Gene-expression analysis identifies novel RBL2/p130 target genes in endemic Burkitt lymphoma cell lines and primary tumors by Giulia De Falco, Eleonora.

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Gene-expression analysis identifies novel RBL2/p130 target genes in endemic Burkitt lymphoma cell lines and primary tumors by Giulia De Falco, Eleonora Leucci, Dido Lenze, Pier Paolo Piccaluga, Pier Paolo Claudio, Anna Onnis, Giovanna Cerino, Joshua Nyagol, Walter Mwanda, Cristiana Bellan, Michael Hummel, Stefano Pileri, Piero Tosi, Harald Stein, Antonio Giordano, and Lorenzo Leoncini Blood Volume 110(4):1301-1307 August 15, 2007 ©2007 by American Society of Hematology

Mutational analysis of the RBL2/p130 gene. Mutational analysis of the RBL2/p130 gene. Exon 21 has been sequenced and reveals an A insertion in the 20–21 intron-exon boundary. Giulia De Falco et al. Blood 2007;110:1301-1307 ©2007 by American Society of Hematology

Morphology and immunohistochemistry of BL primary tumors and cell lines. Morphology and immunohistochemistry of BL primary tumors and cell lines. (A) May-Grunwald Giemsa staining of cytologic smears showing the characteristic intracytoplasmic vacuoles. (B) Immunohistochemistry of BL primary tumors showing cytoplasmic localization of the pRb2/p130 protein suggestive of mutations within the nuclear localization signal. Immunohistochemistry of BL cells transfected with either the empty vector (C) or RBL2/p130 (D). Cytoplasmic localization of the pRb2/p130 protein is observed in empty vector–transfected cells whereas RBL2/p130-transfected BL cells show normal nuclear expression of pRb2/p130. Images were viewed by means of a Zeiss Aski Axioskop 40 microscope equipped with a Zeiss Axiocam H Rc (panels A, C, and D, 40×/0.65 NA; panel B, 63×/0.80 NA) (Zeiss, Vertrieb, Germany) and processed using Axiovision (Zeiss) software. Original magnification: panels A, C, and D, × 750; panel B, × 970. Giulia De Falco et al. Blood 2007;110:1301-1307 ©2007 by American Society of Hematology

Proliferation rate of BL cell lines either transfected with the empty vector or with RBL2/p130. Proliferation rate of BL cell lines either transfected with the empty vector or with RBL2/p130. Cells were counted for 4 days, and proliferation rate was assessed. Each experiment was performed in duplicate, and the average of 5 different experiments is reported. P = .006. Error bars represent standard deviation. Giulia De Falco et al. Blood 2007;110:1301-1307 ©2007 by American Society of Hematology

Analysis of the cell cycle of BL cell lines, transduced either with RBL2/p130 (B) or with the empty vector (A), as a negative control, obtained by FACS analysis. Analysis of the cell cycle of BL cell lines, transduced either with RBL2/p130 (B) or with the empty vector (A), as a negative control, obtained by FACS analysis. Briefly, each cell-cycle phase is distinguishable on the basis of DNA content because propidium iodide (PI) stoichiometrically binds to DNA, making it possible to discriminate between dividing and nondividing cells. Each cell-cycle stage is graphically represented as a distinct peak on the graph. Percentages of cells in each cell-cycle stage are reported in Table 2. Notably, RBL2/p130 induces an increase in the G0/G1 fraction, thus suggesting a reduction of the proliferation rate. Giulia De Falco et al. Blood 2007;110:1301-1307 ©2007 by American Society of Hematology

Gene-expression analysis of RBL2/p130 transfected vs untransfected cells. Gene-expression analysis of RBL2/p130 transfected vs untransfected cells. (A,B) Supervised analysis (t test ANOVA, P < .01), wild-type versus RBL2-transfected Raji cells, 103 genes modulated after transfection (A). Genes with Pearson correlation of at least 0.95 to RBL2/p130 (B). Giulia De Falco et al. Blood 2007;110:1301-1307 ©2007 by American Society of Hematology

Quantitative RT-PCR of BL cell lines, either transfected with the empty vector or RBL2/p130, versus BL primary tumors to validate microarray analysis upon RBL2/p130 reintroduction. Quantitative RT-PCR of BL cell lines, either transfected with the empty vector or RBL2/p130, versus BL primary tumors to validate microarray analysis upon RBL2/p130 reintroduction. Error bars represent standard deviation. Giulia De Falco et al. Blood 2007;110:1301-1307 ©2007 by American Society of Hematology