Gene transfer to ankyrin-deficient bone marrow corrects spherocytosis in vitro.

OBJECTIVE: The goal of this study was to transfer by retroviral vector the cDNA for ankyrin to progenitors from normal bone marrow and from the nb/nb spherocytosis mutant deficient in expression of full-length ankyrin to achieve erythroid expression of functional ankyrin protein. MATERIALS AND METHODS: A minigene composed of the human ankyrin promoter, murine ankyrin cDNA, and the 3' human domain corresponding to the ankyrin 2.2 isoform was assembled in the retroviral vector, pG1. Murine erythroleukemia (MEL) cells, normal murine bone marrow cells, 3T3 fibroblasts, and nb/nb mutant bone marrow and spleen cells were transduced with the retroviral supernatant. Transduced mutant cells were induced to differentiate in liquid culture. Gene transfer was assessed by colony polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR, immunofluorescence, and Southern, Northern, and Western blot analysis. RESULTS: MEL cells, normal bone marrow progenitors, and nb/nb cells were all successfully transduced and expressed ankyrin by RT-PCR and Western blot. Transduced murine 3T3 fibroblasts and MEL cells exhibited cell membrane staining by immunofluorescence. Colony RT-PCR demonstrated dependence of expression on erythropoietin. In vitro, the transduced nb/nb cells matured to polychromatophils, whereas nontransduced nb/nb cells matured to microspherocytes. CONCLUSION: Retroviral transfer of ankyrin corrected the defect leading to formation of microspherocytes in erythroid differentiation cultures from the nb/nb mutant. The human ankyrin promoter conferred erythropoietin-dependent expression in normal and mutant erythroid progenitors, which could have implications for the gene therapy of human hemolytic anemias.

Immunofluorescence characterization of key extracellular matrix proteins in murine bone marrow in situ.

The mechanism of hemopoietic stem cell homing to the bone marrow involves molecular interactions that mediate the recognition and interaction of these cells with the marrow microenvironment, including the extracellular matrix. On selective binding, this environment, in combination with soluble cytokines, regulates stem cell proliferation and differentiation. Using immunofluorescence labeling, we analyzed the location of the prominent extracellular matrix proteins fibronectin, collagen Types I, III, and IV, and laminin in sections of murine femoral bone marrow. Collagen Types I, IV, and fibronectin were localized to the endosteum, the region of the femoral microenvironment for which homing stem cells have a high affinity. The results further demonstrated a strong spatial association of collagen Type IV and laminin with the bone marrow vessels, including arterioles, veins, and sinuses. Fibronectin was distributed throughout the central marrow region, and all the proteins analyzed except collagen Type III were present in the bone, although at different levels. Fibronectin, collagen Types III and IV, and laminin were also present in the periosteum. The distinct locations of particular extracellular matrix proteins support the notion that they may play an important mechanistic role in the homing of engrafting cells.

Cytokine-facilitated transduction leads to low-level engraftment in nonablated hosts.

Using a murine bone marrow transplantation model, we evaluated the long-term engraftment of retrovirally transduced bone marrow cells in nonmyeloablated hosts. Male bone marrow was stimulated in a cocktail of interleukin-3 (IL-3), IL-6, IL-11, and stem cell factor (SCF) for 48 hours, then cocultured on the retroviral producer line MDR18.1 for an additional 24 hours. Functional transduction of hematopoietic progenitors was detected in vitro by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of multiple drug resistance 1 (MDR1) mRNA from high proliferative potential-colony forming cell (HPP-CFC) colonies. After retroviral transduction, male bone marrow cells were injected into nonablated female mice. Transplant recipients received three TAXOL (Bristol-Myers, Princeton, NJ) injections (10 mg/kg) over a 14-month period. Transplant recipient tissues were analyzed by Southern blot and fluorescence in situ hybridization for Y-chromosome-specific sequences and showed donor cell engraftment of approximately 9%. However, polymerase chain reaction amplification of DNAs from bone marrow, spleen, and peripheral blood showed no evidence of the transduced MDR1 gene. RT-PCR analysis of total bone marrow RNA showed that transcripts from the MDR1 gene were present in a fraction of the engrafted donor cells. These data show functional transfer of the MDR1 gene into nonmyeloablated murine hosts. However, the high rates of in vitro transduction into HPP-CFC, coupled with the low in vivo engraftment rate of donor cells containing the MDR1 gene, suggest that the majority of stem cells that incorporated the retroviral construct did not stably engraft in the host. Based on additional studies that indicate that ex vivo culture of bone marrow induces an engraftment defect concomitantly with progression of cells through S phase, we propose that the cell cycle transit required for proviral integration reduces or impairs the ability of transduced cells to stably engraft.

Cannabinoid photolabelling of a G-protein gamma-subunit in mouse peritoneal cells.

Binding and photoactivation of a cannabinoid-derived ligand to intact mouse peritoneal cells has resulted in the labelling of a G-protein gamma-subunit. The assignment of structure is based on the product's physical characteristics and its ability to react with a polyclonal antiserum raised against the partial amino acid sequence for a gamma-subunit expressed in spleen cells. The binding characteristics of the ligand to the cells suggests that this gamma-subunit, and its associated alpha and beta subunits, are located in close proximity to one of the transmembrane cannabinoid receptors. Our findings further suggest a possible experimental approach to identifying receptor-G-protein complexes in intact cells.

Phospholipase participation in cannabinoid-induced release of free arachidonic acid.

The exposure of cells in culture to cannabinoids results in a rapid and significant mobilization of phospholipid bound arachidonic acid. In vivo, this effect has been observed as a rise in eicosanoid tissue levels that may account for some of the pharmacological actions of delta 9-tetrahydrocannabinol (THC), the major psychoactive cannabinoid. Fluoroaluminate pretreatment of mouse peritoneal cells potently reduced the cannabinoid response, while promoting arachidonate release on its own, consistent with earlier observations that this effect may be a receptor/G-protein-mediated process. Further support for receptor mediation was the demonstration of saturable, high-affinity cannabinoid binding in these cells. THC potency was reduced in the presence of ethanol, and was accompanied by significant increases in phosphatidylethanol (PdEt) levels, a unique product of phospholipase D (PLD) activity. THC-dependent arachidonate release was reduced partially in similar amounts by either propranolol or wortmannin, further implicating PLD as a mediator of THC action. A central role for diacylglyceride (DAG), a secondary product of PLD metabolism, in this THC-induced process, both as a source of arachidonate and as a stimulator of protein kinase C (PKC), is supported by the data in this report. Cells exposed to phorbol ester for 18 hr prior to THC challenge became less responsive, indicating a possible role for PKC. The involvement of PKC further suggests participation by phospholipase A2 (PLA2) whose activity may be regulated by the former. Treatment of cells with interleukin-1 alpha, an agent known to elevate PLA2 levels, caused an increase in the THC response, supporting a role for this enzyme in the release reaction. Direct evidence, by immunoblotting, for the activation and phosphorylation of PLA2 by THC was also obtained. In summary, the evidence presented in this report indicates that THC-induced arachidonic acid release occurs through a series of events that are consistent with a receptor-mediated process involving the stimulation of one or more phospholipases.