Defective expression of Notch1 and Notch2 in connection to alterations of c-Myc and Ikaros in
-radiation-induced mouse thymic lymphomas
P. López-Nieva,
J. Santos and
J. Fernández-Piqueras1
Laboratorio de Genética Molecular Humana, Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, E-28049 Madrid, España
1 To whom correspondence should be addressed Email: jf.piqueras{at}uam.es
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Abstract
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Gamma-radiation-induced thymic lymphomas constitute a heterogeneous group of T-cell lymphomas. Some tumour suppressor genes and oncogenes have been shown to be defective in a fraction of such lymphomas, yet a considerable number of these remain elusive in terms of gene alterations. In the present work we present evidence that
-radiation-induced thymic lymphomas in (C57BL/6 J x BALB/c) F1 hybrid mice often exhibit increased levels of Notch1 expression, but, contrary to what was expected, they also exhibit a clearly reduced Notch2 mRNA expression, suggesting a cooperative antagonism of these genes. These results represent the first reported instance for the involvement of Notch2 inactivation in the development of thymic primary tumours while confirming the role of Notch1 as an activated oncogene. Additional analyses revealed that c-Myc over-expression and partial inactivation of Znfn1a1/Ikaros appear to be relevant events some how coupled to alterations in Notch genes inducing these kinds of tumours.
Abbreviations: RTPCR, reverse transcriptionPCR
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Introduction
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Gamma-radiation-induced thymic lymphomas consist of a heterogeneous group of T-cell lymphoblastic lymphomas characterized by the presence of immature T lymphocytes. Several papers have shown inactivation of p53 (1), p15/INK4b and p16/INK4a (2,3), p73 (4), p19/ARF (5), Pten and Cd95/Fas (6), Znfn1a1/Ikaros (7) and Rit1/Bcl11b (8) genes, in
-radiation-induced mouse lymphomas.
In terms of oncogene activation, a minor fraction of these tumours were found to have their N- and K-ras oncogenes activated, and most of them exhibited trisomy of chromosome 15, where c-Myc is located (911). Still oncogene activation remains unknown in a considerable fraction of these tumours. There exists previous evidence of the involvement of Notch1 in human and mouse T-cell lymphomagenesis (see ref. 12 for a review). Additionally, Tsuji et al. (13) have shown that Notch1 also participates in the development of mouse thymic lymphomas induced by ionizing radiation. However, other Notch family genes, such as Notch2, which is over-expressed in feline leukaemia virus-induced thymic lymphomas (14), appears to have little or no effect on radiation-induced thymic lymphomagenesis.
On the other hand, there exists experimental evidence in transgenic mouse models that Notch1 may collaborate with some partners such as c-Myc (15) and Ikaros (16) to cause T-ALL. These partners have not been identified as of this writing in primary tumours induced in genetically unmodified mice.
The aim of this work was to find out whether Notch1 and Notch2 are involved in the development of primary thymic lymphomas induced by
-radiation in a panel of (C57BL/6 J x BALB/c)F1 hybrid mice, and if so, to analyse c-Myc and Ikaros in order to explore the nature of this cooperative effect in primary thymic-lymphomas.
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Material and methods
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Mice and tumour induction
C57BL/6 J and BALB/c mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Thymic lymphomas were induced in (C57BL/6 J x BALB/c)F1 mice, aged 3565 days, by whole body
-irradiation split into 4 weekly doses of 1.75 Gy, starting with 3560-day-old mice. Thymic lymphoma development was observed at weekly intervals for 12 weeks following
-irradiation. Mice were killed when they showed obvious signs of thymic lymphomas, such as enlargement of spleen or lymph nodes detectable by palpation, hunched posture, and shortness of breath, ruffled fur or lethargy. Survivors were killed at the end of the latency period (26 weeks). Thymus tissue obtained from
-treated animals was staged for thymic lymphoma development by using thymus weight, histology and serology of T-cell markers as described in the literature (9,17). Animals with primary tumours were identified at necropsy by the presence of an enlarged thymus mass (>100 mg), T-cell lymphoblastic lymphoma, and a serological profile indicating higher accumulation of immature double positive (CD4+ CD8+) or double negative (CD4 CD8) T-lymphocytes (data not shown).
