Pre-B cell proliferation and lymphoblastic leukemia/high-grade lymphoma in Eμ-miR155 transgenic mice

Production and Characterization of Eμ-mmu-miR155.

We generated transgenic mice in which the expression of mmu-miR155 (mouse miR155) is under the control of a V_H promoter-Ig heavy chain Eμ enhancer, which becomes active at the late pro-B cell stage of the B cell development. Fifteen transgenic founders were identified by Southern blot hybridization, seven on C57BL/B6 and eight on FVB/N backgrounds. These were bred to wild-type mice of the same strain to produce 15 independent transgenic lines.

Northern blot and real-time PCR analysis (data not shown) performed on total RNA extracted from transgenic and wild-type spleens showed a very good expression of miR155, described in Fig. 1c. All other founder lines but one also expressed the transgene. Wild-type mice did not express mature miR155 in the spleen, as reported previously (16).

Eμ-mmu-miR155 Exhibited Splenomegaly as Early as 3 Weeks of Age.

Spleens of transgenic mice were enlarged, with a spleen weight/body weight ratio three to four times greater than that of wild-type mice at 3 weeks of age and did not increase significantly with age (Table 1 and Fig 2).

Transgenic Mice Overexpressing miR155 Are Leukopenic.

The white blood cell count (WBC) of transgenics 3 months of age was 10 × 10⁶ ± 1 × 10⁶/ml compared with 40 × 10⁶ ± 1.5 × 10⁶/ml for normal age-matched mice. The WBC for transgenic mice 6 months of age was even lower, at 6 × 10⁶ ± 0.5 × 10⁶/ml, whereas that of wild type was unchanged.

Histological and Immunohistochemical Analysis of Eμ-mmu-miR155 Mice.

The histopathology of the spleens [hematoxylin/eosin (H&E) stain] from 3-week-old mice featured a consistent atypical lymphoid population invading the red pulp and expanding it; the lymphoid follicles were unaffected, and there were multiple foci of secondary hematopoiesis (Fig. 3a). Mice at 6 months of age presented histologically a greatly increased malignant lymphoid population with marked atypia and blastic appearance, proliferating in the vascular channels of the red pulp and gradually replacing the white pulp. The number of lymphoid follicles was decreased, and the overall architecture of the spleen was distorted by lymphoid proliferation (Fig. 3 b and c). A histologically similar lymphoid population was present in the bone marrow of 6-month-old mice. Expression of the proliferation Ki67 antigen showed a marked lymphoid proliferation in transgenic mice, not observed in wild-type mice (Fig. 3f).

Immunohistochemistry performed to identify the immunophenotype of the lymphoid proliferation showed low positivity of the atypical expanded lymphocytes for B220 and VpreB1 (CD179a), although CD43 was negative (data not shown). The IgM staining of the paraffin-embedded sections of the transgenic spleens showed the presence of μ chains in the cytoplasm of the proliferating lymphocytes (Fig. 4), whereas flow cytometry analysis failed to identify the expression of IgM on the surface of these cells, indicating that the expanded lymphoid cells expressed cytoplasmic μ chain but did not express surface IgM.

Flow Cytometry Analysis of Eμ-mmu-miR155 Reveals an Expansion of the B220^low/CD10^low/IgM⁻/CD5⁻/TCR⁻/CD43⁻ Population.

Flow cytometry analysis, performed on single-cell suspensions of WBC from spleens and bone marrows of transgenic and wild-type mice of 3, 6, and 7 weeks and 6 months of age, showed an increase of B220^low/CD10^low/IgM⁻/CD5⁻/TCR⁻/CD43⁻ lymphoid cells in both spleen and bone marrow in the transgenic mice compared with their wild-type counterparts. This phenotype resembles the phenotype of proliferating lymphocytes observed in human acute lymphoblastic leukemia or lymphoblastic lymphoma. Our findings indicate that the B220^low/CD19^low/CD10^low/IgM⁻/TCR⁻/CD43⁻ lymphoid population in the spleens of transgenic mice at 3 weeks old (assessed on one transgenic and one wild-type mouse) is 9% out of the entire gated lymphoid population and only 1.65% in the wild type; this becomes 6.6 ± 1.4% in the spleen of 6-week-old transgenic mice (two transgenics analyzed from two different founding lines and two wild-type mice) and 4.7 ± 0.3% for mice 7 weeks of age (two transgenics and one wild type), while remaining unchanged for the wild type. In the bone marrow of transgenic mice 6 months old, we found an increase of the pre-B cell population as defined by B220^low/IgM⁻ expression, compared with the wild type (Figs. 5 and 6).

