The Effects Of 12-O-tetradecanoylphorbol-13-acetate Research Articles

Human erythroleukemia cell line (HEL) undergoes a drastic macrophage- like shift with TPA

We investigated the effect of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the human erythroleukemia cell line, HEL, and found that TPA addition (10(-6)-10(-8) M) to HEL cell cultures induces morphological, functional, and biochemical changes in HEL cells that are characteristic for macrophage-like cells. Apart from the drastic changes in morphology, the cells greatly enhance their phagocytic ability and acquire receptors for binding and degradation of chemically modified lipoproteins. At the biochemical level, a newly synthesized 85K glycoprotein is observed, and the cells are unresponsive to inducers of globin synthesis. Comparative observations with K562 cells indicate that TPA inhibits, as in HEL cells, spontaneous and induced globin synthesis, but induces minimal macrophage-like properties in these cells. The results with HEL cells are interpreted to indicate that TPA uncovers a latent monocyte-like phenotype in these cells.

Human Erythroleukemia Cell Line (HEL) Undergoes a Drastic Macrophage-Like Shift With TPA

We investigated the effect of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the human erythroleukemia cell line, HEL, and found that TPA addition (10(-6)-10(-8) M) to HEL cell cultures induces morphological, functional, and biochemical changes in HEL cells that are characteristic for macrophage-like cells. Apart from the drastic changes in morphology, the cells greatly enhance their phagocytic ability and acquire receptors for binding and degradation of chemically modified lipoproteins. At the biochemical level, a newly synthesized 85K glycoprotein is observed, and the cells are unresponsive to inducers of globin synthesis. Comparative observations with K562 cells indicate that TPA inhibits, as in HEL cells, spontaneous and induced globin synthesis, but induces minimal macrophage-like properties in these cells. The results with HEL cells are interpreted to indicate that TPA uncovers a latent monocyte-like phenotype in these cells.

Opposing effects of 12‐o‐tetradecanoylphorbol 13‐acetate on human myeloid and lymphoid cell proliferation

The effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on human hematopoietic cells has been investigated. It was found that 1-10 ng/ml of TPA totally abrogated erythroid and granulocytic colony growth and, simultaneously in the presence of PHA, stimulated T-lymphocyte colony formation. TPA concentrations insufficient to inhibit myeloid colony growth also failed to stimulate lymphocyte colony formation. Optimal culture conditions for the growth of these colonies required the presence of TPA, PHA, and leukocyte-conditioned medium in the cultures. Cells within the colonies were 80-90% E-rosette positive and by monoclonal antibody characterization contained 45-66% OKT3-positive cells. Colony-forming cells were found in both E-rosette-positive and-negative fractions. Although by cell surface marker characterization the cells within the colonies had properties of T-cells, the exact relationship of cells forming colonies under these conditions to those detected in other T-cell colony assays remain to be determined.

Role of intercellular communication in the promotion of C3H/10T1/2 cell transformation.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) upon intercellular communication and promotion were studied in cultures of C3H/10T1/2 mouse embryo fibroblasts. Cell-to-cell communication was quantitated by autoradiographic analysis of [3H]uridine transfer from prelabelled donor cells to an excess of unlabelled recipient cells during a 4 h co-cultivation. Extensive transfer of label was observed from donor to recipient cells in contact. Treatment of non-transformed C3H/10T1/2 cultures with 250 ng/ml TPA at co-cultivation of donor and recipient cells markedly inhibited intercellular communication during the 4 h incubation, producing an 80% reduction in uridine exchange relative to solvent-treated control cultures. Concentrations of TPA ranging from 0.25 to 25 ng/ml were also effective in inhibiting [3H]uridine exchange in a dose dependent fashion from 13% to 74%. This inhibition of intercellular communication was transient; cells exposed to 250 ng/ml TPA for 1.5 h prior to co-cultivation with TPA exhibited a 60% inhibition and the exchange of uridine had increased to control values in cultures pretreated for 12-72 h. An examination of label transfer between non-transformed and transformed C3H/10T1/2 cells indicated that both the extent of inhibition by TPA and the kinetics of communication inhibition were similar to that observed for non-transformed cells. Initiation and promotion experiments demonstrated that exposure to 250 ng/ml TPA for 5 weeks, but not to reduced concentrations of 0.25, 2.5 or 25 ng/ml, was capable of promoting morphological transformation. The lack of correlation between the dose responses of TPA for promotion and for reduction of cell-to-cell communication, and the transient nature of intercellular communication inhibition by TPA, suggests that an inhibition of cell-to-cell communication is not a sufficient event for promotion of oncogenic transformation in these cells.

