Fritz Hölzel

Stimulatory effects of androgen and antiandrogen on the in vitro proliferation of human mammary carcinoma cells

SummaryThe proliferation of three mammary carcinoma cell lines was explored for the effectiveness of dihydrotestosterone (DHT) and the antiandrogenic substances cyproterone acetate (CPA) or hydroxyflutamide. The cell growth, determined in multiple experimental cultures of the estrogen-sensitive lines MCF-7 and EFM-19, was stimulated by 10-9M to 10-6M DHT, whereas estrogen-resistant MFM-21 cells were unresponsive to the hormonal factors applied. Growth-promoting effects of 10-8M to 10-6M CPA were detected in cultures of those cell lines which were sensitive to estrogen and androgen. Competition experiments with DHT and the antiandrogens suggested involvement of the androgen receptor in the stimulation of cell growth by CPA. Participation of the estrogen receptor was excluded by lack of competition between CPA and the enhancement of proliferation by estradiol-17β. At the receptor level the anti-androgens were able to compete with androgen binding. The results of the study demonstrate androgenic properties of CPA in regard to the growth of human mammary carcinoma cells.

Karyotype alterations in human ovarian carcinoma cells during long-term cultivation and nude mouse passage

Cytogenetic analyses of the ovarian carcinoma line EFO-27 demonstrated a limited amount of chromosomal aberrations during long-term maintenance. Between passage 16 and 179 of the cultivation the karyotype shifted to a more homogeneous pattern of tetraploid cells, the occurrence of marker chromosomes mar1 [del(X)] and mar2 [t(X;2)], already present in low-passage cells, was stabilized and marker mar3 [del(9)] was added. After passage through the nude mouse, only near-tetraploid cells were preserved; mar1 to mar3 were abandoned and three additional marker chromosomes mar4 [t(4;9)], mar5 [t(8;9)], and mar6 [t(10;14)] were acquired. Marker 7, consisting of chromosome #16 with an amplified heterochromatic region was characteristically present in nearly all cells and passages of the cultivation. Pericentric variation of the heterochromatin position was found in chromosomes #9 with the exception of nude mouse tumor cells. Hetermorphism of chromosomes #9 and #16 appears to have a prominent role as structural aberration in EFO-27 cells. The results suggest the influence of in vitro and in vivo conditions acting as selection mechanisms concomitant with acquisition or loss of chromosomal markers.

Down-regulation of androgen receptor by progestins and interference with estrogenic or androgenic stimulation of mammary carcinoma cell growth

SummaryThe regulatory influence of medroxyprogesterone acetate (MPA) on estrogen and androgen receptors of the human breast cancer cell lines MCF-7 and EFM-19 was explored in conjunction with the growth-promoting properties of these steroids. In the absence of steroidal stimulation, up to 1 μM MPA had no effect on the proliferation of the MCF-7 cell strain used and of EFM-19 cells. Under stimulation with 10 nM 17β-estradiol or 1 μM dihydrotestosterone, dose-dependent inhibition of the cell proliferation rates by 0.1–1 μM MPA was observed. Binding of MPA to the androgen receptor (Kd=2.1 nM) but not to the estrogen receptor was demonstrable. During incubation of MCF-7 or EFM-19 cells with 1 μM MPA for 7 days, the estrogen and androgen receptor contents were down-regulated by approximately 50% and 60%, respectively. Likewise, the number of androgen-binding sites was reduced to 35% of the untreated controls after incubation of MCF-7 cells with 1 μM synthetic progestin R5020 for 7 days. The results indicate down-regulation of estrogen and androgen receptors by progestins in the absence of stimulatory effects on the proliferation of mammary carcinoma cells.

