Targeted expression of Tert to the thymus: Lck-Tert mice.
We generated Lck-Tert mice, transgenic mice that express the murine Tert cDNA under the control of the p56lck proximal promoter (see Materials and Methods), which targets Tert overexpression to thymocytes and peripheral T cells (Fig. ). A total of four independent Lck-Tert transgenic lines (M15, M17, M40, and M41) were obtained. (For mouse generation, genotyping, and mouse housing, see Materials and Methods.)
To determine Tert mRNA levels in thymocytes from both transgenic and wild-type littermates, we performed real time RT-PCR using primers directed against the mouse Tert coding sequence (Fig. , A primers) (see Materials and Methods). These primers amplify both endogenous and transgenic Tert mRNA. Lck-Tert mice of the M15 line showed Tert mRNA levels that were only slightly (1.5 ± 0.9-fold) increased over those for wild-type controls (not statistically significant), suggesting that the transgene is not expressed in this Lck-Tert line (Fig. ). The absence of transgenic Tert expression in the M15 line could be due to the insertion of the transgene in the Y chromosome (with a high heterochromatin content), as suggested by the fact that only males carried the transgene (data not shown). In contrast, Lck-Tert mice of the M17, M40, and M41 lines showed 129- to 789-fold more Tert mRNA in the thymus than their wild-type littermates (Fig. ), indicating that the transgene was being vastly overexpressed (see below).
To study the whole-body expression pattern of the Lck-Tert transgene, Tert mRNA levels were determined in a variety of tissues from Lck-Tert mice and wild-type littermates. Figure shows Tert mRNA levels for the M40 line. Tert overexpression was largely specific to the thymus, although it was also found in other lymphoid tissues from Lck-Tert mice, such as the spleen (7-fold increase in Tert expression over that for wild-type mice) and lymph nodes (40-fold increase), as well as in the brain (16-fold increase) (Fig. ). The rest of the Lck-Tert tissues analyzed showed no or very low increases in Tert mRNA levels over those in the controls (Fig. ). To demonstrate that the transgenic Lck-Tert mRNA was expressed in these different tissues, we performed real-time RT-PCR using primers specific for the transgenic Lck-Tert mRNA that do not amplify the endogenous Tert mRNA in Lck-Tert mice (hGHp2 primers) (see Fig. and Materials and Methods). Transgenic Lck-Tert mRNA was abundantly expressed in the thymuses, spleens, and brains of M17 Lck-Tert mice, showing 100,000, 45,100, and 1,000 arbitrary units of expression, respectively (Fig. ), but was undetectable in wild-type littermates of the same line (data not shown). We next determined Tert mRNA levels in isolated T- and B-cell populations from the different lymphoid organs of Lck-Tert mice (see Materials and Methods). As shown for the M17 line in Fig. , Tert mRNA levels were increased in all T-cell populations of the Lck-Tert thymus compared to the wild-type thymus, and double-positive T cells (CD4+
) showed the highest overexpression of Tert mRNA levels. In the case of the spleen and lymph nodes, Tert mRNA levels were increased in T cells (both CD4+
) compared to wild-type controls (Fig. ) (see Materials and Methods). In addition, we performed real-time RT-PCR using hGHp2 primers. Transgenic Lck-Tert mRNA was abundantly expressed in the different T-cell populations of the thymus, with the highest abundance in double-positive T cells, as well as in the T-cell populations of the spleen and lymph nodes (both CD4+
) (Fig. ). Importantly, B cells (positive for the B220 marker) and immune cells that did not stain with B- or T-cell markers (non-B, non-T cells) showed only background levels of Lck-Tert mRNA expression (Fig. ). These results indicate that transgenic Lck-Tert expression is specifically targeted to thymocytes and peripheral T cells, in agreement with the known expression pattern of the p56lck proximal promoter (30
Increased telomerase activity in Lck-Tert thymocytes compared to control wild-type thymocytes.
