Hairy Cell Line Research Articles

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

AbstractHairy cell leukemia (HCL) is a chronic lymphoproliferative disease, the cause of which is unknown. Diagnostic of HCL is abnormal expression of the gene that encodes the β2 integrin CD11c. In order to determine the cause of CD11cgene expression in HCL theCD11c gene promoter was characterized. Transfection of theCD11c promoter linked to a luciferase reporter gene indicated that it is sufficient to direct expression in hairy cells. Mutation analysis demonstrated that of predominant importance to the activity of the CD11c promoter is its interaction with the activator protein-1 (AP-1) family of transcription factors. Comparison of nuclear extracts prepared from hairy cells with those prepared from other cell types indicated that hairy cells exhibit abnormal constitutive expression of an AP-1 complex containing JunD. Functional inhibition of AP-1 expressed by hairy cells reducedCD11c promoter activity by 80%. Inhibition of Ras, which represents an upstream activator of AP-1, also significantly inhibited the CD11c promoter. Furthermore, in the hairy cell line EH, inhibition of Ras signaling through mitogen-activated protein kinase/extracellular signal–regulated kinase kinases 1 and 2 (MEK1/2) reduced not only CD11c promoter activity but also reduced both CD11c surface expression and proliferation. Expression in nonhairy cells of a dominant-positive Ras mutant activated the CD11cpromoter to levels equivalent to those in hairy cells. Together, these data indicate that the abnormal expression of the CD11cgene characteristic of HCL is dependent upon activation of the proto-oncogenes ras and junD.

Expression of a functional inducible nitric oxide synthase in hairy cell leukaemia and ESKOL cell line.

The expression of nitric oxide synthase (NOS) isoforms was investigated in the established ESKOL hairy cell line and in leukemic cells of patients with hairy cell leukemia (HCL). By reverse transcription-polymerase chain reaction (RT-PCR), these cells were found to spontaneously express inducible NOS (iNOS)-specific mRNA, but not endothelial constitutive NOS (ecNOS) mRNA. The iNOS protein was detected by immunofluorescence in the cytoplasm of permeabilized leukemic cells and ESKOL cells, using different anti-iNOS monoclonal antibodies. A protein of 135 kDa was identified by Western blotting in ESKOL and HCL lysates, confirming the presence of an iNOS in these cells. Cytosolic homogenates displayed NOS catalytic activity, as measured by the conversion of 14C-labelled L-arginine into 14C L-citrulline and by detection in situ using the DAF-2DA (diaminofluorescein diacetate) NO-sensitive fluorescent probe. Ligation of CD23 (low affinity IgE receptor) was found to increase iNOS expression in ESKOL and conversely to decrease the percentage of cells undergoing apoptosis, as measured by the percentage of cells expressing annexin V. These results indicate that, as in chronic B cell lymphocytic leukemia cells (B-CLL) a functional iNOS is expressed constitutively in hairy cells that contributes to protecting these tumoral cells from apoptosis.

Phosphorylation of CD20 in cells from a hairy cell leukemia cell line. Evidence for involvement of calcium/calmodulin-dependent protein kinase II.

Hairy cell leukemia is an uncommon B cell lymphoproliferative disease of unknown etiology. We previously observed that CD20, a membrane protein involved in B cell activation, is hyperphosphorylated on hairy cells and that these cells have unusually high levels of intracellular free Ca2+. Therefore, we used a hairy cell line, HCLL-7876, to study the potential involvement of Ca(2+)-activated protein kinases in CD20 phosphorylation. Addition of the Ca2+ ionophore, ionomycin, increased CD20 phosphorylation both in activated B cells and in cells from the hairy cell line; addition of EGTA to either cell type decreased basal levels of CD20 phosphorylation. Ionomycin treatment of these cells resulted in increased kinase activity of cytosolic extracts toward syntide-2, a synthetic peptide substrate for calcium/calmodulin-dependent kinase II (CaM-KII), with kinetics similar to those of CD20 phosphorylation in the cell line. CD20 isolated from the cell line was a substrate for purified CaM-KII in vitro. Phosphopeptide maps of CD20 from untreated hairy cells or ionomycin-treated HCLL-7876 cells were similar to maps of CD20 that had been phosphorylated in vitro by CaM-KII. These results suggest that the unusually high levels of intracytoplasmic Ca2+ in hairy cells may enhance the phosphorylation of key surface proteins.