Macrophage Differentiation of Human Precursor B-Cell Line by Phorbol Ester and Colony-Stimulating Factor

A human round cell line, KLM-2, is considered to be precursor B-cell by immunologic surface marker analysis and histochemical studies. The effect of 12-O-tetradecanoyl phorbol-13-acetate (TPA) and/or colony-stimulating factor (CSF) on KLM-2 cells was investigated. KLM-2 cells became adherent to the bottom of the flask on day 2 after liquid culture with TPA, and the number of macrophage-like adherent cells (mAC) with pseudopodia showed a TPA dose-dependent increase to the peak level on day 3 or 4. The mAC had a phagocytic activity. The suspending cells were positive for alpha-naphthyl acetate esterase (NAE) up to about 50% in culture with TPA. A TPA concentration of 100 ng/ml was optimal to provide a maximum number of mAC and NAE-positive suspending cells. Particularly, such effects of TPA on KLM-2 cells were markedly accelerated in the presence of CSF. These findings indicate macrophage differentiation of KLM-2 cells in the presence of TPA and CSF. KLM-2 cells may be a bipotential stem cell line differentiating into B-cell and monocyte-macrophage lineages.

Macrophage differentiation of human precursor B-cell line by phorbol ester and colony-stimulating factor

A human round cell line, KLM-2, is considered to be precursor B-cell by immunologic surface marker analysis and histochemical studies. The effect of 12-O-tetradecanoyl phorbol-13-acetate (TPA) and/or colony- stimulating factor (CSF) on KLM-2 cells was investigated. KLM-2 cells became adherent to the bottom of the flask on day 2 after liquid culture with TPA, and the number of macrophage-like adherent cells (mAC) with pseudopodia showed a TPA dose-dependent increase to the peak level on day 3 or 4. The mAC had a phagocytic activity. The suspending cells were positive for alpha-naphthyl acetate esterase (NAE) up to about 50% in culture with TPA. A TPA concentration of 100 ng/ml was optimal to provide a maximum number of mAC and NAE-positive suspending cells. Particularly, such effects of TPA on KLM-2 cells were markedly accelerated in the presence of CSF. These findings indicate macrophage differentiation of KLM-2 cells in the presence of TPA and CSF. KLM-2 cells may be a bipotential stem cell line differentiating into B-cell and monocyte-macrophage lineages.

Tumor promoters increase the synthesis of a 32,000-dalton protein in BALB/c 3T3 cells.

The effect of 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent tumor promoter, on the synthesis of proteins in BALB/c 3T3 mouse fibroblasts has been studied. When [35S]methionine-labeled proteins synthesized after the addition of TPA were analyzed by one- or two-dimensional polyacrylamide gel electrophoresis, the increased synthesis of a 32,000-dalton protein (designated p32) was noted as one of the earliest changes. The synthesis of p32 increased approximately 2-fold within 2 hr at a promoter concentration of 20 ng/ml. Phorbol 12,13-didecanoate, another potent tumor promoter, showed the same effect as TPA, whereas 4-O-methyl-TPA, which has little promoting activity, did not enhance the synthesis of p32 at the same concentration but did effect a slight increase at 1 microgram/ml. p32 differed immunologically from a major excreted protein discovered by Gottesman [Gottesman, M. M. (1978) Proc. Natl. Acad. Sci. USA 75, 2767-2771]. The increased rate of the synthesis of p32 was considered to be regulated at the transcriptional level, because the increase in synthesis of p32 was inhibited by actinomycin D, and TPA-treated cells were shown to contain a higher level of translatable mRNA coding for p32 than do control cells. A possible relationship between the increase in p32 synthesis and transformation is discussed.

