Spatial dynamics of GFP-tagged proteins investigated by local fluorescence enhancement.

We describe a method of monitoring the spatial dynamics of proteins in intact cells by locally enhancing the blue excited fluorescence of green fluorescent protein (GFP) using a spatially focused ultraviolet-laser pulse. GFP fusion proteins were efficiently expressed by micro-electroporation of in vitro synthesized mRNA into adherent mammalian cells. We found that the diffusion coefficient of cycle 3 mutant GFP was 43 microns2/sec, compared to 4 microns2/sec for wild-type GFP, suggesting that cycle 3 GFP diffuses freely in mammalian cells and is ideally suited as a fusion tag. The local fluorescence enhancement method was used to study the membrane dissociation rate of GFP-tagged K-ras, a small GTP binding protein that localizes to plasma membranes by a farnesyl lipid group and a polybasic region. Our data suggest that K-ras exists in a dynamic equilibrium and rapidly switches between a plasma membrane bound form and a cytosolic form with a plasma membrane dissociation time constant of 1.5 sec.

Monophasic epithelial synovial sarcoma arising in the temporomandibular joint.

Synovial sarcoma is a mesenchymal spindle-cell tumour that occurs infrequently in the head and neck. It originates from unknown stem cells differentiating into mesenchymal and/or epithelial structures. Most synovial sarcomas are biphasic in character, consisting of epithelial and spindle-cell elements. Here is reported a case of monophasic epithelial synovial sarcoma arising in the temporomandibular joint. The tumour was of a predominantly epithelial pattern, although a minute area of sarcomatous cells was found. The primary mode of treatment was wide en-bloc excision. Two years after surgery, the patient died of hepatocellular carcinoma, but there was no evidence of synovial sarcoma recurrence.

Low levels of NPM gene expression in UV-sensitive human cell lines.

Nucleophosmin (NPM) is a major nuclear matrix protein associated with neoplastic growth in various cell types. We recently suggested that expression of the NPM gene is involved in an increased resistance to UV irradiation in human cells against the cell-killing effects of UV (mainly 254nm wavelength far-ultraviolet ray) [Y. Higuchi, K. Kita, H. Nakanishi, X-L. Wang, S. Sugaya, H. Tanzawa, H. Yamamori, K. Sugita, A. Yamaura, N. Suzuki, Biochem. Biophys. Res. Commun. 248 (1998) 597-602]. In the present study, expression levels of the NPM gene were examined in human cell lines with a high sensitivity to UV cell-killing. Cockayne syndrome patient-derived cell lines, CSAI and CSBI, and the Xeroderma pigmentosum patient-derived cell line, XP2OS(SV), XP13KY, XP3KA, XP6BE(SV), XP101OS and XP3BR(SV), have been investigated for their NPM mRNA expression with Northern blotting analysis. All of these UV-sensitive cells demonstrated lower expression levels compared with those of normal fibroblast cells, FF, or an UV-resistant cell line, UH(r)-10; quite a lower level of expression in XP205(SV) cells after UV irradiation in contrast to a distinguishable increase in the expression in UV(r)- cells. These results confirmed an intimate correlation between degree of UV sensitivity and expression levels of the NPM gene in human cells.

Mutational state of p16/cdkn2 and vhl genes in squamous-cell carcinoma of the oral cavity.

Two new tumor-suppressor genes, the cyclin dependent kinase 4 inhibitor gene (p16/CDKN2) and the von Hippel-Lindau disease gene (VHL), have been cloned and mapped on chromosomes 9p and 3p respectively, where putative tumor-suppressor genes of the oral squamous-cell carcinoma (SCC) may be present. In order to elucidate whether abnormalities of these genes could contribute to the tumorigenesis of oral SCC, genomic DNAs from 62 tissue samples of tumors (32 primary SCCs and 30 pre-cancerous lesions) and from 7 oral SCC cell lines were examined by polymerase chain reaction-single strand conformation polymorphism analysis and direct DNA sequencing. Four of 7 (57%) cell lines contained nonsense mutations or missense mutations in the p16/CDKN2 gene and 2 of 32 (6%) primary oral SCCs had nonsense mutations. Particularly, 3 of 4 nonsense mutations detected in the present study were found in codon 80 (CGA-->TGA; Arg-->Stop), suggesting that codon 80 was a mutational hot spot of the p16/CDKN2 gene. No VHL gene mutation was found in any subject. These results suggest that mutation of the VHL gene is not a common factor in the development of human oral SCC. In contrast, the p16/CDKN2 gene may be correlated with the progression of a subtype of this cancer.

