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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2002 Oct;83(5):247–255. doi: 10.1046/j.1365-2613.2003.00233.x

A novel animal model of thymic tumour: Development of epithelial thymoma in transgenic rats carrying human T lymphocyte virus type I pX gene

Kazunori Kikuchi *,†, Hitoshi Ikeda *, Takahiro Tsuchikawa *, Takahiro Tsuji *, Satoshi Tanaka *, Kazunori Fugo *, Toshiaki Sugaya *, Yuetsu Tanaka ‡, Masatoshi Tateno §, Naoki Maruyama †, Takashi Yoshiki *
PMCID: PMC2517683  PMID: 12641821

Abstract

The pX region encodes a major product of human T lymphocyte virus type I (HTLV-I) that has been implicated previously in tumour formation. To investigate the pathogenesis of pX gene in lymphoid tissues, we established a series of novel transgenic rats carrying the pX gene under the control of a rat lymphocyte-specific protein tyrosine kinase (p56lck) proximal promoter. The transgene was constructed with the −269 to +26 region of a rat p56lck proximal promoter and the pX cDNA, and was microinjected into fertilized ova of Fischer 344/jcl female rats. Six transgenic lines from 114 pups were established. Integration and expression of the transgene were detected by polymerase chain reaction (PCR) and Southern hybridization or by reverse transcriptase-PCR, northern hybridization, and immunostaining.

 Thymic tumours with lethal expansion occurred in 4 of 6 transgenic lines. The tumour consisted of spindle shaped cells. Immunohistochemical and ultra-structural analysis characterized the tumour cells to as epithelial cell type, and in the tumour arose in the medulla. Therefore, the tumour is classified into predominantly epithelial and spindle cell of medullary thymoma (type A of the new World Health Organization classification), as based on the human classification. Tumor occurrence increased in proportion to levels of the pX transcription in the thymus, for each line, and sex distinction was evident regarding rates related to tumour expansion. The transgenic rat model described here is suitable as a model for analysing tumorigenesis in epithelial thymoma occurring in humans.

Keywords: animal model, epithelial thymoma, transgenic rat, HTLV-I pX gene, tumorigenicity

Introduction

Human T lymphocyte virus type I (HTLV-I) is an aetiologic agent of adult T cell leukaemia (Poiesz et al. 1980; Yoshida et al. 1982). Tax protein encoded by a unique pX gene of HTLV-I is a potent transcriptional activator of its own self promoter and is a transactivator or repressor of several host cellular genes (Sodroski et al. 1984; Fujisawa et al. 1985; Yoshida & Fujisawa 1992). Primary T cells transfected with the pX gene can be immortalized (Grassmann et al. 1989), and fibroblast cell lines transfected with the tax gene are transformed in vitro (Tanaka et al. 1990). Tumours can be induced in transgenic animal models carrying pX genes, under the control of various promoters. These include mesenchymal tumours, neurofibromas, mammary carcinomas, and large granular lymphocytic leukaemias (Hinrichs et al. 1987; Nerenberg et al. 1987; Grossman et al. 1995; Yamada et al. 1995). This variety of tumours occurring in these transgenic animals may relate to promoter and/or strain differences.

To investigate the tumorigenicity of the HTLV-I pX gene in lymphoid tissues, we developed a novel transgenic rat model (lck-pX rats), using a rat lymphocyte-specific protein-tyrosine kinase (p56lck) proximal promoter to express the pX gene in lymphoid tissues preferentially. This promoter is mainly active in thymic T lymphocytes (Garvin et al. 1988). When this promoter has been used with oncogenic factors, the development of thymic lymphoma has occurred in transgenic mice (Abraham et al. 1991; McGuire et al. 1992). In our transgenic model, spindle cell thymoma but not thymic lymphoma occurred. We report the characteristics tumour here and discuss the correlation between HTLV-I pX expression and tumorigenicity in the thymus.

