Table 1.
Primer sequences.
Fig 1.
Identification of CLDN1 transcript variants in invasive human breast cancer.
PCR analysis was carried out on reverse transcribed RNA from 12 breast tumors using primers (Table 1) flanking the coding regions of CLDN1, 3, and 4. The expected full length cDNA products (representing the classical transcripts) for CLDN1 and CLDN4 (700 bases and 706 bases respectively) was evident in all tumors. However, no CLDN3 transcript was detected in some of the breast tumors (middle panel; lanes 4, 10, 11). Colored arrows indicate CLDN1 PCR products which were verified by Sanger sequencing: yellow arrow, transcript variant 1 (V1) is 615 bp; red arrow, transcript variant 2 (V2) is 440bp; blue arrow, transcript variant 3 (V3) is 362 bp; and green arrow, transcript variant 4 (V4) is 217 bp. In the lower panel, the product indicated by the black arrow, was a non-specific band and had no sequence homology to CLDN4.
Fig 2.
CLDN1 transcript variants reveal alternate splicing within exons.
The shorter CLDN1 transcript variants were aligned with the full length coding region (636 bp). Translation of the putative protein of each variant resulted in alignment only in the N-terminal of claudin 1 (exon 1). Remaining sequence was out of frame and translated into a non-functional claudin 1 protein. * = location of stop codon.
Fig 3.
CLDN1 transcript variants in human breast tumors versus normal breast.
A. Primer Design. Forward primers were designed to specifically amplify V1 and V2 CLDN1 transcripts. The reverse primer flanked the coding region of CLDN1 mRNA and was identical to the one used for the PCR amplification shown in Fig 1. A reverse primer was specifically designed to amplify the V3 transcript variant and used in conjunction with the CLDN1 forward primer (Table 1). B. Differential expression of transcript variants. V1 was amplified in both breast cancer (2/6 tumors) and normal tissue samples (3/6). However, V2 was only evident in the breast tumors (3/6). V3 was detected in both tumors (3/6 tumors) and normal tissue (4/6). Gapdh was used as a control for the RT-PCR reaction. (blk = no template).
Table 2.
Clinico-pathological characteristics and claudin 1 protein levels in human breast tumors.
ER α and PR status, determined by the ligand binding assay (ER+, >3fmol/mg protein; PR+, >10fmol/mg protein), tumor size, grade and nodal status were obtained from the Manitoba Breast Tumor Bank. Claudin 1 protein levels were evaluated by immunohistochemistry. H scores: % claudin 1 positive tumor cells multiplied by the intensity of staining (scale 1–3). H scores were further categorized as 0–15 = —(neglible); 20–75 = + (low); 80–150 = ++ (moderate); 155–300 = +++ (high). There was a trend towards high claudin 1 levels (3/4) being associated with ER- tumors, as previously described [18], but no direct correlation with transcript variant and ER status was evident.
Fig 4.
Identification of SNPs in human breast cancers within CLDN1 exons.
Several SNPs were identified by sequencing of the PCR products (see Table 1). The cDNA position of the SNPs are shown and designated by their corresponding Reference SNP (rs) numbers. The frequency of the missense SNP detected in exon 2, was significantly higher in the breast tumors than predicted for the normal population (p< 0.5; two-tailed Fisher’s Exact Test).
Fig 5.
Representative immunostaining of breast tumors with claudin 1 antibody.
A human breast tumor exhibiting high (A; tumor #11) and one exhibiting low (B; Tumor #3) levels of the claudin 1 protein. Scale bars = 60 μm.