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Keywords:

  • MLL;
  • AF10;
  • MPD;
  • cytokine

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Several mouse models studying the MLL fusion-induced leukemic transformation showed that a myeloproliferation stage precedes leukemia or occurred as the only phenotype of hematological disorder in mice. We established 6 MLL/AF10(OM-LZ)-immortalized cell lines by retrovirally transducing the fusion gene into bone marrow cells from B6 or congenic GFP-B6 mice. Immunophenotypic and cytological analyses revealed that the immortalized cell lines could be divided into 2 types. Type I had a high percentage of cells expressing monocytic lineage marker CD115 in the medium containing IL3 and could terminally differentiate into granulocytes and monocytes in response to granulocyte colony-stimulating factor (G-CSF) and macrophage colony-stimulating factor (M-CSF) treatments, respectively. On the other hand, type II had a low percentage of cells expressing CD115. The type II cell lines could not differentiate into granulocytes by G-CSF treatment and died rapidly in response to M-CSF treatment. Transplantation of both types I and II cells induced lethal myeloproliferative disease (MPD)-like myeloid leukemia in most of the sublethally irradiated B6 mice. Flow cytometric analysis of GFP and lineage markers of the peripheral blood cells from MPD mice revealed that the monocytes and granulocytes were generated not only from the donor cells but also from the host cells. RT-PCR analysis revealed that the MLL/AF10(OM-LZ)-immortalized cells expressed mRNAs encoding colony-stimulating factors (CSFs) of M-CSF and GM-CSF and inflammatory cytokines of IL-1α, IL-1β and TNF-α. Our results showed that the MLL/AF10(OM-LZ)-immortalized cells could induce host cell proliferation in the transplanted mice, probably through stimulation by CSFs or cytokines produced by the donor cells.

MLL gene on chromosome 11q23 is a common target of chromosomal translocations in acute myeloid leukemia (AML), acute lymphoblastic leukemia and myelodysplastic syndrome.1 Till now, more than 50 MLL translocation partners encoding nuclear or cytoplasmic proteins with heterogeneous functions have been identified.2 An individual MLL translocation is usually linked to a specific subtype of leukemia with prognostic relevance. Acute myelomonocytic leukemia (AMMoL, M4) and monocytic leukemia (M5) are the most frequent subtypes found in AML patients with MLL translocations.3–6MLL/AF10 is 1 of the 3 MLL translocations that mostly associated with AML-M4/-M5 subtypes (the other 2 are MLL-AF9 and MLL-ELL).7–9 It remains to be elucidated how these MLL translocations lead to the lineage-specific disease phenotype.

By using a retroviral transduction/transplantation mouse model, DiMartino et al. showed that the murine bone marrow cells expressing MLL/AF10 caused hyperleukocytosis with circulating myeloblasts in syngeneic and severe combined immunodeficiency mice.10 They also demonstrated that an octapeptide motif and a leucine zipper domain (OM-LZ) of AF10 were necessary and sufficient for the immortalization of murine myeloid progenitors by MLL/AF10. In our study, we used the same strategy to establish multiple MLL/AF10(OM-LZ)-immortalized cell lines. The in vitro differentiation potential of these cell lines in induction of the lineage-specific disease phenotype was characterized. In vivo leukemogenesis study showed that most of the mice transplanted with MLL/AF10(OM-LZ) immortalized cells developed a myeloproliferative disease (MPD)-like myeloid leukemia. Further experiments were performed to demonstrate how the MLL/AF10(OM-LZ)-immortalized cells induced myeloproliferation in the transplanted mice.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Plasmid construction

Complementary DNA (cDNA) fragments of the MLL/AF10(OM-LZ) (encoding amino acids [aa] 1–1362 of MLL and aa 643–800 of AF10), truncated MLL (tMLL, encoding aa 1–1362 of MLL) and truncated AF10 (tAF10, encoding aa 643–800 of AF10) were amplified from a patient with t(10;11)(p12;q23)9 by reverse transcription-polymerase chain reaction (RT-PCR). The cDNAs were inserted into the EcoRI and XhoI sites of a retroviral vector pMSCVneo (Clontech, Palo Alto, CA). The nucleotide sequences of all constructs were reconfirmed to exclude mutations introduced by PCR.

