Biological behavior of myoepithelial cells in the regeneration of rat atrophied sublingual glands following release from duct ligation.

The present study aimed to clarify how myoepithelial cells behave during regeneration of an atrophied sublingual gland by investigating cell proliferation and ultrastructure. Atrophy of rat sublingual glands was induced by unilateral ligation of the excretory duct near the hilum with metal clips, which were then removed after one week of ligation for regeneration. The sublingual glands 0-14 days after unligation were examined with single immunohistochemistry for actin as a marker of myoepithelial cells, double immunohistochemistry for actin and proliferating cell nuclear antigen (PCNA) as a marker of proliferating cells, and transmission electron microscopy (TEM). The single immunohistochemistry and TEM showed that myoepithelial cells surrounded residual ducts in the atrophied glands and immature and mature acini in the regenerating glands. Although PCNA-positive myoepithelial cells were identified during regeneration, PCNA labeling indices of myoepithelial cells were low at all time points except at day 7. Ultrastructurally, myoepithelial cells showing bizarre shaped structures in the atrophy changed with maturation of differentiating acinar cells and appeared normal in the regenerated glands. There was no differentiation of the remaining duct cells to myoepithelial cells. These observations suggest that proliferation of myoepithelial cells and differentiation to myoepithelial cells do not commonly participate in the regeneration of atrophied sublingual glands and that the bizarre shaped myoepithelial cells in the atrophied sublingual glands recover the original shapes with acinar cell regeneration.

Induction of calcification in MC3T3-E1 cells by inorganic polyphosphate.

Relatively large amounts of inorganic polyphosphate [poly(P)] (400 microM) have been found in normal osteoblasts. The effect of poly(P) with an average chain length of 65 phosphate residues on cell calcification was therefore investigated with the use of MC3T3-E1 cells. Expression of both osteopontin and osteocalcin was induced by poly(P) (0.1 approximately 1 mM), and cells treated with poly(P) were strongly stained by alizarin red. In addition, the level of alkaline phosphatase activity induced in poly(P)-treated cells was two-fold higher than that in either orthophosphate-treated or control cells but not higher than that in cells treated with beta-glycerophosphate and ascorbic acid. In contrast, however, polyphosphatase activities were activated by poly(P) treatment to levels up to six-fold greater than that in controls. MC3T3-E1 cells may utilize poly(P) as a phosphate source for calcification rather than phosphate sources that are mainly produced by ALPase. Poly(P)-dependent induction of polyphosphatase activities may therefore promote calcification in MC3T3-E1 cells.

Foreign body giant cell induction in the CSF-1-deficient osteopetrotic (op/op) mouse.

The osteopetrosis (op) mutation in mice is characterized by generalized skeletal sclerosis; reduced numbers of osteoclasts, macrophages, and monocytes; and failure to be cured by bone marrow transplantation. This mutation has been shown to result from an absence of colony-stimulating factor-1 (CSF-1) and reported to be cured by treatment with CSF-1. Macrophage polykaryons are known to be formed by fusion of mononuclear precursors and the presence of subcutaneous implants can elicit the formation of macrophage polykaryons. In order to determine if recruitment of foreign body giant cells is also impaired in osteopetrotic mice, tissue reactions to subcutaneously implanted polyvinyl sponges were studied and compared with normal mice. Our result showed that, in the op mouse, recruitment of macrophages and foreign body giant cells in response to the implants was quantitatively not different from that of normal mice. However, these cells were smaller and did not migrate as deeply into the implant as those seen in normal littermates. In contrast, resident macrophages obtained by peritoneal lavage were significantly reduced in op mice. These data indicate that there is a deficiency in the ability of op mice to mount a foreign body giant cell response to an implanted sponge characterized by a deficiency in the recruitment of precursor cells that are capable of either full development and spreading or migration into the implanted sponge. These data add to the emerging appreciation of the regional differences among macrophage populations in their dependence on CSF-1 for differentiation and survival.

