utImmunohistochemical Detection of the Alternate INK4a-Encoded Tumor Suppressor Protein p14ARF in Archival Human Cancers and Cell Lines Using Commercial Antibodies: Correlation with p16INK4a Expression

Abstract

The INK4a locus encodes two structurally unrelated tumor suppressor proteins, p16 INK4a and p14 ARF . Although the former is one of the most common targets for inactivation in human neoplasia, the frequency of p14 ARF abrogation is not established. We have developed an immunohistochemical assay that allows the evaluation of p14 ARF expression in formalin-fixed, paraffin-embedded tissues, using commercially available antibodies. p14 ARF positive cells showed nuclear/nucleolar staining, which was absent in all cell lines and tumors with homozygous deletions of the INK4a gene. The assay was applied to 34 paraffin-embedded cell buttons, 30 non-small cell lung cancers and 28 pancreatic carcinomas, and the staining results were correlated with p16 INK4a expression. Loss of p14 ARF expression was common but less frequent than down-regulation of p16 INK4a (53% versus 76% of all specimens). The p14 ARF and p16 INK4a expression pattern was concordant in 65 of 92 cases (71%). Significantly, 24 cases were p16 INK4a ؊/p14 ARF ؉, while the opposite staining pattern was observed in three cases, consistent with the notion that the two proteins have nonredundant functions. The immunohistochemical assay described here may facilitate studies on the prevalence and significance of aberrant p14 ARF expression in human tumors.

p16INK4A and p14ARF expression pattern by immunohistochemistry in human papillomavirus-related cervical neoplasia

“…12,18,28,[32][33][34] In our material, p14ARF staining was predominantly nuclear. Although, p14ARF presents it as a nucleolar protein in human tumor cell lines, 35 localization of the protein has also been detected in the nucleoplasm as reported elsewhere and in some cases 36 it is associated with aggressive growth and a malignant phenotype. 34 Sano et al 12,18 described different types of p14ARF expression patterns: those staining the top half layer of the epithelium (the superficial type) and those staining the full thickness type.…”

Methylation of CpG islands of p16INK4a and cyclinD1 overexpression associated with progression of intraductal proliferative lesions of the breast

“…12-16) proteins [22,23] were located in the nuclei. For the detection of p16 INK4a protein expression as a tumor suppressor product, cytoplasmic reactivity was disregarded, and only nuclear staining above any cytoplastic background was considered evidence of p16 INK4A protein expression [24,25]. At least 10 fields in each specimen were randomly selected and examined under high-power magnification, and more than 500 cells were counted to determine the percentage of positive cells.…”

“…At least 10 fields in each specimen were randomly selected and examined under high-power magnification, and more than 500 cells were counted to determine the percentage of positive cells. Based on the criteria Geradts et al [24,25] established, with minor modification, the case wherein the percentage of positive cells was 10% or greater was considered as p16 INK4a -positive and cyclinD1-positive.…”

The status of CDKN2A alpha (p16INK4A) and beta (p14ARF) transcripts in thyroid tumour progression

“…A specimen was considered positive for p14 ARF or p16 INK4A if there was nuclear staining above any cytoplasmic background. Cytoplasmic staining itself was noted but not included in the scoring (Geradts et al, 2001). The immunohistochemical results were evaluated using a semiquantitative analysis: no staining reaction ¼ 0, o10% positive-stained cells ¼ þ , 10-80% positive cells or focal staining ¼ þ þ , 480% positive cells…”