Tongue Carcinogenesis Research Articles

Collagenase-2 (matrix metalloproteinase-8) plays a protective role in tongue cancer

Squamous cell carcinoma (SCC) of the tongue is the most common cancer in the oral cavity and has a high mortality rate. A total of 90 mobile tongue SCC samples were analysed for Bryne's malignancy scores, microvascular density, and thickness of the SCC sections. In addition, the staining pattern of cyclooxygenase-2, αvβ6 integrin, the laminin-5 γ2-chain, and matrix metalloproteinases (MMPs) -2, -7, -8, -9, -20, and -28 were analysed. The expression of MMP-8 (collagenase-2) was positively associated with improved survival of the patients and the tendency was particularly prominent in females. No sufficient evidence for a correlation with the clinical outcome was found for any other immunohistological marker. To test the protective role of MMP-8 in tongue carcinogenesis, MMP-8 knockout mice were used. MMP-8 deficient female mice developed tongue SCCs at a significantly higher incidence than wild-type mice exposed to carcinogen 4-Nitroquinoline-N-oxide. Consistently, oestrogen-induced MMP-8 expression in cultured HSC-3 tongue carcinoma cells, and MMP-8 cleaved oestrogen receptor (ER) α and β. According to these data, we propose that, contrary to the role of most proteases produced by human carcinomas, MMP-8 has a protective, probably oestrogen-related role in the growth of mobile tongue SCCs.

A novel rasH2 mouse carcinogenesis model that is highly susceptible to 4-NQO-induced tongue and esophageal carcinogenesis is useful for preclinical chemoprevention studies

We investigated the susceptibility of 4-nitroquinoline 1-oxide (4-NQO)-induced tongue carcinogenesis in male CB6F1-Tg-rasH2 @Jcl mice (Tg mice). The Tg mice were administered 4-NQO (20 p.p.m. in drinking water) for 2, 4, 6 or 8 weeks, and thereafter they were untreated up to week 24. At week 24, a higher incidence (80%) of tongue neoplasm with dysplasia was noted in the mice that received 4-NQO for 8 weeks in comparison with the other groups (20% incidence for each) treated with 4-NQO for 2, 4 and 6 weeks. Esophageal tumors also developed in the Tg mice were 4-NQO. Immunohistochemical observation revealed that the EP receptors, especially EP(1) and EP(2), expressed in the tongue and esophageal lesions induced by 4-NQO, thus suggesting the involvement of prostaglandin (PG) E(2) and EP(1,2) receptors in the tongue and esophageal carcinogenesis. Using this animal model, we investigated the potential chemopreventive ability of pitavastatin (1, 5 and 10 p.p.m. in diet for 15 weeks), starting 1 week after the cessation of 4-NQO-exposure (20 p.p.m. in drinking water for 8 weeks). Dietary pitavastatin at 10 p.p.m. significantly reduced the incidence and multiplicity of the tongue, but not esophageal neoplasms by the modulation of prostaglandin E2 biosynthesis, EP(1) and EP(2) expression and proliferation. Our results thus suggest that a rasH2 mouse model of 4-NQO-induced tongue and esophageal carcinogenesis can be utilized for investigating the pathogenesis of cancer development in these tissues and may well prove to be useful for identifying candidate cancer chemopreventive agents for the upper digestive organs.

Gingival Changes in Wistar Rats after Oral Treatment with 4-Nitroquinoline 1-Oxide

4-nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinogenesis is a useful model for studying oral squamous cell carcinoma. However, gingival changes following 4NQO administration via drinking water are absent in the literature. The aim of this study was to investigate gingival changes concomitant to tongue carcinogenesis induced by 4NQO by means of morphological analysis. Male Wistar rats were distributed into 3 groups of 10 animals each and treated with 50 ppm 4NQO solution by drinking water for 4, 12 or 20 weeks. Thirty animals were used as negative control. Regarding tongue mucosa, the primary histopathological change i.e., hyperplasia and dysplasia was evidenced after 12 weeks treatment with 4NQO. At 20 weeks, squamous cell carcinoma was found in the majority of animals. Gingival squamous hyperplasia was induced by 4NQO after 20-weeks of treatment. Dysplastic changes appeared in some animals (two cases) as well. Taken together, our results support the notion that 4NQO is more effective in rat tongue mucosa than gingival tissue. Probably, this discrepancy depends strongly on route of administration and the susceptibility with respect to animals species. Certainly, such data will contribute when using this experimental test-system for understanding oral cancer pathogenesis.

