Production of platelet-derived growth factorlike protein(s) by a human carcinoma cell line.

Human adrenal carcinoma cells (SW-13) produce and secrete autocrine growth factors. We now report that these cells secrete a factor that is biologically related to platelet-derived growth factor (PDGF). Serum-free medium conditioned by SW-13 cells stimulated incorporation of tritiated thymidine into DNA by quiescent Balb/c 3T3 cells in the presence of platelet-poor plasma. The conditioned media also competed with 125I-labeled PDGF for binding to PDGF receptors on human fibroblasts. Hybridization studies identified mRNA transcripts for PDGF-2/sis chains in logarithmically growing SW-13 cells. These data suggest secretion of PDGF-like mitogens by SW-13 cells.

Inhibition of mitogen stimulated growth of human colon cancer cells by interferon.

Recombinant human interferon alpha inhibits growth of a human colon cancer cell line, Colo 205. To explore the mechanisms of IFN induced growth inhibition, quiescent Colo 205 cells were stimulated to proliferate in serum-free media by defined growth factors. Addition of insulin, transferrin and selenium (ITS) stimulated DNA synthesis, as measured by 3H-thymidine incorporation, in a dose-dependent manner. IFN-alpha (at concentrations greater than 100 U ml-1) inhibited ITS stimulated DNA synthesis by 63%. Inhibition of cell cycle traverse was confirmed by flow cytometric analysis. Although IFN inhibited growth of ITS-treated cells, steady state levels of c-myc mRNA remained above levels observed in unstimulated cells. IFN inhibited DNA synthesis only when added prior to mitogen stimulation. IFN, added 6 h after exposure of quiescent cells to ITS, failed to inhibit cell growth. Addition of increasing concentrations of ITS failed to overcome the IFN-induced growth inhibition. These results suggest IFN may inhibit cell growth in part by antagonizing the action of growth factors.

Stimulation of anchorage-independent growth of human tumor cells by interleukin 1.

Human peripheral blood monocytes and tumor-associated macrophages release a factor that enhances the clonal growth of a human epithelial tumor cell line (SW-13) in soft agar. We now demonstrate that purified interleukin 1 (IL-1) may account for part of this colony-stimulating activity. Purified IL-1 (0.5 to 8 units/ml) was added to SW-13 cells cultured in soft agar. IL-1 increased colony growth in a dose-dependent manner and did not inhibit colony formation at the highest doses tested. Other purified human monocyte products (alpha-interferon, tumor necrosis factor, transforming growth factor beta, fibronectin) did not stimulate colony growth. Antibody to IL-1 only partially inhibited the ability of monocyte-conditioned medium to stimulate SW-13 colony growth. This antibody did, however, completely inhibit the ability of purified IL-1 to support the growth of SW-13 colonies in soft agar. IL-1 increased growth of quiescent SW-13 cells cultured in monolayers as assessed by tritiated thymidine incorporation assays. The results of this study indicate that IL-1 can enhance clonogenic growth of an epithelial cell line in soft agar. However, other uncharacterized activities in monocyte conditioned medium also promote colony growth. These studies add to an increasing body of evidence indicating that inflammatory products play a role in maintaining the transformed phenotype.

Enhancement of survival and proliferation of clonogenic cells by monocyte-derived growth factors.

Human peripheral blood monocytes release a factor that enhances the clonal growth of human epithelial tumor cells in soft agar. The monocyte-derived growth factor was needed for both cellular proliferation and survival. Survival of SW-13 colony-forming cells decreased linearly in the absence of monocyte-conditioned media (MO-CM). Cells failed to respond to MO-CM after four days in culture. Although MO-CM enhanced growth when cells were plated at low density, growth was also enhanced when cell density was not a limiting factor. MO-CM increased DNA synthesis of SW-13 cells growing in monolayer culture as measured by tritiated-thymidine incorporation. These findings support evidence indicating inflammatory products may play a role in maintenance of the transformed phenotype.

Recombinant human interferon alpha enhancement of retinoic-acid-induced differentiation of HL-60 cells.

