DNA Hypermethylation Involves in the Down-Regulation of Chloride Intracellular Channel 4 (CLIC4) Induced by Photodynamic Therapy.

The altered expression of chloride intracellular channel 4 (CLIC4) was reported to correlate with tumor progression. Previously, we have shown that the reduced cellular invasion induced by photodynamic therapy (PDT) is associated with suppression of CLIC4 expression in PDT-treated cells. Herein, we attempted to decipher the regulatory mechanisms involved in PDT-mediated CLIC4 suppression in A375 and MDA-MB-231 cells in vitro. We found that PDT can increase the expression and enzymatic activity of DNA methyltransferase 1 (DNMT1). Bisulfite sequencing PCR further revealed that PDT can induce hypermethylation in the CLIC4 promoter region. Silencing DNMT1 rescues the PDT-induced CLIC4 suppression and inhibits hypermethylation in its promoter. Furthermore, we found tumor suppressor p53 involves in the increased DNMT1 expression of PDT-treated cells. Finally, by comparing CLIC4 expression in lung malignant cells and normal lung fibroblasts, the extent of methylation in CLIC4 promoter was found to be inversely proportional to its expression. Taken together, our results indicate that CLIC4 suppression induced by PDT is modulated by DNMT1-mediated hypermethylation and depends on the status of p53, which provides a possible mechanistic basis for regulating CLIC4 expression in tumorigenesis.

Expression of the human telomerase reverse transcriptase gene is modulated by quadruplex formation in its first exon due to DNA methylation.

DNA secondary structures and methylation are two well-known mechanisms that regulate gene expression. The catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT), is overexpressed in ∼90% of human cancers to maintain telomere length for cell immortalization. Binding of CCCTC-binding factor (CTCF) to the first exon of the hTERT gene can down-regulate its expression. However, DNA methylation in the first exon can prevent CTCF binding in most cancers, but the molecular mechanism is unknown. The NMR analysis showed that a stretch of guanine-rich sequence in the first exon of hTERT and located within the CTCF-binding region can form two secondary structures, a hairpin and a quadruplex. A key finding was that the methylation of cytosine at the specific CpG dinucleotides will participate in quartet formation, causing the shift of the equilibrium from the hairpin structure to the quadruplex structure. Of further importance was the finding that the quadruplex formation disrupts CTCF protein binding, which results in an increase in hTERT gene expression. Our results not only identify quadruplex formation in the first exon promoted by CpG dinucleotide methylation as a regulator of hTERT expression but also provide a possible mechanistic insight into the regulation of gene expression via secondary DNA structures.

Knockout of ho-1 protects the striatum from ferrous iron-induced injury in a male-specific manner in mice.

Men have worse survival than premenopausal women after intracerebral hemorrhage (ICH). After ICH, overproduction of iron associated with induction of heme oxygenase-1 (HO-1) in brain was observed. Rodent ICH model using ferrous citrate (FC)-infusion into the striatum to simulate iron overload, showed a higher degree of injury severity in males than in females. However, the participation of HO-1 in sex-differences of iron-induced brain injury remains unknown. The present results showed a higher level of HO-1 expression associated with more severe injury in males compared with females after FC-infusion. Estradiol (E2) contributed to lower levels of FC-induced HO-1 expression in females compared with males. Heterozygote ho-1 KO decreased the levels of FC-induced injury severity, histological lesions, behavioral deficits, autophagy and autophagic cell death in the striatum of males but not in females. Moreover, ho-1 deficiency enhanced the neuroprotection by E2 only in males. These results suggested that over induction of HO-1 plays a harmful role in FC-induced brain injury in a male-specific manner. Suppression of HO-1 combined with E2 exhibits a synergistic effect on neuroprotection against FC-induced striatal injury in males. These findings open up the prospect for male-specific neuroprotection targeting HO-1 suppression for patients suffering from striatal iron overload.

Characterization of Cell Wall Proteins in Saccharomyces cerevisiae Clinical Isolates Elucidates Hsp150p in Virulence.

The budding yeast Saccharomyces cerevisiae has recently been described as an emerging opportunistic fungal pathogen. Fungal cell wall mannoproteins have been demonstrated to be involved in adhesion to inert surfaces and might be engaged in virulence. In this study, we observed four clinical isolates of S. cerevisiae with relatively hydrophobic cell surfaces. Yeast cell wall subproteome was evaluated quantitatively by liquid chromatography/tandem mass spectrometry. We identified totally 25 cell wall proteins (CWPs) from log-phase cells, within which 15 CWPs were quantified. The abundance of Scw10p, Pst1p, and Hsp150p/Pir2p were at least 2 folds higher in the clinical isolates than in S288c lab strain. Hsp150p is one of the members in Pir family conserved in pathogenic fungi Candida glabrata and Candida albicans. Overexpression of Hsp150p in lab strain increased cell wall integrity and potentially enhanced the virulence of yeast. Altogether, these results demonstrated that quantitative cell wall subproteome was analyzed in clinical isolates of S. cerevisiae, and several CWPs, especially Hsp150p, were found to be expressed at higher levels which presumably contribute to strain virulence and fungal pathogenicity.

