ALBERT

Description: The stem cell inhibitor, macrophage inflammatory protein-1 alpha (MIP-1 alpha) or LD78, protects multipotent hematopoietic progenitors in murine models from the cytotoxic effects of chemotherapy. Clinical use of human MIP-1 alpha during chemotherapy could therefore lead to faster hematologic recovery and may allow dose intensification. We have also shown that human MIP-1 alpha causes the rapid mobilization of hematopoietic cells, suggesting an additional clinical use in peripheral blood stem cell transplantation. However, the clinical evaluation of human MIP-1 alpha is complicated by its tendency to associate and form high molecular weight polymers. We have produced a variant of rhMIP-1 alpha, BB-10010, carrying a single amino acid substitution of Asp26 〉 Ala, with a reduced tendency to form large polymers at physiologic pH and ionic strength. This greatly increases its solubility, facilitating its production and clinical formulation. We confirmed the potency of BB-10010 as a human MIP-1 alpha-like agonist in receptor binding, calcium mobilization, inhibition of colony formation, and thymidine suicide assays. The myeloprotective activity of BB-10010 was shown in a murine model of repeated chemotherapy using hydroxyurea. BB-10010 is therefore an ideal variant with which to evaluate the therapeutic potential of recombinant human MIP-1 alpha.

Description: Long-term culture-initiating cells (LTC-IC) are arguably the most primitive human hematopoietic cells detectable by in vitro functional assays. We have investigated the mobilization of these cells into the blood of patients with ovarian carcinoma randomized to receive granulocyte colony-stimulating factor (G-CSF; 5 micrograms/kg) plus different doses of stem cell factor (SCF; c-kit ligand) after chemotherapy or G-CSF alone after chemotherapy. We have shown a significant SCF dose response for the mobilization of LTC-IC, with a 5.8-fold increase in LTC-IC mobilization in those patients receiving chemotherapy, G-CSF, and 20 micrograms/kg of SCF, the highest dose used, compared with the patients receiving chemotherapy and G-CSF alone. We have shown a threefold increase in CD34+ cells and up to a 64-fold increase in CD34+/33- cells was seen in patients treated with chemotherapy, G-CSF, and 20 micrograms/kg of SCF compared with those patients treated with chemotherapy and G-CSF alone. However, significant numbers of CD34+/38- cells were only found in the patients receiving 20 micrograms/kg of SCF as part of their mobilization regimen. Patients receiving chemotherapy plus G-CSF and SCF have enhanced mobilization of primitive cells and of the more committed progenitor cells compared with those patients receiving chemotherapy followed by G-CSF alone.

Description: BB-10010 is a variant of the human form of macrophage inflammatory protein-1 (MIP-1), which has been shown in mice to block the entry of hematopoietic stem cells into S-phase and to increase their self-renewal capacity during recovery from cytotoxic damage. Its use may constitute a novel approach for protecting the quality of the stem cell population and its capacity to regenerate after periods of cytotoxic treatment. Thirty patients with locally advanced or metastatic breast cancer were entered into the first randomized, parallel group controlled phase II study. This was designed to evaluate the potential myeloprotective effects of a 7-day regimen of BB-10010 administered to patients receiving six cycles of 5-fluorouracil (5-FU), adriamycin, and cyclophosphamide (FAC) chemotherapy. Patients were randomized, 10 receiving 100 μg/kg BB-10010, 11 receiving 30 μg/kg BB-10010, and nine control patients receiving no BB-10010. BB-10010 was well-tolerated in all patients with no severe adverse events related to the drug. Episodes of febrile neutropenia complicated only 4% of the treatment cycles and there was no difference in incidence between the treated and nontreated groups. Studies to assess the generation of progenitor cells in long-term bone marrow cultures were performed immediately preceding chemotherapy and at the end of six dosing cycles in 18 patients. Circulating neutrophils, platelets, CD 34+ cells, and granulocyte/macrophage colony-forming cell (GM-CFC) levels were determined at serial time points in cycles 1, 3, and 6. The results showed similar hemoglobin and platelet kinetics in all three groups. On completion of the six treatment cycles, the average pretreatment neutrophil levels were reduced from 5.3 to 1.7 × 109/L in the control patients and from 4.3 to 1.9 and 4.5 to 2.5 × 109/L in the 30/100 μg/kg BB-10010 groups, respectively. Relative to their pretreatment values, 50% of the patients receiving BB-10010 completed the treatment with neutrophil values significantly higher than any of the controls (P = .02). Mobilization of GM-CFC was enhanced by BB-10010 with an additional fivefold increase over that generated by chemotherapy alone, giving a maximal 25-fold increase over pretreatment values. Bone marrow progenitor assays before and after this standard regimen of chemotherapy indicated little long-term cumulative impairment to recovery from chemotherapy. Despite the limited cumulative damage to the bone marrow, which may have minimized the protective value of BB-10010 during this regimen of chemotherapy, better recovery of neutrophils in the later treatment cycles with BB-10010 was indicated in a number of patients. © 1998 by The American Society of Hematology.

