Strain difference of susceptibility to 4-nitroquinoline 1-oxide-induced tongue carcinoma in rats.

Strain difference of susceptibility to 4-nitroquinoline 1-oxide (4NQO)-induced squamous cell carcinomas of the tongue among Dark-Agouti, Long-Evans, Sprague-Dawley, ACI/Ms, Fischer 344, Donryu and Wistar/Furth rats was surveyed by evaluating the survival times, incidences and sizes of developed tumors as markers of susceptibility. Administration of 4NQO dissolved in drinking water induced squamous cell carcinomas in various sites of the upper digestive tract mucosa of all the experimental male and female rats of the seven strains. Regarding the mean survival times, Wistar/Furth rats survived much longer than any other strain of rats, and Dark-Agouti showed the shortest survival. The incidence of large, mass-type carcinomas of the tongue of Dark-Agouti rats was higher than in any other strain of rats, while that of Wistar/Furth rats was the lowest. Subsequently the mitotic activity and bromodeoxyuridine incorporation in the tongue epithelium of Dark-Agouti and Wistar/Furth rats were estimated after a short-term administration of 4NQO. There was a pronounced difference between the two strains of rats, because the proliferative responses of the tongue epithelium of Dark-Agouti rats to the 4NQO stimulation were much higher than those of Wistar/Furth rats. These results indicated that there are marked differences in the susceptibility to 4NQO-induced tongue carcinoma among the seven strains of rats, and that Dark-Agouti and Wistar/Furth rats could be useful as models of highly and poorly susceptible strains, respectively, for further genetic analysis.     

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 (DAxWF)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.     

Selective loss of resistant alleles at p15INK4B and p16INK4A genes in chemically-induced rat tongue cancers

We previously reported that susceptibility to 4-nitroquinoline 1-oxide (4NQO)-induced tongue cancer in Dark-Agouti (DA) and Wistar/Furth (WF) rats was determined by a number of quantitative trait loci. In this article, we further scrutinized one of the quantitative trait loci at a suggestive level on rat chromosome 5. Analyzing a DNA panel of 130 (DAxWF) F2 rats treated with 4NQO showed a quantitative trait loci, containing p15INK4B and p16INK4A. To study the possible relevance of these genes in the development of tongue cancer, we examined 45 4NQO-induced tongue cancers in 100 (DAxWF) F1 rats for loss of heterozygosity. The incidence of loss of heterozygosity at p15INK4B and p16INK4A genes in large advanced tongue cancers was 37.8% and 40.0%, respectively, and the WF allele was selectively lost. Accumulation of loss of heterozygosity and methylation of the promoter regions in the tumour suppressor genes in advanced tumours suggests that they may play a role in tongue cancer progression.     

Five Quantitative Trait Loci Affecting 4-Nitroquinoline 1-Oxide-induced Tongue Cancer in the Rat

In our previous study, Dark-Agouti (DA) rats were found to be highly susceptible to 4-nitroquino-line 1-oxide (4NQO)-induced tongue carcinoma (TC), whereas Wistar/Furth (WF) rats were barely susceptible. Interval mapping analysis of reciprocal backcross rats showed two quantitative trait loci (QTL) on rat chromosomes (RNO) 1 and 19. In the present study, a composite interval mapping analysis was applied to 4NQO-induced TC in 130 (DAxWF) F2 rats, demonstrating five independent QTL, Tongue squamous cell carcinoma 1-5 (Tscc1-5 ), responsible for phenotypic differences in the size and number of TCs in the two strains. Two of these QTL were mapped on RNO1, and the others were mapped on RNO4, 14, and 19. The DA allele at these loci consistently yielded semidominant susceptibility to TC. Out of the five loci detected in this F2 generation, Tscc1 and 2 were identical to Stcl and Rtc1 described in our previous study, but the other three were novel. We propose a new nomenclature consistent with their function. Genome-wide screening of the F2 progeny also suggested the presence of three additional QTL on RNO5, 6, and 10. The possible roles of these loci in tongue carcinogenesis are discussed.     

Five quantitative trait loci affecting 4-nitroquinoline 1-oxide-induced tongue cancer in the rat.

