concentrations of INSL3, without statistical significance and no correlation with UCHL1 levels.ConclusionsUchl1 concentrations in the blood plasma of boys with cryptorchidism, may reflect the heat-induced apoptosis of germ cells. Higher UCHL1 concentrations in older boys with undescended testicles, probably express intensity of germ cell apoptosis, more extensive when testicles are subjected to heat-stress for longer period. Further analyses of UCHL1 may help to elucidate its role in mechanisms influencing spermatogenesis.

Plant Nucleolar Stress Response, a New Face in the NAC-Dependent Cellular Stress Responses.

The nucleolus is the most prominent nuclear domain, where the core processes of ribosome biogenesis occur vigorously. All these processes are finely orchestrated by many nucleolar factors to build precisely ribosome particles. In animal cells, perturbations of ribosome biogenesis, mostly accompanied by structural disorders of the nucleolus, cause a kind of cellular stress to induce cell cycle arrest, senescence, or apoptosis, which is called nucleolar stress response. The best-characterized pathway of this stress response involves p53 and MDM2 as key players. p53 is a crucial transcription factor that functions in response to not only nucleolar stress but also other cellular stresses such as DNA damage stress. These cellular stresses release p53 from the inhibition by MDM2, an E3 ubiquitin ligase targeting p53, in various ways, which leads to p53-dependent activation of a set of genes. In plants, genetic impairments of ribosome biogenesis factors or ribosome components have been shown to cause characteristic phenotypes, including a narrow and pointed leaf shape, implying a common signaling pathway connecting ribosomal perturbations and certain aspects of growth and development. Unlike animals, however, plants have neither p53 nor MDM2 family proteins. Then the question arises whether plant cells have a nucleolar stress response pathway. In recent years, it has been reported that several members of the plant-specific transcription factor family NAC play critical roles in the pathways responsive to various cellular stresses. In this mini review, we outline the plant cellular stress response pathways involving NAC transcription factors with reference to the p53-MDM2-dependent pathways of animal cells, and discuss the possible involvement of a plant-unique, NAC-mediated pathway in the nucleolar stress response in plants.

In search of chondrocyte-specific antigen.

The purpose of this work was to establish, whether rat chondrocyte associated antigen, transmembrane Tmp21 protein belonging to the p24 protein family may immunize rats and thus be included into the panel of immunogens potentially involved in cartilage pathology. For immunization of rats extract from cultured chondrocytes containing surface chondrocyte proteins suspended in incomplete Freund’s adjuvant was used. Control animals were injected with incomplete Freund’s adjuvant without chondrocyte extract. Morphological observations indicated that both in control and experimental animals occurred subperiosteal resorption of bone, suggesting that it arised as the response to adjuvant. In trachea, however, resorption of cartilage and inflammatory changes in the respiratory epithelium and lamina propria were present only in animals exposed to antigen. Unexpectedly, sera from immunized rats strongly reacted with other antigen, which we were able to identify by Western blot and protein sequencing as cartilage oligomeric matrix protein (COMP). COMP is attached to chondrocyte membrane by integrins and its presence in chondrocyte extract is not surprising. Antibody response to COMP raises a question whether the observed changes in tracheal cartilage and epithelium represent anti-COMP reaction or were caused by some other, no specified factors. COMP is used as the marker of osteoarthritis progression, but its role in polychondritis, cartilage pathology involving i.a. tracheal cartilage resorption remains unknown. Thus, our observations may serve as the starting point for future studies in this direction.

Myricetin enhances on apoptosis induced by serum deprivation in PC12 cells mediated by mitochondrial signaling pathway

Polyphenols have many beneficial effects and an effective disease therapeutic auxiliary drug. Previously, myricetin, a polyphenol, had been reported to possess various biological effects on human physiology. However, mechanism of myricetin on apoptosis induced in PC12 cells is still unclear. PC12 cells were treated with myricetin in two concentration levels comprising 0.1 and 1 μM under serum-free condition. As a result, morphological changes were observed using trypan blue assay. DNA fragmentation was determined by DNA ladder assay to evaluate DNA damage levels. Western blotting results showed that cytosolic cytochrome c which was released from mitochondria. Subsequently, tumor suppressor gene p53, pro-apoptotic and anti-apoptotic Bcl-2 family proteins Bax and Bcl-2 were expressed. The caspase cascade reaction was induced through caspase 3 and 9 expression. From these results, it is suggested that myricetin significantly enhanced the apoptosis induced by serum deprivation in a dose-dependent manner in PC12 cells.