Biomedical subjects

C Knabbe

Publications and source records attributed to C Knabbe.

At least 37 records · Page 2Linked to original sources

PubMed1993

[Androgen regulation of secreted growth factors in prostate carcinoma cell and tumor lines].

Previous studies have shown that part of steroid hormone action on hormone dependent carcinoma cells is mediated through secreted autocrine and paracrine growth factors. Coculture experiments using the androgen receptor positive human prostate carcinoma cell line LNCaP as feeder cells and the androgen receptor negative prostate cell line DU 145 as indicator cells, such as experiments with conditioned medium suggest that androgens might regulate proliferation of prostate carcinoma through a similar mechanism. LNCaP and DU 145 cells express high affinity EGF-receptors and show an increased growth rate under treatment with EGF, TGF alpha and FGF. The growth stimulating potential of LNCaP-conditioned medium can be enhanced by androgens. The polyanionic compounds suramin and dextran sulfates which have been shown to inactivate a variety of growth factors e.g. EGF/TGF alpha inhibit growth of LNCaP cells and DU 145 cells in a dose dependent and reversible fashion. Growth stimulation of LNCaP cells by EGF/TGF alpha can be completely reversed by simultaneous addition of polyanions but they inhibit androgen stimulation only partially. These data suggest the existence of at least two different mechanisms of growth regulation by androgens which can be distinguished by their different sensitivity of prostatic carcinoma cells to growth factor inhibitory agents. In order to investigate the therapeutic potential of these substances in complex, heterogeneous cell systems of solid tumors we treated 8 representative human prostate cancer lines in the nude mouse model. Systemic applications of polyanions revealed significant growth inhibition in hormone dependent as well as hormone independent xenografts. In androgen responsive lines growth inhibition was intensified by additional androgen withdrawal.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens↗

PubMed1992

Regulation of fibroblast growth factor-like protein(s) in the androgen-responsive human prostate carcinoma cell line LNCaP.

Growth of the normal and malignant prostate is known to be regulated by androgens. Part of their effect has been suggested to be mediated through coordinated regulation of secreted growth factors with autocrine function. We now examine the biological role of preferentially paracrine acting factors in growth control of prostate cancer, i.e. fibroblast growth factor(s) (FGF). Coculture experiments using the androgen-responsive human prostate carcinoma cell line LNCaP as feeder cells and the FGF-dependent human adrenal carcinoma SW-13 cell line as target cells show that (i) LNCaP cells induce growth of SW-13 cells, (ii) even higher stimulation of SW-13 cells is seen in the presence of androgen treated LNCaP cells and (iii) a specific anti-bFGF antibody inhibits growth of SW-13 cells induced by androgen treated LNCaP cells; no proliferation of SW-13 cells occurs in the absence of LNCaP cells. Partial purification of the secretory products of LNCaP cells was performed by affinity chromatography using a heparin sepharose column. Fractions were tested for biological activity in a soft agar assay with SW-13 cells. Several activities could be detected, the main activity was eluted with about 1.5 M NaCl. These data suggest that androgen treatment of LNCaP cells leads to enhanced secretion of proteins which belong to the FGF-family.

Adrenal Gland Neoplasms↗

PubMed1991

Post-transcriptional destabilization of estrogen receptor mRNA in MCF-7 cells by 12-O-tetradecanoylphorbol-13-acetate.

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the regulation of the estrogen receptor (ER) was investigated in this study. After treatment with 100 nM TPA the concentration of receptor protein was measured using an enzyme immunoassay. By 24 h the receptor protein declined by about 80% from a level of approximately 236 fmol of ER/mg of protein in control cells to 50 fmol of ER/mg of protein in cells treated with TPA. Similar results were obtained with an estrogen receptor ligand binding assay. After removal of TPA, the level of ER returned to control values. 4-alpha-Phorbol, a compound related to TPA, had no effect on ER. The effects of TPA on ER expression appear to be mediated by activation of protein kinase C as H-7, an inhibitor of protein kinase C, blocks these effects. In addition to the effect on ER protein, TPA treatment also resulted in a decrease in the steady-state level of ER mRNA as determined by a RNase protection assay. The metabolic inhibitor cycloheximide was unable to prevent the TPA-induced decrease in ER mRNA. Transcription run-off experiments demonstrated that TPA had no effect on ER gene transcription. A half-life study demonstrated that TPA decreased ER mRNA half-life by a factor of 6 from approximately 4 h in control cells to 40 min in TPA-treated cells. These data suggest that the decline in ER expression is mediated by post-transcriptional destabilization of ER mRNA.

