Sleeping beauty-mediated somatic mutagenesis implicates CSF1 in the formation of high-grade astrocytomas - PubMed (original) (raw)

. 2010 May 1;70(9):3557-65.

doi: 10.1158/0008-5472.CAN-09-4674. Epub 2010 Apr 13.

Lara S Collier, Fausto J Rodriguez, Christina Tieu, Jon D Larson, Chandralekha Halder, Eric Mahlum, Thomas M Kollmeyer, Keiko Akagi, Gobinda Sarkar, David A Largaespada, Robert B Jenkins

Affiliations

Sleeping beauty-mediated somatic mutagenesis implicates CSF1 in the formation of high-grade astrocytomas

Aaron M Bender et al. Cancer Res. 2010.

Abstract

The Sleeping Beauty (SB) transposon system has been used as an insertional mutagenesis tool to identify novel cancer genes. To identify glioma-associated genes, we evaluated tumor formation in the brain tissue from 117 transgenic mice that had undergone constitutive SB-mediated transposition. Upon analysis, 21 samples (18%) contained neoplastic tissue with features of high-grade astrocytomas. These tumors expressed glial markers and were histologically similar to human glioma. Genomic DNA from SB-induced astrocytoma tissue was extracted and transposon insertion sites were identified. Insertions in the growth factor gene Csf1 were found in 13 of the 21 tumors (62%), clustered in introns 5 and 8. Using reverse transcription-PCR, we documented increased Csf1 RNAs in tumor versus adjacent normal tissue, with the identification of transposon-terminated Csf1 mRNAs in astrocytomas with SB insertions in intron 8. Analysis of human glioblastomas revealed increased levels of Csf1 RNA and protein. Together, these results indicate that SB-insertional mutagenesis can identify high-grade astrocytoma-associated genes and they imply an important role for CSF1 in the development of these tumors.

(c)2010 AACR.

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Conflict of interest statement

Potential Conflict of Interest: None

Figures

Figure 1

Figure 1. SB-Induced Brain Tumors Display Characteristics of High Grade Astrocytomas

(A) Left: Low power image of large, SB-induced astrocytoma including mitotic figure (inset). Note invasive edge (black arrowheads). Right: Image depicting pseudopallisading necrosis (white arrowheads), a classic histologic feature of human GBM. (B, C, D,) A subset of tumor cells stain positive for glial markers GFAP (black arrowheads, B), S100 (Black arrowheads, C), and Olig2 (D).

Figure 2

Figure 2. Clustering of SB insertions at the Csf1 locus as well as elevated expression suggests a functional connection to pathogenesis of high grade astrocytomas

(A) SB insertions at the Csf1 locus cluster in the 5th and 8th introns and are largely oriented in the same direction as Csf1 transcription (arrows). Underlined insertions denote chimeric transcripts. Blue arrows indicate samples used for comparative PCR analysis in B. (B) qRT (above) and semi-quantitative (below) PCR analysis of primary murine GBM samples showing elevation of Csf1 transcript in tumor (T) vs. adjacent normal (N) tissue. Error bars represent standard deviation of fold-change. (C) Top: Neoplastic cells from mouse tumor 333 display immunohistochemical staining of Csf1 (arrowheads) and its receptor CSF1R (arrows) respectively. Bottom: In contrast, adjacent normal tissue from the same specimen did not show appreciable staining for either Csf1 or Csf1R.

Figure 3

Figure 3. Over-expression of CSF1 in human GBM

(A) Immunohistochemical staining showing CSF1 (Arrows, right) and CSF1R (Arrows, left) staining in human GBM tissue sections. (B) Western blot of whole cell lysates from normal (post-mortem) brain (N1-N5) as well as from primary human GBMs (T1-T15). Bars represent the ratio of CSF1 to GAPDH signal intensity. (C) qRT-PCR result showing elevated levels of CSF1 transcript in normal (n=5) vs. GBM (n=18) tissue (p-value generated using Wilcoxon rank-sum test). (D) Top: Dual label immunofluorescence images illustrating tumor cells (white arrows) expressing both CSF1 (Red) and the glial marker GFAP (Green). Bottom: Separate immunofluorescence experiment illustrating distinct CSF1 staining in (red) tumor cells (arrowheads) and CD45 staining in (green) infiltrating lymphoid/myeloid cells.

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