Rare mutations of the growth suppressor genes involved in negative regulation of the cell cycle.

The growth suppressing activity of the retinoblastoma susceptibility gene product, pRb, is down regulated by cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) whose activity is negatively regulated by CDK inhibitors of the p16 family. We have previously reported point mutations of the p16/CDKN2 gene in 4 (57%) of 7 oral squamous cell carcinoma (SCC) cell lines. In the current study, we examined the mutational status of CDK inhibitors, including 3 genes of the p16 family (p16, p15 and p18), in 50 human oral SCCs, and also additional results concerning their loss of heterozygosity in the regions of the p16, p15 and p18 genes. Our results demonstrated that 2 of 50 (4%) primary oral SCCs had nonsense mutations of the p16 gene, and 2 of 50 (4%) showed frameshift mutations of the p18 gene. However, we detected no mutation of the p15 gene in any of the 50 oral SCCs. In addition, no evidence of hypermethylation of the p16 gene was found in our series. To better understand the extent of alterations affecting chromosomes 9p21 (location of the p15/p16 genes) and 1p32 (location of the p18 gene), loss of heterozysity (LOH) on these locations was examined. LOH was detected in 16 of 34 (47%) informative samples that had no detectable mutation of the p15/p16 genes on 9p21, but we found no LOH at 1p32. These results strongly suggest that a putative tumor suppressor gene for oral SCC may be present on chromosome 9p21-22, while the p16, p15 and p18 genes play a minor role in the oncogenesis of this cancer.

Induction of polyploidization in the human erythroleukemia cell line (HEL) by protein kinase inhibitor (K252a) and the phorbol-ester TPA.

Endomitosis (polyploidization) is a distinctive feature of megakaryocyte differentiation. We examined this mechanism in an erythromegakaryocytic cell line, HEL, using a protein kinase inhibitor K252a or a phorbol-ester TPA. HEL cells treated with K252a showed a marked increase in the proportion of CD41 positive cells and polyploid cells as well as in cellular size and nuclear size. TPA showed similar results but induced multi-nucleation instead of enlargement of nuclear size. K252a added at the G1/S boundary phase did not inhibit the first and second round DNA synthesis, but inhibited cell division. K252a did not inhibit the expression of genes involved in mitosis such as cyclin B, cdc25B and cdc2, in the first round S phase. However, the cyclin B associated Cdc2 kinase activity needed for mitosis during the G2/M phase was reduced by K252a. TPA delayed DNA synthesis and expression of these genes, and suppressed Cdc2 kinase activity in the second round G2/M phase. These results suggest that the polyploidization induced by K252a results from inhibiting mitosis possibly caused by suppression of Cdc2 kinase activity. TPA may induce the multi-nucleation through a different mechanism.

Expression of an inhibitor of apoptosis, survivin, in oral carcinogenesis.

A novel inhibitor of apoptosis, survivin, plays a role in oncogenesis. To determine the potential involvement of survivin in oral carcinogenesis, we investigated the distribution of survivin protein expression in oral squamous cell carcinomas (OSCCs) and oral pre-malignant lesions. The mRNA expression level and methylation status of the gene also were evaluated in OSCCs and OSCC-derived cell lines. In immunohistochemistry, 58% of tumors and 37% of pre-malignant lesions examined were positive for survivin, while no immunoreaction was observed in corresponding normal tissues. The reverse-transcription/polymerase chain-reaction revealed similar changes in survivin gene expression levels. Furthermore, of the 9 normal oral tissues with no survivin gene expression, 4 showed methylation of the gene, while no methylation was detected in the corresponding tumorous tissues. The results suggest that survivin plays an important role during oral carcinogenesis, and that the gene expression may be regulated by an epigenetic mechanism.

Frequent microsatellite instability in oral cancer.