We measured telomerase activity in thymuses from at least two pairs of Lck-Tert and wild-type littermates for each of the different transgenic lines by using the TRAP assay (Fig. ) (see Materials and Methods). Mice of line M15, with null transgenic Lck-Tert mRNA expression, did not show higher TRAP activity in the thymus than wild-type littermates (Fig. ). TRAP activity was increased in the thymuses of M40, M41, and M17 Lck-Tert mice compared to that in their wild-type littermates from the same lines, as indicated by increases in the intensities of the TRAP products (Fig. ) (see Materials and Methods). TRAP assays were performed under linear conditions of product amplification, which allowed for quantification of TRAP activity levels. The Lck-Tert/wild-type TRAP signal ratios were 0.65 ± 0.14, 1.53 ± 0.08, 2.08 ± 0.76, and 3.27 ± 2.62 for the M15, M40, M41, and M17 lines, respectively (see Materials and Methods).
FIG. 2. Telomerase activity as detected by the TRAP assay in the thymuses of wild-type and Lck-Tert littermates of the indicated mouse lines. The S-100 extract concentrations assayed are indicated. − or +, extracts not treated or treated with (more ...)
The fact that TRAP activity is approximately twofold higher in Lck-Tert thymuses than in wild-type controls is surprising given the fold overexpression of Tert mRNA in transgenic thymuses, described above (Fig. ). This fact could be explained if one considers that wild-type thymocytes already contain active telomerase, and it is likely that the vast overexpression of Tert in transgenic cells is saturating the formation of active telomerase complexes with the telomerase RNA component (Terc), which may be rate-limiting under these conditions.
Similar telomere length distributions in wild-type and Lck-Tert lymphoid tissues.
We measured telomere lengths in thymocytes and peripheral T cells derived from age-matched (6 week-old) Lck-Tert mice and wild-type littermates. For this purpose, we determined telomere fluorescence after hybridization with a peptide nucleic acid-telomeric probe by using Q-FISH and flow-FISH (see Materials and Methods). Q-FISH measures the lengths of all individual telomeres on metaphase spreads and allows distributions of telomere length frequencies to be obtained (Fig. ). Measurement of the telomere length of each individual chromosome is essential, because telomerase overexpression may alter the normal telomere length distribution in Lck-Tert cells from that in wild-type controls. In particular, short telomeres are more likely to be deregulated as a consequence of telomerase overexpression, since telomerase has been reported to act preferentially on critically short telomeres (19
). In turn, the shortest telomeres are critical for cell viability and chromosome stability (19
). The histograms of telomere length frequencies for primary thymocytes and lymph node cells from Lck-Tert and wild-type mice, however, showed no significant differences between genotypes (Fig. ). In particular, in both thymocytes and lymph node cells, the frequencies of short and undetectable telomeres (signal-free ends) are similar for Lck-Tert and wild-type mice (Fig. ). These results indicate that Tert overexpression does not alter the normal distribution of telomere lengths in Lck-Tert primary thymocytes.
FIG. 3. Telomere lengths in lymphoid tissues from wild-type and Lck-Tert littermates from the indicated mouse lines. (A) Histograms showing telomere length frequencies in lymphocytes and lymph node cells derived from wild-type and Lck-Tert mice as determined (more ...)
We also measured telomere length in lymphoid tissues from Lck-Tert and wild-type littermates by using telomere flow-FISH, a technique that combines flow cytometry with telomere Q-FISH and that has been proven to be useful in determining telomere length in lymphoid cells (see Materials and Methods). Flow-FISH was performed on freshly isolated thymocytes, splenocytes, and lymph node cells from age-matched Lck-Tert mice and wild-type littermates of each of the indicated transgenic lines (Fig. ). In this case, 24-month-old animals were chosen for the analysis so as to detect any possible telomere length differences between genotypes with aging. Telomere fluorescence values for individual Lck-Tert and wild-type mice from the M40 and M41 mouse lines, however, showed no significant differences between genotypes, in agreement with the Q-FISH results (P > 0.05 by Student′s t test in all cases) (Fig. ).
As an independent technique, not based on fluorescence, to estimate telomere length, we performed TRF analysis, which is based on visualization of telomeric DNA fragments by Southern blotting (see Materials and Methods). The average size of the TRF smear is indicative of the average telomere length. Again, no obvious differences in TRF product size were detected between Lck-Tert mice and wild-type littermates for any of the lymphoid tissues studied (Fig. ; littermates from the different genotypes are grouped for better comparison).
