Haruhiko Kashiwazaki

High frequency of p53 mutations in human oral epithelial dysplasia and primary squamous cell carcinoma detected by yeast functional assay

To determine the timing and actual incidence of p53 mutations in oral epithelial lesions, we examined 33 primary squamous cell carcinomas (SCCs), 14 dysplasias and six hyperplasias from Japanese patients by a combination of yeast functional assay and DNA sequencing. The assay detects mutations of p53 mRNA between codons 67 and 347 on the basis of the DNA-binding activity of the protein. Twenty-six SCCs (79%) and five dysplasias (36%) were positive for p53 mutation, while all six hyperplasias were negative for the mutation. Human papillomavirus type 16 E6 mRNA was detected in one of seven p53 mutation-negative SCCs by reverse transcription polymerase chain reaction (RT – PCR). We further examined p53 mutations in 17 Sri Lankan oral SCCs using the yeast functional assay and the single-strand conformation polymorphism analysis of PCR-amplified DNA fragments (PCR – SSCP) of exon 5 – 8. The mutations were confirmed by DNA sequencing and the detection sensitivity was compared between the two methods. Six samples (35%) were positive for p53 mutation in PCR – SSCP analysis, while nine samples (53%) were positive in yeast functional assay. This suggests that the incidence of p53 mutations has been considerably underestimated in the conventional SSCP analysis. The present data indicate that p53 mutations are extremely frequent in oral cancers in the Japanese, and suggest that the timing and significance of p53 mutation in oral tumor progression vary in different ethnic populations and areas.

Replicating Adenoviruses That Target Tumors with Constitutive Activation of the wnt Signaling Pathway

ABSTRACT Despite important advances in understanding the molecular basis of cancer, few treatments have been devised which rationally target known causal oncogenic defects. Selectively replicating viruses have a major advantage over nonreplicating viruses to target these defects because the therapeutic effect of the injected virus is augmented by virus produced within the tumor. To permit rational targeting of colon tumors, we have developed replicating adenoviruses that express the viral E1B and E2 genes from promoters controlled by the Tcf4 transcription factor. Tcf4 is constitutively activated by mutations in the adenomatous polyposis coli and β-catenin genes in virtually all colon tumors and is constitutively repressed by Groucho and CtBP in normal tissue. The Tcf-E2 and Tcf-E1B promoters are active in many, but not all, cell lines with activation of the wnt pathway. Viruses with Tcf regulation of E2 expression replicate normally in SW480 colon cancer cells but show a 50- to 100-fold decrease in replication in H1299 lung cancer cells and WI38 normal fibroblasts. Activation of wnt signaling by transduction of a stable β-catenin mutant into normal fibroblasts renders the cells permissive for virus replication. Insertion of Tcf4 sites in the E1B promoter has only small effects on replication in vitro but significantly reduces the inflammatory response in a rodent lung model in vivo. Replicating adenoviruses with Tcf regulation of both E1B and E2 transcription are potentially useful for the treatment of liver metastases from colorectal tumors, but additional changes will be required to produce a virus that can be used to treat all colon tumors.

Specific p53 mutations predict poor prognosis in oral squamous cell carcinoma

In this study, we focused on p53 mutations in specific regions, including DNA-binding surface regions, to clarify the correlation between mutations within the specific regions of p53 and clinical outcomes of patients with oral cancers. We analyzed p53 mutations in 121 fresh primary oral squamous cell carcinomas (SCCs) by polymerase chain reaction-single-strand conformation polymorphism or a yeast functional assay. p53 mutations were detected in 51/121 (42%) cases. Mutation of p53 was not associated with any clinicopathological parameters; however, tumors containing specific p53 mutations, e.g. DNA-binding surface regions (L2, L3 and the LSH motif) and conserved regions (II-V), had significantly poorer prognoses than tumors with mutations outside of those regions. Moreover, locoregional failure, lymph node metastasis and the occurrence of subsequent distant metastasis were also significantly associated with mutations within DNA-binding surface regions. These data indicate that specific mutations of p53 could be important prognostic factors in oral SCCs.

