-
Sung-Young Kim,
Dongki Yang,
Jongyoun Myeong,
Kotdaji Ha,
Su-Hwa Kim,
Eun-Jung Park, In-Gyu Kim,
Nam-Hyuk Cho,
Kyu Pil Lee,
Ju-Hong Jeon,
Insuk So
[show abstract]
[hide abstract]
ABSTRACT: Ca(2+) is a critical factor in the regulation of signal transduction and Ca(2+) homeostasis is altered in different human diseases. The level of Ca(2+) in cells is highly regulated through a diverse class of regulators. Among them is the transient receptor potential vanilloid 6 (TRPV6), which is a Ca(2+) selective channel that absorbs Ca(2+) in the small intestine. TRPV6 is overexpressed in some cancers and exhibits oncogenic potential, but its exact mechanism is still poorly understood. The Numb protein is a cell fate determinant that functions in endocytosis and as a tumor suppressor via the stabilization of p53. Numb protein consisted of four isoforms. Here, we showed a novel function of Numb1, which negatively regulates TRPV6 activity. The expression of Numb1 decreased cytosolic Ca(2+) concentrations in TRPV6-transfected HEK293 cells. When all the isoforms of Numb were depleted using siRNA in a TRPV6 stable cell line, the levels of cytosolic Ca(2+) increased. We observed an interaction between Numb1 and TRPV6 using co-immunoprecipitation. We confirmed this interaction using Fluorescence Resolution Energy Transfer (FRET). We identified the TRPV6 and Numb1 binding site using TRPV6 C-terminal truncation mutants and Numb1 deletion mutants. The binding site in TRPV6 was an aspartic acid at amino acid residue 716, and that binding site in Numb1 was arginine at amino acid residue 434. A Numb1 mutant, lacking TRPV6 binding activity, failed to inhibit TRPV6 activity. Every isoform of Numb knockdown, using an siRNA-based approach in MCF-7 breast cancer cells, not only showed enhanced TRPV6 expression but also both the cytosolic Ca(2+) concentration and cell proliferation were increased. The down-regulated expression of TRPV6 using siRNA increased Numb protein expression; however, the cytosolic influx of Ca(2+) and proliferation of the cell were decreased. To examine downstream signaling during Ca(2+) influx, we performed Western blotting analysis on TRPV6 upregulated cancer cells (MCF-7, PC-3). Taken together, these results demonstrated that Numb1 interacts with TRPV6 through charged residues and inhibits its activity via the regulation of protein expression. Moreover, we provided evidence for a Ca(2+)-regulated cancer cell signaling pathway and that the Ca(2+) channel is a target of cancer cells.
Cell calcium 11/2012; · 4.29 Impact Factor
-
Chae Ryun Lee,
Jung Nyeo Chun,
Sang-Yeob Kim,
Soonbum Park,
Su-Hwa Kim,
Eun-Jung Park,
In-San Kim,
Nam-Hyuk Cho, In-Gyu Kim,
Insuk So,
Tae Woo Kim,
Ju-Hong Jeon
[show abstract]
[hide abstract]
ABSTRACT: Cyclosporin A (CsA) has antitumor effects on various cancers including prostate cancer. However, its antitumor mechanism is poorly understood. In this study, we showed that AMP-activated protein kinase (AMPK) mediates the antitumor effect of CsA on prostate cancer cells. CsA attenuated cell growth by inducing a G1 arrest through the inhibition of mTOR complex 1 (mTORC1) signaling. In this context, Akt was paradoxically activated downstream of the EGF receptor (EGFR)-mediated increase in phosphatidylinositol 3,4,5-trisphosphate (PIP₃) production. However, CsA also caused a Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ)-dependent activation of AMPK, which inhibits mTORC1 signaling; this led to ineffective Akt signaling. An EGFR or Akt inhibitor increased the growth suppressive activity of CsA, whereas the combination of an AMPK inhibitor and CsA markedly rescued cells from the G1 arrest and increased cell growth. These results provide novel insights into the molecular mechanisms of CsA on cancer signaling pathways.
