Yayoi Karasawa

PTHrP and PTH/PTHrP Receptor 1 Expression in Odontogenic Cells of Normal and HHM Model Rat Incisors

Parathyroid hormone related peptide (PTHrP) was discovered as a causative factor of humoral hypercalcemia of malignancy (HHM). We examined PTHrP and its receptor (PTHR1) expression patterns in odontogenic cells in normal and HHM model rat incisors. Nontreated nude rats serving as the normal control and HHM model rats produced by implantation of PTHrP-expressing tumor (LC-6) cells were prepared. HHM rats fractured its incisor, and histopathologically, restrict population of odontoblasts showed findings classified as “shortening of high columnar odontoblasts” and “dentin niche.” The incisors were immunostained against PTHrP and PTHR1. In normal rats, PTHrP and PTHR1 colocalized in ameloblasts, cementoblasts, and odontoblastic cells from mesenchymal cells to columnar odontoblasts. In high columnar odontoblasts, PTHrP solely expressed. In the HHM animals, although the expression patterns were identical to those of the normal rats in normal area, the shortened high columnar odontoblasts maintained PTHR1 expression and dentin niche comprising odontoblastic cells expressed both proteins. In the HHM model, the protein expression patterns changed in the odontoblastic cells with histological anomalies, and thus direct relations between the anomalies and PTHrP/PTHR1 axis are suggested.

Optimization of tissue processing for immunohistochemistry for the detection of human glypican-3

Glypican-3 (GPC3) is frequently upregulated in hepatocellular carcinoma (HCC) and data on the expression profile in HCC might be useful for therapeutic decision-making and prognostic prediction. This study was performed using HepG2 xenograft tissues to optimize the tissue processing method for GPC3 immunohistochemistry. The optimization was conducted in terms of using GPC3 immunohistochemistry for biological study of GPC3 (Experiment 1) and as a diagnostic tool (Experiment 2). In Experiment 1, GPC3 immunoreactivity (IR) and tissue architecture were compared among differently fixed and embedded specimens. In Experiment 2, using conventional formalin-fixed paraffin-embedded (FFPE) procedures, the effects of different fixation times and antigen retrieval treatments were assessed. In Experiment 1, the periodate-lysine-paraformaldehyde (PLP)-fixed and AMeX method-embedded (PLP-AMeX) specimen showed superior immunoreactivity and excellent tissue architecture preservation. In contrast, the other specimens, especially frozen specimens, resulted in poor IR. In Experiment 2, specimens fixed for 24h showed better IR than those fixed for 7 days and the most remarkable improvement in IR was achieved after protease treatment. These findings indicate that with GPC3 immunohistochemistry for biological studies, the PLP-AMeX specimen is preferable. For diagnostics using FFPE specimens, the fixation time should not be too long and protease should be used for the antigen retrieval treatment.

Histopathological study on the PTHrP-induced incisor lesions in rats.

Parathyroid hormone related peptide (PTHrP) was discovered as a causative factor of humoral hypercalcemia of malignancy (HHM). The present study elucidates the histopathological characters of incisor lesions in the HHM rat model. Nude rats were implanted with PTHrP-expressing tumor (LC-6) cells, maintained for 12 weeks, after which the mandibular incisors were collected. Incisor fractures were observed grossly. Microscopically, hypercalcified dentin, dentin niche with osteodentin, and thinning of dentin were observed. Hypercalcified dentin was observed as a basophilic line of calcified dentin without associated odontoblastic changes, whereas dentin niche and thinning of dentin occurred with osteodentin and loss of cell height, respectively. In contrast with hypercalcified dentin, which was distributed throughout the dentin, dentin niche and thinning of dentin were localized to the labial area of the apical and middle region, and to the labial and lingual areas of the middle and incisal region, respectively. These results suggest that hypercalcemia affected the entire calcification process resulting in hypercalcified dentin, and that high PTHrP concentrations affected selective populations of odontoblasts resulting in formation of dentin niche and thinning of dentin. The localization of dentin niche and thinning of dentin also suggest that PTHrP may also be involved odontoblastic development in the rat.

Histopathological Study of Time Course Changes in PTHrP-Induced Incisor Lesions of Rats

Parathyroid hormone related peptide (PTHrP) was discovered as a causative factor of humoral hypercalcemia of malignancy (HHM). In the present study using HHM model rats, the time course of odontoblastic response to PTHrP and its relation to incisal fracture were elicited. Nude rats were implanted with PTHrP-expressing tumor (LC-6) cells, mandibular incisors were collected at several time points. Microscopically 3 distinctive types of odontoblastic/dentin lesions were observed. Hypercalcfied dentin, which was reported as hypercalcemia-induced lesion in previous reports, observed in all areas of the dentin from week 5–10 samplings. Dentin niche, observed solely in week-10 sampling point, exhibited a nature identical to that of reparative odontoblast reported in the literatures of various cytotoxic agents. Since cytotoxicites were neither observed prior to the lesions nor reported as a role of PTHrP, the reparative response may have derived from highly sustained levels of PTHrP. Loss of columnar odontoblasts height was initially observed at week-5 time point in the middle section of the incisor. This primary loss of cell height prior to incisor fracture was considered to be the earliest response to the increased PTHrP levels of this model.