Hisako Fujihara

Parp-1 deficiency implicated in colon and liver tumorigenesis induced by azoxymethane

Poly(ADP‐ribose) polymerase‐1 (Parp‐1) is activated by DNA strand breaks and functions in the maintenance of genomic integrity and cell death control. On the other hand, Parp‐1 is also involved in transcriptional regulation of various genes, and the relationship between Parp‐1 deficiency and susceptibility to tumorigenesis has not been fully elucidated. In the present study, Parp‐1−1− mice, harboring exon 1 disruption in Parp‐1, and Parp‐1+l+ animals were administered azoxymethane (AOM) at a dose of 10 mg/kg body weight once a week for 6 weeks. At 30 weeks after the first carcinogen treatment, mice were sacrificed. The incidence of animals bearing either adenomas or adenocarcinomas in the colon and the average number of colon tumors per mouse were significantly higher in Parp‐1−1− mice than in Parp‐1+l+ animals. β‐Catenin accumulation was observed in 43/44 of Parp‐1−1− tumors and 19/21 of the Parp‐1+l+ tumors and was not statistically different between the genotypes. This suggests that most tumors developed through a pathway involving the alteration of Wnt‐β‐catenin signaling in both Parp‐1−1‐ and Parp‐1+l+ mice. In the liver, where AOM is primarily activated, the incidence of animals bearing nodules and the average number of nodules per section were significantly increased in Parp‐1−1‐ mice compared with Parp‐1+l+ mice. Therefore, the results indicate that susceptibility to AOM‐induced tumorigenesis in the colon and also in the liver is enhanced in Parp‐1−1‐ mice, and Parp‐1 could have a substantial role in colon and liver tumorigenesis.

Poly(ADP-ribose) Glycohydrolase Deficiency Sensitizes Mouse ES Cells to DNA Damaging Agents

Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme for degradation of poly(ADP-ribose) by splitting ribose-ribose bonds. Parg-deficient (Parg(+/-) and Parg(-/-)) mouse ES cell lines have been established by disrupting both alleles of Parg exon 1 through gene-targeting. A transcript encoding a full length isoform of Parg was eliminated and only low amounts of Parg isoforms were detected in Parg(-/-) embryonic stem (ES) cells. Poly(ADP-ribose) degradation activity was decreased to one-tenth of that in Parg(+/+) ES cells. Parg(-/-) ES cells exhibited the same growth rate as Parg(+/+) ES cells in culture. Sensitivity of Parg(-/-) ES cells to various DNA damaging agents, including an alkylating agent dimethyl sulfate, cisplatin, gemcitabine, 5-fluorouracil, camptothecin, and gamma-irradiation was examined by clonogenic survival assay. Parg(-/-) ES cells showed enhanced lethality after treatment with dimethyl sulfate, cisplatin and gamma-irradiation compared with wild-type (Parg(+/+)) ES cells (p<0.05, respectively). In contrast, a sensitization effect by Parg-deficiency was not observed with gemcitabine and camptothecin. These results suggest the possibility that functional inhibition of Parg leads to sensitization of tumor cells to some chemo- and radiation therapies.

Controlled delivery of bFGF to recipient bed enhances the vascularization and viability of an ischemic skin flap

Therapeutic angiogenesis is a promising approach to treat ischemic skin flaps. We delivered basic fibroblast growth factor (bFGF) to the recipient bed of a rat dorsal skin flap by a drug delivery system with acidic gelatin hydrogel microspheres (AGHMs), and assessed augmentation of neovascularization and flap viability. An axial skin flap was elevated on the back of male Sprague–Dawley rats, and bFGF solution or bFGF‐impregnated AGHMs were injected into the recipient bed. The dose of bFGF in the bFGF solution was set to 15 (Sol‐15 group), 50 (Sol‐50 group), or 150 μg (Sol‐150 group). Correspondingly, 2 mg AGHMs were impregnated with 15 (AGHM‐15 group), 50 (AGHM‐50 group), or 150 μg (AGHM‐150 group) bFGF. Other groups of animals received phosphate‐buffered saline (Sol‐Cont group) or phosphate‐buffered saline‐impregnated AGHMs (AGHM‐Cont group) as controls. Seven days later, analyses of the area of necrosis, microangiographic findings, and histological findings in the flap were carried out. The area of necrosis in the AGHM‐150 group was significantly smaller than that in the other groups. Microangiographic and histological analyses showed that neovascularization of the ischemic skin flap significantly increased in the AGHM‐150 group as compared with the Sol‐150 group and the AGHM‐Cont group. These findings suggest that continuous delivery of bFGF to the recipient bed by bFGF‐impregnated AGHMs enhances the viability of an ischemic skin flap.