A recurrent case of odontogenic ghost cell tumour of the mandible.

Odontogenic ghost cell tumour (OGCT), also referred to as dentinogenic ghost cell tumour, is an extremely rare tumour classified as a neoplastic variant of calcifying ondontogenic cyst (COC). To date, only 13 cases of OGCT arising in the maxilla or mandible have been reported. We describe an OGCT that recurred after segmental resection of the mandible in a 59-year-old man. Histopathological examination revealed tumour invasion of the surrounding cortical bone, areas containing numerous calcifying flaky cell nests, and dentinoid matrix near epithelial cell nests. No atypical mitosis was found. There has been no evidence of recurrence or metastasis in the 4 years after operation.

nm23-H1 suppresses invasion of oral squamous cell carcinoma-derived cell lines without modifying matrix metalloproteinase-2 and matrix metalloproteinase-9 expression.

nm23-H1 is a candidate gene for the suppression of cancer metastasis. Several studies on human breast, hepatocellular, gastric, ovarian, and colon carcinomas and melanomas have shown that reduced nm23-H1 expression was closely related to metastatic progression with poor prognosis. However, the biochemical mechanism by which nm23-H1 suppresses the metastasis has yet to be elucidated. In this study, we analyzed the correlation between nm23 expression, cell motility, and the invasive abilities of six different oral squamous cell carcinoma cell lines (HSC2, HSC3, HSC4, KB, OSC19, and OSC20). Reduced mRNA/protein expression of the nm23-H1 was observed in three cell lines (HSC2, HSC3, and HSC4). These cell lines exhibited increased cell motility and an invasive character on organotypic raft culture. On the other hand, the cell lines (KB, OSC19, and OSC20) that showed a higher expression of nm23-H1 exhibited a threefold to fivefold reduced motility and also reflected fewer invasions compared to the former three cell lines. Because the HSC3 cells demonstrated the lowest nm23-H1 expression with the highest cell motility and invasive character, we established nm23-H1-transfected HSC3 cell lines to investigate whether exogenous nm23-H1 protein could inhibit cell migration and invasive activity. These transfectants showed a significant reduction in cell motility with exogenous nm23-H1 in a dose-dependent manner, and exhibited a noninvasive character. An immunofluorescence study demonstrated a distinct stress-fiber distribution at peripheral region of these transfectants. However, no significant difference of matrix metalloproteinase (MMP)-2 and MMP-9 expression was observed between mock transfectant and nm23-H1-transfected cells. These findings suggest that nm23-H1 inhibits the invasive activity of oral squamous cell carcinoma by suppression of cell motility without altering the MMP-2 and MMP-9 status.

Geometric effect of matrix upon cell differentiation: BMP-induced osteogenesis using a new bioglass with a feasible structure.

A new biocompatible glass, which is composed of CaO, P2O5, SiO2, and Al2O3 (abbreviated CPSA) and is characterized by higher elasticity than previous bioglass products, was molded into fibers with a diameter of 9 microm. With CPSA fibers, two geometrically different structures, balls and bundles (each 20 mg in weight), were prepared, combined with 2.2 microg of rhBMP-2 (a gift from Yamanouchi Co., Japan) and implanted subcutaneously into rats. The histology showed remarkably higher bone formation in the ball-CPSA/BMP at 2 and 4 weeks than in the bundle-CPSA/BMP. The ball-CPSA/BMP showed 10 times higher alkaline phosphatase (ALP) activity at the second week and 5 times higher osteocalcin content at the fourth week than the bundle-CSPA/BMP. Vascular development in the implants was evaluated by mRNA expression of Flt-1 and KDR, two receptors for vascular endothelial growth factor (VEGF). Both receptors showed higher expression in the case of the ball, while they were not detected in the bundle. It is concluded that the BMP-induced bone formation depends highly upon the porous vasculature-inducing geometry of the matrix, which can be constructed with the new CPSA fibers.

