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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 × × × ×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. Key words: Genetic susceptibility -QTL -Tongue cancer -Rat -4-Nitroquinoline 1-oxideTongue carcinoma (TC) is one of the most frequent malignancies in the head and neck region. An increase in TC has been reported in the Western world over the past ten years. TC has a poor prognosis because therapeutic strategies provide limited effects.1-4) For effective prevention of TC, it would be necessary to identify the genetically predisposed risk group. However, virtually nothing is known about the genetic predisposition to TC.Recently, we found that the Dark-Agouti (DA) strain of rats had an extremely high susceptibility to 4-nitroquinoline 1-oxide (4NQO)-induced squamous cell carcinoma of the tongue. Virtually 100% of DA rats given drinking water containing 0.001% 4NQO developed one or more large TC within 180 days of administration, whereas the Wistar/Furth (WF) strain of rats was much less susceptible. [5][6][7][8] In the previous study with reciprocal backcrosses between DA and WF rats, 7,8) DA rats were shown to have a semidominant susceptibility locus, Stc1 (Susceptibility to TC-1) on rat chromosome (RNO) 19, and WF rats, another semidominant resistance gene Rtc1 (Resistance to TC-1) on RNO1.In this study, to scrutinize further the genetic predisposition to TC in the rat, we studied F2 progeny between DA and WF strains by means of a composite interval mapping analysis. After several statistical trials, we employed a quantitative parameter for tumor number, TC >3 mm in diameter, rather than TC >5 mm. In addition to two quantitative trait loci (QTL) described in the previous study, three novel QTL have been found to affect TC susceptibility. These five QTL explain almost all phenotypic variations between the two strains. MATERIALS AND METHODS Animals and phenotypeThe origin of DA rats is related to Copenhagen rats, although there is n...

For isolation of mouse mtDNA-less ( 0 ) cell lines, we searched for various antimitochondrial drugs that were expected to decrease the mtDNA content and found that treatment with ditercalinium, an antitumor bis-intercalating agent, was extremely effective for completely excluding mtDNA in all the mouse cell lines we tested. The resulting 0 mouse cells were successfully used for trapping the mtDNA of living nerve cells into dividing cultured cells by fusion of the 0 cells with mouse brain synaptosomes, which represent synaptic endings isolated from nerve cells. With neuronal mtDNA obtained, all of the cybrid clones restored mitochondrial translation activity similarly regardless of whether the mtDNA was derived from young or aged mice, thus at least suggesting that defects in mitochondrial genomes are not involved in the age-associated mitochondrial dysfunction observed in the brain of aged mice. Furthermore, we could trap a very small amount of a common 5823-base pair deletion mutant mtDNA (⌬mtDNA 5823 ) that was detectable by polymerase chain reaction in the cybrid clones. As the amount of mutant mtDNA with large scale deletions was expected to increase during prolonged cultivation of the cybrids, these cells should be available for establishment of mice containing the deletion mutant mtDNA.

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.

Rats of the DA strain are highly susceptible to 4NQO-induced TCs, whereas WF rats are barely susceptible. In (DA ؋ WF)F 2 rats, 5 QTL, Tscc1-5, are responsible for most of the phenotypic variations, though they do not account for all of the phenotypic differences between WF and DA rats. Analysis of 40 tongue tumors >5 mm in diameter from (DA ؋ WF)F 1 rats for LOH at the Tscc loci revealed a high frequency of LOH in chromosomal regions where the Tscc2, -3 and -4 loci map. In most cases of LOH, the allele of the barely susceptible WF strain was lost, suggesting that these loci in the WF strain encode tumor-suppressor genes. Analysis of the same tumors for somatic mutations in oncogenes indicated frequent alteration of Ha-ras, which maps in the Tscc3 region, but rare mutation of the p15 INK4B and p16 INK4Agenes or the p53 and Msh2 genes. Frequent LOH was also found on rat chromosomes 5 (RNO5) and 6 (RNO6 Squamous cell carcinoma of the oral cavity is the sixth most common solid tumor in the Western world. The overall survival rate of patients with this carcinoma has not improved significantly in the last decade, and the disease continues to be a serious health problem. 1,2 Rat TC induced by 4NQO is an experimental model for human TC since both tumors share several morphologic and molecular biologic properties. For instance, mutations in Ha-ras in rat or mouse TCs are as prevalent as those in human TCs. 3,4 Our recent studies have shown that susceptibility to 4NQO-induced rat tongue carcinogenesis is determined by several QTL, Tscc1-5. 5-7 DA strain rats are very susceptible to TCs, and DA-derived alleles at Tscc loci are the primary determinants of disease susceptibility. 8,9 The gene products encoded by the Tscc loci as well as the molecular mechanisms responsible for their allele-specific cancer-modifying activities are, for the most part, unknown. The difference in the Tscc alleles may rest in genetic polymorphisms, in either the regulating or the coding regions of the candidate genes. To test whether Tscc genes display either an oncogene or allele-specific tumor-suppressor-like activity on TC, we examined 4NQO-induced TCs in (DA ϫ WF)F 1 hybrid rats for LOH at these loci and for mutations at genes involved in cancer to characterize genetic alterations in TC and their possible relationship with the Tscc loci. MATERIAL AND METHODS AnimalsDA rats were purchased from the Shizuoka Laboratory Animal Center (Hamamatsu, Japan). The origin and history of these rats have been described. 10 WF rats were originally obtained from Hiroshima University (Hiroshima, Japan) and maintained by brother-sister matings for more than 90 generations in our laboratory. A total of 88 F 1 rats (40 females, 48 males) were derived by reciprocal mating between DA and WF rats. All rats were weaned at 35 days after birth, individually numbered and housed in plastic cages at 22°C in an air-conditioned room. Animals were fed on a commercial CE2 pellet diet (Nippon Clea, Tokyo, Japan) and given tap water ad libitum. No spontaneous tumors were obse...