Chromosomal Mapping of Genetic Locus Associated with Thymus-size Enlargement in BUF/Mna Rats

The thymoma-prone rat of the BUF/Mna strain is a useful model for human thymoma. In this strain thymoma development is regulated by a single autosomal susceptible gene, Tsr-1. At pre-thymoma age, BUF/Mna rats have extremely large thyrauses, when compared to those of other strains of rats. Genetic studies in crosses between BUF/Mna rats with large thymuses and WKY/NCrj rats with small thymuses suggested the presence of a major autosomal gene, Ten-1 , which contributes to thymus enlargement in a backcross population. Linkage studies between Ten-1 and microsatellite markers in backcross rats of (WKY/NCrj×BUF/Mna)Fl×BUF/Mna have led to the localization of Ten-1 in chromosome 1. This result may provide an approach to clone Tsr-1 , which could be allelic to Ten-1.     

Nodular Development of Spontaneous Epithelial Thymoma in (ACI/NMs × BUF/Mna)F1 Rats

The BUF/Mna strain is a high thymoma line of rats, and virtually all rats develop overt thymomas by the age of 40 weeks. To reveal the early morphologic changes in this thymomagenesis, thymuses and thyraomas were studied in (ACI/NMs × BUF/Mna)Fl (ABF1) rats, which inherit a thymoma susceptibility gene ( Tsr-1 ) from the BUF/Mna strain. At 50 weeks of age, 18% of ABF1 rats had developed medium to large thymomas, 54% had just began to develop multiple, small round nodules in their involuted thymuses, and the remaining 29% had involuted thymus only. The nodules were, microscopically, composed of cortex-like tissues with a starry-sky pattern, showing a quite similar structure to that of the large macroscopic thymomas of predominantly lymphocytic type seen in 104-week-old ABF1 or BUF-Mna rats. Thus, the nodule was actually a small thymoma. In fact, their epithelial cells often had larger atypical nuclei than those in the adjacent involuted thymus cortex. At 104 weeks of age, the incidences of the medium to large thymomas and the small thymoma nodules in ABF1 rats were 64 and 19%, respectively. These results suggest that the thymoma of ABF1 rats occurs initially as multiple small nodules which develop further into medium to large overt thymomas as a result of growth and fusion.     

Nodular development of spontaneous epithelial thymoma in (ACI/NMs x BUF/Mna)F1 rats

The BUF/Mna strain is a high thymoma line of rats, and virtually all rats develop overt thymomas by the age of 40 weeks. To reveal the early morphologic changes in this thymomagenesis, thymuses and thymomas were studied in (ACI/NMs x BUF/Mna)F1 (ABF1) rats, which inherit a thymoma susceptibility gene (Tsr-1) from the BUF/Mna strain. At 50 weeks of age, 18% of ABF1 rats had developed medium to large thymomas, 54% had just began to develop multiple, small round nodules in their involuted thymuses, and the remaining 29% had involuted thymus only. The nodules were, microscopically, composed of cortex-like tissues with a starry-sky pattern, showing a quite similar structure to that of the large macroscopic thymomas of predominantly lymphocytic type seen in 104-week-old ABF1 or BUF-Mna rats. Thus, the nodule was actually a small thymoma. In fact, their epithelial cells often had larger atypical nuclei than those in the adjacent involuted thymus cortex. At 104 weeks of age, the incidences of the medium to large thymomas and the small thymoma nodules in ABF1 rats were 64 and 19%, respectively. These results suggest that the thymoma of ABF1 rats occurs initially as multiple small nodules which develop further into medium to large overt thymomas as a result of growth and fusion.     

An intrinsic thymic epithelial abnormality is responsible for the spontaneous development of predominantly lymphocytic thymomas in BUF/Mna rats.

