Results: 5

1.
Figure 4.

Figure 4. From: Activation-induced cytidine deaminase acts as a mutator in BCR-ABL1-transformed acute lymphoblastic leukemia cells.

Tracing the clonal evolution of Ph+ ALL cells by VH region gene mutations. Genealogic trees of ongoing SHM of VH gene segments amplified from the Ph+ ALL cell lines NALM1 and BV173 are shown. Numbers indicate the mutated codons within the rearranged V region. Each circle represents one VH sequence amplified from a leukemia subclone, and a and b denote distinct mutations within the same codon.

Niklas Feldhahn, et al. J Exp Med. 2007 May 14;204(5):1157-1166.
2.
Figure 2.

Figure 2. From: Activation-induced cytidine deaminase acts as a mutator in BCR-ABL1-transformed acute lymphoblastic leukemia cells.

AID expression in patient-derived Ph+ ALL cells depends on BCR-ABL1 kinase activity in vivo. Matched sample pairs from four patients with Ph+ ALL before the onset and during continued treatment with the BCR-ABL1 kinase inhibitor STI571 were analyzed for AID mRNA levels by semiquantitative RT-PCR (A). The content of Ph+ ALL cells in all samples was normalized by BCR-ABL1 fusion transcripts (A), with “p190” and p210" indicating the two different breakpoints. Protein lysates from the same Ph+ ALL cases were also subjected to Western blot analysis for AID expression using EIF4E as a loading control (B). Protein lysates from CD19+ tonsillar B cells were used as a positive control.

Niklas Feldhahn, et al. J Exp Med. 2007 May 14;204(5):1157-1166.
3.
Figure 5.

Figure 5. From: Activation-induced cytidine deaminase acts as a mutator in BCR-ABL1-transformed acute lymphoblastic leukemia cells.

AID induces DNA-SSB in Ig and non-Ig genes. To establish a causative link between AID function and the occurrence of DNA-SSB, AID mRNA expression was silenced in three Ph+ ALL cell lines by RNA interference using fluorochrome-labeled siRNAs against AID or nontargeting siRNA duplexes as a control. Fluorochrome-labeled cells were sorted and analyzed for silencing efficiency and specificity by RT-PCR (A), and for DNA-SSB within rearranged VH gene segments and the CDKN2B gene by LM-PCR (B). For BV173, Nalm1, and SD1 cells, DNA-SSB intermediates in rearranged VH3-21, VH3-9, and VH3-30 gene segments were amplified, respectively. As a loading control for genomic DNA, VH gene rearrangements and a genomic fragment of the CDKN2B gene were amplified (B). The CDKN2A gene at chromosome 9p21 immediately adjacent to CDKN2B was partly or entirely deleted in BV173 and Nalm1 cells, precluding LM-PCR analysis of this locus in these cell lines (not depicted).

Niklas Feldhahn, et al. J Exp Med. 2007 May 14;204(5):1157-1166.
4.
Figure 3.

Figure 3. From: Activation-induced cytidine deaminase acts as a mutator in BCR-ABL1-transformed acute lymphoblastic leukemia cells.

BCR-ABL1–mediated up-regulation of AID involves repression of ID2, a negative regulator of AID. Two Ph+ ALL cell lines (BV173 and SUP-B15) were incubated in the presence or absence of 10 μmol/l STI571 for 16 h and subjected to microarray analysis using the Affymetrix U133A 2.0 platform as described in Materials and methods (A). mRNA levels of ID2 were compared with those of AID and its positive regulators E2A and PAX5. As controls, known STI571-inducible genes (IGKC, RAG1, RAG2, and BACH2) are shown. (B) The two Ph+ ALL cell lines were cultured for 48 h in the presence or absence of STI571, and protein levels of ID2 (top) and AID (bottom) were measured by flow cytometry. (C) To test the functional relevance of BCR-ABL1–mediated down-regulation of ID2 in Ph+ ALL cells, the effect of ID2 overexpression on AID mRNA levels was measured in Ph+ ALL cells. Therefore, the two Ph+ ALL cell lines were stably transduced with a vector encoding only GFP (left) or both GFP and ID2 (right). Overexpression of ID2 was monitored together with mRNA levels of AID. GAPDH mRNA levels were used for normalization of cDNA amounts.

Niklas Feldhahn, et al. J Exp Med. 2007 May 14;204(5):1157-1166.
5.
Figure 1.

Figure 1. From: Activation-induced cytidine deaminase acts as a mutator in BCR-ABL1-transformed acute lymphoblastic leukemia cells.

AID expression in Ph+ ALL cells. mRNA expression of AID was measured in normal human pro–B, pre–B, B1, naive, germinal center, and memory B cells as well as plasma cells by RT-PCR (A). In a semiquantitative RT-PCR analysis, AID mRNA expression in Ph+ ALL cells was compared with germinal center B cells and Ph ALL cells. GAPDH was used for normalization of cDNA amounts (A). Ph+ ALL cell lines (BV173 and Nalm1; 10 μmol/l STI571) and v-abl–transformed mouse pre–B cells (300-19; 1 μmol/l STI571) were treated with or without STI571 for 24 h and subjected to semiquantitative RT-PCR analysis for human AID and GAPDH or murine Aid and Hprt mRNA expression (B). Protein lysates from STI571-treated or untreated Ph+ ALL cells (BV173) were used for Western blotting (C) together with antibodies against AID and EIF4E (used as a loading control). IL-3–dependent murine pro–B cells carrying a doxycycline-inducible BCR-ABL1 transgene were incubated with or without 1 μg/ ml doxycycline for 24 h and subjected to RT-PCR analysis of Aid, Oct2, and Obf1 mRNA expression (C). Ph ALL cells were transiently transfected with a pMIG vector encoding GFP and/or GFP and BCR-ABL1. After 24 h, GFP-expressing cells were sorted and subjected to RT-PCR analysis for AID expression (C).

Niklas Feldhahn, et al. J Exp Med. 2007 May 14;204(5):1157-1166.

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