The Absence of Oligonucleosomal DNA Fragmentation during Apoptosis of IMR-5 Neuroblastoma Cells

Abstract

Caspase-activated DNase is responsible for the oligonucleosomal DNA degradation during apoptosis. DNA degradation is thought to be important for multicellular organisms to prevent oncogenic transformation or as a mechanism of viral defense. It has been reported that certain cells, including some neuroblastoma cell lines such as IMR-5, enter apoptosis without digesting DNA in such a way. We have analyzed the causes for the absence of DNA laddering in staurosporine-treated IMR-5 cells, and we have found that most of the molecular mechanisms controlling apoptosis are well preserved in this cell line. These include degradation of substrates for caspases, blockade of cell death by antiapoptotic genes such as Bcl-2 or Bcl-X L , or normal levels and adequate activation of caspase-3. Moreover, these cells display normal levels of caspase-activated DNase and its inhibitory protein, inhibitor of caspase-activated DNase, and their cDNA sequences are identical to those reported previously. Nevertheless, IMR-5 cells lose caspase-activated DNase during apoptosis and recover their ability to degrade DNA when human recombinant caspase-activated DNase is overexpressed. Our results lead to the conclusion that caspase-activated DNase is processed during apoptosis of IMR-5 cells, making these cells a good model to study the relevance of this endonuclease in physiological or pathological conditions.

Characterization of splice variants of human caspase-activated DNase with CIDE-N structure and function

“…IMR-5 cells expressed normal levels of CAD but upon STP-induced cell death, CAD rapidly disappeared from cytosolic fractions. This fact explains the absence of oligonucleosomal degradation of DNA, since forced overexpression of CAD restored the laddering during apoptosis [36].…”

“…Caspase-activated DNase gene shows alterations in the splicing of the second intron STP is a powerful and non-specific inhibitor of protein kinases that has been established so far as an effective inductor of apoptotic cell death [35][36][37][38][39]. During the characterization of the STP-induced apoptotic process in neuroblastoma cells, we observed that IMR-5 cells did not show internucleosomal DNA fragmentation irrespective of the times and concentrations of STP used [35].…”

“…Unless otherwise stated, all media and reagents were purchased from Sigma (St. Louis, MO). Apoptosis induction using STP was performed as described previously [35,36]. Human peripheral blood leukocyte samples from 10 healthy individuals were purified with an HistoPaque gradient (Sigma) following the manufacturer's instructions.…”

BCL-XL regulates TNF-α-mediated cell death independently of NF-κB, FLIP and IAPs

“…For transient overexpression, the cDNA for human Bcl-x L was extracted from pcDNA3 (Invitrogen) [42] and subcloned into pWPI [43,44]. PC12 cells stably transfected with IκBα super-repressor (SR-IκBα)-pcDNA3 or empty-pcDNA3 vectors were obtained as described by Sole et al [45].…”

“…Bcl-x L levels were assessed by western blot and, after 3 days of infection, viruses carrying Bcl-x L or shBcl-x L efficiently induced Bcl-x L protein overexpression or reduced Bcl-x L protein levels, respectively. Selection of pools of cells transfected with human Bcl-x L or empty pcDNA3 [42] was performed by adding G-418 to the medium at a final concentration of 500 µg/ml (Gibco).…”