PIC-1/SUMO-1-modified PML-retinoic acid receptor alpha mediates arsenic trioxide-induced apoptosis in acute promyelocytic leukemia - PubMed (original) (raw)

PIC-1/SUMO-1-modified PML-retinoic acid receptor alpha mediates arsenic trioxide-induced apoptosis in acute promyelocytic leukemia

T Sternsdorf et al. Mol Cell Biol. 1999 Jul.

Abstract

Fusion proteins involving the retinoic acid receptor alpha (RARalpha) and PML or PLZF nuclear protein are the genetic markers of acute promyelocytic leukemia (APL). APLs with PML-RARalpha or PLZF-RARalpha fusion protein differ only in their response to retinoic acid (RA) treatment: the t(15;17) (PML-RARalpha-positive) APL blasts are sensitive to RA in vitro, and patients enter disease remission after RA treatment, while those with t(11;17) (PLZF-RARalpha-positive) APLs do not. Recently it has been shown that complete remission can be achieved upon treatment with arsenic trioxide (As2O3) in PML-RARalpha-positive APL, even when the patient has relapsed and the disease is RA resistant. This appears to be due to apoptosis induced by As2O3 in the APL blasts by poorly defined mechanisms. Here we report that (i) As2O3 induces apoptosis only in cells expressing the PML-RARalpha, not the PLZF-RARalpha, fusion protein; (ii) PML-RARalpha is partially modified by covalent linkage with a PIC-1/SUMO-1-like protein prior to As2O3 treatment, whereas PLZF-RARalpha is not; (iii) As2O3 treatment induces a change in the modification pattern of PML-RARalpha toward highly modified forms; (iv) redistribution of PML nuclear bodies (PML-NBs) upon As2O3 treatment is accompanied by recruitment of PIC-1/SUMO-1 into PML-NBs, probably due to hypermodification of both PML and PML-RARalpha; (v) As2O3-induced apoptosis is independent of the DNA binding activity located in the RARalpha portion of the PML-RARalpha fusion protein; and (vi) the apoptotic process is bcl-2 and caspase 3 independent and is blocked only partially by a global caspase inhibitor. Taken together, these data provide novel insights into the mechanisms involved in As2O3-induced apoptosis in APL and predict that treatment of t(11;17) (PLZF-RARalpha-positive) APLs with As2O3 will not be successful.

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Figures

FIG. 1

(A) Structure of the t(11;17) and t(15;17) fusion proteins. RARα is subdivided into its conserved functional domains. C and E indicate the DNA binding and the ligand binding domains, respectively. In the fusion, PML retains three novel zinc fingers, the RING domain (R) and the B boxes 1 and 2 (B1 and B2). In the α helix, PML presents a coiled-coil region, which is its homodimerization interface. The PLZF POZ domain and the retained two zinc fingers are also shown. The breakpoints (bp) where PML and PLZF fuse to RARα are indicated by black arrows. (B) Zn-induced PML-RARα and PLZF-RARα expression in U937 cells. Western blot analysis from U937 cells stably transfected with a Zn-inducible PLZF-RARα or PML-RARα expression vector in the presence (+) or absence (−) of Zn induction. Blots were stained with an anti-RARα polyclonal antibody directed against the RARα F domain. Molecular weight markers are given to the left (in thousands). Each lane was loaded with lysates from 2 × 105 cells. The positions of PLZF-RARα and PML-RARα polypeptides are indicated.

FIG. 2

(A) Apoptotic effect of As2O3 on NB4 cells (cell line derived from an APL patient [30]) and NB4 cells treated with As2O3 in the presence of ZVAD, as shown by 7-AAD analysis (one of three experiments that gave nearly identical results). (B) Apoptotic effect of As2O3 on PML-RARα- and PLZF-RARα-expressing U937 cells, as shown by 7-AAD analysis (one of three experiments that gave nearly identical results): MTB45-control cells transfected with the empty MT expression vector; B412, PLZF-RARα-expressing cells; P/R9, PML-RARα-expressing cells. The U937 cells are treated with Zn alone (Zn+) and with As2O3 (As+) in the absence or presence of Zn-induced protein expression. Also represented are Zn-induced P/R9 cells exposed to As2O3 in the presence of ZVAD-FMK (ZVAD). (C) bcl-2 and PARP expression of the P/R9 clone in the absence of As treatment (As−) and after 12, 24, and 48 h of As2O3 (As+) or 12 and 24 h of RA treatment (t-RA + or −) as a control for PARP cleavage. The PARP and bcl-2 proteins are indicated.

