p16INK4a Promoter mutations are frequent in primary sclerosing cholangitis (PSC) and PSC-associated cholangiocarcinoma  Makiko Taniai, Hajime Higuchi, Lawrence J. Burgart, Gregory J. Gores  Gastroenterology  Volume 123, Issue 4, Pages 1090-1098 (October 2002) DOI: 10.1053/gast.2002.36021 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Visualization of procured cells by LCM. LCM using Arcturus PixCell II was performed to selectively transfer only desired cells to the polymer cap. (A) A bile duct of a PSC specimen before LCM is depicted. (B) The cholangiocytes have been removed from the bile duct by LCM. Note the vacancy left by the removal of selected cells. (C) Cholangiocytes transferred to the transfer cap surface are shown (original magnification 60×). Gastroenterology 2002 123, 1090-1098DOI: (10.1053/gast.2002.36021) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Detection of mutations in the p16INK4a gene from cholangiocytes. Chromosomal DNA was extracted from cells procured by LCM. Genomic DNA was amplified by PCR employing the conditions and primers shown in Table 1. (A) The final amplified product sizes are 1072 bp for the promoter, 335 bp for exon 1, 383 bp for exon 2, and 265 bp for exon 3. The amplified products were separated on 1% agarose gel, stained with ethidium bromide and photographed using ultraviolet illumination. (B) PCR products amplified from cholangiocyte chromosomal DNA were sequenced by an ABI prism 377 fluorescent DNA sequencer. In this sequence of the promoter region of p16, an arrow shows a G to A base change at −317 bp from the initiation site (right panel) compared with an arrowhead shown in the sequence obtained from control DNA (left panel). Gastroenterology 2002 123, 1090-1098DOI: (10.1053/gast.2002.36021) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Several point mutations significantly reduced p16INK4a promoter activity. HuH-7 cells were cotransfected with 20 ng of TK-Renilla and 1 μg of the indicated reporter plasmid. Twelve hours after transfection, cell lysates were prepared. Both firefly and Renilla luciferase activites were quantitated. The data are expressed as the firefly luciferase/Renilla luciferase activity ratio and normalized to values obtained for the wild-type promoter. Assays were performed in triplicate, and error bars indicate standard deviations. Gastroenterology 2002 123, 1090-1098DOI: (10.1053/gast.2002.36021) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Immunostaining of nuclear p16INK4a protein in PSC cholangiocytes. (A) p16INK4a expression (nuclear immunoreactivity) in cholangiocytes (arrow) in a patient without a p16INK4a promoter mutation. (B) Loss of p16INK4a protein expression in cholangiocytes (dotted arrow) in a patient with a p16INK4a promoter mutation (G to A in position – 437 bp from the initiation site). Note that p16INK4a nuclear immunoreactivity was observed in the surrounding hepatocytes (arrowhead) indicating specificity of the observation for cholangiocytes (original magnification 60×). Gastroenterology 2002 123, 1090-1098DOI: (10.1053/gast.2002.36021) Copyright © 2002 American Gastroenterological Association Terms and Conditions