Phosphatidylinositol glycan-A (PIGA) is a gene that encodes an element required for the first step in glycosylphosphatidylinositol (GPI) anchor assembly. Because PIGA is X-located, a single mutation is sufficient to abolish cell surface GPI-anchored protein expression. In this study, we investigated whether mutation of the PIGA gene could be exploited to identify mutator (Mut) phenotypes in cancer. We examined eight Mut colon cancer lines and four non-Mut colon cancers as controls. In every case, flow cytometric analyses of cells sorted for low fluorescence after staining for GPI-linked CD59 and CD55 revealed negative peaks in the Mut lines but not in the controls. Single cell cloning of purged and sorted GPI-anchor-HCT116 cells and sequencing of the PIGA gene in each clone uniformly showed mutations. Pretreatment of the Mut lines with anti-CD55 or anti-CD59 antibodies and complement or with the GPI-anchor-reactive bacterial toxin aerolysin enriched for the GPI-anchor-populations. Expansion of purged GPI-anchor+cells in the Mut lines and analyses using aerolysin in conjunction with flow cytometry yielded PIGA gene mutation frequencies of 10-5to 10-4values similar to the mutation frequencies of the hprt gene. This novel approach allows for the detection of as yet undescribed repair or replication defects and in addition to its considerably greater ease of use than existing techniques and in principle would not require the production of cell lines.
|Original language||English (US)|
|Number of pages||5|
|State||Published - Jan 15 2001|
ASJC Scopus subject areas
- Cancer Research