Male-Specific Alleviation of Iron-Induced Striatal Injury by Inhibition of Autophagy.

Men exhibit a worse survival rate than premenopausal women after intracerebral hemorrhage (ICH), however, no sex-specific management has been concerned. In a rat model involving infusion of ferrous citrate (FC) that simulates iron accumulation after hemorrhage, a higher degree of autophagy associated with higher injury severity was observed in striatum of males than in females. Since the imbalance between the levels of autophagy and energy demand may lead to cell death, we proposed that FC-induced autophagy is detrimental in a male specific manner and autophagy modulation affects injury severity in a sex-dependent manner. Rapamycin, an autophagy inducer, and conditional knockout gene of autophagy-related protein 7 (Atg7) in dopamine receptor D2 (DRD2) neurons were used to test our hypothesis using a mouse model with striatal FC infusion. The result showed that the levels of autophagic cell death and injury severity were higher in male than in female mice. Pre-treatment of FC-infused females with rapamycin increased the FC-induced behavioral deficit and DRD2 neuron death. However, DRD2 neuron-specific knockout of Atg7 decreased FC-induced injury severity and the number of TUNEL(+) DRD2 neurons in males. These results suggest that autophagy in FC-infusion males is overactive with maladaptive consequences and inhibition of autophagy decreases the severity of FC-induced striatal injury in males. These findings present prospects for male-specific therapeutic strategy that targets autophagy in patients suffering from iron overload.

Impact of ammonia exposure on coagulation in white shrimp, Litopenaeus vannamei.

Ammonia (un-ionized plus ionized ammonia as nitrogen), the end product of protein catabolism, is produced by decomposing organic matter. In aquaculture, shrimp are commonly exposed to high concentrations of ammonia that induces immunological and histological changes. The purpose of this study was to evaluate the effects on hemolymph coagulation time, transglutaminase (TG) activity as well as TG and clottable protein (CP) genes expressions in Litopenaeus vannamei when exposed to ambient ammonia-nitrogen (N) at 0, 1, 5, and 10mg/L for 0, 2, and 7 days. The actual concentrations in control and tests solution were 0.001, 1.15, 5.11, and 11.68mg/L for ammonia-N, and 7×10(-5), 0.080, 0.357, and 0.815mg/L for NH3-N (unionized ammonia). Delayed coagulation time following exposure to 5 and 10mg/L of ambient ammonia-N for 7 days, and increased transglutaminase (TG) activity following exposure to 5 and 1mg/L of ambient ammonia-N for 2 and 7 days, respectively, were observed. Downregulated TG expression and upregulated clottable protein (CP) expression in the hemocytes of L. vannamei exposed to 10 and 5mg/L of ambient ammonia-N for 2 and 7 days, respectively, were shown. These results indicated that ambient ammonia-N (>5mg/L) and NH3-N (>0.357mg/L) interrupted coagulation and down-regulated TG gene expression in L. vannamei, which caused ecotoxicity on immune deficiencies and may contribute the increased susceptibility to infection by pathogens.

Chloride intracellular channel 4 involves in the reduced invasiveness of cancer cells treated by photodynamic therapy.

