Establishment of three permanent human leukaemia cell lines producing immunoreactive calcitonin

Abstract

Three permanent human leukaemia cell lines, designated EW2, LG3 and MS6, were established from bone marrow aspirates of a patient with acute myelomonocytic leukaemia (EW2), acute monocytic leukaemia (LG3) and acute myeloblastic leukaemia (MS 6). In vitro all lines exhibited a myelomonocytoid marker profile in terms of classical staining reactions and cell-surface antigen structure. In supernatants and extracts of EW2 and MS 6 cells immunoreactive human calcitonin was found in raised levels. No significant levels of immunoreactive human calcitonin were found in supernatants of LG3 cells, while extracts of LG3 cells and the patients sera at diagnosis contained high levels, suggesting the in vitro selection of a non-secreting clone of leukaemic cells. Gel-chromatography showed several peaks of ihCT with higher molecular weight than normal human calcitonin, suggesting the production of precursor molecules. In addition to ihCT raised levels of immunoreactive calcitonin gene related peptide (ICGRP) was found in supernatants of EW2. These data support the concept of ectopic immunoreactive human calcitonin production by leukaemic cells.

Peptide hormones in patients with acute leukaemia

“…Results of previous studies [23] demonstrated that immunoreactive h-CT in sera and cell extracts of individual patients exhibited molecular weights ranging from 3500 to 60000 daltons. The normal low molecular weight fraction (3500 d) was found beside high molecular h-CT only in sera of patients, whereas in cell extracts of leukaemia blast from patients [23] and leukaemic cell lines [16], only high molecular weight forms of immunoreactive CT were present. These findings, and the in-vitro production of h-CT by leukaemic cells, strongly suggest that elevated serum levels of h-CT in patients with leukaemia originate from the leukaemic cells rather than from other sources including non-malignant and physiological states.…”

“…The present investigation shows no significant coincidence of these two hormones. Of 52 patients, only 16 exhibited elevated levels of both peptides, 14 had normal levels for both hormones and 21 showed high serum levels of one compound only. This distribution indicates that the pathway of alternative RNA splicing also occurs in leukaemic blasts producing CT gene-related peptides 'ectopically'.…”

“…Three cell lines (EW 2, LG 3, and MS 6) established in our laboratory were maintained in serum-free medium (RPMI 1640supplemented with selenium, insulin, and transferrin) [16]. The cell lines were characterized by means of cytochemical reactions, functional tests and by immunological detection of surface antigens [16]. In addition, the established cell lines HL-60, KG-l and K-562 were used.…”

Bombesin‐like immunoreactivity in eosinophils. A light and electron microscopic study: effect of fixatives and cross‐reactivity with various compounds

“…As for binding sites, the storage and/or synthesis of neuropeptide(s) seem(s) to occur mostly in iymphocytes but, to our knowledge, nothing has been described for substance P or bombesin in contrast with descriptions for ACTH [19,48,62,69,70], preproenkephalin [87] or calcitonin [35]. On the other hand, bombesin was identified in alveolar macrophages by HPLC, RIA and immunofluorescence without any cross-reactivity with substance P [85].…”