Expression analysis of Notch1, Notch2 and c-Myc
The determination of the transcript levels of these genes was performed by quantification assays on the basis of real-time reverse transcriptionPCR (RTPCR). RTPCR of the gene encoding the tubulin beta 5 chain (Tubb5) (http://www.informatics.jax.org) was used as internal control of the RNA quality and amplification.
The quantification of mRNAs from Notch1 and Notch2 was performed with a LightCycler instrument (Roche Diagnostics, Penzberg, Germany) by use of the SYBR Green I method. PCR products were labelled with the SYBR Green I fluorophores that binds to all double-stranded DNA. Primers for Notch1 and Notch2 were those described in Liu et al. (22). Primers for Tubb5 were: 5'-TGGGACTATGGACTCCGTTC-3' (forward), and 5'-AAAGCCTTGCAGGCAATCA-3' (reverse). 400 ng of total cellular RNA, extracted by the TriPure Isolation Reagent method (Roche), were used to generate cDNA in a reaction containing 3.25 mM of Mn(OAc)2, 0.3 µM of each primer, and 1x LC RNA Master SYBRGreen I mix (Roche). The cDNAs of Notch1 or Notch2 and Tubb5 were subsequently co-amplified under the following PCR conditions: 95°C for 2 min followed by 40 cycles with 95°C for 10 s, 55°C for 30 s and 72°C for 15 s (in this step the online measurements took place). The Notch expression level in each sample was calculated with the calibrator normalized relative quantification method of the LightCycler Relative Quantification software (Roche). In this quantification method the result is expressed as the target (Notch1 or Notch2 in our case)/reference (Tubb5 in our case) of each sample divided by the target/reference ratio of the calibrator (a sample that is used for the normalization of the results).
For determination of the c-Myc transcript levels the quantification was performed by use of the fluorescence-resonance energy-transfer (FRET) hybridization technique. This method works with two oligonucleotide probes that bind to the target DNA. In our case the 3' probe had a donor fluorophore (fluorescein) at the 5' end whereas the 5' probe had an acceptor fluorophore (LightCycler-Red 640 or LightCycler-Red 705) at the 3' end. When the probes bind to target DNA next to each other, the dyes are close enough for FRET to occur. Primers and FRET probes were as follows: for c-Myc, 5'-CACCAGCAGCGACTCTGA-3' (forward), 5'-GTTGTGCTGGTGAGTGGAGA-3' (reverse), 5'-GCCACAGCAAACCTCCGCACA-3' (fluorescein) and 5'-CCCACTGGTCCTCAAGAGGTGC-3' (LCRed640); for Tubb5, 5'-TGGGACTATGGACTCCGTTC-3' (forward), 5'-AAAGCCTTGCAGGCAATCA-3' (reverse), 5'-GGCCTTTAGCCCAGTTGTTGCCT-3' (fluorescein) and 5'-CCCCAGACTGACCGAAAACGAAGTT-3' (LCRed705). The hybridization probes were designed by TibMolBiol (Berlin, Germany). RTPCR reactions were performed in thymus samples by using the one-step LightCycler (Roche) kit. 400 ng of total cellular RNA were used to generate cDNA in a reaction containing 3.25 mM of Mn(OAc)2, 0.5 µM of each primer, 0.2 µM of each probe and 1x LC RNA Master Hybridization Probes mix (Roche). The cDNAs of c-Myc and Tubb5 were subsequently co-amplified using the same PCR conditions as in the former case. The c-Myc expression level in each sample was calculated with the calibrator normalized relative quantification method of the LightCycler Relative Quantification software (Roche).
LOH analysis of Ikaros
DNA was extracted by standard methods from normal thymuses or
-radiation-induced thymic lymphomas, and amplified by PCR using a set of primers tailored in exon 4 as described in Okano et al. (18). Amplifications were performed in a volume of 25 µl with a final concentration of 10 mM TrisHCl pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 200 µM of each deoxynucleotide triphosphate, 0.2 µM of each primer and 0.5 U of Taq-polymerase (Finnzymes, Finland). Samples of 100 ng of genomic DNA were used as templates in each PCR reaction. All PCR amplifications were carried out with 35 cycles of 1 min at 95°C, 1 min at 55°C and 2 min at 72°C. Detection of the PCR products was performed by gel electrophoresis and silver staining. Amplified DNA fragments of exon 4 were subsequently digested with HpaII (18) and characterized by SSCP.
