DNA Fragmentation Factor Research Articles

Induced Cell Death Pathway Post Photodynamic Therapy Using a Metallophthalocyanine Photosensitizer in Breast Cancer Cells

Zinc phthalocyanine (ZnPcSmix) was used as the photosensitizer (PS) in this study to investigate the cell death patterns as a result of photodynamic therapy (PDT) in a breast cancer cell line (MCF-7) in vitro using a 680 nm diode laser at a fluence of 5 J/cm(2). PDT is a noninvasive form of cancer therapy, successfully applied for the treatment of various cancer types. Flow cytometry using Annexin V-fluorescein isothiocyanate (FITC), a cell death immunosorbent assay (ELISA), and gene expression analysis following ZnPcSmix mediated PDT were performed to determine the induced cell death pathways. The apoptotic cells abounded after the treatment, nuclear fragmentation was seen as oligonucleosomal degradation and increased expression of the B-cell lymphoma 2 (Bcl-2), DNA fragmentation factor alpha (DFFA1), and caspase 2 (CASP2) genes, indicated that apoptosis is the main induced mode of cell death. ZnPcSmix mediated PDT led to an apoptotic cell death pathway and the PS used showed its ability to stimulate and initiate programmed cell death.

Cloning and characterization of an apoptosis-related DNA fragmentation factor (DFF) from oyster, Crassostrea hongkongensis

Apoptosis plays an important pathophysiological role in the homeostasis of immune systems. DNA fragmentation factors (DFFs) have been shown to be essential for DNA fragmentation, and the resultant DNA fragments follow a laddering pattern during apoptosis in vertebrates. In invertebrates, the functions of the DFF orthologs are not well characterized; therefore, we cloned and characterized a bivalve DFFA ortholog from the Hong Kong oyster Crassostrea hongkongensis (designated ChDFFA). The full-length cDNA of ChDFFA is 1186 bp in length and encodes a putative protein of 200 amino acids that contains an N-terminal CAD domain and a DFF-C domain at its C-terminus. Real-time RT-PCR results showed that ChDFFA is ubiquitously expressed in several tissues, and its highest expression is in gill. Following a 3- to 48-h challenge by microbial infection, the expression of ChDFFA increased in hemocytes. Using fluorescence microscopy, ChDFFA was localized in nuclei when exogenously expressed in HeLa cells. In addition, over-expression of ChDFFA inhibited the transcriptional activities of p53/p21-Luc reporter genes in HEK293T cells. These results suggest that ChDFFA may be involved in immune response reactions in the Hong Kong oyster C. hongkongensis.

Maternal diet amplifies the hepatic aging trajectory of Cidea in male mice and leads to the development of fatty liver

The importance of the early environment on long-term heath and life span is well documented. However, the molecular mechanisms mediating these effects remain poorly understood. Male offspring from a maternal protein restriction model, in which animals are exposed to a low-protein diet while in utero and then are cross-fostered to normally fed dams, demonstrate low birth weight, catch-up growth, and reduced life span (recuperated offspring). In the current study, we used microarray analysis to identify hepatic genes that changed with age. Cell death-inducing DNA fragmentation factor, α subunit-like effector A (Cidea), a transcriptional coactivator that has been implicated in lipid accumulation demonstrated one of the largest age-associated increases in expression (200-fold, P<0.001). This increase was exaggerated ∼3-fold in recuperated offspring. These demonstrated increased hepatic lipid accumulation, higher levels of transcription factors important in lipid regulation, and greater oxidative stress. In vitro analysis revealed that Cidea expression was regulated by oxidative stress and DNA methylation. These findings suggest that maternal diet modulates the age-associated changes in Cidea expression through several mechanisms. This expression affects hepatic lipid metabolism in these animals and thus provides a mechanism by which maternal diet can contribute to the metabolic health and ultimately the life span of the offspring.

DNase γ is the effector endonuclease for internucleosomal DNA fragmentation in necrosis.

Apoptosis and necrosis, two major forms of cell death, can be distinguished morphologically and biochemically. Internucleosomal DNA fragmentation (INDF) is a biochemical hallmark of apoptosis, and caspase-activated DNase (CAD), also known as DNA fragmentation factor 40 kDa (DFF40), is one of the major effector endonucleases. DNase γ, a Mg2+/Ca2+-dependent endonuclease, is also known to generate INDF but its role among other apoptosis-associated endonucleases in cell death is unclear. Here we show that (i) INDF occurs even during necrosis in cell lines, primary cells, and in tissues of mice in vivo, and (ii) DNase γ, but not CAD, is the effector endonuclease for INDF in cells undergoing necrosis. These results document a previously unappreciated role for INDF in necrosis and define its molecular basis.