FIG. 3

Western blot analysis of PML-RARα- and PLZF-RARα-expressing U937 cells. (A) P/R9 and B412 clones in the presence of Zn-induced PML-RARα or PLZF-RARα expression, respectively, in the presence or absence of 12 and 24 h of RA treatment (t-RA − or +). (B) P/R9 and B412 clones in the absence or presence of Zn induced PML-RARα or PLZF-RARα expression (Zn − or +), respectively, and in the absence or presence of 12 h of As2O3 exposure (As − or +). Blots were stained with an anti-RARα polyclonal antibody (α-RARα). The positions of PLZF-RARα and PML-RARα polypeptides are indicated. (C) PML-RARα lanes of panel B stained with anti-Sp100 antibody (α-Sp100). The positions of Sp100 and PIC-1/SUMO-1-modified Sp100 are indicated.

FIG. 4

PIC-1/SUMO-1 modification of PML/RARα upon As2O3 exposure. (A) P/R9 clone in the absence or presence of Zn-induced PML-RARα expression (Zn − or +) and in the absence or presence of 12 h of As2O3 exposure (As − or +). Blots were stained with an anti-RARα polyclonal antibody (α-RARα) and anti-SUMO-1 monoclonal antibody (α-Sumo1). (B) Electronic juxtaposition of the lanes from panel A stained with anti-RARα antibody with that stained with anti-SUMO-1 antibody in the absence or presence of Zn-induced PML-RARα expression in the P/R9 clone (Zn − or +). The SUMO-1-conjugated bands are indicated by bars. (C) Electronic quantification of wild-type and As2O3-modified PML-RARα protein from the lanes stained with anti-RARα antibody shown in panel A. (D) Electronic comparison between single wild-type PML-RARα bands and the smear of PIC-1/SUMO-1-modified PML-RARα resulting from As2O3 treatment.

FIG. 5

Immunofluorescence analysis of PML, PML-RAR, PLZF-RARα, and PIC-1/SUMO-1 protein localization in the U937 P/R9 and B412 cell clones and the NB4 cells. Results for the P/R9 and B412 clones are reported in the absence (−Zn) and presence (+Zn) of Zn-induced PML-RARα or PLZF-RARα expression with or without As2O3 (+ or −As) NB4 cells are reported in the presence or absence of As2O3. Cells were stained with the indicated antibodies: the anti-PML (α-PML) and anti-PLZF (α-PLZF) polyclonal antibodies (green fluorochrome) and the anti-SUMO-1 MAb (α-SUMO) (anti-GMP-1 21C7) (red fluorochrome). Colocalization images of PIC-1/SUMO-1 and PML or PLZF were obtained by electronic overlapping of the images recorded (for merge, colocalization of fluorochromes yields a yellow color). Phaco, phase-contrast images.

FIG. 6

7-AAD analysis of the U937 clones B321 and B327 expressing the PML-ΔRARα mutant (one out of three experiments that gave nearly identical results). MTB45, control cells transfected with the empty MT expression vector; P/R9, PML-RARα-expressing cells; B321 and B327, PML-ΔRARα-expressing clones. The U937 cells are treated with Zn alone (+Zn) and with As2O3 (− or + As) in the absence or presence of Zn-induced protein expression.

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PIC-1/SUMO-1-modified PML-retinoic acid receptor alpha mediates arsenic trioxide-induced apoptosis in acute promyelocytic leukemia - PubMed (original) (raw)