Immunohistochemical detection of the pro-apoptotic Bax∆2 protein in human tissues.

The pro-apoptotic Bax isoform Bax∆2 was originally discovered in cancer patients with a microsatellite guanine deletion (G8 to G7). This deletion leads to an early stop codon; however, when combined with the alternative splicing of exon 2, the reading frame is restored allowing production of a full-length protein (Bax∆2). Unlike the parental Baxα, Bax∆2 triggers apoptosis through a non-mitochondrial pathway and the expression in human tissues was unknown. Here, we analyzed over 1000 tissue microarray samples from 13 different organs using immunohistochemistry. Bax∆2-positive cells were detected in all examined organs at low rates (1-5%) and mainly scattered throughout the connective tissues. Surprisingly, over 70% of normal colon samples scored high for BaxΔ2-positive staining. Only 7% of malignant colon samples scored high, with most high-grade tumors being negative. A similar pattern was observed in most organs examined. We also showed that both Baxα and Bax∆2 can co-exist in the same cells. Genotyping showed that the majority of Bax∆2-positive normal tissues contain no G7 mutation, but an unexpected high rate of G9 was observed. Although the underlying mechanism remains to be explored, the inverse correlation of Bax∆2 expression with tissue malignancy suggests that it may have a clinical implication in cancer development and treatment.

Detection of pro-apoptotic Bax∆2 proteins in the human cerebellum.

Bax∆2 is a pro-apoptotic protein originally discovered in colon cancer patients with high microsatellite instability. Unlike most pro-apoptotic Bax family members, Bax∆2 mediates cell death through a non-mitochondrial caspase 8-dependent pathway. In the scope of analyzing the distribution of Bax∆2 expression in human tissues, we examined a panel of human brain samples. Here, we report four cerebellar cases in which the subjects had no neurological disorder or disease documented. We found Bax∆2 positive cells scattered in all areas of the cerebellum, but most strikingly concentrated in Purkinje cell bodies and dendrites. Two out the four subjects tested had strong Bax∆2-positive staining in nearly all Purkinje cells; one was mainly negative; and one had various levels of positive staining within the same sample. Further genetic analysis of the Purkinje cell layer, collected by microdissection from two subjects, showed that the samples contained G7 and G9 Bax microsatellite mutations. Both subjects were young and had no diseases reported at the time of death. As the distribution of Bax∆2 is consistent with that known for Baxα, but in a less ubiquitous manner, these results may imply a potential function of Bax∆2 in Purkinje cells.

The functional domains for Bax∆2 aggregate-mediated caspase 8-dependent cell death.

Bax∆2 is a functional pro-apoptotic Bax isoform having alterations in its N-terminus, but sharing the rest of its sequence with Baxα. Bax∆2 is unable to target mitochondria due to the loss of helix α1. Instead, it forms cytosolic aggregates and activates caspase 8. However, the functional domain(s) responsible for BaxΔ2 behavior have remained elusive. Here we show that disruption of helix α1 makes Baxα mimic the behavior of Bax∆2. However, the other alterations in the Bax∆2 N-terminus have no significant impact on aggregation or cell death. We found that the hallmark BH3 domain is necessary but not sufficient for aggregation-mediated cell death. We also noted that the core region shared by Baxα and Bax∆2 is required for the formation of large aggregates, which is essential for BaxΔ2 cytotoxicity. However, aggregation by itself is unable to trigger cell death without the C-terminus. Interestingly, the C-terminal helical conformation, not its primary sequence, appears to be critical for caspase 8 recruitment and activation. As Bax∆2 shares core and C-terminal sequences with most Bax isoforms, our results not only reveal a structural basis for Bax∆2-induced cell death, but also imply an intrinsic potential for aggregate-mediated caspase 8-dependent cell death in other Bax family members.