SiRNA-mediated knock-down of DFF45 amplifies doxorubicin therapeutic effects in breast cancer cells

RNA interference (RNAi) has become a promising tool for cancer therapy. Small interfering RNAs (siRNAs) can synergistically enhance the cell killing effects of drugs used in cancer treatment. Here we examined the effects of siRNA-mediated DNA fragmentation factor 45 (DFF45) gene silencing on breast cancer cell viability, cell cycle arrest, and apoptosis in the presence and absence of doxorubicin. We designed three siRNAs, which target different regions of the DFF45 mRNA. Gene silencing was confirmed by real time RT-PCR and Western blot analyses. The impact of DFF45 siRNA, doxorubicin, and their combination on the viability, cell cycle and apoptosis of T-47D and MDA-MB-231 breast cancer cells were determined by MTT, PI staining, annexin V binding, caspase-3 activity, DNA laddering, and chromatin condensation assays. Based on flow cytometric analyses, we found that silencing of DFF45 alone had little effect on apoptosis, especially in T-47D cells. However, when used in combination with doxorubicin (0.33μM) a significant increase (P < 0.05) in apoptosis was observed in T-47D and MDA-MB-231 cells, i.e., ~2.5- and 3-fold, respectively. Caspase-3 activity, chromatin condensation, as well as DNA laddering supported increased apoptosis in the combinatorial treatment. Cell cycle arrest in both cell lines occurred at lower levels after siRNA + doxorubicin treatment compared to doxorubicin only. Our data indicate that DFF45 gene silencing, when applied in combination with doxorubicin, may offer a novel therapeutic strategy for the treatment of breast cancer.

Inhibition of c-Jun N-terminal kinase (JNK) suppresses porcine oocyte ageing in vitro

Oocyte ageing is a complex of processes that occur when matured in vitro oocytes are, after reaching the metaphase II stage, exposed to further in vitro culture. Aged oocytes remaining at the metaphase II stage undergo spontaneous parthenogenetic activation, or cellular death, through apoptosis (fragmentation) or lysis. The key factor in apoptotic pathway regulation is c-Jun-N-terminal kinase (JNK), stress kinase from the mitogene-activated protein kinase (MAPK) family. To investigate the effect of JNK inhibition on porcine oocytes ageing, cleavage rate, and embryonic development after parthenogenetic activation, DNA fragmentation, and pro-apoptotic factor Bax expression, we cultured in vitro matured oocytes for another 1&amp;ndash;4 days in the presence of a JNK inhibitor. The inhibition of JNK significantly protected the oocytes from fragmentation (0% of fragmented oocytes under JNK inhibition vs. 13.4% of fragmented oocytes in the control group, 2&lt;sup&gt;nd&lt;/sup&gt;&amp;nbsp;day of&amp;nbsp;ageing) and increased the percentage of parthenogenetically activated oocytes (82 vs 57.7%, 2&lt;sup&gt;nd&lt;/sup&gt; day of ageing). The embryonic development of oocytes parthenogenetically activated after 24 h of ageing was influenced by JNK inhibition as well. The percentage of oocytes at the morula stage, after seven days of cultivation, was significantly increased when oocytes aged in the presence of a JNK inhibitor (42.5%) by comparison to the percentage of oocytes exposed to ageing in an inhibitor-free medium (23.3%). DNA fragmentation was significantly suppressed by JNK inhibition from the 1&lt;sup&gt;st&lt;/sup&gt; day of ageing, but the expression of pro-apoptotic factor Bax in the oocytes was not influenced. On the basis of our experiments, we can conclude that JNK inhibition suppresses apoptosis and DNA fragmentation of aged oocytes and improves their embryonic development following the parthenogenetic activation. However, to completely eliminate all ageing related processes is insufficient.

