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.     

Action Site of the Gene Determining Susceptibility to Propylnitrosourea-induced Thymic Lymphomas in F344 Rats

To clarify whether the determining effect of the thymic lymphoma susceptible-1 (Tls-1) gene is on putative N-propyl-N-nitrosourea (PNU) target cells among T-lineage cells or on other host factors, we investigated the PNU-induced lymphomagenesis in transplantation chimeras between susceptible F344 and resistant LES strain rats. Administration of PNU to lethally irradiated (F344 X LES)F1 rats reconstituted with bone marrow cells from either F344 or LES parental rats invariably led to development of donor-origin thymic lymphomas. On the other hand, thymic lymphomas were induced in thymectomized Fl rats grafted with neonatal LES thymtis, of which 4 out of 8 were of the donor origin. These observations indicate that the target cells of thymic lymphomagenesis of F344 and LES rats are equally susceptible to PNU provided they are in susceptible hosts and the LES thymus seems capable of supporting thymic lymphomagenesis, although this capability wanes with aging of the thymus. The effect of the Tls-1 gene, therefore, is neither on PNU susceptibility of the target cells nor on the capability of the thymus to support lymphomagenesis, but on other host factors either in or out of the thymus.     

Genetic and Epigenetic Resistance of SL/Ni Mice to Lymphomas

The murine spontaneous B lymphoma is etiologically related to the expression of endogenous ecotropic marine leukemia virus (ETV). Although both SL/Kh and SL/Ni mouse strains show a high level of expression of ETV from early in life, the former is a pre-B lymphoma-prone strain and the latter is rather lymphoma-resistant. In order to identify the host background difference related to the lymphomagenesis, we performed a genetic cross study between these two strains. In the reciprocal F₁ generation, the length of the lymphoma latent period was slightly but significantly longer in (SL/Ni XSL/Kh)F₁ than in (SL/Kh × SL/Ni)F₁ (P<0.05). The incidence of overall lymphomas and that of acute pre-B lymphomas was lower in (SL/Ni × SL/Kh)F₁ than in (SL/Kh × SL/Ni)F₁, although the difference was not statistically significant. These observations indicate that an epigenetic maternal resistance mechanism of SL/Ni mice plays a role in the lymphoma resistance. Furthermore, in the backcross combinations without maternal influence of SL/Ni, we observed a genetic mechanism of lymphoma resistance: an SL/Ni-derived recessive lymphoma-resistance gene mapped in the proximal segment of Chr. 4. We named this gene nir-1 (SL/Ni-lymphoma resistance-1). Thus, we have demonstrated epigenetic and genetic mechanisms of lymphoma resistance of the SL/Ni mouse with the high expression of endogenous ETV.     

Action site of the gene determining susceptibility to propylnitrosourea-induced thymic lymphomas in F344 rats

To clarify whether the determining effect of the thymic lymphoma susceptible-1 (Tls-1) gene is on putative N-propyl-N-nitrosourea (PNU) target cells among T-lineage cells or on other host factors, we investigated the PNU-induced lymphomagenesis in transplantation chimeras between susceptible F344 and resistant LES strain rats. Administration of PNU to lethally irradiated (F344 x LES)F1 rats reconstituted with bone marrow cells from either F344 or LES parental rats invariably led to development of donor-origin thymic lymphomas. On the other hand, thymic lymphomas were induced in thymectomized F1 rats grafted with neonatal LES thymus, of which 4 out of 8 were of the donor origin. These observations indicate that the target cells of thymic lymphomagenesis of F344 and LES rats were equally susceptible to PNU provided they are in susceptible hosts and the LES thymus seems capable of supporting thymic lymphomagenesis, although this capability wanes with aging of the thymus. The effect of the Tls-1 gene, therefore, is neither on PNU susceptibility of the target cells nor on the capability of the thymus to support lymphomagenesis, but on other host factors either in or out of the thymus.     

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 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.     

Hereditary Low Level of Plasma Ceruloplasmin in LEC Rats Associated with Spontaneous Development of Hepatitis and Liver Cancer

Both young (5 weeks old) and old (61 100 weeks old) hereditary hepatitis LEC rats showed a markedly low level of plasma ceruloplasmin (Cp) ferroxidase activity as compared with that of age-matched LEA and BN strain rats. This trait was genetically examined hy the use of (BN × LEC) F₁ hybrid and (F₁× LEC) backcross rats. The F₁ hybrids never developed hepatitis and showed a similar level of Cp to that found in the parental BN rats. Among the backcross rats with about 1:1 segregation rate for hepatitis, affected rats had a remarkably decreased level of Cp, as found in LEC rats, whereas unaffected rats exhibited a similar level of Cp to that of BN, F₁ and LEA rats. These results indicate that the low level of Cp is heritable in a single autosomal recessive mode in LEC rats. The observed tight link between the low Cp level and the hepatitis in LEC rats suggests that defective copper metabolism may he associated with the occurrence of hepatitis in LEC rats, since Cp is a copper-binding protein primarily involved in copper transport from the liver.     