In oder to confirm whether microsatellite instability (MI) contributes to oral carcinogenesis, we studied 30 unrelated patients with oral cancer by PCR-MI assay with 14 microsatellite markers. MI was detected in 60% of 30 primary tumors. Tn particular, 12 of these cases presented at least two loci with MI, which were considered as patients with replication error (RER). Moreover, an additional MI, which was not observed in the primary tumor and normal tissue, was observed in one lymph node metastasis. We found significant correlation between RER and lymph node metastasis. These results suggest that RER is a common event in the oncogenesis of oral cancer and may be correlated with the progression of this disease.

Frequent allelic loss/imbalance on the long arm of chromosome 21 in oral cancer: evidence for three discrete tumor suppressor gene loci.

Frequent allelic imbalances including loss of heterozygosity (LOH) and microsatellite instability (MI) on the long arm of chromosome 21 (21q) have been found in several types of human cancer. This study was designed to identify tumor suppressor locus (or loci) associated with oral squamous cell carcinoma (SCC) on 21q. Among 38 patients with oral SCC tested, 15 (44%) of 34 informative cases showed LOH at one or more loci. Deletion mapping of these 15 tumors revealed three discrete commonly deleted regions on the chromosome arm. A minimal region with frequent LOH was found at the marker D21S236 mapped on 21q11.1. Another region of frequent deletion was identified between markers D21S11 and D21S1436 on 21q21, and a further commonly deleted region was found at D21S1254 on 21q22.1. In addition, we have detected MI on the chromosome arm in our oral SCC samples with significant correlation with tumor stage. Thus, our results strongly suggest that allelic imbalances on 21q may be involved in the development of oral SCC; and that at least three different putative tumor suppressor genes contributing to the pathogenesis of this disease are present on 21q.

Allelic loss on the short arm of chromosome 8 in oral squamous cell carcinoma.

Allelic deletions on the short arm of chromosome 8 (8p) are frequent events in many different types of malignant tumors, including head and neck tumors and oropharyngeal cancers. These regions are thought to harbor tumor suppressor genes. However, there has been no detailed analysis of loss of heterozygosity (LOH) on the chromosome arm 8p in oral squamous cell carcinoma (SCC). In order to estimate details of 8p loci involved in oral SCC, 32 patients with oral SCCs were examined for the LOH state 8p by PCR-LOH assay using 14 microsatellite markers. Based on our results a detailed deletion map of 8p showed LOH in at least one of the loci tested in 62.5% (20 of 32) of patients; and microsatellite instability (MI) was observed in 50% (16 of 32). The frequent LOH on this chromosome arm was identified at D8S87 and D8S258, mapped on 8p12 and 8p22, respectively. Fisher's exact test revealed no significant statistical correlation between the incidence of LOH or MI and clinicopathological features. Our observations indicate that the short arm of chromosome 8 may play a role in the pathogenesis of oral SCC; and that the two loci identified in this study may be tumor suppressor gene loci on 8p.

Establishment of a new human megakaryoblastic cell line, CMY, with chromosome 17p abnormalities.

A new megakaryoblastic cell line CMY was established from a Down's syndrome patient suffering from acute megakaryoblastic leukemia. The karyotypes of CMY showed deletion of chromosome 17 or the translocation of 17p, whereas the blasts of the patient did not reveal these abnormalities of chromosome 17 by conventional karyotype analysis. Blasts of the patient failed to respond to chemotherapy and complete remission could not be attained. The abnormalities of 17p became progressively predominant in the patient. These results suggest that the blasts of a minor clone which had the abnormalities of chromosome 17p might have existed in the patient from the beginning and CMY was established from the minor clone. Investigation of p53 gene by PCR-SSCP analysis revealed that blasts of the patient showed normal patterns, while CMY showed an abnormally migrating band in exon 5 alone. This result suggests that another novel oncogenic factor(s) besides p53 might be present on chromosome 17p and other tumor suppresser genes need to be studied.

Protease activity induced by nicotine in human cells.

Nicotine has a wide range of biological effects, and proteases have been extensively studied for their biological roles in living creatures. The aim of this study is to determine whether nicotine can induce proteolytic protease activity in cultures of various human cell lines. Plasminogen activator-like fibrinolytic protease activity, using 125I-fibrin as substrate in the presence of plasminogen, was estimated in cells with and without nicotine treatment. Among 16 cell lines tested, APr-1 cells were found to have the highest induced protease activity. Partial purification of the proteases was carried out by high performance liquid chromatography gel filtration on TSKG2000SW. Protease inhibitor tests indicated that the proteases induced by nicotine are serine proteases.