Biochemical pharmacology 05/2012; 84(4):425-31. · 4.25 Impact Factor
-
Su-Hwa Kim,
Sanghoon Lee,
Stephen R Piccolo,
Kristina Allen-Brady,
Eun-Jung Park,
Jung Nyeo Chun,
Tae Woo Kim,
Nam-Hyuk Cho, In-Gyu Kim,
Insuk So,
Ju-Hong Jeon
[show abstract]
[hide abstract]
ABSTRACT: Menthol, a naturally occurring monoterpene, is used in foods, cosmetic products, and topical therapeutic preparations. It also exerts cytotoxic activity against several cancer cell types, including prostate cancer cells. However, little is known about the mechanism of menthol action against prostate cancer cells. In this study, we investigated the effect of menthol on the gene expression profile of PC-3 prostate cancer cells using DNA microarray analyses. Gene set enrichment analysis revealed that menthol primarily affects the expression of cell cycle-related genes. Subsequent experimental analyses validated that menthol induces G2/M arrest. Particularly, menthol markedly down-regulated polo-like kinase 1 (PLK1), a key regulator of G2/M phase progression and inhibited its downstream signaling. Our computational analyses and experimental validation provide a basis for future investigations that are aimed at elucidating the action of menthol on cell cycle control in prostate cancer cells.
Biochemical and Biophysical Research Communications 05/2012; 422(3):436-41. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The ubiquitous transient receptor potential canonical (TRPC) channels function as non-selective, Ca(2+)-permeable channels and mediate numerous cellular functions. It is commonly assumed that TRPC channels are activated by stimulation of Gα(q)-PLC-coupled receptors. However, whether the Gα(q)-PLC pathway is the main regulator of TRPC4/5 channels and how other Gα proteins may regulate these channels are poorly understood. We previously reported that TRPC4/TRPC5 can be activated by Gα(i). In the current work, we found that Gα(i) subunits, rather than Gα(q), are the primary and direct activators of TRPC4 and TRPC5. We report a novel molecular mechanism in which TRPC4 is activated by several Gα(i) subunits, most prominently by Gα(i2), and TRPC5 is activated primarily by Gα(i3). Activation of Gα(i) by the muscarinic M2 receptors or expression of the constitutively active Gα(i) mutants equally and fully activates the channels. Moreover, both TRPC4 and TRPC5 are activated by direct interaction of their conserved C-terminal SESTD (SEC14-like and spectrin-type domains) with the Gα(i) subunits. Two amino acids (lysine 715 and arginine 716) of the TRPC4 C terminus were identified by structural modeling as mediating the interaction with Gα(i2). These findings indicate an essential role of Gα(i) proteins as novel activators for TRPC4/5 and reveal the molecular mechanism by which G-proteins activate the channels.
Journal of Biological Chemistry 03/2012; 287(21):17029-39. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The activation of transglutaminase 2 (TG2), an enzyme that catalyzes post-translational modifications of proteins, has been implicated in apoptosis, cell adhesion and inflammatory responses. We previously reported that intracellular TG2 is activated under oxidative stress conditions, such as ultraviolet irradiation, ischemia-reperfusion, and hypoxia. In this study, we examined the effect of genotoxic stress on the intracellular activity of TG2 using doxorubicin which generates reactive oxygen species that lead to double-strand breakage of DNA. We demonstrated that doxorubicin elicits the persistent activation of TG2. Doxorubicin-induced TG2 activity was suppressed by treatment with caffeine at the early phase, N-acetylcysteine at the mid-phase, and EGTA at the late phase. However, treatment with a blocking antibody against TGFβ or toll-like receptor 2 showed no effect on TG2 activity, indicating that at least three different signaling pathways may be involved in the process of TG2 activation. In addition, using MEF cells defective for TG2 and cells overexpressing an activesite mutant of TG2, we revealed that doxorubicin-induced cell death is inversely correlated with TG2 activity. Our findings indicate that the persistent activation of TG2 by doxorubicin contributes to cell survival, suggesting that the mechanism-based inhibition of TG2 may be a novel strategy to prevent drug-resistance in doxorubicin treatment.