Metastasis of Hepatocellular Carcinoma into the Mandible with Radiographic Findings Mimicking a Radicular Cyst: A Case Report

INTRODUCTION Hepatocellular carcinoma (HCC) is a common neoplasm worldwide, with more than half of the tumors associated with regional metastasis. Extrahepatic metastasis is also common, and the most frequently affected sites are the lungs, abdominal lymph nodes, diaphragm, and bone. However, HCC metastasis to the mandible is rare, with approximately 50 cases reported in the literature. METHODS In this report, we describe a case of HCC metastasis to the mandible at the apex of #18 root in a 62-year-old man. This patient had already been diagnosed with metastasis to pancreatic caput lymph node. The radiographic features of the mandible resembled radicular cyst and did not show typical findings of malignancy. RESULTS Under the first diagnosis of radicular cyst, root canal treatment was initially performed, and then surgical treatment of the removal of the cystic lesion and #18 extraction were performed. Finally, the lesion was diagnosed as HCC metastasis from pathological examination. Consequently, he received constitutional chemotherapy in the hepatitis unit and is now in remission. CONCLUSION This case shows the importance of considering the differential diagnosis of malignancy.

Cyclooxygenase-2 activity is important in craniofacial fracture repair.

The aim of this study was to examine the effect of cyclooxygenase (COX)-2 on bone repair after craniofacial fracture in mice. A 4-mm fracture was created in the parietal bone of 8-week-old male COX-2 wild-type (COX-2(+/+)) and knockout (COX-2(-/-)) mice. Ribonucleic acid was extracted from the fractured bone and analysed. For morphological and histological analysis, the mice were killed 8 and 12 weeks after treatment, and sections were prepared. Three-dimensional computed tomography was performed, and the sections were stained with hematoxylin-eosin for histological examination. Expression of COX-2 messenger ribonucleic acid was induced in COX-2(+/+) mice, but not in COX-2(-/-) mice. Ossification at the fracture site was almost complete 12 weeks after fracture in COX-2(+/+) mice. In COX-2(-/-) mice, incomplete union had occurred at the fracture site. In both types of mice, the fracture site contained no cartilaginous tissue, and the callus formed from the periosteal side. These results suggest that COX-2 plays an important role in craniofacial fracture repair and that COX-2-selective non-steroidal anti-inflammatory drugs might interfere with fracture repair of the membranous viscerocranium in the clinical setting.

Synergetic Effects of PARP Inhibitor AZD2281 and Cisplatin in Oral Squamous Cell Carcinoma in Vitro and in Vivo.

Cisplatin is a commonly used chemotherapeutic drug for treatment of oral carcinoma, and combinatorial effects are expected to exert greater therapeutic efficacy compared with monotherapy. Poly(ADP-ribosyl)ation is reported to be involved in a variety of cellular processes, such as DNA repair, cell death, telomere regulation, and genomic stability. Based on these properties, poly(ADP-ribose) polymerase (PARP) inhibitors are used for treatment of cancers, such as BRCA1/2 mutated breast and ovarian cancers, or certain solid cancers in combination with anti-cancer drugs. However, the effects on oral cancer have not been fully evaluated. In this study, we examined the effects of PARP inhibitor on the survival of human oral cancer cells in vitro and xenografted tumors derived from human oral cancer cells in vivo. In vitro effects were assessed by microculture tetrazolium and survival assays. The PARP inhibitor AZD2281 (olaparib) showed synergetic effects with cisplatin in a dose-dependent manner. Combinatorial treatment with cisplatin and AZD2281 significantly inhibited xenografted tumor growth compared with single treatment of cisplatin or AZD2281. Histopathological analysis revealed that cisplatin and AZD2281 increased TUNEL-positive cells and decreased Ki67- and CD31-positive cells. These results suggest that PARP inhibitors have the potential to improve therapeutic strategies for oral cancer.

Evaluation and analysis of formation of bone at the palate in patients with cleft lip and palate after palatoplasty based on computed tomograms and three-dimensional data

Abstract There are various techniques for palatoplasty, but no studies of postoperative osteogenesis at the palatal fissure. In the cranial and maxillofacial region it is thought to develop from the periosteum, so palatoplasty with mucoperiosteal flaps may encourage new bone to form at the fissure. We evaluated the status of osteogenesis in the hard palate after palatoplasty on computed tomograms (CT). We studied 29 patients (22 boys and 7 girls) with unilateral cleft lip and palate who had pushback palatoplasty with the use of CT obtained between May 2003 and March 2007. Age at the time of operation was recorded. The width of the palatal fissure at the first premolar, the first molar, and the maxillary posterior region were measured on coronal CT. The mean (SD) age at the time of palatoplasty was 16 (2) months. The mean (SD) width of the fissure at the first molar was 3.96 (3.1) mm, and bony union was seen in four patients. The width of the fissure was significantly less at the first molar than at the other sites (p = 0.006). The shape of the margin of the fissure was irregular in nearly all patients. The width of the fissure at the first molar became significantly less, suggesting that osteogenesis had occurred. In some patients the height of the fissure differed. Given the results of previous studies, bony regeneration from the periosteum most likely happens together with regeneration from the margins of the fissure.