Aberrant expression of HOX genes in oral dysplasia and squamous cell carcinoma tissues

Human HOX genes consist of 39 genes and encode transcription factors that function as master developmental regulators. We hypothesized that the misexpression of HOX genes was associated with carcinogenesis and malignant progression. The expression levels of 39 HOX genes in 31 human oral squamous cell carcinoma (SCC), 11 dysplasia, and 10 normal mucosa tissues were quantified by the real-time RT-PCR method. The expression levels of 18 HOX genes in the SCC tissues were significantly higher than those in the normal mucosa tissues. The dysplasia tissues showed higher expression of HOXA2, A3, B3, and D10 than normal mucosa tissues whereas they showed lower expression of HOXA1, B7, B9, and C8 than SCC. The SCC with lymph node metastasis showed high expression of HOXC6 compared to the SCC without it. These results suggest that misexpressions of particular HOX genes are implicated in the development of oral dysplasia and SCC.

p73 is not mutated in meningiomas as determined with a functional yeast assay but p73 expression increases with tumor grade.

The p53 gene is normally wild type in meningiomas. Since all three members of the p53 gene family recognize the same DNA sequence, tumors containing wild type p53 could decrease transactivation of p53 target genes by mutating either p63 or p73. In meningiomas the most likely target is p73, because loss of heterozygosity of the chromosomal band containing p73 is the commonest genetic lesion in these tumors. To screen p73 for mutations we have developed a functional assay which tests the ability of p73 to activate transcription from a p53‐responsive promoter in yeast. The assay correctly identified p73 mutants with mutations equivalent to hotspot mutations in p53, demonstrating that the assay can detect transcriptionally inactive p73. No mutations in p73 were identified in meningiomas. p73 RNA level was higher in more advanced tumors, but there was no correlation between the expression level of p73 and p21, a known p53 target gene. The yeast assay was also used to measure the intrinsic sensitivity of the p73 protein to mutagenesis. Like p53, p73 is exceptionally easy to inactivate as a transcription factor by point mutation. Taken together, these results indicate that p53 and p73 serve very different functions in tumors.

Professional oral health care reduces oral mucositis and febrile neutropenia in patients treated with allogeneic bone marrow transplantation

Goal of workLittle is known about the effects of professional oral health care (POHC) on the outcome of hematopoietic stem cell transplantation (HSCT). We evaluated the effects of POHC given by dentists and dental hygienists on the development of oral mucositis and febrile neutropenia (FN) after allogeneic bone marrow transplantation (BMT).Patients and methodsWe retrospectively studied 140 adult patients who had received allogeneic BMT, with or without POHC, in our hospital consecutively between February 2002 and December 2009. Oral mucositis was evaluated according to the World Health Organization scale.Main resultsThe incidence of oral mucositis was 66.7% (52/78) in the patients who had received POHC, compared to 93.5% (58/62) in the non-POHC group (P < 0.001). The incidence of FN and the maximal level of CRP were also significantly lower in the POHC group. Multivariate analysis revealed that the POHC was significantly associated with the incidence of oral mucositis (odds ratio, 7.58; 95%CI, 2.45–23.34; P < 0.001).ConclusionsWe concluded that POHC reduced the incidences of oral mucositis and FN by upgrading the overall oral hygiene during HSCT.

Characteristics of mutations in the p53 gene of oral squamous-cell carcinomas associated with betel-quid chewing in Sri Lanka

Oral squamous‐cell carcinoma (SCC) is the most common neoplasm in Sri Lanka, accounting for approximately 30% of all cancers in males. Epidemiologic evidence indicates that there is an unequivocal relationship between betel chewing and oral carcinogenesis, suggesting that there may be specific genetic targets of betel‐quid ingredients. The p53 gene has been indicated to be a tumor‐suppressor gene that is found in mutated form in common human cancers; however, there are few reports about “carcinogen‐specific” p53 mutation. Because of this background, primary resected specimens from 23 oral SCCs, 7 leukoplakias and 2 oral submucous fibrosis were collected from oral SCC patients in Sri Lanka and were used for p53 mutation analysis. Exons 5 through 8 of the p53 gene were examined by polymerase chain reaction‐single‐strand conformation polymorphism (PCR‐SSCP) and direct sequencing. Mutations in the p53 gene were frequent (10/23) in oral SCC specimens from Sri Lanka. Moreover, the mutations clustered significantly in exon 5 (7/10) of the p53 gene, and small deletions and inclusions other than point mutations were observed. These results indicate that 1) betel‐quid chewing may cause specific genetic changes, including mutation in the p53 gene; 2) mutations in the p53 gene are not rare events in SCC patients who are betel‐quid chewers, which contrasts with other reports; 3) exon 5 of the p53 gene could be one of the specific targets for some betel‐quid ingredients; and 4) betel‐quid chewing may be a critical environmental factor in the development of oral SCC. Int. J. Cancer 77:839–842, 1998.© 1998 Wiley‐Liss, Inc.

Distinct prognostic values of p53 mutations and loss of estrogen receptor and their cumulative effect in primary breast cancers.