PCRSSCP and sequencing of coding regions of Ikaros
PCRSSCP and direct sequence analyses for coding regions of the Ikaros gene were conducted with the primers indicated in Table I. For PCRSSCP analyses, amplifications were performed with 100 ng of genomic DNA in 25 µl vol under the PCR conditions described above. Following PCR, samples were mixed (1:1) in a stop solution containing 94% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol, 20 mM EDTA (pH 8), 10 mM NaOH and 0.1% SDS, denatured 10 min at 95°C, followed by an incubation at 65°C 5 min and then cooled on ice. Next, samples were loaded onto 0.5x non-denaturing polyacrylamide-derived matrix, MDE (Mutation Detection Enhancement) gels (BioWhittaker Molecular Applications, Rockland, ME) in 0.6x TBE buffer, and run at 8 W constant power for 17 h at room temperature. After electrophoresis the DNA fragments were stained using conventional silver staining. Sequencing reactions were set up directly using 100 ng of purified PCR fragment as a template and 10 pmol of a primer, following the dye terminator protocol with BigDye (Applied Biosystems, Foster City, CA). The samples were analysed on an ABI Prism 310 Automated Sequencer (Applied Biosystems).
Ikaros isoforms characterization
Total RNA (400 ng) extracted by the TriPure Isolation Reagent method (Roche) was used to produce cDNAs using the Enhanced Avian HS RTPCR-100 kit (Sigma, St Louis, MO) following the indications of the manufacturer. Specific amplification of the Ikaros cDNA was performed with a pair of primers located at exons 2 and 7 (as described in Table II), in 40 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min. RTPCR products were characterized in non-denaturing polyacrylamide (8%) or agarose gels (2%) and DNA for specific bands (isoforms) were isolated using the GenClean II Kit (Q-BIO Gene Systems). Finally, purified DNA of each band (isoform) was cloned with a TA Cloning Kit Dual Promoter (Invitrogen, Prat del Llobregat, Barcelona, Spain) and sequenced as described above.
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Table II. Real-time RTPCR expression of Notch1, Notch2 and c-Myc genes. RT-data represent an average from two independent experiments (mean values ± standard deviation)
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Western blotting
For further analysis of Ikaros proteins, thymuses were lysed in NP-40 Cell Lysis buffer (Amersham Biosciences, Cerdanyola, Barcelona, Spain) with one tablet added of Protease Inhibitor Cocktail (Roche). Electrophoresis was performed on 100 µg of protein/sample on 6% SDSPAGE. The separated gels were transferred to an immobilon membrane (Millipore, Billerica, MA) overnight at 4°C. The membrane was incubated in blocking buffer containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 0.1% Tween 20 and 5% dry milk for 1 h at room temperature. The primary antibody was a mouse monoclonal anti-Ikaros 4E9 antibody (kindly provided by Dr K.Georgopoulos). Membranes were incubated for 2 h at room temperature, then washed and incubated with the antimouse F(ab')2 secondary antibody labelled with horseradish peroxidase (Amersham), diluted (1:3000) in blocking solution for 1 h. Subsequently, the membrane was washed and incubated in LUMINOL detection reagent (Santa Cruz Biotechnology, Santa Cruz, CA) and exposed to X-ray film. The membrane used for the blot was stained with Ponceau S solution (Sigma, St Louis, MI) to ascertain that an equal amount of protein was loaded to each lane. In addition, the filters were reproved with anti-ß-actin antibody as a loading control. Western blots were repeated twice.
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Results
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Expression analysis of Notch1, Notch2 and c-Myc genes
Since there exists substantial evidence indicating that some members of the Notch family play a direct role in leukemogenesis, we have studied the level of expression of both Notch1 and Notch2 genes in a random selection of 16 tumours. A fluorescent real-time RTPCR was chosen to analyse the mRNA expression levels. In this kind of methodology, the crossing point (threshold cycle number) is the parameter used for calculating the expression levels for each sample. In the case of Notch1, the online fluorescence curves from tumours showed crossing points earlier than that of the normal sample, indicating transcriptional over-expression for this gene in tumours. In contrast, the online fluorescence curves for Notch2 mRNA amplification exhibited crossing points later than that of the normal sample, meaning that tumours exhibit lower Notch2 levels than the normal thymus sample. Quantification data of the real-time RTPCR experiments for all of the analysed tumours are indicated in Table II. We found that Notch1 mRNA expression increased in the vast majority of tumours (14 out of 16, 87.50%) ranging from 1.73- to 14.47-fold. In contrast, mRNA expression of Notch2 is greatly decreased in all tumours (ranging from 0.02- to 0.41-fold).