Description: The effects of direct activators of protein kinase C (PKC) (the phorbol ester tetradecanoyl phorbol myristic acid [TPA] or bryostatin) on the ability of a highly enriched population of granulocyte-macrophage colony-forming cells (GM-CFC) to proliferate and develop in soft agar was assessed. In the absence of colony stimulating factors, the PKC activators did not stimulate colony formation. However, in the presence of optimal concentrations of granulocyte colony-stimulating factor (G- CSF) or interleukin-6 (IL-6), TPA or bryostatin markedly elevated the number of colonies formed from the GM-CFC. In the absence of TPA, IL-6, and G-CSF, respectively, both stimulated the formation of about 3% of the colonies observed when IL-3 was present. When TPA plus G-CSF or IL- 6 were added together, this figure increased to 48% and 54%, respectively. In both instances, the types of mature cells formed was altered from colonies of mature neutrophilic cells to a mixture consisting predominantly of macrophages with some neutrophils. Similar results were observed when bryostatin replaced TPA in these assays. When single cell colony-forming assays were performed, the same results were obtained. The presence of G-CSF, or IL-6, and the activator of PKC used (TPA or bryostatin) was required throughout the colony-forming assay for an optimal synergistic effect to be observed. These data indicate that agents that activate PKC can promote the proliferation and development of GM-CFC via a synergistic interaction with G-CSF or IL-6. Furthermore, there is an apparent role for PKC in development and possibly lineage commitment of GM-CFC.

Description: To examine the potential role of stem-cell factor (SCF) in cancer chemotherapy, we have administered it to mice either before or after 5- fluorouracil (5-FU). When polyethylene glycolated (PEG-ylated) SCF was administered to mice before 5-FU, it had a significant sensitizing effect on primitive bone marrow cells. Examination of the hematopoietic status of these mice showed that the damage caused by 5-FU to both bone marrow and spleen hematopoiesis was exaggerated when it was preceded by SCF. SCF given before each of two 5-FU treatments at 7-day intervals resulted in the death of all treated mice. The time of death and hematopoietic status of these animals are compatible with the onset of hypoplastic marrow failure leading to pancytopenia and death. SCF given after 5-FU had little impact either on the initial degree of hematopoietic damage or subsequent recovery. Gut populations were similarly sensitized to 5-FU by prior treatment with SCF, and the damage caused to intestinal populations was greater than that resulting from 5-FU alone. This indicates that the different tissues may be similarly sensitized by SCF. The sensitizing effect of SCF was reversed by concurrent administration of transforming growth factor (TGF)-beta 3, and survival of the majority of the mice was ensured. Examination of hematopoiesis in mice treated concurrently with SCF and TGF-beta 3 showed that the degree of marrow and spleen damage had reverted to that caused by 5-FU alone. In further experiments, 100% survival and normal hematopoiesis could be attained by transplantation of 1 million syngeneic bone marrow cells 24 hours after 5-FU treatment following SCF sensitization. These data indicate that PEG-ylated SCF can sensitize normally resistant hematopoietic and gut stem cells to the effects of 5- FU. This sensitization resulted in effective eradication of hematopoiesis in SCF-pretreated/5-FU-treated animals and their subsequent death from marrow failure. These findings imply that SCF pretreatment may represent a novel method of increasing the effectiveness of conventional chemotherapy, making marrow ablation more effective without drug dose escalation and perhaps sensitizing some tumor cells to the effects of therapy.

