Induction of cell cycle progression and acceleration of apoptosis are two separable functions of c-Myc: transrepression correlates with acceleration of apoptosis

Abstract

Analysis of amino-terminus mutants of c-Myc has allowed a systematic study of the interrelationship between Myc's ability to regulate transcription and its apoptotic, proliferative, and transforming functions. First, we have found that c-Myc-accelerated apoptosis does not directly correlate with its ability to transactivate transcription using the endogenous ornithine decarboxylase (ODC) gene as readout for transactivation. Furthermore, deletion of the conserved c-Myc box I domain implicated in transactivation does not inhibit apoptosis. Second, the ability of c-Myc to repress transcription, using the gadd45 gene as a readout, correlates with its ability to accelerate apoptosis. A conserved region of c-Myc implicated in mediating transrepression is absolutely required for c-Myc-accelerated apoptosis. Third, a lymphoma-derived Thr58Ala mutation diminishes c-Myc-accelerated apoptosis through a decreased ability to induce the release of cytochrome c from mitochondria. This mutation in a potential phosphorylation site does not affect cell cycle progression, providing genetic evidence that induction of cell cycle progression and acceleration of apoptosis are two separable functions of c-Myc. Finally, we show that the increased ability of Thr58Ala mutant to elicit cellular transformation correlates with its diminished ability to accelerate apoptosis. Bcl-2 overexpression blocked and the lymphoma-associated Thr58Ala mutation decreased c-Myc-accelerated apoptosis, and both led to a significant increase in the ability of Rat1a cells to form colonies in soft agar. This enhanced transformation was greater in soft agar containing a low concentration of serum, suggesting that protection from apoptosis is a mechanism contributing to the increased ability of these cells to proliferate in suspension. Thus, we show here for the first time that, in addition to mutations in complementary antiapoptotic genes, c-Myc itself can acquire mutations that potentiate neoplastic transformation by affecting apoptosis independently of cell cycle progression.

FIG. 1

Expression levels of wild-type and mutant c-Myc proteins in stably infected Rat1a polyclonal cell lines. (A) Schematic illustration of wild-type and mutant c-MycER fusion proteins. Hatched boxes indicate MB I and MB II; potential phosphorylation sites (Thr58 and Ser62) in MB I are also indicated. In the VP16-Myc construct, the activation domain of VP16 replaces aa 47 to 263 and is fused in frame with the DNA binding domain of c-Myc. (B) Western blot analysis of MycER proteins using the ER-specific H222 antibody.

FIG. 2

Induction of apoptosis by c-Myc mutants. Time course of percentages of apoptotic Rat1a cells scored by DAPI staining. Cells (10⁵) were plated in 30-mm wells, allowed to adhere overnight, and then serum deprived in the presence of 4-OHT in order to activate c-Myc. Cells were then fixed directly in tissue culture plates, stained with DAPI, and scored for nuclear condensation. Averages of at least 300 cells from three independent experiments are shown ± the standard error (SE).

FIG. 3

Induction of ODC mRNA by c-Myc mutants. (A) Northern blot analysis of ODC mRNA was performed using 10 μg of Rat1a RNA from cell pools expressing various mutant c-Myc proteins. Cells were made quiescent with serum deprivation, and c-Myc was activated by the addition of 4-OHT. RNA was harvested 6 h later and assayed for induction of ODC mRNA. (B) ODC mRNA fold induction relative to control (GAPDH) RNA, as quantified by densitometry. The fold induction following c-Myc activation with 4-OHT was averaged from three individual experiments.

FIG. 4

Repression of gadd45 mRNA by c-Myc mutants. RNA (15 μg) from Rat1a cell pools deprived of serum for 60 h and then exposed to 4-OHT was analyzed by RNase protection assay to detect the presence of endogenous gadd45 and GAPDH-specific sequences. Experiment is a representative of three independent experiments.

FIG. 5

Cell cycle analysis after activation of c-Myc mutants. (A) Cells were deprived of serum for 60 h until ∼80% were in G₀/G₁. The percentage of cells was determined by FACS in three independent experiments, and the SE was calculated. (B) Histogram showing cell cycle distributions of c-Myc pools prior to and following activation of Myc by 4-OHT.

FIG. 6

Acceleration of apoptosis in MEF by wild-type and T58A mutant c-Myc. The percentages of apoptotic MEF were scored by DAPI staining. Cells (3 × 10⁴) were plated in 30-mm wells, allowed to adhere overnight, and then placed in either 0.5 or 2% FCS in the presence of 4-OHT in order to activate c-Myc. Cells were then fixed directly in tissue culture plates for 24 or 48 h after addition of 4-OHT, stained with DAPI, and scored for nuclear condensation. Averages of at least 300 cells from three independent experiments are shown ± the SE.

FIG. 7

Induction of cytochrome c release by c-Myc. The percentages of cells demonstrating diffuse cytochrome c immunostaining were quantitated. Rat1a, Rat1a/MycER, Rat1a/T58AMycER, Rat1a/dl106-143MycER, and Rat1a/MycER/Bcl-2 cells were incubated overnight in DMEM with 2.5% FCS with 1 μM 4-OHT. The next day cells were placed in DMEM with 0.5% FCS and 100 μM zVAD for 3 h. Cells were then fixed and immunostained for cytochrome c. Averages (±SE) of at least 100 cells from four independent experiments are shown.

FIG. 8

Anchorage-independent growth on soft agarose of Rat1a polyclonal cell lines expressing different c-Myc alleles. Cells were plated in 2 or 10% FCS in the presence of 4-OHT and allowed to grow for 21 days in soft agarose. Colonies were counted by projecting plates on a white board using an overhead projector. Only colonies 1 mm in diameter or larger were scored. (A) Bar graph of the results as a relative percentage of growth of cell lines in soft agarose, where the number of colonies formed by wild-type-Myc-expressing cells is 100%. (B) Light microscopy pictures of soft agarose colonies formed in 2% FCS by either wild-type c-Myc, T58A-expressing cells, or cells infected with empty retrovirus.