MCLI , a gene expressed in programmed myeloid cell differentiation , has sequence similarity to BCL 2

During their lifespan, immature cells normally pass through sequential transitions to a differentiated state and eventually undergo cell death. This progression is aberrant in cancer, although the transition to differentiation can be reestablished in inducible leukemia cell lines. This report describes a gene, MCLl, that we isolated from the ML-1 human myeloid leukemia cell line during phorbol ester-induced differentiation along the monocyte/macrophage pathway. Our results demonstrate that expression ofMCLI increases early in the induction, or "programming," of differentiation in ML-1 (at 1-3 hr), before the appearance of differentiation markers and mature morphology (at 1-3 days). They further show that MCLI has sequence similarity to BCL2, a gene involved in normal lymphoid development and in lymphomas with the t(l4;18) chromosome translocation. MCLI and BCL2 do not fall into previously known gene families. BCL2 differs from many oncogenes in that it inhibits programmed cell death, promoting viability rather than proliferation; this parallels the association of MCL1 with the programming of differentiation and concomitant maintenance of viability but not proliferation. Thus, in contrast to proliferation-associated genes, expression of MCLI and BCL2 relates to the programming of differentiation and cell viability/death. The discovery of MCLI broadens our perspective on an emerging MCLI/BCL2 gene family and will allow further comparison with oncogene families. Normal tissues are characterized by a balance between cell proliferation, cell differentiation, and cell death. The molecular programs that control this balance are aberrant in cancer. Gaining an understanding of these programs involves identifying genes that influence proliferation and the transitions to differentiation and death (e.g., oncogenes, tumor-suppressor genes). Our research focuses on the molecular events that underlie the transition from proliferation to differentiation in a hematopoietic cell line, the ML-1 human myeloid leukemia cell line (1-3). ML-1 cells proliferate as immature myeloblasts and can be induced to differentiate to monocytes/ macrophages with phorbol 12-myristate 13-acetate ["12-0tetradecanoylphorbol 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 (1-3). In sum, immature, proliferative cells convert to a differentiated, viable, nonproliferative phenotype. In ML-1 cells, the initial induction or "programming" of this conversion can be separated from the ensuing phenotypic transition: upon exposure to TPA under optimal conditions, ML-1 cells become induced (i.e., irreversibly committed, or programmed) at -3 hr and then undergo phenotypic differentiation at 1-3 days (1-3). 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 ofDNA 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. The p53 tumor-suppressor gene product had previously been shown (4) to increase in differentiation, but this occurred at later times (21 day). Our rationale in seeking mRNAs increased within 3 hr was that (i) some of these "early-induction" genes might influence later phenotypic changes and (ii) a similar strategy had been successful in identifying interesting "early-response" genes expressed upon the stimulation of cell proliferation (5). We describe here an early-induction gene, MCLJt, isolated from maturing ML-1 myeloid cell leukemia. We found MCLI to have sequence similarity to BCL2, a gene that was originally identified in B-cell lymphomas. Most follicular B-cell lymphomas have a t(14;18) chromosome translocation that juxtaposes BCL2 with the immunoglobulin heavy-chain locus; this results in deregulated expression of an unaltered BCL2 gene product (6-9). BCL2 had not previously been found to have sequence similarity to known cellular oncogenes; it does not contain motifs characteristic of other gene families, although it does have distant similarity to a viral gene, BHRF1 (8, 10, 11). Expression of BCL2 relates to inhibition of programmed cell death (12-14), while that of BHRF1 occurs early in the lytic cycle of Epstein-Barr virus (10, 11). MCLI provides an example ofa cellular gene related to BCL2. This allows us to begin to define the characteristics of what appears to be a gene family represented by MCLI, BCL2, and BHRF1. MATERIALS AND METHODS Preparation of a cDNA Library from TPA-Induced ML-1 Cells. ML-1 cells (1-3) were induced with TPA by a protocol that optimizes the programming of differentiation (3): cells were exposed to 0.5 nM TPA for 3 hr, after preincubation under reduced serum conditions for 3 days. Poly(A)+ RNA was isolated (2, 3) and used in oligo(dT)-primed first-strand cDNA synthesis, which was carried out with Moloney murine leukemia virus reverse transcriptase (BRL). After second-strand cDNA synthesis, the cDNA was treated with Si nuclease (BRL), EcoRI methylase (New England Biolabs), and Klenow fragment of DNA polymerase (BRL). After addition of EcoRI linkers, cDNA of >500 bp was isolated (Bio-Gel ASOM column, BioRad), cloned into the AgtlO vector, packaged (Packagene, Promega), and infected into Escherichia coli C600hfl. The yield was 3 x 104 plaqueforming units/ng of cDNA, with an average insert size of 1.5 kb. Screening for cDNAs Representing Early-Induction Genes. For differential screening, recombinant phage from the Abbreviation: TPA, "12-0-tetradecanoylphorbol 13-acetate" (phorbol 12-myristate 13-acetate). *To whom reprint requests should be addressed. tThe nucleotide sequence reported in this paper has been deposited in the GenBank data base (accession no. L08246). 3516 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 90 (1993) 3517 cDNA library were plated and transferred to nitrocellulose filters. Replicate filters were hybridized to 32P-labeled cDNA probes derived either from TPA-induced or uninduced cells. [Induction with TPA was carried out for 3 hr with the above-described optimized protocol. The cDNA probes were synthesized by reverse transcription of poly(A)+ RNA, using random hexamers as primers. ] Primary and secondary screenings were carried out to identify clones exhibiting preferential hybridization to the cDNA probe from induced cells. One of the positive clones obtained, dif8C, was subcloned into the pBluescript plasmid (Stratagene). Clone dif8C contained nucleotides 3150-3934 of MCLJ followed by 11 adenine residues. This clone was used in obtaining additional cDNAs, both from the above cDNA library and from acDNA library from TPA-induced U-937 cells (Clontech). Characterization of the Early-Induction cDNAs Obtained by Differential Screening. Northern blotting (2, 3) was carried out using probes for MCLJ, /3-actin (15), c-myb [pCM8 from R. Dalla-Favera (16)], and CD11b [from D. G. Tenen (17)]. After densitometric scanning, the values for expression of MCLI (the more abundant, 3.8-kb MCLI transcript) were normalized by dividing by the corresponding value for 3-actin. Relative expression of MCLI was then estimated as the ratio of expression in TPA-induced cells to that in uninduced controls. Differentiation markers were monitored by flow cytometry on a FACScan using phycoerythrin-conjugated antibodies to CD11b and CD14 (Becton Dickenson). Corrected mean fluorescence intensity was calculated by subtraction of background, which was determined by using isotype-matched controls (IgG2a from Becton Dickinson and IgG2b from Coulter Immunology). Sequencing was carried out by the dideoxy chaintermination method (18, 19) with a modified T7 polymerase (Sequenase, United States Biochemical). The MCLI coding region was sequenced on both strands, and the 3' untranslated region on one strand. Computer analysis was carried out using the Genetics Computer Group sequence-analysis software (Madison, WI), the PESTFIND program (from Martin Rechsteiner, University of Utah), and the SIGSEQ programs (from Jeffrey Gordon, Washington University).