We have previously demonstrated that a combination of interferon beta and a differentiation agent, dimethyl sulfoxide (DMSO), is cytotoxic for HL-60 cells, a human promyelocytic leukemic cell line. We now report that a combination of recombinant interferon alpha (Intron; Schering) and retinoic acid is synergistically cytostatic for HL-60 cells. Retinoic acid (RA) induced the differentiation of HL-60 cells into granulocytes. Interferon (IFN) alone at 1-1000 IU/ml had no effect on either differentiation or proliferation of HL-60 cells. The addition of 1000 IU/ml of IFN and 10(-7) M RA at the initiation of culture reduced the number of viable cells to 28% of that observed for cells treated with RA alone. The decreased number of cells was a result of decreased cellular proliferation, rather than of a cytotoxic effect of the combination. IFN-RA-treated cells differentiated more rapidly than cells treated with RA alone. In addition, the final percentage of mature cells was increased at day 7 in IFN-RA-treated cultures, as compared with RA-treated cells. Simultaneous treatment of the cells with IFN and RA decreased the concentration of RA needed to induce differentiation or to exert a cytostatic effect. Significant changes in the nuclear structure of RA-treated HL-60 cells after 24 h have been reported. Cells were pulsed with RA for 24 h, washed, and IFN added. At day 7, cell growth was inhibited to the same extent as that of cells continuously exposed to IFN-RA. However, while 70% of the continuously exposed cell differentiated, cells pulsed with RA and subsequently treated with IFN did not differentiate. The results of this investigation further support our findings that combinations of IFN and inducers of differentiation may be of importance in the treatment of leukemia.

Switch in differentiative response to maturation inducers of human promyelocytic leukemia cells by prior exposure to alkaline conditions.

The myeloid lineage to which HL-60 promyelocytic leukemia cells will differentiate in response to a chemical differentiation inducer can be switched by altering the pH of the growth medium. Cells passaged previously at pH 7.2 become neutrophiles, and those passaged previously at pH 7.6 become eosinophiles after 5 to 7 days of culture in the presence of 0.5 mM butyric acid. Butyric acid and its analogues are unique in that all other chemical maturation inducers tested, such as dimethyl sulfoxide and retinoic acid, promote neutrophilic differentiation regardless of the prior culture history of the cells. This suggests that lineage commitment and maturational commitment are mechanistically separate processes in this multipotential cell line and can be independently manipulated experimentally.

Angiotensin-converting enzyme inhibitors: importance of the amide carbonyl of mercaptoacyl amino acids for hydrogen bonding to the enzyme.

A series of mercaptoacyl amino acids and related compounds was synthesized and evaluated for inhibition of angiotensin-converting enzyme (ACE) in order to determine the nature and importance of the putative interaction between ACE and the amide moiety of inhibitors such as captopril (3-mercapto-2-methylpropanoyl-L-proline). It was concluded that the interaction involves a hydrogen bond from a donor site on ACE to the oxygen of the amide carbonyl. Compounds in which the amide moiety is replaced by other groups (ester, ketone, sulfonamide) capable of accepting a hydrogen bond are effective inhibitors, but compounds in which only the geometrical features of the amide are retained are ineffective inhibitors. The presence of an NH group is not necessary for effective inhibition. The activity of a series of mercaptoacyl cycloalkyl carboxylic acids parallels the activity of the isosteric series of mercaptoacyl imino acids.

Design of potent and specific inhibitors of carboxypeptidases A and B.

The combination in one molecule of functional groups that can interact specifically with different substrate binding areas at the active site of carboxypeptidases A and B has led to the development of potent and specific inhibitors of these enzymes. 2-Benzyl-3-mercaptopropanoic acid (SQ 14,603) has a Ki of 1.1 x 10(-8) M vs. carboxypeptidase A and a Ki of 1.6 x 10(-4) M vs. the B enzyme. 2-Mercaptomethyl-5-guanidinopentanoic acid (SQ 24,798) has a Ki of 4 x 10(-10) M vs. carboxypeptidase B and a Ki of 1.2 x 10(-5) M vs. carboxypeptidase A. It is proposed that the sulfhydryl groups of these inhibitors bind to the catalytically important zinc ions of these enzymes, and that, in conjunction with the benzyl and guanidinopropyl side chains, they are responsible for their specificity.

Nondepressant beta-adrenergic blocking agents. 1. Substituted 3-amino-1-(5,6,7,8-tetrahydro-1-naphthoxy)-2-propanols.

A series of 3-amino-1-(5,6,7,8-tetrahydronaphthoxy)-2-propanols was synthesized and investigated for beta-adrenergic blocking activity and direct myocardial depressant action. The cis- and trans-diols 12--15 were found to retain the beta-blocking potency of propranolol but to lack its myocardial depressant action. Compound 15 (nadolol) is currently undergoing extensive clinical evaluation as a potential antianginal, antiarrhythmic, and antihypertensive agent.