Taken together, these findings indicate that constitutive Tert expression in T cells from Lck-Tert mice does not result in a significant lengthening of telomeres compared to those from age-matched wild-type littermates. These results agree with those obtained for a different Tert transgenic mouse model, K5-Tert mice, with a telomere length similar to that of the corresponding wild-type controls (16
). The absence of telomere lengthening in Lck-Tert cells agrees with the notion that telomerase preferentially elongates those telomeres that have lost telomere capping but does not result in further elongation of normal-length telomeres (19
Increased frequency of spontaneous lymphoma in Lck-Tert mice compared to that in age-matched wild-type littermates.
To analyze the impact of Tert constitutive expression in T cells, we maintained large mouse colonies of the M15, M40, and M41 mouse lines (including both Lck-Tert mice and wild-type littermates) for at least 2 years (Table ; Fig. ). Lck-Tert mice of the M15 line were used as negative transgenic controls with null Lck-Tert mRNA expression. Age-matched (2-year-old) Lck-Tert and wild-type littermates of the different lines were sacrificed in parallel and subjected to full-body histopathological analysis (see Materials and Methods). This approach allowed comparison of spontaneous tumor rates and tumor progression between age-matched Lck-Tert mice, which expressed (M40 and M41) or did not express (M15) transgenic Tert, and the corresponding wild-type littermate controls.
Tumor spectrum in 24-month-old wild-type and Lck-Tert micea
FIG. 4. Spontaneous-lymphoma incidence in 24-month-old wild-type and Lck-Tert mice. (A) Percentage of mice of each genotype affected with lymphoma in the indicated mouse lines. The average number of wild-type mice affected with lymphoma from the different transgenic (more ...)
Neoplastic lesions were classified according to the cell type of origin and the type of lesion (Table ). The percentage of mice that presented each type of lesion was determined for the different mouse lines and genotypes. Only the incidence of lymphomas was reproducibly higher in Lck-Tert mice than in their wild-type littermates; no differences were observed for other tumor types (see Fig. for statistical calculations). The incidence of bronchoalveolar carcinoma was increased in Lck-Tert mice of the M15 and M40 lines compared with that for their wild-type littermates; however, this lesion did not coincide with Lck-Tert mRNA expression (i.e., the lesion is present in M15 Lck-Tert mice, which do not express transgenic Lck-Tert mRNA [Fig. ]) and thus is not a direct consequence of Tert constitutive expression. In contrast, increased lymphoma incidence was observed in Lck-Tert mice of the M40 and M41 lines, with high transgene expression, but not in the M15 line, which did not express the transgene. In particular, 21.7 and 14.2% of Lck-Tert and wild-type mice, respectively, were affected with lymphoma in the M41 line; for the M40 line, these percentages were 33.3 and 11.1%, respectively (Table ; Fig. ). In addition, most of the lymphomas present in Lck-Tert mice were macroscopic at the time of sacrifice, while lymphomas in wild-type mice could be diagnosed only after histopathological analysis (see Fig. for thymus and spleen sizes, as well as for histopathological diagnoses, of wild-type and Lck-Tert mice of the M40 line at the time of sacrifice). None of the Lck-Tert mice of the M15 line, which were null for transgene expression, showed spontaneous lymphoma, compared with an average of 14.2% of wild-type mice (Table ; Fig. ). In addition, wild-type and Lck-Tert mice from the M40 line were left to age in the animal facility in order to study spontaneous-lymphoma incidence at the time of death. In agreement with the results obtained for mice sacrificed at the age of 2 years, 33.3% of Lck-Tert mice showed macroscopic lymphoma lesions at the time of death, compared to only 18% of the wild-type controls. Importantly, telomere lengths were similar in tumor cells isolated from wild-type and Lck-Tert T-cell lymphomas (see Fig. ), suggesting that the increased lymphoma incidence in Lck-Tert mice is not the consequence of longer telomeres in lymphoma cells in these mice.
FIG.5. (A) Quantification of thymus and spleen weights (corrected by body size), with histopathological diagnosis, for each 2-year-old wild-type and Lck-Tert mouse of the M40 line. The average organ weight and standard deviation for each genotype is shown above (more ...)