Hairy cell transformation of human peripheral blood lymphocytes by Coxiella burnetii.

Hairy cell (HC) transformation of human peripheral blood lymphocytes (PBL) by Coxiella burnetii was studied to clarify the significance of persistency of C. burnetii in a hairy cell line (designated "TOL"). TOL cells which exhibited HC characteristics in hairy cell leukemia (HCL) were persistently infected with C. burnetii. Two strains of C. burnetii, our isolate from TOL cells and the original isolate in 1935, the Nine Mile strain from American Type Culture Collection (ATCC, U.S.A), were inoculated to PBL cultures. HC transformation not only by our isolates (87%) but also by Nine Mile strain (100%) was demonstrated in an average of 20 days. The original observation that Coxiella induced HC transformation in vitro was also confirmed in experiments with PBL exposed to C. burnetii in vivo. Spontaneous development of HC were observed in cultures of PBL only from coxiellemic cases (12/24) but not from C. burnetii negative cases (0/57). All HC cell lines (34) as determined by their morphology and cytochemical markers of HC in HCL remained infected with C. burnetii invariably.

Cytochemical, cytogenetic, immunophenotypic and tumorigenic characterization of two hairy cell lines

Two cell lines (EH and HK) were derived from two patients with hairy cell leukemia (HCL). Both patients exhibited a clinical picture characteristic of HCL, including splenomegaly, cytopenias, and tartrate- resistant acid phosphatase (TRAP)-positive “hairy” lymphocytes in blood and marrow. EH and HK were demonstrably of B lineage, as judged by cytochemistry and immunophenotype, including expression of B1, B2, and LEU-12 antigens and of monoclonal surface immunoglobulins (slgs). Monoclonality was confirmed by clonal karyotype abnormality demonstrated in cell line HK. HCL parentage suggested by cytochemistry and electron microscopy was confirmed by the immunophenotypic observation that HCL lines expressed antigens alpha S-HCL1, alpha S- HCL3, and cCLLa. While alpha S-HCL1 and alpha S-HCL3 are nonspecific, their co-expression is characteristic of HCL cells. The cCLLa is a novel 69-kd membrane HCL-associated polypeptide antigen not shared by circulating normal T or B lymphocytes nor by malignant cells from unrelated lymphoid or nonlymphoid malignancies. The doubling time of EH and HK was 24 and 36 hours, respectively. While HK included a small subset of Epstein-Barr virus (EBV) nuclear antigen-positive cells, EH cells were homogeneously negative for the presence of this antigen. Both cell lines were consistently implantable in irradiation- preconditioned immunodeficient mice giving rise to primary tumors and widespread metastasis.

Cytochemical, Cytogenetic, Immunophenotypic and Tumorigenic Characterization of Two Hairy Cell Lines

Two cell lines (EH and HK) were derived from two patients with hairy cell leukemia (HCL). Both patients exhibited a clinical picture characteristic of HCL, including splenomegaly, cytopenias, and tartrate-resistant acid phosphatase (TRAP)-positive "hairy" lymphocytes in blood and marrow. EH and HK were demonstrably of B lineage, as judged by cytochemistry and immunophenotype, including expression of B1, B2, and LEU-12 antigens and of monoclonal surface immunoglobulins (slgs). Monoclonality was confirmed by clonal karyotype abnormality demonstrated in cell line HK. HCL parentage suggested by cytochemistry and electron microscopy was confirmed by the immunophenotypic observation that HCL lines expressed antigens alpha S-HCL1, alpha S-HCL3, and cCLLa. While alpha S-HCL1 and alpha S-HCL3 are nonspecific, their co-expression is characteristic of HCL cells. The cCLLa is a novel 69-kd membrane HCL-associated polypeptide antigen not shared by circulating normal T or B lymphocytes nor by malignant cells from unrelated lymphoid or nonlymphoid malignancies. The doubling time of EH and HK was 24 and 36 hours, respectively. While HK included a small subset of Epstein-Barr virus (EBV) nuclear antigen-positive cells, EH cells were homogeneously negative for the presence of this antigen. Both cell lines were consistently implantable in irradiation-preconditioned immunodeficient mice giving rise to primary tumors and widespread metastasis.