In our previous study, Dark-Agouti (DA) rats were found to be highly susceptible to 4-nitroquinoline 1-oxide (4NQO)-induced tongue carcinoma (TC), whereas Wistar / Furth (WF) rats were barely susceptible. Interval mapping analysis of reciprocal backcross rats showed two quantitative trait loci (QTL) on rat chromosomes (RNO) 1 and 19. In the present study, a composite interval mapping analysis was applied to 4NQO-induced TC in 130 (DA x WF) F2 rats, demonstrating five independent QTL, Tongue squamous cell carcinoma 1 - 5 (Tscc1 - 5), responsible for phenotypic differences in the size and number of TCs in the two strains. Two of these QTL were mapped on RNO1, and the others were mapped on RNO4, 14, and 19. The DA allele at these loci consistently yielded semidominant susceptibility to TC. Out of the five loci detected in this F2 generation, Tscc1 and 2 were identical to Stc1 and Rtc1 described in our previous study, but the other three were novel. We propose a new nomenclature consistent with their function. Genome-wide screening of the F2 progeny also suggested the presence of three additional QTL on RNO5, 6, and 10. The possible roles of these loci in tongue carcinogenesis are discussed.     

Genetic Controls of Susceptibility and Resistance to 4-Nitroquinoline 1-Oxide-induced Tongue Carcinomas in Rats

We analyzed the incidence of infiltrative mass-type tongue carcinomas (IMTC) induced in 550 rats by continuous oral administration of 0.001% 4-nitroquinoline 1-oxide solution for 180 days. The study included various crosses of susceptible Dark-Agouti rats (DA) and resistant Wistar/Furth rats (WF). DA showed a 93.6% incidence of IMTC measuring more than 5 mm in their largest diameter, while WF showed only a 4% incidence. Reciprocal F₁ and F₂ hybrids mated by DA and WF showed 47.5% and 45.8% incidences, respectively. Meanwhile, reciprocal backcrossed hybrids to DA and WF showed 73.7%, and 24.6% incidences, respectively. Segregation of the incidences suggests that there are two autosomal dominant genes, one linked to the susceptibility of DA and the other to the resistance of WF.     

Loss of epithelial cell surface carbohydrates during experimental oral carcinogenesis in the rat

Cell surface glycoconjugates were investigated in a rat model of oral chemical carcinogenesis. The lectins Griffonia simplicifolia (GS-I-B4; specific for alpha-D-galactosyl end groups) and Ulex europeus (UEA-I; specific for alpha-L-fucosyl groups) were examined microspectrofluorimetrically in the oral epithelium of rats painted with the carcinogen 4-nitroquinoline N-oxide (4NQO) and compared with those treated with solvent alone. After labelling with GS-I-B4, the fluorescent intensity of the basal and parabasal epithelial cells was significantly less after 9 months of 4NQO treatment and in overt squamous cell carcinomas compared to controls. The fluorescent activity of the spinous epithelial cells in the non-invasive tissues treated with 4NQO and in the well differentiated (sites of keratin elaboration) malignant epithelium of squamous cell carcinomas was unchanged after labelling with UEA-I. UEA-I failed to stain undifferentiated (areas lacking keratin) malignant epithelium. The findings indicate that alpha-D-galactosyl residues are diminished on the membranes of premalignant and malignant rat epithelial cells. The expression of alpha-L-fucosyl groups, however, remains unchanged in premalignant rat oral epithelium and is closely correlated to the presence of keratin in the malignant epithelium of squamous cell carcinomas.     

Epithelial dendritic cells and connective tissue macrophages in oral carcinogenesis and the effects of systemic Corynebacterium parvum