In summary, an increase in spontaneous-lymphoma incidence was detected in two independent lines of Lck-Tert mice over that in age-matched wild-type controls or in the M15 Lck-Tert line, with null transgene expression. Lymphomas were the only tumor type whose incidence was increased in Lck-Tert mice compared to wild-type mice; no reproducible differences in the incidences of other tumor types were observed between genotypes (Table ). These findings suggest that constitutive Tert expression targeted to T cells specifically promotes lymphoma in the context of the organism in the absence of significant differences in telomere length and in agreement with the specific pattern of Tert overexpression.
We also studied whether transgenic Tert expression in Lck-Tert mice resulted in abnormal blood cell counts in these mice. For this purpose, we analyzed peripheral blood in Lck-Tert and wild-type mice at the age of 22 to 27 weeks (Table ). We reproducibly detected slight increases in the numbers of white blood cells and lymphocytes (leukocytosis and lymphocytosis) in Lck-Tert mice over those in wild-type controls (Table ); however, these differences were not statistically significant (P = 0.18 by the chi-square test). Similarly, we detected no differences in red blood cell counts or the blood formula between genotypes (Table ). In addition, we found no detectable organ damage or pathology in these Lck-Tert mice that could be related to abnormal T-cell counts. In particular, no significant differences in the incidence of glomerulonephritis or other autoimmune diseases, associated with abnormal lymphocyte proliferation and accumulation, were detected between genotypes (data not shown).
Hematology analyses of wild-type and Lck-Tert mice
Transgenic telomerase increases the dissemination of Lck-Tert lymphoma to both lymphoid and nonlymphoid tissues.
Histopathological analysis of all the spontaneous-lymphoma cases showed increases in total numbers of both lymphoid and nonlymphoid organs (Fig. ; for examples, see Fig. and Fig. ) affected with lymphoma in LcK-Tert mice compared to those in wild-type controls. Figure shows significant increases in the numbers of both lymphoid and nonlymphoid organs affected with lymphoma in Lck-Tert mice when all Lck-Tert mice that developed lymphoma in lines M40 and M41 are compared with all wild-type controls. In particular, a larger variety of lymphoid tissues per mouse were affected with lymphoma in Lck-Tert mice than in wild-type controls (Fig. ; P
= 0.047 by Student's t
test). In addition, lymphoma in Lck-Tert mice not only affected lymphoid tissues (see Fig. and Fig. for examples) but also affected various nonlymphoid organs such as the liver (Fig. ), lungs, and kidney (Fig. ), which were never affected in the age-matched wild-type mice that developed lymphoma (see Fig. for quantifications; P
= 0.019 by Student's t
test when all Lck-Tert mice of lines M40 and M41 that developed lymphoma are compared with all wild-type controls that developed lymphoma). It is noteworthy that infiltrating lymphomas were distinguished from lymphoproliferative disease based on Bethesda's proposed parameters (24
). First, we evaluated organ architecture, cytology, and dissemination. As can be observed in Fig. , lymphoma lesions typically showed (i) loss of normal tissue architecture (Fig. ), (ii) monomorphic or pleiomorphic lymphoid cell populations containing numerous mitotic figures (Fig. ), and (iii) invasive dissemination of tumor cells in various lymphoid and nonlymphoid tissues (Fig. ). It is noteworthy that we also detected reactive expansions of lymphocytes, which were diagnosed as lymphoid hyperplasias, in 2-year-old mice (see Fig. for examples); however, no significant differences between genotypes were observed in this pathology (data not shown).
Next, we examined the clonal nature (clonality) of lymphomas appearing in Lck-Tert mice, an important parameter for distinguishing between lymphoproliferative disorders and lymphoma (24
). In particular we studied clonality for three different genes: TCRβ, TCRγ, and IgH (Fig. ). For this purpose, we used specific sets of primers and performed PCR analysis of genomic DNA derived from lymphoma-affected organs (see Materials and Methods). In particular, we used a set of primers for TCRβ that amplifies rearrangements in Vβ-DJβ and Dβ-Jβ gene segments (13
), a set of primers for TCRγ that amplifies rearrangements of Vγ-Dγ gene segments (13
), and a set of primers for IgH that amplifies rearrangements of DH-JH gene segments (21
). All Lck-Tert lymphomas showed a clear monoclonal profile for the TCRβ gene (Fig. ), and two out of four Lck-Tert lymphomas were monoclonal for TCRγ (Fig. ). In addition, one out of four Lck-Tert lymphomas was monoclonal for IgH as well as monoclonal for TCRβ (Fig. ). Simultaneous monoclonality for IgH and TCRβ has been described for a small proportion of T-cell malignancies (24
). Taken together, these results indicate a clear monoclonal character for Lck-Tert lymphomas, reinforcing the notion that Lck-Tert mice develop malignant T-cell lymphoma rather than a lymphoproliferative disorder (24
). In addition, the remarkable TCRβ monoclonality of Lck-Tert lymphomas emphasizes their clear T-cell origin.