Forward-scatter analysis of the B220^low/IgM⁻ cell population showed that these cells are large blastoid cells (data not shown).

Cytogenetics Indicated Genomic Alterations.

Cytogenetic studies of the karyotype of the splenocytes failed to identify consistent chromosomal abnormalities in the transgenic spleens compared with the normal littermates but showed occasionally some genomic alterations (Fig. 7). These results indicate that the expanded population of pre-B cells is diploid and cytogenetically quasinormal.

Southern Blot Analysis for Ig Heavy Chain Rearrangements Showed a Polyclonal Pre-B Cell Proliferation at Least Until 6 Weeks of Age.

Southern blot analysis for clonality by V(D)J rearrangements on splenocyte mouse DNA 3–6 weeks of age using multiple digestion enzymes did not show the presence of clonally rearranged bands in the transgenic compared with wild-type mice (except for one transgenic mouse that had a single prominant rearranged band on all of the Southern blots performed with different restriction enzymes; data not shown). This shows that the B cell population in mice of this age range was usually polyclonal, indicating that the lymphoproliferation was, for the most part, polyclonal, at least until 6 weeks of age (Fig. 8).

Microarray Analysis Confirmed the Up-Regulation of VpreB1 mRNA, Specific for the Pre-B Cells.

Microarray analysis of miRNAs was performed on the total RNA extracted from the splenic white cells of five transgenics (among which one did not have the expression of the transgene) and six wild-type littermate counterparts, revealing a 10- to 20-fold increase in expression of miR155, -194, -224, -217, and -151 (Table 2, which is published as supporting information on the PNAS web site) and a decrease of expression of miR146 and -138, 2- to 3-fold in transgenic mice overexpressing miR155. Using an Affymetrix microarray chip, we studied the differential expression of mRNAs in the same group of transgenics, compared with their littermate controls. The statistical analysis of the Affymetrix microarray showed that 200 proliferation genes were up-regulated, whereas 50 genes were down-regulated in the miR155-overexpressing mice (data not shown). Interestingly enough, the VpreB1 mRNA was up-regulated, as one would expect when the proliferation of pre-B cells takes place. These data complement the data from flow cytometry analysis and immunohistochemistry (Table 3, which is published as supporting information on the PNAS web site).

Eμ-mmu-miR155 Transgenic Mice Present Pre-B Cell Proliferation and Lymphoblastic Leukemia/High-Grade Lymphoma.

We concluded, based on the flow cytometry, histological and immunohistochemical analyses, that a pre-B cell proliferation defined as IgM⁻/CD43⁻/TCR⁻ occurred in the spleens and bone marrows of transgenic mice, already detectable at 3 weeks of age. This proliferation led later on to splenomegaly, bone marrow replacement, and marked lymphopenia, features often associated with high-grade B cell malignancies. By the age of 6 months, seven of seven transgenic mice developed high-grade B cell neoplasms (seven of seven transgenic mice, 6 months old) compared with the wild-type controls, which were all healthy (11 of 11 wild-type mice). The transgenic mouse line not overexpressing miR155 was also normal.

Eμ-mmu-miR155 Transgenic Mice.