Placental glutathione S-transferase correlates with cellular proliferation during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

Taking into consideration that glutatione S-transferase (GST) and cellular proliferation play a crucial role during carcinogenesis, the goal of this study was to investigate the expression of placental GST, called GST-P, and proliferating cellular nuclear antigen (PCNA) by means of immunohistochemistry during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide (4NQO). This is a useful model for studying oral squamous cell carcinoma phase by phase. Male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution by drinking water for 4, 12 or 20 weeks. Ten animals were used as negative control. GST-P positive foci were detected in non-neoplastic oral cells at 4 weeks of 4NQO administration. In the same way, GST-P positive cells were detected in pre-neoplastic lesions and squamous cell carcinomas induced after 12 and 20 weeks-treatment, respectively. None of the control animals expressed GST-P positive cells. Regarding cellular proliferation, PCNA positive nuclei were higher at 12 and 20 weeks following 4NQO exposure ( p<0.05) when compared to negative control. These results suggest that the expression of GST-P is correlated with cellular proliferation, in which GST-P is associated with risk and progression of oral cancer, whereas PCNA is closely involved during neoplastic conversion.

P134 Placental gutathione S-transferase correlates with cellular proliferation during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

P134 Placental gutathione S-transferase correlates with cellular proliferation during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

Cytoplasmic Localization of BAG-1 in Leukoplakia and Carcinoma of the Tongue: Correlation with p53 and C-Erbb2 in Carcinoma

The present study evaluated the clinical significance of BAG-1, an antiapoptotic protein, in leukoplakia and carcinoma of the tongue. BAG-1 expression was evaluated by immunohistochemistry in paraffin-embedded tissues of leukoplakia (n=25) and carcinoma of the tongue (n=61). Cytoplasmic expression was predominantly seen in 80% and 70% of patients with leukoplakia and carcinoma, respectively. BAG-1 expression was found to be significantly lower in tobacco users than in non-tobacco users. BAG-1 expression in tobacco-using leukoplakia and carcinoma patients was compared by grouping the carcinoma patients according to lymph node status and disease stage. Carcinoma patients with tumor-positive lymph nodes had significantly lower BAG-1 expression than patients with negative lymph nodes and leukoplakia. Further, a trend towards an inverse correlation was observed with p53 and c-erbB2. In univariate and multivariate survival analysis, patient subgroups with 2+ or 3+ marker positivity (BAG-1 negativity, p53 and c-erbB2 positivity) had a reduced overall survival compared with patient subgroups with 1+ marker positivity or negativity. BAG-1 negativity in association with p53 and c-erbB2 positivity identified a subgroup of tongue cancer patients with an aggressive phenotype. Hence, an antiapoptotic protein, BAG-1, was found to be down-regulated in chewing-tobacco-mediated tongue carcinogenesis.

Survivin and inducible nitric oxide synthase production during 4NQO-induced rat tongue carcinogenesis: A possible relationship

This study was undertaken to investigate, by immunohistochemistry, the expression of survivin and inducible nitric oxide synthase during 4NQO-induced rat tongue carcinogenesis. Male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution through their drinking water for 4, 12, and 20 weeks. Ten animals were used as negative control. Although no histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure, survivin and iNOS were expresssed ( p < 0.05) in some cells of the ‘normal’ oral epithelium. In pre-neoplastic lesions at 12 weeks following carcinogen exposure, the levels of survivin and iNOS were increased ( p < 0.05) when compared to negative control, being the strongest effect observed to iNOS. In well-differentiated squamous cell carcinoma induced after 20 weeks of treatment with 4NQO, survivin and iNOS were expressed in some tumor cells. Lack of immunoreactivity for both markers was observed in the negative control group. Taken together, our results support the belief that expression of survivin and iNOS are early events during malignant transformation and conversion of the oral mucosa.