To evaluate differences between genotypes in the incidence and severity of lymphomas, we calculated a score on the lymphoma severity index for each mouse included in the study (see Materials and Methods) (Fig. ). Lck-Tert mice showed significantly higher lymphoma severity index scores than wild-type controls (Fig. ; P = 0.033 by Student's t test upon comparison of severity index scores of all Lck-Tert and wild-type littermates from lines M40 and M41).
Taken together, these results show that lymphomas appearing in Lck-Tert mice are more aggressive than those in wild-type controls, as indicated by significant increases in the numbers and varieties of lymphoid and nonlymphoid organs affected with lymphoma. These results suggest that Tert overexpression confers a more malignant phenotype on murine lymphoma progression, revealing a novel role of telomerase in sustaining tumor dissemination.
Spontaneous lymphomas in transgenic mice are predominantly of a mature T-cell origin.
If constitutive Tert expression in T cells was responsible for promoting malignant lymphoma in Lck-Tert mice, we should expect the nonlymphoid tissues affected with lymphoma to be highly positive for T-cell markers, such as CD3 staining, but negative for B-cell markers such as B220 and IgM staining. To investigate this question, we first performed immunohistochemistry with anti-CD3, anti-B220, and anti-IgM antibodies on various tissues affected with lymphoma from individual Lck-Tert mice of both the M40 and M41 lines. In addition, to determine whether the lymphomas had a mature origin, we performed immunohistochemistry with an anti-TdT antibody, since TdT is a marker of precursor T- and B-cell lymphomas. The spontaneous-lymphoma lesions present in different tissues from Lck-Tert mice were predominantly positive for the CD3 marker and negative for B220 and IgM, suggesting that they had originated mostly from T cells (Fig. ). For wild-type mice, some of the lymphoma lesions were predominantly positive for B220 staining (Fig. ), suggesting that they had originated mostly from B cells. Furthermore, the negative staining for the anti-TdT antibody in all Lck-Tert lymphomas suggests a mature T-cell origin for these lymphomas (Fig. ).
FIG. 7. Lymphomas were characterized by immunohistochemistry using anti-CD3, anti-B220, anti-TdT, and anti-IgM antibodies, as well as by FACS analysis using anti-CD3, anti-CD4, anti-CD8, and anti-B220 antibodies. (Top and bottom left) Representative images of (more ...)
We confirmed these findings by performing FACS analysis on some of the Lck-Tert lymphomas by using anti-CD4, anti-CD8, anti-B220, and anti-CD3 antibodies. FACS analysis showed that Lck-Tert lymphomas were CD3 positive, B220 negative, and predominantly single positive for the anti-CD4 or anti-CD8 antibody (CD4+ CD8− or CD4− CD8+), again suggesting their mature T-cell origin (Fig. ). In summary, both immunohistochemistry and FACS analyses suggest that Lck-Tert lymphomas are predominantly mature T-cell lymphomas.
T-cell lymphomas in Lck-Tert mice preserve Lck-Tert expression at a higher rate than corresponding normal Lck-Tert tissues.
We next studied whether T-cell lymphomas in Lck-Tert mice preserved transgenic Tert expression. For this purpose, we performed real-time RT-PCR using primers that amplify endogenous or transgenic Tert (A primers or hGHp2 primers, respectively). Most of the lymphoma lesions present in various organs from Lck-Tert mice showed increased Tert mRNA expression levels with A primers compared to those in the corresponding normal Lck-Tert tissues; a ratio of 1 indicates similar Tert mRNA expression in normal and lymphoma tissues (Fig. ). Therefore, Tert expression is preserved or increased in lymphoma lesions relative to expression in corresponding normal tissues in Lck-Tert mice. Similarly, when Lck-Tert-specific primers were used, transgenic Tert mRNA levels were increased in most lymphoma lesions present in Lck-Tert mice compared with those in the corresponding normal Lck-Tert tissues (Fig. ), as would be expected if these lymphoma lesions were mostly derived from Lck-Tert-expressing T cells.