The association of TP53 mutations with the resistance of colorectal carcinoma to the insulin-like growth factor-1 receptor inhibitor picropodophyllin

BackgroundThere is growing evidence indicating the insulin-like growth factor 1 receptor (IGF-1R) plays a critical role in the progression of human colorectal carcinomas. IGF-1R is an attractive drug target for the treatment of colon cancer. Picropodophyllin (PPP), of the cyclolignan family, has recently been identified as an IGF-1R inhibitor. The aim of this study is to determine the therapeutic response and mechanism after colorectal carcinoma treatment with PPP.MethodsSeven colorectal carcinoma cell lines were treated with PPP. Following treatment, cells were analyzed for growth by a cell viability assay, sub-G1 apoptosis by flow cytometry, caspase cleavage and activation of AKT and extracellular signal-regulated kinase (ERK) by western blot analysis. To examine the in vivo therapeutic efficacy of PPP, mice implanted with human colorectal carcinoma xenografts underwent PPP treatment.ResultsPPP treatment blocked the phosphorylation of IGF-1R, AKT and ERK and inhibited the growth of TP53 wild-type but not mutated colorectal carcinoma cell lines. The treatment of PPP also induced apoptosis in TP53 wild-type cells as evident by the presence of sub-G1 cells and the cleavage of caspase-9, caspase-3, DNA fragmentation factor-45 (DFF45), poly (ADP-ribose) polymerase (PARP), and X-linked inhibitor of apoptosis protein (XIAP). The loss of BAD phosphorylation in the PPP-treated TP53 wild type cells further suggested that the treatment induced apoptosis through the BAD-mediated mitochondrial pathway. In contrast, PPP treatment failed to induce the phosphorylation of AKT and ERK and caspase cleavage in TP53 mutated colorectal carcinoma cell lines. Finally, PPP treatment suppressed the growth of xenografts derived from TP53 wild type but not mutated colorectal carcinoma cells.ConclusionsWe report the association of TP53 mutations with the resistance of treatment of colorectal carcinoma cells in culture and in a xenograft mouse model with the IGF-1R inhibitor PPP. TP53 mutations often occur in colorectal carcinomas and could be used as a biomarker to predict the resistance of colorectal carcinomas to the treatment by this IGF-1R inhibitor.

Exploring the pathophysiology behind the more common genetic and acquired lipodystrophies

Lipodystrophies are an immense group of genetic or acquired metabolic disorders that are characterized by varying degrees of body fat loss and in some instances localized accumulation of subcutaneous fat. Lipodystrophies are often tightly linked with profound metabolic complications; this strong bond emphasizes and reinforces the significance of adipose tissue as a dynamic endocrine organ. The extent of fat loss determines the severity of associated metabolic complications such as diabetes mellitus, hypertriglyceridemia and hepatic steatosis. The lipodystrophies can be divided into generalized, partial or local, depending on the degree and locality of the observable fat loss; moreover, the generalized and partial divisions can be partitioned further into inherited or acquired forms. The major genetic factors in the generalized forms of the lipodystrophies, particularly Congenital generalized lipodystrophy (CGL)-Berardinelli-Seip syndrome, are the AGPAT2, BSCL2, caveolin 1 (CAV1) and polymerase-I-and-transcriptrelease factor (PTRF) genes. In the acquired forms, genes such as LMNA, PPARG, CIDEC (cell-death-inducing DNA fragmentation factor a-like effector c) and PLIN1 are heavily involved in familial partial lipodystrophy (FPLD) type 2 (also known as the Dunnigan-Variety) and WRN along with RECQL5 in Werner Syndrome (WS). Autoimmune causes are particularly noted in acquired partial lipodystrophy (APL)-Barraquer-Simons syndrome and in AGL-Lawrence syndrome; panniculitis has been shown to have a substantial role in the former as well as in other forms of localized lipodystrophies. Patients with human immunodeficiency virus (HIV) exposed to protease inhibitors, nucleoside reverse transcriptase inhibitors (NRTIs) (for example, zidovudine and stavudine) or non-nucleoside reverse transcriptase inhibitors (NNRTIs) (for example, efavirenz) while undergoing Highly Active Antiretroviral Therapy (HAART) have led to the current most-prevalent form of the lipodystrophies: lipodystrophy in HIV-infected patients (LD-HIV) and HAART-associated lipodystrophy syndrome (HALS).