Description: We have identified a gene that has a high level of mRNA expression in undifferentiated, multipotential hematopoietic cells (FDCP-Mix) and that downregulates both transcript and protein, as these cells are induced to differentiate into mature myeloid cells. Sequence analysis of this gene has identified it as a serine protease inhibitor EB22/3 (serpin 2A). Constitutive expression of serpin 2A in FDCP-Mix cells was associated with an increase in the clonogenic potential of the cells and with a delay in the appearance of fully mature cells in cultures undergoing granulocyte macrophage differentiation when compared with control cells. Serpin 2A was also found to be expressed in bone marrow-derived bipotent granulocyte macrophage progenitor cells (GM-colony forming cell [CFC]), but not in erythrocyte progenitor cells from day 15 fetal liver. Expression of serpin 2A also showed a marked up regulation during the activation of cytotoxic suppressor CD8+ T cells, with a clear lag between the appearance of transcript and detection of protein.

Description: A human O6-alkylguanine-DNA-alkyltransferase (ATase) cDNA-containing retrovirus was used to infect murine long-term primary bone marrow cultures. High levels of ATase expression were obtained, and colony- forming cells of the granulocyte-macrophage lineage from the cultures transduced with the human ATase retrovirus were three times more resistant to the alkylating agent, N-methyl-N-nitrosourea (MNU), than control cultures. Furthermore, expression of the human ATase protected long-term hematopoiesis, measured as the output of progenitor cells to the nonadherent fraction of the culture, against the cytotoxic effects of repeated exposures to MNU. These results clearly show that a human ATase cDNA-containing retrovirus can be used to infect long-term primary bone marrow cultures and that this attenuates their sensitivity to nitrosoureas.

Description: Maintenance of myelopoiesis and pluripotential stem cell production for prolonged periods in vitro hitherto has been limited to mouse bone marrow culture. In an effort to adapt the system for use in higher species, particularly in human and non-human primates, studies were undertaken using the prosimian species, Tupaia glis (tree shrew). In a number of experiments the duration of sustained normal hematopoiesis observed in cultures of this species, following a single inoculum of 5 X 10(6)--10(7) bone marrow cells, with or without addition of fresh allogeneic bone marrow exceeded 1 yr. Analysis of suspension cells obtained by weekly demidepopulation of such cultures revealed production of CFU-C, differentiating neutrophils, and basophils at high levels. Direct comparison with murine cultures indicated that in both species a complex series of cellular interactions takes place within an adherent environment of marrow-derived endothelial cells, macrophages, and fat-containing cells. Certain functional and ultrastructural features served to distinguish murine from Tupaia marrow cultures, and the prolonged duration of in vitro hematopoiesis in the latter species could be attributed to a regenerative capacity possessed by its adherent hematopoietic microenvironment. The availability of this primate marrow culture system should facilitate studies of hematopoiesis, viral leukemogenesis, and transplantation biology, which have more direct relevance to man than that provided by the existing murine system.

Description: The isolation of a DNA synthesis inhibitor (NBME fraction IV) and stimulator (RBME fraction III) specific for the hemopoietic stem cell (CFU-s) from freshly isolated normal adult and regenerating murine bone marrow, respectively, has been well documented. We have utilized long- term liquid bone marrow cultures in a further analysis of the role of these factors in the regulation of CFU-s proliferation. Our results show that shortly after feeding, at a time when the cultured CFU-s are actively proliferating, high levels of the hemopoietic stem cell proliferation stimulator fraction III can be isolated from the culture medium. In contrast, the presence of essentially noncycling CFU-s found in cultures fed 8–10 days previously correlates with high levels of the hemopoietic stem cell inhibitor fraction IV. These results suggest that a certain balance between these factors determines CFU-s proliferation in the long-term cultures. In support of this, DNA synthesis in actively cycling CFU-s in the long-term cultures is inhibited for at least 3 days by the addition of excess NBME fraction IV (inhibitor). Furthermore, DNA synthesis in noncycling cultured CFU-s is stimulated for at least 5 days by the addition of RBME fraction III (stimulator).

Description: In long-term marrow cultures, proliferation and differentiation of hemopoietic stem cells occurs for several months. Normally, only the most primitive erythroid progenitor cells are produced (the BFU-E). Following treatment with anemic mouse serum (AMS) or normal mouse serum plus erythropoietin, the BFU-E mature into CFU-E, which then go to produce mature nonnucleated red cells. This development is associated with the production of adult type hemoglobin. Furthermore, erythropoiesis and granulopoiesis occur in association with discrete cellular elements of the adherent cell layer in the long-term culture. Following treatment with AMS, erythropoiesis is enhanced while granulopoiesis is depressed, with no apparent competition at the stem cell or progenitor cell level.