Toshiro Tagawa

p53 gene therapy of human osteosarcoma using a transferrin-modified cationic liposome

Gene delivery via transferrin receptors, which are highly expressed by cancer cells, can be used to enhance the effectiveness of gene therapy for cancer. In this study, we examined the efficacy of p53 gene therapy in human osteosarcoma (HOSM-1) cells derived from the oral cavity using a cationic liposome supplemented with transferrin. HOSM-1 cells were exposed to transferrin-liposome-p53 in vitro, and the growth inhibition rate, expression of p53 and bax, and induction of apoptosis were measured 48 hours later. Treatment of HOSM-1 cells with transferrin-liposome-p53 resulted in 60.7% growth inhibition. Wild-type p53 expression and an increase in bax expression were observed following transfection with transferrin-liposome-p53, and 20.5% of the treated HOSM-1 cells were apoptotic. In vivo, the HOSM-1 tumor transplanted into nude mice grew to 5 to 6 mm in diameter. Following growth of the tumor to this size, transferrin-liposome-p53 was locally applied to the peripheral tumor (day 0) and then applied once every 5 days for a total of six times. During the administration period, tumor growth did not occur, and the mean tumor volume on the last day of administration (day 25) was 10.0% of that in the saline control group. These results suggest that p53 gene therapy via cationic liposome modification with transferrin is an effective strategy for treatment of osteosarcoma.

Bax mRNA therapy using cationic liposomes for human malignant melanoma.

Bax is a pro‐apoptotic molecule that functions as a tumor suppressor and Bax gene therapy has been examined for various cancers. Gene transfer by mRNA lipofection is more efficient than plasmid DNA lipofection and, in the present study, we examined the anti‐tumor effects in human malignant melanoma cells (HMGs) using Bax mRNA lipofection.

Calmodulin-Dependent Cyclic Nucleotide Phosphodiesterase (PDE1) Is a Pharmacological Target of Differentiation-Inducing Factor-1, an Antitumor Agent Isolated from Dictyostelium

The differentiation-inducing factor-1 (DIF-1) isolated from Dictyostelium discoideum is a potent antiproliferative agent that induces growth arrest and differentiation in mammalian cells in vitro. However, the specific target molecule(s) of DIF-1 has not been identified. In this study, we have tried to identify the target molecule(s) of DIF-1 in mammalian cells, examining the effects of DIF-1 and its analogs on the activity of some candidate enzymes. DIF-1 at 10–40 μm dose-dependently suppressed cell growth and increased the intracellular cyclic AMP concentration in K562 leukemia cells. It was then found that DIF-1 at 0.5–20 μm inhibited the calmodulin (CaM)-dependent cyclic nucleotide phosphodiesterase (PDE1) in vitro in a dose-dependent manner. Kinetic analysis revealed that DIF-1 acted as a competitive inhibitor of PDE1 versus the substrate cyclic AMP. Because DIF-1 did not significantly affect the activity of other PDEs or CaM-dependent enzymes and, in addition, an isomer of DIF-1 was a less potent inhibitor, we have concluded that PDE1 is a pharmacological and specific target of DIF-1.

Myofibroma of the mandible: Clinicopathologic study and review of the literature

A case of mandibular myofibroma in a 2-month-old boy is presented. Including this case, 24 pediatric and 11 adult patients with maxillofacial myofibroma have been reported since 1981. Of the 24 pediatric patients, 15 (62.5%) had lesions affecting the mandible. The adult cases had no mandibular involvement. Histologic evaluation of the tissue specimen revealed an interlacing pattern of spindle-shaped cells with long oval nuclei. Tissue immunohistochemical staining found it to be reactive for antibodies directed against vimentin and alpha-smooth muscle actin, but not desmin, S-100 protein, neuron-specific enolase, or myoglobin. Electron microscopy examination revealed the following cells: myofibroblast-like cells, fibroblast-like cells, and intermediate cells that were similar to the fibroblast-like cells except for the presence of a few microfilaments. Myoblast-like cells were not seen.

Clear cell odontogenic carcinoma: A case report and literature review of odontogenic tumors with clear cells

Clear cell odontogenic carcinomas are uncommon epithelial neoplasms that have metastatic potential. We describe such a tumor in a 67-year-old Japanese man with a well-defined, unilocular radiolucent area in the mandible. The tumor was enucleated and has not recurred or metastasized 3 years after surgery. Histologically, the tumor was composed of a predominant clear cell area and a plexiform ameloblastic pattern. The latter was located at the periphery of the tumor. In the English literature, 19 cases of clear cell odontogenic carcinoma, clear cell odontogenic tumor, and clear cell ameloblastoma have been reported. The mean age of the patients at diagnosis was 56.4 years and the gender ratio (M:F) was 8:11. Fourteen tumors were located in the mandible and 5 in the maxilla. Recurrence or metastasis occurred in approximately one half of these patients, and five of the patients died of their disease. More than one half recurrent or metastatic tumors contained palisading clear cells.

An immuno-light- and electron-microscopic study of the expression of bone morphogenetic protein-2 during the process of ectopic bone formation in the rat.

