Analysis of phorbol ester receptors in phorbol ester unresponsive 3T3 cell variants

The phorbol ester tumor promoters induce multiple cellular responses in cell culture, including mitogenesis. We have analyzed 3 variants of mouse 3T3 cells mitogenically unresponsive to the phorbol esters for phorbol ester receptors. All resembled control 3T3 cells in their specific [³H]phorbol 12,13-dibutyrate binding. The variants thus appear to be altered at steps distal to receptor occupancy in the mitogenic response to the phorbol esters.     

Phorbol ester-induced differentiation permits productive human cytomegalovirus infection in a monocytic cell line

The susceptibility of four different human cell lines (HUT 102, THP-1, MOLT-4, and HL-60) to infection by human CMV (HCMV) was studied. Only HUT 102 was susceptible and only immediate-early gene products were produced. However, THP-1, a monocytic cell line, could be infected by HCMV with a full cycle of replication after treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which produced differentiation of the cell line into cells with characteristics of mature macrophages. Late (structural) Ag were demonstrated, as were infectious virions as detected by electron microscopy and infectious center assay. HL-60, a promyelocytic cell line, was not susceptible to HCMV infection after treatment with TPA despite differentiation into adherent cells with properties of macrophages, suggesting that cellular lineage was important. Treatment with TPA after infection resulted in a greatly reduced frequency of infected cells, suggesting that pretreatment was essential. Furthermore, continued presence of TPA was unnecessary after differentiation was induced. This study establishes the precedent of productive HCMV infection in human monocytic cells. The potential mechanism and relevance of enhanced replication induced by TPA are discussed.     

A human T lymphoblastic cell line lacks lamins A and C

Lamins A, B and C, the three major proteins of nuclear envelope, constitute a class of intermediate filament polypeptides. We have compared the amount of these polypeptides in two human cell lines, epithelial HeLa cells and T lymphoblasts KE 37. It was found that the three lamins were present in roughly equimolar stoichiometry in HeLa cells, while lamin B was the unique lamin component in T lymphoblasts. Moreover, 3-kb mRNA of lamin A and 2.1-kb mRNA of lamin C were detected with a human cDNA probe in HeLa cells but not in T lymphoblasts. These results suggest that (i) lamin B can build up the lamina structure in actively dividing somatic cells by itself, and (ii) lamin expression in lymphoid cells may be subject to important quantitative variations. Comparison of the lamin composition of human cloned T lymphocytes and Epstein-Barr virus-transformed human B lymphocytes confirmed this statement. The lamin B level was nearly equivalent in both cells but the content of lamins A and C varied to a large extent, being low in T cells and high in B cells.     

Phorbol ester induction of plasmacytoid and hairy cell leukemia features in B-type lymphocytic leukemias: the relation to B-cell differentiation and maturation

Mononuclear cells concentrated from 11 patients with chronic lymphocytic leukemia (CLL), 7 with non-Hodgkin's lymphoma in leukemic phase (NHL), 5 with hairy cell leukemia (HCL), 1 with prolymphocytic leukemia (PLL), and 1 with plasma cell leukemia (PCL) were induced to differentiate with various doses of TPA. The degree of induction was followed for up to 6 days by measuring the expression of surface membrane markers (SmIg and GP-70) and Ig secretion, the induction of tartrate-resistant acid phosphatase (TRAP) and by recording ultrastructural changes as seen by electronmicroscopy. The results show a dose and time dependency of the TPA effect and a great heterogeneity in the cellular response, particularly in cells obtained from B-CLL patients. TPA induced two main features, namely the development of "plasmacytoid" or "hairy cell" leukemia features that clearly depended on the dose and duration of treatment with the phorbol ester. The plasmacytoid features were more frequently encountered with lower doses (1 ng/ml) of TPA and were more evident after shorter exposures to TPA (1-2 days). Nevertheless, the hairy cell features were more striking after incubation with higher concentrations of TPA (10-100 ng/ml) after longer periods of incubation (up to 6 days) with lower doses of TPA. The various features of differentiation measured including cell morphology, surface membrane markers, Ig secretion, and TRAP staining, were frequently independent of each other, suggesting an autonomous pathway of differentiation for some of these features. Furthermore, in most of the cases, hairy cell leukemia features were obtained more frequently following TPA exposure than plasmacytic changes.     