Molecules and Cells 02/2012; 33(3):235-41. · 2.18 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Herpesvirus saimiri (HVS), a T-lymphotropic monkey herpesvirus, induces fulminant T-cell lymphoma in non-natural primate hosts. In addition, it can immortalize human T-cells in vitro. HVS tyrosine kinase-interacting protein (Tip) is an essential viral gene required for T-cell transformation both in vitro and in vivo. In this study, we found that Tip interacts with the STAT6 transcription factor and induces phosphorylation of STAT6 in T-cells. The interaction with STAT6 requires the Tyr(127) residue and Lck-binding domain of Tip, which are indispensable for interleukin (IL)-2-independent T-cell transformation by HVS. It was also demonstrated that Tip induces nuclear translocation of STAT6, as well as activation of STAT6-dependent transcription in Jurkat T-cells. Interestingly, the phosphorylated STAT6 mainly colocalized with vesicles containing Tip within T-cells, but was barely detectable in the nucleus. However, nuclear translocation of phospho-STAT6 and transcriptional activation of STAT6 by IL-4 stimulation were not affected significantly in T-cells expressing Tip. Collectively, these findings suggest that the constitutive activation of STAT6 by Tip in T-cells may contribute to IL-2-independent T-cell transformation by HVS.
Journal of General Virology 02/2012; 93(Pt 2):330-40. · 3.36 Impact Factor
-
Sang-Mok Lee,
Eui Man Jeong,
Jinho Jeong,
Dong-Myung Shin,
Hyun-Ju Lee,
Hyo-Jun Kim,
Jisun Lim,
Jin-Haeng Lee,
Sung-Yup Cho,
Mee-Kum Kim,
Won-Ryang Wee,
Jin-Hak Lee, In-Gyu Kim
[show abstract]
[hide abstract]
ABSTRACT: The activation of transglutaminase 2 (TG2) by oxidative stress through TGFβ has been reported to play a crucial role in cataract formation. The authors investigated whether TG2 is involved in selenite-induced cataract formation in rats and whether cysteamine, a chemical inhibitor of TG2, can prevent cataract formation in this model.
Intracellular TG2 activity was monitored in a human lens epithelial cell (HLE-B3) line and cultured rat lenses after treatment with selenite. Rat pups (13 days old) were injected subcutaneously with sodium selenite (Na(2)SeO(3); 20 μmol/kg) and intraperitoneally with cysteamine (30, 40, and 60 mg/kg) for 14 days. Lenses were evaluated photographically at days 7 and 14. The concentrations of malondialdehyde and glutathione in the lenses were determined.
In HLE-B3 cells or rat lenses, selenite induced intracellular TG activity, which was inhibited by cysteamine. In selenite-treated rats, the rate of cataract formation was significantly reduced by cysteamine (P < 0.001). The mean cataract area in the lenses of cysteamine-treated rats was smaller than that of control rats (P < 0.01). The levels of total and reduced glutathione in the lenses of cysteamine-treated rats extracted at day 14 were higher than those of control rats.
Cysteamine suppresses cataract formation induced by selenite in rats, suggesting that cysteamine can be used as a pharmaceutical intervention to prevent or delay cataract formation.
