MBM-1, a differentiation marker of mouse hemopoietic cells defined by a rat monoclonal antibody.

Hybridoma clones were isolated after the fusion of mouse myeloma cells with spleen cells from a rat immunized with mouse bone marrow. One of them produced a monoclonal antibody reacting with a murine cell surface differentiation antigen that we have termed MBM-1 (Mouse Bone Marrow-1). This antigen is present on eosinophils, on neutrophils, and on subpopulations of lymphocytes and macrophages, but appears to be absent from erythroid cells. Precursor cell analysis, after sorting of bone marrow cells using a fluorescence-activated cell sorter, suggests that the antigen is absent from most progenitors with the exception of certain cells in the macrophage lineage.

A simple, single-step technique for selecting and cloning hybridomas for the production of monoclonal antibodies.

A simple method is described by which hybridomas can be selected and cloned in a single step immediately after fusion. This is done by plating the cells in semi-solid medium containing methylcellulose and the components of the HAT selection system. A number of variables have been examined in order to optimise the technique. The system is particularly suitable for isolating large numbers of hybridomas secreting different monoclonal antibodies. Evidence is presented to show that the colonies which grow in the system are in all probability clones. Thus, the need for routine recloning of the hybridomas is eliminated. In this way, the technique cuts down on the amount of tissue culture work associated with the production of monoclonal antibodies. Using this technique, it is easier to plate out large numbers of cells and to recover many independent hybridoma clones, than is the case when using cloning by limiting dilution.

Regulation of 40S ribosomal protein S6 phosphorylation in Swiss mouse 3T3 cells.

Addition of serum to resting cultures of Swiss mouse 3T3 cells causes an immediate multiple phosphorylation of 40S ribosomal protein S6. After 60 min of stimulation, changing to medium containing no serum led to the net dephosphorylation of S6. During this same period, a second protein, as yet unidentified, became increasingly phosphorylated. Incubation of cells with cycloheximide prior to the addition of serum almost completely blocked the activation of protein synthesis. There was no effect on the serum-induced phosphorylation of S6. If cells were stimulated in the presence of cAMP phosphodiesterase inhibitors theophylline or SQ 20006, both S6 phosphorylation and the activation of protein synthesis were inhibited. Stimulation of cells with serum also led to an immediate drop in total intracellular cAMP levels. This was blocked by prostaglandin E1 (PGE1), which caused a 10 fold increase in total intracellular cyclic AMP. However, PGE1 had no effect on protein synthesis or S6 phosphorylation.

The stimulation of the initiation of DNA synthesis by fibroblast growth factor in Swiss 3T3 cells: interactions with hormones during the pre-replicative phase.

Fibroblast Growth Factor (FGF) stimulates quiescent Swiss 3T3 cells to initiate DNA synthesis and divide. Cells begin to enter the S-phase after a lag of 13--15 hr, and the rate of initiation of DNA synthesis in the population can be quantified by a first order rate constant, k. A subsaturating concentration of FGF may establish the lag phase, while the value of k is dependent on the FGF concentration present during the second half of the lag phase. Insulin and hydrocortisone enhance the effect of FGF by increasing k without changing the lag phase, and they can act when added at any time after FGF. Prostaglandin E1 (PGE1) causes a decrease in k and a lengthening of the lag phase, and acts only when added during the first 8 hr. None of these agents stimulate DNA synthesis in the absence of FGF.

Cytoskeleton-disrupting drugs enhance effect of growth factors and hormones on initiation of DNA synthesis.

Addition of growth factors, such as prostaglandin F2 alpha or fibroblastic growth factor, to quiescent Swiss mouse 3T3 cells resulted in an abrupt increase in the rate of initiation of DNA synthesis after a lag phase of 13-15 hr. This increase could be quantified by a rate constant k. Addition of colchicine, Colcemid, or vinblastine had a synergistic effect on the initiation of DNA synthesis triggered by PGF2 alpha or FGF by increasing the value of k. These drugs alone had no effect. Colchicine had a synergistic effect only if added within 8 hr of the PGF2 alpha or FGF addition. Also, colchicine exerted its full effect when it was present only for the first 5 hr with either growth factor. These results suggest that an intact cytoskeleton is not required for the initiation of DNA synthesis. Furthermore, cytoskeleton-disrupting drugs enhance the stimulatory effect of the growth factors.