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.     

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.     

A single dominant susceptible gene determines spontaneous development of thymoma in BUF/Mna rat

Rats of the BUF/Mna strain developed spontaneous epithelial thymomas morphologically indistinguishable from human homologues at virtually 100% incidence. Segregation of thymoma development among crosses between BUF/Mna and ACI/NMs, which has 0% thymoma incidence, indicated that thymoma susceptibility was determined principally by a single autosomal dominant gene Tbm-1 (thymoma in BUF/Mna rats). In these crosses, another autosomal dominant or semidominant gene(s) contributed by ACI/NMs parents moderately reduced the thymoma incidence.     

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.     

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.     

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.     

Incidence, morphology, and ultrastructure of spontaneous thymoma--the most common neoplasm in W/Nhg rats

Spontaneous thymoma was observed with an incidence of 97 and 36% in female and male rats, respectively, from an inbred Wistar/Neuherberg strain (W/Nhg). The thymomas often caused dyspnea and were occasionally the direct cause of death. The neoplasms resembled human thymomas and showed a variable cell composition, ranging from mainly lymphocytic to mainly epithelial. The detailed ultrastructural findings are described and compared with those in other rat thymomas and in human thymomas. A characteristic feature of all dividing lymphocytes was the presence of often multilayered, confronting cisternae. As in more than 50% of human thymomas, W/Nhg rat thymomas were not associated with myopathies or any other possibly autoimmune diseases. They may thus offer a useful model for the study of thymoma without associated parathymic syndromes.     

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.     

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.     

Immunohistological analysis of thymic tumors with PE-35 monoclonal antibody reactive with medullary thymic epithelium

PE-35 mouse monoclonal antibody (MoAb) (IgG1) detecting an epithelial antigen with a molecular weight of 35,000 was characterized serologically. Immunoperoxidase staining and double immunoenzymatic staining showed that PE-35 antigen is predominantly on nonlymphoid cells in the medulla of thymus. By immunoelectron microscopy, thymic epithelial cells in the medulla were positive with PE-35 MoAb, but macrophages, interdigitating reticulum cells, and thymocytes were negative with this MoAb, which demonstrated that PE-35 is a valuable marker for medullary epithelium. Using PE-35 and other MoAbs detecting thymic epithelial antigens (TE-3A, RFD-4, TE-4, and HLA-DR), 25 thymomas were studied, together with 6 other tumors of thymic origin. Among 25 thymomas, all 6 cases of epithelial type and 8 of 14 mixed lymphoepithelial type were positive with PE-35 MoAb, but only one of 5 lymphocytic type was positive. PE-35 antigen has a tendency to be expressed in the cases retaining medullary type thymocytes, with the phenotype of cluster of differentiation (CD) 1-/CD3+/CD6+, and also in the area of medullary differentiation. TE-3A, RFD-4, and TE-4 MoAbs reacted with most thymoma cases regardless of the types. HLA-DR was, however, expressed on a part of thymomas and the phenotype combined with that of PE-35 was as follows: PE-35+/HLA-DR+, 8 cases; PE-35+/HLA-DR-, 8 cases; PE-35-/HLA-DR+, 8 cases; PE-35-/HLA-DR-, one case. The results suggested that thymoma may originate from different subsets and/or different stages of thymic epithelium.     

An ultrastructural study of nine thymomas.

Nine thymomas, a normal adult thymus, and tissue culture of one thymoma were studied ultrastructurally. The histologic types of thymoma included lymphocytic, epithelial, mixed and spindle cell varieties. Two of the tumors were invasive and one was associated with myasthenia gravis. Despite the histologic dissimilarities, the complex anatomic interrelationships involving lymphocytes, epithelial cells, and blood vessels as seen in the thymus tended to be preserved in all but one of the thymomas. The exception was the spindle-cell thymoma which contained only rare lymphocytes and simpler vascular structures. The findings include variations in the frequency of demosomes and cytoplasmic fibrils of the epithelial cells, the occasional presence of lymphocytes within the laminated venules, and the unusual finding of a gland-like structure in one of the tumors. Correlations between ultrastructural changes and clinical behavior could not be made.     

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 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.     

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.     

Preoperative steroid pulse therapy for invasive thymoma: clinical experience and mechanism of action

BACKGROUND: Glucocorticoid was used in thymomas. The purpose of the study was to evaluate the efficacy of intravenous high-dose glucocorticoid (steroid pulse) therapy in patients with previously untreated advanced thymoma. Causes were also sought for a possible underlying mechanism of the effect of steroid on thymoma. METHODS: Seventeen patients with invasive thymoma who had not received previous chemotherapy or radiation therapy were enrolled in the study. All cases were treated with 2 courses of glucocorticoid therapy before surgery. Tumor response was assessed by computed tomography (CT) scan 1 week after the steroid pulse therapy. Lymphocytes associated with thymoma were analyzed for their CD4/CD8 phenotype and glucocorticoid receptor (GR). TdT-mediated dUTP-biotin nick-end labeling (TUNEL) staining was used to analyze the apoptotic lymphocytes and epithelial cells. RESULTS: The overall response rate to the steroid pulse therapy was 47.1% (8 of 17). The reduction in tumor size was most prominent in type B1 thymomas; there were significant differences between type AB and type B1 thymomas (P = .0234) and type B1 and type B3 thymomas (P = .0068). The reduction in tumor size was accompanied with a marked reduction in the CD4+8+ double-positive immature thymocytes that expressed higher levels of glucocorticoid receptor. Apoptotic changes were observed in both neoplastic epithelial cell and lymphocyte components after glucocorticoid therapy. CONCLUSIONS: The efficiency of preoperative steroid pulse therapy in type B1 thymoma was most prominent, which is probably related to the specific effect on GR-rich CD4+8+ double-positive immature lymphocytes, which are abundant in this type of thymoma.