In vitro immortalization assay

Transformation of murine hematopoietic progenitor cells was performed as described previously10 with some modifications. Viral supernatants were collected 2 days after transfection of EcoPack2-293 cells (Clontech) with pMSCV constructs. Retroviral titers were determined by infection of NIH3T3, a murine fibroblast cell line, with serial diluted viral supernatants. Enriched hematopoietic progenitor cells were harvested from femurs and tibiae of 6-week-old C57BL/6JNarI (B6) or C57BL/6J-Tg(Pgk1-EGFP) 03N (GFP-B6) (National Laboratory Animal Center, Taiwan) mice that had been injected intravenously with 5-flurouracil (150 mg/kg) 5 days ago. After coculturing with virus at a low multiplicity of infection of 1–3, the transduced hematopoietic progenitor cells were selected in the methylcellulose medium [1% MethoCult M3231 (Stem Cell Technologies, Vancouver, BC) supplemented with 20 ng/ml stem cell factor and 10 ng/ml each of interleukin (IL)-3, IL-6 and granulocyte macrophage-colony stimulating factor (GM-CSF) (R&D Systems, Minneapolis, MN)] in the presence of 1 mg/ml G418. After 10 days, colonies consisting of >100 cells were scored. Single-cell suspensions (1 × 104 cells) pooled from the colonies in the methylcellulose medium were replated in the same medium without G418. Plating was repeated every 7 days for a total of 4 rounds. Immortalized cell lines were generated by culturing the pooled quaternary colonies in a RPMI complete medium (RPMI 1640, 10% fetal calf serum, 2 mM L-glutamine, 100 μM 2-mercaptoethanol and penicillin G-streptomycin) supplemented with IL-3. Cells were maintained at an initial number of 2 × 105 per milliliter and subcultured every 2 days.

In vivo tumorigenicity assay

The retroviral MLL/AF10(OM-LZ)-transduced cells (4 × 104) pooled from the 1st round methylcellulose medium or the MLL/AF10(OM-LZ)-immortalized cells (1 × 106) were injected intraperitoneally into 6-week-old syngeneic B6 mice that had received a sublethal dose of irradiation [1 dose of 5.25 Gy total body γ irradiation (135Cs)] to study their in vivo tumorigenicity.11 Peripheral blood (PB) from the retro-orbital plexus of transplanted mice was collected and counted weekly for 4 weeks post-transplantation until moribund to monitor complete blood cell counts using a hemocytometer (Hemavet 950, Drew scientific, Oxford, CT). Mice were killed when moribund; their PB and bone marrow (BM) cells were Liu-stained (Handsel Technologies, Taipei, Taiwan); their livers and spleens were fixed in buffered formalin, paraffin embedded, sectioned and stained with hematoxylin and eosin using standard techniques.

Phenotype analyses

Immunophenotypic analysis was performed by staining cells with phycoerythrin (PE)-Mac-1, PE-CD115, fluorescein isothiocyanate (FITC)-c-kit, FITC-CD4, FITC-CD14, allophycocyanin (APC)-Sca-1, APC-Gr-1, APC-B220 (eBioscience, San Diego, CA) and APC-Ly-6G (Miltenyi biotec, Auburn, CA). Stained cells were analyzed on a flow cytometer, FACS-Calibur (Becton-Dickinson, Mountain View, CA). The EGFP fluorescence was captured in the FITC channel. To study cell proliferation and differentiation, IL3 was substituted by10 ng/ml granulocyte colony-stimulating factor (G-CSF) or macrophage colony-stimulating factor (M-CSF) (R&D Systems). Cell proliferation and viability were measured by using a quick cell proliferation kit to monitor the activity of mitochondrial dehydrogenase (WST-1 assay, BioVision, Mountain View, CA). For WST-1 assay, the cells (1 × 105 cells/100 μl/well) were incubated in the RPMI complete medium without or with cytokines for 24 hr. Then, 10 μl of WST reagent was added and incubated for 1 hr. Absorbance was measured at 440 nm against a reference wavelength of 650 nm using a microplate reader (SpectraMax 190 microplate reader; Molecular Devices, Sunnyvale, CA) for every 1 hr for a total of 4 hr. Immortalized cells treated with colony-stimulating factors (CSFs) were cytospined, air dried and stained with Liu stain for morphological examination.