Expression of Receptor for Advanced Glycation End Products during Rat Tongue Carcinogenesis by 4-Nitroquinoline 1-Oxide and Effect of a Selective Cyclooxygenase-2 Inhibitor, Etodolac

Objectives: The expression of a receptor for advanced glycation end products (RAGE) in lesions developed during rat tongue carcinogenesis by 4-nitroquinoline 1-oxide (4-NQO) and the effect of a selective cyclooxygenase-2 inhibitor, etodolac, were investigated. Methods: The tongue lesions were induced in Fischer 344 rats given 20–30 ppm 4-NQO in their drinking water for 12 weeks and then fed the basal diet containing 150 and 300 ppm of etodolac for 16 weeks, respectively. Results: The incidence of carcinomas in the 4-NQO-alone group was 100%. Etodolac significantly reduced the incidences of carcinomas to 66.7% (p < 0.05) and 50% (p < 0.01) at doses of 150 and 300 ppm, respectively. RAGE protein was immunohistochemically expressed in dysplastic cells and cancer cells of dysplasias and carcinomas. Etodolac significantly decreased the stainability of RAGE protein in dysplasias (p < 0.02) and carcinomas (p < 0.01). The expression of RAGE mRNA analyzed by RT-PCR was clearly detected in carcinomas developing in the 4-NQO-alone group. In carcinomas developing in the etodolac-treated group, RAGE mRNA expression significantly decreased (p < 0.03). Conclusions: These results indicate that RAGE is involved in rat tongue carcinogenesis by 4-NQO and suggest that the chemopreventive effect exerted by etodolac is partly related to the inhibition of RAGE expression.

Cytoplasmic localization of BAG-1 in leukoplakia and carcinoma of the tongue: correlation with p53 and c-erbB2 in carcinoma

The present study evaluated the clinical significance of BAG-1, an antiapoptotic protein, in leukoplakia and carcinoma of the tongue. BAG-1 expression was evaluated by immunohistochemistry in paraffin-embedded tissues of leukoplakia (n=25) and carcinoma of the tongue (n=61). Cytoplasmic expression was predominantly seen in 80% and 70% of patients with leukoplakia and carcinoma, respectively. BAG-1 expression was found to be significantly lower in tobacco users than in non-tobacco users. BAG-1 expression in tobacco-using leukoplakia and carcinoma patients was compared by grouping the carcinoma patients according to lymph node status and disease stage. Carcinoma patients with tumor-positive lymph nodes had significantly lower BAG-1 expression than patients with negative lymph nodes and leukoplakia. Further, a trend towards an inverse correlation was observed with p53 and c-erbB2. In univariate and multivariate survival analysis, patient subgroups with 2+ or 3+ marker positivity (BAG-1 negativity, p53 and c-erbB2 positivity) had a reduced overall survival compared with patient subgroups with 1+ marker positivity or negativity. BAG-1 negativity in association with p53 and c-erbB2 positivity identified a subgroup of tongue cancer patients with an aggressive phenotype. Hence, an antiapoptotic protein, BAG-1, was found to be down-regulated in chewing-tobacco-mediated tongue carcinogenesis.

Possible enhancing activity of diacylglycerol on 4-nitroquinoline 1-oxide induced carcinogenesis of the tongue in human c-Ha- ras proto-oncogene transgenic rats