A novel JNK2/SREBP-1c pathway involved in insulin-induced fatty acid synthesis in human adipocytes

Insulin plays important roles in apoptosis and lipid droplet (LD) formation, and it is one of the determinants involved in increasing fat mass. However, the mechanisms underlying insulin-induced enlargement of fat mass remain unclear. Our previous study suggested that insulin-induced increases in LDs are related to c-Jun N-terminal kinase (JNK)2-mediated upregulation of cell death-inducing DNA fragmentation factor-α-like effector (CIDE)C in human adipocytes. However, other genes involved in insulin/JNK2-induced LD formation are unknown. Here, we explored insulin/JNK2-regulated genes to clarify the mechanism of enlargement of LDs. Microarray analysis revealed that an insulin/JNK2 pathway mostly regulates expression of genes involved in lipid metabolism, including sterol regulatory element binding protein (SREBP)-1, a key transcription factor of lipogenesis. The JNK inhibitor SP600125 blocked insulin-induced upregulation of SREBP-1c expression. Small interfering RNA-mediated depletion of JNK2 suppressed insulin-induced nuclear accumulation of the active form of SREBP-1 protein and upregulation of SREBP-1c. Furthermore, depletion of JNK2 attenuated insulin-induced upregulation of SREBP-1c target lipogenic enzymes, leading to reduced de novo fatty acid synthesis. In addition, JNK2 coimmunoprecipitated with SREBP-1, reinforcing the correlation between JNK2 and SREBP-1. These results suggest that SREBP-1c is a novel insulin/JNK2-regulated gene and that the JNK2/SREBP-1c pathway mediates insulin-induced fatty acid synthesis, which may lead to enlargement of LDs in human adipocytes.

FSP27 is a potent player regulating lipid storage in liver as well as adipose tissue

Obesity has increased dramatically worldwide and leads to numerous disorders including cardiovascular diseases, type 2 diabetes, dyslipidemia and cancers. These pathological states are closely associated with hyperinsulinemia or insulin resistance. Obesity itself is characterized by excessive triacylglycerol (TAG) storage as lipid droplets (LDs) in adipose tissue due to an increase in nutrient intake and insufficient energy expenditure. Excessive TAG storage in adipocytes causes adipose tissue inflammation and deregulation of adipokines, both of which induce systemic insulin resistance. At the same time, a hallmark of obesity is TAG accumulation in the liver and skeletal muscle, which is also important in inducing insulin resistance in these tissues. These findings underline the importance of lipid accumulation in insulin-sensitive organs in the development of insulin resistance and type 2 diabetes. Lipid droplets are comprised of a central core of neutral lipids containing TAG and cholesterol ester and surrounded by a phospholipid monolayer. This monolayer of LDs is decorated with a variety of proteins, which contribute to the formation of the droplets, the synthesis and hydrolysis of lipids, and the movement of theses lipids to specific intracellular and secretory pathways. The ability to store TAG in LDs is evolutionarily conserved in yeast, plants, invertebrates and vertebrates. In mammals, excess energy is primarily stored as TAG in LDs in adipose tissue. Especially white adipocytes are specifically differentiated cells for storing TAG as the large unilocular LDs that occupy most of the cell volume. The LD size can be in the 100 lm range. Such TAG storage in LDs serves a vital role as the body’s major stored energy supply. In the case of energy demand, such as starvation and exercise, TAG reserves are hydrolyzed by lipolysis to supply free fatty acid (FFA) to a variety of tissues. LDs are also recognized in non-adipose cells; however, non-adipocyte LDs are usually much smaller than those of adipocytes. Such TAG storage in liver, skeletal muscle and heart is thought to serve as a local energy supply. Recently, a fat-specific protein of 27 kDa (FSP27) was found to be the determining factor for LD size and critical for large unilocular LD formation in white adipocytes [1–3]. FSP27 was first identified in 1992 as a fat-specific protein whose expression was regulated by C/EBP, although its function in adipocytes remained elusive then [4]. Subsequently, FSP27 was reported to be a member of the CIDE family of proteins, comprised of CIDE-A, CIDEB and FSP27, which had homology with the amino-terminal domain of DFF-45 (45-kDa subunit of the DNA fragmentation factor) [5]. Later, CIDE-A was shown to be expressed in brown adipose tissue in mice and to inhibit energy expenditure [6]. CIDE-B is expressed mainly in the liver and kidney and is important for the maturation of VLDL as apolipoprotein B binding protein in the liver [7]. As for FSP27, functional analysis of FSP27 has robustly progressed since knockout (KO) mice were generated in 2008 [1, 3], although FSP27 was already found to be a LD Y. Tamori (&) Division of Metabolism and Endocrinology, Department of Internal Medicine, Chibune Hospital, 2-2-45 Tsukuda, Nishiyodogawa-ku, Osaka 555-0001, Japan e-mail: tamori@med.kobe-u.ac.jp