Materials and methods

Construction of the transgene

The HTLV-I pX expression vector, pH 2/tax.rex (Yamada et al. 1995), was used as the source of the lck-pX transgene construct. The KpnI/BamHI fragment of a mouse H-2Kd promoter in the pH 2/tax.rex was replaced with the KpnI/BamHI fragment containing a rat p56lck proximal promoter (−269 to +26 bases). The p56lck type I promoter was prepared by polymerase chain reaction (PCR) amplification with a sense primer including KpnI site at the 5′ end (5′-gcaggtaccttgtactgagaagggacagacacc-3′) and an antisense primer including BamHI site at the 5′ end (5′-gaaggatccaattctagtttccactgggtttact-3′) (referred from GenBank accession number Z15029). The adjacent region of the murine p56lck proximal promoter (−269 to +26 bases) was reported to have marked promoter activity (Allen et al. 1992). Figure 1 shows the construct of 3.5 kb KpnI/SalI fragment of the lck-pX transgene used for microinjection.

Figure 1.

Figure 1

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Construction of the lck-pX transgene. The p56lck proximal promoter (−269 to +26) was obtained using PCR. The H-2Kd promoter (KpnI to BamHI) of pH 2/tax.rex (Yamada et al. 1995) was replaced by the p56lck proximal promoter. The 3.5 kb fragment digested from the plasmid by KpnI and SalI was used for microinjection. Positions of pX cDNA hybridization probe and pX PCR primers were as indicated. Hybridization size of the transgene in the host genome with BamHI digestion should be detected at over 3.2 kb, using the pX cDNA probe.

Establishment of transgenic rats

Inbred Fischer 344/jcl (F344) and Sprague-Dawley (SD) strain rats were purchased from SLC Japan (Shizuoka, Japan). Microinjections were given as described elsewhere (Yamada et al. 1995; Yamazaki et al. 1997). Briefly, the lck-pX transgene was microinjected into fertilized one-cell eggs from F344 female rats. Embryos were kept in modified Krebs-Ringer bicarbonate medium (Toyoda & Chang 1974) and transferred to oviducts of pseudo pregnant female SD rats. Integration of the transgene was confirmed by PCR and Southern hybridization, as described (Yamada et al. 1995). Genomic DNA prepared from rat-tails was digested with BamHI and analysed by Southern hybridization. Positions of the pX primers for PCR (Kinoshita et al. 1989) and hybridization probe are given in Fig. 1. Founder rats were mated with normal F344 rats and were subsequently established as transgenic lines in closed colonies. All animal experiments were done according to the Guide for the Care and Use of Laboratory Animals, Hokkaido University Graduate School of Medicine.

Histopathology and immunohistochemistry

Tissues of transgenic rats and nontransgenic rats were fixed in 10% phosphate-buffered formaldehyde and embedded in paraffin blocks. Each 4 µm section was stained with haematoxylin and eosin. For immunohistochemical analysis, we used avidin-biotin immunoperoxidase kits (DAKO, Glostrup, Denmark). Three specific antibodies for epithelial cells, two polyclonal rabbit antikeratin/cytokeratin (DAKO and Nichirei, Tokyo, Japan) and one monoclonal anticytokeratin (MNF116, DAKO), a monoclonal antivimentin antibody (Vim 3B4, DAKO) for nonepithelial cell marker, and a monoclonal anti-Tax antibody (Lt-4: Tanaka et al. 1991) for detecting the product of the pX transgene were used as primary antibodies. After immunostaining, tissue sections or cultured cells were counter-stained with Mayer's haematoxylin (Merck, Darmstadt, Germany).

Electron microscopy

Preparation of samples for electron microscopy was according to a standard procedure using 1% glutaraldehyde and 4% formaldehyde solution for fixation, and 1% osmium tetroxide solution for post-fixation. After preparing ultra-thin section from tissue embedded in Epon 812, the section was stained with lead citrate and uranyl acetate and examined under an electron microscope (H600, Hitachi, Tokyo, Japan).