Effects of oestrogen and the antioestrogens, tamoxifen and LY117018, on four oestrogen-regulated RNAs in the EFM-19 breast cancer cell line

The EFM-19 cell line is a new breast cancer cell line whose proliferation has been reported to be stimulated by oestrogens and inhibited by the antioestrogen tamoxifen. Oestrogen receptor mRNA levels are higher in EFM-19 cells than in other oestrogen-responsive cell lines. The levels of four oestrogen-inducible RNAs [pNR-1, pNR-2, pNR-25 and pNR-100] were measured in EFM-19 cells. Oestradiol treatment increased the levels of the four regulated RNAs between 3-fold (pNR-100) and greater than 100-fold (pNR-2). The induction was half maximal between 1.5 x 10(-11) and 1.5 x 10(-10) M oestradiol. The effects of two antioestrogens, tamoxifen and LY117018, were measured on the expression of the oestrogen-regulated RNAs. Tamoxifen was a partial oestrogen agonist for the induction of the pNR-1 and pNR-25 RNAs but had very little effect on the pNR-2 and pNR-100 RNA levels. The pNR-2 RNA levels were less induced by tamoxifen in EFM-19 cells than in MCF-7 cells. LY117018 did not increase the levels of any RNA. The oestrogen-induced levels of the four RNAs were reduced by both antioestrogens to the RNA levels present in cells treated with the antioestrogens alone. LY117018 was at least 100-fold more potent than tamoxifen as an oestrogen antagonist.

Effect of continuous vs intermittent application of 3-OH-tamoxifen or tamoxifen on the proliferation of the human breast cancer cell line MCF-7 M1

SummaryThe antiproliferative potency of 5x10-7M tamoxifen (TAM) and 3-hydroxytamoxifen (3-OH-TAM) was investigated during continuous (8 days) or intermittent (2 h every 2nd or 3rd day, respectively) application to the oestrogen-receptor-positive, estradiol-sensitive human mammary carcinoma cell line MCF-7 M1, a variant of MCF-7 wild type. Growth modulation was evaluated in parallel by counting cells and by measuring DNA content. Continuous incubation resulted in a growth inhibition to 21.8±3.2% by 3-OH-TAM and to 39.5±4.8% by TAM when compared with control cultures defined as 100%. Intermittent addition induced a growth reduction to 23.0±2.1% by 3-OH-TAM and to 41.2±2.4% by TAM in relation to 100% controls. Addition of 3-OH-TAM for 2 h only at day 1 resulted in an inhibition to 70.3±3.2%, again in relation to 100% controls. When TAM was administered once for 2 h at day 1 it induced an inhibition to 79.0±4.9% at day 8. The in vitro results indicate that (a) at 5x10-7M3-OH-TAM has a better antiproliferative effectiveness than TAM, (b) the intermittent application is as effective as continuous application (no significant difference), and (c) the addition once a week reveals only a slight growth reduction after 8 days of culture.Application of the long-living TAM results in continuously high serum concentrations, which have been shown to create resistant cell clones. Compared to TAM the 3-OH metabolite has a considerably shorter half-life and its application in vivo reveals rise and fall of its serum concentrations. Since the presented data demonstrate that 3-OH-TAM is more potent than TAM and that the intermittent application is as effective as the continuous form, interval therapy with 3-OH-TAM may slow down the process of acquiring resistance to antioestrogens.

Effectiveness of antineoplastic drugs on the proliferation of human mammary and ovarian carcinoma cells in monolayer culture.

SummaryProcedures for the in vitro determination of the drug-induced inhibition of mammary and ovarian carcinoma cell growth were established. In monolayer cultures derived from advanced tumors, separation of epithelial carcinoma cells from concomitant cells of fibroblast-like or mesothelial appearance was achieved by differential trypsinization. The carcinoma cell character of the stock cultures was verified by chromosome analyses showing a high degree of aneuploidy for the epitheloid cell lines and euploidy for cells of apparently mesenchymal origin. When cultured carcinoma cells were injected in nu/nu mice, the tissue and cell cultures obtained from the heterotransplantation tumors closely resembled the original tumors and cell cultures in morphology, karyotype, and expression of tumor markers. The action of carcinostatic drugs in the logarithmic phase of the carcinoma cell proliferation was tested by kinetics experiments in multiple experimental cultures. In cell proliferation assays based on cell counts the 50% inhibition dose (ID50) of the drug effects was determined from the dose-response curves. Comparison of the ID50s revealed highly differential effectiveness of the drugs examined. The inhibitory effects were reproducible, rendering the procedures used suitable for testing the chemosensitivity of newly explanted gynecological carcinoma cells by proliferation assays.

Alteration of steroid hormone sensitivity during the cultivation of human mammary carcinoma cells.

