Ways formed metastases in newborn scid mice (permissive host), yet never did so in adult ones (nonpermissive host). This model made it possible to examine the melanoma `fingerprint’ during the metastatic processes. In vivo expression patterns were evaluated on two human melanoma cell lines HT199 and HT168M1. We performed our PCR reaction series on theprimary subcutaneous tumour, circulating tumour cells obtained from blood and lung metastases from transplanted newborn scid mice, as well as the primary subcutaneous tumours from transplanted adult mice. In addition lung tumours were generated in adult animals by intravenous injection (Fig. S4). For HT199 we found that the CD44 fingerprint Title Loaded From File demonstrated in vitro was unchanged throughout the sampled sites (Fig. 6B). These findings do not explain published observations, that the expression of certain CD44 exons correlate with metastatic potential. Our results suggest that the CD44 ASP behind the `fingerprint’ is the same in all these cases, meaning that 18325633 the same isoforms are present. The cited quantitative expression changes of single variable exons should therefore be explained differently.We made a further quantitative PCR analysis with our variable exon specific primers on the same samples. We examined the quantitative changes of the individual variable exons (VE) during tumour progression in the in vivo animal models of two genetically different human melanoma cell lines (HT199, HT168M1). It has become clear after the first measurements, that the two cell lines have 3 orders of magnitude difference in their CD44 VE expression relative to beta-actin housekeeping gene, but despite this, their malignant potential was practically identical. CD44 in HT199, the cell line with a low base VE expression, behaved as a ‘classical’ metastasis gene. The non-metastatic adult primary (AP), the metastatic newborn primary (NP) and the lung colony formed after intravenous injection into adult animals (IVLC), which is also a form of primary tumour, all expressed the VEs within the same order of magnitude (Fig. 6A). The circulating tumour cells (NCTC) and lung metastases (NM) from the animal implanted as a newborn showed 21 times and 9 times increased expression respectively. In the case of HT168M1, which expresses the CD44 VEs in 3 times larger order of magnitude than HT199, the role ofCD44 Alternative Splicing Pattern of MelanomaFigure 3. The CD44 alternative splice pattern of different human tumour cell lines demonstrated by virtual gels and Title Loaded From File electropherograms generated by Experion DNA Capillary Electrophoresis System and corresponding agarose gel picture. A. HT199 human melanoma cell line B. HT29 human colorectal adenocarcinomacell line C. K562 human erythromyeloblastoid leukemia cell line D. MDA-MB-231 human breast carcinoma cell line. doi:10.1371/journal.pone.0053883.gCD44 Alternative Splicing Pattern of MelanomaFigure 4. The CD44 alternative splice pattern of different human tumours is different, but preserved throughout samples from the same the tumour type as it is demonstrated by the agarose gel electropherograms of human melanoma (A 2058, WM983B, WM35 and HT168M), colorectal adenocarcinoma (HT25 and HCT116), oral squamous cell carcinoma (PE/CA PJ15 and PE/CA PJ41) and vulval squamous cell carcinoma (A431) cell lines. The melnanoma CD44 fingerprint also differs from that of non neoplastic melanocyte, keratinocyte and fibroblast cell lines as constituents of the microenvironment. doi:10.1371/journal.Ways formed metastases in newborn scid mice (permissive host), yet never did so in adult ones (nonpermissive host). This model made it possible to examine the melanoma `fingerprint’ during the metastatic processes. In vivo expression patterns were evaluated on two human melanoma cell lines HT199 and HT168M1. We performed our PCR reaction series on theprimary subcutaneous tumour, circulating tumour cells obtained from blood and lung metastases from transplanted newborn scid mice, as well as the primary subcutaneous tumours from transplanted adult mice. In addition lung tumours were generated in adult animals by intravenous injection (Fig. S4). For HT199 we found that the CD44 fingerprint demonstrated in vitro was unchanged throughout the sampled sites (Fig. 6B). These findings do not explain published observations, that the expression of certain CD44 exons correlate with metastatic potential. Our results suggest that the CD44 ASP behind the `fingerprint’ is the same in all these cases, meaning that 18325633 the same isoforms are present. The cited quantitative expression changes of single variable exons should therefore be explained differently.We made a further quantitative PCR analysis with our variable exon specific primers on the same samples. We examined the quantitative changes of the individual variable exons (VE) during tumour progression in the in vivo animal models of two genetically different human melanoma cell lines (HT199, HT168M1). It has become clear after the first measurements, that the two cell lines have 3 orders of magnitude difference in their CD44 VE expression relative to beta-actin housekeeping gene, but despite this, their malignant potential was practically identical. CD44 in HT199, the cell line with a low base VE expression, behaved as a ‘classical’ metastasis gene. The non-metastatic adult primary (AP), the metastatic newborn primary (NP) and the lung colony formed after intravenous injection into adult animals (IVLC), which is also a form of primary tumour, all expressed the VEs within the same order of magnitude (Fig. 6A). The circulating tumour cells (NCTC) and lung metastases (NM) from the animal implanted as a newborn showed 21 times and 9 times increased expression respectively. In the case of HT168M1, which expresses the CD44 VEs in 3 times larger order of magnitude than HT199, the role ofCD44 Alternative Splicing Pattern of MelanomaFigure 3. The CD44 alternative splice pattern of different human tumour cell lines demonstrated by virtual gels and electropherograms generated by Experion DNA Capillary Electrophoresis System and corresponding agarose gel picture. A. HT199 human melanoma cell line B. HT29 human colorectal adenocarcinomacell line C. K562 human erythromyeloblastoid leukemia cell line D. MDA-MB-231 human breast carcinoma cell line. doi:10.1371/journal.pone.0053883.gCD44 Alternative Splicing Pattern of MelanomaFigure 4. The CD44 alternative splice pattern of different human tumours is different, but preserved throughout samples from the same the tumour type as it is demonstrated by the agarose gel electropherograms of human melanoma (A 2058, WM983B, WM35 and HT168M), colorectal adenocarcinoma (HT25 and HCT116), oral squamous cell carcinoma (PE/CA PJ15 and PE/CA PJ41) and vulval squamous cell carcinoma (A431) cell lines. The melnanoma CD44 fingerprint also differs from that of non neoplastic melanocyte, keratinocyte and fibroblast cell lines as constituents of the microenvironment. doi:10.1371/journal.
