The extremely high sequence identity among the four TEM7 splice variants poses a daunting task for designing PCR primers with the goal of obtaining variant-specific amplification products. To design primers, we first aligned the four cDNA sequences as published, and manually scanned for locations (sequences) specific for a variant. We failed to find sequences for any variant, which could be used to design a primer set (two primers) that would, theoretically, amplify a given variant only. Consequently, concessions were allowed to generate best possible variant-specific primer pairs. Primer sequences and other relevant information are presented in .
Two primers Ti-14-26D and Ti-263-24U were designed for amplifying a segment from the T-i variant mRNA. The sequence of the primer Ti-263-24U is common to all the variants; however, that for the primer Ti-14-26D is specific for the intracellular variant. Consequently, specific amplification product was expected with this specific primer pair. Creating appropriate primers for the T-s1 variant posed the greatest challenge: Two primers Ts1-862-24D and Ts1-1090-24U were designed for amplifying a segment for this transcript. As presented in , the first primer is common to all the variants, while the first 18 nucleotides of the second primer are common to T-s1, T-s2 and T-i variants but not to T-m (the sequence is absent in T-m). The last five nucleotides of this primer are also common to all the variants; however, the 19th nucleotide (a ‘C’) is specific for the T-s1 variant. We speculated that these primer pairs would produce amplification products for the T-s1 and T-i variants but not the other two variants. Two primers, Ts2-939-22D and Ts2-1094-24U, were designed for specific amplification of a segment from the T-s2 variant. While sequence of the first primer is common to all the variants, the sequence of the second primer is completely specific for the T-s2 variant and consequently a T-s2-specific PCR product was expected. A specific amplification product was also expected for the T-m variant because although the primer Tm-901-25D has a sequence common to all the variants and the first 19 nucleotides of the primer Tm-1090-23U is also common for all the variants, the last five nucleotides of the latter are specific for the T-m variant, consequently we expected a specific amplification product since the nucleotides at the 3′-end of a primer are among the most important determinants for specific annealing and elongation of DNA synthesis during PCR. It is important to note that even though a variant-specific primer pair may amplify the specific as well as segments from other variants, the identity of a specific product can be deduced from the length of the amplified DNA (see ).
We first evaluated expression of the four TEM7
transcript variants in five OS-derived cell lines and in FOB cells by semi-quantitative RT-PCR. Based on our animal model of spontaneous lung metastasis of OS (11
), MG63 is a non-tumorigenic cell line, SAOS and TE85 are tumorigenic but poorly metastatic cell lines, and LM7 (a derivative of SAOS) and 143B (a derivative of TE85) are highly metastatic cell lines. LM7 and 143B cells have been observed in our laboratory to cause lung metastasis in 100% of animals (11
Representative RT-PCR results are presented in . The T-m variant had a high expression in SAOS cells only (panel A); the FOB cells also appeared to express the variant but at a very low level. The T-s1 variant was expressed in all cell lines including FOB (panel B), whereas the T-s2 variant is expressed in all 5 OS-derived cells but not in FOB (panel C). The T-i variant was expressed by FOB cells as well as by 3 out of 5 OS-derived cells (panel D).
Figure 1 Semi-quantitative RT-PCR to evaluate the expression level of the four alternatively spliced TEM7 transcripts in cell lines. A, T-m variant; B, T-s1 variant; C, T-s2 variant; D, T-1 variant; E, GAPDH mRNA. M, 100 bp DNA ladder. Cell line identifications (more ...)
The pattern of expression of the four TEM7 alternatively spliced variants in normal bone, as determined by semi-quantitative RT-PCR, is presented in . As can be seen, only the intracellular mRNA variant appeared to be expressed in bone (4 out of 7 samples). The T-m and T-s1 variants were expressed at a very low level in one sample only (ABA055), whereas low-level expression of the T-s2 variant was seen in 4/7 samples. Thus, the intracellular TEM7 variant mRNA appears to be the predominant form expressed in normal bone.
Figure 2 Semi-quantitative RT-PCR to evaluate the expression level of the four alternatively spliced TEM7 transcripts in cultured osteoblasts prepared in the laboratory from bone biopsy specimens. A, T-m variant; B, T-s1 variant; C, T-s2 variant; D, T-1 variant; (more ...)
We also examined the expression pattern of the four TEM7 mRNA variants in 9 primary OS tumor specimens, and the results are presented in . As can be seen, the T-m variant was expressed in 3 out of 9 tumor specimens (intense PCR-amplified product in JH21 and GS53, less intense product in SC14), whereas the T-s1 and T-s2 variants were expressed in 8 of 9 and in 7 out of 9 samples, respectively. The T-i variant mRNA appears to be expressed in all 9 tumor samples. Thus, except for the T-m variant, expression of the other TEM7 mRNA variants was frequent in these tumor samples. Note that for GAPDH, we routinely obtain a clean amplification product from cell lines, however, we sometimes obtained two amplified DNA bands from some whole tissue samples (normal or tumor tissues, containing cells of diverse types) – upon sequencing, one of these ‘doublet’ bands was found to originate from a GAPDH pseudogene (data not shown).
Figure 3 Semi-quantitative RT-PCR to evaluate the expression level of the four alternatively spliced TEM7 transcripts in OS tumor specimens. A, T-m variant; B, T-s1 variant; C, T-s2 variant; D, T-1 variant; E, GAPDH mRNA. M, 100 bp DNA ladder. Tumor specimen identifications (more ...)
A summary of the RT-PCR results for all the TEM7 mRNA variants in OS-derived cell lines, normal bone specimens and OS tumors is presented in . It can be inferred from these results that although expression of the T-m variant is infrequent, it is tumor specific; expression of the T-i variant is not tumor specific since it was also expressed in 4 out of 7 bone samples. However, expression of the secreted forms appears to be tumor specific since they were not expressed in any of the normal bone samples tested.
Summary of expression of TEM7 variants in various samples by RT-PCR.
We have reported previously a significant association of TEM7 expression with OS metastasis. From results presented above, it appears that the T-s and/or the T-m forms of TEM7 may underlie this association. We performed quantitative PCR to compare the level of these three forms of TEM7
mRNA in non-metastatic, poorly metastatic and highly metastatic OS-derived cell lines; results from these experiments are presented in . All three TEM7
mRNA variants were highly expressed in the metastatic LM7 cells compared to the poorly metastatic SAOS cells (>20-fold). The non-tumorigenic MG63 cells and the immortalized FOB cells had very little expression of the mRNAs. These results provide further support for a relationship between TEM7 expression and metastasis (1
Figure 4 Quantitative RT-PCR to determine the expression level of T-m, T-s1 and T-s2 transcripts in OS cell lines. FOB, An immortalized human fetal osteoblast cell line; MG-63, a non-tumorigenic OS cell line; SAOS-2, a poorly metatstatic OS cell line; LM7, a highly (more ...)