Epidermal growth factor (EGF) inhibited the growth of A431 human epidermoid carcinoma cells. The tumor promoting, phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) also retarded A431 cell growth. Addition of both TPA and EGF inhibited cell growth in an additive or synergistic manner depending upon the initial plating density of the cultures. EGF increased the production of diacylglycerol (60-70%) and stimulated the synthesis of phosphatidylinositol (PI) from 3H-inositol (three- to fourfold increase). Both of these responses were attenuated in the presence of TPA. TPA alone stimulated the production of diacylglycerol (DG) but had little effect on PI synthesis. The biological effect of TPA appeared to be mediated by the presence of a high-affinity receptor for phorbol esters on A431 cells. Moreover, the binding of 125I-EGF to A431 cells was unaffected by TPA, suggesting that the antagonistic effects of TPA were occurring distal to the EGF receptor. These findings also indicated that although TPA and EGF both inhibited A431 cell growth, this effect could be dissociated from changes in PI synthesis but may be dependent upon transient changes in DG production.

The monoclonal antibody OX7 recognizes an epitope expressed on the Thy‐1 glycoprotein, OX22 recognizes the high molecular weight forms(s) on leukocyte common antigen, and W3/13 recognized determinants found on certain sialoglycoproteins. Recently, the rat colony‐forming unit spleen (CFU‐S) was characterized as being OX7 upper 20% positive (OX7u20%), OX22 negative (OX22−), and W3/13 weakly positive (W3/13+). In the present study these observations have been extended to include the hematopoietic stem cell (HSC). Rat marow cells were incubated with allophycocyanine‐OX7 Fab' (APC‐OX7 Fab') and phycoerythrin (B‐OX22) Fab' (PhyB‐OX22 Fab'). The cells were sorted with a FACS‐ii instrument by using a Krypton laser tuned to the 530 nm spectral line for phycobiliprotein excitation. It was found that marrow cells capable of protecting lethally irradiated Lewis rats (9.5 Gy total body radiation, 0.4 Gy/min Co60) had the phenotype OX7u20%, OX22−. The percentage of cells in the marrow with this phenotype was found to be 0.34 ± 0.01 (mean ± S.E.). Three thousand of these cells were required to rescue 50% of lethally irradiated recipients (30‐d survival), while the number of unsorted bone marrow cells required was 1.05 × 106. Thus, a 350‐fold purification of the HSC was realized. Although CFU‐S copurified with HSC, purification of only 105‐fold was obtained. This might indicate that purified HSC have a reduced capacity to generate splenic hematopoietic colonies. The OX7u20%, OX22−‐enriched HSC population could be further divided into W3/13 dimand W3/13+ subpopulations by three‐parameter immunofluorescence analysis with the use of a new optical bench arrangement.