The effect of phorbols on metabolic cooperation between human fibroblasts

Autoradiography has been used to study the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), 4-O-methyl TPA, and phorbol on metabolic cooperation between human diploid fibroblasts. When the donors, hypoxanthine-guanine phosphoribosyl transferase+ (HGPRT+) cells, and recipients, HGPRT- cells, were plated together in the presence of [3H]hypoxanthine and either 4-O-methyl TPA or phorbol, nearly all interactions that developed in 4 h were positive for metabolic cooperation whereas when high concentrations of TPA were used, the number of positive interactions was significantly less than the control. If the phorbol analogs were added after the donors and recipients had made contact, the number of positive interactions was the same as the control in all cases. However, although primary recipients in the cultures that had been treated with phorbol had the same number of grains as those in the control, primary recipients in cultures that had been treated with TPA or high concentrations of 4-O-methyl TPA had significantly fewer grains than those in the control. TPA treatment for 4 h had no effect on total [3H]hypoxanthine incorporation or incorporation into acid-soluble and acid-insoluble fractions. Thus, the effect of TPA on metabolic cooperation is interpreted as a reduction in the transfer of [3H]nucleotides and is an indication of an interference with intercellular communication.

Growth Stimulation of Microenvironment-Dependent Mouse Leukemias by Tumor-Promoting Phorbol Esters<xref ref-type="fn" rid="fn2">2</xref>

The effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) and related tumor-promoting phorbol esters on 23 symbiotic cell lines from AKR thymic leukemias were studied. The phorbol derivatives with in vivo cocarcinogenic activity increased the viable yield of leukemia cells of most symbiotic cell lines in the absence of growth-supporting adherent cells. Particularly, in 10 cell lines remarkable growth stimulation was observed with 1 to 10 ng TPA/ml. The effect of TPA was reversible, since the leukemia cells exposed to TPA for 48 hours failed to grow without TPA or adherent cells. The leukemia cells from a symbiotic cell line could be maintained in TPA-containing medium for as long as 8 weeks without losing dependence. The leukemia cells were refractory to growth stimulation by TPA after they acquired the capability to grow independently. Furthermore, by affecting the leukemia cells, TPA and active tumor promoters inhibited pseudoemperipolesis (i.e., localization of leukemia cells under or between adherent cells), which is the basic cell interaction in symbiotic complexes. These effects of tumor-promoting phorbol esters are discussed with a special reference to the multistage hypothesis of leukemogenesis.

Tumor promoters alter gene expression and protein phosphorylation in avian cells in culture.

We have investigated the effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on the synthesis and modification of polypeptides in normal avian cells and cells infected by wild-type and temperature-sensitive Rous sarcoma virus (RSV). Using two-dimensional gel electrophoresis, we have detected alterations in both the abundance of cellular polypeptides and in their phosphorylation that seem unique to TPA treatment. However, the state of phosphorylation of the major putative substrate for the action of the src gene-associated protein kinase, the 34- to 36-kilodalton protein, was not altered. Moreover, examination of the phosphorylated amino acid content of total cellular phosphoproteins revealed that the response to TPA was not associated with detectable increases in their phosphotyrosine content. These results make it unlikely that TPA acts by the activation of the phosphorylating activity of the cellular proto-src gene or by the activation of other cellular phosphotyrosine-specific kinases. We have shown previously that temperature-sensitive RSV-infected cells at nonpermissive temperature demonstrate an increased sensitivity to TPA treatment [Bissell, M. J., Hatie, C. & Calvin, M. (1979) Proc. Natl. Acad. Sci. USA 76, 348-352]. Our present results indicate that this is not due to reactivation of the phosphorylating activity of the defective src gene product or to its leakiness, and they lend support to the notion of multistep viral carcinogenesis.