Cycloheximide↗

PubMed1991

Transforming growth factor beta 1 induces cachexia and systemic fibrosis without an antitumor effect in nude mice.

While stimulating the growth of fibroblasts, transforming growth factor beta 1 (TGF-beta 1) inhibits the growth of various normal and malignant cell lines in vitro. We studied the effects of TGF-beta 1 in vivo. The level of TGF-beta 1 in serum was maximally elevated 2 h after injecting 1 muCi of 125I-TGF-beta 1 into the peritoneal cavity of nude mice. Five h after the i.p. administration of 10 micrograms of unlabeled TGF-beta 1, 20 ng/ml of TGF-beta-like material in serum were detected by a radioreceptor assay on A549 lung carcinoma cells. Trichloracetic acid-precipitable 125I-TGF-beta 1 was taken up by liver, spleen, lungs, kidneys, and tumor tissue but not by the brain. At doses exceeding 2 micrograms/day, TGF-beta 1 induced a generalized interstitial fibrosis and a cachexia, which was not mediated by elevated serum levels of tumor necrosis factor alpha as determined by Western blot analysis and enzyme-linked immunosorbent assay. A total of 200,000 cells of the estrogen receptor-negative human breast cancer line MDA-MB-231, which had been shown to be maximally growth inhibited in vitro by 40 pM TGF-beta 1 and to have high-affinity receptors (9, 11, 12), were injected into the mammary fat pad of each nude mouse. The duration of treatment was 16 days with ten animals in the control group and five animals in the treated groups. The dose ranged from 1 to 4 micrograms per animal daily. The treatment was started 24 h after the injection of the tumor cells. Tumor growth was not significantly affected at either nontoxic or toxic doses of TGF-beta 1. Thus, we have demonstrated that TGF-beta 1, apart from being a local growth factor, has systemic effects, such as cachexia and multiple fibrosis. Its role as an antitumor agent may be limited.

Animals↗

PubMed1991

Growth factors in human prostate cancer cells: implications for an improved treatment of prostate cancer.

It has been previously shown that estrogens may exert their action on human breast cancer cells through coordinated control of secreted growth factors which act in an autocrine and paracrine fashion. Growth stimulation of the androgen receptor negative prostate carcinoma cell line DU-145 by dihydrotestosterone in the presence of the androgen-responsive human prostate carcinoma cell line LNCaP now indicates that androgens may regulate growth of prostate carcinoma cells through related mechanisms. A variety of androgen-regulated growth modulatory activities with autocrine and paracrine potential can be detected in conditioned media from LNCaP cells partially purified by ion exchange chromatography. Androgen-induced growth of LNCaP cells is partially inhibited by the polyanions suramin and dextran sulfates which antagonize growth factor action. These data suggest the existence of at least two different mechanisms of growth regulation by androgen which can be distinguished by their different sensitivity to growth factor inhibitory agents. We conclude that the combination of antipeptidergic substances and androgen withdrawal would represent a new and promising strategy for treatment of human prostate cancer.

Cell Division↗

PubMed1991

Tissue culture conditions determine the effects of estrogen and growth factors on the anchorage independent growth of human breast cancer cell lines.

We determined the effects of epidermal growth factor, insulin-like-growth-factor-1 and estradiol on the anchorage independent growth of the estrogen receptor positive human breast cancer cell lines MCF7 and T-47D. In serum free conditions growth factors but not estrogen induced a dose dependent stimulation of growth in both cell lines. The ability of estrogen to induce colony formation of early passage MCF7 cells (less than 100) was strictly correlated to the concentration of sulfatase and charcoal treated calf serum (CCS) with a maximal effect at a concentration of 5% CCS and 10 nM estradiol. CCS alone had no stimulatory effect on the anchorage independent growth of early passage MCF7 cells, but increased colony formation in late passage (greater than 1000) MCF7 and T-47D cells. The growth of late passage MCF7 cells was inhibited by antiestrogen. Thus, the presence of serum components is necessary for the effect of estrogen but not for the effects of growth factors on the anchorage independent growth of estrogen receptor positive human breast cancer cell lines; after a prolonged period of tissue culture serum components switch their function from indirectly modulating estrogen effects to directly stimulating growth in the absence of estrogen.

