Related Experiment Video
Updated: Sep 27, 2026
Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex
Published on: August 2, 2021
CD95 (APO-1/FAS) deficiency in infant acute lymphoblastic leukemia: detection of novel soluble Fas splice variants
Carla M Wood1, Patricia A Goodman, Alexei O Vassilev
1Department of Molecular Genetics, Parker Hughes Institute, St Paul, MN, USA.
Insights
Infant acute lymphoblastic leukemia (ALL) cells show low Fas protein and resistance to apoptosis due to alternatively spliced Fas mRNA variants, including novel forms. These findings suggest a role for aberrant Fas splicing in ALL pathogenesis.
Area of Science:
- Molecular biology
- Immunology
- Oncology
Background:
- Fas (APO-1/CD95) is a key regulator of apoptosis in lymphoid cells.
- Resistance to apoptosis is a hallmark of cancer, including acute lymphoblastic leukemia (ALL).
Purpose of the Study:
- To investigate Fas expression and its regulation in primary leukemic cells from infants with ALL.
- To identify molecular mechanisms underlying resistance to Fas-mediated apoptosis in infant ALL.
Main Methods:
- Analysis of Fas protein expression in primary ALL cells.
- Reverse transcription and polymerase chain reaction (RT-PCR) for Fas mRNA analysis.
- Nested RT-PCR to detect alternatively spliced Fas transcripts.
Main Results:
- Infant ALL cells exhibited low Fas protein expression and resistance to anti-Fas antibody-induced apoptosis.
- Full-length Fas mRNA was undetectable by standard RT-PCR.
- Nested RT-PCR revealed alternatively spliced Fas transcripts in 60% of patients, including novel variants lacking transmembrane domains.
- Identified variants included a soluble Fas lacking exon 6 with an exon 7B insertion and an exon 4Del variant with a deletion at the exon 5/6 splice junction.
Conclusions:
- Low expression of functional full-length Fas transcripts and protein contributes to CD95-mediated apoptosis resistance in infant ALL.
- Aberrant splicing of Fas mRNA represents a novel mechanism contributing to the pathogenesis of infant ALL.
Abstract:
Fas (APO-1/CD95) is a 45-kDa membrane protein which regulates apoptosis in many lymphoid cell types. In the present study, FAS expression was examined in primary leukemic cells from infants with acute lymphoblastic leukemia (ALL). The cells were resistant to apoptosis induction by an anti-FAS antibody and expressed nearly undetectable amounts of FAS protein. Molecular analysis of FAS transcripts in these cells revealed no detectable expression of full-length Fas mRNA after a single round of reverse transcription and polymerase chain reaction (PCR) amplification (RT-PCR). However, a more sensitive nested RT-PCR analysis revealed alternatively spliced Fas transcripts in three of five infants (60%) with the remaining two infants showing no detectable Fas mRNA expression. The primary sequence variation of Fas mRNA seen in the samples was a previously described variant lacking exon 6 encoding soluble FAS. However, we also detected the presence of several novel alternatively spliced FAS transcripts in the ALL cells. In one patient, we observed a novel spliced form of soluble Fas, which not only lacked exon 6 but also contained an insertion of an alternative exon 7 (exon 7B). In another, a novel exon 4Del FAS mRNA variant was observed, which contained an additional 4-bp deletion at the exon 5/6-splice junction. These variants lack intact transmembrane domains and thus are predicted to encode soluble FAS variants. The low level of expression of functional full length FAS transcripts with corresponding low level of FAS protein expression in the ALL cells contribute to their resistance to CD95-mediated apoptosis.