Solitary plasmacytoma of the mandible. Case report and review of the literature.

A case of solitary plasmacytoma of the mandible is presented. Review of the literature disclosed the following characteristics regarding the clinical course and prognosis. The patients ages ranged from 34 to 76 years, with a mean of 53 years; there was a marked preponderance of males. The site of predilection was the posterior portion of the mandible. The common symptom was a non-painful swelling of the mandible of long duration, and radiological features were non-specific. Monoclonal immunoglobulin was initially detected in 42% of the evaluated patients. The majority of patients were treated with radiation therapy with a mean dose of 48Gy with or without surgery. The period of follow-up ranged from 4 months to 12 years, and 17% of the patients progressed to multiple myeloma within 1 year.

Gingival squamous cell carcinoma in a patient with chronic graft-versus-host disease.

This article describes a gingival squamous cell carcinoma that developed in a 21-year-old woman who received a bone marrow transplant at the age of 16 from her human leukocyte antigen-identical sister as treatment for severe aplastic anemia. Thirty days after transplantation, she presented with cutaneous erythema as a result of acute graft-versus-host disease, and this subsequently evolved into chronic graft-versus-host disease. A lichenoid white plaque of the gingiva developed shortly thereafter, and it began to increase in size rapidly 4 years posttransplantation. Biopsy indicated squamous cell carcinoma arising in this region, apparently associated with chronic graft-versus-host disease. Few reports have described a secondary solid malignancy involving the oral cavity of young adults after bone marrow transplantation.

[Antitumor effects of newly synthesized analogues of platinum (NK121) and bleomycin (NK313) in nude mice implanted human tumor].

In order to exploit the optimal administration schedules of newly synthesized analogues of cisplatin (NK121) and bleomycin (NK313), antitumor effects of three different injection schedules, namely (A) an injection every fourth day for 3 times in 9 days, (B) once daily injection for 9 days, and (C) continuous infusion for 5 days, were examined in nude mice bearing human squamous cell carcinoma. The tumor growth delay was employed as an experimental endpoint. Antitumor effects of both analogues showed schedule dependencies showing the highest effect with schedule (A) of NK121 and schedule (C) of NK313, respectively. In the combination chemotherapy of NK121 with NK313, the highest antitumor effect was observed when mice were given NK121 by schedule (A) followed by NK313 by schedule(C). This combination sequence also was less toxic with respect to the change of the body weight, while simultaneous treatment was with weak clinical significance because of its low antitumor activity and severe toxicity.

Clinical observations of postoperative delirium after surgery for oral carcinoma.

The aim of the present study was to clarify the clinical characteristics of postoperative delirium and to determine appropriate postoperative management for its prevention. The authors analysed 132 cases of primary surgery for oral carcinoma and observed 24 (18%) cases of postoperative delirium. Univariate analysis revealed that significant risk factors for postoperative delirium were older age, male gender, extensive surgery and morphine pain control. Logistic regression analysis showed that older age and male gender were significant risk factors for postoperative delirium, while patient-controlled analgesia with fentanyl was effective for prevention of postoperative delirium. There was a trend for postoperative delirium to be associated with extensive surgery. In those who had delirium, blood tests revealed that alkaline phosphatase, total protein, sodium, chlorine, red blood cell count, haemoglobin and haematocrit were significantly diminished after surgery. These results indicate that general condition is closely related to the onset of postoperative delirium, and suggest that appropriate postoperative management can reduce the incidence of this complication.

Network-based analysis of calcium-binding protein genes identifies Grp94 as a target in human oral carcinogenesis.

To characterise Ca(2+) -binding protein gene expression changes in oral squamous cell carcinomas (OSCCs), we compared the gene expression profiles in OSCC-derived cell lines with normal oral tissues. One hundred Ca(2+) -binding protein genes differentially expressed in OSCCs were identified, and genetic pathways associated with expression changes were generated. Among genes mapped to the network with the highest significance, glucose-regulated protein 94 kDa (Grp94) was evaluated further for mRNA and protein expression in the OSCC cell lines, primary OSCCs, and oral premalignant lesions (OPLs). A significant (P<0.001) overexpression of Grp94 protein was observed in all cell lines compared to normal oral epithelium. Immunohistochemical analysis showed highly expressed Grp94 in primary OSCCs and OPLs, whereas most of the corresponding normal tissues had no protein immunoreaction. Real-time quantitative reverse transcriptase-PCR data agreed with the protein expression status. Moreover, overexpression of Grp94 in primary tumours was significantly (P<0.001) correlated with poor disease-free survival. The results suggested that Grp94 may have potential clinical application as a novel diagnosis and prognostic biomarker for human OSCCs.