PARP Inhibitor PJ34 Suppresses Osteogenic Differentiation in Mouse Mesenchymal Stem Cells by Modulating BMP-2 Signaling Pathway

Poly(ADP-ribosyl)ation is known to be involved in a variety of cellular processes, such as DNA repair, cell death, telomere regulation, genomic stability and cell differentiation by poly(ADP-ribose) polymerase (PARP). While PARP inhibitors are presently under clinical investigation for cancer therapy, little is known about their side effects. However, PARP involvement in mesenchymal stem cell (MSC) differentiation potentiates MSC-related side effects arising from PARP inhibition. In this study, effects of PARP inhibitors on MSCs were examined. MSCs demonstrated suppressed osteogenic differentiation after 1 µM PJ34 treatment without cytotoxicity, while differentiation of MSCs into chondrocytes or adipocytes was unaffected. PJ34 suppressed mRNA induction of osteogenic markers, such as Runx2, Osterix, Bone Morphogenetic Protein-2, Osteocalcin, bone sialoprotein, and Osteopontin, and protein levels of Bone Morphogenetic Protein-2, Osterix and Osteocalcin. PJ34 treatment also inhibited transcription factor regulators such as Smad1, Smad4, Smad5 and Smad8. Extracellular mineralized matrix formation was also diminished. These results strongly suggest that PARP inhibitors are capable of suppressing osteogenic differentiation and poly(ADP-ribosyl)ation may play a physiological role in this process through regulation of BMP-2 signaling. Therefore, PARP inhibition may potentially attenuate osteogenic metabolism, implicating cautious use of PARP inhibitors for cancer treatments and monitoring of patient bone metabolism levels.

Detection accuracy for epithelial dysplasia using an objective autofluorescence visualization method based on the luminance ratio

The autofluorescence visualization method (AVM) uses blue excitation light to assist in the diagnosis of epithelial dysplasia. It detects epithelial dysplasia as a black area, which is known as fluorescence visualization loss (FVL). In this study, we evaluated the detection accuracy for epithelial dysplasia of the tongue using the objective AVM and assessed its possible clinical utility. Seventy-nine tongue specimens clinically suspected to have leukoplakia or squamous cell carcinoma (SCC) were analyzed. First, the AVM was subjectively performed using the Visually Enhanced Lesion scope (VELscope), and the iodine-staining method was then performed. After biopsy, the histopathological results and the luminance ratio between the lesion and healthy tissue were compared, and a receiver operating characteristic curve was created. The cutoff value for the objective AVM was determined; the lesion was considered FVL-positive or -negative when the luminance ratio was higher or lower than the cutoff value, respectively. The histopathological diagnoses among the 79 specimens were SCC (n=30), leukoplakia with dysplasia (n=34), and leukoplakia without dysplasia (n=15). The cutoff value of the luminance ratio was 1.62, resulting in 66 FVL-positive and 13 FVL-negative specimens. The luminance ratio was significantly higher in the epithelial dysplasia-positive than -negative group (P<0.000 1). The objective AVM showed much higher consistency between histopathological results than did the two methods (kappa statistic=0.656). In conclusion, objective autofluorescence visualization has a potential as an auxiliary method for diagnosis of epithelial dysplasia.

Necrotising Ulcerative Stomatitis in a Neutropenic Patient with Malignant Lymphoma

Abstract Necrotising ulcerative stomatitis is a very rapid, destructive disease of the alveolar bone and gingiva. The rapid destruction leads to devastating facial defects and death, if it is not treated promptly. Candida albicans and Pseudomonas aeruginosa are frequent causes of disseminated infections among patients who are immunocompromised. This report is of a 72-year-old woman with dose large B-cell lymphoma, who developed acute necrotising ulcerative stomatitis and septic shock together with histopathological and microbiological evidence of C. albicans and P aeruginosa infections. Gingival necrosis was managed by debridement to remove slough and irrigation with povidone iodine. All oral symptoms subsided following the initiation of antimicrobial therapy and debridement. The patient completed chemotherapy and achieved prompt remission. This report highlights the importance of prompt recognition, debridement, teeth extraction, scrupulous oral hygiene, appropriate antibiotic therapy, and nutritional support for immunocompromised patients with necrotising ulcerative stomatitis.