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.     

Genetic control of resistance to 3-methylcholanthrene-induced T-cell lymphoma in mice

Different strains of inbred mice exhibit different levels of susceptibility to T-cell lymphoma induced by the carcinogen 3-methylcholanthrene (MCA). Resistance to MCA-induced lymphoma was dominant over susceptibility in all crosses tested, and in inbred strain mice MCA resistance was found to correlate with resistance to lymphoma induced by a fractionated dose of gamma irradiation. The susceptible RF/J strain was also found to be extraordinarily sensitive to lymphoma induction by low doses of X-irradiation; this low dose sensitivity was also recessive. Experiments on both first and second generation backcross populations supported the hypothesis that a single locus is the main determinant of MCA resistance. Studies examining the possible linkage of low lymphoma incidence to a number of loci failed to establish any clear association, but linkage was seen between the Lyt-2 locus and a significant delay in MCA-induced lymphoma development. We also examined the strain distribution of several activities which might have been expected to have an effect on susceptibility to induced lymphoma. No correlation was seen between resistance and either DNA repair ability, thymic superoxide dismutase levels, or natural killer activity.     

A new locus for resistance to gamma-radiation-induced thymic lymphoma identified using inter-specific consomic and inter-specific recombinant congenic strains of mice

Mice of the C57BL/6J inbred strain develop thymic lymphomas at very high frequency after acute gamma-irradiation, while mice of several inbred strains derived from the wild progenitor of the Mus spretus species and their F1 hybrids with C57BL/6J appear extremely resistant. Analysis of the genetic determinism of the gamma-radiation-induced thymic lymphoma (RITL) resistance with the help of inter-specific consomic strains (ICS), which carry a single introgressed Mus spretus chromosome on a C57BL/6J genetic background, provide significant evidence for the existence of a thymic lymphoma resistance (Tlyr1) locus on chromosome 19. The subsequent analysis of the backcross progeny resulting from a cross between consomic mice heterozygous for the Mus spretus chromosome 19 and C57BL/6J mice, together with the study of inter-specific recombinant congenic strains (IRCS), suggest that this Tlyr1 locus maps within the D19Mit60-D19Mit40 chromosome interval. In addition to the discovery of a new locus controlling RITL development, our study emphasizes the value of ICS and IRCS for the genetic analysis of cancer predisposition.     

The D4Mit12 locus on mouse chromosome 4 provides susceptibility to both gamma-ray-induced and N-methyl-N-nitrosourea-induced thymic lymphomas

Low-penetrance genes control different susceptibilities to γ-ray-induced thymic lymphomas in mouse strains. Our previous genetic analyses with backcross mice between BALB/c and MSM strains and congenic lines localized one such gene near the D4Mit12 locus on chromosome 4. N-Methyl-N-nitrosourea (MNU) is a guanine base-alkylating agent and differs from γ-radiation in its mechanism of mutagenic action. Accordingly, in this study, we examined whether or not the locus also provides susceptibility to MNU-induced thymic lymphomas using 84 offsprings derived from congenic mice for D4Mit12 . Association analysis provided a suggestive linkage at D4Mit12 (P=0.0075) and the linkage was sustained by the peak of likelihood ratio statistical values being at the same position as that for the γ-ray-induced lymphomas. The results strongly suggest that the BALB/c allele near D4Mit12 is associated with susceptibility to lymphomas induced by two carcinogenic agents having different mechanisms of mutagenic action.     