BAG-1 expression correlates highly with the malignant potential in early lesions (T1 and T2) of oral squamous cell carcinoma.

BAG-1 is a Bcl-2-binding protein that functions as an anti-apoptotic molecule. In this report we show a possible correlation between BAG-1 expression levels and the probability of oral squamous cell carcinoma (SCC) progression. We investigated BAG-1 expression levels in 22 patients diagnosed with early lesions (T1 and T2) of oral SCCs using immunohistochemistry and western blotting. High steady-state levels of BAG-1 were detected in 13 out of 22 cases (59%). High BAG-1 expression was observed more frequently in cases with nodal metastasis (89%) than in those without nodal metastasis (38%) (P<0. 03), suggesting that BAG-1 expression levels may correlate with the pathological stage of oral SCCs. Furthermore, BAG-1 expression levels correlated with the WHO grade, i.e. 45% in grade-I cases as opposed to 72% in grade-II cases (P<0.02). These data suggest that an analysis of BAG-1 expression may be useful in establishing a prognosis for patients with oral SCCs, and especially in predicting the metastatic potential of SSCs.

Effects of geometry of hydroxyapatite as a cell substratum in BMP-induced ectopic bone formation.

Three different types of porous hydroxyapatite with pore sizes of 100-200 micrometer in diameter-porous particles of hydroxyapatite (PPHAP), porous blocks of hydroxyapatite (PBHAP), and honeycomb-shaped hydroxyapatite (HCHAP)-were compared in terms of their abilities to induce osteogenesis when implanted subcutaneously with recombinant human BMP-2 into rats and extracted at 1, 2, 3, and 4 weeks. Histologically, direct bone formation occurred in PPHAP and PBHAP while only endochondral ossification took place in HCHAP. Interestingly, cartilage in the central zones and bone in the orifice zones of the tunnels of the HCHAP were observed at 2 weeks. After 3 weeks, the cartilage disappeared and bone formation occurred throughout the inner surface of the tunnels of the HCHAP, always leaving space for capillaries within the tunnels. Alkaline phosphatase activity and osteocalcin content were the highest in HCHAP among the three hydroxyapatite implants. These results clearly indicate that BMP-induced bone formation is highly dependent on the geometry of the carrier, which provides feasible structural factors for vascularization.

Hepatocyte growth factor upregulates E1AF that induces oral squamous cell carcinoma cell invasion by activating matrix metalloproteinase genes.

Hepatocyte growth factor (HGF) is thought to play a role in cell motility and invasion. Matrix metalloproteinases (MMPs) have been implicated in invasion and metastasis of tumor cells. We have previously reported that the Ets-oncogene family transcription factor E1AF positively regulates transcription of MMP genes in transient expression assays and that overexpression of the E1AF gene confers an invasive phenotype on breast cancer cells. Here we examined the effect of HGF on E1AF and MMP gene expression in terms of the invasive potential of the oral squamous cell carcinoma cell line HSC3. HGF stimulated expression of the E1AF gene. The levels of MMP-1, -3 and -9 mRNAs increased in cells treated with HGF and correlated with E1AF upregulation. In contrast, no obvious upregulation of MMP-1 and -9 mRNA was observed in ASE1AFHSC3 cells transfected with the antisense E1AF expression vector into parental HSC3 cells. The wild-type MMP-9 gene promoter was activated by endogenous E1AF in HSC3 cells, and chloramphenicol acetyltransferase (CAT) activities increased when HGF was added to transfected cells. On the other hand, CAT activity was reduced to almost two-thirds of the wild-type activity when HSC3 cells were transfected with a CAT reporter plasmid driven by a mutant MMP-9 promoter lacking the Ets-binding site, and induction of CAT activity was not observed upon addition of HGF. Analysis of organotypic raft cultures revealed that HSC3 cells invaded and degraded collagen gel actively upon addition of HGF. These results suggest that HGF induces expression of the Ets-related E1AF transcription factor gene whose product in turn activates MMP genes and leads to oral cancer cell invasion.

