2005 Fiscal Year Final Research Report Summary
Regeneration of salivary gland and differentiation-inducing therapy for salivary gland tumors
| Project/Area Number |
16390587
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Surgical dentistry
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| Research Institution | Osaka University |
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Principal Investigator |
OKURA Masaya Osaka University, Graduate School of Dentistry, Associate Professor, 歯学研究科, 助教授 (10281130)
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| Project Period (FY) |
2004 – 2005
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| Keywords | salivary gland / salivary gland tumor / differentiation / anoikis / apoptosis |
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| Research Abstract |
HSG cells formed lobules on laminin1 gels, and expressed salivary α-amylase and cystatin C. On the other hand, HSG-Src297M (dominant negative Src) cells failed to form the lobules on laminin1 gels, and they did not express these salivary acinar proteins. HSG-Src297M cells did not have tight cell-cell junction, and there were fissures between cells. HSG cells expressed both CEACAM1-4L and -4S with using real-time PCR. HSG cells on laminin1-coated dishes (2D culture) expressed 2.8-folds of CEACAM1-4L mRNA on poly-L-lysine coated dishes. On gels (3D culture), laminin1 and fibronection inereased CEACAM1-4L expression of HSG cells. Furthermore, catalytic activity of src tyrosine kinase mediate CEACAM1-4L expression levels. Real-time quantitative RT-PCR amplification of total RNA showed that the levels of CEACAM1-4L expression was greatly reduced to 14% in HSG-Src297M cells (dominant negative c-Src expressing cells) on laminin1 gels and fibronectin gels compared with the parental HSG cells.
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Cells on gelatin gels expressed similar amounts of CEACAM-4L mRNA to -4S. Immunocytochemical stainings revealed CEACAM1 strongly localized at cell-cell junctions of HSG cells on laminin1 gels, whereas HSG-Src297M cells had no the expression at cell-cell contacts.
cDNA fragment that encode a full-length CEACAM1-4L was kindly gift from Dr.Motomu Kuroki (Fukuoka Urniversity, Japan) and was subcloned into pcDNA3.1(+) (Invitrogen). PCR-based mutagenesis was performed with Pfu DNA polymerase (Stratagene) to introduce mutation (Y488F). The three times of mutagenesis with different primers, however did not reach success, likely due to primer helix. On this account, site-directed mutagenesis was performed with the GeneTailor^<TM> site-directed mutagenesis system. (Invitrogen). The accuracy of the mutated constructs was confirmed by DNA sequencing, and we successfully got a mutant, CEACAM1-4L-Y488F.
SCC Cells aggregated and grew on low density laminin-1 (200μg/mL) gels, whereas the cells resulted in cell apoptosis cultured on high density laminin-1 (600μg /mL) gels. The nuclear condensation was apparent, cytoplasm was shrunken. The cells had discrete clumping and condensation of the chromatin, margination of condensed chromatin at nuclear membrane (crescent formation). Dissolution of the nuclear membrane, and breaking apart of the highly condensed nucleus into fragments surrounded by a small rim of cytoplasm and plasma membranem, the so-called apoptotic bodies. In addition, PPAR-γ antagonists induced SCC cell aniokis, inhibiting intracellular FAK signaling pathways. Less
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Research Products
(2 results)