Phorbol ester reduces number of heparin-binding growth-factor receptors in human adult endothelial cells

Heparin-binding growth factors (HBGF) are essential and key mitogens for human adult large vessel endothelial cells. At 170 pg/ml, the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) caused a 50% inhibition of heparin-binding growth factor type one (HBGF-1)-stimulated DNA synthesis in human adult large vessel endothelial cells. TPA at 1 ng/ml completely inhibited HBGF-1-stimulated proliferation. TPA at 5 ng/ml reduced specific HBGF-1 receptor sites from 6600 per cell to 3200 per cell without affecting receptor affinity. Since phorbol esters are potent activators of protein kinase C, desensitizes both animal capillary and human adult large vessel endothelial cells to the mitogenic effects of HBGF by down-regulation of specific HBGF receptors.--HOSHI, H; KAN, M.; MIOH, H.; CHEN J.-K.; McKEEHAN, W. L. Phorbol ester reduces number of heparin-binding growth factor receptors in human adult endothelial cells.     

Phorbol ester causes desensitization of gonadotropin-responsive adenylate cyclase in a murine Leydig tumor cell line

The murine Leydig tumor cell line, MLTC-1, contains gonadotropin receptors and a gonadotropin-responsive adenylate cyclase system that became refractory (desensitized) when exposed to human chorionic gonadotropin (hCG). MLTC-1 cells also contain phorbol ester receptors with a Kd of 53 nM for [3H]phorbol dibutyrate. Exposing cells to 12-O-tetradecanoyl phorbol 13-acetate (TPA) also causes desensitization of the hCG response. TPA-induced desensitization was similar to hCG-induced desensitization by every criteria tested. Both TPA- and hCG-induced desensitization caused approximately 50% loss of the hormone response within 30 min. Neither TPA or hCG altered receptor affinity for hCG. The dose response of adenylate cyclase to hCG or GTP in isolated membranes was not affected by either hCG- or TPA-induced desensitization. Similarly the dose response to hCG of cAMP accumulation in intact cells was not altered by desensitization with hCG or TPA. It was determined that MLTC-1 cells have Ca2+/phospholipid-dependent protein kinase activity that displayed a dose-dependent response to TPA. The concentration of TPA required to activate the protein kinase was similar to that required for desensitization. Phorbol esters that were unable to activate protein kinase C were also unable to desensitize MLTC-1 cells. The protein kinase from MLTC-1 cells was also activated by diacylglycerol. In addition, diacylglycerols caused desensitization of the hCG response. TPA- and diacylglycerol-induced desensitization is probably mediated by protein kinase C, and the similarities between hCG- and TPA-induced refractoriness suggests a convergence of mechanisms at some point of MLTC-1 cell desensitization.     

High radiosensitivity of the MOLT-4 leukaemic cell line

Radiation survival of MOLT-4, a leukaemic T-lymphocyte cell line, was measured by counting colonies formed in 0.8 per cent methyl cellulose. The survival curve was a simple exponential and showed the cells to be radiation sensitive, with D0 = 0.49 +/- 0.02 Gy and extrapolation number n = 0.92 +/- 0.09. No increase in survival as measured by colony-forming ability or trypan blue dye exclusion was seen when the dose was split into two fractions, separated by a 5 h incubation period. Electron microscopy and trypan blue dye exclusion showed that 5 h after exposure to high doses, MOLT-4 cells began to die and displayed condensed, marginated chromatin and cellular vesiculation.     

Phorbol ester regulation of the human gamma-glutamyltransferase gene promoter

In the present study the molecular mechanisms underlying tetradecanoylphorbol-13-acetate (TPA) mediated regulation of the human gamma-glutamyltransferase (GGT) gene were examined. TPA challenge of HeLa cells resulted in an increase of GGT mRNA and enzyme activity. Deletion analysis of the promoter revealed that the -348 to +60 fragment was able to mediate TPA induced expression. Gel shift and supershift analyses showed that TPA treatment increased nuclear protein binding to a putative AP-1 site (-225 to -214) and that c-Jun was part of the complex. This AP-1 element, when cloned either in its native arrangement or as tandem repeat 5' of the minimal thymidine kinase promoter, mediated a significant increase of luciferase activity after TPA treatment of transfected HeLa cells, while its mutated counterpart abolished the induction. The same AP-1 element was able to mediate TPA induced expression in HepG2 cells. Collectively these results indicate that like other GSH metabolising enzymes, GGT too is a target for AP-1 mediated regulation.     