BACKGROUND AND OBJECTIVES: The mechanisms of photodynamic therapy (PDT) have been studied on the cellular and tissue levels. However, the cellular behaviors of cancer cells survived from PDT are still not clear. Previously, we have found that PDT-derived variants A375/3A5 and A375/6A5 have reduced invasion ability. This study attempted to further elucidate the possible molecules associated with the altered invasiveness in the PDT-derived variants and cancer cells treated with PDT. STUDY DESIGN/MATERIALS AND METHODS: Scratch wound healing assay and invasion assay were performed to evaluate the migration and invasion ability of human A375 melanoma and MDA-MB-231 breast adenocarcinoma cells. Single colony selection and microarray analysis were performed to examine the differentially expressed transcripts in parental A375 and PDT-derived variants. RT-PCR and Western blots analysis were performed to examine the expression levels of matrix metalloproteinase 9 (MMP9) and chloride intracellular channel 4 (CLIC4). The MMP9 activity was examined by Zymography assay. CLIC4 expressing construct was used to examine the influence on MMP9 expression and invasion ability of cancer cells treated with PDT. RESULTS: Correlated with the reduced invasiveness, we found that A375/3A5 and A375/6A5 cells have decreased production of MMP9. Microarray analysis and RT-PCR showed CLIC4 was down-regulated in the PDT-derived variants. Furthermore, down-regulation of CLIC4 and MMP9 was found in cancer cells treated with PDT. Transfection of surviving cancer cells with a plasmid vector encoding CLIC4 increased MMP9 expression and cell invasion. Furthermore, overexpression of CLIC4 in A375 and MDA-MB-231 cancer cells constrains PDT-induced suppression of invasiveness. CONCLUSION: Our results showed that the reduced expression of CLIC4 could further down-regulate MMP9 and result in the suppression of invasion in cancer cells treated with PDT. These results provide an insight into a new mechanism by which PDT affects the metastatic potential of cancer cells through down-regulation of MMP9 by CLIC4.

The decline of autophagy contributes to proximal tubular dysfunction during sepsis.

Severe sepsis associated with overproduction of tumor necrosis factor α and reactive oxygen species leads to energy depletion and cellular damage. Both reactive oxygen species and damaged organelles induce autophagy for recycling nutrients to combat pathological stress. To study whether autophagy plays a beneficial role in the pathogenesis of renal failure during sepsis, rats were subjected to cecal ligation and puncture (CLP) or sham operation. Temporal relationship of autophagy and renal dysfunction were examined in vivo. The results showed that the level of lipidated microtubule-associated protein light chain 3 (LC3-II), a marker of autophagy, elevated transiently at 3 h but declined at 9 h until 18 h after CLP. Light chain 3 aggregation in renal tissue showed a similar trend to the change of LC3-II protein. High levels of blood urea nitrogen and creatinine as well as low tubular sodium reabsorption occurred at 18 h after CLP. The distribution of autophagy located primarily in angiotensin-converting enzyme-positive, which is concentrated in proximal tubule, but calbindin D28k (calcium-binding protein D28K, a marker of distal tubule)-negative cells in renal cortex. Therefore, NRK-52E (proximal tubule epithelial cell line) cells were used to further examine cell viability and DNA fragmentation after silencing or inducing autophagy. We found that knockdown of Atg7 (autophagy-related gene 7) exaggerates, whereas preincubation of rapamycin (an autophagy inducer) diminishes tumor necrosis factor α-induced cell death. These results suggest that the decline of sepsis-induced autophagy contributes to the proximal tubular dysfunction, and maintenance of sufficient autophagy prevents cell death. These data open prospects for therapies that activate autophagy during sepsis.

Suppression of autophagy in rat liver at late stage of polymicrobial sepsis.

Sepsis develops as a result of the host response to infection, and its mortality rate in ICU remains high. Severe inflammation usually causes overproductions of proinflammatory cytokines, i.e., TNF-α and reactive oxygen species, which lead to mitochondrial damage and energetic depletion. Autophagy is a survival mechanism for eukaryote to recycle intracellular nutrients and maintain energy homeostasis. We hypothesize that autophagy plays a beneficial role in the pathogenesis of organ failure during sepsis. A rat model of cecal ligation and puncture (CLP) that simulate peritonitis-induced sepsis was used, and indicators for liver dysfunction, serum glutamic oxaloacetic, serum glutamic pyruvic, alkaline phosphatase, and bilirubin were measured. Levels of LC3-II and LC3 aggregation were quantified by Western blot analysis and by immunohistochemistry, respectively. The tissue localization of autophagy was identified by immunohistochemistry and transmission electron microscopy. Our results showed that (a) increase in LC3-II level in liver tissue occurs at 3 h, peaks at 6 h, and then surprisingly declines quickly until 18 h after CLP (CLP18h); (b) significant hepatic dysfunction was observed at CLP18h; (c) ratio of LC3 aggregation is significantly higher in hepatocytes than in Kupffer cells, and (d) loss of Atg7, an essential gene for autophagosome formation, exaggerates the TNF-α-induced cell death, depletion of ATP, and decrease of albumin production in hepatocytes. These results indicate that autophagy occurs transiently in hepatocytes at early stage, and the decline in autophagy at late stage may contribute to the functional failure in liver during polymicrobial sepsis.

ALA-PDT results in phenotypic changes and decreased cellular invasion in surviving cancer cells.