Cytokine expression analysis

Total RNAs were prepared from cells using Trizol reagent (Gibco BRL, Gaithersburg, MD). cDNA was synthesized by reverse transcription of the RNA using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) and random hexamers. To detect the expression of genes encoding CSFs and cytokines, cDNAs were amplified using mouse specific primers for IL-3 (5′-GTT CTT GCC AGC TCT ACC AC and 5′-AGA GAG GGT CCT TCA TCA TC), IL-6 (5′-GAA GTT CCT CTC TGC AAG AG and 5′-GTT ATC TTT TAA GTT GTT CT), GM-CSF (5′-GGC TGC AGA ATT TAC TTT TC and 5′-GTG AAA TTG CCC CGT AGA CC), M-CSF (5′-CGC TGC CCT TCT TCG ACA TG and 5′-GGT AAA AAT GTT CCA GTC CT), G-CSF (5′-GCT AAT GGC CCT GCA GCT GC and 5′-AGG CAC TGT GTC TGC TGC AG), IL-1α (5′-GTT ACA GTG AAA ACG AAG AC and 5′-TAG TGT TTG TCC ACA TCC TG), IL-1β (5′-TCC ATG AGC TTT GTA CAA GG and 5′-GGT GCT GAT GTA CCA GTT GG), interferon-γ (IFN-γ) (5′-TGA ACG CTA CAC ACT GCA TC and 5′-CCA CTC GGA TGA GCT CAT TG), tumor necrosis factor-α (TNF-α) (5′-GTG GAG GAG CAG CTG GAG TG and 5′-CAA AGT AGA CCT GCC CGG AC) with AmpliTaq Gold polymerase (Applied Biosystems). The Escherichia coli-activated RAW264.7, a murine monocyte/macrophage cell line, was used as the CSF and cytokine expression control. RAW264.7 was maintained in Dulbecco's modified Eagle's medium with 4 mM L-glutamine, 100 μM 2-mercaptoethanol, penicillin G-streptomycin and 10% fetal calf serum. E. coli-activated RAW264.7 was generated by adding 1% (vol/vol) phosphate- buffered saline-washed, autoclave-killed E. coli suspension into RAW264.7 culture for a total of 4 hr.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Morphology and immunophenotype of the MLL/AF10(OM-LZ)-immortalized cell lines

The tMLL, tAF10 and MLL/AF10(OM-LZ) genes (Fig. 1a) were introduced into murine BM cells by retroviral transduction. We found that cells transduced with tMLL or tAF10 ran out of their proliferation capacity in the 2nd or 3rd round of plating. On the contrary, cells transduced with MLL/AF10(OM-LZ) exhibited an enhanced proliferation potential and were able to generate colonies in the 3rd and 4th rounds of plating (Fig. 1b). Six cell lines were generated by culturing the colonies pooled from the 4th round of methylcellulose plates in the RPMI complete medium containing IL3 in the independent transduction experiments. Lines 2, 4, 6, 8 and 10 were derived from the BM cells of B6 mice, and line 12G was from GFP-B6 mice. Liu staining of the cytospin preparations of the suspension cells showed that these cell lines were composed of immature myeloid cells with varying degrees of maturation and rare monocyte/macrophage cells, except line 6, which also contained 2% granulocytes (Fig. 1c).