RNA analysis

Expression of the transgene was analysed using northern hybridization and reverse transcriptase (RT)-PCR with DNase pretreatment, as described (Yamada et al. 1995; Yamazaki et al. 1997). Total RNAs were extracted from each tissue using RNA extraction kits (ISOGEN, Nippon Gene, Toyama, Japan). A hybridization probe and a pX PCR primer pair used to detect the transgene were also used as probe and primer pair for mRNA detection. Northern hybridization was done according to a standard procedure. A BglII fragment of the rat β-actin cDNA (Nudel et al. 1983) was used as a hybridization probe, as an internal control. Rat β-actin primers (sense 5′-atgggtcagaaggactcctacgt-3′, antisense 5′-aggcagctcatagctcttctccag-3′), as an internal control, were used for RT-PCR. For semiquantification of the pX mRNA expression, a competitive RT-PCR, using a PCR MIMIC construction kit (CLONTECH, Palo Alto, CA) was done as described by Siebert & Larrick (1992) and Katsumata et al. (1999). The pX and β-actin MIMIC DNA competitors were obtained by PCR amplification, using composite primers for the pX(sense 5′-atcccgtggagactcctcaacaagtttcgtgagctgattg-3′, antisense 5′-aacacgtagactgggtatcctctgtcaatgcagtttgtag-3′) and for β-actin (sense 5′-atgggtcagaaggactcctacgtcgcaagtgaaatctcctccg-3′, antisense 5′-aggcagctcatagctcttctccaggggacaagatactcatctgc-3′). An equal volume of target cDNA was added to each of a series of tubes containing serially diluted MIMIC DNA competitors. The DNA mixture was amplified by PCR containing pX or β-actin specific primers (described above). The density of PCR products (originating from the target cDNA and MIMIC DNA competitors) on an agarose gel stained with ethidium bromide was measured and analysed using a flatbed scanner (HP Scan Jet 3C/T, Hewlett-Packard, Palo Alto, CA) and NIH image. After measuring molecules of target pX and β-actin cDNA in each competitive RT-PCR according to the protocol given the MIMIC kit, the ratio of molecules between pX and β-actin mRNAs was calculated, for each sample.

Statistical analysis

Comparisons of survival time among transgenic rats were made using the Kaplan-Meier method and were evaluated using the log-rank test. The total white blood cells, segmented neutrophils, and lymphocytes in peripheral blood samples were counted and statistically analysed using one factor anova.

Results

Establishment of transgenic rat lines

Using a microinjection technique, we introduced the lck-pX transgene (Fig. 1) into ova of F344 strain rats and 114 pups were obtained. To detect the transgene, genomic DNAs of their tails were analysed by polymerase chain reaction (PCR), using pX specific primers. Eight rats were pX positive and 6 transgenic lines (Tg3, 33, 38, 64, 80, and 99) were established in each closed colony. The transgene segregated according to Mendelian rules, in each line. Southern hybridization with the pX probe showed various sizes and number of bands among the lines, except for Tg80 (Fig. 2). Accumulation of the 3.5 kb common band, which is the same molecular size as the transgene (see Fig. 1), indicated the tandem integration of a number of transgenes in each host genome. At least one band of other sizes included the last transgene of the tandem integrated transgene. Other bands indicated that the transgene was integrated into multiple sites of the host genome. Results of Southern hybridization suggested that the transgene was integrated into at least two sites, including tandem integration, in these transgenic lines. In the Tg80 line, the transgene was detected by PCR but not by Southern hybridization. Since the region detected by PCR primers is not identical with that of the pX probe (Fig. 1) and no PCR product was amplified using other pX primers designed in the pX probe region (data not shown), the transgene would probably be truncated in the Tg80 line.

Figure 2.

Figure 2

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Southern analysis of the transgene in lck-pX rats. DNAs extracted from the tails of each line were digested with BamHI. The radioactively labelled pX cDNA was used as a hybridization probe. Various numbers and sizes of bands are shown and the 3.5 kb band (arrowhead) is prominent in all lck-pX lines except Tg80. λDNA with HindIII digestion was used as a marker of molecular size.