Investigative ophthalmology & visual science 01/2012; 53(3):1452-9. · 3.43 Impact Factor
-
Su-Hwa Kim,
Eun-Jung Park,
Chae Ryun Lee,
Jung Nyeo Chun,
Nam-Hyuk Cho, In-Gyu Kim,
Sanghoon Lee,
Tae Woo Kim,
Hyun Ho Park,
Insuk So,
Ju-Hong Jeon
[show abstract]
[hide abstract]
ABSTRACT: Geraniol, an acyclic dietary monoterpene, suppresses prostate cancer growth and enhances docetaxel chemosensitivity in cultured cell or xenograft tumor models. However, the mechanisms of the geraniol action against prostate cancer are largely unknown. In this study, we investigated the cellular and molecular mechanisms of geraniol-induced cell death in PC-3 prostate cancer cells. Among the examined structurally and functionally similar monoterpenes, geraniol potently induced apoptosis and autophagy. Although independent processes, apoptosis and autophagy acted as cooperative partners to elicit geraniol-induced cell death in PC-3 cells. At a molecular level, geraniol inhibited AKT signaling and activated AMPK signaling, resulting in mTOR inhibition. Combined treatment of AKT inhibitor and AMPK activator markedly suppressed cell growth compared to either treatment alone. Our findings provide insight into future investigations that are aimed at elucidating the role of apoptosis and autophagy in prostate cancer therapy and at developing anticancer strategies co-targeting AKT and AMPK.
International Journal of Oncology 12/2011; 40(5):1683-90. · 2.40 Impact Factor
-
Tae-Ho Jang,
Sung-Jun Lee,
Chang-Hoon Woo,
Kyung Jin Lee,
Ju-Hong Jeon,
Dong-Sup Lee,
Kihang Choi, In-Gyu Kim,
Young Whan Kim,
Tae-Jin Lee,
Hyun Ho Park
[show abstract]
[hide abstract]
ABSTRACT: Genotoxic stress induced apoptosis is mediated by the formation of PIDDosome, which is a caspase-2 activating complex composed of three protein components, PIDD, RAIDD, and caspase-2. Here, synthetic TAT-fused peptides designed by the structure of PIDD and RAIDD, TAT-Y814A and TAT-R147E, respectively, were produced and tested for their ability to inhibit PIDDosome formation in vitro as well as to attenuate genotoxic stress-induced apoptosis in human renal cancer cells. The results show that TAT-Y814A and TAT-R147E have the potential to inhibit formation of the PIDDosome in a dose-dependent manner. Furthermore, both peptides partially inhibit genotoxic stress mediated apoptosis and activation of caspase2 and caspase3 in Caki cells. These results suggest that TAT-Y814A (also TAT-R147E) is a novel inhibitor of genotoxic stress-induced apoptosis that may serve as a prototype for anti-apoptotic drug development.
Biochemical pharmacology 10/2011; 83(2):218-27. · 4.25 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Pulmonary fibrosis is a potentially life-threatening disease that may be caused by overt or asymptomatic inflammatory responses. However, the precise mechanisms by which tissue injury is translated into inflammation and consequent fibrosis remain to be established. Here, we show that in a lung injury model, bleomycin induced the secretion of IL-6 by epithelial cells in a transglutaminase 2 (TG2)-dependent manner. This response represents a key step in the differentiation of IL-17-producing T cells and subsequent inflammatory amplification in the lung. The essential role of epithelial cells, but not inflammatory cells, TG2 was confirmed in bone marrow chimeras; chimeras made in TG2-deficient recipients showed reduced inflammation and fibrosis, compared with those in wild-type mice, regardless of the bone marrow cell phenotype. Epithelial TG2 thus appears to be a critical inducer of inflammation after noninfectious pulmonary injury. We further demonstrated that fibroblast-derived TG2, acting downstream of transforming growth factor-β, is also important in the effector phase of fibrogenesis. Therefore, TG2 represents an interesting potential target for therapeutic intervention.