A 318-bp fragment was amplified by PCR from the 129SvJ mouse genome (The Jackson Laboratory) containing the precursor sequence of the miR155 and cloned into the EcoRV and SalI sites of the pBSVE6BK (pEμ) plasmid, which had been previously used by our group for the development of chronic lymphocytic leukemia in Eμ-TCL1 transgenic mice (18), which contains the Eμ enhancer V_H promoter for Ig heavy chains, alongside the 3′ UTR and the poly(A) of the human β-globin gene (Fig. 1a) The transgene was isolated by cutting the construct with BssHII and PvuI and injected into the male pronucleus of fertilized oocytes of pregnant FVB/N and C57BL/6 mice. Pups were screened for the presence of the transgene by Southern blot performed on tail-extracted DNA and digested with BamHI, using a probe designed in the Eμ enhancer sequence. Fifteen founders were identified (seven for C57BL/6, marked F1–7, and eight for the FVB/N strain, marked F8–15) and bred to age-matched wild-type mice (Fig. 1b). Transgenic hemizygous mice were born, studied, and compared with their wild-type counterparts. Mice were genotyped by PCR performed on tail-extracted DNA (data not shown).

Northern Blot Analysis of Transgenic Offspring.

Spleens were dissociated between two frosted slides, and the lysate was washed in PBS, depleted of red cells by hypotonic lysis with ammonium chloride (NH₄Cl), centrifuged, and resuspended in PBS. Total RNA was extracted with TRIzol (GIBCO, Invitrogen), loaded and denatured on SDS/PAGE, and blotted on a Hybond N⁺ membrane (Amersham Pharmacia). The membrane was hybridized with a γ-³²P radioactive probe represented by the antisense of the mature mmu-miR155 sequence, incubated overnight, washed, and exposed to a PhosphorImager screen (Molecular Dynamics). The image was processed by using a Typhoon image processing system (Amersham Biosciences) (Fig. 1c).

Somatic Measurements.

Mice were weighed after being killed, and the spleens were measured and weighed.

WBC and Smear Preparation.

Blood was drawn from retroorbital blood vessels, smeared on frosted slides, and Giemsa-stained; part of it was centrifuged, washed in PBS, and treated with ammonium chloride, then cells were counted with a cell-counter chamber.

Flow Cytometry Analysis.

Single-cell suspension of splenocytes and bone marrow cells was depleted of mature red blood cells by hypotonic lysis (0.165 M NH₄Cl) and stained with the following conjugated antibodies: anti-B220-phycoerythrin, anti-IgM-FITC, anti-TCR-phycoerythrin cy5, anti-CD5-phycoerythrin, and anti-CD-43-FITC (all antibodies were from BD PharMingen). Flow cytometry was carried out on a Becton Dickinson FACSCalibur, and data were analyzed by using the Becton Dickinson FACS convert 1.0 for Mac software.

Histology and Immunohistochemistry.

Mice were necropsied, and spleens, femurs, and sternums were fixed in 10% buffered formalin, included in paraffin, and then cut at 4 μm. Sections were stained with H&E according to standard protocols. For the dewaxing step, sections were heated for 1 h at 55°C, followed by rehydration steps through a graded ethanol series and distilled water, immersed in PBS, and then treated with 0.1% trypsin solution in Tris buffer for 30 min at 37°C. Endogenous peroxidase was blocked with 10% normal serum. CD43, B220, and VpreB1 (CD179a) were used as primary antimouse antibody (BD PharMingen). Secondary antibodies and diaminobenzidine were added according to the manufacturer’s instruction.

Ig Heavy Chain Gene Rearrangements.

A probe was designed and amplified in the JH4 fragment of the Ig heavy chain region on the mouse genomic, using the following primers: forward, 5′-TGAAGGATCTGCCAGAACTGAA-3′, and reverse, 5′-TGCAATGCTCAGAAAACTCCAT-3′.

Spleens of the transgenic and wild-type mice were dissociated between frosted slides in PBS, treated with ammonium chloride for the erythrocyte lysis, centrifuged, and resuspended in PBS. DNA was extracted from the spleen white cells and digested with EcoRI, StuI, BglII, BamHI, and HindIII. Digested DNA was blotted on a Hybond N⁺ membrane, hybridized with the JH4 probe radioactively labeled with ³²P, then exposed to a PhosphorImager screen, and processed by using a Typhoon scanner.

Microarray Analysis.

Cytogenetics.

Femur bone marrow was flushed with RPMI medium 1640/20% FBS and collected into 5 ml of RPMI medium 1640/20% FBS with heparin 1%. Cells were grown and assessed for chromosomal deletions, translocations, inversions, and number of metaphases.