Macrophage Inflammatory Protein-1α Mediated Growth Inhibition in a Haemopoietic Stem Cell Line is Associated with Inositol 1,4,5 Trisphosphate Generation

Abstract

Macrophage Inflammatory Protein-1 alpha (MIP-1 alpha) can inhibit the proliferation of multipotent haemopoietic cells. Using the FDCP-Mix A4 multipotent stem cell line, MIP-1 alpha was shown to inhibit 1L-3 stimulated cell cycling (assessed using the [3H]-thymidine "suicide" assay). Furthermore, MIP-1 alpha can inhibit 1L-3-stimulated [3H]-thymidine incorporation in FDCP-Mix cells, with half maximal inhibition observed at 3 ng/ml MIP-1 alpha. Prostaglandin E2, but not MIP-1 alpha was able to elevate cyclic AMP levels in FDCP-Mix A4 cells although both agents can cause growth inhibition. However, MIP-1 alpha addition resulted in a pertussis-toxin-insensitive increase in the level of the second messenger inositol 1,4,5 triphosphate (Ins 1,4,5P3). This response was both rapid (maximal at 5 seconds) and transient. A half maximal effect was observed at 5 ng/ml MIP-1 alpha and the dose dependency correlated with that for MIP-1 alpha mediated growth inhibition. A rapid increase in cytosolic Ca2+ levels was also observed in response to MIP-1 alpha. Inositol lipid hydrolysis and an increase in cytosolic Ca2+ (signals normally associated with proliferation) may therefore be implicated in growth inhibitory mechanisms in multipotent cells.

Transforming Growth Factor-β1 Induces Apoptosis Independently of p53 and Selectively Reduces Expression of Bcl-2 in Multipotent Hematopoietic Cells

“…FDCP-Mix cells are karyotypically normal and have previously been shown to be responsive to the growth inhibitory effects of MIP-1␣ (40). We report that TGF-␤1 is able to reversibly inhibit the growth of FDCP-Mix cells cultured in high concentrations IL-3 that promote proliferation, while in lower IL-3 concentrations TGF-␤1 induces apoptosis independent of p53, and this is inhibited by Bcl-2 expression.…”

Abl protein kinase abrogates the response of multipotent haemopoietic cells to the growth inhibitor macrophage inflammatory protein-1 alpha

“…We have expressed a temperature sensitive abl PTK gene in a growth factor dependent, multipotential stem cell line (FDCP-Mix) (Spooncer et al, 1986) in which growth is normally suppressed by MIP-1a (Heyworth et al, 1995). These ts-abl FDCP-Mix show a marked increase in the levels of protein tyrosine phosphorylation when grown at the permissive (338C) compared with the non-permissive (38.58C) temperatures although the abl PTK protein is expressed at the same levels at both these temperatures (Spooncer et al, 1994): the tyrosine kinase activity and not the protein levels are in¯uenced by the temperature shift (Spooncer et al, 1986).…”

“…Cells were washed and incubated with 10 ng/ml IL-3 and the appropriate concentration of MIP-1a for 2 h. Cells were then pulsed either with Iscoves, 1 mg/ml cytosine arabinofuranoside (Ara-c) or 5 mg/ml 5-¯uorouracil (5-FU) or high speci®c activity [ 3 H]thymidine at 20 mCi/ml ([Ca 2+ ] i ) was determined using fura-2 Ca 2+ indicator using the dual wavelength spectro¯uorometric method. Cells were loaded with fura-2-acetoxymethylester (5 mM for 30 min at either 38.58C or 338C, then washed and taken for [Ca 2+ ] i measurements as previously described (Heyworth et al, 1995) at either 38.58C or 338C.…”

“…Thus, a possible explanation for the aberrant response to MIP-1a is that this occurs as a consequence of constitutive activation of abl PTK activity. To test this hypothesis and to`model' the e ects of a disregulated abl protein tyrosine on primitive haemopoietic cells, we have expressed a temperature sensitive abl PTK gene in the IL-3 dependent stem cell line, FDCP-Mix, in which growth is suppressed by MIP-1a (Heyworth et al, 1995). We found that expression of the abl PTK activity did not lead to growth autonomy nor to a block in the cells undergoing myeloid development.…”

Retroviral transfer and expression of human MDR-1 in a murine haemopoietic stem cell line does not alter factor dependence, growth or differentiation characteristics

“…15 As well as responding to growth stimulatory growth factors this cell line will respond to negative regulators of haemopoiesis in an appropriate manner. [16][17][18] These characteristics make this cell line a suitable model for the investigation of the effects of expression of exogenous MDR-1 on the survival and myeloproliferation of haemopoietic progenitor cells.…”