1,2-Diacylglycerol (1,2-DAG) is involved in cell proliferation as an activator of protein kinase C (PKC) and has been shown to stimulate growth of cancer cells, raising the possibility of a role in tumor promotion. Ingested DAG oil, containing 70% 1,3-DAG and 30% 1,2-DAG, is digested and considered to be safe as edible oil. However, DAG may directly contact with oral cavity mucosa in undigested form. The present study was conducted to examine the effects of DAG oil on carcinogenesis in c-Ha- ras proto-oncogene transgenic (Tg) rats administered 4-nitroquinoline 1-oxide (4NQO, 10 ppm) in their drinking water for 10 weeks for initiation of mainly upper digestive organs. DAG oil added in basal diet at 5.5%, 2.75%, 1.38% and 0% with total fat made up to 5.5% with triacylglycerol (TAG) was administered during the initiation and post-initiation period. The study was terminated at week 12 (Tg females) and 20 (Tg males, wild females and males). The fatty acid composition of DAG oil was similar to TAG (linoleic acid 46.6% and oleic acid 38.9%). In Tg male rats, DAG oil administration was associated with significant increase ( P < 0.05) in the incidence of squamous cell carcinomas (SCC) of the tongue (5.5% DAG, 43.8%; 2.75% DAG, 20%; 1.38% DAG, 14.3%; 0%, 12.3%) with the Cochran–Armitage trend test and also number of tumors in coefficients for linear contrast trend tests. Tongue SCC induction of wild males and all females was not significant. The present results suggest that DAG oil may have enhancing and/or promotion potential for tongue carcinogenesis in male Tg featuring elevated ras expression.

Frequent mutation of p16CDKN2A exon 1 during rat tongue carcinogenesis induced by 4‐nitroquinoline‐1‐oxide

In this study we explored the mutation types of p16(CDKN2A) exon 1 and the corresponding frequencies in experimental rat tongue carcinogenesis. Twenty barrier Sprague-Dawley (SD) rats were divided into the control (n = 5) and experimental group (n = 15), to which 4-nitroquinoline-1-oxide (4-NQO) in drinking water was administered. Two samples of normal, three samples of moderate/severe dysplasia and four samples of invasive squamous cell carcinoma lesions were selected following strict histopathological examination in double-blind manner. The PCR products of p16(CDKN2A) exon 1 amplified from these tissues were sequenced. Point mutations of p16(CDKN2A) exon 1 were found in all of the precancerous and cancerous lesions. Half of the mutations were detected on guanine (G). Twenty mutations, including a missense mutation of the start codon resulting in alternative reading frame of p16(CDKN2A) exon 1, were also identified. These preliminary results suggested that mutation of p16(CDKN2A) exon 1 might be an early molecular event of rat tongue carcinogenesis induced by 4NQO and G was the mutation hotspot.

Medium-term tongue carcinogenesis assays: A comparative study between 4-nitroquinoline 1-oxide (4NQO)-induced rat and dimethylbenzanthracene (DMBA)-induced hamster carcinogenesis

The most used animal models in oral cancer research are the hamster treated by dimethylbenzanthracene (DMBA), and the rat treated by 4-nitroquinoline 1-oxide (4NQO). The purpose of this study was to compare the DMBA-induced hamster tongue carcinogenesis and 4NQO-induced rat tongue carcinogenesis by means of morphological analysis. Male Wistar rats were distributed into three groups of ten animals each and treated with 50 ppm 4NQO solution by drinking water for 4, 12 or 20 weeks. A total of 18 Syrian golden hamsters were submitted to 0.5% DMBA (dissolved in acetone) topical application three times/week for 4, 12 and 20 weeks. The primary histopathological change i.e., hyperplasia and hyperkeratosis, was evidenced after 4 weeks treatment with DMBA. Regarding 12 weeks treatment, 4NQO and DMBA were able to induce morphological changes as depicted by hyperplasia and dysplasia. At 20 weeks, squamous cell carcinoma was found in the majority of animals for both carcinogens used. Taken together, our results suggest that the hamster experimental model disclosed aspects related with tongue carcinogenesis in lesser time than rats. Probably, such discrepancies depend strongly on route of administration and the susceptibility with respect to animal species.