High malignant transformation rate of widespread multiple oral leukoplakias.

OBJECTIVE: This study was undertaken in order to investigate clinicopathologic characteristics and malignant potential of widespread multiple oral leukoplakias. PATIENTS AND METHODS: The present study includes 12 patients with widespread multiple leukoplakias (widespread patients) and 99 with localized lesions (localized patients), and all patients were followed for more than 6 months with the mean follow-up period of 4 years. RESULTS: Gingiva and tongue were the major affected sites of leukoplakias in the localized patients, whereas gingiva and buccal mucosa were predominantly affected in the widespread patients. The rate of developing carcinoma was significantly (P < 0.02) higher in the widespread patients (3/12) than in the localized patients (5/99), although there was no significant difference in the rate of dysplastic lesions between these groups. CONCLUSION: These results indicate that the widespread leukoplakias have a higher potential for the development of carcinoma than do the localized lesions.

Pathological evaluation of the effects of intentional disocclusion and overloading occlusion in odontogenesis disorders in N-methylnitrosourea-treated hamsters.

This study compares the effects of disocclusion and overloading occlusion on dental lesions. Ten-day-old Syrian hamsters were divided into 4 groups: group I, untreated animals; group II, animals whose hemilateral incisors were disoccluded; group III, N-methylnitrosourea (MNU)-treated animals; and group IV, MNU-treated animals whose hemilateral incisors were disoccluded. The ipsilateral maxillary and mandibular incisors were repetitively cut with diamond discs. The hamster is easier to anesthetize. Animals received a 0.2% solution of MNU (10 mg/kg body weight) intragastrically twice a week for 16 wk. All the cut mandibular incisors and the MNU-treated uncut mandibular incisors showed lack of iron deposition on the enamel surface. The eruption rate was significantly higher in the cut disoccluded incisors of groups II and IV (p < 0.05) and significantly lower in the uncut overloaded incisors of groups II and IV (p < 0.05). In the cut mandibular incisors of group IV, the degree of the disturbance of odontogenesis and the atypical proliferation of odontogenic epithelium were more prominent (p < 0.02), and the dental lesions occurred earlier. Histologically, the disturbed Hertwig's epithelial sheath and the Hertwig's epithelial sheath-like transformed U-shaped part and enamel organ seemed to lead to disturbances of amelogenesis and detinogenesis as well as to atypical proliferation of odontogenic epithelium nests. Thus, this method of disocclusion of the incisors of rodents may represent a useful model for the investigation of the effects of various agents on tooth formation over a short experimental period.

The enhancing effect of excess retinol palmitate on induction of odontogenic tumors and inhibitory effect on squamous cell carcinoma of the gingiva in hamsters treated with N-methylnitrosourea.

The influence of excess retinol palmitate on induction of tumors in the oral region was examined histopathologically. Sixty-three weanling Syrian golden hamsters were divided into five groups and received either 0.2% N-methylnitrosourea (MNU) (1 mg/100 g body weight) or retinol palmitate (RP) (25,000 IU/100 g body weight) twice a week for 16 weeks, singly or in combination. Animals received RP intraperitoneally or intragastrically and then, 6 hours later, the animals received intragastric administration of MNU. To accelerate the cell activity of the incisal tooth buds, intentional disocclusion of the left upper and lower incisor of all hamsters was carried out by repeated cutting with cooled diamond disks to a level just above the inter-dental papilla twice a week for 12 weeks. The right incisors were left in occlusion. In all animals exposed to RP + MNU, while the induction of squamous cell carcinomas of the gingiva and forestomach were prevented, the notable findings were a significantly increased incidence of odontogenic tumors in cut incisal regions of the animals with intragastric administration of RP + MNU and an induction of maxillary neurogenic tumors. The incidence of MNU-induced disturbances in odontogenesis in the incisors was reduced but marked disturbances were increased. RP seemed to have opposite effects of prevention and enhancement for development of neoplastic changes in the oral region.