Journal of Experimental Medicine 08/2011; 208(8):1707-19. · 13.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Ribosomal proteins are a major component of ribosomes, which catalyze protein synthesis. One ribosomal protein, L7a (RPL7a), which is a component of the 60S large ribosomal subunit, has additional functions involved in cell growth and differentiation that occur via interaction with human thyroid hormone receptor (THR) and retinoic acid receptor (RAR) and in turn inhibit the activities of the two nuclear hormone receptors. In this study, the N-terminal domain of human RPL7a was overexpressed in Escherichia coli using an engineered C-terminal His tag. The N-terminal domain of human RPL7a was then purified to homogeneity and crystallized at 293 K. X-ray diffraction data were collected to a resolution of 3.5 Å from a crystal belonging to the tetragonal space group P4(1)22 or P4(3)22 with unit-cell parameters a = 92.28, b = 92.28, c = 236.59 Å.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 04/2011; 67(Pt 4):510-2. · 0.51 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Chondrocyte apoptosis has been implicated in the pathogenesis of osteoarthritis. Transglutaminase 2 (TG2), the expression of which is higher in osteoarthritis patients, has been shown to be up-regulated during apoptosis in many experimental models. This study investigated the expression and role of TG2 in human chondrocytes undergoing apoptosis induced by hydrogen peroxide (H₂O₂). Human chondrocytes were obtained from the knee articular cartilage of patients undergoing total joint arthroplasty. Apoptosis was induced by H₂O₂ and was measured with Annexin-V flow cytometry, DNA Fragmentation ELISA and DAPI staining. Western Blot, an in situ activity assay and immunocytochemistry were used to examine TG2 expression. The role of TG2 was evaluated by TG-specific siRNA transfection and monodansylcadaverine (MDC), a competitive substrate for TG2. H₂O₂ induced apoptosis of human chondrocytes in a dose- and time-dependent manner. The level of TG2 expression was higher in the chondrocytes undergoing H₂O₂-induced apoptosis. Inhibition of TG2 by siRNA or MDC increased the level of apoptosis in the H₂O₂-treated chondrocytes. TG2 expression is higher in human chondrocytes undergoing apoptosis, and inhibition of TG2 leads to increased apoptosis. These results may raise the possibility of TG2 as a modulator of cartilage damage in osteoarthritis by offering protection against chondrocyte apoptosis.
Journal of Orthopaedic Research 02/2011; 29(2):252-7. · 2.81 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: An abrupt increase of intracellular Ca(2+) is observed in cells under hypoxic or oxidatively stressed conditions. The dysregulated increase of cytosolic Ca(2+) triggers apoptotic cell death through mitochondrial swelling and activation of Ca(2+)-dependent enzymes. Transglutaminase 2 (TG2) is a Ca(2+)-dependent enzyme that catalyzes transamidation reaction producing cross-linked and polyaminated proteins. TG2 activity is known to be involved in the apoptotic process. However, the pro-apoptotic role of TG2 is still controversial. In this study, we investigate the role of TG2 in apoptosis induced by Ca(2+)-overload. Overexpression of TG2 inhibited the A23187-induced apoptosis through suppression of caspase-3 and -9 activities, cytochrome c release into cytosol, and mitochondria membrane depolarization. Conversely, down-regulation of TG2 caused the increases of cell death, caspase-3 activity and cytochrome c in cytosol in response to Ca(2+)-overload. Western blot analysis of Bcl-2 family proteins showed that TG2 reduced the expression level of Bax protein. Moreover, overexpression of Bax abrogated the anti-apoptotic effect of TG2, indicating that TG2-mediated suppression of Bax is responsible for inhibiting cell death under Ca(2+)-overloaded conditions. Our findings revealed a novel anti-apoptotic pathway involving TG2, and suggested the induction of TG2 as a novel strategy for promoting cell survival in diseases such as ischemia and neurodegeneration.
