Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Dec 1;106(12):3932-9.
doi: 10.1182/blood-2005-03-1113. Epub 2005 Aug 18.

Insertional mutagenesis identifies genes that promote the immortalization of primary bone marrow progenitor cells

Yang Du  1 , Nancy A Jenkins, Neal G Copeland
Affiliations

Insertional mutagenesis identifies genes that promote the immortalization of primary bone marrow progenitor cells

Yang Du et al. Blood. .

Abstract

Retroviruses can induce hematopoietic disease via insertional mutagenesis of cancer genes and provide valuable molecular tags for cancer gene discovery. Here we show that insertional mutagenesis can also identify genes that promote the immortalization of hematopoietic cells, which normally have only limited self-renewal. Transduction of mouse bone marrow cells with replication-incompetent murine stem cell virus (MSCV) expressing only neo, followed by serial passage in liquid culture containing stem cell factor (SCF) and interleukin-3 (IL-3), produced immortalized immature myeloid cell lines with neutrophil and macrophage differentiation potential in about 50% of the infected cultures. More than half of the lines have MSCV insertions at Evi1 or Prdm16. These loci encode transcription factor homologs and are validated human myeloid leukemia genes. Integrations are located in intron 1 or 2, where they promote expression of truncated proteins lacking the PRDI-BF1-RIZ1 homologous (PR) domain, similar to what is observed in human leukemias with EVI1 or PRDM16 mutations. Evi1 overexpression alone appears sufficient to immortalize immature myeloid cells and does not seem to require any other cooperating mutations. Genes identified by insertional mutagenesis by their nature could also be involved in immortalization of leukemic stem cells, and thus represent attractive drug targets for treating cancer.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Immature myeloid progenitor cells with biphenotypic differentiation potential can be immortalized by infection with replication-defective MSCV. (A) Schematic representation of the immortalization procedure. Horse serum was included in the cultures along with the indicated growth factors after infection. (B) Wright-Giemsa staining of uninfected (left panel) and infected (right panel) bone marrow (BM) cells after 1 month in culture. Original magnification × 400. (C) Wright-Giemsa staining of 2 immortalized cell lines (BM-7 and BM-39) before and after treatments with G-CSF (3 days), GM-CSF (5 days), or PMA (2 days). BM-7 harbors a single MSCV Evi1 integration, while BM-39 harbors a Prdm16 integration. Both cell lines respond similarly to cytokine stimulation. Original magnification × 400. Images were obtained with the use of an Olympus Vanox AHBS3 microscope and a Nikon DXM1200F digital camera. Images were processed using Image-Pro Plus 5.1.
Figure 2.
Figure 2.
Immortalized cell lines are often clonal. (A) Southern blot analysis of the MSCV integrations present in 15 immortalized cell lines. DNA (8 μg) from each immortalized line was digested with HindIII and hybridized with a neo probe isolated from the MSCV vector. Each band represents a separate MSCV integration. (B) Southern blot analysis of 1 representative immortalized immature myeloid parental line (BM-2a) and its 7 single-cell subcloned lines (BM-2a-1-BM-2a-7).
Figure 3.
Figure 3.
Evi1 and Prdm16 are frequent targets of MSCV integration in immortalized immature myeloid progenitor cell lines. Evi1 MSCV integrations (top panel) and Prdm16 integrations (bottom panel). Yellow boxes depict exons, while red triangles indicate the location and orientation of integrated MSCV proviruses. Cell line numbers are listed above the red triangles.
Figure 4.
Figure 4.
Evi1 and Prdm16 mRNA expression is activated by MSCV integrations. Northern blot analysis of Evi1 and Prdm16 expression in immortalized cell lines using probes specific for each gene. β-actin was used as a loading control. Lines BM-62, BM-72, and BM-83 have confirmed Evi1 integrations, while lines BM-60, BM-67, BM-69, BM-75, BM-80, and BM-81 have confirmed Prdm16 integrations. Lines BM-37 and BM-70 have neither integrations.
Figure 5.
Figure 5.
MSCV integrations promote the expression of Evi1 and Prdm16 isoforms lacking the PR domain. (A) Mouse Mds1 locus and partial Evi1 locus with MSCV integrations are shown in the top panel. Mds1 and Evi1 exons are depicted as blue and yellow boxes, respectively. Red triangles indicate the location and orientation of integrated MSCV. Asterisks indicate translational start sites. The bottom panel depicts the generation of the Mds1-Evi1 fusion transcript and shows the location of the PR domain. (B) Mouse Prdm16 locus (partial) with MSCV integrations and the transcript encoding the long Prdm16 isoform containing the PR domain. Exons are depicted as yellow boxes. Translational start sites for the long and short isoforms are indicated by asterisks. (C) The nature of the fusion transcript between MSCV and Prdm16 exon 2 is shown. SD indicates splice donor in MSCV.
Figure 6.
Figure 6.
Evi1 can immortalize immature myeloid cells with high efficiency. Southern blot analysis of MSCV integrations in 5 immortalized cell lines (P1 to P5) generated by MSCV-Evi1 infection (10 μg/lane) and 11 cell lines subcloned from the P3 line (P3-1 to P3-11) (5 μg/lane) digested with BglII.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. 1997;3: 730-737. - PubMed
    1. Cobaleda C, Gutierrez-Cianca N, Perez-Losada J, et al. A primitive hematopoietic cell is the target for the leukemic transformation in human philadelphia-positive acute lymphoblastic leukemia. Blood. 2000;95: 1007-1013. - PubMed
    1. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003;100: 3983-3988. - PMC - PubMed
    1. Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature. 2001;414: 105-111. - PubMed
    1. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100: 57-70. - PubMed

Publication types

MeSH terms