Flow cytometric analysis of cell cycle fractions in oral leukoplakia.

Flow cytometric analysis of cell cycle fractions and DNA ploidy was performed on 39 biopsy specimens of oral leukoplakia. The aneuploidy rate of these leukoplakias was 9/39 (23.1%) and the mean DNA index of the aneuploid lesions was 1.34. The aneuploidy rate was significantly higher in severely dysplastic lesions (8/17) than in mildly dysplastic (1/15, P<0.02) and nondysplastic (0/7, P<0.05) lesions. No significant differences in the percentages of each cell cycle fraction were seen between the diploid and the aneuploid leukoplakias. However, the S-phase fraction of the severely dysplastic lesions (23.0%) among the diploid leukoplakias was higher than those of the mildly dysplastic (12.4%) and nondysplastic (15.5%) lesions, and the difference between the severely dysplastic and the mildly dysplastic lesions was statistically significant (P<0.001). These results suggest that flow cytometric analysis of cell cycle fractions and DNA ploidy might offer additional information for assessing the malignant potential of oral leukoplakias.

Comparison between submucosal (extra-nodal) and nodal non-Hodgkin's lymphoma (NHL) in the oral and maxillofacial region.

Fifty-two cases of non-Hodgkin's lymphoma (NHL) in the oral and maxillofacial region, comprising 31 submucosal (extra-nodal) and 21 cervical node NHLs, were investigated. The patients' ages ranged from 5 to 86 years, with a bimodal age distribution among young people below 12 years of age (average 8 years) and in those aged 30 years or older (average 60.3 years). The male-to-female gender difference ratio was 1.3:1. Patients presented with swelling as the major symptom. Histologically, diffuse, large cell malignant lymphoma was the most frequent type and 67.9% of lymphomas were of intermediate malignancy as defined by the Working Formulation for Clinical Usage. All submucosal lymphomas showed diffuse proliferation patterns, although follicular proliferation was identified in 5 of the 21 nodal lymphomas. Immunohistochemistry showed that the B-cell type was predominant, especially in nodal lymphomas.

Early bone formation around calcium-ion-implanted titanium inserted into rat tibia.

Rat tibia tissue into which calcium ion (Ca2+)-implanted titanium was surgically placed was histologically analyzed to investigate the performance of the Ca(2+)-implanted titanium as a biomaterial. Calcium ions were implanted into only one side of titanium plates at 10(17) ions/cm2 and the Ca(2+)-treated titanium was surgically implanted into rat tibia for 2, 8, and 18 days. Tetracycline and calcein were used as hard-tissue labels. After excision of the tibia, the tissues were fixed, stained, embedded in polymethyl methacrylate, and sliced. The specimens were observed using a fluorescence microscope. A larger amount of new bone was formed on the Ca(2+)-treated side than on the untreated side, even at 2 days after surgery. In addition, part of the bone made contact with the Ca2(+)-treated surface. On the other hand, bone formation on the untreated side was delayed and the bone did not make contact with the surface. Mature bone with bone marrow formed in 8 days. Neither macrophage nor inflammatory cell infiltration was observed. The results indicated that Ca(2+)-implanted titanium is superior to titanium alone for bone conduction.

Antisense E1AF transfection restrains oral cancer invasion by reducing matrix metalloproteinase activities.