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Figure 1. Immortalization of the murine BM cells by MLL/AF10(OM-LZ). (a) Schematic representations of MLL, AF10, tMLL, tAF10 and MLL/AF10(OM-LZ). AT-H, AT hooks; MT, DNA methyltransferase domain; Zn-F, zinc finger domain; OM, octopeptide; LZ, leucine zipper. (b) Transduction efficiency of the MLL/AF10(OM-LZ) fusion gene in 6 experiments. The bars indicate the colonies generated per 104 transduced murine BM cells. (c) Liu staining of the MLL/AF10(OM-LZ)-immortalized cell lines in medium containing IL3. (d, e) The immunophenotypic characteristics of the MLL/AF10(OM-LZ)-immortalized cell lines. The dot plots of d and the left dot plot of e were the results from line 4, which was used as a type II representative cell line. The right dot plot of e was from line 6 and used as a type I representative cell line. Percent of cells expressing the surface antigen CD115 or Ly-6G was indicated.

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Immunophenotyping analysis revealed that these cell lines had a similar surface antigen expression profile of the early myeloid lineage, near 100% of cells were positive for Mac-1, and the majority of the cells expressed c-kit but negative for Sca-1 (Fig. 1d). All of these cell lines were negative for the lineage-specific surface antigen of T-lymphocyte (CD4), B-lymphocyte or granulocyte (Ly-6G) (B220) (Figs. 1d and 1e). The expression of monocytic lineage surface antigen CD115 in the cell lines could be divided into 2 types. Type I cell lines, lines 2, 6, 8 and 12G, had a high percentage of CD115+ cells, and type II cell lines, lines 4 and 10, had a low percentage of CD115+ cells (Fig. 1e).

Differentiation potentials of the MLL/AF10(OM-LZ)-immortalized cell lines in response to G-CSF and M-CSF

The immortalized cell lines of both types I and II showed a very low proliferation rate in the medium without any CSFs using WST-1 assay (Fig. 2a), and no alive cells could be detected under microscopic examination after 4–6 days of culture. This observation indicated that the MLL/AF10(OM-LZ)-immortalized cells were absolutely dependent on the cytokines for their survival. Cells cultured in the medium containing G-CSF grew more slowly than those in the medium containing IL3 (Fig. 2a). Flow cytometric study revealed that the percentage of CD115+ cells in type I cell lines was decreased and that of the Ly-6G+ cells was slightly increased after 4 days treatment of G-CSF when compared with that was cultured in the medium containing IL3 (Fig. 2b upper and Fig. 1e). Cytological analysis showed that type I cell lines could terminally differentiate into granulocytes in response to G-CSF treatment (Fig. 2b lower). On the other hand, the percentage of CD115+ cells was increased in type II cell lines, and there were no apparent changes in Ly-6G expression when the cells were cultured in the medium containing G-CSF for 4 days (Fig. 2b, upper). Cytological analysis revealed that type II cells had a more mature morphology but no terminally differentiated granulocytes could be detected after 4 days G-CSF treatment (Fig. 2b, lower).

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Figure 2. Phenotypes of the MLL/AF10(OM-LZ)-immortalized cell lines in response to G-CSF and M-CSF treatments. (a) Proliferation rate of the MLL/AF10(OM-LZ)-immortalized cells in medium with or without colony-stimulating factors was determined by WST-1 assay. (b) FACS analysis of the MLL/AF10(OM-LZ)-immortalized cells in medium containing G-CSF (upper panel) for 4 days and then double stained with PE-CD115 (a monocytic-lineage marker) and APC-Ly-6G (a granulocytic-lineage marker), followed by the flow cytometer analysis. Liu staining (lower panel) of the MLL/AF10(OM-LZ)-immortalized cell lines in medium containing G-CSF. (c) FACS analysis of the MLL/AF10(OM-LZ)-immortalized cells in medium containing M-CSF (upper panel) for 2 days and then double stained with PE-CD115 and FITC-CD14 (a macrophage marker), followed by the flow cytometer analysis. Liu staining (lower panel) of the MLL/AF10(OM-LZ)-immortalized cell lines in medium containing M-CSF.