Experimental and Molecular Medicine 09/2010; 42(9):639-50. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Transglutaminase 4 (TG4) is a member of the enzyme family that catalyzes the calcium-dependent post-translational modification of proteins via cross-linking, polyamination, or deamidation. TG4 exhibits prostate-specific expression pattern and plays a crucial role in the formation of the copulatory plug in rodents. However, the physiological function(s) of human TG4 remains speculative. Human TG4 has been postulated to participate in the maturation process of sperm by modifying its cell surface, which results in suppression of sperm antigenicity in the female genital tract. To better understand the pathophysiological role of TG4 in prostate tissue, we generated monoclonal antibodies (MAb) against human TG4 in mice by repeated injections with the recombinant human TG4. Western blot analysis demonstrated that the selected MAbs react specifically with TG4, but not with other isoenzymes of the TG family. Immunocytochemical and immunohistochemical analyses showed that specific staining is observed with the cells overexpressing TG4 and with the paraffin-embedded prostate tissue specimens obtained from the benign prostate hyperplasia and prostate cancer patients, respectively. Our results indicate that these MAbs are suitable for detecting TG4 in the cultured cells or prostate tissues for investigating the biological functions of human TG4.
Hybridoma (2005) 06/2010; 29(3):263-7. · 0.42 Impact Factor
-
Sung-Yup Cho,
Kyungho Choi,
Ju-Hong Jeon,
Chai-Wan Kim,
Dong-Myung Shin,
Jong Bouk Lee,
Sang Eun Lee,
Choung-Soo Kim,
Jeong-Soo Park,
Eui Man Jeong,
Gi-Yong Jang,
Kye-Yong Song, In-Gyu Kim
[show abstract]
[hide abstract]
ABSTRACT: Transglutaminase 4 is a member of enzyme family that catalyzes calcium-dependent posttranslational modification of proteins. Although transglutaminase 4 has been shown to have prostate-restricted expression pattern, little is known about the biological function of transglutaminase 4 in human. To gain insight into its role in prostate, we analyzed the expression status of human transglutaminase 4 in benign prostate hyperplasia (BPH) and prostate cancer (PCa). Unexpectedly, RT-PCR and nucleotide sequence analysis showed four alternative splicing variants of transglutaminase 4: transglutaminase 4-L, -M (-M1 and -M2) and -S. The difference between transglutaminase 4-M1 and -M2 is attributed to splicing sites, but not nucleotide size. The deduced amino acid sequences showed that transglutaminase 4-L, -M1 and -M2 have correct open reading frames, whereas transglutaminase 4-S has a truncated reading frame. RT-PCR analysis of clinical samples revealed that transglutaminase 4-M and -S were detected in all tested prostate tissue (80 BPH and 48 PCa). Interestingly, transglutaminase 4-L was found in 56% of BPH (45 out of 80) and only in 15% of PCa (7 out of 48). However, transglutaminase 4-L expression did not correlate with serum prostate-specific antigen (PSA) level, prostate volumes or PSA densities. These results will provide a clue to future investigation aiming at delineating physiological and pathological roles of human transglutaminase 4.
Experimental and Molecular Medicine 02/2010; 42(4):310-8. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Transglutaminase 2 (TG2) is a calcium-dependent enzyme that catalyzes the transamidation reaction. There is conflicting evidence on the role of TG2 in apoptosis. In this report, we show that TG2 increases in response to low level of oxidative stress, whereas TG2 diminishes under high stress conditions. Monitoring TG2 expression, activity and calcium concentration in cells treated with A23187 revealed that the initial rise of calcium activates TG2 but subsequent calcium-overload induces the degradation of TG2 via calcium-mediated polyubiquitination. These results indicate that the role of TG2 in apoptosis depends on the level of calcium influx triggered by oxidative stress.