FIG. 8. Real-time quantitative RT-PCR analysis of both Lck-Tert mRNA and total Tert mRNA expression in Lck-Tert lymphomas. (A) Lck-Tert mRNA was quantified by using hGHp2 primers. Histogram shows the ratio of Lck-Tert expression between lymphomas and corresponding (more ...) Normal telomere capping but increased chromosomal damage in Lck-Tert thymocytes compared to wild-type controls.
To investigate the mechanisms by which Tert constitutive expression could be promoting T-cell lymphoma in Lck-Tert mice independently of the known role of telomerase in net telomere elongation, we first studied whether primary thymocytes derived from Lck-Tert mice showed a normal telomere capping function. It is plausible that an excess of Tert in Lck-Tert thymocytes could interfere with the formation of a proper telomere cap in these cells. A hallmark of defective telomere capping is a higher incidence of spontaneous end-to-end chromosome-type and chromatid-type fusions involving telomeric sequences (2
). In the mouse, the most frequent type of end-to-end fusion associated with telomere dysfunction is RLF (7
). In addition, it has recently been shown that loss of telomere capping interferes with proper repair of DNA double-strand breaks (DSB) in the genome, as indicated by the fact that telomerase-deficient mice with critically short or uncapped telomeres are hypersensitive to ionizing radiation (IR) (18
). In particular, critically short telomeres in these mice illegitimately join to IR-induced DSB in the genome, thus increasing chromosomal instability (22
). Furthermore, there has been recent speculation that Tert overexpression may directly interfere with proper DNA repair of lesions in the genome in a way that is independent of telomere capping (5
To address these possibilities, we performed a full karyotypic analysis of ~100 metaphases of primary thymocytes isolated from both wild-type and Lck-Tert mice of two independent mouse lines (M17 and M40) before or after whole-body IR with a single dose of gamma rays (4 Gy) (see Materials and Methods). In the absence of IR, Lck-Tert thymocytes showed increased frequencies of chromatid breaks and TA compared to those for the corresponding nonirradiated wild-type controls (Table ; Fig. ; for examples of aberrations, see Fig. ). However, we did not detect significantly increased frequencies of end-to-end fusions involving the telomeres (Robertsonian-like fusions [RLF] plus multicentrics plus rings) in Lck-Tert thymocytes compared to those in controls (Table ; Fig. ). Significantly, no RLF or dicentrics were detected in nonirradiated Lck-Tert thymocytes from two different Lck-Tert mouse lines (M17 and M40) (Table ; Fig. ), indicating that telomere function was intact in these cells and that, therefore, overexpressed Tert does not interfere with the formation of a proper telomere capping structure.
Chromosomal aberrations in primary thymocytes from irradiated or nonirradiated Lck-Tert and wild-type mice
FIG. 9. (A) Frequencies of chromosomal aberrations per metaphase. Lck-Tert (blue bars) and wild-type (red bars) thymocytes from two independent mouse lines (M17 and M40) were analyzed in the absence of IR (0 Gy) or after IR (4 Gy). Asterisks indicate highly significant (more ...)
Upon gamma irradiation, Lck-Tert thymocytes from two independent mouse lines (M17 and M40) showed significantly increased frequencies of fusions (particularly dicentrics and tricentrics) and fragments compared to those for similarly irradiated wild-type controls (Table ; Fig. ; see Fig. for representative examples). However, these fusions never showed telomeric TTAGGG repeats at the fusion, again indicating proficient telomere capping.
Altogether, Lck-Tert thymocytes show greater spontaneous and IR-induced chromosomal instability than wild-type controls. In addition, a higher percentage of metaphases showed chromosomal aberrations in Lck-Tert thymocytes than in wild-type controls (Table ). However, the fact that telomeric sequences are not involved in these aberrations indicates that Tert overexpression does not directly interfere with proper telomere capping; instead, Tert overexpression may interfere with DNA repair of DSB in the genome or with the cellular response to DNA damage.