Defective RNA-mediated c-myc gene silencing pathway in Burkitt’s lymphoma Deepak Kaul * and Kavleen Sikand Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research, Chandigarh 160 012, India Received 31 October 2003 Abstract Keeping in view the fact that molecular basis of Burkitt’s lymphoma (BL) is poorly understood, we attempted to explore the small interfering RNA (siRNA) mediated c-myc gene regulation using BL-derived EB-3 cell line as archetype cellular model. Such a study revealed that EB-3 cells possess 4-fold higher expression of Dicer gene coupled with 2-fold higher activity of RNA polymerase III than that observed in normal human lymphocytes. siRNAs derived from EB-3 cells had the inherent capacity to suppress c-myc gene expression in normal cells but not in native cells. Based on these findings we have proposed a novel RNA-mediated c-myc gene regulation pathway that may be responsible for BL. Ó 2003 Elsevier Inc. All rights reserved. Keywords: RNA pol-III; Dicer; siRNA; Silencing; C-myc; Burkitt’s lymphoma Burkitt’s lymphoma, recognized to be highly aggres- sive malignancy of B cells, is invariably associated with chromosomal translocations that dysregulate the ex- pression of c-myc gene, which consists of three exons [1,2]. Exons 2 and 3 code for the c-myc protein and exon 1 encodes an unusually long 5 0 untranslated sequence that is responsible for blockage of transcriptional elongation [1]. DNA sequence analysis in various types of lympho- mas has revealed that the c-myc regulatory regions within exon 1 and intron 1 are either mutated or removed re- sulting in the abrogation of negative regulation [1]. In- terestingly, RNA polymerase III (Rp-III) promoter sequences have been shown to be present in exon 1/intron 1 of the c-myc gene [1,3] and thereby Rp-III has the in- herent capacity to transcribe short hairpin RNAs (shR- NAs). Further, there exists a general recognition of the fact that genes can be regulated by RNA interference (RNAi), which depends on the accidental or deliber- ate expression of double-stranded RNA (dsRNA). These dsRNAs are processed by Dicer into small interfering RNAs (siRNAs) that either bind to and degrade any mRNA transcripts with the same sequence or regulate heterochromatin formation [4,5]. Based on these find- ings, the present study was addressed to explore whether or not the deregulated expression of c-myc gene in BL derived EB-3 cell line is because of the defect in the RNA- mediated gene regulation pathway proposed in Fig. 1. Materials and methods Cellular models employed. The human Burkitt’s lymphoma cell line, EB-3, was obtained from the National Centre for Cell Science, Pune (India). It was maintained in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) in humidified 5% CO 2 atmosphere at 37 °C. Lymphocytes were obtained from normal healthy volunteers, who were fasting for 12 h and abstained from any medication for 2 weeks before blood donation. Lymphocytes from blood were isolated using Ficoll–Hypaque gradient centrifugation [6]. Isolated lymphocytes were cultured using the same growth medium and conditions as those of EB-3 cell line. Quantitative gene expression. Total cellular RNA was extracted by the acid–guanidinium–phenol–chloroform method [7]. RNA yield and purity was determined spectrophotometrically at 260–280 nm and RNA integrity was verified by agarose gel electrophoresis. The isolated RNA was subjected to RT-PCR using specific primers for genes coding for c-myc, Dicer, PPAR-c, and b 2 -microglobulin (b 2 M) under the optimized conditions [8–10]. The numbers of cycles were determined in preliminary experiments to be within the exponential range of PCR amplification. b 2 M gene expression was used as a control for RNA loading and efficiency of reverse transcription. The PCR products were * Corresponding author. Fax: +91-172-744401. E-mail address: dkaul_24@hotmail.com (D. Kaul). 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.12.002 Biochemical and Biophysical Research Communications 313 (2004) 552–554 BBRC www.elsevier.com/locate/ybbrc