7-Hydroxymethotrexate formation in a human lymphoblastic cell line

7-Hydroxymethotrexate is a major metabolite of methotrexate in patients undergoing chemotherapy with this drug. It has now been demonstrated that when cultures of CCRF-CEM, a human lymphoblastic cell line, are incubated in the presence of 100 microM methotrexate, significant quantities of 7-hydroxymethotrexate are formed. This finding is relevant to clinical use of the drug since it is now clear that during the treatment of acute lymphocytic leukemia conversion of methotrexate to the relatively inactive 7-hydroxy derivative probably occurs not only in the liver but also in the target cells.     

Dexamethasone regulation of terminal differentiation in 3T3-F442A preadipocyte cell line

The 3T3-F442A preadipocyte cell line was previously shown to possess specific glucocorticoid receptors whose number increased in the time course of differentiation. We have examined the effects of a three day dexamethasone treatment, added at confluence, on cells differentiated in the presence or absence of insulin. Triglyceride accumulation, polyamine content as well as glycerophosphate dehydrogenase and fatty acid synthetase activities were measured during the adipose conversion. We have also determined 2-deoxyglucose uptake in non-differentiated and differentiated cells. Dexamethasone was shown to decrease the adipose conversion by 3T3-F442A cells in the presence or absence of insulin. Intracellular spermidine content in differentiating cells was sensitive to dexamethasone and insulin in the same way as an enzymatic marker of terminal differentiation, glycerophosphate dehydrogenase. Dexamethasone decreases the 2 deoxyglucose uptake in non-differentiated and differentiated cells while insulin increases this uptake only in differentiated cells. This work shows that glucocorticoids inhibit adipocyte metabolism at distinct levels and suggests that these hormones might play an important role in the regulation of adipose tissue mass.Abbreviations DEX dexamethasone - FAS fatty acid synthetase - GPDH glycerophosphate dehydrogenase - MIX 1-methyl-3-isobutylxanthine     

Phorbol and calcium decreased atriopeptin response in a human renal cell line.

We investigated regulation of atrial natriuretic factor (ANF)-stimulated cellular cGMP accumulation (ANF-s-cGMP) in an ANF-responsive human renal cell line, SK-NEP-1. Dose-response data indicated that the EC50 for ANF(99-126) was 1.1 x 10(-9) M. Brain natriuretic peptide (10(-6) M) increased cGMP to a level indistinguishable from that of ANF (10(-6) M). [Met-(O)]ANF was only half as potent as ANF, and atriopeptin I (10(-6) M) did not increase cGMP over basal levels. Preincubation of SK-NEP-1 cells with ANF, but not atriopeptin I (API), for two hours or longer, caused a concentration-dependent down-regulation of ANF-s-cGMP. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, and A23187 and its 4-bromo derivative, calcium ionophores, inhibited ANF-s-cGMP in a dose-dependent manner. A23187 inhibition was calcium dependent and promoted net cGMP degradation. Thirty-six hour preincubation with PMA, a procedure used to down-regulate PKC, abolished acute PMA inhibition of ANF-s-cGMP without having an effect on ANF-s-cGMP or on 4-bromo-A23187 inhibition thereof. These data indicate that PKC activation specifically inhibited ANF-s-cGMP but that PKC was not required for ANF-s-cGMP in SK-NEP-1 cells. Thus structurally related ANF peptides, protein kinase C (PKC) activators, calcium ionophores are potential modulators of ANF-s-cGMP in cells from this human renal cell line.     

Characterization of human interleukin-3 receptors on a multi-factor-dependent cell line

Recombinant human interleukin-3 (hIL-3) was radioiodinated by Bolton-Hunter method with maintenance of biological activity. Using 125I-hIL-3, hIL-3 receptors were characterized on a multi-factor-dependent cell line TF-1. Equilibrium binding studies revealed the existence of a single class of binding sites (667 +/- 306 sites/cell) with a Kd of 173 +/- 25 pM. Affinity labeling of TF-1 cells with 125I-IL-3 yielded two bands of 150 kDa and 85 kDa, implying molecular weights of 135 kDa and 70 kDa for the hIL-3 receptors.     