Epithelial dendritic cells (EDC) and connective tissue macrophages were examined during the induction and growth of oral squamous cell carcinomas in Sprague-Dawley rats treated with the carcinogen 4-nitroquinoline N-oxide (4NQO) and the immune potentiator Corynebacterium parvum. Splenomegaly was induced in all animals receiving C. parvum. Acetone-fixed frozen sections of the palate and tongue were stained using an indirect immunoperoxidase technique and monoclonal antibodies to rat Ia (MRC OX-6) and macrophage subpopulations (ED1, ED2, ED3). EDC were predominantly Ia+, ED1-, ED2- and ED3-. The lamina propria contained Ia+, ED1+ and ED2+ cells; ED3-reactive cells were rare. ED2+ cells predominated in the interstitial connective tissue of deeper muscle. In the non-invasive tissues, the number of positive cells (Ia+EDC and connective tissue Ia+, ED1+ and ED3+ cells) increased significantly throughout the experimental period (0-9 months), were significantly more prevalent in the test tissues (4NQO, 4NQO + C.parvum, C.parvum) compared to untreated controls and, at 9 months, the carcinogen-treated rats (4NQO, 4NQO + C.parvum) had significantly more Ia+ EDC and connective tissue Ia+ cells than C.parvum controls. Irrespective of the marker under study, there were no significant differences between rats treated with 4NQO or 4NQO + C.parvum at any time during the experimental period. Similarly, intra-epithelial Ia+ and ED1+ cells increased significantly throughout the experimental period in all test groups compared to untreated controls, but no significant differences were evident between carcinogen-treated animals (4NQO, 4NQO + C.parvum) and C.parvum controls. Significant positive correlations between connective tissue Ia+ and ED1+ cells and also intra-epithelial Ia+ and ED1+ cells were present in all experimental groups; connective tissue ED2+ and ED3+ cell numbers did not correlate with any of the other phenotypes and intra-epithelial ED2+ and ED3+ cells were rare/absent. Palatal and/or lingual tumours developed in 80% of carcinogen-treated rats by 9 months and the tumour incidence was similar in rats treated with either 4NQO or 4NQO + C.parvum. There were no significant differences in the number of Ia+ EDC between the infiltrating and the non-invasive overlying epithelium of the lingual carcinomas and the non-invasive lingual epithelium treated with either 4NQO or 4NQO + C.parvum.(ABSTRACT TRUNCATED AT 400 WORDS)     

Xeroderma pigmentosum group A gene action as a protection factor against 4-nitroquinoline 1-oxide-induced tongue carcinogenesis

To test the hypothesis that nucleotide excision repair (NER) plays a protective role in chemical carcinogenesis in internal organs, xeroderma pigmentosum group A gene-deficient (XPA(-/-)) mice, heterozygous (XPA(+/-)) and wild-type (XPA(+/+)) mice were orally administered 0.001% 4-nitroquinoline 1-oxide (4NQO) in their drinking water and compared. After 50 weeks of 4NQO exposure, tongue squamous cell carcinomas (SCCs) occurred in XPA(-/-) mice only, no tumors being observed in XPA(+/-) and XPA(+/+) animals. Of the XPA(-/-) mice 86% had tumors and 100% demonstrated multiple foci of dysplastic epithelium in the tongue. Accumulation of p53 protein was immunohistochemically detected in 56% of the SCCs. Mutational analysis of the p53 gene (exons 4-10) in carcinoma DNA revealed missense mutations in exons 5 and 9 in four of 20 samples. Our results clearly demonstrate that the NER gene XPA acts as a defensive factor against 4NQO-induced tongue carcinogenesis in vivo.     

Comparison of 7,12-dimethylbenz[a]anthracene metabolism and DNA binding in mammary epithelial cells from three rat strains with differing susceptibilities to mammary carcinogenesis

The capacity of polycyclic aromatic hydrocarbons such as 7,12-dimethylbenz[a]anthracene(DMBA) to induce mammary carcinomas has been studied in threerat strains. Wistar/Furth (WF) rats are highly susceptible toDMBA-induced mammary carcinogenesis, Copenhagen (Cop) rats arecompletely resistant, and Fischer 344 (F344) rats have an intermediatesusceptibility. We have previously shown that WF rats possess‘enhancer genes’, which enhance susceptibility toinduced mammary cancer. Cop rats, however, possess a single‘suppressor’ gene which confers complete resistanceto mammary cancer. Both gene types are apparently absent inF344 rats. In order to determine possible mechanisms of actionof these enhancer and suppressor genes, we have examined DMBAmetabolism and DNA binding in mammary epithelial cells isolatedfrom each rat strain. Quantitative analyses of both metabolismand DNA binding indicate no significant differences among thestrains. In addition, HPLC analyses of DMBA metabolites andDMBA-DNA adducts were essentially identical. These data suggestthat the genes controlling susceptibility and resistance tomammary carcinogenesis in these rat strains are likely to beactive at later stages of the carcinogenic process.     

Candida albicans as a promoter of oral mucosal neoplasia.