Anticarcinogenesis effect of Gynura procumbens (Lour) Merr on tongue carcinogenesis in 4NQO-induced rat

In Indonesia Gynura procumbens (Lour) Merr leaves have been long used as various cancers medication. Many in vitro and in vivo studies have demonstrated anticarcinogenesis of ethanol extract of Gynura procumbens leaves. The aim of this study was to investigate the anticarcinogenesis of the ethanol extract of Gynura procumbens leaves on 4 nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinogenesis. Fifty six 4 week old male Sprague Dawley rats were used in this study and divided into 7 groups. Group 1, 2 and 3 were lingually induced by 4NQO for 8 weeks. In groups 2 and 3 the extract was given simultaneously with or after 4NQO induction finished, each for 10 weeks and 26 weeks, respectively. Groups 4, 5 and 6 were induced by 4NQO for 16 weeks. However, in groups 5 and 6 the extract was given as well simultaneously with or after the 4NQO induction, each for 18 weeks, respectively. Group 7 served as the as untreated control group. The results from microscopical assessment showed that tongue squamous cell carcinomas (SCC) developed in 100% (3/3) of group 1. However, only 33.3% (2/6) and 25% (2/8) of rats in groups 2 and 3, respectively demonstrated tongue SCC. Among groups 4, 5 and 6, no significant difference of tongue SCC incidence was observed. From these results it is apparent that the ethanol extract of Gynura procumbens leaves could inhibit the progression of 4NQOinduced rat tongue carcinogenesis in the initiation phase.

A prostaglandin E2 receptor subtype EP1-selective antagonist, ONO-8711, suppresses 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis

We previously reported that certain cyclooxygenase (COX) inhibitors could inhibit chemically induced tongue carcinogenesis. In the present study, we investigated the effects of prostaglandin E(2) (PGE(2)) receptor EP(1)-selective antagonist ONO-8711 on 4-nitroquinoline 1-oxide (4-NQO)-induced oral carcinogenesis to know whether an EP(1) receptor involves in oral carcinogenesis. Male Fischer 344 rats were given drinking water containing 4-NQO for 8 weeks (20 p.p.m. for the initial 2 weeks, 25 p.p.m. for 2 weeks, and then 30 p.p.m. for 4 weeks). After 4-NQO treatment, animals were given 400 or 800 p.p.m. ONO-8711 containing diets for 23 weeks. The incidence of tongue squamous cell carcinomas (SCC) in the 4-NQO-treated rats was 64%, while that in the rats given ONO-8711 after 4-NQO exposure was 29 (P<0.05) and 29% (P<0.05) in the 400 and 800 p.p.m. of ONO-8711, respectively. The multiplicity of tongue cancer was also smaller in the 4-NQO + ONO-8711 (400 p.p.m. ONO-8711, 0.35 +/- 0.61; and 800 p.p.m. ONO-8711, 0.29 +/- 0.47; P<0.05), when compared with the 4-NQO alone group (0.88 +/- 0.88). Feeding with ONO-8711 significantly reduced PGE(2) level and cell proliferation activity in the non-tumorous epithelium of the tongue. Also, treatment with ONO-8711 resulted in the decrease in EP(1) immunohistochemical expression in the tongue lesions induced by 4-NQO. The results suggest that EP(1) receptor involves in oral carcinogenesis, and that an EP1-selective antagonist ONO-8711 exerts the cancer chemopreventive effects through the suppression of EP(1) expression, PGE(2) biosynthesis and cell proliferation.

Activation of ERK1/2 and cyclin D1 expression in oral tongue squamous cell carcinomas: Relationship between clinicopathological appearances and cell proliferation

We report an immunohistochemical investigation of the expression of activated extracellular signal-regulated kinase (ERK1/2) and cyclin D1 protein in both oral tongue squamous cell carcinomas (OTSCCs) and normal tongue epithelium. The expression of Ki-67 labeling index (LI) was also examined in order to evaluate cell proliferation activity. The expression of activated ERK1/2, cyclin D1 protein and Ki-67 LI were significantly stronger in OTSCCs than in normal oral mucosa (P<0.05). Both over-expression of activated ERK1/2 and positive expression of Ki-67 in OTSCCs were significantly associated with a moderately or poorly differentiated grade of carcinoma (P<0.05). Cyclin D1 immunostaining showed statistically significant association with both lymph node metastasis (P<0.05) and a tumor thickness >5mm (P<0.05). Over-expression of activated ERK1/2 was positively correlated with cyclin D1 protein expression (P<0.05, r=0.624) and cell proliferation-related indexes Ki-67 (P<0.05, r=0.723). Our results suggest that over-expression of activated ERK1/2 and cyclin D1 protein are involved in oral tongue carcinogenesis, and that activation of ERK1/2 might be related to cell cycle regulation and cell proliferation in OTSCCs.

