Robyn D. Clutterbuck

A Comparison of the Effect of the 3-Hydroxy-3-Methylglutaryl Coenzyme A (HMG-CoA) Reductase Inhibitors Simvastatin, Lovastatin and Pravastatin on Leukaemic and Normal Bone Marrow Progenitors

Simvastatin is an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and also selectively inhibits the growth of leukaemic progenitor cells. The antileukaemic action of simvastatin was compared in vitro with that of lovastatin and pravastatin, chemically related compounds which are also competitive inhibitors of HMG-CoA reductase. After 18 hours incubation with 2.5-20 microM of inhibitor, no effect was observed by any of the compounds on the subsequent clonogenic growth of normal bone marrow (BM) progenitor cells from 4 donors and BM cells from one patient in remission. However, simvastatin and lovastatin produced inhibition of acute myeloid leukaemia (AML) progenitor cell growth of between 25% and 100% in 5 populations tested (4 primary AMLs and the HL60 cell line). Pravastatin showed similar growth inhibitory effects to simvastatin and lovastatin in 2 out of 3 primary AMLs but was less active against one primary AML cell population and HL60 cells. These results indicate that, in addition to simvastatin, lovastatin and pravastatin are also selective inhibitors of leukaemic cell growth, however simvastatin was chosen for clinical trial in patients with leukaemia.

Inhibitory effect of simvastatin on the proliferation of human myeloid leukaemia cells in severe combined immunodeficient (SCID) mice

SCID mice were inoculated intravenously with cells from the human HL60 myeloblastic leukaemia cell line and then treated with the 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase inhibitor, simvastatin, by subcutaneous continuous infusion. The effect of the drug was measured by subsequent colony formation of surviving HL60 cells in vitro and flow cytometry. The number of clonogenic HL60 cells was reduced in the bone marrow of mice that received simvastatin compared with control mice by 65% and 68% in two separate experiments. The number of clonogenic, normal, murine, bone marrow progenitor cells concomitantly exposed to simvastatin in vivo, was not affected in either experiment. Flow cytometric analysis of bone marrow and spleen cells confirmed these results by showing that simvastatin had reduced the percentage of human leukaemia cells in these tissues by 70% and 88% respectively.  The data show that the reported selective effect of simvastatin against acute myeloid leukaemia cells in vitro, can be extended to this in vivo model. HL60 bears an N‐ras mutation. In further in vitro studies, ketoconazole, an inhibitor of cholesterol biosynthesis post farnesyl pyrophosphate synthesis, had a similar effect to simvastatin on HL60 colony development. Furthermore, the clonogenicity of a population of N‐ras mutated, primary acute myeloid leukaemia (AML) cells was no more sensitive to simvastatin than a population without the mutation. The data suggest that the inhibition of AML cell proliferation by simvastatin may be independent of the RAS signalling pathway.

Studies on the development of human acute myeloid leukaemia xenografts in immune-deprived mice: comparison with cells in short-term culture.

Human AML cells from the blood of a series of patients have been implanted subcutaneously into mice immune-suppressed by thymectomy and total-body irradiation. Solid tumours resulted from 18 out of 19 samples and their growth was compared with the proliferation of AML cells in culture. In 17 cases tumours grew to a maximum size and then spontaneously regressed. Cells from one patient produced tumours which did not regress and could be retransplanted into freshly immune-suppressed mice. Cells from a human promyelocytic cell line (HL60) also produced nonregressing and retransplantantable tumours. Normal human mononuclear bone marrow cells implanted s.c. produced a growth pattern similar to that of the majority of AML cells. A second inoculum of AML cells into animals with regressing tumours also produced tumours and thus regression cannot be accounted for on the basis of returning immunity. AML cells placed into short-term suspension culture invariably matured to monocyte/macrophage type cells and/or granulocytic cells as identified by cytochemical staining. However, no correlation was observed between proliferation or maturation of cells in culture, and tumour growth in vivo. Cells derived from disaggregated AML tumours also showed evidence of myeloid differentiation suggesting that tumour regression is due to maturation of leukaemic cells.

Growth of Primary Human Acute Lymphoblastic and Myeloblastic Leukemia in SCID Mice

The in vivo growth of 8 human primary acute lymphoblastic (ALL) and 17 primary acute myeloblastic (AML) cell populations was investigated in severe combined immunodeficient (SCID) mice. Bone marrow (BM) or peripheral blood (PB) samples, either fresh or cryopreserved, were implanted i.v. into irradiated SCID mice. The cells from 5/8 patients with ALL resulted in engraftment with systemic proliferation and dissemination leading to morbidity and mortality of the animals within 12-18 weeks from implantation. In contrast, none of the 17 AML samples resulted in sustained engraftment. Four of the 5 engrafted ALL populations have been successfully passaged into fresh recipients and phenotypic and karyotypic characteristics remain unaltered. The data presented indicate that the SCID mouse may be a useful model for studying the pathogenesis of ALL and may also enable the development and investigation of new therapies for this disease.

Interleukin-6 and other gp130-dependent cytokines selectively inhibit proliferation of macrophage-lineage hemopoietic progenitor cells

OBJECTIVE Hemopoiesis is regulated by cytokines with positive or negative effects on proliferation of lineage-committed or multipotent hemopoietic stem cells. We have investigated the roles of interleukin-6 and other gp130-dependent ligands on the proliferation of macrophage-lineage hemopoietic progenitor cells. METHODS The responses of human and murine hemopoietic cells to combinations of cytokines involving interleukin-6 or related factors were assessed in short-term culture by clonogenic assay. RESULTS Interleukin-6, leukemia inhibitory factor, and ciliary neurotrophic factor inhibited formation of colonies stimulated by macrophage colony-stimulating factor. These effects were dose dependent and selective for macrophage-lineage precursors. Progenitors from murine peripheral blood were inhibited by 37-93% in cultures containing interleukin-6 (11 experiments; median, 68%). Macrophage progenitors from murine bone marrow were also inhibited by interleukin-6 but were less sensitive (seven experiments; median, 48%). In cultures costimulated with leukemia inhibitory factor, peripheral blood and bone marrow progenitors were inhibited by 82% and 58%, respectively. Ciliary neurotrophic factor inhibited macrophage colonies by 66%. Multilineage bone marrow colony formation was not affected. In cultures of human bone marrow cells stimulated with macrophage colony stimulating factor and stem cell factor, interleukin-6 inhibited colony formation by 51-74%. Bone marrow colonies stimulated by granulocyte-macrophage colony stimulating factor were not inhibited by costimulation with interleukin-6. CONCLUSIONS These results suggest a novel mechanism for the negative regulation of macrophage-lineage hemopoietic cells. They also demonstrate new properties of interleukin-6 and certain other gp 130-dependent ligands.

Comparison of the Growth of Xenografted Human Bone Marrow with the Growth of Xenografted Acute Myeloid Leukemia Cells and Marrow Repopulating Capacity Following Transplantation into Allogeneic Recipients

Blast cells from the peripheral blood of patients with acute myeloid leukemia (AML) have been shown to be capable of forming discrete subcutaneous tumours when inoculated into immune-deprived mice [2]. The proliferative potential of these tumours is limited and in the great majority of cases, regression ensues. One possible cause for tumour regression is the maturation of blast cells, producing a cell population with an abolished capacity for proliferation [7,1]. This process is to a certain extent analogous to the cell kinetics of normal haemopoiesis. It was considered that normal human bone marrow cells might therefore also undergo a phase of proliferation when implanted into immune-deprived mice.