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Blood, Vol. 113, Issue 5, 1086-1096, January 29, 2009
RAS oncogene suppression induces apoptosis followed by more differentiated and less myelosuppressive disease upon relapse of acute myeloid leukemia
Blood Kim et al.
113: 1086
Supplemental materials for: Kim et al
Immunofluorescent detection of MLL
4 µm formalin fixed paraffin sections of murine tissues were stained for MLL. The sections were deparaffinized and rehydrated, followed by antigen retrieval using a pressure cooker with citrate buffer pH 6.0. Slides were steamed for 30 minutes then cooled for 20 minutes. Next, protein block (Serum-Free Protein Block, Dako, Carpinteria, CA) was applied for 15 minutes at room temperature. Goat anti-human MLL antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was diluted at 1:20 and incubated for 60 minutes at room temperature. A conjugated donkey anti-goat–Alexa Fluor diluted, 1:400, was applied for 60 minutes at room temperature in the dark. Slides were counterstained with Vectashield DAPI (Vector Laboratories, Burlingame, CA) and coverslipped. Slides were viewed using Nikon C1si Laser Scanning Confocal Microscope. Fluorescence-activating cell sorting (FACS) and RT-PCR
To analyze the mRNA expression patterns of endogenous Mll, Mll-AF9, endogenous Nras, and NRASG12V in GFP-positive and GFP-negative leukemia cells, we sorted bone marrow cells of transplanted SCID mice before/after treating Dox. 0.5–1 × 107 bone marrow cells were cultured in media including mouse SCF, IL-3, and IL-6 for 12 hours and suspended in 200 µl FACS buffer (PBS, 2% fetal bovine serum, 0.1% sodium azide). The bone marrow cells were filtered through 70 µm cell strainers (BD Falcon, Bedford, MA) and then sorted by the fluorescence of GFP. The cell sorting was performed by FACSDiva Cell-Sorting System (BD Biosciences, San Jose, CA) through 90 µm nozzle under cooling conditions. All procedures were performed in ice or pre-chilled 4°C centrifuge and protected from light. Using a direct mRNA micro kit (Qiagen Inc., Valencia, CA), we prepared mRNA from the sorted bone marrow cells. To detect the expression of mRNAs, we used a pair of DNA primers. Endogenous GAPDH transcripts as a loading control. RT-PCR was performed by using the Robust-RT kit (Finnzymes; MJ Research, Waltham, MA). We also performed RT-PCR without reverse transcriptase to ensure the RT-PCR products were not amplified from contaminated chromosomal DNA.
Files in this Data Supplement:
- Figure S1. Phenotypic profiles of the triply- or doubly-transgenic mice generated by crossing Vav-tTA; TRE-NRASG12V co-transgenic FVB/n to Mll-AF9 knock-in C57BL/6J mice (JPG, 33.3 KB)
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Table shows survival time, WBC concentration, size/weight of spleen, and size of lymph node in TRM-, TM-, and TR-transgenic mice when they were moribund or died after the development of AML, mastocytosis, or both.
- Figure S2. Dox treatment during prenatal periods does not have an effect on survival time (JPG, 22.4 KB)
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Vav-tTA; TRE-NRASG12V; Mll-AF9 triply-transgenic and Vav-tTA; Mll-AF9 doubly-transgenic FVB/n × C57BL/6J F1 mice developed AML and had similar survival time regardless whether they were or were not treated with Dox during the prenatal period (Dox treatment indicated with bidirectional arrow).
- Figure S3. Histopathology of bone marrow and spleen in transgenic F1 mice (JPG, 141 KB)
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Bone marrow of a Vav-tTA; Mll-AF9; TRE-NRASG12V triply-transgenic mouse with AML contains large numbers of myeloid cells in various stages of differentiation (including ~30% of blasts) suggestive of myeloid neoplasia. Spleen contains large number of mast cells consistent with diagnosis of mastocytosis. Bone marrow and spleen of a Vav-tTA; Mll-AF9 doubly-transgenic mouse with AML contain large numbers of blasts suggestive of myelomonocytic lineage constituting 50–80% of total cell population. Bone marrow and spleen from a Vav-tTA; TRE-NRASG12V doubly-transgenic mouse contain large numbers of mast cells occupying 80–90% of sections consistent with systemic mastocytosis. All sections were stained with hematoxylin and eosin stain.
