Inhibitor of caspase-activated DNase expression enhances caspase-activated DNase expression and inhibits oxidative stress-induced chromosome breaks at the mixed lineage leukaemia gene in nasopharyngeal carcinoma cells

Abstract

Background: Nasopharyngeal carcinoma (NPC) is commonly found in Asia, especially among the Chinese ethnic group. Chromosome rearrangements are common among NPC patients. Although the mechanism underlying the chromosome rearrangements in NPC is unclear, various mechanisms including activation of caspase-activated DNase (CAD) were proposed to contribute to chromosome rearrangements in leukaemia. Activation of CAD can be initiated by multiple agents, including oxidative stress, which is well implicated in carcinogenesis. CAD is the main enzyme that causes DNA fragmentation during apoptosis, and CAD is also implicated in promoting cell differentiation. In view of the role of oxidative stress in carcinogenesis and CAD activation, and since CAD was suggested to contribute to chromosome rearrangement in leukaemia, we hypothesise that oxidative stress-induced CAD activation could be one of the mechanisms that leads to chromosome rearrangements in NPC.

Methods: SUNEI cells were treated with various concentrations of H2O2 for different period of time to ensure that cells undergo H2O2-induced MLL gene cleavage. Transfections with hCAD, mCAD, mutant hCAD, or cotransfection with hCAD and mICAD, and cotransfection with mutant hCAD and mICAD were performed. Gene expression was confirmed by Western blotting and MLL gene cleavage was assessed by inverse polymerase chain reaction (IPCR).

Results: Treatment with H2O2 clearly induces cleavages within the MLL gene which locates at 11q23, a common deletion site in NPC. In order to investigate the role of CAD, CAD was overexpressed in SUNE1 cells, but that did not result in significant changes in H2O2-induced MLL gene cleavage. This could be because CAD requires ICAD for proper folding. Indeed, by overexpressing ICAD alone or co-expressing ICAD with CAD, Western blotting showed that CAD was expressed. In addition, ICAD overexpression also suppressed H2O2-induced MLL gene cleavage, suggesting a possible role of CAD in initiating chromosome cleavage during oxidative stress.

Conclusions: Oxidative stress mediated by H2O2 induces cleavage of the MLL gene, most likely via the caspase-activated DNase, CAD, and CAD expression requires ICAD. Since the MLL gene is located at 11q23, a common deletion site in NPC, thus stress-induced CAD activation may represent one of the mechanisms leading to chromosome rearrangement in NPC.

Keywords: CAD; Chromosome breaks; ICAD; MLL; Nasopharyngeal carcinoma; Oxidative stress.

Fig. 1

Hydrogen peroxide (H₂O₂) induces MLL gene cleavage in NPC cells. SUNE1 cells were treated with either 0 μM, 10 μM, 50 μM, 100 μM, 500 μM, 1 mM or 10 mM of H₂O₂ for 20 h (lanes 1–7 respectively). Genomic DNA was extracted and modified for IPCR as described in Materials and Methods. DNA was digested with Msc I during the modification. Side arrow shows the 2.2 kb band resulting from amplification of the intact MLL gene. Side bracket shows bands smaller than 2.2 kb resulting from amplification of cleaved MLL gene. M₁ represents 1 kb DNA marker

Fig. 2

Transient transfection with normal and mutant CAD do not enhance H₂O₂-induced MLL gene cleavage. a Microscopic morphology of transiently transfected SUNE1 cells. SUNE1 cells were transfected with either the vector pTracer, T; pTracer-hCAD, h; pTracer-mCAD, m; or pTracer-hCAD (k157Q), k. A mock transfection (no DNA), N, was included as control. Transfected cells were either treated with 50 μM of H₂O₂ for 8 h at 17 h post-transfection, or left untreated. Observation was then carried out with light microscopy and fluorescence microscopy. b Nested IPCR detection of MLL gene cleavage. SUNE1 cells were transfected with either the vector pTracer, T (Lanes1 and 6), pTracer-hCAD, h (Lanes 2 and 7), pTracer-mCAD, m (Lanes 3 and 8) or pTracer-hCAD (k157Q), k (Lanes 4 and 9). A mock transfection (no DNA), N (Lanes 5 and 10) was included as control. The cells were either non-treated (Lanes 1–5) or treated with 50 μM of H₂O₂ for 8 h at 17 h post-transfection. Genomic DNA extracted was modified for IPCR as described in Materials and Methods. Side arrow shows the 2.2 kb band resulting from amplification of the intact MLL gene. Side bracket shows bands smaller than 2.2 kb resulting from amplification of cleaved MLL gene. M₁ represents 1 kb DNA marker. M₂ represents 100 bp DNA marker

Fig. 3

ICAD expression enhances CAD expression. Two sets of SUNE1 cells were transiently transfected with vector pTracer (Lane 2), pcDNA (Lane 3), pTracer-hCAD (Lane 4), pcDNA-mICAD-L (Lane 5), or cotransfection with pTracer-hCAD/pcDNA-mICAD-L (Lane 6) and pTracer-hCAD (K157Q)/pcDNA-mICAD-L (Lane 7). A mock transfection (No DNA) was included as control (Lane 1). Transfected cells were either not treated (Panel a) or treated with 50 μM of H₂O₂ for 6 h at 17 h post-transfection (Panel b). Protein was extracted as detailed in Materials and Methods. Expression of CAD, ICAD and GAPDH was each analysed by anti-CAD, anti-ICAD and anti-GAPDH respectively

Fig. 4

ICAD expression inhibited H₂O₂-induced MLL gene cleavage. SUNE1 cells were transiently transfected with vector pTracer (Lane 2), pcDNA (Lane 3), pTracer-hCAD (Lane 4), pcDNA-mICAD-L (Lane 5), or cotransfection with pTracer-hCAD/pcDNA-mICAD-L (Lane 6) and pTracer-hCAD (K157Q)/pcDNA-mICAD-L (Lane 7). A mock transfection (No DNA) was included as control (Lane 1). Transfected cells were treated with 50 μM of H₂O₂ for 6 h at 17 h post-transfection. Genomic DNA was extracted and processed for IPCR as detailed in Materials and Methods. Side arrow shows the 2.2 kb band resulting from amplification of the intact MLL gene. Side bracket shows bands smaller than 2.2 kb resulting from amplification of cleaved MLL gene. M₁ represents 1 kb DNA marker. M₂ represents 100 bp DNA marker

Fig. 5

Flow chart showing DNA modification and IPCR. The arrow heads indicate the forward and reverse primers that were designed in opposite direction. BamH I digestion yielded a mixture of intact chromosome and cleaved chromosome. Klenow fill-in produced blunt ended chromosome fragments which were then cyclilsed by T4 DNA ligase. The intact chromosome will become a large circle while the cleaved chromosome will become a smaller circle. Upon cyclisation, the primers are now in correct orientation for amplification. Msc I digestion cleaved both circles outside the amplification region, thus merely linearise the molecule. Amplification from intact MLL gene will produce longer PCR products while amplification from cleaved MLL gene will yield shorter PCR products