In order to determine what other gene/s are cooperating with Notch in these tumours, we estimated the levels of mRNA expression for c-Myc by a similar procedure. As in the case of Notch1, the fluorescence run profiles for tumours exhibited crossing points earlier than that of the normal sample, indicating a transcriptional over-expression for c-Myc in these thymic lymphomas. Quantification data of the real-time RTPCR experiments showed that all tumours exhibited increased levels of mRNA expression ranging from 1.40- to 5.57-fold (Table II).
LOH and mutational analysis at the Znfn1a1/Ikaros locus
LOH was determined in a sample of 75 thymic lymphomas (including those used for expression analysis of Notch and c-Myc) with an HpaII polymorphism at exon 4 as described Okano et al. (18). The allelic loss frequency was 32/75 (42.66%), the BALB/cJ allele exhibiting a higher frequency loss relative to the C57BL/6 J allele (84.37 versus 15.62%). Homozygous deletion was only detected in one tumour (BLB60) (Figure 1). We next performed mutational analysis of the coding regions of Znfn1a1/Ikaros by SSCP/sequencing analysis from genomic DNA samples from normal and tumour samples, with and without allelic losses, using the primers described in Table I. We found a new polymorphism between C57BL/6 J and BALB/c [located at position 132 (G
A) in exon 4] that was also used for LOH analysis. The same pattern of LOH and homozygous deletion was obtained with both polymorphisms. In addition, we identified a new point mutation in exon 4 of tumour BLB88 at position 124 (C
T), which changed an amino acid residue (Cys
Arg) at the N-terminal zinc-finger domain (Figure 2).

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Fig. 1. LOH analysis in a sample of thymic lymphomas using primers for exon 4 and digestion with HpaII as described by Okano et al. (18). B6 and BALB, represent parental strains C57BL/6 J and BALB/c, respectively. BALB44-95, thymic lymphomas induced in (C57BL/6 J x BALB/c)F1 mice. HD, homozygous deletion. LOH, loss of heterozygosity by the absence of the allele of the parental BALB/c strain.
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Fig. 2. SSCP and sequentiation analyses at the Ikaros locus. (a) Polymorphic allele variation between the two parental strains at position 132 of exon 4. (b) Missense mutation at position 124 of exon 4.
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Expression analyses of the Ikaros gene: cloning and sequencing of the detected isoforms
The same panel of 16 thymic lymphomas used for expression analysis of Notch and c-Myc genes, were also analysed for Ikaros expression by RTPCR (Figure 3a). RTPCR products were characterized in acrylamide and agarose gels and the different bands (isoforms) were subsequently cloned and sequenced. Normal samples and most of the tumours predominantly expressed the Ik1 as well as the Ik2/3 isoforms. However, tumours exhibited a slight increase in the expression of the shorter sized isoforms. One tumour (BLB60) expressed four isoforms that had different exon configurations with respect to the control samples (Ik9, 7, 11 and 6). It is worth mentioning that the isoform Ik11, which encodes all exons except exons 3, 4 and 6 (thus lacking three internal DNA-binding zinc finger motifs), has not been described previously. We have named this new isoform as Ik11 following on the nomenclature of the different Ikaros isoforms described to date from Ik1 to Ik10 (Figure 3b).

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Fig. 3. Expression of Ikaros in a sample of thymic lymphomas. (a) RTPCR analysis with indication of the Ikaros isoforms detected in this study. Four tumours (BLB 30, 31, 74 and 95) failed to produce detectable mRNA expression in this experiment. (b) Schematic representation of the Ik11 isoform, which is described here for the first time. (c) Western blot analysis of Ikaros on whole cell lysates from the same panel of tumour samples. ß-Actin was used as control reference.
