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.

BCL2 family members are subject to regulation at multiple levels, providing checks on their ability to contribute to tumorigenesis. However, findings on posttranslational BCL2 phosphorylation in different systems have been difficult to integrate. Another antiapoptotic family member, MCL1, exhibits a difference in electrophoretic mobility upon phosphorylation induced by an activator of PKC (12-O-tetradecanoylphorbol 13-acetate; TPA) versus agents that act on microtubules or protein phosphatases 1/2A. A multiple pathway model is now presented, which demonstrates that MCL1 can undergo distinct phosphorylation events -mediated through separate signaling processes and involving different target sites -in cells that remain viable in the presence of TPA versus cells destined to die upon exposure to taxol or okadaic acid. Specifically, TPA induces phosphorylation at a conserved extracellular signal-regulated kinase (ERK) site in the PEST region (Thr 163) and slows turnover of the normally rapidly degraded MCL1 protein; however, okadaic acid and taxol induce ERK-independent MCL1 phosphorylation at additional discrete sites. These findings add a new dimension to our understanding of the complex regulation of antiapoptotic BCL2 family members by demonstrating that, in addition to transcriptional and post-transcriptional regulation, MCL1 is subject to multiple, separate, post-translational phosphorylation events, produced in living versus dying cells at ERKinducible versus ERK-independent sites.

An immortalized cell line was created from a primary culture of bronchial epithelia isolated from a patient with cystic fibrosis. The culture was transformed with a hybrid virus, adeno-12-SV40, which has been used successfully on a number of different human epithelial tissues. The transformed bronchial epithelial cells have the following characteristics. (1) Cyclic adenosine monophosphate (cAMP) is stimulated by beta-adrenergic agonists. (2) Outwardly rectifying Cl- channels are present on the apical cell membrane. These channels can be activated by depolarizing voltages but not by protein kinase A or C. (3) Keratin is present by immunofluorescence, and this is consistent with the epithelial origin of the cells. (4) The SV40 large T antigen is present as demonstrated by immunofluorescence. (5) Multiple karyotype analyses show modal chromosome number to be 80 to 90. There are an average of four chromosome 7 per cell. (6) The phenylalanine508 deletion in the gene coding for the cystic fibrosis transmembrane regulator is present on at least one chromosome. The cells can be grown in multiple passages, contain the abnormal regulation of the secretory Cl- channel, and should be an appropriate substrate for studies of the mutant cystic fibrosis transmembrane regulatory protein and its interaction with the Cl- channel.

Abstract.A family of genes related to the bcl-2 protooncogene has recently emerged. One member of this family, mcl-1, was cloned from a human myeloblastic leukemia cell line (ML-1) undergoing differentiation. The intracellular localization of mcl-1, as well as the kinetics of its expression during differentiation, have now been studied. These studies show that the intracellular distribution of mcl-1 overlaps with, but is not identical to, that of bcl-2:mcl-1 is similar to bcl-2 in that the mcl-1 protein has a prominent mitochondrial localization, and in that it associates with membranes through its carboxyl hydrophobic tail. mcl-1 differs from bcl-2, however, in its relative distribution among other (nonmitochondrial/heavy membrane) compartments, mcl-1 also being abundant in the light membrane fraction of immature ML-1 cells while bcl-2 is abundant in the nuclear fraction. Similarly, in differentiating ML-1 cells, the timing of expression of mcl-1 overlaps with, but is not identical to, that of bcl-2: the mcl-1 protein increases rapidly as cells initiate differentiation, and mcl-1 is a labile protein. In contrast, bcl-2 decreases gradually as cells complete differentiation. Overall, the mcl-1 and bcl-2 proteins have some properties in common and others that are distinct. A burst of expression of mcl-1, prominently associated with mitochondria, complements the continued expression of bcl-2 in ML-1 cells differentiating along the monocyte/macrophage pathway. MCL-1, a member of the bcl-2 family, was cloned from the ML-1 human myeloblastic leukemia cell line (26). ML-1 cells proliferate as immature myeloblasts, and they differentiate to monocytes and macrophages upon exposure to the phorbol ester, 12-0-tetradecanoylphorbol 13-acetate (TPA) I (9). The progression to differentiated phenotype is accompanied by a loss of proliferative capacity without substantial loss of viability, mcl-1 was isolated by screening for genes that increase in expression early in the switch from proliferative to differentiating phenotype (26).It is the carboxyl portion of mcl-1 that has sequence similarity to bcl-2 (the carboxyl 139 out of the 350 amino acid residues of mcl-1 [25]; bcl-2 sequence [8,52]). At the extreme carboxyl terminus, both mcl-1 and bcl-2 have a hydrophobic stretch (19-20 amino acids); in bcl-2, this stretch mediates the association of the protein with membranes (6, 7, 38, 51). bcl-2 was initially reported to localize to mitochondrial membranes and has since also been found in other intraceUular membrane-containing compartments, including the nuclear envelope and endoplasmic reticulum (21,27,33). Recent data show that bcl-2 associates with the outer