Breast Neoplasms↗

PubMed1991

Induction of transforming growth factor beta by the antiestrogens droloxifene, tamoxifen, and toremifene in MCF-7 cells.

We have previously shown that transforming growth factor beta (TGF beta) is a hormonally regulated negative growth factor in estrogen responsive MCF-7 human breast cancer cells. We have now compared the antiestrogens tamoxifen, droloxifene (3-hydroxytamoxifen), and toremifene in their ability to induce the secretion of autoinhibitory TGF beta by MCF-7 cells. The main results are as follows: induction of TGF beta secretion by droloxifene is about two to three times higher than by identical concentrations of tamoxifen or toremifene. A 5-10 times higher concentration of tamoxifen or toremifene than droloxifene is necessary to reach a similar induction of TGF beta secretion. In contrast to tamoxifen, intermittent application of droloxifene is as effective as continuous treatment in inducing TGF beta secretion. We conclude from these data that TGF beta proteins represent markers of antiestrogen action and might also play a pivotal role in their mechanism of action. Droloxifene is a more effective inducer of TGF beta and a more potent growth inhibitor for estrogen responsive human breast cancer cells than tamoxifen and toremifene in vitro. Therefore, droloxifene might also possess a higher antiestrogenic potential in treatment of human breast cancer.

Antineoplastic Agents↗

PubMed1989

Transforming growth factors type beta 1 and beta 2 are equipotent growth inhibitors of human breast cancer cell lines.

At least one member of the TGF-beta family, TGF-beta 1, has been previously shown to inhibit the anchorage-independent growth of some human breast cancer cell lines (Knabbe et al., 1987; Arteaga et al., 1988). Members of the TGF-beta family might, therefore, provide new strategies for breast cancer therapy. We have studied the inhibitory effects of TGF-beta 1 and TGF-beta 2 on the anchorage-independent growth of the oestrogen receptor-negative cell lines MDA-MB-231, SK-BR-3, Hs578T, MDA-MB-468, and MDA-MB-468-S4 (an MDA-MB-468 clone not growth inhibited by EGF) and the estrogen receptor-positive cell lines MCF7, ZR-75-1, T-47D. TGF-beta 1 and TGF-beta 2 caused a 75-90% growth inhibition of MDA-MB-231, SK-BR-3, Hs578T, and MDA-MB-468 cells and a 50% growth inhibition of ZR-75-1 and early passage (less than 100) MCF7 cells. T-47D cells responded to TGF-beta only in serum-free conditions in the presence of IGF-1 or EGF. The growth of MDA-MB-468-S4 cells and late passage (greater than 500) MCF7 cells was not inhibited by TGF-beta 1 or TGF-beta 2. TGF-beta-sensitive MCF7 and MDA-MB-231 cells did not respond to Muellerian inhibiting substance (MIS), a TGF-beta-related polypeptide. TGF-beta 1 or TGF-beta 2 were mutually competitive for receptor binding with a similar affinity (Kd 25-130 pM, 1,000-13,000 sites per cell). To determine the time course of the TGF-beta effect, an anchorage-dependent growth assay was carried out using MDA-MB-231 cells. Growth inhibition occurred at 6 days, and cell-cycle changes were seen 12 hr after the addition of TGF-beta. Cells accumulated in the G1 phase and were thus inhibited from entering the S-phase. These data indicate that TGF-beta is a potent growth inhibitor in most breast cancer cell lines and provide a basis for studying TGF-beta effects in vivo.

Anti-Mullerian Hormone↗

PubMed1989

Role of transforming growth factor-alpha in human prostate cancer cell growth.