FEBS letters 02/2009; 583(4):648-54. · 3.54 Impact Factor
-
Dong-Myung Shin,
Ju-Hong Jeon,
Chai-Wan Kim,
Sung-Yup Cho,
Hye-Jin Lee,
Gi-Yong Jang,
Eui Man Jeong,
Dong-Sup Lee,
Ja-Heon Kang,
Gerry Melino,
Sang-Chul Park, In-Gyu Kim
[show abstract]
[hide abstract]
ABSTRACT: Transglutaminase 2 (TGase2) is a ubiquitously expressed enzyme that catalyzes irreversible post-translational modification of protein, forming cross-linked protein aggregates. We previously reported that intracellular TGase2 is activated by oxidative stress. To elucidate the functional role of TGase2 activation in cells under the oxidatively stressed condition, we identified the mediator that activates TGase2. In this study, we showed that low levels of oxidative stress trigger the release of TGFbeta, which subsequently activates TGase2 through the nuclear translocation of Smad3. Analysis of substrate proteins reveals that TGase2-mediated protein modification results in a decrease of protein solubility and a collapse of intermediate filament network, which leads to aggregation of proteins. We confirm these results using lens tissues from TGase2-deficient mice. Among several antioxidants tried, only N-acetylcysteine effectively inhibits TGFbeta-mediated activation of TGase2. These results indicate that TGFbeta mediates oxidative stress-induced protein aggregation through activation of TGase2 and suggest that the formation of protein aggregation may not be a passive process of self-assembly of oxidatively damaged proteins but may be an active cellular response to oxidative stress. Therefore, TGFbeta-TGase2 pathway may have implications for both the pathogenesis of age-related degenerative diseases and the development of pharmaceutics.
The FASEB Journal 08/2008; 22(7):2498-507. · 5.71 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Transglutaminase2 (TGase2) activates Rho-associated kinase (ROCK), an important mediator of ischemia-reperfusion (IR) injury, through polyamination of RhoA. Cystamine, an oxidized dimer of cysteamine inhibits the transamidation activity of TGase2. We examined whether addition of cystamine to an organ preservation solution protects rat cardiomyocyte cells (H9C2) from cell death in IR injury. H9C2 cells were stored under hypoxic conditions at 4 degrees C in laboratory-made preservation solution (SNU) or SNU solution supplemented with cystamine (SNU-C1), and cell preservation in the two solutions was compared by measuring the release of lactate dehydrogenase. The cells were preserved more effectively in SNU-C1 than in SNU solution. Cystamine inhibited the intracellular activity of TGase2 which increased during cold storage or reoxygenation. The inhibition of TGase2 by cystamine reduced the polyamination of RhoA, the interaction between RhoA and ROCK2, and F-actin formation. Cystamine also prevented the activation of caspases during cold storage. These results suggest that addition of cystamine to the organ preservation solution significantly enhances cardiomyocytes preservation apparently by inhibiting TGase2-mediated RhoA-ROCK pathway and that TGase2 may play an important role in IR injury by regulating ROCK.
Biochemical and Biophysical Research Communications 02/2008; 365(3):509-14. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cervical cancer is characterized by a long period of preclinical dysplasia or carcinoma in situ progressing into invasive cancer. Although Papanicolaou (Pap) smear test has contributed significantly to the early detection of precursor lesions, the cytological screening has inherent problems that produce considerable false negative/positive results. Since the infection of high-risk type of human papillomavirus (HPV) is strongly associated with cervical cancer, we investigated the feasibility of an immunostaining test to detect cells infected by HPV in cervical smear. We produced monoclonal antibodies against HPV16 E7 in mice by repeated injections with the recombinant HPV16 E7. Western blot analysis and immunocytochemical assay demonstrated that the selected monoclonal antibody, mAb (130-9-7), reacts specifically with cultured cervical cancer cell lines infected by HPV16. Specific staining was observable with the HPV16-positive smear specimens obtained from the cervical cancer patients, whereas no staining was detected with the HPV-negative smear specimens. To achieve the desired sensitivity, specificity and reproducibility, we modified and optimized the conventional immunocytochemical procedure for cervical smear specimens. Our results suggest that this immunostaining method for detecting high-risk HPV in cervical smear may be used as a strategy to distinguish a high-risk group, especially those patients with low grade cytological abnormality.
Experimental and Molecular Medicine 11/2007; 39(5):621-8. · 2.48 Impact Factor
-
Analytical Biochemistry 02/2006; 348(2):327-9. · 3.00 Impact Factor