The nature of tumorigenesis of predominantly lymphocytic thymoma was examined using an animal model. Rats of the inbred BUF/Mna strain were found spontaneously to develop predominantly lymphocytic thymomas, histologically indistinguishable from their human counterparts, at an incidence of virtually 100%. Thymic rudiments of BUF/Mna rats grafted 17 months previously under the renal capsule of young athymic ACI/NMs-rnu/rnu rats also gave rise to similar lesions. The lymphocytes in the thymomas expressed T-cell antigens (rat Lyt-1 and Lyt-2.3), as in the normal case, and ACI rat specific antigen. When BUF/Mna rats of thymoma age were irradiated with a lethal dose of 12 Gy and then received a single injection of bone marrow cells (8 x 10(7)) from BALB/c-nu/nu mice, thymomas were re-formed three weeks later (in 2 of 5 rats) with the replacement lymphocytes expressing mouse Thy-1.2 antigen. These results indicate that an intrinsic thymic epithelial abnormality is responsible for the development of predominantly lymphocytic thymomas in BUF/Mna rats.     

An Intrinsic Thymic Epithelial Abnormality Is Responsible for the Spontaneous Development of Predominantly Lymphocytic Thymomas in BUF/Mna Rats

The nature of tumorigenesis of predominantly lymphocytic thymoma was examined using an animal model. Rats of the inbred BUF/Mna strain were found spontaneously to develop predominantly lymphocytic thymomas, histologically indistinguishable from their human counterparts, at an incidence of virtually 100%. Thymic rudiments of BUF/Mna rats grafted 17 months previously under the renal capsule of young athymic ACI/NMs- rnu/rnu rats also gave rise to similar lesions. The lymphocytes in the thymomas expressed T-cell antigens (rat Lyt-1 and Lyt-2.3), as in the normal case, and ACI rat specific antigen. When BUF/Mna rats of thymoma age were irradiated with a lethal dose of 12 Gy and then received a single injection of bone marrow cells (8 × 10⁷) from BALB/c- nu/nu mice, thymomas were re-formed three weeks later (in 2 of 5 rats) with the replacement lymphocytes expressing mouse Thy-1.2 antigen. These results indicate that an intrinsic thymic epithelial abnormality is responsible for the development of predominantly lymphocytic thymomas in BUF/Mna rats.     

Possible single dosage effects of the nude gene: suppression of spontaneous development of thymoma and nephropathy in BUF/Mna-rnu/+ rats

Single dosage effects of the rat nude gene (rnu) on spontaneous development of epithelial thymoma, muscle atrophy and nephrotic syndrome were studied by comparing littermates of rnu /+ and +/+ rats on a high thymoma strain, BUF/Mna, background. Heterozygous rnu/+ rats had a significantly smaller thymus than the +/+ littermates at 6 weeks of age. The incidence of thymoma at 12 months of age was extremely low in the female rnu/+ rats (3%) as compared with that of the +/+ rats (94%). Development of the nephrotic syndrome but not of the muscle atrophy was also suppressed in the heterozygotes. The results suggest that a recessive mutant gene, rnu, in a single dosage, interfered with critical steps of the disease processes of the thymoma and nephrotic syndrome in BUF/Mna-background rats.     

Establishment of transplantable tumor lines and in vitro cell lines of malignant thymoma developed in a BUF/Mna rat.

A spontaneous malignant thymoma was found in an 18-month-old female BUF/Mna rat and serially transplanted subcutaneously in both syngeneic BUF/Mna rats (designated as MTH-R) and KSN nude mice (MTH-NM) for more than 5 years. Both tumors shared the histological appearance of sarcomatoid carcinoma as seen in the original tumor. However, MTH-NM grew faster than MTH-R in the respective hosts. The MTH-NM grew in both KSN-nude mice and BUF/Mna-rnu/rnu rats but not in BUF/Mna rats, the host of the original tumor. Three continuous tissue culture cell lines (MTHC-1, MTHC-2 and MTHC-3) were established from the MTH-NM tumors at the 2nd, 15th and 17th transplantation generations, respectively. The MTH-NM tumors and latter two tissue culture cell lines carried one or more mouse chromosomes, probably acquired by cell fusion with mouse cells during passages in vivo. The presence of the mouse chromosomes was confirmed by the presence of mouse DNA and of antibodies to the MTHC-2 and MTHC-3 cells in the sera of BUF/Mna rats transplanted with MTH-NM.     