Development of thymomas in lck-pX rats

At over 8 weeks of age, severe dyspnea usually led to death of the lck-pX rats, especially in the Tg38 line. In these rats, large tumours, which expanded in the anterior mediastinum (Fig. 3a), compressed adjacent mediastinal organs, such as heart, lung, trachea and bronchi yet invasion was not evident in these neighbouring organs. Cross-sections showed that the tumour was well defined and lobulated by fibrous septa. Several cystic cavities were also present (Fig. 3b). Microscopically, tumour cells showed medullary growth with small nodular, storiform and whirly patterns (Fig. 3c) and were predominantly composed of spindle-shaped cells similar to thymic epithelium. Although infiltrating lymphocytes in the tumour were few in number, they were present in marginal areas of the tumour. On the other hand, small tumour nodules were observed in the thymus of young transgenic rats before the occurrence of dyspnea, and they were located exclusively in thymic medulla. Hence the tumour may have developed in the medulla, de novo (Fig. 3d). Thymomas occurred in 4 of 6 lines (Tg3, 38, 64, and 99), at different frequencies but were microscopically similar. Metastasis of the tumour was never evident in any organ, regardless of lck-pX lines.

Figure 3.

Figure 3

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Development of thymoma in lck-pX rats. A lethally expanded tumour in the anterior mediastinum of a Tg38 male rat at 13 weeks of age is shown (a, arrowhead). Cross section of the tumour shows that the tumour is well circumscribed with multiple cystic spaces and thick septa (b). Microscopically, the tumour shows small lobular, storiform and whirly patterns and tumour cells were predominantly composed of spindle-shaped cells. A few small lymphocytic cells are intermingled within spindle tumour nests (c) (HE staining, original magnification: ×60). A small tumour (arrowhead) located in the medulla in a 15-week-old-lck-pX rat before development of any clinical symptoms (d) (HE staining, original magnification: ×25).

In lck-pX rats with large thymomas, marked neutrophilia was evident in peripheral blood samples (Fig. 4). There was no proliferation of lymphocytes and morphologically abnormal lymphocytes were not seen.

Figure 4.

Figure 4

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Neutrophilia in peripheral blood of lck-pX rats with thymoma. Number of total white blood cells (WBC), segmented neutrophils (Seg) and lymphocytes (Ly) between Tg38 rats with thymomas (n = 16; black bars), Tg38 rats without thymomas (n = 8; grey bars), and age-matched control rats (n = 35; open bars) were compared. There was a significant number of WBC and Seg in the Tg38 line with thymoma compared with findings in other rats (one-factor anova; p < 0.0001). Abnormal lymphocytes were not found in the cytological examination.

Immunohistochemical and ultra-structural characteristics of the thymoma

Immunohistochemically, a product of the pX transgene was evident in tumour cells in the thymus, detected using the anti-Tax monoclonal antibody (Fig. 5a). Although the transgene was integrated and was detected via the pX message in the thymus without thymoma (described below), no immunohistochemical positive staining was evident in non-tumour cells. The tumour cells stained positively with all three antibodies against the epithelial cells used (Fig. 5b), but not with an antivimentin monoclonal antibody (data not shown). Electron microscopically, the thymoma cells had a spindle to polygonal appearance and a round to irregularly shaped nucleus with a heterochromatic pattern (Fig. 5c). Desmosomes and tonofibrils were evident (Fig. 5d). The collective evidence indicated that the tumour was of thymic epithelial origin.

Figure 5.

Figure 5

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Immunohistochemistry and ultrastructure of the thymoma. Spindle-shaped tumour cells were uniformly stained using the anti-Tax monoclonal antibody (a) (original magnification: ×120). Positive staining using a monoclonal anticytokeratin antibody (MNF116) staining is shown (b) (original magnification: ×60). Electron microscopic findings of tumour cells (c) (original magnification: ×2500). Desmosomes between tumour cells (arrowhead) and tonofibrils (arrow) in the cytoplasm are clearly evident (d) (original magnification: ×50 000).

Incidence of thymomas with lethal expansion among lck-pX lines between the sexes

Survival curves among the lck-pX lines differed significantly. The mortality at age 30 weeks for rats in each line was 1%, 10%, 24%, and 78% in Tg3, 99, 64, and 38, in that order of frequency (Fig. 6a). In all the dead rats, the lethal expansion of large thymomas was evident (Fig. 3a), thus indicating that the mortality usually reflected the incidence and growth of thymomas in the lck-pX rats. Thymoma occurred most frequently and developed rapidly in the Tg38 line, and over 90% of the rats died within 12 months. The incidence of thymoma in these lck-pX rats also differed with gender as the male rats had a significantly higher rate of thymoma than the females in the Tg38 line (Fig. 6b, log-rank test, p = 0.002), and in Tg3 and 64 lines (data not shown).