Abnormal expression of bcl‐2 and bax in rat tongue mucosa during the development of squamous cell carcinoma induced by 4‐nitroquinoline 1‐oxide

4-Nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinogenesis is a useful model for studying oral squamous cell carcinoma. The aim of this study was to investigate the expression of bcl-2 and bax during tongue carcinogenesis induced by 4NQO. Male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution through their drinking water for 4, 12 or 20 weeks. Ten animals were used as negative control. Although no histological changes were induced in the epithelium after 4 weeks of carcinogen exposure, bcl-2 and bax were over-expressed (P < 0.01) in all layers of the 'normal' epithelium. The expression levels were the same in all layers of epithelium for both the antibodies used (bcl-2 or bax). In dysplastic lesions at 12 weeks following carcinogen administration, the levels of bcl-2 and bax expression did not increase when compared to negative control with the immunoreactivity for bcl-2 being restricted to the superficial layer of epithelium. In well-differentiated squamous cell carcinoma induced after 20 weeks of treatment with 4NQO, bcl-2 was expressed in some cells of tumour islands. On the other hand, immunostaining for bax was widely observed at the tumour nests. The labelling index for bcl-2 and bax showed an increase (P < 0.05) after only 4 weeks of 4NQO administration. In conclusion, our results suggest that abnormalities in the apoptosis pathways are associated with the development of persistent clones of mutated-epithelial cells in the oral mucosa. Bcl-2 and bax expression appears to be associated with a risk factor in the progression of oral cancer.

An animal model for the rapid induction of tongue neoplasms in human c-Ha- ras proto-oncogene transgenic rats by 4-nitroquinoline 1-oxide: its potential use for preclinical chemoprevention studies

Oral squamous cell carcinoma is one of the most common human neoplasms, and prevention of this malignancy requires a better understanding of its carcinogenesis process. To this end, we tried to establish an animal model using the human c-Ha-ras proto-oncogene-carrying transgenic (Tg) rats and the carcinogen 4-nitroquinoline 1-oxide (4-NQO). 4-NQO (20 p.p.m.) was administered to Tg and non-Tg rats for 8 weeks in their drinking water, and then the occurrence of tongue carcinogenesis was compared during the experimental period of 22 weeks. In addition, we determined the DNA ploidy in tongue lesions and examined the immunohistochemical expression of five biomarkers such as cyclin D1, glutathione S-transferase placental form, cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) and beta-catenin. Next, the cancer chemopreventive effects of nimesulide, pioglitazone and a synthetic geranylated derivative, which have been reported to be inhibitors of tongue carcinogenesis, were examined in Tg rats treated with 4-NQO. Either during or after treatment with 4-NQO in the drinking water, tongue dysplasia and tumors were observed on the tongues of both Tg and non-Tg rats, with a greater incidence and multiplicity in Tg rats. Histopathologically, squamous cell dysplasia, papilloma and carcinoma with or without invasion were present in the tongue. Immunohistochemistry revealed that expression levels against five biomarkers increase with disease progression, and the changes correlated with those of the DNA ploidy pattern. Interestingly, a strong expression of COX-2, iNOS and beta-catenin was observed on the invasive front of squamous cell carcinomas. A subsequent chemoprevention study using Tg rats showed that the chemicals tested suppressed the occurrence of tongue carcinomas when they were administered after 4-NQO-exposure. These results may thus indicate that our 4-NQO-induced Tg rat tongue carcinogenesis model simulates many aspects of human oral carcinogenesis and it can be applied for an analysis of oral cancer development while also helping to identify potentially effective cancer chemopreventive agents against oral cancer.

