Characterization of Two DNase γ-Specific Monoclonal Antibodies and the in Situ Detection of DNase γ in the Nuclei of Apoptotic Rat Thymocytes

Shiokawa, Daisuke; Tanaka, Mayumi; Kimura, Tetsunori; Hashizume, Kaoru; Takasawa, Ryoko; Ohyama, Harumi; Fujita, Kazuko; Yamada, Takeshi; Tanuma, Sei Ichi

doi:10.1006/bbrc.2000.3249

Cited by 14 publications

(19 citation statements)

References 33 publications

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“…Indirect Immunofluorescence Analysis-Activated DNase ␥ located in apoptotic nuclei was detected in situ using hg302 anti-DNase ␥ mAb (34). Proliferating C2C12 cells, grown on collagen-coated coverslips, were cultured in DM for 2 days or treated with 0.5 M STS for 24 h. The resulting cells were fixed with 3.5% formaldehyde and soaked in ethanol, and after blocking with DPBS containing 5% fetal calf serum, the resulting cells were incubated with hg302 (2 g/ml) at room temperature for 1 h in a humid sealed chamber.…”

Section: Methodsmentioning

confidence: 99%

“…To examine whether DNase ␥ is located in the nuclei of apoptotic myoblasts, we performed indirect immunofluorescence analysis for DNase ␥. For this purpose, we used an anti-DNase ␥ monoclonal antibody (mAb), hg302, which has been shown to recognize activated DNase ␥ located within apoptotic nuclei (34). Fig.…”

Section: Apoptosis In C2c12 Cells Under Differentiating and Proliferamentioning

confidence: 99%

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Involvement of DNase γ in Apoptosis Associated with Myogenic Differentiation of C2C12 Cells

Shiokawa¹,

Kobayashi²,

Tanuma³

2002

Journal of Biological Chemistry

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Nucleosomal DNA fragmentation is detected in myoblasts only when apoptosis is induced under differentiating conditions. However, the molecular mechanisms and the DNase responsible for the differentiation-dependent apoptotic DNA laddering are poorly understood. Here we show that a Ca 2؉ /Mg 2؉ -dependent endonuclease, DNase ␥, is induced in C2C12 myoblasts during myogenic differentiation and catalyzes apoptotic DNA fragmentation in differentiating myoblasts. A Ca 2؉ /Mg 2؉ -dependent, Zn 2؉ -sensitive endonuclease activity appears in C2C12 myoblasts during myogenic differentiation. The enzymatic properties of the inducible DNase were found to be quite similar to those of DNase I family of DNases. Reverse transcriptase-PCR analysis revealed that the induction of DNase ␥, a member of the DNase I family of DNases, is correlated with the appearance of inducible DNase activity. The induction of DNase ␥ occurs simultaneously with myogenin induction but precedes the up-regulation of p21. A high level of DNase ␥ expression was also detected in differentiated myotubes but not in skeletal muscle fibers in which DNase X is highly expressed. The role of DNase ␥ in myoblast apoptosis was evaluated in the following experiments. Proliferating myoblasts acquire DNA ladder producing ability by the ectopic expression of DNase ␥, but not DNase X, suggesting that the expression level of DNase ␥ is the determinant of the differentiation-dependent apoptotic DNA laddering observed in myoblasts. DNA fragmentation during differentiation-induced apoptosis is strongly suppressed by the antisensemediated down-regulation of DNase ␥. Importantly, the extent of DNA laddering is well correlated with the level of endogenous DNase ␥ activity. Our data demonstrate that DNase ␥ is the endonuclease responsible for DNA fragmentation in apoptosis associated with myogenic differentiation.

