Ceramide engagement in apoptotic pathways has been a topic of controversy. To address this controversy, we tested loss-of-function (lf) mutants of conserved genes of sphingolipid metabolism in Caenorhabditis elegans. Although somatic (developmental) apoptosis was unaffected, ionizing radiation-induced apoptosis of germ cells was obliterated upon inactivation of ceramide synthase and restored upon microinjection of long-chain natural ceramide. Radiation-induced increase in the concentration of ceramide localized to mitochondria and was required for BH3-domain protein EGL-1-mediated displacement of CED-4 (an APAF-1-like protein) from the CED-9 (a Bcl-2 family member)/CED-4 complex, an obligate step in activation of the CED-3 caspase. These studies define CEP-1 (the worm homolog of the tumor suppressor p53)-mediated accumulation of EGL-1 and ceramide synthase-mediated generation of ceramide through parallel pathways that integrate at mitochondrial membranes to regulate stress-induced apoptosis.
Androgens inhibit the growth of breast cancer cells in vitro and in vivo by mechanisms that remain poorly defined. In this study, treatment of asynchronously growing MCF-7 breast cancer cells with the androgen, 5 -dihydrotestosterone (DHT), was shown to inhibit cell proliferation and induce moderate increases in the proportion of G1 phase cells. Consistent with targeting the G1-S phase transition, DHT pretreatment of MCF-7 cultures impeded the serum-induced progression of G1-arrested cells into S phase and reduced the kinase activities of cyclin-dependent kinase (Cdk)4 and Cdk2 to less than 50% of controls within 3 days. DHT treatment was associated with greater than twofold increases in the levels of the Cdk inhibitor, p27 Kip1 , while p21 Cip1/Waf1 protein levels remained unchanged. During the first 24 h of DHT treatment, levels of Cdk4-associated p21 Cip1/Waf1 and p27Kip1 were reduced coinciding with decreased levels of Cdk4-associated cyclin D3. In contrast, DHT treatment caused increased accumulation of Cdk2-associated p21 Cip1/Waf1 , with no significant alterations in levels of p27Kip1 bound to Cdk2 complexes. These findings suggest that DHT reverses the Cdk4-mediated titration of p21 Cip1/Waf1 and p27 Kip1 away from Cdk2 complexes, and that the increased association of p21Cip1/Waf1 with Cdk2 complexes in part mediates the androgen-induced growth inhibition of breast cancer cells.
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