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Expression of anion exchanger 1 sequestrates p16 in the cytoplasm in gastric and colonic adenocarcinoma - PubMed

Expression of anion exchanger 1 sequestrates p16 in the cytoplasm in gastric and colonic adenocarcinoma

Wei-Wei Shen et al. Neoplasia. 2007 Oct.

Abstract

p16(INK4A) (p16) binds to cyclin-dependent kinase 4/6 and negatively regulates cell growth. Recent studies have led to an understanding of additional biologic functions for p16; however, the detailed mechanisms involved are still elusive. In this article, we show an unexpected expression of anion exchanger 1 (AE1) in the cytoplasm in poorly and moderately differentiated gastric and colonic adenocarcinoma cells and in its interaction with p16, thereby sequestrating the protein in the cytoplasm. Genetic alterations of p16 and AE1 were not detectable. Forced expression of AE1 in these cells sequestrated more p16 in the cytoplasm, whereas small interfering RNA-mediated silencing of AE1 in the cells induced the release of p16 from the cytoplasm to the nucleus, leading to cell death and growth inhibition of tumor cells. By analyzing tissue samples obtained from patients with gastric and colonic cancers, we found that 83.33% of gastric cancers and 56.52% of colonic cancers coexpressed AE1 and p16 in the cytoplasm. We conclude that AE1 plays a crucial role in the pathogenesis of gastric and colonic adenocarcinoma and that p16 dysfunction is a novel pathway of carcinogenesis.

Keywords: Anion exchanger 1; colonic adenocarcinoma; gastric adenocarcinoma; p16; siRNA.

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Figures

Figure 1
Figure 1

Cytoplasmic expression of AE1 and p16. (A) The expression of AE1, AE2, and p16 in tumor cell lines was identified by Western blot analysis. AE2 was expressed in all cells as indicated. Positive staining of AE1 and p16 was observed in MKN45 and SW1116 cells. (B) The proportion of p16 in the nucleus and cytoplasm of MKN45 and SW1116 cells was identified, and the cytoplasmic distribution of p16 was dramatically observed. W, whole-cell extract; C, cytoplasmic fraction; N, nuclear fraction. (C) Immunochemistry of endogenous AE1 and AE2 in MKN45 (a) and SW1116 (c) cells. Secondary Texas red - or fluorescein isothiocyanate-labeled antibodies were added to the samples before mounting the coverslips on slides. Blue: Nuclear staining of the cells (b to a; d to c). (D) The expression of AE1 and p16 in human gastric adenocarcinoma tissues. Sample slices were probed with primary anti-AE1 or anti-p16 antibodies. (a) Negative staining of AE1 in noncancer tissue. (b) Nuclear staining of p16 in noncancer tissue (brown). (c) Cytoplasmic staining of AE1 in gastric adenocarcinoma (brown). (d) Cytoplasmic expression of p16 in gastric adenocarcinoma (brown).

Figure 2
Figure 2

AE1 and p16 interaction. Whole-cell lysates from MKN45 or SW1116 cells were immunoprecipitated with mouse anti-human AE1 or rabbit anti-human p16 antibodies, followed by immunoblotting for associated p16 (A) or AE1 (B) antibodies. Mouse and rabbit preimmune sera were used as negative controls.

Figure 3
Figure 3

The cytoplasmic distribution of p16 in MKN45 and SW1116 cells was not affected by the low pH of the culture, and expression occurred in the three other gastric adenocarcinoma cell lines. (A) p16 expression in whole-cell extract (upper panel) and nucleus (lower panel) from two tumor cell lines cultured at pH 6.0 (for 12, 24, and 48 hours) and pH 7.4. (B) Direct comparison of the nuclear-cytoplasmic distribution of p16 in the two tumor cell lines cultured at pH 6.0 and 7.4. (C) The expression of AE1 and AE2 in the three other gastric adenocarcinoma cell lines. (D) Cytoplasmic distribution of p16 in the three other gastric adenocarcinoma cell lines. W, whole-cell extract; C, cytoplasmic fraction; N, nuclear fraction. MKN28, AGS, and SGC7901 represent well, moderately, and poorly differentiated gastric adenocarcinoma cell lines, respectively.

Figure 4
Figure 4

Forced expression or knockdown of AE1 in AGS cells. (A) Forced expression of AE1 in AGS cells at 0 to 72 hours posttransfection of the pcDNA3-AE1 construct. The expression of endogenous p16 in whole-cell lysates was synchronously detected as indicated. (B) Cytoplasmic sequestration of p16 in AGS cells transfected with pcDNA3-AE1 (lower panel), compared with the transfection of an empty vector (upper panel). C, cytoplasmic fraction; N, nuclear fraction. (C) The expression of endogenous AE1 in AGS cells transiently transfected with P1-pSilencer, P2-pSilencer, and P3-pSilencer 3.1-H1 neo constructs for 48 hours; AE1 was detected by immunoblots. (D) Movement of p16 from the cytoplasm to the nucleus after the transfection of P2-pSilencer 3.1-H1 neo constructs.

Figure 5
Figure 5

Forced expression or blockage of AE1 in SW1116 cells. The experimental design was the same as that in Figure 4.

Figure 6
Figure 6

AE1 knockdown induced cell death and growth inhibition in AGS and SW1116 cells. (A) AGS and SW1116 cells were stably transfected with P2-pSilencer or scrambled-pSilencer 3.1-H1 neo constructs, respectively. After selection by G418, 12 single clones of P2 vector or scrambled vector transfectants—numbered 3, 22, and 36 (AGS; scrambled vector transfectants); 7, 8, and 47 (AGS; P2 vector transfectants); 2, 5, and 7 (SW1116; scrambled vector transfectants); and 1, 4, and 6 (SW1116; P2 vector transfectants)—were isolated and expanded in 96-well plates. Viable cells were counted after 17 days. One thousand cells were then isolated from the same clones and expanded for another 8 days (total, 25 days), and the cells were counted. *Number of cells, < 104. **Number of cells, 0. (B) Another six clones of P2 vector or scrambled vector transfectants of AGS and SW1116 were isolated and expanded. The cell deaths of the P2 vector AGS (a) and SW1116 (c) transfectants or scrambled vector AGS (b) and SW1116 (d) transfectants were observed. The morphologic features of the P2 vector AGS (e) and SW1116 (g) transfectants or the scrambled vector AGS (f) and SW1116 (h) transfectants were examined under a microscope following Wright's staining of the cells.

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