Regulation of CD 9 Expression during 1 2 - O - Tetradecanoyl - phorbol - 1 3 - acetate - induced Differentiation of Human Myeloid Leukemia ( HL - 60 ) Cells 1

CD9 antigen is a member of the tetra spans superfamily of proteins which are expressed on the surface of a variety of hematopoietic and epithelial cell types. CD9 appears to play a role in platelet activation and to enhance sensitivity of cells to diphtheria toxin through its association with the diphtheria toxin receptor. Although several studies indicate that treatment of specific hematopoietic cells with the tumor promoter, 1 2-O-tetradecanoylphorbol-1 3-acetate (TPA), induces CD9 expression, the mechanisms by which CD9 expression is regulated have not been elucidated. Here, we provide evidence that, in HL-60 cells, increases in the level of CD9 protein occur in parallel with TPAinduced differentiation. More than 80% of HL-6O cells exposed to 1 7 nM TPA become CD9 positive within 24 h. CD9 mRNA levels increase within 8-1 0 h after starting TPA treatment. Activation of CD9 transcription occurs during the same time period. Both transcriptional activation and accumulation of CD9 mRNA require protein synthesis. However, once CD9 mRNA has accumulated, inhibition of protein synthesis has no effect on its level or rate of turnover. These results suggest that CD9 expression in TPA-treated HL-60 cells is regulated at the transcriptional level and that activation of transcription occurs subsequent to the production of proteins induced as an immediate-early response to TPA. Since CD9 expression is not induced in HL-GOTR cells, which respond to TPA but are resistant to its differentiating effects, the results also indicate that CD9 expression may serve as a marker for monocyte/ macrophage differentiation of HI-GO cells. Introduction CD9 is a cell surface protein with an apparent molecular weight of 24,000. It is posttranslationally modified by glycosylation and palmitylation (1 , 2). CD9 was first identified as a cell surface protein using monoclonal antibody Ba-2, Received 6/3/94; accepted 8/18/94. 1 Supported by National Cancer Institute/NIH Grant CA50909 and the Lloyd and Marilyn Smith Fund. Oligomer primers used in this study were synthesized by the Molecular Oncology Core of the Meyer L. Prentis Comprehensive Cancer Center of Metropolitan Detroit, which is supported in part by National Cancer Institute Grant CA22453. 2 To whom requests for reprints should be addressed, at Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, NE 681 98-4525. which was produced against an acute lymphoblastic leukemia cell line (3). Analysis ofthe sequence of cloned CD9 cDNA3 (4, 5) indicated that it belongs to a superfamily of cell surface molecules expressed on various cell types including lymphocytes (CD37, CD53, and TAPA-i ; Refs. 6-8), melanoma cells (ME491 ; Ref. 9), intestinal cells (CO029; Ref. 10), activated human T cells, Epstein-Barr virustransformed or Burkitt B-lymphocyte lines (R2 or IA4; Refs. 1 1 and 1 2), and Schistosoma mansoni (Sm23; Ref. 1 3). This group of proteins is known as the “tetra spans transmembrane protein family” since all members contain four transmembrane spanning domains. Although the function of CD9 has not been fully elucidated, it appears to play a role in platelet activation. Incubation of human platelets with mAbs to CD9 results in their activation, stimulating aggregation (1 4-1 7), granule secretion (1 7, 1 8), thromboxane synthesis (1 6-i 8), and phosphorylation of myosin light chains (18). Binding of some anti-CD9 mAbs also increases Ca2 uptake and intracellular free calcium concentration (1 9-21 ). However, this effect may be mediated indirectly through the action of secreted ADP and thromboxane (21 ). It has also been suggested that association of CD9 with the diphtheria toxin receptor enhances the sensitivity of cells to diphtheria toxin (22). CD9 antigen is expressed in both hematopoietic (23, 24) and nonhematopoietic tissues and cell lines (25). CD9 expression is increased over a period of 4-6 days in small tonsilB cellsactivated in vitro with TPA by a process that is dependent on activation of protein kinase C (26). Other differentiating agents, such as cis-retinoic acid, do not induce production of CD9 in these cells. Similarly, B-cell chronic lymphocytic leukemia cells and a variety of hematopoietic cell lines of lymphoid and monocytoid lineages have been shown to express CD9 after exposure to high concentrations of TPA for 3 days (27). HL-60 cells, a line derived from a patient diagnosed with acute promyelocytic leukemia (28), do not express CD9 under normal growth conditions (25) or after differentiation to a granulocyte-like phenotype (27) but become CD9 positive after 3 days of treatment with 160 nM TPA (28). While these results clearly demonstrated increased levels of cell surface CD9 antigen in response to TPA treatment, they did not define the mechanisms by which CD9 antigen expression was regulated or the relationship between CD9 expression and differentiation. Since adherent monocyte/ macrophage-like cells can be detected in cultures of HL-60 cells within 4-6 h after treatment with 1 0-20 nM TPA and more than 80% of the cells differentiate within 24 h (29, 30), we reasoned that if CD9 expression was linked to monocyte/macrophage differentiation, it should either 3 The abbreviations used are: cDNA, complementary DNA; mAb, monoclonal antibody; TPA, 12-O-tetradecanoylphorbol-1 3-acetate; poly(A) , polyadenylated; PCR, polymerase chain reaction; CSF, colony-stimulating factor; PBS, phosphate-buffered saline. Results A 1 2 3 4 5 6 7