GM-CSF-DFF40: a novel humanized immunotoxin induces apoptosis in acute myeloid leukemia cells

DNA fragmentation factor 40 (DFF40) is an endonuclease that acts downstream in the apoptotic cascade. The objective of this study was to generate a novel humanized chimeric protein with human DFF40 fused with GM-CSF for targeting acute myeloid leukemia (AML) cells. cDNA cloning of human DFF40 and GM-CSF was done and the chimeric gene GM-CSF-DFF40 was generated by overlap extension PCR. The fusion protein was expressed in E.coli, purified, refolded and characterized. In vitro cytotoxicity was evaluated on various AML cell lines. Treated cell lines were screened for various morphological and biochemical changes that are characteristic of apoptosis, by different assays like annexin V-FITC staining, TUNEL assay, JC-1 staining and immunocytochemistry of pro-apoptotic proteins and caspases. Cell cycle analysis of treated cells was done to quantify the percentage of apoptotic cells. The chimeric protein was found to be cytotoxic to AML cells in a dose and time dependent manner. Morphological changes such as formation of apoptotic bodies were revealed by microscopic examination of treated cells after staining. Immunocytochemical staining demonstrated biochemical changes such as changes in mitochondrial membrane potential, mitochondrial co-localization of Bax, cytochrome c release, presence of activated caspase-3 and DNA fragmentation. FACS analysis proved the presence of apoptotic cells following treatment. The chimeric protein GM-CSF-DFF40 was found to mediate targeted killing of AML cells by inducing apoptosis. Thus, this chimeric construct can act as a prospective candidate for targeted therapy of AML and other malignancies where GM-CSF receptor expression is upregulated.

Chromatin Collapse during Caspase-dependent Apoptotic Cell Death Requires DNA Fragmentation Factor, 40-kDa Subunit-/Caspase-activated Deoxyribonuclease-mediated 3′-OH Single-strand DNA Breaks

Apoptotic nuclear morphology and oligonucleosomal double-strand DNA fragments (also known as DNA ladder) are considered the hallmarks of apoptotic cell death. From a classic point of view, these two processes occur concomitantly. Once activated, DNA fragmentation factor, 40-kDa subunit (DFF40)/caspase-activated DNase (CAD) endonuclease hydrolyzes the DNA into oligonucleosomal-size pieces, facilitating the chromatin package. However, the dogma that the apoptotic nuclear morphology depends on DNA fragmentation has been questioned. Here, we use different cellular models, including MEF CAD(-/-) cells, to unravel the mechanism by which DFF40/CAD influences chromatin condensation and nuclear collapse during apoptosis. Upon apoptotic insult, SK-N-AS cells display caspase-dependent apoptotic nuclear alterations in the absence of internucleosomal DNA degradation. The overexpression of a wild-type form of DFF40/CAD endonuclease, but not of different catalytic-null mutants, restores the cellular ability to degrade the chromatin into oligonucleosomal-length fragments. We show that apoptotic nuclear collapse requires a 3'-OH endonucleolytic activity even though the internucleosomal DNA degradation is impaired. Moreover, alkaline unwinding electrophoresis and In Situ End-Labeling (ISEL)/In Situ Nick Translation (ISNT) assays reveal that the apoptotic DNA damage observed in the DNA ladder-deficient SK-N-AS cells is characterized by the presence of single-strand nicks/breaks. Apoptotic single-strand breaks can be impaired by DFF40/CAD knockdown, abrogating nuclear collapse and disassembly. In conclusion, the highest order of chromatin compaction observed in the later steps of caspase-dependent apoptosis relies on DFF40/CAD-mediated DNA damage by generating 3'-OH ends in single-strand rather than double-strand DNA nicks/breaks.

General interaction mode of CIDE:CIDE complex revealed by a mutation study of the Drep2 CIDE domain

The CIDE domain is a well known protein–protein interaction module that is initially detected at the apoptotic DNA fragmentation factor (DFF40/45). The interaction mechanism via the CIDE domain is not well understood. To elucidate CIDE domain mediated interactions in the apoptotic DNA fragmentation system, we conducted biochemical and mutational studies and found that the surface of CIDE domains can be divided into an acidic side and a basic side. In addition, a mutagenesis study revealed that the basic surface side of Drep2 CIDE is involved in the interaction with the acidic surface side of Drep1 CIDE and Drep3 CIDE. Our research supports the idea that a charge–charge interaction might be the general interaction mode of the CIDE:CIDE interaction. Structured summary of protein interactionsDrep2andDrep2bindbymolecular sieving(View Interaction:1,2)Drep1andDrep2bindbymolecular sieving(View interaction)Drep3andDrep2bindbymolecular sieving(View interaction)Drep2andDrep3bindbyblue native page(View interaction)Drep2andDrep1bindbyblue native page(View interaction)