SummaryDuring early cultivation steps of the newly derived and karyotyped human mammary carcinoma line EFM-19, the cells developed faster growth rates and became increasingly less responsive to the presence of serum in the culture medium. No drastic alterations of the morphology and of the karyotype were observed, and carcinoembryogenic antigen remained expressed during the course of the cultivation. In experimental incubations at various time intervals after the explantation, the cell proliferation was analyzed for dose-dependent effects of estradiol, cortisol, progesterone, and testosterone. After 16 wk of cultivation of the stock culture in the presence of estradiol, the cells had acquired a distinct sensitivity to estradiol resulting in permanent growth enhancement. The withdrawal of cortisol from the medium of the stock culture subsequently provoked the loss of the initially noted stimulation of the proliferation by cortisol. The stimulatory effect of progesterone on the proliferation was reversed to inhibition when the stock culture was deprived of cortisol in the growth medium. The results indicate that the choice of steroid hormones in the stock culture medium was determining the quality of the cellular growth responses.

Individual chemosensitivity of in vitro proliferating mammary and ovarian carcinoma cells in comparison to clinical results of chemotherapy

SummaryCell lines established from advanced mammary and ovarian carcinomas were assayed for the inhibition of in vitro proliferation by various antineoplastic drugs. The assays were performed with multiple experimental cultures derived from stock cultures of the tumor cell lines in early passages of the cultivation. As determined by comparison of the 50% inhibition of in vitro growth, differential sensitivity of the individual cell lines was observed. Based on the 2-h plasma level of the drugs as discriminatory threshold between resistance and sensitivity, the in vitro effectiveness of each, drug on the individual cell lines was compared with the clinical results of chemotherapy applied to the corresponding patients. In total, positive in vitro/in vivo correlations were observed in 39 of 42 cases. The 17 cell lines evaluated retrospectively were resistant to those drugs which had been tried unsuccessfully during chemotherapy. Among the 25 cases tried prospectively 11 cases showed sensitivity in vitro and in vivo, and furthermore 11 prospective cases were resistant in vitro and in vivo.

Influence on parathyroid hormone storage in normal and adenomatous parathyroid tissue by stimulation and inhibition in vitro.

The aim of the study was to elucidate morphological and secretory differences between normal and adenomatous parathyroid tissue after incubations with neutral, stimulatory, and inhibitory culture medium for 2, 6, and 24 hr. The glandular response was determined 1) immunohistochemically by revealing alterations of the parathyroid hormone (PTH) distribution pattern, and 2) radioimmunologically by measuring the amounts of PTH secreted into the medium. Normal parathyroids contained varying amounts of intracellular PTH, even in adjacent cells. Before incubation, 350/c of the parathyroid cells displayed high, 25% medium, and 40% low amounts of PTH. Hypocaicemic stimulation reduced the intracellular PTH content in normal tissue markedly within 2 hr, indicating rapid depletion of the PTH stores. Simultaneously, the PTH secretion into the culture medium was enhanced by a factor of 2.9. Hypercalcemic inhibition induced an increase of the intracellular PTH within 2 hr; this increase continued during incubation for up to 6 hr. The elevated storage of the hormone is interpreted as the result of the reduction by 40% of the PTH secretion, and of the ap-

IFN-γ-Induced Change in Microtubule Organization and α-Tubulin Expression During Growth Inhibition of Lung Squamous Carcinoma Cells

In cultures of KNS-62 cells derived from a human lung squamous cell carcinoma, the initial growth arrest in the continuous presence of interferon-γ (IFN-γ) turned to cytopathic effects after 2 days of treatment. The remaining viable cells showed grossly distorted morphology, with enlargement and extensions up to 5 cell diameters. The presence of apoptotic cells was shown 3 days after treatment with IFN-γ. Immunocytochemically, the microtubular structures appeared augmented and highly aggregated. The level of α-tubulin-specific mRNA was distinctly increased after administration of IFN-γ, and the amount of extractable α-tubulin protein was reduced. In parallel kinetics experiments, growth arrest by serum depletion or by contact inhibition during confluence resulted in reduced levels of α-tubulin-specific mRNA and in slightly elevated α-tubulin protein. The IFN-γ-induced effects suggest interference with assembly or maintenance of the tubulin cable network, presumably associated with cell deformation and cyt...