Yoshikazu Hirayama

Rare mutations of p53, Ki-ras, and beta-catenin genes and absence of K-sam and c-erbB-2 amplification in N-methyl-N'-nitro-N-nitrosoguanidine-induced rat stomach cancers.

Rat stomach cancers induced by N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG) have been widely used as a model for human stomach cancers of the differentiated type. However, there has been little information regarding their molecular basis. In this study, we examined the genetic alterations reported in human stomach cancers in 10 rat stomach cancers that had been induced in male ACI/N rats by administering MNNG in the drinking water. One of the 10 cancers had a mutation of the p53 gene at the second position of codon 171 (Val→Glu). However, none of the 10 cancers had mutations in codons 12, 13, or 61 of Ki‐ras or in the N‐terminal phosphorylation sites of the β‐catenin gene. Southern blot analysis showed no amplification of K‐sam or c‐erbB‐2 in the seven cancers examined. Finally, we searched for microsatellite alterations in 12 loci in nine cancers, but no alterations were observed. As these genetic alterations are observed in only a minor fraction of human stomach cancers, further analysis of genetic and epigenetic alterations in MNNG‐induced rat stomach cancers is needed to disclose the major mechanisms of stomach carcinogenesis. Mol. Carcinog. 25:42–47, 1999.

Glutathione S-transferase π-class as a tumour marker in lingual preneoplastic and neoplastic lesions of rats and humans

Abstract Immunocytochemical expression of the π class glutathione S-transferase (GST) was investigated in preneoplastic and neoplastic lingual lesions in a 4-nitroquinoline 1-oxide (4NQO)-induced rat genetic model [Wistar/Furth rats (WF) and Dark-Agouti rats (DA)] and in human surgical material [fibrous polyp, mild to moderate dysplasia, severe dysplasia, carcinoma in situ (CIS), squamous cell carcinoma (SCC)]. Two polyclonal antibodies raised against rat (GST-P) and human (GST-π) antigens were used. In the rat model, DA and WF rats showed contrasting susceptibility to 4NQO, DA rats having a much higher tumour incidence and a significantly shorter survival time than WF rats. While the established lingual SCC in DA and WF rats all expressed GST-P, the number of GST-P+ foci in the preneoplastic lingual epithelium was significantly higher in DA (14.5 ± 6.5) than in WF rats (5.5 ± 2.6; P < 0.0001). In contrast, GST-π epithelial staining in human specimens was more variable and the results overlapped in different groups. More frequent nuclear and/or basal cell staining was detected in severe dysplasia, CIS and SCC than in benign and mild to moderate dysplastic lesions. Although the π class GST may be a useful marker for rat lingual carcinogenesis, its value in clinical applications is unclear. GST-π staining patterns and their distribution may be helpful in identifying high-risk lingual lesions in humans.

Expressions of junB and c‐fos are enhanced in 4‐nitroquinoline 1‐oxide‐induced rat tongue cancers

Activator protein‐1 (AP‐1) is a transcription factor activated in many tumors. Using 4‐nitroquinoline 1‐oxide (4NQO)‐induced rat tongue cancers (TC), the present study investigated the expression levels of genes that encode the components of AP‐1, the jun gene family (c‐jun, junB and junD) and the fos gene family (c‐fos, fra‐1, fra‐2 and fosB). Expression levels of junB and c‐fos mRNAs in TC were significantly elevated compared with those in epithelial tissue of control rat tongue, although only c‐fos mRNA levels tended to be elevated in dysplastic tongue epithelium. Histologically, all 4NQO‐induced rat TC were well‐differentiated squamous cell carcinomas. Immunostaining for JunB and c‐Fos proteins was positive in the nuclei of tumor cells of all TC. It is noteworthy that JunB was negative, but c‐Fos was positive in the dysplastic tongue epithelium of the 4NQO‐treated rats. Immunostaining for both proteins was negative in tongue mucosal epithelium of control rats. There were no mutations in the coding regions of either junB or c‐fos in all the TC examined. These results suggest the possibility that the expressions of junB and c‐fos were enhanced stepwise in 4NQO‐induced carcinogenesis of rat tongue, and that the coexpression of JunB and c‐Fos might play an important role in the establishment of TC.

Increased Fractal Complexity of the Epithelial-connective Tissue Interface in the Tongue of 4NQO-treated Rats

The effect of the carcinogen 4-nitroquinoline 1-oxide (0.001% in drinking water) on the irregularity of the epithelial connective tissue interface (ECTI) of the ventral surface of the tongue was quantified in Dark Agouti and Wistar Furth rats. Histological tongue sections stained with the Azan-Mallory method were digitised (111 images, resolution 1 pixel = 3.1 microns), and the limit between epithelium and stroma of the ventral surface was extracted and analysed using a fractal geometry technique (local connected dimension). The results showed that although none of the images included carcinomas on the ventral surface of the tongue (all cases had other oral carcinomas), the epithelial profiles of the treated cases showed a statistically significant increase in irregularity when compared to controls. Canonical discriminant analysis of the parameters describing the irregularity of the ECTI classified 81.1% of the images in the original groups (treated or control). Fractal analysis is capable of detecting subtle architectural changes in the oral epithelium of the rat occurring after exposure to the carcinogen, even when full malignant transformation has not yet taken place. Fractal analysis, which may prove useful for monitoring the progression of carcinogenesis in this animal model, is a morphometrical parameter in the diagnosis of oral epithelial dysplasia.

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.

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.

Case of adult T-cell leukemia with pulmonary involvement presenting as nodular shadows

A 60‐year‐old man with a diagnosis of smoldering adult T‐cell leukemia (ATL) had been treated successfully for 4 years with psoralen and ultraviolet A therapy, γ‐interferon, oral etoposide and sobuzoxane. He subsequently developed rapidly‐growing skin nodules over his entire body. Chest X‐ray and thoracic computed tomography showed nodular shadows in the right lower lung field and nodules in both lower lung lobes. Despite combined chemotherapy, he died. Upon autopsy, numerous nodules were found in the bilateral lower lobes; microscopically, the nodules were diffusely infiltrated by ATL cells. Our review of the published work found only two previously reported cases of ATL with pulmonary involvement manifested as nodular shadows. Herein, we present details on the third case.