Improved coupling between proliferation‐arrest and differentiation‐induction in ML‐1 human myeloblastic leukemia cells

Abstract

Proliferation and differentiation are coupled in normal cells and are aberrant in leukemia cells. The studies reported here were aimed at more effectively coupling proliferation-arrest and differentiation-induction in a human myeloblastic leukemia cell line (ML-1). This was accomplished by using reduced serum conditions in conjunction with a differentiation-inducing agent: cells were first incubated in reduced serum [0.3% fetal bovine serum (FBS)] instead of standard conditions (7.5% FBS) and, second, exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). The effects of this protocol were as follows: first, cell proliferation was slowed and cells accumulated in G0/G1 phase of the cell cycle; this occurred with only a minimal decrease in viability [to approximately 88-92% (0.3% FBS) from greater than or equal to 96% (7.5% FBS)]. Second, the induction of differentiation was accelerated; this allowed the time of exposure to TPA to be decreased. Acceleration of induction was very pronounced when cells were maintained in 0.3% FBS both before and during exposure to TPA, with TPA at concentrations above the minimum sufficient for induction but below those causing significant cytotoxicity; as little as 1 hour of TPA exposure resulted in near-maximal induction (approximately 80%) with this protocol, compared to the greater than or equal to 1 day required with previous standard protocols. In sum, conditions that slow ML-1 cell proliferation (0.3% FBS) enhance TPA-induced differentiation, substantially narrowing the time frame of induction; these conditions should be useful for studying the molecular mechanisms that underlie the induction process.

MCL1 provides a window on the role of the BCL2 family in cell proliferation, differentiation and tumorigenesis

“…The BCL2 family member MCL1 was originally identified based on increased expression in ML-1 human myeloblastic leukemia cells that had committed to, but had not yet undergone, differentiation in response to the phorbol ester TPA. Peak expression of MCL1 occurs within 3 h after the application of TPA, while phenotypic differentiation occurs over days [1][2][3]. This early increase in expression of MCL1 serves to promote the viability of myeloblasts initiating differentiation in response to other gene products.…”

“…Commitment to differentiation occurs rapidly -within a window of several hours -upon the application of TPA to growth-arrested cells. 2 The 'commitment window' precedes phenotypic differentiation, which proceeds over the next several days. ML-1 cells that have passed through the commitment window do not difCorrespondence: RW Craig, Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH, USA; Fax: 603 650 1129…”

MCL1 transgenic mice exhibit a high incidence of B-cell lymphoma manifested as a spectrum of histologic subtypes

“…The cells obtained were then washed with cold phosphate buffered saline (PBS) and were assayed for cell number by hemacytometer. 48 Gel electrophoresis and immunoblotting were carried out as in previous work, 43 except that a monoclonal mouse antihuman MCL1 antibody was used at a dilution of 1:5000 (antibody preparation described below). After probing with the anti-MCL1 antibody, blots were reprobed for expression of Bcl-2 using previously described methods 43 or for expression of Bax using an antibody from Santa Cruz Biotechnology (Santa Cruz, CA).…”

MCL1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to BCL2.

“…The initial induction probably involves the combined action of a variety of genes, some increasing and others decreasing in expression. Previous work showed the c-myb oncogene mRNA to decrease within 3 hr [i.e., during induction and before loss of DNA synthesis or overt differentiation (2,3)]. In the work described here, we set out to identify genes that increase in expression within this early time frame.…”

“…ML-1 cells proliferate as immature myeloblasts and can be induced to differentiate to monocytes/ macrophages with phorbol 12-myristate 13-acetate ["12-0-tetradecanoylphorbol 13-acetate" (TPA)] (1-3). The differentiated cells lose proliferative capacity and accumulate in the Go/G1 phase of the cell cycle, while remaining viable and capable of carrying out normal monocyte/macrophage functions (1)(2)(3). In sum, immature, proliferative cells convert to a differentiated, viable, nonproliferative phenotype.…”