'Loop' domain is necessary for taxol-induced mobility shift and phosphorylation of Bcl-2 as well as for inhibiting taxol-induced cytosolic accumulation of cytochrome c and apoptosis

Guofu Fang, Brian S. Chang, Caryn N. Kim, Charles Perkins, Craig B. Thompson, Kapil Bhalla

Research output: Contribution to journalArticle

119 Scopus citations

Abstract

Taxol, 1-β-D-arabinofuranosylcytosine (ara-C), and etoposide induce apoptosis in HL-60 cells that is blocked by overexpression of Bcl-2 or Bcl- x(L). A ~60-amino acid 'loop' domain of Bcl-2 and Bcl-x(L) that contains phosphorylation sites is known to negatively regulate their antiapoptotic function. In the present studies, Taxol-, ara-C-, or etoposide-induced apoptosis was examined in HL-60/Bcl-2Δ and HL-60/Bcl-x(L)Δ cells that express the loop-deletional mutant cDNA constructs p19Bcl-2Δ32-80 and p18Bcl-x(L)Δ26-83, respectively. This was compared with control HL-60/neo cells as well as HL-60/Bcl-2 and HL-60/Bcl-x(L) cells. The latter two cell lines overexpress full-length Bcl-2 and Bcl-x(L), respectively. Immunoblot analyses showed that HL-60/neo and HL-60/Bcl-2Δ cells express similar levels of p26Bcl-2. In contrast, as compared with HL-60/neo, HL-60/Bclx(L)Δ cells expressed significantly lower levels of p26Bcl-2. p29Bcl-x(L) and p21Bax levels were similar in all cell types. Exposure to etoposide (50 μM) or ara- C (100 μM) for 4 h induced apoptosis in HL-60/neo cells, but not in HL- 60/Bcl-2, HL-60/Bcl-x(L), HL-60/Bcl-2Δ, or HL-60/Bcl-x(L)Δ cells. In contrast, Taxol treatment (500 nM for 24 h) triggered the molecular cascade of apoptosis, represented by the cytosolic increase of cytochrome c and poly(ADP-ribose) polymerase or the DNA fragmentation factor cleavage activity of caspase-3 in HL-60/neo cells as well as in HL-60/Bclx(L)Δ and HL-60/Bcl- 2Δ cells, but not in their counterparts overexpressing full-length Bcl-2 and Bcl-x(L). Equal amounts of p26Bcl-2 were coimmunoprecipitated with apoptosis protease-activating factor 1 (APAF-1) in HL60/neo and HL-60/Bcl-2Δ cells, whereas a markedly higher level of p26Bcl-2 coimmunoprecipitated with APAF-1 in HL-60/Bcl-2 cells. In association with Taxol-induced apoptosis, the levels of Bcl-2 that were coimmunoprecipitated with APAF-1 declined in HL-60/neo and HL-60/Bcl-2Δ cells. This was not observed in HL-60/Bcl-2 cells, in which Taxol-induced apoptosis was blocked. Previous studies have demonstrated that Taxol induces phosphorylation of Bcl-2 in association with Taxol-induced apoptosis of HL-60/neo cells. Immunoblot analysis demonstrated a Taxol- induced mobility shift of Bcl-2 but not p19Bcl-2Δ. Taxol also increased [32P]P(i) incorporation in p26Bcl-2, but not in p19Bcl-2A or p18Bcl-x(L). These findings indicate that the loop domain is necessary for the Taxol- induced mobility shift and phosphorylation of Bcl-2. Loop domain also seems to be necessary for the antiapoptotic effect of Bcl-2 against Taxol-induced apoptosis but not ara-C- or etoposide-induced apoptosis.

Original languageEnglish (US)
Pages (from-to)3202-3208
Number of pages7
JournalCancer research
Volume58
Issue number15
StatePublished - Aug 1 1998

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

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