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Section: Methodsmentioning

confidence: 99%

Section: Apoptosis In C2c12 Cells Under Differentiating and Proliferamentioning

confidence: 99%

Involvement of DNase γ in Apoptosis Associated with Myogenic Differentiation of C2C12 Cells

Shiokawa¹,

Kobayashi²,

Tanuma³

2002

Journal of Biological Chemistry

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“…We used a monoclonal antibody (mAb) that recognizes activated DNase g located in apoptotic nuclei. 27,26 Therefore, the negative staining of living cells does not mean the absence of DNase g in control cells.…”

Section: Involvement Of Cad In Sts-induced Apoptosis In Proliferatingmentioning

confidence: 99%

“…A cDNA fragment containing the entire open reading frame of rat DNase g was excised from prDNase g-Myc-His vector 27 by XhoI digestion. The fragment was then subcloned into pCAG/puro in the reverse orientation and an antisense RNA expression vector for rat DNase g (pCAG-rDNase gAS) was obtained.…”

Section: Establishment Of N1e-115/icad-cr Cellsmentioning

confidence: 99%

Differential DNases are selectively used in neuronal apoptosis depending on the differentiation state

Shiokawa

Tanuma

2004

Cell Death Differ

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In this study, we investigate the roles of two apoptotic endonucleases, CAD and DNase c, in neuronal apoptosis. High expression of CAD, but not DNase c, is detected in proliferating N1E-115 neuroblastoma cells, and apoptotic DNA fragmentation induced by staurosporine under proliferating conditions is abolished by the expression of a caspaseresistant form of ICAD. After the induction of neuronal differentiation, CAD disappearance and the induction of DNase c occur simultaneously in N1E-115 cells. Apoptotic DNA fragmentation that occurs under differentiating conditions is suppressed by the downregulation of DNase c caused by its antisense RNA. The induction of DNase c is also observed during neuronal differentiation of PC12 cells, and apoptotic DNA fragmentation induced by NGF deprivation is inhibited by the antisense-mediated downregulation of DNase c. These observations suggest that DNA fragmentation in neuronal apoptosis is catalyzed by either CAD or DNase c depending on the differentiation state. Furthermore, DNase c is suggested to be involved in naturally occurring apoptosis in developing nervous systems.

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“…Anti-DNase g antibody is used as described previously, 34) and diluted to a final concentration of 0.001%. Antibodies for poly-(ADP-ribose) polymerase (PARP), caspase 8, caspase 9, and b-actin were purchased from Santa Cruz (Santa Cruz, CA, U.S.A.).…”

Section: Methodsmentioning

confidence: 99%

Cladribine Enhances Apoptotic Cell Death in Lung Carcinoma Cells Over-Expressing DNase .GAMMA.

Hayakawa

Suzuki

Kamei

et al. 2011

Biological & Pharmaceutical Bulletin

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Worldwide, lung cancer is the most common form of cancer and often has a poor prognosis. Establishment of effective therapies for lung cancer is a major concern in clinical cancer research. We compared the cytotoxic effects of chemotherapeutic agents including cisplatin, 5-fluorouracil, vinorelbine and cladribine, on a human lung cancer cell line, A549, and its derivative transfected with the DNase g g gene. We observed selective cytotoxicity of cladribine on the DNase g g-expressing sub-cell line of A549. Cladribine induces selective apoptosis in DNase g g-expressing A549 cells, which depends on activation of caspases. These results suggest that a combination therapy that includes cladribine along with the introduction of DNase g g has potential as a new therapeutic strategy for lung cancer.

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Characterization of Two DNase γ-Specific Monoclonal Antibodies and the in Situ Detection of DNase γ in the Nuclei of Apoptotic Rat Thymocytes

Cited by 14 publications

References 33 publications

Involvement of DNase γ in Apoptosis Associated with Myogenic Differentiation of C2C12 Cells

Involvement of DNase γ in Apoptosis Associated with Myogenic Differentiation of C2C12 Cells

Differential DNases are selectively used in neuronal apoptosis depending on the differentiation state

Cladribine Enhances Apoptotic Cell Death in Lung Carcinoma Cells Over-Expressing DNase .GAMMA.

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