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 Mapping and Allelic Loss Analysis in Mouse Thymic Lymphomas of Helios and Aiolos Belonging to the Ikaros Gene Family

The Ikaros gene undergoes bi-allelic changes at a high frequency in γ-ray-induced mouse thymic lymphomas, suggesting the relevance of Ikaros to the lymphoma development. Here we test whether Helios and Aiolos , two other members of the Ikaros gene family, are also involved in lymphomagenesis. Genetic mapping showed that Helios is located between D1Mit531 and D1Mit19 on chromosome 1 and Aiolos is between D11Mit222 and D11Mit332 on chromosome 11. Analysis using polymorphic markers around the two regions revealed that neither locus exhibited allelic loss in the 78 lymphomas that were induced in _p53 wild-type mice, whereas in 102 _p53(KO/+ ) mouse-derived lymphomas Helios and Aiolos loci showed allelic loss in 8% (8/102) and 33% (34/102), respectively. However, 33 of the 34 lymphomas showing allelic loss at Aiolos were/j53(KO/-) and were accompanied by loss of the _p53 wild-type allele on the same chromosome. Homozygous deletion and mutation analyses failed to detect bi-allelic alterations. These results do not suggest any obvious contribution of Helios or Aiolos to oncogenesis of the mouse thymic lymphomas.     

Two-dimensional Electrophoretic Analysis of Hepatitis-associated Polypeptides in Liver of LEC Rats Developing Spontaneous Hepatitis

High-resolution two-dimensional polyacrylamide gel electrophoresis in combination with silver staining was used to analyze between 800 and 1000 cytosolic and particulate polypeptides from age-matched livers of normal male Long-Evans rat with Agouti coat color (LEA) and Long-Evans rat with Cinnamon-like coat color (LEC) rats with hereditary trait of hepatitis at ages long before, immediately prior to, and just after the onset of hepatitis. Although the electrophoretic patterns of polypeptide expression were very similar with respect to the overall spot patterns, a number of polypeptides which differed either qualitatively or quantitatively were noted. Two constitutively expressed cytosolic polypeptides, P29.5 (M_r 29.5 kDa/pI 6.73) and P30 (30 kDa/6.70), were not detected in livers of LEC animals at any age. In the normal LEA rats both P29.5 and P30 were detected as early as one day after birth and both were expressed at similar concentrations at all ages. In the LEC rats P30-C (30 kDa/6.68) was constitutively expressed in close proximity to the expected position of P30, and P30-C was not detected in the LEA rats. By means of non-equilibrium pH gradient electrophoresis two relatively basic polypeptides were detected in the LEC rats. P18ne was detected immediately prior to and P27ne immediately after the clinical manifestation of hepatitis. Experiments in F₁ backcross ([LEA × LEC] × LEC) animals, however, failed to demonstrate any genetic link between either the expression or lack of expression of P29.5, P30, P30-C, or P18ne and hepatitis development. P27ne was detected in all backcross animals exhibiting hepatitis, but was never observed in LEC rats prior to the onset of hepatitis. Although we were unable to identify any unique loss of expression of polypeptides which are genetically linked to hepatitis susceptibility in LEC rats, specific subsets of quantitatively modulated polypeptides were detected.     

Chromosome mapping of multiple loci affecting the genetic predisposition to rat liver carcinogenesis

Previous studies on (BNxF344)F1 (BFF1) rat model of genetic predisposition to hepatocarcinogenesis led to the identification, in BFF1xF344 backcross progeny, of two hepatocarcinogenesis susceptibility (Hcs) and three resistance (Hcr) loci affecting the progression of neoplastic liver nodules. To evaluate the presence of other hepatocarcinogenesis-related loci in the BFF1 genome, nodule induction by resistant hepatocyte model in 116 male BFF2 rats 32 weeks after initiation with diethylnitrosamine was subjected to quantitative trait loci analysis. The rats were typed with 179 genetic markers, and linkage analysis identified three loci on chromosomes 1, 16, and 6, in significant linkage with nodule mean volume (V), volume fraction, and number, respectively, and two loci on chromosomes 4 and 8 in suggestive linkage with V. These loci were differently positioned with respect to Hcs and Hcr loci mapped previously in backcross rats. On the basis of phenotypic and allele distribution patterns of BFF2 rats, loci on chromosomes 1 and 16 were identified as Hcs3 and Hcs4, and loci on chromosomes 4, 8, and 6 as Hcr4, Hcr5, and Hcr6. Additive interactions occurred between Hcs3 and Hcs4, and Hcr4 and a locus on chromosome 3 with less than suggestive linkage with V. All of the loci were in chromosomal regions syntenic to mouse and/or human chromosomal segments showing allelic gain or loss in hepatocellular carcinomas. These data indicate that inheritance of predisposition to rat liver tumor is characterized by the interplay of several genetic factors and suggest some possible mechanisms of polygenic control of human liver cancer.     