ICAD Deficiency in Human Colon Cancer and Predisposition to Colon Tumorigenesis: Linkage to Apoptosis Resistance and Genomic Instability

We previously showed that DNA fragmentation factor, which comprises a caspase-3-activated DNase (CAD) and its inhibitor (ICAD), may influence the rate of cell death by generating PARP-1-activating DNA breaks. Here we tested the hypothesis that ICAD-deficient colon epithelial cells exhibiting resistance to death stimuli may accumulate additional genetic modifications, leading to a tumorigenic phenotype. We show that ICAD deficiency may be associated with colon malignancy in humans. Indeed, an examination of ICAD expression using immunohistochemistry in an array of both colon cancer and normal tissues revealed that ICAD expression levels were severely compromised in the cancerous tissues. Upon DNA damage caused by a low dose of irradiation, ICAD cells acquire a tumorigenic phenotype. Colon epithelial cells derived from ICAD mice showed a significant resistance to death induced by the colon carcinogen dimethylhydrazine in vitro and in mice. Such resistance was associated with a decrease in PARP-1 activation. In an animal model of dimethylhydrazine-induced colon tumorigenesis, ICAD−/− mice developed significantly higher numbers of tumors with markedly larger sizes than the wild-type counterparts. Interestingly, the phenotype of the ICAD−/− mice was not associated with a significant increase in the precancerous aberrant crypt foci suggesting a potential link to tumor progression rather than initiation. More importantly, ICAD deficiency was associated with severe genomic instability as assessed by array comparative genomic hybridization. Such genomic instability consisted most prominently of amplifications but with sizable deletions as compared to the wild-type counterparts affecting several cancer-related genes including RAF-1, GSN, LMO3, and Fzd6 independently of p53. Altogether, our results present a viable case for the involvement of ICAD deficiency in colon carcinogenesis and show that apoptosis and genomic instability may comprise the means by which such deficiency may contribute to the process of increasing susceptibility to carcinogen-induced tumorigenesis.

Induction of apoptosis through caspase-independent or caspase-9-dependent pathway in mouse and human osteosarcoma cells by a new nitroxyl spin-labeled derivative of podophyllotoxin

Previous study has found that a new nitroxyl spin-labeled derivative of podophyllotoxin, 4-[4"-(2",2",6",6"-tetramethyl-1"-piperidinyloxy)amino]-4'-demethyl-epipodophyllotoxin (GP7), can induce apoptosis in human leukemia cells. However, there have been no studies about the effects of GP7 on osteosarcoma (OS) cells. Here, we observed the anti-OS effects of GP7 in mouse and human OS cells with the comparison of etoposide. GP7 and etoposide inhibited the proliferation of a panel of mouse and human OS cells in a concentration- or time-dependent manner, and the inhibitory effect of GP7 on the proliferation of mouse LM8 or human U2OS cells was 1.28- or 1.35-fold higher than that of etoposide. GP7 or etoposide augmented the anti-OS effects of methotrexate, adriamycin, cisplatin, or their combination, and the combined inhibitory effects of GP7 with MTX on the proliferation of LM8 cells was higher than those of etoposide with MTX. GP7 arrested the cell cycle in S phase but etoposide in G(2)/M phase. GP7 or etoposide induced sub-G(1) peak, apoptotic DNA fragmentation, activations of caspase-3, -8, -9, and DNA fragmentation factor, downregulation of Bcl-2 and Bcl-xL, upregulation of Bax and Bak, and cytochrome-c release from mitochondria in both mouse and human OS cells. GP7 or etoposide also induced endonuclease G translocation from mitochondria into cytosol in mouse cells. GP7- or etoposide-induced apoptotic DNA fragmentation of human OS cells was inhibited by the pan caspase inhibitor and caspase-9 inhibitor, not by caspase-8 inhibitor whereas it was not inhibited by the pan caspase inhibitor in mouse OS cells. Our findings indicate that GP7 is effective against mouse and human OS cells in vitro. The apoptotic DNA fragmentation in mouse OS cells may be mediated by caspase-independent pathway with the involvement of endonuclease G whereas in human OS cells by caspase-9-dependent pathway downstream of the cytochrome-c-initiated caspase cascade.