Figure 6.

Figure 6

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Survival curves for 4 lck-pX rat lines, Tg3 (n = 244), Tg38 (n = 142), Tg64 (n = 181) and Tg99 (n = 64) are shown (a). Lethally expanded large thymomas were confirmed in all the dead rats and mortality was significantly different among each line (log-lank test; p < 0.0001). Survival curves of males (n = 47) and females (n = 65) of the Tg38 line are shown (b). The mortality rates differed, significantly (log-lank test; p = 0.002).

Expression of the transgene in thymus and thymoma

To examine the influence of the transgene on the induction of thymoma, we investigated pX mRNA expression in various tissues of the lck-pX rats, using northern hybridization. The expression was detected only in thymoma and thymus (Fig. 7). The pX mRNA expressed very high levels in thymomas but very low levels in the unaffected thymus. While no significant signal was detected in other organs, the pX mRNA expression was evident in all organs tested, determined using RT-PCR (data not shown). To determine if the incidence of thymoma would correlate with pX expression among lck-pX lines, we quantified levels of the pX mRNA expression in the thymus, using competitive RT-PCR (Fig. 8). The pX mRNA was detected in all lines including 2 lines with no thymoma (Tg33 and 80). However, the degree of pX expression relatively correlated with the incidence of thymomas among lines, Tg3, 99, 64 and 38 (Figs 6a and 8).

Figure 7.

Figure 7

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Detection of mRNA expression of the transgene by northern hybridization. Total RNAs from each organ, except thymoma, were prepared from 6 week old Tg38 male rats. Thymoma samples were prepared from 30 week old Tg38 male rats. A 2.1 kb band of the pX mRNA (arrowhead) is clear in case of thymoma and faintly in the thymus, but not in other organs tested. A β-actin probe was used as an internal control. Lane 1; liver, 2; spleen, 3; heart, 4; lung, 5; kidney, 6; brain, 7; lymph node, 8 and 9; thymus, 10 and 11; thymoma.

Figure 8.

Figure 8

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Semi-quantification of the pX mRNA expression in thymuses of each line. Levels of the pX mRNA expression were measured by competitive RT-PCR. Total RNAs were extracted from the thymus of male rats of each line at 6 weeks of age and from thymoma (38T) of a Tg38 male rat at 30 weeks of age. All these tissues were histopathologically confirmed to be thymoma-free. The ratios of pX and β-actin molecules in each sample are shown. The percent mortality of each line at 30 weeks of age is shown in parentheses.

Discussion

With transfer of the lck-pX transgene in rats, we established a unique rat model of thymoma which has several characteristics that are seen in human thymomas. Few animal models are available to study the development of human thymoma. As a spontaneous thymoma model, the BUF/Mna rat has been described (Yamada et al. 1973). This model has a lymphocytic thymoma, including a large number of non-neoplastic lymphocytes. Other animal thymoma models have been generated by gene manipulation and most are transferred with an oncogene such as myc or SV40 T antigen (Spanopoulou et al. 1989; Lee et al. 1998). Recently, Nakajima et al. (2000) demonstrated that heritable lympho-epithelial thymoma was induced in a line of transgenic mice by insertional mutation of a transgene. However, our thymoma model is unique in terms of morphological and functional features. Spindle cell thymoma with marked neutrophilia in our rats was not noted in other models.