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To determine whether the Znfn1a1/Ikaros mRNA profile of T-cell lymphomas correlated well with protein expression, we performed a western blot analysis on the same panel of tumours, using a monoclonal antibody (4E9) that recognizes the C-terminal fragments of all Ik isoforms (16). Using as reference the expected molecular weights of the different isoforms, the pattern of expression of normal samples, and that of tumour BLB60 (which exhibited a very clear pattern with four characteristic mRNA isoforms), it might be deduced that all of the analysed samples predominantly expressed a band of approximately the same size as the Ik1 isoform, with the exception of BLB60, which harbours instead a rare Ik9 isoform lacking exon 4 (13). In contrast to the normal control samples, most of the tumours (15 out of 16, 93.75%) not only expressed the active isoform Ik1, but also expressed variable levels of dominant inactive isoforms of Ikaros (Figure 3c).
Taken together, these results and those showed in Table II demonstrate that 13 out of 16 tumours (81.25%) simultaneously experience over-expression of both Notch1 and c-Myc, down-expression of Notch2 and diminished Znfn1a1/Ikaros DNA-binding activity.
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Discussion
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As mentioned above,
-radiation-induced thymic lymphomas constitute a heterogeneous group of T-cell lymphoblastic lymphomas. Despite previous studies dealing with the inactivation of some tumour suppressor genes and the activation of certain oncogenes, a considerable number of these tumours remain to be elucidated. In relation to oncogene activation, a minor fraction of
-radiation-induced thymic lymphomas were found to have activated N- and K-ras oncogenes (9,11). Constitutive activation of the Notch1 gene also predisposes mice to T-cell leukaemia (1921). Furthermore, radiation induced deletions in the 5' end region of Notch1 lead to the formation of truncated proteins involved in the development of mouse thymic lymphomas (13). However, whereas the expression of Notch1 was frequently rearranged in mouse radiation-induced thymus lymphomas or greatly enhanced in leukemic T-cells, expression of Notch2 either remained unaffected (13) or increased slightly (3.5-fold with respect to normal T-cells) in leukemic T-cells (22), indicating that this gene has little or no effect on thymic lymphomagenesis.
To elucidate the involvement of Notch1 and Notch2 alterations in
-radiation-induced primary thymic lymphomas we analysed the pattern of transcriptional expression of these genes by real-time RTPCR. As expected, most of the tumours (87.50%) showed over-expression of Notch1, suggesting that Notch1 functions as a major oncogene. Yet, surprisingly, Notch2 mRNA expression was considerably reduced in all tumours (Table II), despite previous data indicating little or no effect of this gene on thymic lymphomagenesis (13). This unexpected result may be indicative of some kind of cooperative antagonism effect of Notch1 and Notch2 in the development of
-radiation-induced thymic lymphomas. Notch signalling impinges on several cellular processes, including the maintenance of stem cells, the specification of cell fate, as well as their differentiation, proliferation and apoptosis, and there is evidence that Notch signalling might not be exclusively oncogenic but likewise tumour suppressive in skin tumours (12). These two facets of Notch signalling might as well be occurring in murine T-cell lymphomagenesis, with Notch1 behaving as an oncogene, and Notch2 as a tumour suppressor gene. In lymphocytes, the transforming ability of Notch1 seems to require its transactivation function. Given that Notch1 contains a strong transactivation domain whereas Notch2 contains a weak one (12), it is reasonable to think that a reduction in the amount of Notch2 protein would cause a lowering of the competence between Notch1 and Notch2 receptors for the limited amount of ligands, leading to a more effective transformation activity of Notch1.
Although the simultaneous activation of Notch1 and the inactivation of Notch2 can promote the process of T-cell lymphomagenesis, it is clear that they need to partner another oncoprotein in order to trigger thymic lymphomas. These partners have not been yet identified in naturally occurring tumours (12). Experimentally, one of these partners could be c-Myc, as frequent proviral-insertional-mutagenesis of Notch1 has been detected in thymomas arising in MMTV/myc transgenic mice (15). Furthermore, a considerable fraction of
-radiation-induced thymic lymphomas exhibited trisomy of chromosome 15, where c-Myc is located (10). Significantly, all of these tumours we have thus far analysed exhibited increased expression of c-Myc, suggesting that the over-expression of Notch1, the inactivation of Notch2 and the over-expression c-Myc may be collaborating in thymic lymphomagenesis (see Table II).