E1AF is a newly identified human ets-family transcription factor. We have reported that E1AF can up-regulate transcription of matrix metalloproteinase (MMP) genes and confers invasive phenotype on human cancer cells. HSC3 is an oral squamous-cell-carcinoma-derived cell line, and it manifests high levels of E1AF and MMP-1 and -9 gene expression that are associated with invasive potential. We reconstructed an E1AF antisense expression vector, transfected HSC3 cells with the vector, and obtained HSC3AS cells that express E1AF antisense RNA. HSC3AS showed decreasing mRNA and protein levels of MMP-1, -3, and -9. Moreover, HSC3AS showed lower invasive potential in vitro three-dimensional raft culture and in vivo implantation into nude mice. These results imply that transfection of antisense E1AF inhibits tumor invasion by down-regulating MMP genes.

Expression of E1AF, an ets-family transcription factor, is correlated with the invasive phenotype of oral squamous cell carcinoma.

E1AF is a newly identified ets-oncogene family transcription factor. Previous reports have noted that E1AF can upregulate promoter activities of several matrix metalloproteinase (MMP) genes and showed that invasive potentials of oral squamous cell carcinoma-derived cell lines are correlated with expression of E1AF and MMPs. The invasive phenotype is restrained by transfection with an antisense E1AF expression vector. Thus, E1AF is thought to be highly correlated with malignant potentials of cancer cells. However, little is known about E1AF expression and cancer cell malignancies in in vivo tumours. In the present study, 27 oral squamous cell carcinoma (SCC) specimens were examined using RT-PCR, Southern blot hybridisation and in situ hybridisation (ISH) and compared to the clinicopathological parameters. Among the 27 patients, E1AF was detected in 15 cases. E1AF mRNA was detected in 13 of 17 invasive SCCs, whereas the majority of SCCs not expressing E1AF showed an expansive growth pattern. Increased prevalence of E1AF-positive oral SCC was observed in cases with nodal metastasis. These results indicate that E1AF may be involved in cancer cell malignancies through its ability to promote invasive potential.

Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) in 23 cases of ameloblastoma.

Ameloblastoma is the most frequent odontogenic tumour. It occurs mainly in the mandible and grows expansively. The treatment of ameloblastoma, which influences the prognosis, is decided in consideration of many factors, especially the age and size of the tumour. Conservative treatment sometimes leads to the recurrence of tumours and poor prognosis, but the relationships between the prognosis and the cytological features of tumour cells are still unclear. In the present study, we examined the immunohistochemical detection of proliferating cell nuclear antigen (PCNA) in 23 cases of ameloblastoma and evaluated the correlation between the positive index of PCNA and the clinical and histological character. Our results revealed the higher the age of the patient the greater was the incidence of a positive index of PCNA. It was also shown that the mean positive PCNA index in the follicular type (34.56 +/- 14.00 S.D.) was higher than that of the plexiform type (24.436 +/- 15.74 S.D., P < 0.10). The cystic type showed a low positive PCNA index (14.75 +/- 8.41 S.D.). In the follicular type, the localisation of PCNA-positive cells was different according to the histological patterns of tumours. Additionally, the positive indices of the same patient differed at different periods of treatment.

Two novel gene mutations (Glu174-->Lys, Phe383-->Tyr) causing the "hepatic" form of carnitine palmitoyltransferase II deficiency.

Carnitine palmitoyltransferase II (CPT II) deficiency has two different clinical forms, one with "hepatic" and the other with "muscular" symptoms. We studied the molecular basis of the "hepatic" form in two Japanese siblings. Their CPT II activity in lymphoblasts was reduced to 3% of the level observed in normal controls. cDNA analysis showed that the proband was a compound heterozygote. One allele carried a new mutation, G621-->A (Glu174-->Lys). The other carried three single-base substitutions; a new mutation, T1249-->A (Phe383-->Tyr), and two previously reported polymorphisms. The brother had the same four substitutions. Neither of the two new mutations in this study was detected in the 60 alleles of 30 Japanese control subjects. Secondary structure prediction analysis of the mutated CPT II protein was different from that of the normal protein. We concluded that these mutations caused the "hepatic" form of CPT II deficiency in the probands.