Establishment of Transplantable Tumor Lines and in vitro Cell Lines of Malignant Thymoma Developed in a BUF/Mna Rat

A spontaneous malignant thymoma was found in an 18-month-old female BUF/Mna rat and serially transplanted subcutaneously in both syngeneic BUF/Mna rats (designated as MTH-R) and KSN nude mice (MTH-NM) for more than 5 years. Both tumors shared the histological appearance of sarcomatoid carcinoma as seen in the original tumor. However, MTH-NM grew faster than MTH-in the respective hosts. The MTH-NM grew in both KSN-nude mice and BUF/Mna- rnu/rnu rats but not in BUF/Mna rats the host of the original tumor. Three continuous tissue culture cell lines (MTHC-1, MTHC-2 and MTHC-3) were established from the MTH-NM tumors at the 2nd, 15th and 17th transplantation generations, respectively. The MTH-NM tumors and latter two tissue culture cell lines carried one or more mouse chromosomes, probably acquired by cell fusion with mouse cells during passages in vivo. The presence of the mouse chromosomes was confirmed by the presence of mouse DNA and of antibodies to the MTHC-2 and MTHC-3 cells in the sera of BUF/Mna rats transplanted with MTH-NM.     

Tumor-cytotoxicity of nitric-oxide produced from alveolar macrophages directly stimulated with tumor-cells

Macrophages activated by lipopolysaccharide or interferon-gamma have been shown to be cytotoxic to tumor cells by releasing nitric oxide. Here, we report that unstimulated rat alveolar macrophages cultured with certain tumor cells produce nitric oxide and are cytotoxic to these tumor cells. Alveolar macrophages were taken from BUF/Mna rats, which were known to produce spontaneous thymoma, and cultured with syngeneic BUF/Mna-derived thymoma cells. They were killed by syngeneic or allogeneic alveolar macrophages and this killing was partially abolished by addition of N(G)-monomethyl-L-arginine. X-ray irradiated, mitomycin C-treated or membranous fragments of BUF/Mna-derived thymoma cells directly stimulated rat alveolar macrophages to produce nitric oxide.     

Cytogenetic studies on rat thymic lymphomas induced by N-propyl-N-nitrosourea

N-Propyl-N-nitrosourea (PNU) was proved to be a strong leukemogen, which induces myelogenous leukemia or thymic lymphoma in rats. BUF/Mna rats and F344 rats were the strain most susceptible to thymic lymphomagenic activity of PNU. In addition, F1 rats between BUF/Mna and WKY rats were also susceptible to PNU-lymphomagenic activity. In the present experiment, karyotypes of 31 thymic lymphomas induced by PNU in BUF/Mna rats and in F1 rats between BUF/Mna and WKY rats were analysed for chromosomal abnormalities. Although no specific chromosomal abnormalities were observed throughout all lymphomas, del(11q) and dup(2q) were observed frequently in BUF/Mna rat lymphomas. Breakpoints and/or fusion-points were frequently observed in chromosome 11, followed by chromosomes 2, 5 and 6. Trisomy of chromosome 7, on which c-myc oncogene is mapped, was observed in seven cases, and monosomy of chromosomes 12, 18, 19, 20 and X was seen in seven or eight cases each, though these changes were generally observed in minor cell population in each case.     

Identification of 29 Rat Genetic Markers by Arbitrarily Primed Polymerase Chain Reaction

The number of genetic markers for the rat is still limited, in spite of its wide use in cancer research. To facilitate accurate mapping of both established and novel rat genetic markers, we constructed a linkage map by genotyping 105 F₂ rats from ACI/N (ACI) and BUF/Nac (BUF) crosses. This map consists of 120 genetic markers that had been previously reported, mainly by two research groups, but had not been integrated. To find new genetic markers, the arbitrarily primed polymerase chain reaction (AP-PCR) was applied to detect polymorphic bands between ACI and BUF rats. After testing 56 single primers and 12 combinations of primers, we found 36 bands produced by 16 single primers and two combinations to be reliably polymorphic between ACI and BUF rats. The 36 bands were typed in the 105 F₂ rats, and 29 of them could be linkage-mapped. AP-PCR is thus useful to detect new genetic markers in laboratory strains of rats.     