A total of 76 primary breast cancers were screened for p53 mutations using the yeast p53 functional assay, and the mutations were determined by DNA sequencing. Clonal mutations of p53 were detected in 30 tumors (39%). Immunohistochemical staining for nuclear p53 accumulation performed on the yeast assay‐positive cases clearly differentiated missense mutations in the DNA binding domain (contact mutant; 17 cases) as positive stain and nonsense‐type mutations or missense mutations that may affect 3D‐structure of p53 protein (structural mutant; 13 cases) as negative stain. Enzyme immunoassay revealed loss of estrogen receptor in 36 tumors (50%). Prognostic values of p53 mutation and loss of estrogen receptor were evaluated after a median follow‐up period of 44 months. p53 mutations were associated with a short overall survival (log rank test, p = 0.0319), whereas it was not related to disease‐free (recurrence‐free) survival. Contact mutants were associated with slightly shorter survival compared with structural mutants. Inversely, loss of estrogen receptor was associated with early recurrence (p = 0.0461) but not with short overall survival. The patients with tumors harboring both p53 mutation and loss of estrogen receptor had the poorest outcome (p = 0.0019 and 0.0075 for overall and disease‐free survivals, respectively), suggesting independent and additive effects of the 2 factors. The independent role of the 2 factors was confirmed by a multivariate analysis using the Cox proportional hazard model stratified according to clinical tumor stages. Although preliminary, due to the small number of patients studied and the relatively short follow‐up time, our results suggest that p53 mutations and loss of estrogen receptor cooperatively affect the prognosis of primary breast cancer patients. Int. J. Cancer (Pred. Oncol.) 89:92–99, 2000.

Presence of dominant negative mutation of TP53 is a risk of early recurrence in oral cancer

Genetic alteration of p53 is a significant determining factor in the carcinogenesis. The loss of function, mutant p53 can possess a dominant negative effect on wild-type p53 and may also exert gain-of-function activity. It is, however, not clear how p53 functional status due to various types of mutation results in outcome of patients with oral cancer. A total of 60 oral SCC samples were subjected to yeast functional assay that screens human p53 function in yeast, and sequencing for determination of p53 mutations. The detected mutants were further investigated for their dominant negative activity using a yeast-based transdominance assay that tests dominant negative activity of a mutant p53 over wild-type p53 by coexpressing the mutant and wild-type p53 in a yeast transcriptional reporter system. p53 mutation was found in 42 out of 60 of which 10 (24%) exhibited dominant negative activity and 32 (76%) without dominant activity (recessive mutation). The remaining 18 (30%) were considered to have wild-type p53. The patients with dominant negative mutation had significantly shorter disease-free survival than patients with no mutation (log-rank test, p<0.001) and those with a recessive mutation (p<0.016). There were slight significant differences in disease-free survival were found between the patients with tumours harbouring a recessive p53 mutation and those with tumours harbouring a wild-type p53 (p<0.038). The presence and absence of a dominant negative p53 mutation may thus provide a predictor of early recurrence in oral SCC patients.

Construction and characterization of a tissue-engineered oral mucosa equivalent based on a chitosan-fish scale collagen composite†

This study was designed to (1) assess the in vitro biocompatibility of a chitosan-collagen composite scaffold (C3) constructed by blending commercial chitosan and tilapia scale collagen with oral mucosa keratinocytes, (2) histologically and immunohistochemically characterize an ex vivo-produced oral mucosa equivalent constructed using the C3 (EVPOME-C), and (3) compare EVPOME-C with oral mucosa constructs utilizing AlloDerm® (EVPOME-A), BioMend® Extend™ (EVPOME-B), and native oral mucosa. C3 scaffold had a well-developed fibril network and a sufficiently small porosity to prevent keratinocytes from growing inside the scaffold after cell-seeding. The EVPOME oral mucosa constructs were fabricated in a chemically defined culture system. After culture at an air-liquid interface, EVPOME-C and EVPOME-B had multilayered epithelium with keratinization, while EVPOME-A had a more organized stratified epithelium. Ki-67 and p63 immunolabeled cells in the basal layer of all EVPOMEs suggested a regenerative ability. Compared with native oral mucosa, the keratin 15 and 10/13 expression patterns in all EVPOMEs showed a less-organized differentiation pattern. In contrast to the β1-integrin and laminin distribution in EVPOME-A and native oral mucosa, the subcellular deposition in EVPOME-C and EVPOME-B indicated that complete basement membrane formation failed. These findings demonstrated that C3 has a potential application for epithelial tissue engineering and provides a new potential therapeutic device for oral mucosa regenerative medicine.