Calcium and calmodulin (CaM) are known to play critical roles in controlling cell cycle progression in a variety of cells. We observed that the CaM antagonist, N-(6-aminohexyl)-5-chloro-1- naphthalensulfonamide hydrochloride (W-7), inhibited 3H-thymidine incorporation into DNA of factor-dependent hematopoietic cells. To delineate the role of CaM in proliferation of hematopoietic cells, we have investigated intracellular distribution of specific CaM-binding proteins (CaM-BPs) in response to hematopoietic growth factors in FDC- P1, 32D, NFS-60, and T1165 cells. Each of these cell lines, when deprived of cytokines for 16 to 18 hours, essentially ceased proliferation, even in the presence of fetal calf serum. Concomitant to the cessation of proliferation, there was a dramatic depletion of a specific CaM-BP of about 68 Kd in both their cytoplasmic and nuclear fractions. Within 6 to 12 hours of reexposure to proliferation-specific cytokines, there was a restoration of the nuclear as well as cytoplasmic 68-Kd CaM-BP. Furthermore, such an induction and nuclear localization of the 68-Kd CaM-BP by the cytokines coincided temporally with the progression of synchronized FDC-P1 cells from G1 to S phase. By contrast, colony-stimulating factor-1 (CSF-1)-dependent bone marrow macrophages and BAC-1 cells did not exhibit 68-Kd CaM-BP in the nuclear or cytoplasmic fractions. These studies suggest that while hematopoietic growth factor granulocyte CSF-, granulocyte-macrophage CSF-, interleukin-3 (IL-3)-, or IL-6-, whose receptors are members of the hematopoietin receptor family, induced cell proliferation is associated with a common mechanism involving nuclear localization of the 68-Kd CaM-BP, the CSF-1-induced proliferation seems to involve 68- Kd CaM-BP-independent pathways.

A specific calmodulin-binding protein of 68 kDa (CaM-BP68) is modulated in response to growth factors that induce proliferative stimulation in a variety of hemopoietic progenitor cells. The nuclear localization of the CaM-BP68 coincided temporally with interleukin 3 (IL-3)-dependent progression of synchronized FDC-P1 cells from G1 to S phase [Reddy et al. (1992) Blood 79, 1946-1956]. To delineate the role of the CaM-BP68 in the onset of DNA synthesis (S phase), this protein was purified to an apparent homogeneity from FDC-P1 cells and its effects on DNA replication in permeabilized FDC-P1 cells were examined. Purified CaM-BP exhibited a single silver-stained protein band of 68 kDa on SDS-polyacrylamide gels. This purified protein, when incubated with permeabilized log-growing FDC-P1 cells, caused a 3-4-fold increase in the rate of [3H]dTTP incorporation into DNA as compared to the controls. There was a direct correlation between the increase in the rate of [3H]dTTP incorporation into DNA and the concentration of the added CaM-BP68 in the incubation mixture. These observations suggest that the CaM-BP68, whose nuclear localization is associated with growth factor dependent proliferative stimulation of myeloid progenitor cells, is involved in the regulation of nuclear DNA synthesis.

Adherent murine stromal cells support long-term in vitro lymphopoiesis or myelopoiesis dependent on the culture conditions used. A cell line, TC-1, isolated from long-term liquid murine marrow cultures under conditions approaching those permissive for lymphoid growth, has been found to produce an activity that acts synergistically with interleukin- 3 (IL-3) or colony-stimulating factor-1 (CSF-1) to stimulate in vitro myeloid colonies, but which has no intrinsic colony-stimulating activity. We report here the presence of multiple growth factors in conditioned medium (CM) from the TC-1 line, including granulocyte- macrophage colony-stimulating factor (GM-CSF) (bioassay with antibody blocking and messenger RNA [mRNA] analysis), granulocyte CSF (G-CSF) and IL-4 (factor-dependent cell line bioassay), and CSF-1 (radioimmunoassay, mRNA) along with a pre-B cell inducing activity, which appears separate from these CSFs and segregates with the myeloid synergizing activity through anion exchange, sizing, and Conconavalin A chromatography. Because these activities are not yet purified to homogeneity, their identity or lack of identity remains an open question. Assays of TC-1 CM or cellular mRNA analysis have given negative results for IL-1, IL-2, IL-3, IL-6, and IL-7, and IL-6 does not stimulate pre-B cells in this assay. However, IL-4 and G-CSF do stimulate in vitro induction of pre-B cells from pre-B and B-cell- depleted Balb/C marrow and are present in CM by selective cell line assay. A monoclonal antibody to IL-4 that inhibited its pre-B inducing activity did not inhibit pre-B inducing activity of TC-1 CM. These data suggest the existence of a unique synergizing and pre-B inducing factor(s) in TC-1 CM. Given the known capacity of subliminal levels of growth factors to act synergistically, an alternate possibility is that these biologic phenomena represent the actions of low concentrations of growth factors acting synergistically and possibly associated with some core protein.

Calcium and calmodulin (CaM) are known to play critical roles in controlling cell cycle progression in a variety of cells. We observed that the CaM antagonist, N-(6-aminohexyl)-5-chloro-1- naphthalensulfonamide hydrochloride (W-7), inhibited 3H-thymidine incorporation into DNA of factor-dependent hematopoietic cells. To delineate the role of CaM in proliferation of hematopoietic cells, we have investigated intracellular distribution of specific CaM-binding proteins (CaM-BPs) in response to hematopoietic growth factors in FDC- P1, 32D, NFS-60, and T1165 cells. Each of these cell lines, when deprived of cytokines for 16 to 18 hours, essentially ceased proliferation, even in the presence of fetal calf serum. Concomitant to the cessation of proliferation, there was a dramatic depletion of a specific CaM-BP of about 68 Kd in both their cytoplasmic and nuclear fractions. Within 6 to 12 hours of reexposure to proliferation-specific cytokines, there was a restoration of the nuclear as well as cytoplasmic 68-Kd CaM-BP. Furthermore, such an induction and nuclear localization of the 68-Kd CaM-BP by the cytokines coincided temporally with the progression of synchronized FDC-P1 cells from G1 to S phase. By contrast, colony-stimulating factor-1 (CSF-1)-dependent bone marrow macrophages and BAC-1 cells did not exhibit 68-Kd CaM-BP in the nuclear or cytoplasmic fractions. These studies suggest that while hematopoietic growth factor granulocyte CSF-, granulocyte-macrophage CSF-, interleukin-3 (IL-3)-, or IL-6-, whose receptors are members of the hematopoietin receptor family, induced cell proliferation is associated with a common mechanism involving nuclear localization of the 68-Kd CaM-BP, the CSF-1-induced proliferation seems to involve 68- Kd CaM-BP-independent pathways.