A comparison of the relative chemosensitivity of human gliomas to tamoxifen and n-desmethyltamoxifen in vitro.

Tamoxifen has been shown to inhibit the proliferation of human gliomas in vitro. This inhibition is independent of tamoxifen's known anti-estrogenic properties. Tamoxifen is an inhibitor of protein kinase C (PKC), a calcium- and phospholipid-dependent serine kinase which plays a critical role in the proliferation of certain cell lines. Gliomas overexpress PCK, and their growth rate is coupled to the level of this key enzyme. As such, the effect of tamoxifen may be mediated by its inhibitory effect on PKC. To further investigate this possibility, we compared the chemosensitivity of cultured glioma lines to both tamoxifen and N-desmethyltamoxifen (DMT). DMT is the major metabolite of tamoxifen in humans and is a ten-fold more potent inhibitor of PKC. Seven lines were tested using the standard MTT assay, which quantitates metabolically active cells colorimetrically using a tetrazolium dye. Four of the seven lines were also tested using a tritiated thymidine uptake assay. In the MTT assay, all seven lines showed significantly greater sensitivity to DMT, while three of the four lines tested in the thymidine uptake assay were more sensitive to DMT. Correlation between the two assays was good. The dose of tamoxifen required to produce a 50% inhibition of optical absorbance or thymidine uptake (ID50) was typically five- to ten-fold greater than the ID50 for DMT, approximating the relative strength of the two compounds as PKC inhibitors. In addition to providing some support for the ypothesis that triphenylethylenes inhibit gliomas via PKC inhibition, these findings have clinical significance.(ABSTRACT TRUNCATED AT 250 WORDS)

Response of low-passage human malignant gliomas in vitro to stimulation and selective inhibition of growth factor-mediated pathways.

The proliferation of many nonglial tumors in vitro depends on the presence of nanomolar concentrations of one or more growth factors. To define the growth factor requirements of malignant glial tumors, the authors examined the response properties of four low-passage human malignant glioma lines to the following mitogens: epidermal growth factor (EGF), acidic and basic fibroblast growth factors (FGF's), insulin-like growth factor I (IGF-I), nerve growth factor (NGF), platelet-derived growth factor (PDGF), 12-O-tetradecanoyl-13-phorbol acetate (TPA), and serum. Each of the tumors showed increased deoxyribonucleic acid (DNA) synthesis (assessed by acid-precipitable [3H]-thymidine incorporation) in response to PDGF with a maximum effect at 50 ng/ml. Three tumors responded to EGF, three to IGF-I, two to acidic FGF, two to basic FGF, and two to TPA with maximum effects at 10, 50, 1, 1, and 10 ng/ml, respectively. None of the tumors responded to NGF. In the responsive tumors, optimum concentrations of EGF, IGF, TPA, acidic FGF, and basic FGF induced, at most, a two- to fourfold increase in [3H]-thymidine incorporation, which was only 30% to 50% of the response seen in 10% serum. In contrast, PDGF increased DNA synthesis eight- to 10-fold, equaling the effect of 10% serum. Measurements of cell proliferation also demonstrated a significant response to PDGF in each of the tumors. Appropriate concentrations of an anti-PDGF neutralizing antibody inhibited baseline DNA synthesis and proliferation in the absence of added growth factors, suggesting the possible role of PDGF in autocrine stimulation of these cells. However, this antibody produced only slight inhibition of serum-induced mitogenesis. Trapidil, an agent reported to inhibit the effects of PDGF, and polymyxin B, an inhibitor of protein kinase C, strongly inhibited baseline as well as PDGF- and serum-induced mitogenesis. It is concluded that, in the malignant gliomas studied, PDGF may be acting as a dominant mitogen to enhance DNA synthesis, and may function in autocrine stimulation. However, other factors contained in serum can also contribute to cell division.

Effect of tamoxifen on DNA synthesis and proliferation of human malignant glioma lines in vitro.

