CD11c gene expression in hairy cell leukemia is dependent upon activation of the proto-oncogenes ras andjunD

Abstract

IntroductionHairy cell leukemia (HCL) or leukemic reticuloendotheliosis represents approximately 2% of adult leukemias and is characterized by pancytopenia, hepatomegaly, splenomegaly, leukocytosis, and neoplastic mononuclear cells in the peripheral blood, bone marrow, liver, and spleen. 1 The name of the disease is derived from the presence of broad-based undulating ruffles on the surface of the leukemic cells that appear under the phase contrast microscope as cytoplasmic projections or "hairs." These cells can be derived both from B and T lymphocytes as demonstrated by their expression of B-or T-cell-specific antigens and are characterized biochemically by their abnormal expression of the integrin heterodimer CD11c/ CD18. [2][3][4] Under normal circumstances the gene encoding the CD18 component of this marker is transcribed both in lymphocytes and myeloid cells, whereas the gene encoding the CD11c component is transcribed primarily in cells of the myeloid lineage. 5 The CD11c/ CD18 heterodimer is, therefore, normally largely restricted in its expression to the surface of myeloid cells dictated by the myeloidspecific transcription of the CD11c gene. In hairy cell leukemia the CD11c/CD18 heterodimer is present on not only myeloid cells but also on the neoplastic lymphocytes. Diagnostic of this disease is, therefore, the abnormal regulation of CD11c gene transcription. Consequently, elucidation of the molecular causes of this abnormal regulation are likely to result in insights into the molecular basis of hairy cell leukemia. Using this rationale we isolated the human CD11c gene and identified the cis-acting elements that control its transcription. The most important of these elements interacts with activator protein-1 (AP-1) encoded by the jun and fos families of proto-oncogenes. In hairy cells an AP-1 complex containing JunD exhibits a constitutive pattern of expression, whereas in other cell types it is functionally expressed only upon induction with phorbol ester. The use of dominant-negative mutants in transfection assays demonstrated that both AP-1 and its upstream activator Ras are necessary for CD11c expression in hairy cells. In addition, exogenous expression of a dominant-positive mutant of Ras was able to activate the CD11c promoter in nonhairy cells to a level equivalent to that seen in hairy cells. Taken together, these results indicate that activation of the proto-oncogenes junD and ras underlie the abnormal expression of the CD11c gene characteristic of HCL. Materials and methods Cell cultureThe cell lines HeLa, IM-9, Mo, and U937 were obtained from the American Type Culture Collection (Manassas, VA) and grown according to their specifications. The promegakaryocytic cell line MEG-01 6 was kindly provided by Dr W. S. May (John Hopkins Oncology Center, Baltimore, MD) through permission of Dr H. Saito (Nagoya University School of Medicine, Nagoya, Japan). MEG-01 were cultured in RPMI 1640 medium supplemented with glutamine, 20% fetal calf serum (FCS), aqueous penicillin G (100 U/mL), and streptomycin ...

Hypoxia: The Force that Drives Chronic Kidney Disease

“…[366][367][368][369][370][371] Using transfection analyses and phorbol myristate acetate, it was established for each β2-integrin gene that the proximal promoter region is sufficient to drive a pattern of inducible expression in vitro which mimics that of the endogenous gene in vivo. 360 In subsequent studies it was determined that GA-binding proteins alpha and beta mediate induction of the CD18 gene, 369 activator protein 1, specificity protein 1 and Purα mediate induction of the CD11c gene, 368,371 myeloid specificity 2 and Purα mediate induction of the CD11a and CD11b genes, 262,370 and Purα mediates induction of the CD11d gene. 262 Despite β2-integrin expression being regulated at the transcriptional level, accepted dogma held that changes in β2-integrin expression relevant to leukocyte function occur almost exclusively after protein translation.…”

Underexpression of RhoH in Hairy Cell Leukemia

“…Previous studies have shown that the Ras family of proto-oncogenes is abnormally active in HCL (13,25). Because RhoH represents a natural inhibitor of Ras in the hematopoietic lineage, we investigated the possibility that HCL may be characterized by RhoH underexpression.…”

“…PCR was used to generate a fragment of the CD11c gene representing nucleotides À128 to +36 relative to the major transcription initiation site. This fragment was then subcloned into the ''filled-in'' HindIII site of pATLuc to generate p11c-Wt (13,44). The ability of RhoH to repress the CD11c promoter was assessed using the expression constructs pCMV-RhoH and pMEP-RhoH.…”

“…Critical to the expression of the CD11c gene is its interaction with the transcription factor activator protein-1 (AP-1) (13). Normally, AP-1 expression is transient and mediates induction of CD11c in response to specific differentiation signals (14).…”

“…Normally, AP-1 expression is transient and mediates induction of CD11c in response to specific differentiation signals (14). However, in the neoplastic lymphocytes of HCL, AP-1 is expressed constitutively, thus driving abnormal concomitant expression of CD11c (13). One of the most important mechanisms controlling AP-1 expression is protein phosphorylation regulated by the Ras family of proto-oncogenes (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25).…”