A model of oral mucosal carcinogenesis using the water-soluble carcinogen 4-nitroquinoline-1-oxide (4NQO) was combined with a model of oral mucosal candidosis to examine the ability of Candida albicans to promote the development of neoplasia in suitably initiated epithelium. Sprague-Dawley rats were initiated by the application of 4NQO to their palatal and tongue mucosa 3 times weekly for 4 weeks. The animals then received either application of a phorbol ester to act as a promoter, induction of experimental oral mucosal infection with C. albicans, or no further procedures. Animals were killed at 34 or 52 weeks and the tongues and palates sectioned for light-microscopic examination. Control groups with no treatment, mucosal infection only, phorbol ester application only, 4NQO with the tetracycline or vehicle application only were also used. The development of carcinoma in the experimental groups was similar to that in the positive control groups, indicating that the particular strain of Candida used had a similar ability to promote neoplastic changes as the known promoter phorbol-12,13-didecanoate and caused neoplastic changes to occur by week 34 with no additional lesions occurring by week 52. This indicated that the speculation that strains of C. albicans may participate in causing neoplastic transformation in humans was well founded.     

Zinc supplementation suppresses 4-nitroquinoline 1-oxide-induced rat oral carcinogenesis

Dietary zinc (Zn) deficiency is implicated in the pathogenesis of human oral-esophageal cancers. In rats, Zn deficiency causes increased cell proliferation and cyclooxygenase-2 (COX-2) overexpression and enhances oral carcinogenesis by 4-nitroquinoline 1-oxide (NQO). Zn replenishment reverses all these effects. We questioned whether Zn has antitumor efficacy in a Zn-sufficient animal by investigating in Zn-sufficient rats (i) the efficacy of Zn supplementation on the progression of tongue squamous cell carcinogenesis induced by drinking water exposure to high (20-30 p.p.m.) and low (10 p.p.m.) doses of NQO and (ii) the modulating effects of Zn supplementation on biomarker expression in tongue lesions by immunohistochemistry. In rats exposed to high doses of NQO, Zn supplementation significantly reduced the incidence of papillomas from 100 to 64.7% (P=0.018) and invasive carcinomas from 93.8 to 52.9% (P=0.017). In rats exposed to low doses of NQO, where only minimally invasive carcinomas developed, Zn supplementation significantly reduced tumor multiplicity, incidence of tumors (1-2 mm), hyperplasia, dysplasia, papillomas and progression to carcinoma. Immunohistochemical analysis of carcinomas showed that Zn supplementation caused a shift to a less proliferative/aggressive cancer phenotype by reducing cell proliferation, stimulating apoptosis and decreasing expression of the key tumor markers cyclin D1, p53 and COX-2. Additionally, Zn supplementation significantly reduced cell proliferation in non-lesional tongue squamous epithelia, thereby suppressing tumor development. Together, the results demonstrate that Zn supplementation has chemopreventive efficacy against oral carcinogenesis in nutritionally complete animals. Our data suggest that Zn supplementation may be efficacious in the chemoprevention of human oral cancer.     

Histomorphologic and morphometric changes in minor salivary glands of the rat tongue during 4-nitroquinoline-1-oxide-induced carcinogenesis

4-Nitroqinoline-1-Oxide (4NQO)-induced tongue carcinogenesis in rats is considered to be a preferred model for study of oral squamous cell carcinoma. Aim of study was to investigate histomorphologic and morphometric 4NQO-induced changes in tongue minor salivary glands. Histopathological examinations of serous and mucous acini and ducts of tongue salivary glands of 26 Wistar-derived rats were performed after 14 (T(1)), 22 (T(2)) and 28 (T(3)) weeks of 0.001% 4NQO administration in drinking water and compared with nine controls. Histomorphological findings were recorded as normal/abnormal acini and as normal/dysplastic ducts. Morphometrical results were expressed as volume fraction (Vv%) of each of the components. Morphometric and histomorphologic changes in the salivary glands were evident only at T(3) and they included a significant (P=0.008) decrease in the Vv of the serous acini compared with the control group accompanied by abnormal acini (Vv=18%). In contrast, mucous acini and ducts did not demonstrate significant changes. In one case (3.8%), dysplastic ducts were found adjacent to islands of invasive squamous cell carcinoma of tongue mucosa origin. The change in saliva composition expected after considerable damage of the serous glands could create a microenvironment that makes entrapment of the carcinogen easier and prolongs exposure of tongue epithelium. Furthermore, the dysplastic changes in the ducts can serve as a reservoir of carcinoma cells. These observations should be considered in human patients diagnosed with oral dysplasia or carcinoma, especially involving the tongue and floor of mouth.     

