by Sanjai Sharma, and Alan Lichtenstein Aberrant splicing of the E-cadherin transcript is a novel mechanism of gene silencing in chronic lymphocytic leukemia cells by Sanjai Sharma, and Alan Lichtenstein Blood Volume 114(19):4179-4185 November 5, 2009 ©2009 by American Society of Hematology

Analysis of exon 11-skipped transcript. Analysis of exon 11-skipped transcript. (A) RT-PCR analysis of untreated chronic lymphocytic leukemia (CLL) cells and cells treated with emetine (e) or emetine plus actinomycin D (e + A) and assayed for expression of E-cadherin, BIK, and DUSP4. In the top panel, E-cadherin cDNA was amplified with primers at the 3′ end of the gene, In the bottom panel, the E-cadherin cDNA is amplified by primers in the region of exons 10 and 12. The upper points toward the band of expected size, and the lower points toward a DNA fragment lacking exon 11. The next 2 panels show amplification of BIK and DUSP4. Glyceraldehyde-3-phosphate dehydrogenase amplification shown as a control. (B) Time course of emetine-induced up-regulation of E-cadherin RNA. CLL cells treated with emetine for 0, 4, 8, 12, and 16 hours. Primers in the region of exons 10 and 12 were used for amplification of E-cadherin cDNA and give rise to 2 fragments that differ by 146 base pairs in emetine-treated cells due to exon 11 deletion. (C) Sequence of the E-cadherin gene in the region of interest. There is an exon 11 deletion resulting in a frame shift and PTC codon. Sanjai Sharma, and Alan Lichtenstein Blood 2009;114:4179-4185 ©2009 by American Society of Hematology

Inactivation of Rent 1. Inactivation of Rent 1. (A) Using a siRNA against Rent 1, this gene was inactivated in Raji cells, a B-cell line. The bar diagram shows relative expression of Rent 1 RNA by real-time RT-PCR analysis in untreated cells, Raji cells transfected with scrambled siRNA, or with Rent 1 siRNA (relative to actin expression). (B) The same cDNA material was analyzed by PCR with primers in exons 10 and 12. An exon 11–skipped transcript is seen in the Rent 1–transfected Raji cells, as indicated by the lower ←. Sanjai Sharma, and Alan Lichtenstein Blood 2009;114:4179-4185 ©2009 by American Society of Hematology

Relative amount of skipped transcript in CLL specimens. Relative amount of skipped transcript in CLL specimens. (A) Schematic showing the binding sites of the 2 probes that detect different E-cadherin transcripts. (B) Bar diagram showing selected examples of relative levels of aberrant transcript in normal B cells and CLL specimens relative to their wild-type expression. Numbers above the bars represent aberrant E-cadherin transcript levels. (C) Dot-plot analysis of normal B cells and 26 CLL specimens with E-cadherin expression levels on x-axis and relative aberrant transcript expression on the y-axis. Significant statistical correlation with P = .018. Sanjai Sharma, and Alan Lichtenstein Blood 2009;114:4179-4185 ©2009 by American Society of Hematology

Wnt reporter activity in CLL specimens. Wnt reporter activity in CLL specimens. Bar diagram with wnt reporter activity. Each CLL sample was transfected with the negative control vector (neg K, firefly luciferase lacking TCF/LEF binding sites), or the wnt reporter (wnt rep, wnt reporter vector with TCF/LEF binding sites and empty vector), or the wnt reporter plus E-cadherin expression vector (wnt rep + E). Constitutively expressing Renilla luciferase was cotransfected in all of the groups as a control for transfection efficiency. Reporter expression of the negative control (firefly to Renilla ratio) was arbitrarily kept as 1 unit for each CLL case. Sanjai Sharma, and Alan Lichtenstein Blood 2009;114:4179-4185 ©2009 by American Society of Hematology