Previous studies in our laboratory have shown that proliferation of human malignant gliomas in vitro depends in part upon the activation of protein kinase C (PKC) and, conversely, can be blocked by inhibitors of PKC. Here, we examined the effect of tamoxifen, a known PKC inhibitor, on DNA synthesis and proliferation of an established human glioma line (U138) and two low passage cultures of explanted human glioblastomas. Tamoxifen produced a profound, dose-dependent inhibition of both [3H] thymidine incorporation and cell proliferation, with a 50% effective dose of 20 ng/ml under serum-free conditions and 50 to 200 ng/ml in the presence of 10% serum. These tumors were estrogen receptor negative and showed no mitogenic response to estradiol. Furthermore, concentrations of estradiol as high as 10 micrograms/ml had no effect on the tamoxifen-induced inhibition. This suggests that the mechanism of growth inhibition by tamoxifen in these gliomas did not involve an estrogen receptor-mediated process but may instead result from its inhibition of PKC. In view of the profound effect of tamoxifen on cultured gliomas at concentrations that can safely be achieved therapeutically, further in vitro and in vivo studies of this agent are warranted.

Response of malignant glioma cell lines to epidermal growth factor and platelet-derived growth factor in a serum-free medium.

The use of a serum-free culture system for assessing the growth factor responsiveness of malignant glial cells is described. The mitogenic properties of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) were examined in three human malignant glioma cell lines (T98G, U87, and U138). Each of the three had high-affinity EGF receptors and all responded in a dose-dependent fashion to physiological concentrations of EGF. These cell lines also showed a pronounced mitogenic response to PDGF which equaled or exceeded that achieved with EGF. Simultaneous stimulation with both factors produced an additive response, which approximated that obtained in medium supplemented with 10% fetal calf serum. The authors conclude that functional EGF and PDGF receptors were present in the human malignant glial tumors studied. The response of the human glioma lines to these growth factors in many respects parallels the response seen in fetal astrocytes tested under similar conditions. In contrast, the behavior of two chemically induced rat gliomas (9L and C6) differed significantly from that seen in the human lines, suggesting that the rat lines may not be entirely acceptable as models for studying the growth characteristics of human malignant glial tumors.

Response of malignant glioma cell lines to activation and inhibition of protein kinase C-mediated pathways.

To evaluate the role of protein kinase C-mediated pathways in the proliferation of malignant gliomas, this study examined the effect of a protein kinase C (PKC)-activating phorbol ester (12-O-tetradecanoyl-13-phorbol acetate or TPA) and a protein kinase C inhibitor (polymyxin B) on deoxyribonucleic acid (DNA) synthesis of malignant glioma cells in vitro. A serum-free chemically defined medium, MCDB 105, was employed for all studies. Two established human malignant glioma cell lines (T98G and U138), two rat glioma lines (9L and C6), and two low-passage human glioma lines (obtained from surgical specimens) were studied. With the exception of the C6 line, all tumors responded in a dose-dependent fashion to nanomolar concentrations of TPA with a median effective dose that varied from 0.5 ng/ml for the U138 glioma to 1 ng/ml for the T98G glioma. At optimal concentrations (5 to 10 ng/ml), TPA produced a two- to five-fold increase in the rate of DNA synthesis (p less than 0.05) as assessed by incorporation of 3H-thymidine. However, TPA had no additive effect on the mitogenic response produced by epidermal growth factor (EGF) or platelet-derived growth factor (PDGF). Inhibition of PKC using the antibiotic polymyxin B (20 micrograms/ml) abolished the TPA-induced mitogenic response in the five responsive lines tested. In two tumors (U138 and 9L), polymyxin B also eliminated EGF-, PDGF-, and serum-induced DNA synthesis as well as abolishing baseline DNA synthesis. These cells remained viable, however, as assessed by trypan blue exclusion; after removal of polymyxin B from the medium, they were able to resume DNA synthesis in response to TPA and serum. In the three other tumors (T98G and the two low-passage human glioma lines), growth factor-induced and serum-induced DNA synthesis were inhibited by approximately 25% to 85%. It is concluded that PKC-mediated pathways affect DNA synthesis in the human malignant glial tumors studied. The response of the glioma cells to TPA is similar to the responses seen in fetal astrocytes, but differs significantly from those reported for normal adult glial cultures. Because the response of the 9L glioma to TPA is similar to the responses seen in the human tumors, the 9L rat glioma model may prove useful for examining the role of PKC-mediated pathways in controlling glioma growth in vivo.