ABSTRACT
OBJECTIVE: TGIF1 homeobox gene involvement in oral cancer has not yet been investigated. This study analyzed the expression of TGIF1 transcripts and protein in oral squamous cell carcinoma (OSCC). STUDY DESIGN: Snap-frozen samples from 16 patients were taken from both OSCC and nontumoral adjacent epithelium (NT) for in situ hybridization (ISH). Forty-six paraffin-embedded samples of OSCC were submitted to immunohistochemistry (IHC). A descriptive analysis of the transcript signal detection was accomplished, and TGIF1 immunoexpression was carried out considering protein levels, localization, and cellular differentiation. RESULTS: ISH reactions showed TGIF1 transcripts with a signal that was frequently intense in NT, and generally weak in OSCC, and that had stronger transcript signal in well-differentiated areas of OSCC when compared with poorly differentiated ones. IHC reactions had poorly differentiated cases associated with TGIF1 protein expression in both the nucleus and cytoplasm (P = .05, Fisher test). CONCLUSIONS: TGIF1 gain or loss of function might possibly play a role in oral cancer cell differentiation.
Subject(s)
Carcinoma, Squamous Cell/genetics , Cell Nucleus/metabolism , Cytoplasm/metabolism , Homeodomain Proteins/genetics , Mouth Neoplasms/genetics , Repressor Proteins/genetics , Adult , Aged , Aged, 80 and over , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Female , Homeodomain Proteins/metabolism , Humans , Immunohistochemistry , Male , Middle Aged , Mouth Mucosa/metabolism , Mouth Neoplasms/metabolism , Mouth Neoplasms/pathology , Repressor Proteins/metabolism , Single-Blind MethodABSTRACT
We have evaluated RECK (reversion-inducing-cysteine-rich protein with Kazal motifs), MMP-2 (matrix metalloproteinase-2), MMP-3, and MMP-9 involvement during palate development in mice by using various techniques. Immunohistochemical features revealed the distribution of RECK, MMP-2, and MMP-3 in the mesenchymal tissue and in the midline epithelial seam at embryonic day 13 (E13), MMPs-2, -3, and -9 being particularly expressed at E14 and E14.5. In contrast, RECK was weakly immunostained at these times. Involvement of MMPs was validated by measuring not only their protein expression, but also their activity (zymograms). In situ hybridization signal (ISH) for RECK transcript was distributed in mesenchymal and epithelial regions within palatal shelves at all periods evaluated. Importantly, the results from ISH analysis were in accord with those obtained by real-time polymerase chain reaction. The expression of RECK was found to be temporally regulated, which suggested possible roles in palatal ontogeny. Taken together, our results clearly show that remodeling of the extracellular matrix is finely modulated during secondary palate development and occurs in a sequential manner.
Subject(s)
Extracellular Matrix Proteins/metabolism , Membrane Glycoproteins/metabolism , Mesoderm/metabolism , Metalloproteases/metabolism , Palate/embryology , Palate/metabolism , Animals , Body Patterning/physiology , GPI-Linked Proteins , Gene Expression Regulation, Developmental/physiology , Immunohistochemistry , In Situ Hybridization , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 3/metabolism , Matrix Metalloproteinase 9/metabolism , Membrane Glycoproteins/genetics , Mesoderm/cytology , Mice , Palate/cytology , RNA, Messenger/analysis , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
In this study, Bmp-4, Wnt-5a and Shh gene expressions were compared during early craniofacial development in mice by comparative non-isotopic in situ hybridization. Wild-type C57BL/6J mice were studied at various stages of embryonic development (from 8.5- to 13.5-day-old embryos--E8.5-13.5). During early odontogenesis, transcripts for Bmp-4, Shh and Wnt-5a were co-localised at the tooth initiation stage. At E8.5, Shh mRNA expression was restricted to diencephalon and pharyngeal endoderm. Before maxillae and mandible ossification, Bmp-4 and Wnt-5a signals were detected in the mesenchymal cells and around Meckel's cartilage. During palatogenesis, Shh was expressed only in the epithelium and Wnt-5a only in the mesenchyme of the elevating palatal shelves. During tongue development, Shh expression was found in mesenchyme, probably contributing to tongue miogenesis, while Wnt-5a signal was in the epithelium, possibly during placode development and papillae formation. Taken together, these findings suggest that Bmp-4, Shh and Wnt-5a gene expressions may act together on the epithelial-mesenchymal interactions occurring in several aspects of the early mouse craniofacial development, such as odontogenesis, neuronal development, maxillae and mandible ossification, palatogenesis and tongue formation.
Subject(s)
Bone Morphogenetic Protein 4/genetics , Embryo, Mammalian/metabolism , Face/embryology , Gene Expression Regulation, Developmental/physiology , Hedgehog Proteins/genetics , Skull/embryology , Wnt Proteins/genetics , Animals , Diencephalon/embryology , Diencephalon/metabolism , Embryo, Mammalian/embryology , Endoderm/embryology , Endoderm/metabolism , Epithelium/embryology , Epithelium/metabolism , Female , Jaw/embryology , Jaw/metabolism , Mesoderm/embryology , Mesoderm/metabolism , Mice , Mice, Inbred C57BL , Mouth/embryology , Mouth/metabolism , Nasal Mucosa/metabolism , Nose/embryology , Palate/embryology , Palate/metabolism , Skull/metabolism , Tongue/embryology , Tongue/metabolism , Tooth/embryology , Tooth/metabolism , Wnt-5a ProteinABSTRACT
A técnica de hibridização in situ (ISH) tem sido usada para identificar mRNA (ou DNA) em amostras de tecido de material humano e animal. Embora uma série de protocolos para essa técnica seja utilizada, as descrições não são bem detalhadas. O objetivo deste trabalho é descrever a reação de hibridização in situ em tecido fresco e sua aplicação em patologia, tornando mais compreensível essa técnica tão importante, que possibilita observar a localização tecidual e a expressão temporal e espacial dos transcritos de um determinado gene (mRNA). Resultados de reações com as ribossondas PITX1, SHH e WNT-5A, realizadas em amostras de tecido congelado, são apresentados.
In situ hybridization (ISH) has been used to identify mRNA (or DNA) in fresh tissue samples of humans and animals. Several protocols describing this technique are available, although its description is not usually detailed enough. The present work describes in situ hybridization reaction on fresh tissue in a way to make understandable this important technique, which allows verifying the cellular localization, and the spatial and temporal expression, of gene transcripts (mRNA). Results with PITX1, TGIF, SHH and WNT-5A riboprobes, in fresh tissue samples, are presented.
