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.     

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.     

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.     

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.     

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.     

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.     

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.     

Molecular biologic characteristics of seven new cell lines of squamous cell carcinomas of the head and neck and comparison to fresh tumor tissue

BACKGROUND: Squamous cell carcinoma (SCC) is the most frequent malignant tumor of the upper aerodigestive tract. Cell lines of these tumors facilitate the investigation of various tumor biological parameters. This study was conducted to compare molecular biologic characteristics between cell lines and fresh tumor tissue. METHODS: In seven SCC-derived cell lines, cytokeratin 5/6 and cytokeratin 19 expression, DNA content, chromosome aberrations and tumorigenicity were assessed in nude rats. Unbalanced numerical and structural chromosomal aberrations were investigated by comparative genomic hybridization (CGH), and results were compared to those obtained in fresh tumor tissues of the same patients. RESULTS: All cell lines expressed cytokeratins 5/6 and 19, indicating their epidermoid origin. Tumor growth after transplantation into nude rats occurred in five of seven cell lines. Routine histology and immunohistochemical examinations confirmed SCC. Aneuploidy was detected in all cell lines, with a 2c deviation index ranging from 1.9 through 9.5 and a 5c exceeding rate ranging from 2.6 through 36.7%. The most frequent chromosomal aberrations in cell lines were overrepresentations of chromosomal material on chromosomes 15q, 7p (5 cases each), 3q, 5p (4 cases each), and 11q and 17q (3 cases each) and losses of chromosomal material on chromosomes 3p, 18q (3 cases each), and 19p and 7q (2 cases each). Comparing these results to CGH analysis of fresh tumor tissue from the same patients, overrepresentations of chromosomal material on 10q, 20q and 21q, along with loss of chromosomal material on 4q was detected more frequently in primary tumors, whereas overrepresentations on 7p and loss of chromosomal material on 7q were more frequently detected in cell lines. Nevertheless, there was a high degree of similarity of chromosomal alterations in cell lines and corresponding fresh tumor tissue. CONCLUSION: The data suggest a high degree of genetic similarity between tumor cells of cell lines and the tumors from which they were derived. Therefore, these cell lines can serve as an accurate model to investigate cell biology of SCC in vitro.     

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.     

Frequent loss of heterozygosity on chromosomes 4, 12 and 19 in radiation-induced lymphomas in mice

We found frequent loss of heterozygosity (LOH) on chromosomes 4, 12 and 19 in radiation-induced lymphomas from (BALB/cHeA x STS/A) F1 hybrid mice by allelotype analysis at polymorphic microsatellite loci. The incidences of LOH were 27% (20 of 74 lymphomas), 57% (42 of 74 lymphomas) and 50% (37 of 74 lymphomas) on chromosomes 4 (at D4Mit31), 12 (at D12Mit17) and 19 (at D19Mit11), respectively. These frequent LOH regions are homologous to human chromosomes 9p and 1p, chromosome 12q32.1 and chromosome 10q, respectively. Strain-specific preferential allele loss was observed only on chromosome 4. However, no bias in the frequency of loss between alleles of maternal and paternal origin was observed, indicating that genomic imprinting may not be predominantly involved in these lymphomas. The results suggest that these three regions might harbor tumor suppressor genes responsible for this lymphomagenesis.     

Comparative genomic hybridization detects many recurrent imbalances in central nervous system primitive neuroectodermal tumours in children

A series of 23 children with primitive neuroectodermal tumours (PNET) were analysed with comparative genomic hybridization (CGH). Multiple chromosomal imbalances have been detected in 20 patients. The most frequently involved chromosome was chromosome 17, with a gain of 17q (11 cases) and loss of 17p (eight cases). Further recurrent copy number changes were detected. Extra copies of chromosome 7 were present in nine patients and gains of 1q were detected in six patients. A moderate genomic amplification was detected in one patient, involving two sites on 3p and the whole 12p. Losses were more frequent, and especially involved the chromosomes 11 (nine cases), 10q (eight cases), 8 (six cases), X (six patients) and 3 (five cases), and part of chromosome 9 (five cases). These recurrent chromosomal changes may highlight locations of novel genes with an important role in the development and/or progression of PNET.     

Chromosomal imbalances in sporadic neuroendocrine tumours of the thymus

Neuroendocrine (carcinoid) tumours of the thymus are rare neoplasms characterized by a highly malignant clinical behavior. Some of these tumors are associated with MEN1. In this study we evaluated 10 cases of sporadic thymic neuroendocrine tumours using immunohistochemistry and comparative genomic hybridization (CGH). All tumours showed a diffuse expression of neuron specific enolase (NSE) and synaptophysin. Chromosomal imbalances were detected in 8/10 cases, the most frequent gains were seen on chromosome Xp (3/10 cases), 7p, 7q, 11q, 12q, and 20q (2/10 each), losses were most frequently detected at 6q (5/10 each), 6p (3/10 each), 4q (3/10 each), 3p, 10q, 11q and 13 q (2/10 each). These CGH data show a degree of overlap with chromosomal imbalances commonly observed in advanced thymomas.     