Based on morphological features, human thymoma has been variously classified (Shimosato & Mukai 1997). (1) In general, thymoma is divided into predominantly lymphocytic, predominantly epithelial, and mixed lymphoepithelial types according to the predominance of cellular components (Katzin et al. 1988). (2) Cytologically, thymoma has also been subclassified into polygonal cell type, spindle cell type, and mixed cell type, based on aspects of the neoplastic epithelial cells. (3) Müller-Hermelink et al. (Marino & Müler-Hermelink 1985; Kirchner & Müller-Hermelink 1989) subtyped thymoma into medullary, mixed, predominantly cortical, cortical, and well-differentiated thymic carcinoma based on the histogenesis of epithelial components. (4) Levine & Rosai (1978) simply classified thymic epithelial tumours according to biological behaviour; circumscribed thymoma (benign), malignant thymoma type I (invasive thymoma with no or minimal atypia), and malignant thymoma type II (thymic carcinoma). Recently, a World Health Organization (WHO) committee achieved consensus to use the morphologic criteria, which was classified into type A, AB, B1, B2, B3 and C (Rosai 1999). According to these classifications, the thymoma in our model can be classified into predominantly epithelial, spindle cell and medullary types, and type A of the WHO-approved Histological Classification of Tumours of the Thymus. Since invasion and metastasis were not evident in our rats, the thymoma is categorized into benign circumscribed thymoma. However, the thymoma in our rats led to a poor prognosis as severe dyspnea and death occurred due to rapid tumour growth in the thoracic cavity.

Thymoma is often associated with a large number of systemic disorders, nearly all of which are immune-related, such as myasthemia graves (MG), hypogammaglobulinemia, pure red cell aplasia (reviewed by Müller-Hermelink & Marx 2000). The most common type is associated with MG which occurs in about 30% of humans with thymomas. This is the WHO type B. Hypogammaglobulinemia (about 12% of thymomas) and pure red cell aplasia (about 5%) are mainly associated with type A thymomas (Rosai 1996; Müller-Hermelink & Marx 2000). However, autoimmune diseases did not develop in our lck-pX rats. We did observe marked neutrophilia in addition to thymoma, as was noted previously in an HTLV-I pX transgenic rat model prepared using a mouse H-2 class I promoter, which was mammary carcinoma with neutrophilia (Yamada et al. 1995). Evidence in this model suggested that up-regulation of the interleukin-8 family cytokines by the HTLV-I pX gene in the tumour cells could account for the marked neutrophilia.

Tax is a major tumorigenic protein encoded by the pX gene (Tanaka et al. 1990). This protein functions as a trans-activator or repressor independent on its integration site. Since histologically identical thymoma developed in 4 different lck-pX rat lines and Tax protein was evident in the thymoma cells, the pX gene expression considered to be responsible for induction of thymoma. The endogenous p56lck proximal promoter is specifically active in thymic lymphocytes (Garvin et al. 1988). The region between positions −565 and −433 is potentially important to express the gene with cell specificity (Allen et al. 1992; Leung et al. 1993). In humans, cell specificity is mainly regulated by repressor binding to the TTTCATCAG sequence located at positions −474 to −466 of the promoter in humans (Muise-Helmericks & Rosen 1995). Since the promoter region of our transgene construct did not contain the region with this repressor sequence, the pX gene could be expressed at transformable levels in thymic medullary epithelial cells and then epithelial thymoma occur and semiquantitative analysis showed a relative correlation between tumour occurrence and levels of pX expression in the thymus. High levels of pX expression in the thymus may augment the probability of transformation and clonal proliferation and may lead to the occurrence of thymoma. The mortality of lck-pX rats as a result of tumour expansion also correlated with levels of pX expression and there were gender differences, male lck-pX rats had a higher mortality than did the females. It has been reported that the thymus is a target organ for sex-related hormones (Nilsson et al. 1990). Seiki & Sakabe (1997) reported that in male BUF/Mna strain rats there was a rapid growth of thymoma and oestrogen inhibited the onset of thymoma. Although the morphological features of the thymoma in our rats is distinct from the lymphocytic thymoma seen in BUF/Mna rats, the gender dependent survival rates in our lck-pX rats may depend on oestrogen or other sex-related hormones.

Thus in summary, the lck-pX rats described here may serve as an animal model to study tumorigenicity of the HTLV-I pX gene, and may also serve as a model for analysing tumorigenic processes of epithelial thymoma originating from medullary epithelial cells in humans.

Acknowledgments

This work was supported in part by grants from the Ministry of Education, Science, Technology, Sports and Culture of Japan. We thank Tsutomu Osanai and the entire staff of the Institute for Animal Experimentation, Hokkaido University Graduate School of Medicine, for maintenance of the transgenic rats, Chisato Sudo and Masayo Tateyama for technical assistance, and Mariko Ohara for language assistance.

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