Ubiquitous mitochondrial creatine kinase downregulated in oral squamous cell carcinoma.

In this study, we performed two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionisation time of fly mass spectrometry to identify the protein(s) associated with the development of oral squamous cell carcinomas (OSCCs) by comparing patterns of OSCC-derived cell lines with normal oral keratinocytes (NOKs), and found that downregulation of ubiquitous mitochondrial creatine kinase (CKMT1) could be a good candidate. Decreased levels of CKMT1 mRNA and protein were detected in all OSCC-derived cell lines examined (n=9) when compared to those in primary normal oral keratinocytes. Although no sequence variation in the coding region of the CKMT1 gene with the exception of a nonsense mutation in exon 8 was identified in these cell lines, we found a frequent hypermethylation in the CpG island region. CKMT1 expression was restored by experimental demethylation. In addition, when we transfected CKMT1 into the cell lines, they showed an apoptotic phenotype but no invasiveness. In clinical samples, high frequencies of CKMT1 downregulation were detected by immunohistochemistry (19 of 52 (37%)) and quantitative real-time RT-PCR (21 of 50 (42%)). Furthermore, the CKMT1 expression status was significantly correlated with tumour differentiation (P<0.0001). These results suggest that the CKMT1 gene is frequently inactivated during oral carcinogenesis and that an epigenetic mechanism may regulate loss of expression, which may lead to block apoptosis.

Overexpression of stathmin in oral squamous-cell carcinoma: correlation with tumour progression and poor prognosis.

Stathmin is an intracellular phosphoprotein that is overexpressed in a number of human malignancies. Our previous study using proteomic profiling showed that significant upregulation of stathmin occurs in oral squamous-cell carcinoma (OSCC)-derived cell lines. In the current study, to determine the potential involvement of stathmin in OSCC, we evaluated the state of stathmin protein and mRNA expression in OSCC-derived cell lines and human primary OSCCs. A significant increase in stathmin expression was observed in all OSCC-derived cell lines examined compared to human normal oral keratinocytes. In immunohistochemistry, 65% of the OSCCs were positive for stathmin, and no immunoreaction was observed in corresponding normal tissues. Real-time quantitative reverse transcriptase-polymerase chain reaction data were consistent with the protein expression status. Moreover, stathmin expression status was correlated with the TNM stage grading. Furthermore, we found a statistical correlation between the protein expression status and disease-free survival (P=0.029). These results suggest that expression of stathmin could contribute to cancer progression/prognosis, and that stathmin may have potential as a biomarker and a therapeutic target for OSCC.

Monitoring of circulating tumour-associated DNA as a prognostic tool for oral squamous cell carcinoma.

Frequent allelic imbalances (AIs) including loss of heterozygosity and microsatellite instability on a specific chromosomal region have been identified in a variety of human malignancies. The objective of our study was to assess the possibility of prognostication and monitoring of oral squamous cell carcinoma (SCC) by microsatellite blood assay. DNA from normal and tumorous tissues and serum DNA obtained at three time points (preoperatively, postoperatively, and 4 weeks postoperatively) from 64 patients with oral SCC was examined at nine microsatellite loci. In all, 38 (59%) DNA samples from tumorous tissues and 52% from serum showed AIs in at least one locus. Patterns of AIs in the serum DNA were matched to those detected in tumour DNA. Of them, AIs were frequently detected preoperatively (44%, 28 of 64), and postoperatively (20%, 13 of 64). Moreover, among 12 cases with AIs during the postoperative period, six had no evidence of an AI 4 weeks postoperatively, and they had no recurrence and were disease free. In contrast, six patients with AI-positive DNA 4 weeks postoperatively have died with distant metastasis within 44 weeks. Thus, our results suggest that the assessment of microsatellite status in the serum DNA could be a useful predictive tool to monitor disease prognosis.