Genomic instability in non-neoplastic oral mucosa cells can predict risk during 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis

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 level of DNA damage induced by 4NQO in oral mucosa cells by the single cell gel (comet) assay. 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. Statistically significant increase of DNA damage was observed in non-neoplastic oral cells at four weeks of 4NQO administration when compared with control (P < 0.05). The level of DNA damage was directly associated with the severity of histological changes. The results suggest that histologically normal tissue is able to harbor genetically unstable cells contributing to the initiation of oral carcinogenesis. Genomic instability appears to be associated with the risk and progression of oral cancer.     

Nasal tumours in rats after short-term exposure to a cytotoxic concentration of formaldehyde

Male Wistar rats were exposed to 0, 10 or 20 ppm formaldehyde vapour for 4, 8 or 13 weeks (6 h/day; 5 days/week), and were then observed for periods up to 126 weeks. Transient growth retardation occurred in both test groups. Death rate was not noticeably affected by formaldehyde. Despite recovery periods of at most 126 weeks, the nasal respiratory and olfactory epithelium of many rats of the 20 ppm group exhibited non-neoplastic histopathological changes. Similar but much less severe changes of the respiratory epithelium were seen in a small number of rats of the 10 ppm group; the olfactory epithelium was not visibly affected in rats of this group. Nasal tumours considered to be induced by formaldehyde were seen only in the 20 ppm group and mainly in rats that had been exposed for 13 weeks, the incidence being 4.5% (6/132). These tumours comprised 3 squamous cell carcinomas, 1 carcinoma in situ and 2 polypoid adenomas, all originating from respiratory epithelium. It was concluded that rat nasal respiratory epithelium severely damaged by formaldehyde vapour often does not regenerate and in some cases develops tumours.     

Strain Differences in N-Butyl-N-(4-hydroxybutyl)nitrosamine Bladder Carcinogenesis in Rats

Differences in susceptibility of the urinary bladder epithelium to N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) in various strains were examined. In experiment 1, 5 strains of male rats were given 0.025% BBN in the drinking water for 8 weeks followed by drinking water without BBN for 32 weeks. Analbuminemic rats (NAR) and ACI rats had high incidences of urinary bladder lesions (papillary or nodular hyperplasia, papilloma and carcinoma), F344 and Wistar rats had low incidences, and Sprague-Dawley (SD) rats showed an intermediate incidence. Carcinoma area was largest in NAR rats followed in decreasing order by SD, ACI and F344 rats. The extent of tumor invasion was higher in NAR and ACI rats than in SD rats. In experiment 2, the 5 strains of male rats were administered 0.025% BBN in the drinking water. Some rats from each group were killed after each of weeks 4 and 8. The urinary bladder of ACI and NAR rats given BBN had the most marked lesions observed by scanning electron microscopy, with less marked changes in SD rats. F344 and Wistar rats showed the weakest response. Cytochrome P-450 content of the liver in ACI rats treated with BBN for 4 weeks was significantly higher than those of the controls. Cytochrome P-450 and Cytochrome b ₅ contents of the control and BBN-treated rats were significantly higher in ACI and SD rats than in Wistar, F344 or NAR rats. These results indicate that there are strain differences in the urinary bladder response to BBN.     

Cyclin D1 overexpression increases susceptibility to 4‐nitroquinoline‐1‐oxide‐induced dysplasia and neoplasia in murine squamous oral epithelium

The cyclin D1 oncogene is frequently amplified/overexpressed in oral squamous cell carcinomas. Mice with overexpression of cyclin D1 targeted to the stratified squamous epithelia of the tongue, esophagus, and forestomach develop a phenotype of epithelial dysplasia at these sites. In this study, we examined the effect of cyclin D1 overexpression on susceptibility of mice to carcinogen‐induced tumorigenesis, using 4‐nitroquinoline‐1‐oxide (4NQO), an established potent oral carcinogen in mice. Cyclin D1 overexpressing mice and nontransgenic littermates were administered 4NQO (20 or 50 parts per million (ppm) in the drinking water) for 8 wk and monitored for an additional 16 wk. Histopathological analyses of the tongue revealed significantly higher severity of dysplasia in the cyclin D1 overexpression mice, compared with nontransgenic controls and with untreated controls. Moreover, only the cyclin D1 overexpression mice developed neoplastic lesions in the oro‐esophageal epithelia. Examination of the dysplastic and neoplastic lesions revealed abnormal proliferation. Our findings suggest that cyclin D1 overexpression enhances susceptibility to carcinogen‐induced oral tumorigenesis. These results underscore the importance of cyclin D1 in the process of oral neoplastic development. Further, they emphasize the value of this transgenic model to study the pathogenesis of oral precancer and cancer and establish it as a model system to test candidate agents for chemoprevention of upper aero‐digestive cancer. © 2009 Wiley‐Liss, Inc.     