RAB-1: A new monoclonal antibody to leukemic hairy cells

RAB-1, a new monoclonal antibody (McAb) to human leukemic hairy cell (HC) was produced. Using indirect immunofluorescence methods and microscopic or flow cytometric analysis, it was found that the RAB-1 antigen was expressed on few resting B cells and not on resting T lymphocytes, platelets, monocytes, erythroid and myeloid cells. RAB-1 expression on malignant cells was as follows: strongly positive in 15/15 hairy cell leukemia (HCL), negative with non-T and T-acute lymphoblastic leukemia and T-chronic lymphocytic leukemia (CLL); weakly expressed on myeloma and Waldenstrom cells; moderately on 10–25% of the cells in 4/10 B-CLL and 6/10 B lymphomas and in 7/7 B-prolymphocytic leukemia (PLL). Amongst human cell lines that were tested, RAB-1 reacted strongly with one HC line, moderately with the EBV-lymphoblastoid Daudi and Raji Burkitt's lines and was not expressed on Ramos, ALL, myeloid and erythroid cell lines. Normal B cells activated with PWM or anti-μ beads, and malignant B cells activated with anti-μ and TPA did not show an increase of expression of RAB-1 antigen. Interestingly, 30–40% of T4-Class II antigen positive cloned cells and T cells activated with PHA and Con.A expressed RAB-1, suggesting that this McAb recognizes surface molecule, newly induced during T-cell activation and constitutively expressed on HC and some B-cell malignancies.

Establishment and characterization of an Epstein-Barr virus nuclear antigen-negative hairy cell line.

Continuous cell lines have been established from spleen cells of patients with confirmed hairy cell leukemia (HCL). One cell line, HCL-Z1, lacks Epstein-Barr virus nuclear antigen (EBNA), grows attached to the substratum and retains typical features of hairy cells as revealed by transmission and scanning electron microscopy. HCL-Z1 differs morphologically from the three other EBNA-positive lymphoblastoid cell lines obtained (HCL-Z2, HCL-Z3, HCL-Z4) as well as from normal spleen cells or lymphocytes. The three lymphoblastoid cell lines derived from HCL patients show similar surface features as a line from a myeloma patient. Therefore, not all cell lines derived from HCL patients may be considered as representative of the patients leukemia cells.

Immunologic Characterization of Hairy Cell Leukemias in Continuous Culture

The immunobiologic characteristics of three continuous cell lines established from hairy cell leukemia cells were investigated. All three cell lines continued to produce tartrate-resistant acid phosphatase, the enzymatic marker of hairy cells. Two of these cell lines were B lymphoid in nature. They carried Fc and C receptors, had surface and internal immunoglobulin, and did not form spontaneous sheep red blood cell rosettes. Experiments employing biosynthetic radiolabeling of immunoglobulin demonstrated distinctive immunoglobulin kinetics for each of these two hairy cell lines. One cell line remained quite similar to the original hairy cells from which it was derived whereas the other B lymphoid hairy cell line had undergone a switch in the immunoglobulin isotype produced. The third hairy cell leukemia line was shown to be of thymic derivation. These cells formed spontaneous sheep red blood cell rosettes and did not carry Fc or C receptors. The spontaneous sheep red blood cell rosette-forming cells contained tartrate-resistant acid phosphatase. They did not possess surface on internal immunoglobulin and did not synthesize immunoglobulin in vitro. Hairy cell leukemia cells maintained in permanent cell culture retain their immunobiologic properties and offer the opportunity for indepth study of these unusual cells.