Infrequent loss of chromosomal heterozygosity in human stomach cancer

By molecular genetic approach using polymorphic DNA markers which detect allelic deletion at specific chromosomal loci, we analyzed 30 human stomach cancers for possible loss of chromosomal heterozygosity. We analyzed 25 loci on 18 different chromosomes covering regions frequently deleted in several types of cancers. Loss of chromosomal heterozygosity was observed only in five of 30 cases examined, and it was infrequently detected at 10 loci on seven different chromosomes including chromosome 1 in two of 12 cases, chromosome 12 in one of four cases and chromosome 13 in three of 27 cases. It was also observed at loci on chromosomes 11, 14, 16, and 19 with very low frequency (less than 10%), but not on other chromosomes: chromosomes 3, 5, 6, 9, 10, 15, 17, 18, 20, and 22. Thus, in human stomach cancer, loss of heterozygosity occurs infrequently even at chromosomal loci often deleted in other types of cancers.     

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.     

Loss of p53 in benzene-induced thymic lymphomas in p53+/- mice: evidence of chromosomal recombination

The purpose of this study was to examine the role of chromosomal recombination in mediating p53 loss in benzene-induced thymic lymphomas in C57BL/6-Trp53 haploinsufficient (N5) mice (p53+/- mice). We characterized loss of heterozygosity (LOH) on chromosome 11 using seven microsatellite markers in 27 benzene-induced and 6 spontaneous thymic lymphomas. Eleven patterns of LOH were found between the induced and spontaneous tumors, with only one pattern being in common between the tumor groups. Nearly 90% (24 of 27) of benzene-induced tumors exhibited loss of the functional p53 allele locus, and 83% (20 of 24) of these tumors retained two copies of the disrupted p53 allele. The results indicate that benzene induces a high frequency of LOH on chromosome 11 in p53+/- mice, likely mediated by aberrant chromosomal recombination.     

Analysis of 14q+ Derivative Chromosomes in Non-Hodgkin's Lymphomas by Fluorescence In-Situ Hybridization

The most common chromosomal abnormality observed in non-Hodgkin's lymphomas (NHL) involves the structural alteration of the q arm of chromosome 14. It is not always possible, however, to fully analyse such derivative chromosomes by Giemsa-banding. Therefore, we have applied the fluorescence in-situ hybridization (FISH) technique of chromosome painting to elucidate the origins of the der(14) chromosomes in 8 cases of NHL. In 2 NHL the der(14) appeared to be the product of the t(14;18)(q32;q21) translocation, but were not accompanied by the reciprocal der(18) chromosome. In 3 cases the breakpoint was at 14q32 but the translocated material appeared not to be from chromosome 18 and in 2 cases the breakpoint was centromeric to 14q32. One case with a t(14;18)(q32;q21) was also analysed as a control. Dual painting was carried out with paints for chromosome 14 and either chromosome 3, 8, 10, 11, 18 or 19. In the control and 2 other cases the translocated material was demonstrated to be from chromosome 18, in two cases it was from chromosome 3 and in 1 case there was an unusual insertion of chromosome 11 material. We were unable to identify the origins of the translocated material in 1 NHL and in the final case the apparent der(14) was demonstrated not to contain chromosome 14 material. These data demonstrated the utility of the FISH technique for analysing malignant cell karyotypes, and in particular indicated the potential of this approach for identifying cases containing putative NHL associated oncogenes that may have been translocated adjacent to the immunoglobulin locus at 14q32.     

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 abnormalities in patients with non-cutaneous T-cell non-Hodgkin's lymphoma

In contrast to non-Hodgkin's lymphomas (NHL) with a B-cell phenotype, almost no data have been reported dealing with correlations between chromosomal abnormalities and characteristics of the disease in patients with T-cell NHL. In a retrospective analysis we studied all patients with a non-cutaneous T-cell NHL and chromosomal abnormalities that were evaluated at our institution; 20 patients could be identified. Numerical abnormalities involving chromosomes 3, 4, 5, 22 and X were observed most frequently. Structural abnormalities involved mainly the breakpoints 1q22–25, 6q23 and 11q13. There appeared to be an association between +7 breakpoints 2p23–24, 4p14–15, 8q21 and the presence of extranodal disease. All patients with +7 had a diffuse mixed histology. Patients with +2, +3, +11, +17, +18, +20 or breakpoint 1q22–25 had an immunoblastic lymphoma and patients with breakpoints 9q32–34 or 14q12 had a lymphoblastic lymphoma. No correlations were observed between chromosomal abnormalities and response to therapy, survival or phenotypic markers. Abnormalities involving the chromosomes containing the T-cell receptor genes and T-cell markers were infrequent. Several breakpoints were identified that correlate with already described oncogenes.