Genetic alterations of RD(INK4/ARF) enhancer in human cancer cells

Abstract

Recent identification of an enhancer element, RD(INK4/ARF) (RD), in the prominent INK4/ARF locus provides a novel mechanism to simultaneously regulate the transcription of p15(INK4B) (p15), p14(ARF) , and p16(INK4A) (p16) tumor suppressor genes. While genetic inactivation of p15, p14(ARF) , and p16 in human tumors has been extensively studied, little is known about genetic alterations of RD and its impact on p15, p14(ARF) , and p16 in human cancer. The purpose of this study was to investigate the potential existence of genetic alterations of RD in human cancer cells. DNAs extracted from 17 different cancer cell lines and 31 primary pheochromocytoma tumors were analyzed for deletion and mutation of RD using real-time PCR and direct DNA sequencing. We found that RD was deleted in human cancer cell lines and pheochromocytoma tumors at frequencies of 41.2% (7/17) and 13.0% (4/31), respectively. While some of these RD deletion events occurred along with deletions of the entire INK4/ARF locus, other RD deletion events were independent of genetic alterations in p15, p14(ARF) , and p16. Furthermore, the status of RD was poorly associated with the expression of p15, p14(ARF) , and p16 in tested cancer cell lines and tumors. This study demonstrates for the first time that deletion of the RD enhancer is a prevalent event in human cancer cells. Its implication in carcinogenesis remains to be further explored.

Keywords: CDC6; RD enhancer; genetic alteration; the INK4/ARF locus.

Figure 1

The schematic structure of the INK4/ARF locus. Promoters for p15, p14^ARF, and p16 genes are indicated by horizontal arrows. Boxes represent exons, and the empty circle represents the regulatory element RD. The minus sign indicates negative regulation. This figure was adapted from Ref. [4].

Figure 2

qPCR assays to detect genetic alterations of RD. (A) Amplification of RD from selected cell lines. FPR1 (N-formyl peptide receptor) was used as an endogenous control [20]. The PCR products of RD and FPR1 are 406 and 500 bp, respectively. (B) The calibration curve for qPCR-based analyses of RD deletion. Multiplex qPCR for RD and ACTB was performed with a series of mixtures of genomic DNAs from TE-1177 (RD^+/+) and UM-SCC-22A (RD^−/−) cells at various ratios. The resulting Cq values were normalized as described in Materials and Methods Section. The ΔΔCq values were plotted against the relative ratio of normal RD in the mixtures (in exponential form) to get a calibration curve with a linear correlation coefficient of −0.996.

Figure 2

qPCR assays to detect genetic alterations of RD. (A) Amplification of RD from selected cell lines. FPR1 (N-formyl peptide receptor) was used as an endogenous control [20]. The PCR products of RD and FPR1 are 406 and 500 bp, respectively. (B) The calibration curve for qPCR-based analyses of RD deletion. Multiplex qPCR for RD and ACTB was performed with a series of mixtures of genomic DNAs from TE-1177 (RD^+/+) and UM-SCC-22A (RD^−/−) cells at various ratios. The resulting Cq values were normalized as described in Materials and Methods Section. The ΔΔCq values were plotted against the relative ratio of normal RD in the mixtures (in exponential form) to get a calibration curve with a linear correlation coefficient of −0.996.

Figure 3

Quantitative determination of CDC6, p16, p14^ARF, and p15 mRNA expression in human cell lines. Brown, CDC6; orange, p16; lime green, p14^ARF; dark green, p15. HPRT1 was used as an endogenous control, and TE-1177 cells were used to represent the reference baseline state for normalization [6]. Quantitative RT-PCR-based assays were conducted in triplicate. Values (y-axis) represent fold-changes (in the log form) calibrated to the expression levels of indicated genes in TE-1177. Error bars represent standard deviations. Dashed lines represent twofold increases (upper) and decreases (lower) in gene expression. ND, not detected.

Figure 4

Western Blot Analyses. Soluble proteins (50 mg) from selected cell lines were subjected to SDS–PAGE analysis, and after electro-transfer to nitrocellulose membrane, the proteins were probed using primary antibodies against β-actin (sc-56459; Santa Cruz Biotechnology), CDC6 (C42F7; Cell Signaling Technologies), P16 (sc-1661; Santa Cruz Biotechnology), and P14^ARF (sc-8340; Santa Cruz Biotechnology). Immunodetection was performed with the Enhanced Chemiluminescence Kit for Western blotting (GE Healthcare Bioscience). TE-1177 was the reference cell line. β-actin was the internal reference protein.