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When the cells were cultured in the medium with M-CSF, both types of cell lines had a cell proliferation rate either slightly higher than (type I) or the same as (type II) they were in the medium without cytokine (Fig. 2a). Flow cytometric study revealed that type I cell lines had a distinct expression pattern of CD115 and CD14, a macrophage surface antigen, from those of the type II cell lines (Fig. 2c upper). Near 100% of type I cells were CD115+ and CD14+, whereas only about 40 and 70% of the type II cells had a CD115low and CD14low expression, respectively, in response to M-CSF treatment after 2 days of culture (Fig. 2c upper). Cytological analysis showed that type I cells had an increased number of monocytes (Fig. 2c lower), but the cells were apoptotic after 6 days culturing in the medium with M-CSF (data not shown). In contrast, the majority of type II cells were apoptotic within 2 days of culture in the medium with M-CSF (Fig. 2c lower).

In vivo leukemogenesis of MLL/AF10(OM-LZ)-immortalized cells

To analyze the transforming potential of the MLL/AF10(OM-LZ) fusion gene, some of the types I and II cell lines were transplanted into the syngeneic mice. All of the mice had leukocytosis with white blood cell (WBC) count ranging from 11.9 × 109/L to 432.7 × 109/L (normal range is 1.8 × 109/L–10.7 × 109/L) (Table 1). Some of the line 6- and line 12G-mice had an extreme hyperleukocytosis (Table 1). All mice showed neutrophilia and monocytosis, 38 (85%) mice had anemia and 41 (93%) mice had thrombocytopenia. PB smear showed that all but 1 (line 4 no. 5) had circulating blasts with varying stages of maturation in the myelomonocytic lineage (Table 1 and Fig. 3a). The percentage of PB or BM blasts in most of the mice ranged from 1 to 17%, 3 mice (line 4-nos. 6 and 93, line 6-no. 64) had more than 20% blasts (Table 1 and Fig. 3a). The time to leukemia and survival time after transplantation were 5–12 weeks and 6–13 weeks, respectively (Table 1 and Fig. 3b). All the transplanted mice revealed splenomegaly (Fig. 3c). Pathological examination showed that the spleens were extensively infiltrated by hematopoietic cells, and the architecture of red pulps and white pulps was disrupted (Fig. 3c). The periportal regions of liver were also infiltrated by hematopoietic cells (Fig. 3d). According to the Bethesda proposals for classification of murine nonlymphoid hematopoietic neoplasms,12 these mice had MPD-like myeloid leukemia (blast cells lower than 20%) or AMMoL (blast cells more than 20%) (Table 1).

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Figure 3. Features of the leukemia mice transplanted with types I and II of the MLL/AF10(OM-LZ)-immortalized cell lines. (a) Liu staining of cytospin preparation of the BM cells (nos. 93 and 64) or PB smears (all others) from leukemia mice. (b) Survival curve of the sublethally irradiated mice transplanted with lines 2, 4, 6 and 12G. (c) Histologic features of spleen (×100) and (d) liver (×100) of the representative leukemia mice. The inset is the size of spleen and liver. Scales of the organs are included.

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Table 1. Phenotypic features of mice transplanted with the MLL/AF10(OM-LZ)-immortalized cell lines
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The peripheral granulocytes and monocytes of the MPD-mice were generated from both donor and host cells

We have demonstrated that the type II cell lines could not terminally differentiate into mature granulocytes in vitro. However, both types of cell lines induced myeloproliferation with excess mature granulocytes and monocytes in vivo. To analyze the cell origin of largely expanded granulocytes and monocytes in the PB of MPD-mice, we collected the peripheral leukocytes from line 12G (GFP+)-transplanted B6 mice (GFP) and followed by flow cytometric analysis. At 4 weeks post-transplantation, the line 12G-mice had a normal number of WBC as that of the control mice (Figs. 4a and 4b, upper panel). Flow cytometric analysis also revealed that line 12G-mice and the control mice had a similar side-scatter/ forward-scatter (SSC/FSC) dot plot (Fig. 4b, upper panel). However, the GFP/Gr-1 (Gr-1 is a surface marker for the myeloid cells) dot plot revealed that a low percentage of the GFP-expressing myeloid cells (GFP+Gr-1+) could be detected in the line 12G-mice (Fig. 4b, upper panel). At 5 weeks post-transplantation, the number of WBC in line 12G-mice was still in a normal range, but a population of SSC/FSC low/medium cells was expanded (Fig. 4b middle panel). Flow cytometric analysis revealed that the percentages of GFPGr-1+ (host cell origin) and GFP+Gr-1+ (donor cell origin) myeloid cells were both increased (Fig. 4b, middle panel). When the mice were moribund, the majority of the peripheral leukocytes were myeloid cells, in which the cells of donor origin became higher than those of the host origin (Fig. 4b, lower panel). To further discriminate between monocytes and granulocytes, the CD115 and Ly-6G expressions in the myeloid cells were examined (Fig. 4c). The result indicated that the percentages of monocytes of donor and host origin and granulocytes of donor and host origin were all increased in the MPD-mice (Fig. 4c). It is of note that the majority of the donor-derived granulocytes (red dots in Ly-6G+ region) showed an extremely low expression of CD115 (1–3) than the host-derived granulocytes did (1–10) (Fig. 4c). The number of monocytes and granulocytes of host or donor origin in PB of the moribund mice were calculated by WBC count times cell proportion in the dot plot (Fig. 4d). This result clearly demonstrated that the hyperproliferated monocytes and granulocytes were generated from both donor and host cells.

