Regulation of c-myc mRNA Decay in Vitro by a Phorbol Ester-inducible, Ribosome-associated Component in Differentiating Megakaryoblasts

Gary Brewer

doi:10.1074/jbc.m006145200

Abstract

The K562 leukemia cell line is bipotential for erythroid and megakaryoblastic differentiation. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) activates a genetic program of gene expression in these cells leading to their differentiation into megakaryoblasts, a platelet precursor. Thus, K562 cells offer a means to examine early changes in gene expression necessary for megakaryoblastic commitment and differentiation. An essential requirement for differentiation of many hematopoietic cell types is the down-regulation of c-myc expression, because its constitutive expression blocks differentiation. TPA-induced differentiation of K562 cells causes rapid down-regulation of c-myc expression, due in part to an mRNA decay rate that is 4-fold faster compared with dividing cells. A cell-free mRNA decay system reconstitutes TPA-induced destabilization of c-myc mRNA, but it requires at least two components for reconstitution. One component fractionates to the post-ribosomal supernatant from either untreated or treated cells. This component is sensitive to cycloheximide and micrococcal nuclease. The other component is polysome-associated and is induced or activated by TPA. Although in dividing cells c-myc mRNA decays via a sequential pathway involving removal of the poly(A) tract followed by degradation of the mRNA body, TPA activates a deadenylation-independent pathway. The cell-free mRNA decay system reconstitutes this alternate decay pathway as well.

Highlights

Specific and timely changes in gene expression are required for cellular differentiation and proper embryonic development
TPA-induced Differentiation and Down-regulation of c-myc mRNA—TPA treatment of K562 cells activates a genetic program that results in changing patterns of gene expression leading to megakaryoblastic differentiation. c-myc mRNA is known to decline in K562 cells after 1 day of TPA treatment [17]
Because K562 cells can lose their capacity for differentiation during long-term culture [33], the levels of ␥-globin, PDGF-B, TGF-␤1, and ␤-actin mRNAs were examined to ensure that the cells have maintained their fidelity for differentiation

Summary

Introduction

Specific and timely changes in gene expression are required for cellular differentiation and proper embryonic development (reviewed in Ref. 1). Reconstitution of TPA-induced mRNA Destabilization by Cell-free Extracts—Our long-term goal is to define the cellular components required for regulation of c-myc mRNA decay in dividing and differentiating cells. To identify cellular factors required to reconstitute TPA-induced destabilization of c-myc mRNA in vitro, polysome and S130 fractions from untreated (control) and TPAtreated cells were mixed in a variety of combinations and incubated for various times in decay reactions.

Results

Conclusion