Pthlh, a promising cancer modifier gene in rat tongue carcinogenesis

Abstract

Susceptibly to the induction of rat tongue cancer (TC) by oral 4-nitroquinoline 1-oxide (4NQO) exposure is a polygenic trait. Among several quantitative trait loci identified by crosses between TC-susceptible Dark Agouti (DA) rats and TC-resistant Wistar-Furth (WF) rats, we focused on tongue cancer susceptibility locus (Tcas3) of chromosome 4. We examined tongue carcinogenesis in the reciprocal congenic strains DA.WF-Tcas3 and WF.DA-Tcas3 and in their parental strains. The Tcas3DA allele, and not the Tcas3WF allele, significantly favored tumor latency, incidence and TC number/size. In genomic DNA of TCs induced in (DA x WF) F1 rats, the resistant Tcas3WF allele was frequently and selectively lost, particularly in larger tumors. Thus, we searched the possible candidate genes in the Tcas3 region using microarray analysis of TCs in F1 rats and revealed significant upregulation of 2 cancer-related genes, parathyroid hormone-like hormone (Pthlh) and Kras2. The relevance of the WF allele of Pthlh as a cancer modifier was indicated by 3 single nucleotide polymorphisms specific to this strain. In contrast, no consistent strain-specific variations were found in Kras2. Moreover, the plasma Ca2+ level was consistently higher in DA rats when compared to the level in WF rats bearing TCs; moreover, the Pthlh-mRNA expression level was >30-fold higher in TCs when compared to this level in the normal tongue mucosa. Immunostaining experiments showed strong PTHrP protein expression in TCs of DA rats, and the signal was intensified in larger TCs. Kras2 was also upregulated in TCs, but to a lesser degree than PTHrP. Thus, Pthlh is a promising candidate modifier gene in the development and progression of rat TCs.

Figure 1

Tcas3 segment of RNO4 in the WF.DA-Tcas3 and DA.WF-Tcas3 rats. The closed column represents a WF-derived segment, the hatched column is undetermined and the open column represents a DA-derived segment. The closed bars represent the loci with the WF allele, and open bars represent loci with the DA allele. Numbers on the left indicate the map location of marker loci listed in the box (CR). RNO4, rat chromosome 4; WF, Wistar-Furth rats; DA, Dark Agouti rats.

Figure 2

Cumulative incidence of 4NQO-induced tongue cancers (TC#5, >5 mm). The solid line represents WF, the dotted line WF.DA-Tcas3, the dashed lines F1, the dotted/dashed lines DA.WF-Tcas3 and the thin solid line DA rats. ^*P<5×10⁻² significantly different from the corresponding values for WF and WF.DA-Tcas3, DA and DA.WF-Tcas3; ^**P<5×10⁻² for DA and WF; F1 and WF; ^***P<1×10⁻⁴ for WF and DA. 4NQO, 4-nitroquinoline 1-oxide; TC, tongue cancer; WF, Wistar-Furth rats; DA, Dark Agouti rats.

Figure 3

Representative LOH data analysis at microsatellite loci D4Wox11 (Pthlh) (left) and D4Got155 (right). The upper panels show the peak heights in arbitrary units of the WF (filled) and DA (unfilled) alleles from a TC in a (DA × WF) F1 rat, and the lower panels show those from the normal tongue tissue of the same rat. LOH, loss of heterozygosity; WF, Wistar-Furth rat; DA, Dark Agouti rat.

Figure 4

Quantitative analysis of Pthlh and Kras2 mRNA levels in F1 tongue cancer (TC): TC >10 mm, TC 5–10 mm, TC <5 mm and non-TC tissue F1 samples. The line within each box represents the median fold change. The upper and lower edges of each box represent the 75th and 25th percentiles, respectively. The upper and lower bars represent the highest and lowest value determined, respectively. ^**P<0.001, ^*P<0.01.

Figure 5

Detection of sequence variants in the Pthlh gene in the WF and DA strains. The WF strain contained 3 SNPs not noted in the DA and 6 other inbred strains of rat. The SNPs were located at positions (A) +2 bp, (B) +17 bp and (C) +513 bp. The sequences of the primers used for PCR amplification before sequencing were: Pthlh 1 F, 5′-GACTCGCTCACT TCTCAGCA-3′ and R, 5′-GGCTCCCATAGCAATGTCTA-3′; Pthlh 2 F, 5′-GCGGTGTCTGAGCACCAGCTA-3′ and R, 5′-GCACAGCGGACAGAC AATACC-3′. Primer pair 1 was used to amplify the SNPs at positions +2 and +17 bp; pair 2 amplified the SNP at position +1485 bp (B). WF, Wistar-Furth rats; DA, Dark Agouti rats.

Figure 6

Hematoxylin and eosin (H&E) staining (left) and anti-PTHrP immunohistochemistry (right) of F1 normal rat tongue (A) and tongue cancers (TC) (<5 mm in diameter (TC <5) for H&E and PTHrP-positive (+) (B); TC of 5–10 mm (TC 5–10) for H&E and PTHrP (+) (C); TC >10 mm (TC >10) for H&E and PTHrP (+) (D). The PTHrP-(+) tissue shows a positive signal in the cytoplasm and nucleus of many cancer cells. Scale bars, 200 μm.