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Figure 4. Cell origin analysis of the expanded peripheral myeloid cells in line 12G-induced MPD-mice. The SSC/FSC and GFP/Gr-1 dot plots of the peripheral myeloid cells in (a) a control mouse (no. 180) 4 weeks post-transplantation and (b) a MPD-mouse (no. 162) at 4 and 5 weeks post-transplantation and when moribund (7 weeks). The WBC count and the percentage of the donor-derived (GFP+) or host-derived myeloid cells were indicated. (c) The percentage of the monocytes (CD115+) or granulocytes (Ly-6G+) in a control mouse (no. 179) and MPD-mouse (no. 162) when moribund. The red dots indicate the monocytes and granulcytes of donor origin, and the blue dots of host origin. (d) The number of donor- and host-derived monocytes and granulocytes per liter of PB in the MPD-mice and control mouse.

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MLL/AF10(OM-LZ)-immortalized cells express GM-CSF, M-CSF, IL-1α, IL-1β and TNF-α

As part of the peripheral monocytes and granulocytes in the MPD-mice were generated from the host cells, we speculated that the MLL/AF10(OM-LZ) cell lines might express CSFs and/or inflammatory cytokines to stimulate host cell proliferation. Expression of the CSFs, including IL-3, IL-6, GM-CSF, G-CSF and M-CSF, and the inflammatory cytokines, including IL-1α, IL-1β, TNF-α and IFN-γ, was analyzed on the MLL/AF10(OM-LZ)-immortalized cell lines by RT-PCR assay. As shown in Figure 5, both of the types I and II cell lines expressed GM-CSF, M-CSF, IL-1α, IL-1β and TNF-α, but not G-CSF and IFN-γ.

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Figure 5. RT-PCR analysis of the transcripts of genes encoding colony-stimulating factors and inflammatory cytokines. Gene expression of IL3, IL6, GM-CSF, G-CSF, M-CSF, IL1α, IL1β, TNF-α or IFN-γ in the MLL/AF10(OM-LZ)-immortalized cell lines. Lane 1, E. coli-activated RAW264.7; lane 2, no template control; lane 3, line 2; lane 4, line 4; and lane 5, line 6. GAPDH was used as an internal control.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

By using retroviral transduction/transplantation technique, we established 6 MLL/AF10(OM-LZ)-immortalized cell lines with different differentiation potentials. Types I cell lines could terminally differentiate into granulocytes and monocytes by G-CSF and M-CSF treatment. On the other hand, type II cell lines had a weak monocytic but not granulocytic differentiation potential in response to G-CSF treatment, and induced growth arrest and rapid apoptosis by M-CSF treatment. It is conceivable that type I cell lines could develop a myelomonocytic phenotype, and type II cell lines could develop a monocytic phenotype in vivo. The presence of inconsistency in terminal differentiation potentials was also reported in some cell lines transformed by the MLL fusion genes. The human cell lines with MLL-AF9, UG3 and THP-1, responded differently to G-CSF and M-CSF with respect to granulocytic and monocytic differentiations.13, 14 In murine cell lines that were generated by retrovirally transducing a constitutive or a Tet-regulated MLL-ENL, 6 of the 7 could respond to G-CSF and undergo granulocytic terminal differentiation, and 2 cell lines could undergo monocytic terminal differentiation upon treatment with M-CSF.15–17 It was proposed that the heterogeneity of the myeloid progenitors susceptible to immortalization might affect their differentiation potentials.17 Whether the varied differentiation potentials in our MLL/AF10(OM-LZ) cell lines were attributed by the heterogeneity of committed progenitors need further investigation.

