Demonstration of a Melanoma-Specific CD44 Alternative Splicing Pattern That Remains Qualitatively Stable, but Shows Quantitative Changes during Tumour Progression

The role of CD44 in the progression of human melanoma has mostly been characterised by qualitative changes in expression of its individual variable exons. These exons however, may be expressed to form a number of molecules, the alternative splice variants of CD44, which may be structurally and functionally different. Using real-time PCR measurements with variable exon specific primers we have determined that all are expressed in human melanoma. To permit comparison between different tumours we identified a stable CD44 variable exon (CD44v) expression pattern, or CD44 ‘fingerprint’. This was found to remain unchanged in melanoma cell lines cultured in different matrix environments. To evaluate evolution of this fingerprint during tumour progression we established a scid mouse model, in which the pure expression pattern of metastatic primary tumours, circulating cells and metastases, non-metastatic primary tumours and lung colonies could be studied. Our analyses demonstrated, that although the melanoma CD44 fingerprint is qualitatively stable, quantitative changes are observed suggesting a possible role in tumour progression

CD44 ‘fingerprint’ of HT168M1 human melanoma cell line growing on different matrices namely plastic (a), fibronectin (b), laminin (c), collagen (d) and matrigel (e).

<p>L stands for molecular weight marker.</p

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.

<p><b>A.</b> HT199 human melanoma cell line <b>B.</b> HT29 human colorectal adenocarcinomacell line <b>C.</b> K562 human erythromyeloblastoid leukemia cell line <b>D.</b> MDA-MB-231 human breast carcinoma cell line.</p

Relative quantitative expression of CD44 variable exons in cell cultures from metastatic (newborn) and non-metastatic human xenograft model (Real-Time PCR measurement) of HT199, a human melanoma cell line of originally low variable exon expression level.

<p><b>A.</b> The relative expression level of all variable exons is raised in circulating metastatic cells (NCTC) and metastatic cells (NM) compared to their levels in primary tumours [newborn primary (NP) and adult primary (AP)] and lung colony (IVLC) <b>B.</b> The qualitative fingerprint (bottom line) remains unchanged.</p

CD44 isoforms validated by next generation sequencing.

<p><b>A.</b> CD44 isoforms from the qualitative picture of pairing the variable exon specific primers with the standard region specific ones both 5′ and 3′ directions in HT168 human melanoma cell line. These isoforms were validated by next generation sequencing. <b>B.</b> Further validated isoforms from next generation sequencing with the primer pairs of the fingerprint.</p