Ndividual variable exons (VE) during tumour progression in the in vivo

Ndividual 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 PTH 1-34 biological activity 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 10457188 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 12926553 constituents of the microenvironment. doi:10.1371/journal.pone.0053883.gCD44 is more complex. In this case, we created cell cultures from different localisations of the primary tumour of individual animals. The individual lung metastases (newborn animals) and lung colonies (adult animals) of the individual animals were also cultured separately. The non-metastatic adult primary tumour (AP) showed a higher expression level of all CD44 VEs than the metastatis newborn primary tumour (Fig. 7A). The reason behind the rather large error bars seen on the GHRH (1-29) measurements from the newborn lung metastases (NM) is that the individual lung metastases showed huge VE expression level differences. Cells from the cell line created from the lung metastasis showing the highest CD44 VE expression level (NM = S1T2,Figure 7B) were then re-implanted subcutaneously into newborn animals. Cell cultures were then created from three different localisations of the primary tumour (PNM) and three random lung metastases (MPNM) of the chosen animal (Fig. S4). They showed no difference in CD44 VE expression level compared to each other, however they showed on average 24 times lower expression than the cell culture (newborn lung metastasis, NM = S1T2) of origin. We also consistently detected lower CD44 VE expression in liver metastases (LMIVLC) from lung colonies (IVLC), which.Ndividual 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 10457188 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 12926553 constituents of the microenvironment. doi:10.1371/journal.pone.0053883.gCD44 is more complex. In this case, we created cell cultures from different localisations of the primary tumour of individual animals. The individual lung metastases (newborn animals) and lung colonies (adult animals) of the individual animals were also cultured separately. The non-metastatic adult primary tumour (AP) showed a higher expression level of all CD44 VEs than the metastatis newborn primary tumour (Fig. 7A). The reason behind the rather large error bars seen on the measurements from the newborn lung metastases (NM) is that the individual lung metastases showed huge VE expression level differences. Cells from the cell line created from the lung metastasis showing the highest CD44 VE expression level (NM = S1T2,Figure 7B) were then re-implanted subcutaneously into newborn animals. Cell cultures were then created from three different localisations of the primary tumour (PNM) and three random lung metastases (MPNM) of the chosen animal (Fig. S4). They showed no difference in CD44 VE expression level compared to each other, however they showed on average 24 times lower expression than the cell culture (newborn lung metastasis, NM = S1T2) of origin. We also consistently detected lower CD44 VE expression in liver metastases (LMIVLC) from lung colonies (IVLC), which.