Evidence that P12, a specific variant of P16INK4A, plays a suppressive role in human pancreatic carcinogenesis
Introduction
The INK4a-ARF locus plays a central role in tumor suppression as reflected by the fact that a significant proportion (∼50%) of all human cancers harbored an inactivated INK4a-ARF locus [1], [2]. As shown in Fig. 1A, the INK4a-ARF locus (CDKN2A) encodes two proteins, P16INK4A (hereafter, P16) and P14ARF [1], [2], [3], [4]. p16 and p14ARF are transcribed from distinctive first exons (exon 1α and exon 1β, respectively) spliced onto common exons 2 and 3 in alternative reading frames. P16 specifically inhibits cyclin-dependent kinases (CDK) 4/6-mediated phosphorylation of the tumor suppressor pRb (the retinoblastoma susceptible gene product) as well as the subsequent E2F-mediated transcription of genes required for entry into S phase; P14ARF functions to prevent the degradation of the tumor suppressor P53 through interacting with MDM2. Due to its prevalence in human cancers, genetic inactivation of p16 as well as p14ARF has been recognized as the primary cause of a variety of neoplasia, including pancreatic cancer [5], [6].
However, recent studies have demonstrated that there are some unique features of the INK4a-ARF locus in pancreas [7], [8]. First, while both p16 and p14ARF are expressed at low levels in most of human tissues, p16 is highly expressed in pancreas [7]. Conversely, p14ARF expression is not detectable in pancreas, indicating that P14ARF is transcriptionally silenced through unknown mechanisms in pancreas [8]. Secondly, an alternatively spliced INK4a transcript, termed p12, is expressed in the pancreas at high levels [7]. In the p12 transcript (Fig. 1A), an additional 274 bp on intron 1, contiguous with the 3′ end of exon 1α, is included in the normal exon 1α sequence followed by exons 2 and 3. An in-frame stop codon in the intron 1-derived sequence results in P12, a polypeptide of 116 residues with an N-terminus (52 residues) identical to P16 and a novel C-terminus (64 residues) (Fig. 1B). It has been reported that over-expression of p12 in Panc-1 (a pancreatic cancer cell line) and C33A (a cervical cancer cell line) results in cell cycle arrest at G1 and G2 phases in a pRB-independent manner [7]. However, knowledge about the biochemical properties of P12 and its potential contributions to pancreatic carcinogenesis remains limited. Here we present our studies on biochemical characterization of P12 and the prevalence of p12 alteration in human pancreatic tumor specimens.
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Cell line, cell culture, cell transfection and selection
Both Panc-1 and AsPc-1 cells were purchased from American Type Culture Collection (ATCC; Manassas, VA) and cultured in Advanced DMEM/F12 medium (Invitrogen) containing 5% fetal bovine serum (FBS; Invitrogen) in a 90% relative humidity incubator at 37 °C supplied with 5% CO2.
pcDNA3.1-p12 plasmid [7] was transfected into Panc-1 and AsPc-1 cells using Lipofectamine (Invitrogen) according to the manufacturer’s recommendation. After selection with G418 (Invitrogene) at 1200 μg/ml for three weeks, five
P12 inhibited the proliferation of pancreatic cancer cells
To investigate the physiological role(s) of P12 in pancreas, we first introduced exogenous p12 into two pancreatic carcinoma cell lines, Panc-1 and AsPc-1 to obtain cells stably expressing p12. Of note, endogenous p12 was expressed in AsPc-1 but not in Panc-1 due to the deletion of the entire INK4a-ARF locus in the latter [7], [10]; over-expression of p12 in transfected Panc-1 and AsPc-1 was confirmed by qRT-PCR (data not shown). Subsequently, we analyzed p12-induced changes in cell cycle
Discussion
The association between alterations in the INK4a-ARF gene and pancreatic cancer is nearly 100%, the highest of any form of human cancer [14]. Given the fact that p14ARF is transcriptionally silenced in normal pancreas [8], the tumor-suppressing activity associated with the INK4a-ARF locus in pancreatic tumors has been primarily attributed to P16 in the past decades. However, recent studies have demonstrated that P16 may not be the only or the primary tumor suppressor associated with alterations
Acknowledgments
pcDNA3.1-p12 plasmid was a generous gift from Dr. Peter A Jones at USC/Norris Comprehensive Cancer Center. Flow cytometry analyses and cDNA microarray assays were conducted in the Analytic Flow Cytometry Shared Resource and the Microarray Core facility at OSU Comprehensive Cancer Center, respectively. This work was partly supported by a research grant from NIH, R01 CA69472 (J.L.).
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