- Figure S4. AML4 bone marrow transplantation into secondary recipient SCID mice is less sensitive to Dox treatment after the establishment of AML (JPG, 20.6 KB)
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Slight and brief reduction in peripheral WBC concentration after treatment with Dox is followed by rapid relapse in secondary recipient SCID mice transplanted with the bone marrow cells of AML4. The first “Dox” in the arrowed square means the start of chronic and 6hr Dox treatments. Other “Dox” in the arrowed squares indicated the repeated 6hr Dox treatment. Mouse numbers are n=3 treated with 6hr Dox, n=3 with chronic Dox, and n=2 with no Dox. Narrow bars show standard deviations at each measurement.
- Figure S5. Histopathology of bone marrow and spleen in secondary recipient SCID mice is compatible with MPD-like AML (JPG, 99.6 KB)
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Bone marrow and spleen of both mice contain predominantly myeloid cells (M) including mature neutrophiles. Blasts are less than 20%. Erythroid cell clusters are present (E). Spleen of the mouse temporary treated with Dox contains a population of mast cells (MC). Based on histologic findings and WBC count data, relapsed disease in Dox-treated mice was diagnosed as MPD-like AML. All sections were stained with hematoxylin and eosin stain.
- Figure S6. Mll-AF9 is constantly expressed in bone marrow of AML-transplanted SCID mice (JPG, 64.5 KB)
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Immunofluorescent microscopy shows the expression of Mll proteins by staining N-terminus of mouse Mll. Bone marrow cells in SCID mice after the establishment of full-blown TRM (Vav-tTA; TRE-NRASG12V; Mll-AF9)–transgenic or TM (Vav-tTA; Mll-AF9)–transgenic AML expresses similar level of Mll proteins that may include Mll-AF9 protein. The expression level of Mll protein in the relapsed MPD-like AML with constant Dox treatment is also similar to TRM-transgenic and TM-transgenic AML. Since comparably fewer number of bone marrow cells in normal SCID mice expresses Mll protein, the most cells expressing Mll proteins are counted for the transplanted AML cells. Because the expressions of both endogenous Mll and Mll-AF9 are regulated by the intrinsic promoter of mouse Mll gene, these results imply that Mll-AF9 is constantly expressed in most TRM-transgenic AML and relapsed MPD-like AML cells independent from the NRASG12V suppression. All sections were counter-stained with DAPI to identify the nuclear.
- Figure S7. NRASG12V expression is dependent on Dox treatment, but Mll-AF9 is independent in bone marrow of AML-transplanted SCID mice (JPG, 35.5 KB)
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Using RT-PCR, NRASG12V and Mll-AF9 transcriptions are detected in TRM (Vav-tTA; TRE-NRASG12V; Mll-AF9)–transgenic GFP positive bone marrow cells. The NRASG12V expression is repressed by Dox treatment in the GFP positive bone marrow cells of remission and relapsed AML. However, Mll-AF9 expression is not altered by Dox treatment. All bone marrow cells were cultured in vitro for 12 hours in media including mouse SCF, IL-3, and IL-6 prior to cell sorting. TRM-transgenic AML cells are GFP positive, and normal bone marrow cells are GFP negative in SCID mice transplanted with TRM-transgenic AML. TM (Vav-tTA; Mll-AF9)–transgenic AML cells are GFP negative because of no GFP gene along with TRE-NRASG12V. GAPDH is used as a loading control. No RT-PCR products were found in the same procedures without reverse transcriptase. SM – DNA size marker; W – water control.
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