Immunolocalization of endogenous bone morphogenetic protein-2 (BMP-2) was investigated during the process of ectopic bone formation induced by recombinant human BMP-2 (rhBMP-2). Pellets consisting of 5 microg of rhBMP-2 and 6 mg of atelopeptide type I collagen (AC) were implanted into the calf muscles of 6-week-old rats. On days 7, 10, 14, 21 and 28 after implantation, tissue specimens were removed and examined immunohistochemically by light and electron microscopy after incubation with anti-BMP-2 monoclonal antibodies. Immunolocalization by light microscopy showed BMP-2 in chondrocytes at the pellet rim on days 7 and 10, in osteocyte-like cells in the chondroid matrix on day 14, and in osteocytes in the newly formed bone on days 21 and 28 after implantation. Ultrastructurally, on days 7 and 10 after implantation, immunolabelling for BMP-2 was aggregated in vesicle-like matrices released from mature chondrocytes in the chondroid matrix. On day 14, immunolabelling against BMP-2 had accumulated in vesicle-like matrices embedded in the calcified cartilage, in the cytoplasmic vacuoles of chondroclasts absorbing the matrix, and at the resorption surface of the calcified cartilage. On days 21 and 28, BMP-2 immunolabelling was seen in the osteoid layer and osteocyte lacunae. These results suggest that the chondrocytes and osteocytes induced by rhBMP-2 produce endogenous BMP-2. It seems that part of the endogenous BMP-2 that accumulated in the chondroid matrix was absorbed by chondroclasts and then participated in the osteoblastic differentiation of immature mesenchymal cells. This study indicates that, in addition to the implanted exogenous rhBMP-2, endogenous BMP-2 plays an important part in the maintenance of the bone-formation cascade during ectopic osteoinduction.

Magnetic resonance imaging of synovial proliferation in temporomandibular disorders with pain.

Objective: The objective of this study was to assess the clinical significance of synovial proliferation in patients with painful temporomandibular disorders based on magnetic resonance imaging findings. Methods: The current study was conducted in 100 joints of 100 patients with unilateral painful temporomandibular disorders. One hundred joints on the contralateral side of patients with unilateral disease were used as nonpain group. Areas in the articular space that showed a low signal intensity on T1-weighted imaging, a high signal intensity on T2-weighted imaging, and high signal intensity on gadolinium-enhanced fat-suppressed T1-weighted imaging were judged to be regions of synovial proliferation. Results: Synovial proliferation alone was observed in 8.0% of the pain group, but in none of the nonpain group. Synovial proliferation + effusion was observed in 33.0% of the pain group and in 7.0% of the nonpain group. Effusion alone was observed in 7.0% of the pain group and in 3.0% of the nonpain group. The mean visual analog scale value of pain was in the order of synovial proliferation alone > synovial proliferation + effusion > effusion alone. The incidence rates of anterior displacement of the disk were 100% for synovial proliferation alone, 93.9% for synovial proliferation + effusion, 57.1% for effusion alone, and 57.7% for “without synovial proliferation/effusion.” Conclusions: Strong correlations were observed between synovial proliferation, pain, and disk displacement. It is considered that evaluating effusion alone provides only limited information on the disease state in painful temporomandibular disorders. Thus, it is essential to include enhanced T1-weighted imaging as a means to judge the disease state as well as to assess disease progression.

Inflammatory pseudotumor in the submandibular region: Clinicopathologic study and review of the literature

A rare case of right submandibular inflammatory pseudotumor in a 63-year-old man is reported. The tumor appeared as a symptomless swelling in the submandibular region and resembled a malignant neoplasm on computed tomographic and magnetic resonance images. Surgical resection was required. The pertinent English and Japanese literature is reviewed, and histologic and electron microscopic findings are discussed.

Phosphodiesterase 3 as a potential target for therapy of malignant tumors in the submandibular gland.

Phosphodiesterase (PDE) 3s have been characterized in human neoplastic submandibular gland intercalated duct HSG cells. There have been no reports on PDE3 in malignant salivary gland cells. PDE3 activity was detected in homogenates of HSG cells. About 75% of PDE3 activity in HSG cells was recovered in supernatant fractions and 25% in particulate fractions. PDE3A and 3B mRNAs were detected by reverse transcription-polymerase chain reaction in RNA from HSG cells. The nucleotide sequences of the fragments were identical to those of human PDE3A and 3B. The PDE3-specific inhibitor, cilostamide, inhibited the growth of HSG cells. Our results indicate that PDE3s may be important in the growth of HSG cells. PDE3 thus appears to be a potential new target for antiproliferative therapies.

Characterization of phosphodiesterase 2A in human malignant melanoma PMP cells.

The prognosis for malignant melanoma is poor; therefore, new diagnostic methods and treatment strategies are urgently needed. Phosphodiesterase 2 (PDE2) is one of 21 phosphodiesterases, which are divided into 11 families (PDE1-PDE11). PDE2 hydrolyzes cyclic AMP (cAMP) and cyclic GMP (cGMP), and its binding to cGMP enhances the hydrolysis of cAMP. We previously reported the expression of PDE1, PDE3 and PDE5 in human malignant melanoma cells. However, the expression of PDE2 in these cells has not been investigated. Herein, we examined the expression of PDE2A and its role in human oral malignant melanoma PMP cells. Sequencing of RT-PCR products revealed that PDE2A2 was the only variant expressed in PMP cells. Four point mutations were detected; one missense mutation at nucleotide position 734 (from C to T) resulted in the substitution of threonine with isoleucine at amino acid position 214. The other three were silent mutations. An in vitro migration assay and a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay revealed that suppressing PDE2 activity with its specific inhibitor, erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA), had no impact on cell motility or apoptosis. Furthermore, the cytotoxicity of EHNA, assessed using a trypan blue exclusion assay, was negligible. On the other hand, assessment of cell proliferation by BrdU incorporation and cell cycle analysis by flow cytometry revealed that EHNA treatment inhibited DNA synthesis and increased the percentage of G2/M-arrested cells. Furthermore, cyclin A mRNA expression was downregulated, while cyclin E mRNA expression was upregulated in EHNA-treated cells. Our results demonstrated that the PDE2A2 variant carrying point mutations is expressed in PMP cells and may affect cell cycle progression by modulating cyclin A expression. Thus, PDE2A2 is a possible new molecular target for the treatment of malignant melanoma.