Although the in vitro differentiation potential of the MLL/AF10(OM-LZ) cell lines suggested that these cell lines could lead to myelomonocytic or monocytic phenotype, the in vivo tumorigenicity study showed that all of the MLL/AF10(OM-LZ)-immortalized cell lines induced similar MPD-like disease in most of the transplanted mice. As not all of the cell lines could terminally differentiate into granulocytes, suggesting that the excess amount of mature granulocytes in the MPD-mice might be generated from the host cells. Flow cytometric analysis on the cell origin of peripheral myeloid cells confirmed that the monocytes and granulocytes were generated not only from the donor cells but also from the host cells in a type I cell line-induced MPD-mice. This result suggested that the MLL/AF10(OM-LZ) cell lines might secrete some transacting factors to overstimulate host cell proliferation. Two groups of cytokines are known to stimulate myeloid cell proliferation. One is the CSFs, including IL-3, IL-6, GM-CSF, G-CSF and M-CSF, which directly regulate the lineage commitment, proliferation, differentiation and survival of granulocytes, monocytes/macrophages and their progenitors.18 The other is a variety of inflammatory cytokines, including IL-1, TNF-α and IFN-γ, which can stimulate or upregulate endothelial cells, fibroblasts, T-lymphocytes and monocytes to produce GM-CSF, G-CSF or M-CSF, and, therefore, indirectly regulate myelopoiesis.19–24 Our RT-PCR analysis demonstrated that the MLL/AF10(OM-LZ)-immortalized cells expressed CSFs of GM-CSF and M-CSF and the inflammatory cytokines of IL1 and TNF-α. These results suggested that MPD developed in the MLL/AF10(OM-LZ)-transplanted mice might be partially caused by cytokine-induced stimulation of host cells.

Several MLL fusions have also been shown to induce myeloproliferation in either the retroviral transduction/transplantation or knock-in mouse models, including MLL/SEPT6,25MLL/AF926 and MLL/CBP.27, 28 However, these studies neither describe the differentiation potentials nor the capability of expressing CSFs and inflammatory cytokines in the MLL fusion cells. Lavau et al. observed that transplantation of BM cells with retrovirally transduced MLL/CBP (EGFP+) induced MPD and later progressed to AML.28 Before AML progression, only a small fraction of the cells in PB of the MLL/CBP mice was EGFP+, which displayed immunophenotypic characteristics of immature myeloid cells. In addition, the cotransduced EGFP BM cells preferentially differentiated into mature myeloid cells (Mac-1+Gr-1+) in MLL/CBP mice than that of the control mice (24 vs. 8%).28 These observations were similar to ours on the aspect that the MLL/CBP-transduced cells could not terminally differentiate into mature myeloid cells, and the BM cells (cotransduced with the MLL/CBP cells) were stimulated to generate more mature myeloid cells in the transplanted mice.

As most of the transplanted mice developed MPD, 3 mice developed AMMoL (line 4-nos. 6 and 93; line 6-no. 64). It is not clear why these mice had a different disease manifestation. As the MLL/AF10(OM-LZ)-immortalized cell lines were established from a pooled retrovirally transduced BM cells, it will be helpful to characterize the subclones of the leukemia cells between the MPD- and AMMoL-mice. Further experiments may be needed to clarify the differences between the cells induced MPD-like myeloid leukemia and AMMoL in the transplanted mice.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Dr. Ming-Ling Kuo and Dr. Yuan-Ji Day for helping with animal experiments and Ms. Hui-Chin Hsu and Ms. Hui-Tze Huang for technical assistance.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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