Immune complex independent activation of complement, Cls secreted from hamster embryo malignant fibroblasts, Nil2C2 in serum free culture medium

Yamaguchi, Kiichiro; Kato, Norio; Sakai, Norie; Matsumoto, Misako; Nagasawa, Shigeharu; Hatsuse, Hiromi; Toyoguchi, Toru; Moriya, Hideshige; Sakiyama, Hisako

doi:10.1016/0167-4838(94)90101-5

Cited by 7 publications

(8 citation statements)

References 32 publications

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“…3), indicating that Cls on the cell surface was proenzyme form. This observation agrees with a previous report that the L chain was not detectable in the membrane fraction of Ni12C2 cells when immunoblotting was carried out under reducing conditions (Yamaguchi et al, 1994).…”

Section: Immunojluorescent Staining Of Ni12c2 Cellssupporting

confidence: 94%

“…The anti-peptide antibody, RK3, which was raised against peptide 3 of the L chain (Toyoguchi et al, 1995), was also used. Reactivity of the antibodies with C l s was tested by Western blotting (Towbin et aZ., 1979) using HEF-and Nil2C2-conditioned media which contained non-activated and activated Cls, respectively (Yamaguchi et al, 1994). Fluorescent staining was carried out by incubating Ni12C2 cells with antibodies at 0°C for 30 min followed by incubation with FITC-conjugated anti-rabbit antibody.…”

Section: Immunization and Immunological Assaysmentioning

confidence: 99%

“…After removal of nuclei by low-speed centrifugation, the supernatant was subjected to centrifugation at 105,OOOg for 30 min at 4°C. The membrane (P-100) and cytosolic soluble fractions (S-100) were isolated as described (Quigley, 1976;Yamaguchi et al, 1994).…”

Section: Cell Fractionationmentioning

confidence: 99%

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Site‐directed mutagenesis of hamster complement C1S: Characterization with an active form‐specific antibody and possible involvement of C1S in tumorigenicity

Sakiyama¹,

Nishida²,

Sakai³

et al. 1996

Intl Journal of Cancer

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We have previously shown that non-transformed mouse A31 cells became tumorigenic when they were transfected with hamster C1s cDNA expression plasmid BCMGSNeoCS. In the present study, mutations were introduced into the cDNA at the activation cleavage site, Arg423(AGG) and the active center Ser617(AGC). These amino-acids were replaced by His423(CAC) and Thr617(ACC), respectively. The mutated cDNAs were inserted into BCMGSNeo and transfected to A31 and its polyoma-virus-transformed SEA7 cells. C1s produced from these transfectants lost their enzyme activity. Transfectants of these mutated C1s cDNA did not form tumors in nude mice, To distinguish between active and inactive C1s in situ, we have developed novel antibodies, one directed to the NH2-terminal neoepitope of the L chain and the other specific for uncleaved inactive C1s. These antibodies were used to characterize C1s produced by transfectants, so as to determine whether or not it was cleaved at the right position.

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Section: Immunojluorescent Staining Of Ni12c2 Cellssupporting

confidence: 94%

Section: Immunization and Immunological Assaysmentioning

confidence: 99%

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Site‐directed mutagenesis of hamster complement C1S: Characterization with an active form‐specific antibody and possible involvement of C1S in tumorigenicity

Sakiyama¹,

Nishida²,

Sakai³

et al. 1996

Intl Journal of Cancer

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“…We have reported previously that C1s produced from established cell lines, irrespective of transformation, is activated in serum-free culture media, but not in that from secondary cells (Yamaguchi et al 1994). We have suggested that serine proteinase(s), which is (are) expressed in established cell lines, could be responsible for this activation (Yamaguchi et al 1994).…”

Section: Discussionmentioning

confidence: 92%

“…The resulting antibody purified on the immobilized peptide column was shown to react with the L chain of hamster and rat C1s (Toyoguchi et al 1995). Since activated C1s is a heterodimer of the L and H chains linked with a disulfide bond, the L chain is expected to be detected under reducing conditions by immunoblotting with RK3 (Yamaguchi et al 1994;Toyoguchi et al 1995). Anti-human C1s monoclonal antibodies M81, M365, and M241 have been characterized previously (Matsumoto and Nagaki 1986;Matsumoto et al 1989).…”

Section: Antibodiesmentioning

confidence: 98%

Coordinated change between complement C1s production and chondrocyte differentiation in vitro

Nakagawa¹,

Sakiyama²,

Fukazawa³

et al. 1997

Cell and Tissue Research

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In vitro synthesis of the first component of complement C1s was examined by using hamster epiphyseal chondrocytes (HAC) and human chondrosarcoma cell line HCS-2/8. Hamster and human C1s produced by the cells were quantified by immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA), respectively. It was possible to measure active and inactive C1s by sandwich ELISA, when we used anti-human C1s monoclonal antibodies, M241 recognizing only active C1s, and M365 and M81 recognizing both active and inactive C1s. Approximately 40% of C1s secreted from HCS-2/8 was found to be activated in the culture medium, whereas C1s from HAC was not. C1s production increased in accordance with chondrocyte differentiation induced by ascorbic acid. In contrast, transforming growth factor-beta1 and basic fibroblast growth factor, which inhibited differentiation, suppressed C1s production. These results confirmed our previous observation showing that C1s synthesis increased with differentiation into hypertrophic chondrocytes in vivo.

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Tumorigenicity of BALB3T3 A31 cells transfected with hamster‐complement‐C1s cDNA

Sakai

Kusunoki²,

Nishida³

et al. 1994

Intl Journal of Cancer

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Hamster-complement-C1s cDNA was inserted into an expression plasmid BCMGSNeo (BCMGSNeoHACS). BALB/c mouse fibroblast A31 cells, which do not produce C1s, were transfected with BCMGSNeoHACS and the transfectants were selected with G418. Normal C1s production by the transfectants was confirmed by Northern and immunoblot analysis and by an esterase assay. To examine the tumorigenicity of the transfectants, 1 x 10(6) cells were injected s.c. into 6-week-old BALB/c nu/nu mice. Three C1s cDNA transfectants (A3CS9, A3CS12, A3CS13) formed tumors whereas both A31 and A31 transfected with the vector alone (A3BCM1 and A3BCM3) did not. The tumors derived from the transfectants showed invasive growth, and many capillaries were observed in the tumors. A tumor derived from A3CS13 was examined immunohistochemically and found to be reactive with an anti-C1s monoclonal antibody. Tumor cells were cultured in vitro again and C1s secreted into the culture medium was examined by immunoblot analysis. C1s synthesized by the tumor cells derived from A3CS13 maintained its biological functions. Tumor cells derived from A3CS9 and A3CS12 cells, however, produced C1s having abnormal disulfide bonds.

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Immune complex independent activation of complement, Cls secreted from hamster embryo malignant fibroblasts, Nil2C2 in serum free culture medium

Cited by 7 publications

References 32 publications

Site‐directed mutagenesis of hamster complement C1S: Characterization with an active form‐specific antibody and possible involvement of C1S in tumorigenicity

Site‐directed mutagenesis of hamster complement C1S: Characterization with an active form‐specific antibody and possible involvement of C1S in tumorigenicity

Coordinated change between complement C1s production and chondrocyte differentiation in vitro

Tumorigenicity of BALB3T3 A31 cells transfected with hamster‐complement‐C1s cDNA

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