BAX But Not BCL2 Is Necessary for Apoptosis in Neuroblastoma Cells Treated With Casiopeína® IIIia.

BACKGROUND/AIM: The emerging antineoplastics Casiopeínas® induce uncoupling of the respiratory chain, production of reactive oxygen species (ROS), entry of Bax into mitochondria, and exit of Ca2+ and Bcl-2 from them, leading to apoptosis. This study aimed to elucidate whether BAX and BCL2 are necessary for apoptosis. MATERIALS AND METHODS: We silenced BAX and BCL2 by CRISPR-Cas9, assessed ROS and calcium retention capacity (CRC) by spectrofluorometry, and caspase-3 with inmunoblotting in neuroblastoma (NB) cells and 3T3-L1 fibroblasts treated with cisplatin and Casiopeína IIIia (CasIIIia). RESULTS: We observed an increase in O2•- production only in BCL2KO NB cells treated with cisplatin (three-fold) and CasIIIia (five-fold), whereas the production of H2O2 in BCL2KO NB cells treated with cisplatin and CasIIIia increased five-fold and three-fold, respectively. The baseline calcium-retention capacity (CRC) was 1.7 relative fluorescence units (RFU) in both cell types. In BAXKO, cisplatin and CasIIIia increased CRC to ~2.3 RFU, and in BCL2KO, they decreased CRC to ~1.1 RFU. We did not detect caspase-3 in BAXKO NB cells. CONCLUSION: Only BAX is essential for CasIIIia-induced apoptosis.

Viviparous Reptile Regarded to Have Temperature-Dependent Sex Determination Has Old XY Chromosomes.

The water skinks Eulamprus tympanum and Eulamprus heatwolei show thermally induced sex determination where elevated temperatures give rise to male offspring. Paradoxically, Eulamprus species reproduce in temperatures of 12-15 °C making them outliers when compared with reptiles that use temperature as a cue for sex determination. Moreover, these two species are among the very few viviparous reptiles reported to have thermally induced sex determination. Thus, we tested whether these skinks possess undetected sex chromosomes with thermal override. We produced transcriptome and genome data for E. heatwolei. We found that E. heatwolei presents XY chromosomes that include 14 gametologs with regulatory functions. The Y chromosomal region is 79-116 Myr old and shared between water and spotted skinks. Our work provides clear evidence that climate could be useful to predict the type of sex determination systems in reptiles and it also indicates that viviparity is strictly associated with sex chromosomes.

Deciphering Ancestral Sex Chromosome Turnovers Based on Analysis of Male Mutation Bias.

The age of sex chromosomes is commonly obtained by comparing the substitution rates of XY gametologs. Coupled with phylogenetic reconstructions, one can refine the origin of a sex chromosome system relative to specific speciation events. However, these approaches are insufficient to determine the presence and duration of ancestral sex chromosome systems that were lost in some species. In this study, we worked with genomic and transcriptomic data from mammals and squamates and analyzed the effect of male mutation bias on X-linked sequences in these groups. We searched for signatures indicating whether monotremes shared the same sex chromosomes with placental mammals or whether pleurodonts and acrodonts had a common ancestral sex chromosome system. Our analyses indicate that platypus did not share the XY chromosomes with placental mammals, in agreement with previous work. In contrast, analyses of agamids showed that this lineage maintained the pleurodont XY chromosomes for several million years. We performed multiple simulations using different strengths of male mutation bias to confirm the results. Overall, our work shows that variations in substitution rates due to male mutation bias could be applied to uncover signatures of ancestral sex chromosome systems.