A putative role of Drep1 in apoptotic DNA fragmentation system in fly is mediated by direct interaction with Drep2 and Drep4

DNA fragmentation is common phenomenon for apoptotic cell death. DNA fragmentation factor, called DFF40 (CAD: mouse homologue), is a main nuclease for apoptotic DNA fragmentation. Nuclease activity of DFF40 is normally inhibited by DFF45 by tight interaction via CIDE domain without apoptotic stimuli. Once effector caspase is activated during apoptosis signaling, it cleave DFF45, allowing DFF40 to enter the nucleus and cleave chromosomal DNA. Unlike mammalian system, apoptotic DNA fragmentation in the fly might be controlled by four DFF-related proteins, known as Drep1, Drep2, Drep3 and Drep4. Although the function of Drep1 and Drep4 is well known as DFF45 and DFF40 homologues, respectively, the function of Drep2 and Drep3 is still unclear. DFF-related proteins contain a conserved CIDE domain of ~90 amino acid residues that is involved in protein-protein interaction. Here, we showed that Drep1 directly bind to Drep2 as well as Drep4 via CIDE domain. In addition, we found that the interaction of Drep2 and Drep4 to Drep1 was not competitive indicating that Drep2 and Drep4 bind different place of Drep1. All together, we suggest that Drep1 might be involved in apoptotic DNA fragmentation of fly system by direct interaction with Drep2 as well as Drep4.

Clinicopathological significance of DNA fragmentation factor 45 and thyroid transcription factor 1 expression in benign and malignant lesions of the gallbladder

Gallbladder cancer (GBC) is one of the most aggressive tumors; we examined the expression level of DNA fragmentation factor 45 (DFF45) and thyroid transcription factor 1 (TTF-1) in benign and malignant lesions of the gallbladder by immunohistochemistry. The results were correlated with clinicopathological features and prognosis. DNA fragmentation factor 45 and TTF-1 expression was significantly higher in gallbladder adenocarcinomas than in the corresponding peritumoral tissues (χ²DFF45 = 6.92, χ²TTF-1 = 8.68, ps < 0.01), polyps (χ²DFF45 = 4.49, χ²TTF-1 = 5.35, ps < 0.05), and chronic cholecystitis (χ²DFF45 = 12.98, χ²TTF-1 = 17.74, ps < 0.01). Negative expression of DFF45 and TTF-1 was significantly associated with tumor differentiation, tumor mass, lymph node metastasis and invasion of adenocarcinomas (p < 0.05). Univariate Kaplan-Meier analysis showed that elevated expression levels of DFF45 and TTF-1 (p < 0.05) were closely associated with increased overall survival. In addition, the average survival time of patients with DFF45(+) TTF-1(+) tumors was significantly higher than those with DFF45(-) TTF-1(-) tumors (p < 0.05). Finally, multivariate Cox regression analysis showed that negative expression of DFF45 and TTF-1 was an independent prognostic predictor in gallbladder adenocarcinoma (p < 0.05). The expression of DFF45 and/or TTF-1 is closely related to the carcinogenesis, progression, clinical behavior and prognosis of gallbladder adenocarcinomas. DNA fragmentation factor 45 and TTF-1 could be progression-associated genes correlating with good prognosis in GBC.

Expression of Hepatic Fat-Specific Protein 27 Depends on the Specific Etiology of Fatty Liver

Fat-specific protein 27 gene (FSP27), isolated by screening for genes specifically expressed in fully differentiated mouse adipocytes, belongs to the cell death-inducing DNA fragmentation factor, alpha subunit-like effector family. FSP27 is induced in not only adipose tissue but also the liver of ob/ob mice, and it promotes the development of fatty liver. The FSP27 gene is expressed in a fatty liver-specific manner and is not detected in the normal mouse liver. FSP27 expression is directly regulated by the induction of the hepatic peroxisome proliferator-activated receptor γ (PPARγ) in ob/ob fatty liver. In the present study, expression of hepatic FSP27 mRNA was determined in non-genetic fatty liver models. The FSP27 gene was markedly induced in the high-fat- or methionine- and choline-deficient (MCD) diet-induced fatty liver, but it was not elevated in alcohol-induced fatty liver. Interestingly, the induction of FSP27 mRNA due to the MCD diet was independent of PPARγ levels and completely absent in the liver from PPARγ-null mice. These results suggest that FSP27 mRNA expression in the liver depends on the etiology of fatty liver.