BACKGROUND AND OBJECTIVES: The mechanisms of photodynamic therapy (PDT) have been studied on the cellular and tissue levels. However, the cellular behaviors of cancer cells survived from PDT are still not clear. This study attempted to investigate the influence of 5-aminolevulinic acid (ALA)-based PDT on the invasion ability as well as molecular changes in surviving cancer cells and their progeny. MATERIALS AND METHODS: The systematic effects of ALA-PDT were evaluated using human invasive carcinoma cells (lung adenocarcinoma CL1-5 cells, melanoma A375 cells and breast carcinoma MDA-MB-231 cells). To study the cellular behaviors of surviving cancer cells, PDT-derived variants were established as stable cell lines after consecutive treatment with ALA-PDT. Scratch wound assay and invasion assay were performed to evaluate the migration and invasion ability in the surviving cancer cells and the established PDT-derived variants. RT-PCR and immunoblot analysis were performed to examine the expression levels of epidermal growth factor receptor (EGFR). RESULTS: Though ALA-PDT caused differential phototoxicity among these invasive carcinoma cells, reduced migration was found in all the surviving cancer cells Compared to parental cancer cells, the established PDT-derived variants exerted significant phenotypic changes of cellular morphology, reduced mitochondrial function and a suppressed cellular invasiveness. Furthermore, correlated with the reduced invasion ability, expression of EGFR was downregulated in these established PDT-derived variants. CONCLUSIONS: Except for direct cell killing, ALA-PDT could reduce EGFR expression and invasion ability of the surviving cancer cells and these effects could further pass to the progeny. The results from this study provide insights into a new mechanism by which PDT might affect cellular behaviors and tumor metastasis.

Expression of clottable protein of tiger shrimp (Penaeus monodon) in gonads and its possible role as nutrient source for the embryo.

We have investigated the expression of clottable protein (CP) in gonad of tiger shrimp (Penaeus monodon) and extent of its phosphorylation. Polyclonal antibodies against purified CP were prepared from rabbit serum. Using this anti-CP antiserum, the temporal expression of CP in gonads of tiger shrimp was analyzed. It was found that the CP occurs only in mature ovaries but not in immature ovaries and testes. Results of RT-PCR confirmed that these tissues expressed low levels of CP mRNA transcripts. Upon eyestalk-ablation, the ovaries in female shrimps were induced to develop, and the CP expression levels in ovaries were traced chronically by RT-PCR analyses. The expression level peaked on day 3 with an increase of about 40 folds relative to the basal level and returned to normal level (as the control shrimp) at day 12. The shrimp embryos at different intervals from spawning to 16h post-spawning were also collected, and it was found that CP contents were gradually decreased in the embryos until the nauplii were hatched. In addition, purified CP was shown to react with specific anti-phosphoserine, anti-phosphothreonine, and anti-phosphotyrosine antibodies suggesting that CP is a phosphoprotein with all types of phosphorylations. Taken together the results suggest that expression of CP in shrimp ovaries is coupled to ovarian development and CP possibly supply nutrition for shrimp embryo.

Cloning and characterization of hemolymph clottable proteins of kuruma prawn (Marsupenaeus japonicus) and white shrimp (Litopenaeus vannamei).

The hemolymph clottable protein (CP) of Marsupenaeus japonica (designated as Mj-CP) was purified by a DEAE anion-exchanger and a Sepharose CL-6B gel filtration column. In the presence of Ca(2+), it formed stable clots in vitro upon the addition of the hemocytes lysate containing transglutaminase. Results of gel filtration chromatography and SDS-PAGE indicated that Mj-CP mainly existed as disulfide-linked homodimers of 390 kDa. Specific primers were designed; PCR as well as RACE help to clone and sequence Mj-CP cDNA of 5660 bp. The predicted CP-precursor contains a signal peptide followed by a subunit of 1671 amino acids (isoelectric point 5.6), including two RGD motifs and three potential N-glycosylation sites. Mj-CP is structurally 80% and 38% identical to the CPs of tiger shrimp and crayfish, respectively. Likewise, CP cDNA of white shrimp (Litopenaeus vannamei) was also cloned and sequenced; the predicted CP has 1666 amino acid residues and an isoelectric point of 5.2. Both CPs bear potential transglutaminase cross-linking sites, i.e. seven Ser-Lys-Thr repeats near the N-terminus, a Ser- and Gln-rich region in the middle, and polyGln (n=8-11) near the C-terminus. Phylogenetic analyses of crustacean CPs and vitellogenins revealed divergent evolution of the two protein families. By RT-PCR, the sub-cuticular epidermis was identified as one of the major tissues that express CP in M. japonica.