Characterization of NOL7 gene point mutations, promoter methylation, and protein expression in cervical cancer

Abstract

NOL7 is a putative tumor suppressor gene localized to 6p23, a region with frequent loss of heterozygosity in a number of cancers, including cervical cancer (CC). We have previously demonstrated that reintroduction of NOL7 into CC cells altered the angiogenic phenotype and suppressed tumor growth in vivo by 95%. Therefore, to understand its mechanism of inactivation in CC, we investigated the genetic and epigenetic regulation of NOL7. NOL7 mRNA and protein levels were assessed in 13 CC cell lines and 23 consecutive CC specimens by real-time quantitative polymerase chain reaction, western blotting, and immunohistochemistry. Methylation of the NOL7 promoter was analyzed by bisulfite sequencing and mutations were identified through direct sequencing. A CpG island with multiple CpG dinucleotides spanned the 5' untranslated region and first exon of NOL7. However, bisulfite sequencing failed to identify persistent sites of methylation. Mutational sequencing revealed that 40% of the CC specimens and 31% of the CC cell lines harbored somatic mutations that may affect the in vivo function of NOL7. Endogenous NOL7 mRNA and protein expression in CC cell lines were significantly decreased in 46% of the CC cell lines. Finally, immunohistochemistry demonstrated strong NOL7 nucleolar staining in normal tissues that decreased with histologic progression toward CC. NOL7 is inactivated in CC in accordance with the Knudson 2-hit hypothesis through loss of heterozygosity and mutation. Together with evidence of its in vivo tumor suppression, these data support the hypothesis that NOL7 is the legitimate tumor suppressor gene located on 6p23.

Figure 1

Analysis of Endogenous NOL7 Expression in CC Cell Lines. (A) Western blotting was performed on 25 μg whole cell lysate from a panel of CC cell lines. Expression was quantified in (B) using QuantityOne software from BioRad and normalized to β-actin. (B) Real-time quantitative PCR was performed on total RNA isolated from 13 CC cell lines and compared to 293T controls. Dashed line represents 50% of control expression.

Figure 2

Immunohistochemical expression of NOL7 in normal (A), CIN I (B), CIN III (C) and malignant cervical mucosa (D). Decreased nucleoplasmic NOL7 protein expression is observed with increasing histologic atypia. Original magnification 200x and 1000x. (E) Quantification of NOL7 expression in cervical mucosa. Normal (n=70), CIN I (n=22), CIN II (n=20), CIN III (n=23) and SCC (n=88). Data are expressed percentage of tumor specimens demonstrating an intensity of staining ranging from 0-2+.

Figure 3

Prediction and Analysis of NOL7 Genomic Elements. (A) The genomic region of chromosome 6 (NC_000006.11) from bases 13,614,790 to 13,621,437 was analyzed using EMBOSS Isochore program to predict regions of high GC content. Dashed lines indicate the designated threshold of 0.5. (B) The 1,451 bp region of NOL7 that demonstrated a greater than 50% GC content was analyzed for the presence of a CpG island using EMBOSS CpGPlot. Dashed lines indicate a threshold of 0.7.

Figure 4

Bisulfite PCR-mediated Methylation Analysis. (A) Schematic of the NOL7 Genomic region with exons (light grey box), promoter region (white box) and CpG Island (dark grey box). Regions A and B indicate the bisulfite primer location. (B) Representative bisulfite PCR products for regions A and B from HEK293T, HeLa and SiHa cell lines. (C) Analysis of the methylation pattern of 47 individual CpG dinucleotides in the NOL7 promoter region. Ten clones were sequenced for the three cell lines, normal and 23 cervical cancer samples (CC-1 to 23) which is represented in parentheses. Open circles represent unmethylated CpG dinucleotides. Ten clones are represented per circle, with the methylated CpGs corresponding to the black shaded fraction.