Because of the influence of transforming growth factor-alpha (TGF alpha) on the cell growth in other cancer cell systems, we investigated the growth-regulatory role of TGF alpha in human prostate cancer cells. TGF alpha (5 ng/ml) stimulated LNCaP cell growth in monolayer to 60% of the level seen with dihydrotestosterone (DHT). Both DHT and TGF alpha increased cloning in soft agar twofold above that in controls. Metabolism of thymidine and uridine was also increased as evidenced by increased uptake of these macromolecule precursors. In addition, intracellular signalling as indicated by phosphatidyl inositol turnover was also increased by TGF alpha and DHT. Conditioned media contained TGF alpha by radioimmunoassay (RIA), transforming activity by rat kidney fibroblast (NRK) colony formation, and epidermal growth factor (EGF) receptor competable activity by radioreceptor assay. EGF receptors were present by binding assay and immunoprecipitation. These data demonstrate the presence of an autostimulatory growth loop in hormone-responsive human prostate cancer cells.

Cell Division↗

PubMed1989

Differential effects of transforming growth factor beta on human prostate cancer cells in vitro.

Transforming growth factor beta (TGF beta) exerts a wide spectrum of activity on many different cell types. Since TGF beta inhibits the growth of a variety of epithelial tumor cells in vitro, we examined the effects of TGF beta on the human prostate cancer cell lines DU145, PC3 and LNCaP for possible inhibitory activity. Growth in monolayer was initially inhibited in a dose-response fashion in the two androgen-independent cell lines, PC3 and DU145 but not in the androgen-dependent LNCaP cells. The rate of growth of the PC3 and DU145 cells treated with TGF beta, however, eventually returned to control levels despite retreatment with TGF beta. Anchorage-independent growth was inhibited to 55% and 16% control levels in PC3 and DU145, respectively. Scatchard analysis showed 1500 and 2900 TGF beta binding sites/cell on DU145 and PC3 cells with Kd = 6.9 and 12 x 10(-12) M, respectively. High-affinity binding could not be demonstrated on LNCaP cells. We also explored the possibility that TGF beta was secreted by these cells. Analysis of conditioned media by immunoprecipitation and a radioreceptor assay showed secretion of TGF beta into the media by DU145 and PC3 but not by LNCaP. Northern analysis showed the presence of TGF beta mRNA in DU145 and PC3, but not in LNCaP. These data indicate that TGF beta might serve as an autocrine inhibitory factor in prostate cancer. In addition, because TGF beta affects a wide range of cell types, TGF beta production by prostate cancer cells may contribute an important paracrine function in the development of tumor stromal tissue and metastases.

Androgens↗

PubMed1988

Growth regulatory peptide production by human breast carcinoma cells.

The mechanisms by which human breast cancers regulate their own growth have been studied by us in an in vitro model system. We showed that specific growth factors (IGF-I, TGF alpha, PDGF) are secreted by human breast cancer cells. A variety of experiments suggest that they are involved in tumor growth and progression. These activities are induced by estradiol in hormone-dependent breast cancer cells and secreted constitutively by estrogen-independent cells. Concentrates of conditioned medium derived from breast cancer cells can induce the growth of hormone-dependent cells in vivo in athymic nude mice. Hormone-dependent breast cancer cells also secrete TGF beta. TGF beta is growth inhibitory. Growth inhibitors such as antiestrogens or glucocorticoids increase TGF beta secretion. An antiestrogen-resistant mutant of MCF-7 cells does not secrete TGF beta when treated with antiestrogen, but is growth inhibited when treated with exogenous TGF beta. Thus, TGF beta functions as a negative autocrine growth regulator and is probably responsible for some of the growth inhibitory effects of antiestrogens.

Breast Neoplasms↗

PubMed1988

Multihormonal regulation of insulin-like growth factor-I-related protein in MCF-7 human breast cancer cells.

MCF-7 human breast cancer cells have been studied for hormonal regulation of secretion of an insulin growth factor-I (IGF-I)-related growth factor. 17 beta-Estradiol, which is required for tumorigenesis of the cell line in the nude mouse and which stimulates proliferation in vitro, was able to significantly induce IGF-I secretion at 10(-13) M, with maximal induction at 10(-11) M. Under optimal conditions IGF-I could be induced 4-fold after 4 days. Demonstration of estrogenic stimulations required removal of phenol red, a weak estrogen, from the cell culture medium. In addition to estrogen, insulin, epidermal growth factor, and transforming growth factor alpha induce both cellular proliferation and IGF-I secretion, while growth inhibitory antiestrogens, transforming growth factor beta, and glucocorticoids have the opposite effect. In each case, modulations in IGF-I secretion preceeded effects on cellular proliferation. IGF-I was not regulated by human GH, basic fibroblast growth factor, platelet-derived growth factor, or PRL, none of which affected proliferation rate. Thus, regulation of IGF-I secretion in human breast cancer is controlled by different hormones from those previously reported in human fibroblasts. Regulation of IGF-I by neither estrogen nor antiestrogen was associated with changes in steady-state mRNA levels; thus regulation may occur at a step beyond mRNA. We conclude that IGF-I production is tightly coupled to growth regulation by estrogens, antiestrogens, and other hormones and may contribute to autocrine and/or paracrine growth regulation by these agents in breast cancer.