Parp-1 deficiency does not increase the frequency of tumors in the oral cavity and esophagus of ICR/129Sv mice by 4-nitroquinoline 1-oxide, a carcinogen producing bulky adducts

The impact of poly(ADP-ribose) polymerase-1 (Parp-1)-deficiency on 4-nitroquinoline 1-oxide (4NQO)-induced carcinogenesis was studied in mice with an ICR/129Sv mixed genetic background. Parp-1(+/+), Parp-1(+/-) and Parp-1(-/-) animals given 4NQO for thirty-two weeks at 0.001% in their drinking water developed papillomas and squamous cell carcinomas of the tongue, palate and esophagus, but with no statistically significant variation with the Parp-1 genotype. Thus Parp-1 deficiency does not elevate susceptibility to carcinogenesis induced by a carcinogen which gives rise to bulky DNA lesions. This study also indicated that the ICR/129Sv mixed genetic background is associated with high yield induction of esophageal tumors by 4NQO.     

K14‐EGFP‐miR‐31 transgenic mice have high susceptibility to chemical‐induced squamous cell tumorigenesis that is associating with Ku80 repression

Squamous cell carcinoma (SCC) occurring in the head and neck region and the esophagus causes tremendous cancer mortality around the world. miR‐31 is among the most eminently upregulated MicroRNAs in SCC, when it occurs in the head and neck region and the esophagus. We established miR‐31 transgenic mouse lines, in which miR‐31 is under the control of the K14 promoter. 4‐nitroquinoline 1‐oxide (4NQO) is a mutagen that causes double strand breaks. The transgenic mice exhibited a higher potential for tumor induction than wild‐type (Wt) mice of the tongue and esophagus after 4NQO treatment. After 4NQO treatment or irradiation, p‐γH2AX expression in squamous epithelium of transgenic mice was increased more than in Wt mice. Exogenous expression of miR‐31 was also found to be associated with the higher p‐γH2AX expression induced by 4NQO in human oral SCC (OSCC) cell lines. The repair genes PARP1 and Ku80 were validated as new targets of miR‐31 in human OSCC cell lines, and were found to be downregulated in the squamous epithelium of the tongue in transgenic mice. However, only the downregulation of Ku80 was essential for maintaining the high level of p‐γH2AX induced by 4NQO in OSCC cells. Inverse expression profiles for miR‐31 and Ku80 were noted in human OSCC tissue. Our study identifies the high sensitivity of K14‐EGFP‐miR‐31 transgenic mice to chemical carcinogen‐induced squamous cell tumorigenesis and shows that this seems to be associated with the downregulation of Ku80 and an impairment of repair activity in squamous cells, which are mediated by miR‐31.     

Characterization of malignant rat keratinocytes in culture following the induction of oral squamous cell carcinomas in vivo

An in vivo model of oral epithelial carcinogenesis in rats has been established successfully in cell culture. Oral carcinomas of the tongue and palate were induced in Sprague-Dawley male rats by painting their palates three times weekly for 7-8 months with 0.5% (w/v) 4-nitroquinoline-N-oxide. Oral keratinocytes from malignant and untreated control tissues were cultivated using 3T3 fibroblast support. Both the normal and malignant cells stained positively with an anti-human keratin polyclonal antibody but malignant keratinocytes were heterogeneous with regard to cell size, shape and intercellular packing, unlike the regular organization of the normal cultures. Malignant keratinocyte cultures differed markedly from their normal counterparts by an increase in their growth rate, the capacity for serial cultivation to the 25th passage (to date) and independence of 3T3 fibroblast support. In contrast, cultures established from healthy tissue showed signs of senescence usually by passage 4 and were totally reliant on 3T3 fibroblast support for growth. Malignant keratinocytes expressed anchorage independence when cultured in a semi-solid medium and gave rise to tumour formation in athymic mice. The development of this specialized cell culture system substantially increases the potential of the rat 4NQO model to investigate the pathogenesis of oral squamous cell carcinomas.