Chromosomal mapping of genes controlling development, histological grade, depth of invasion, and size of rat stomach carcinomas

Rat stomach cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are widely used as a model of differentiated-type human stomach cancers. ACI/N (ACT) rats are susceptible and BUF/Nac (BUF) rats are resistant to MNNG-induced stomach carcinogenesis, and the presence of an autosomal gene with a dominant BUF allele has been suggested. In this study, we performed a carcinogenicity test by giving MNNG in drinking water to 117 male ACI x (ACIxBUF)F1 backcross rats. Each of 100 effective rats was diagnosed for its "carcinoma development" and when it was bearing stomach carcinoma(s), for histological grade, depth of invasion, and size and number of tumors. Carcinoma development was diagnosed based both on the age of the rat and on the presence of stomach carcinoma(s). Linkage analysis was performed with the genotypes of 161 loci, covering 1637 cM of the rat genome. Contrary to our original expectations, the most influential gene was the one on chromosome (chr.) 15, Gastric cancer susceptibility gene 1 (Gcs1), which confers susceptibility to stomach carcinogenesis (LOD, 3.8) with a dominant BUF allele by promoting conversion from adenomas to carcinomas. Two resistance genes on chr. 4 and chr. 3, Gastric cancer resistance gene 1 (Gcr1) and Gcr2, were shown to confer dominant resistance (LOD, 2.7 and 2.6, respectively). Gcs1, Gcr1, and Gcr2 exerted additive effects on the development of stomach carcinomas. A gene on chr. 16, Gcr3, was indicated to reduce the depth of invasion (LOD, 2.2) and sizes of tumors (LOD, 1.9). No linkage was obtained using the number of tumors. These findings show that the coordinate effect of a susceptibility gene, Gcs1, and two resistance genes, Gcr1 and Gcr2, is responsible for the development of MNNG-induced stomach carcinomas and that Gcr3 is responsible for the growth of a stomach carcinoma, reflected in the depth of invasion and in the tumor size.     

Genetically determined susceptibility of Fischer 344 rats to propylnitrosourea-induced thymic lymphomas

Administration of propylnitrosourea p.o. by our protocol induced a high incidence of hematolymphatic neoplasms in all six rat strains studied. Remarkable strain differences in susceptibility to thymic lymphomas were observed. The incidence of thymic lymphomas was high in Fischer 344 (98%) and Wistar/Furth (71%) but low in Sprague-Dawley (29%), ACI/Ms (23%), Donryu (24%), and Long-Evans (10%) strains. Segregation of thymic lymphoma incidence among crosses between highly susceptible Fischer and poorly susceptible Long-Evans rats indicated that the increased susceptibility to thymic lymphomas of Fischer rats was determined by a dominant gene TIs-1 (thymic lymphoma susceptible) and that this gene was linked to the coat color loci, p and c, in Linkage Group I in the order of TIs-1 - c - p. The presence of another independently assorting dominant gene, TIs-2, was also suggested to accelerate the thymic lymphoma-genesis. Expression of the group-specific antigen of murine leukemia virus as well as infectious viruses was not detected in nine propylnitrosourea-induced thymic lymphomas of Fischer rats.     

Genetic Regulation of Development of Thymic Lymphomas Induced by N-Propyl-N-nitrosourea in the Rat

To clarify the linkage between Hbb and Tls-1 (thymic lymphoma susceptible-1) loci and to investigate other loci concerned in thymic lymphomagenesis, the BUF/Mna rat, which is highly sensitive to the lymphomagenic activity of N -propyl- N -nitrosourea (PNU), the WKY/NCrj rat, reported to be resistant, and their cross offspring were subjected to genetic analysis. F₁ hybrid and backcross generations were raised from the 2 strains, and 6 genetic markers including Hbb were analyzed in individuals of the backcross generation. However, no linkage between Hbb and Tls-1 loci could be demonstrated since WKY rats also developed a high incidence of thymic lymphomas in response to PNU. Nevertheless, thymic lymphomas developed more rapidly and reached a larger size in the BUF rats. F₁ rats expressed a rather rapid and large tumor growth phenotype, while the [(WKY × BUF) × WKY] backcross generation consisted of rats with either rapidly growing or slowly growing tumors. It was thus concluded that rapid development of thymic lymphomas is determined by a gene, provisionally designated Tls-3 . Analysis of the relationship between 6 genetic markers and development of thymic lymphoma in the backcross generation demonstrated that the Tls-3 locus is loosely linked to the Gc locus, suggesting a possible location on rat chromosome 14. Tls-3 may not be identical with Tls-1 and other genes known to be relevant to thymic tumors, but its relationship with Tls-2 remains obscure.     