Breast Neoplasms↗

PubMed1987

Effect of v-rasH oncogene transfection on estrogen-independent tumorigenicity of estrogen-dependent human breast cancer cells.

Spontaneous or therapeutically induced progression of hormone-dependent human breast cancer to a form not amenable to endocrine treatment has been frequently recorded in clinical settings. In an experimental model system, we have changed the estrogen-dependent tumorigenicity of a human breast cancer cell line, MCF-7, to an independent state by stably introducing a model oncogene, v-rasH, into this cell line by means of DNA transfection. We now show that the oncogene-transfected hormone-independent MCF-7 cells may secrete diffusible tumorigenic factors that not only support their own tumor growth in vivo, but are also humorally active in partially triggering the tumor growth of wild type previously nontumorigenic MCF-7 cells, even when the wild type cells are implanted at a distant anatomical site in the same animal. Estrogen-independent tumor formation by MCF-7 cells was also induced in 50% of animals given injection by continuous administration of conditioned media from MCF-7-ras cells. However, the wild type tumors had limited tumor growth. Tumors were verified as adenocarcinomas and by Southern blotting were shown to be derived from the cells injected. In an in vitro coculture assay, a 5- to 7-fold enhancement in anchorage-independent growth of MCF-7 cells was observed in the presence of MCF-7-ras feeder cell layer. These data suggest that v-rasH-induced estrogen-independent tumorigenicity of human breast cancer cells occurs by secretion of mitogens which may function in an endocrine manner.

Animals↗

PubMed1987

Evidence that transforming growth factor-beta is a hormonally regulated negative growth factor in human breast cancer cells.

The hormone-dependent human breast cancer cell line MCF-7 secretes transforming growth factor-beta (TGF-beta), which can be detected in the culture medium in a biologically active form. These polypeptides compete with human platelet-derived TGF-beta for binding to its receptor, are biologically active in TGF-beta-specific growth assays, and are recognized and inactivated by TGF-beta-specific antibodies. Secretion of active TGF-beta is induced 8 to 27-fold under treatment of MCF-7 cells with growth inhibitory concentrations of antiestrogens. Antiestrogen-induced TGF-beta from MCF-7 cells inhibits the growth of an estrogen receptor-negative human breast cancer cell line in coculture experiments; growth inhibition is reversed with anti-TGF-beta antibodies. We conclude that in MCF-7 cells, TGF-beta is a hormonally regulated growth inhibitor with possible autocrine and paracrine functions in breast cancer cells.

Breast Neoplasms↗

PubMed1987

Growth regulation of human breast carcinoma occurs through regulated growth factor secretion.

We describe studies on human breast cancer in which it is shown that specific growth factors (IGF-I, TGF alpha, PDGF) are secreted by human breast cancer cells and likely to be involved in tumor growth and progression. These activities are regulated by estradiol in hormone-dependent breast cancer and secreted constitutively by hormone-independent cells. These growth factor activities can induce the growth of hormone-dependent cells in vivo in athymic nude mice. Hormone-dependent breast cancer cells also secrete TGF beta, a growth-inhibitory substance, when treated with antiestrogens. TGF beta functions as a negative autocrine growth regulator and is responsible for some of the growth-inhibitory effects of antiestrogens.

Animals↗

PubMed1987

Activation of growth factor secretion in tumorigenic states of breast cancer induced by 17 beta-estradiol or v-Ha-ras oncogene.