Phorbol ester receptors in cerebral cortex of cats with GM1 gangliosidosis

The pathogenesis of neuronal dysfunction in the gangliosidoses is poorly understood. Studies of the feline gangliosidoses and in vitro experiments implicate ganglioside inhibition of protein kinase C (PKC) in the pathogenesis of these neurological diseases. Therefore, in the present study, the binding of [³H]phorbol-12, 13 dibutyrate was measured to determine the levels of PKC in cerebral cortex of cats with GM₁ gangliosidosis (mutant) and age matched normal siblings. This binding of ([³H]PDB) to cerebral cortex homogenates in both normal and mutant cats was highly specific. The specificity of receptors was ascertained also from displacement studies using nonradioactive phorbol ester analogues to displace [³H]PDB bound to its receptors. In both mutant and normal cat brain, phorbol 12, 13-dibutyrate (PDB), 4--phorbol 12,13-didecanoate (-PDA) and 4--phorbol 12,13-dibenzoate (-PDBz) were highly potent (approximately to same degree) and effective in displacing [³H]PDB. On the other hand, 4- phorbol 12,13-diacetate (-PDA) was a weak displacer and 4--phorbol did not displace the bound [³H]PDB in either normal or mutant brain. Scatchard analysis of the binding data indicated a homogenous single class of binding sites in normal and mutant brain (Normal: K_d=1.42×10^–7 M, B_max=8.40 pmoles/mg protein. Mutant: K_d=1.60×10^–7 M, B_max=10.00 pmoles/mg protein). Sphingosine inhibited the binding to approximately the same extent in normal and mutant cortex. These studies demonstrate the presence of highly specific, homogenous, single type phorbol ester receptors in cerebral cortex of cats with GM₁ gangliosidosis which are qualitatively and quantitatively similar to normal cat brain.     

Phorbol ester stimulates calcium sequestration in saponized human platelets

When platelets are activated by agonists, calcium (Ca2+) is released from an intracellular storage site. Recent studies using fura-2 show that, after thrombin stimulation, the rise in free calcium is transient and returns to base-line levels in 2-3 min, while the transient following ADP stimulation lasts only 15-20 s. We reported previously that the phorbol ester 12,13-phorbol myristate acetate (PMA), added at nanomolar levels after thrombin, immediately accelerated the rate of return of calcium to the base line severalfold (Yoshida, K., Dubyak, G., and Nachmias, V.T. (1986) FEBS Lett. 206, 273-278). In the present study, we used both intact and saponized platelets to determine whether this is due to stimulation of calcium sequestration. Using fura-2 and intact platelets, we found 1) that PMA stimulated the restoration of free Ca2+ levels after ADP as well as after thrombin, and 2) that H-7, an inhibitor of protein kinase C (Ca2+/phospholipid-dependent enzyme), slowed the return of Ca2+ to baseline levels. Using saponized platelets, we also found 3) that pretreatment of platelets with PMA before saponin treatment increased the ATP-dependent 45Ca2+ uptake 2-fold, with a half-maximal effect at 5 nm; 4) that most of the Ca2+ released by ionomycin or by myoinositol 1,4,5-trisphosphate; and 5) that a GTP-binding protein inhibitor, guanosine 5'-O-(2-thiodiphosphate), decreased basal or PMA-stimulated 45Ca2+ uptake in saponin-treated platelets. Our data suggest that activation of protein kinase C stimulates the sequestration of Ca2+ independently of cAMP or myoinositol 1,4,5-trisphosphate.     

Phorbol ester stimulates expression of the human tryptophanyl-tRNA synthetase gene

The effect of the phorbol ester phorbol 12-myristate 13-acetate (PMA) on expression of the human interferon (IFN)-inducible tryptophanyl-tRNA synthetase (WRS) gene was studied. PMA caused an increase in the basal and IFN-induced WRS protein content in HeLa and HEK293 cultured cells. Besides, PMA upregulated WRS mRNA level in HeLa cells. Since PMA is known as a selective activator of protein kinase C (PKC) and is widely used to study the PKC-related pathways, these results show possible PKC involvement in regulation of the WRS gene expression. PKC inhibition by staurosporine (10 and 100 nM) had no effect on either basal or IFN-induced expression of WRS in either cell line. Consequently, PKC is not an indispensable element in WRS induction by IFN. Rather, PKC may activate WRS gene expression only by a distinct pathway.