Sequential histologic changes during gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in susceptible ACI and resistant BUF rats

Sequential histologic changes of the stomach during carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG; CAS: 70-25-7) were studied in susceptible ACI and resistant BUF strain rats. Rats were given MNNG at a concentration of 83 micrograms/ml in their drinking water for 32 weeks and then tap water and were sacrificed sequentially between weeks 1 and 57. In ACI rats, erosions, regenerative changes, focal and slightly atypical changes, and diffuse and severe atypical changes were observed sequentially in the pyloric region during the period of MNNG administration, where adenocarcinomas were observed after the cessation of MNNG treatment. In BUF rats, the main histologic changes induced by MNNG were erosions and hyperplasia of the glandular portion of pyloric glands at the margin of erosions. After the cessation of MNNG treatment, the hyperplasia of the pyloric glands subsided and was followed by atrophy of these glands. The results suggested that the responses of the gastric mucosa to MNNG in ACI and BUF rats were qualitatively different.     

Profiling and selection of genes differentially expressed in the pylorus of rat strains with different proliferative responses and stomach cancer susceptibility

Rat stomach cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are widely used as a model of differentiated-type human stomach cancers. ACI/NJcl (ACI) rats show persistent and strong cell proliferation in response to gastric mucosal damage by MNNG while BUF/NacJcl (BUF) rats show transient and limited cell proliferation. This difference is considered as one of the mechanisms for the high susceptibility of ACI rats to MNNG-induced stomach carcinogenesis. To identify genes involved in the differential induction of cell proliferation, cDNA subtraction was performed using RNA isolated from the pylorus of ACI and BUF rats treated with MNNG. By the temporal patterns of their expressions, the isolated 16 genes were overviewed and clustered into groups. Expression of the genes in group 1 (such as MHC class I and class II genes and interferon-inducible genes Iigp, Mx2 and Ubd) was induced by MNNG treatment, and the genes in group 2 (such as cellular retinoic acid-binding protein II (CrabpII)) were constantly expressed regardless of MNNG treatment. Then, expression profiles among multiple rat strains were compared with the extents of induction of cell proliferation. Iigp, CrabpII and EST222005 were found to show relatively good accordance, and these three genes were considered as candidates for genes that control differential induction of cell proliferation. Presence of polymorphisms at the genomic DNA level was indicated for CrabpII and EST222005, and these two genes were considered to be better candidates than IIGP: It was shown that the temporal profiles and profiles among strains, taking advantage of animal models, are useful to select candidate genes from a collection of genes isolated by various genome-wide scanning methods.     

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.     

Susceptibility to 1,2-dimethylhydrazine-induced colonic tumors and epithelial cell proliferation characteristics of F1, F2, and reciprocal backcrosses derived from SWR/J and AKR/J parental mouse strains