Alkylation of DNA in F344 rat thymus following administration of 1-n-propyl-1-nitrosourea in vivo and comparison of O6-alkylguanine DNA alkyltransferase activity in thymus from F344 and Long-Evans rats in vitro

The tissue distribution of radioactivity 1 h after i.p. injectionof [n-propyl-2,3-³H]1-n-propyl-1-nitrosourea (PNU) (100 mg/kg)was studied in male F344 rats. This treatment results in a highincidence of thymic lymphomas. The ³H concentration in the thymus,testis and brain was significantly higher than that in blood.7-n-propylguanine and O⁶-n-propylguanine were detected in thymusDNA of F344 rats treated with PNU; the ratio of O⁶-n-propylguanine/7-n-propylguaninewas 0.35, lower than following DNA alkylation in vitro. Thissuggests the presence of O⁶-alkylguanine DNA alkyltransferase(AGT) in thymus. AGT activity in F344 and Long-Evans rats wascompared by using a ³H-propylated DNA as a substrate. AGT activityin the thymus of F344 rats was lower than that in the liver.The AGT activity in the thymus of Long-Evans strain, which hada low incidence of PNU-induced thymic lymphomas, was higherthan that of F344 strain. The high level of DNA alkylation byPNU and the low activity of AGT in the thymus may contributeto the high incidence of thymic lymphoma in F344 rat.     

Bone marrow progenitor cells of AKR mice give rise to thymic lymphoma cells

Bone marrow cells from one-month-old AKR mice give rise to thymic lymphomas when grafted into (AKR × DBA/2)F₁, hybrid mice. This finding implicates a bone marrow progenitor as a “target cell” for the age-related development of thymic lymphomas in AKR mice.     

Quantitative Trait Loci Associated with Promoting Effects of Sodium l-Ascorbate on Two-stage Bladder Carcinogenesis in Rats

In the two-stage rat bladder carcinogenesis model using N -butyl- N -(4-hydroxybutyl)nitrosamine (BBN) as an initiator and sodium l -ascorbate (SA) as a promoter, we found a notable strain difference between F344/DuCrj (F344) and WS/Shi (WS) rats in susceptibility to the promoting effect of SA. Twenty each of F344, WS and reciprocal F₁ hybrid rats were given 0.05% BBN in their drinking water for 4 weeks and then a basal diet with (BBN-SA group) or without (BBN group) a 5% SA supplement for 32 weeks. In F344 and also in reciprocal F₁ hybrids, the number of tumors per rat was significantly higher in the BBN-SA group than in the BBN group ( P <0.0001). In contrast, WS rats were not significantly affected by either treatment ( P =0.8). These findings indicate that F344 rats are highly susceptible to the promoter effect of SA, but WS rats are not. Linkage analysis of 108 WS X (WSXF344) F₁ backcrosses revealed that this difference was related to a quantitative trait locus mapped on rat Chr. 17 (maximum LOD score, 3.86) named Bladder Tumor Susceptible-I and possibly another locus on Chr. 5 (maximum LOD score, 2.39). This study has provided the first evidence that host genes influence the risk of bladder cancer development.     

Propylnitrosourea-induced T-lymphomas in LEXF RI strains of rats: genetic analysis.

Oral administration of propylnitrosourea (PNU) in drinking water induces high incidence of lympho-haemopoietic malignancies in rats. Previously we reported that F344 strain rats were highly susceptible to T-lymphomas, and LE/Stm rats, to erythro- or myeloid leukaemias. For analysis of the genetic factors determining types of diseases, we have established LEXF recombinant inbred strains of rats comprising 23 substrains, each derived from intercross between F344 and LE/Stm rats. Rats of 23 LEXF substrains were given PNU, and the development of tumours was observed. The overall incidence of haemopoietic tumours ranged from 100% to 66.7%, and the fractions of T-lymphomas, from 100% to 4%, showing a continuous spectrum. Based on the genetic profile published as a strain distribution pattern table for the LEXF, we screened the potential quantitative trait loci involved in determination of the types of disease and length of the latency period. Statistical calculation was performed using the Map Manager QT software developed by Manly. Four loci, on chromosome 4, 7, 10 and 18, were suggested to associate with the T-lymphoma susceptibility and three loci, on chromosome 1, 5 and 16, with the length of the latency period. These putative loci were further examined in backcross (F344 x LE)F1 x LE. Among seven loci suggested by the recombinant inbred study, three loci, on chromosome 5, 7 and 10, were significantly associated with T-lymphomas and another locus on chromosome 1, just weakly. These observations indicate that PNU-induced lymphomagenesis is a multifactorial genetic process involving a number of loci linked with susceptibility and resistance.