Abstract
Deregulated expression of c-Myc can sensitize cells to a variety of death stimuli, including loss of growth factors and oxygen. In this study, we examined whether rodent fibroblasts that conditionally express c-Myc undergo a similar mechanism of cell death in response to serum or oxygen deprivation. Our results demonstrate that murine embryonic fibroblasts from bax-/-bak-/- mice that conditionally express c-Myc did not die in response to either oxygen or serum deprivation. Fibroblasts from p53-/- mice that conditionally express c-Myc died in response to oxygen (but not serum) deprivation. The inability of p53 to regulate oxygen deprivation-induced cell death was due to the lack of induction of p53 target genes Puma, Noxa, and Pten. In contrast, serum deprivation transcriptionally induced Puma and Pten in cells that conditionally express c-Myc. The failure of p53 to regulate oxygen deprivation-induced cell death led us to hypothesize whether hypoxia-inducible factor (HIF) might be a critical regulator of cell death during oxygen deprivation. Fibroblasts from HIF-1beta-/- cells that conditionally express c-Myc were not able to transcriptionally activate HIF during oxygen deprivation. These cells died in response to oxygen deprivation. Thus, oxygen deprivation-induced cell death in fibroblasts with deregulated expression of c-Myc is independent of p53 or HIF-1 status, but is dependent on the Bcl-2 family member Bax or Bak to initiate mitochondrial dependent cell death.
Highlights
The proto-oncogene c-Myc is a transcription factor that forms a heterodimer with Max and activates genes involved in proliferation [1]
We examined whether rodent fibroblasts that conditionally express c-Myc undergo a similar mechanism of cell death in response to serum or oxygen deprivation
Our results demonstrate that murine embryonic fibroblasts from bax؊/؊bak؊/؊ mice that conditionally express c-Myc did not die in response to either oxygen or serum deprivation
Summary
Cell Culture—Rat1a fibroblasts and murine embryonic fibroblasts (MEFs) were cultured to 30 – 40% confluence in Dulbecco’s modified essential medium supplemented with 25 mM HEPES, 1 mM pyruvate, 100 units/ml penicillin, 100 g/ml streptomycin, and 10% heat-inactivated fetal bovine serum (Invitrogen). Measurement of Cytochrome c Release and Caspase-9 Activation— Rat1a/MycER cells were plated on 60-mm culture dishes at 30 – 40% confluence and exposed to experimental conditions. Both adherent and non-adherent cells were collected and centrifuged for 5 min at 200 ϫ g. The cells were incubated at 20 °C for 5 min in methanol/acetone (1:1) and blocked for 45 min in phosphate-buffered saline containing 0.1% bovine serum albumin (Sigma). The cells were washed for 4 ϫ 15 min in phosphate-buffered saline containing 0.1% bovine serum albumin, air-dried, and incubated for 1 h with a 1:50 dilution of a rhodamine-conjugated secondary antibody (Chemicon International, Inc.). Cell lysates were prepared using cell lysis buffer (New England Biolabs Inc.) supplemented with 1 mM phenylmethylsulfonyl fluoride
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