Fat-specific protein 27 modulates nuclear factor of activated T cells 5 and the cellular response to stress

Fat-specific protein 27 (FSP27), a member of the cell death-inducing DNA fragmentation factor α-like effector (Cide) family, is highly expressed in adipose tissues and is a lipid droplet (LD)-associated protein that induces the accumulation of LDs. Using a yeast two-hybrid system to examine potential interactions of FSP27 with other proteins, a direct interaction with the N-terminal region of nuclear factor of activated T cells 5 (NFAT5) was identified. NFAT5 is a transcription factor that induces osmoprotective and inflammatory genes after its translocation to the nucleus. The interaction between FSP27 and NFAT5 was confirmed by bimolecular fluorescence complementation and coimmunoprecipitation. Using immunocytochemistry, NFAT5 is detected in the cytoplasm and in the nucleus under isotonic conditions; however, overexpression of FSP27 inhibited the hypertonic-induced nuclear translocation of NFAT5. Consistent with the suppression of NFAT5 nuclear translocation, in cells transfected with a reporter construct containing the NFAT5 response element from the monocyte chemoattractant protein 1 (MCP1) promoter, FSP27 overexpression repressed hypertonic-induced luciferase activity and the expression of NFAT5 target genes. Knockdown of FSP27 in differentiated 3T3-L1 adipocytes increased the NFAT5-mediated rise in MCP1. These results suggest that FSP27 not only modulates LD homeostasis but also modulates the response to osmotic stress via a physical interaction with NFAT5 at the LD surface.

Induction of Endothelial Cell Apoptosis by Cleaved High Molecular Weight Kininogen Is Mediated Through a Lck and p53-Dependent Pathway

AbstractAbstract 3305 Background and objective:High molecular weight kininogen (HK) is an abundant plasma protein that serves as an important component of the intrinsic pathway of coagulation. HK normally circulates as in the single chain form, but may be cleaved by plasma kallikrein to release the nonapeptide bradykinin, resulting in the formation cleaved high molecular weight kininogen (HKa) that consists of a heavy and light chain linked by a single disulfide bond. Conformational changes occurring after kallikrein cleavage result in increased exposure of histidine and glycine-rich regions with kininogen domain 5 that impart HKa with unique properties, including the ability to inhibit angiogenesis by causing selective apoptosis of proliferating endothelial cells. However, the receptors that mediate the antiangiogenic activity of HKa remain controversial, and the signaling pathways that lead to apoptosis have not been defined. Previous studies suggested possible involvement of SRC family kinases (SFK) in this process, and the purpose of this work was to further define the activation of SFKs and their downstream targets during HKa-induced endothelial cell apoptosis. Results:We first assessed the activation of SFKs in proliferating endothelial cells stimulated with bFGF before and after incubation with HKa (6–20 nM). SFKs are maintained in an inactive state through tyrosine phosphorylation of their C-terminal region mediated by the negative regulator C-terminal Src kinase (Csk). Exposure of endothelial cells to HKa caused downregulation of Csk in a dose-dependent manner within 60 minutes. In parallel, we observed a significant increase in expression of the proapoptotic SFK Lck in endothelial cells exposed to HKa, though expression of other SFKs including Lyn, Fyn, Src, Hck and Blk were not significantly altered. Increased expression of Lck was associated with activation of p53 and increased expression of the pro-apoptotic Bcl-2 family members Bax and Bak. Endothelial cell lysates prepared within 60 minutes of exposure to HKa demonstrated significant increases in the activity of caspases 3 and 7, as well as depletion of DNA fragmentation factors (DFF) 45 and 35, which cleave and inactivate DFF40, a major endonuclease involved in apoptosis. In parallel studies, endothelial cells depleted of Lck by treatment with Lck siRNA displayed loss of p53 phosphorylation, caspase 3 and 7 activity, and expression of Bax and Bad with no effects on the expression of Bad and Bid. Conclusion:These findings demonstrate a critical role for Csk in regulation of SFK activation and endothelial homeostasis, and demonstrate that downregulation of Csk by HKa leads to activation of a Lck-dependent, p53-mediated apoptotic pathway. Increasing the expression of Lck may represent a novel mechanism for regulation of aberrant angiogenesis. Disclosures:No relevant conflicts of interest to declare.