A speed congenic rat strain bearing the tongue cancer susceptibility locus Tscc1 from Dark-Agouti rats

We previously reported that Dark-Agouti (DA) rats are highly susceptible to 4-nitroquinoline 1-oxide (4NQO)-induced tongue cancer (TC), whereas Wistar/Furth (WF) rats are barely susceptible. Linkage analysis of reciprocal (DA×WF)F2 rats demonstrated five quantitative trait loci, Tongue squamous cell carcinoma 1–5 ( Tscc1–5) determining the size and number of the TCs. The major susceptibility locus Tscc1 is mapped on rat chromosome 19. In the present study, we used a marker-assisted speed congenic procedure to construct WF.DA- Tscc1 (WF-T1D) rats, i.e. WF rats carrying a DA-derived Tscc1 chromosomal segment, and evaluated the effect of a single Tscc1 on 4NQO-induced tongue carcinogenesis. In WF-T1D rats, the incidence, number and size of 4NQO-induced TCs were significantly higher than those in WF rats, indicating that the introgressed segment contains one of the susceptibility loci for 4NQO-induced TCs from DA rats. Detection of a single nucleotide polymorphism in NQO1, one of the Tscc1 candidate genes, enabled us to map NQO1 in the Tscc1 segment between D19Wox8 and D19Wox7 on chromosome 19. Possible relevance of NQO1 polymorphism to TC susceptibility is discussed.

Aberrant expression of RAB1A in human tongue cancer

This study was designed to identify specific gene expression changes in tongue squamous cell carcinomas (TSCCs) compared with normal tissues using in-house cDNA microarray that comprised of 2304 full-length cDNAs from a cDNA library prepared from normal oral tissues, primary oral cancers, and oral cancer cell lines. The genes identified by our microarray system were further analysed at the mRNA or protein expression level in a series of clinical samples by real-time quantitative reverse transcriptase–polymerase chain reaction (qRT–PCR) analysis and imuunohositochemistry. The microarray analysis identified a total of 16 genes that were significantly upregulated in common among four TSCC specimens. Consistent with the results of the microarray, increased mRNA levels of selected genes with known molecular functions were found in the four TSCCs. Among genes identified, Rab1a, a member of the Ras oncogene family, was further analysed for its protein expression in 54 TSCCs and 13 premalignant lesions. We found a high prevalence of Rab1A-overexpression not only in TSCCs (98%) but also in premalignant lesions (93%). Thus, our results suggest that rapid characterisation of the target gene(s) for TSCCs can be accomplished using our in-house cDNA microarray analysis combined with the qRT–PCR and immunohistochemistry, and that the Rab1A is a potential biomarker of tongue carcinogenesis.

Genetic Predisposition to 4NQO-Induced Tongue Carcinogenesis in the Rat

Objective: This study aims to elucidate the genetic basis of predisposition to 4-nitroquinoline 1-oxide (4NQO)-induced tongue cancers (TCs). Materials and Methods: We have reported that inbred Dark-Agouti (DA) strain rats were highly susceptible to 4NQO-induced TCs, whereas Wistar/Furth (WF) rats were resistant to tongue squamous cell carcinomas induced by oral administration of 4NQO. Using size and number of the tumours as quantitative parameters, responsible host loci were analysed by an interval mapping of F₂ intercross of DA and WF given carcinogenic regimen. Also, loss of heterozygosity (LOH) at these loci was analysed in tongue cancers in (DA × WF) F₁. Results: We identified and mapped 5 significant quantitative trait loci (QTL), the Tongue squamous cell carcinoma 1–5 (Tscc1–5), and several other suggestive QTL that determine susceptibility to 4NQO-induced TC. Study of TCs induced in (DA × WF)F₁ rats revealed a high frequency of LOH in the chromosomal regions of Tscc2, 3, and 4 and also of suggestive QTL on chromosomes 5 and 6. The fact that LOH was found only in larger TCs indicates that LOH occurred in the process of tumour progression. In most LOH, the allele of the resistant WF strain was lost, suggesting that these loci may encode tumour suppressor genes. In larger TCs, in addition to LOH, point mutations and the methylation of possible candidate genes were accumulated. Conclusion: These observations indicate that the 4NQO-induced TC in the rat is a multifactorial disease of a polygenic trait. This model will be useful to understand the complicated genetic basis of predisposition to oral cancers.