Abstract

In vitro growth of acute myelogenous leukemia blasts as an established cell line is an uncommon event and molecular mechanisms allowing permanent in vitro proliferation have not been defined. In particular, few acute monoblastic leukemia (AMoL) cell lines have been described. Here we report a new AMoL cell line expressing a combination of growth factors and growth factor receptor that might be involved in an autocrine stimulation loop. AMoL (M5a according to F.A.B) was diagnosed in November 2004 in a 74 old woman. Caryotypic analysis revealed a combination of 46,XX, 47,XX,+8, 47,XX,+8,6q− caryotypes. A complete remission was achieved by an age- adapted FLAG-Ida regimen but caryotype analysis still revealed about 50% mitosis with 47,XX,+8 and in February 2005 the patient presented an isolated cutaneous relapse. In spite of treatment with low dose cytarabine, etoposide and retinoids, hematologic progression occurred in May 2005. Further treatments with vinblastine, hydroxyurea and gentuzumab-ozogamycine were only temporary effective and in June 2005 the patient died, with hyperleukocytosis, extensive cutaneous and possibly CNS involvement. A 10 ml peripheral blood sample was taken, after informed consent, at the time of the last progression and mononuclear cells were collected. Part the cells were cryopreserved and part cultured in RPMI medium + 15% fetal bovine serum (FBS). Cultured cells survived without significant expansion for 4 weeks, then progressive growth started. After a few passage the cell line (named PI-BR) has been established, with a doubling time of approximately 48 hours. During early passages cell growth was density dependent, impaired at concentrations lower than 3 ×10e4/ml. Morphology and cytochemical stains were in agreement to M5a F.A.B. subtype. Immunophenotypic analysis revealed an antigenic expression comparable to that of patient’s blasts: CD13++, CD33++, HLA-DR ++, CD64++, CD117 +, CD15+/−, CD34−, CD19−, CD9−. Caryotypic analysis was performed on patient’s cells cryopreserved when culture was initiated: only 4 mitosis were found: 2 with normal 46,XX, 2 with 47,XX,+8 caryotype. Caryotypic analysis, repeated on PI-BR cell line at passage 9, revealed a mixture of mitosis with 47,XX,+8, 47,XX,+ ring, 48,XX,+8,+ ring, normal 46,XX and hypodiploid (42–44 chromosome) caryotype. Therefore, although some chromosome abnormalities were acquired during in vitro culture, they do not seem responsible for the capability of continuous in vitro growth, since about 55% of cells at passage 9 maintained the same caryotypes of patient’s cells in vivo. Real time PCR revealed expression of genes codifying for some monocytic cytokines (IL6, IL8, IL10, IL18, TGF-beta1), Vascular Endotelium Growth Factor (VEGF)-A, VEGF-D and its receptor Flt-4. Immunoistochemistry confirmed the synthesis of Flt-4, VEGF-A and VEGF-D. VEGF-D was also detected by ELISA assay in cell line supernatant (14.7 pg/ml) at early passages. VEGF A and C and VEGF receptors have been reported to be expressed in some AML cell lines and in some fresh AML cell samples, displaying anti-apoptotic and proliferative activity. In the case of PI-BR cell line, synthesis of VEGF-D and of its receptor Flt-4 may have established an autocrine loop involved in continuous cell growth.

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