The MCF-7 human breast cancer cell line responds to estrogen stimulation in vitro by increased secretion of growth factors and proliferation and in vivo by tumor formation in the nude mouse. To test a possible role of growth factor secretion in expression of the tumorigenic phenotype, we stably transfected MCF-7 cells with the v-Ha-ras oncogene to produce the MCF-7ras cell line. The MCF-7ras cell line was tumorigenic in the absence of estrogens and secreted 3- to 5-fold elevated levels of a high molecular weight form of a type alpha transforming growth factor-like growth factor, type beta transforming growth factor, and insulin-like growth factor I. MCF-7ras cells, in contrast to MCF-7, were less sensitive to further growth stimulation by estrogen, type alpha transforming growth factor, and insulin-like growth factor I and showed little change in receptor levels for these hormones. Conditioned medium from MCF-7ras cells as well as two of its component growth factors (insulin-like growth factor I and type alpha transforming growth factor) replaced estrogen in stimulating MCF-7 colony formation in vitro. A coordinate increase in growth factor secretion by human breast cancer may contribute to its escape from estrogen dependence.

Breast Neoplasms↗

PubMed1986

8th San Antonio Breast Cancer Symposium--Plenary lecture. Autocrine and paracrine growth regulation of human breast cancer.

We consider the hypothesis that estrogen control of hormone dependent breast cancer is mediated by autocrine and paracrine growth factors secreted by the breast cancer cells themselves. Though we show direct, unmediated effects of estrogen on specific cell functions, we also provide evidence that human breast cancer cells secrete a collection of growth factors (IGF-I, TGF alpha, TGF beta, a PDGF-like competency factor, and at least one new epithelial colony stimulating factor). Some of these are estrogen-regulated in hormone dependent cells, and are constitutively increased in cells which acquire independence either spontaneously or by ras transfection. Collectively, the secreted growth factors are capable of promoting tumor formation by MCF-7 cells in nude mice, though not to the same extent as estrogens. There would seem to be potential for clinical intervention in the autocrine and paracrine control of breast cancer cells, including some cells which are no longer dependent on estrogens.

Breast Neoplasms↗

PubMed1983

Regulation and compartmentalization of androgens in rat prostate and muscle.

Male rats were castrated and treated with various doses of testosterone (T) or 5 alpha-dihydrotestosterone (DHT) by implantation of steroid releasing polydimethylsiloxane depots. After 14 days, the animals were killed and the concentration of T, DHT and 5 alpha-androstane-3 alpha,17 beta-diol (Diol) was measured by radioimmunoassay in plasma, in the homogenates of the prostate (PR), the bulbocavernosus/levator ani muscle (BCLA), skeletal muscle (SM) and heart muscle (HM), as well as in the cytosol and the nuclear fraction of these tissues. Additionally, the weight and DNA content of PR and the levator ani muscle were measured. The main results were: After T or DHT administration the main androgens in plasma were T or Diol, respectively, the concentration of each being linearly dependent from the administered steroid dose (T after T-: r = 0.97, P less than 0.001. Diol after DHT administration: r = 0.97, P less than 0.001). T in a dose of 60 micrograms/24 h maintained the concentrations of T, DHT and Diol in plasma in the range of untreated controls. Irrespective of the T doses administered, its concentration in PR tissue was comparatively low. T administration resulted in a significant accumulation of DHT in PR, when compared to plasma T. The steepest increase of DHT in PR was found with low doses of T administered (0.6-12 micrograms/24 h). At higher doses (12-600 micrograms/24 h) the further increase in the tissue was rather parallel to that of plasma T. A comparable picture was found for the DHT concentration in cytosol and in nuclei. The Diol concentration in the cytosol was linearly related to the administered T dose (r = 0.80, P less than 0.001). This steroid could not be detected in nuclei (less than 0.5 pmol/mg DNA). Administration of DHT in principle mimicked the DHT data observed after T treatment. In muscle homogenates no accumulation of neither T, DHT nor Diol was observed. In the nuclear fractions only after the highest doses of T measurable amounts of T and DHT were found. Whereas a positive correlation between DHT values in nuclei and weight increases in prostate was present, no such parallelism could be demonstrated in muscular tissue. The data are discussed with respect to the present knowledge of androgen binding and metabolism in different organs and to a possible specific role of nuclear DHT accumulation in mediating androgen dependent processes.

Androgens↗