Hybrid crosses were performed between SWR/J, a strain highly sensitive to 1,2-dimethylhydrazine (DMH), and AKR/J, a strain highly resistant to the carcinogen. F1 and F2 and reciprocal backcrosses were tested to determine if proliferative characteristics such as high activity, wide compartment (PC), and a large S-phase population in the middle third of crypts were linked to susceptibility and inherited as a dominant autosomal trait as was reported for DMH tumor response. A blend of resistant and sensitive tumor and proliferative characteristics was observed in the F1 and F2 crosses. A tumor incidence of 43.7% in the F1 and 52% in the F2 was obtained rather than the respective 100% and 75% expected frequencies. One week after the sixth injection of DMH, the incidence of focal areas of atypism (FAA) in the backcross to resistance (BCR) and the backcross to sensitivity (BCS) was the same (4.1 per FAA-bearing animal). This suggested that the response to the carcinogen was similar in both groups up to this point. Yet 20 weeks later, the BCR had a 7.3% tumor incidence, far lower than the 50% incidence expected. The BCS had an incidence of 98.6%, not unlike SWR frequencies and close to the expected 100% tumor incidence. Proliferative characteristics in the backcrosses, however, did not revert to parental levels. Instead, the labeling index (LI) or percentage of S-phase cells to total cells scored was significantly higher in the BCR than in the BCS (10.2% +/- 3.2% versus 8.1% +/- 2.2%, P less than 0.02). This study has shown that in crosses between these two strains (SWR/J and AKR/J), susceptibility to DMH-induced tumor is not inherited as a dominant trait. Neither are the proliferative characteristics of the colonic mucosa inherited in a simple Mendelian manner nor are the kinetic properties of the epithelial cells linked to DMH tumor susceptibility. It is suggested that the parental AKR/J strain may contribute a protective or resistant factor, that is, a repressor gene, which impedes the progression of carcinogen-induced foci of dysplasia to colonic neoplasia.     

Genetic bases of estrogen-induced tumorigenesis in the rat: mapping of loci controlling susceptibility to mammary cancer in a Brown Norway x ACI intercross

Exposure to estrogens is associated with an increased risk of breast cancer. Our laboratory has shown that the ACI rat is uniquely susceptible to 17beta-estradiol (E2)-induced mammary cancer. We previously mapped two loci, Emca1 and Emca2 (estrogen-induced mammary cancer), that act independently to determine susceptibility to E2-induced mammary cancer in crosses between the susceptible ACI rat strain and the genetically related, but resistant, Copenhagen (COP) rat strain. In this study, we evaluate susceptibility to E2-induced mammary cancer in a cross between the ACI strain and the unrelated Brown Norway (BN) rat strain. Whereas nearly 100% of the ACI rats developed mammary cancer when treated continuously with E2, BN rats did not develop palpable mammary cancer during the 196-day course of E2 treatment. Susceptibility to E2-induced mammary cancer segregated as a dominant or incompletely dominant trait in a cross between BN females and ACI males. In a population of 251 female (BN x ACI)F(2) rats, we observed evidence for a total of five genetic determinants of susceptibility. Two loci, Emca4 and Emca5, were identified when mammary cancer status at sacrifice was evaluated as the phenotype, and three additional loci, Emca6, Emca7, and Emca8, were identified when mammary cancer number was evaluated as the phenotype. A total of three genetic interactions were identified. These data indicate that susceptibility to E2-induced mammary cancer in the BN x ACI cross behaves as a complex trait controlled by at least five loci and multiple gene-gene interactions.     

Genetic and epigenetic factors that influence the occurrence of spontaneous lymphoid tumors in crosses of mice of high- and low-incidence strains

AKR mice, which spontaneously develop greater than 90% incidence of lymphocytic leukemia (LL), crossed with SJL mice, which show greater than 80% incidence of Hodgkin's-like reticulum-cell sarcoma (RCS), produced F1 progeny showing incidences of 30% LL and 0% RCS. Thus, each strain possesses one or more dominant genes capable of interfering with the emergence of the tumor type typical of the other strain. Although mice of reciprocal F1 crosses showed a profound difference in expression of endogenous ecotropic murine leukemia virus (E-MuLV) due to a maternal resistance factor transmitted by SJL females but not males, the two populations did not differ detectably in LL incidence. Like AKR mice, mice of 5 other strains studied (C58, DBA/2, PL, RF and ST/b) possessed one or more genes conferring resistance to RCS in F1 crosses with SJL. Analysis of LL incidences in F1 generations of all possible crosses among these 7 strains revealed 4 different categories of strains with respect to susceptibility/resistance to LL; only ST/b mice, which show no significant incidence of spontaneous LL, lacked genes that could suppress the disease in crosses with high- or moderate-incidence strains. SJL mice treated topically with 3-methylcholanthrene (MCA) developed a 50% incidence of LL, mostly before one year of age; treated mice surviving after one year of age developed a high incidence of RCS.