Another partner of Notch might be the Znfn1a1/Ikaros gene. This gene is a transcriptional regulator that has been reported as essential in the development of all lymphoid-derived cells (23). This gene is expressed as multiple isoforms that arise from alternative splicing and have a variable number of DNA-binding zinc fingers (24). All reported isoforms contain two dimerization zinc finger motifs at the carboxylic terminus, as well as a variable number of internal DNA-binding zinc fingers motifs. Ikaros isoforms that do not contain the internal motifs (Ik5Ik10) are able to dimerize with DNA-binding isoforms (Ik1-4) and behave as DN inhibitors (16,25). Actually, heterozygous mice for DN isoforms develop T-cell leukaemia (26) and, vice versa, a majority of human T-cell leukaemias show increased DN isoform levels (27). Beverly and Capobianco (16) have proposed that the biochemical basis for the cooperation between Notch1 and Ikaros is due to de-repression of Ikaros-regulated genes that can be consequently activated by Notch1 (i.e. cyclin D1, IL2Rb, etc.).
Radiation-associated LOH at the Znfn1a1/Ikaros locus on chromosome 11 has been described in thymic lymphomas induced by radiation in (BALB/c x MSM)F1 (23%) and (C57BL/6 J x C3H)F1 (46%) hybrid mice and, interestingly, the alleles derived from MSM or C3H were preferentially lost (18,28). In the present work we have demonstrated that LOH at Znfn1a1/Ikaros was also a very frequent event in thymic lymphomas from (C57BL/6 J x BALB/c)F1 hybrid mice, which carry the weak allele variants that were preferentially retained in tumours from (BALB/c x MSM)F1 or (C57BL/6 J x C3H)F1 mice (Figure 1). This suggests that a single copy of the C57BL/6 J or BALB/cJ alleles is not sufficient to prevent tumorigenesis. However, the loss of the BALB/cJ allele would more likely lead to tumorigenesis. Under these premises it is not surprising that the spectrum of Znfn1a1/Ikaros inactivation that we found is clearly different from that reported by Kakinuma et al. (7). While these authors demonstrated a significant frequency of point mutations (22% of their tumours), we have only found a missense mutation in one tumour out of 75 (1.33%), although it is possible that this frequency is an underestimation due to the limitations of the SSCP technique. Interestingly, the particular tumour carrying a point mutation in Ikaros also had LOH, thus being consistent with the Knudson's two hit model (Figure 2).
With regard to the pattern of Ikaros gene expression, Kakinuma et al. (7) reported expression of DM isoforms in 11% of their tumours, and a lack of the isoform Ik1 due to the creation of a stop codon by insertion of a dinucleotide in exon 3 in 3% of those tumours. We, on the other hand, detected a skewed distribution towards DN isoforms in all tumours with the exception of BLB95. Tumour BLB60 has been of particular interest because it only expressed four of the DN isoforms (Ik9, 7, 11 and 6), Ik11 being one not described previously. We presume that Ik11 acts in a DN fashion as it lacks the three internal zinc finger motifs corresponding to exons 3 and 4 (Figure 3b). The pattern of transcriptional expression was essentially confirmed by western blotting (Figure 3c). These results raise the question of the biological effectiveness of low levels for the DN isoforms of Ikaros in thymic lymphomagenesis.
As 81.25% of the tumours simultaneously experience activation of Notch1, inactivation of Notch2, activation of c-Myc and diminished Znfn1a1/Ikaros DNA-binding activity, we therefore propose that these events might be collaborating in promoting primary thymic lymphomas in mice treated with
-irradiation.
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Acknowledgments
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We thank Dr Katia Georgopoulos for kindly providing the mouse monoclonal anti-Ikaros 4E9 antibody and Dr A.Morales for useful comments. This work was supported in part with funds from the Spanish Ministry of Science and Technology (SAF2003-05048), the Lymphoma Network from Ministry of Health and Consumer (G03/179), and the Madrid Autonomous Community (CAM 08.1/0025.1/2003), to J.Fernández-Piqueras.
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Received January 12, 2004;
revised February 9, 2004;
accepted February 10, 2004.