Biomedical subjects

M E Gleave

Publications and source records attributed to M E Gleave.

61 records · Page 4Linked to original sources

PubMed1993

Epidermal growth factor receptor-mediated autocrine and paracrine stimulation of human transitional cell carcinoma.

Stromal-epithelial interactions may play a key role in tumor growth and metastasis. We have established a model to study the cellular and molecular basis of this paracrine interaction both in vivo and in vitro using a human transitional cell carcinoma cell line (WH). s.c. coinoculation of 1 x 10(6) WH cells with 1 x 10(6) nontumorigenic fetal rat urogenital sinus mesenchymal (rUGM) cells in athymic mice accelerated carcinoma growth 20 times faster than isolated WH cell inoculations and 4 times faster than coinoculations of the same number of NIH-3T3 or human bladder fibroblasts. Characterization of these chimeric tumors with immunohistochemical and DNA dot-blot analyses documented their predominantly human component. To evaluate the underlying mechanisms involved in this paracrine-mediated in vivo tumor growth acceleration, Northern analyses for growth factors (GFs) and extracellular matrix (ECM) expression in the different cell lines, as well as in vitro mitogenic assays, were performed. Northern analysis revealed basic fibroblast growth factor, transforming growth factor alpha, and epidermal growth factor receptor expression by WH cells but not rUGM cells; ECM components (fibronectin and collagens I and IV) were expressed only in the fibroblast cell lines. Cell type-specific paracrine growth factors are produced by cultured stromal and epithelial cells with a 2-3-fold bidirectional increase in WH and rUGM cell growth when cultured with reciprocal cell-type conditioned medium. An autocrine growth loop was observed for WH but not rUGM cells. WH cell growth is stimulated in vitro by low concentrations of transforming growth factor alpha and epidermal growth factor, while rUGM cell growth is stimulated 3-fold by basic fibroblast growth factor. Antiepidermal growth factor receptor antibodies completely inhibited autocrine and paracrine pathways stimulating WH cell growth, while anti-basic fibroblast growth factor antibodies had no inhibitory effect. These observations suggest that autocrine and paracrine growth factor stimulation of WH bladder carcinoma cell growth is most likely mediated by an epidermal growth factor receptor-related pathway. The predominant expression of ECM by fibroblasts in this model suggests that stromal cell ECM components may modulate tumor cell growth and angiogenesis possibly through mechanisms involving cellular adhesion, chemotaxis, or growth factor action.

3T3 Cells↗

PubMed1993

Autocrine regulation of prostate-specific antigen gene expression in a human prostatic cancer (LNCaP) subline.

Prostate-specific antigen (PSA), a M(r) 34,000 serine protease, is recognized as a useful marker for the detection and prognosis of patients with prostate cancer. Although serum PSA is an excellent prognostic indicator, an increasing number of factors were found to regulate the PSA expression of prostatic cancer cells, which include androgenic steroids, the growth factors (GFs) and the extracellular matrix. The purpose of this study is to define a novel protein factor that may be responsible for regulating PSA expression by androgen-independent (AI) human prostate cancer cells. We have established a LNCaP subline (C4) from a parental LNCaP tumor grown in a castrated host. The C4 subline overexpressed PSA mRNA and protein. Serum-free conditioned medium (CM) isolated from the C4 subline is able to stimulate PSA gene expression in parental LNCaP cells in a concentration-dependent manner. This autocrine PSA-inducing activity was found to be organ specific because CMs from other fibroblast cell lines (such as bone, prostate, kidney, and lung fibroblasts) and the CMs from several prostatic carcinoma cell lines (such as parental LNCaP, PC-3, DU-145) and a bladder transitional carcinoma cell line (WH) fail to exhibit similar activity. The activity of the CM from the C4 subline cannot be substituted by GFs such as TGF-alpha, TGF-beta, bFGF, HGF, KGF, or NGF; neuropeptide (bombesin/GRP); secondary messenger analogue (dibutyryl cAMP); beta 2-adrenergic agonist (isoproterenol); or alpha 1-adrenergic agonist (phenylephrine), indicating that the factor(s) may be a novel prostate-specific autocrine factor (PSAF). Both androgen and PSAF exhibit an additive effect on up-regulating PSA gene expression, suggesting that the signal transduction pathway elicited by PSAF may differ from that mediated by the androgen receptor. Further characterization of PSAF by heat, acid, and trypsin digestion revealed that the PSAF may be a protein factor with a unique amino acid composition. These observations suggest that a novel autocrine pathway mediated by PSAF may be responsible for the overexpression of PSA mRNA and protein in a human prostatic cancer cell line. The potential clinical significance of this factor will be discussed.

Castration↗

PubMed1992

Serum prostate specific antigen levels in mice bearing human prostate LNCaP tumors are determined by tumor volume and endocrine and growth factors.

The ability of prostate-specific antigen (PSA) to predict tumor volume and stage in patients with prostate cancer would be improved if factors regulating its production and clearance were better defined. A thorough understanding of the pharmacokinetics (regulation of production, metabolism, and excretion) of PSA has been precluded, however, by the absence of an in vivo animal model. The purposes of this study are to develop a murine model for evaluating PSA pharmacokinetics in vivo and to assess factors that influence PSA production in vitro. The human prostate cancer cell line, LNCaP, was chosen because it is androgen sensitive and PSA positive. Although LNCaP cells are usually nontumorigenic when inoculated s.c. in athymic mice, coinoculation of 1 x 10(6) LNCaP cells with 1 x 10(6) human bone fibroblasts reliably produces PSA-secreting carcinomas. This LNCaP model provides accurate correlation between tumor volume and serum PSA levels (r = 0.94) and demonstrates that tumor volume and androgens are codeterminants of circulating PSA levels. Following castration, serum PSA levels decrease rapidly up to 8-fold and increase up to 20-fold following androgen supplementation, without detectable castration-induced tumor cell death or concomitant changes in tumor volume. Serum PSA levels increase 0.24 ng/ml/mm3 of tumor, which is approximately 5-fold less than that estimated for humans. Most likely this reduced PSA index (PSA:tumor volume ratio) results from a 7-fold faster clearance of PSA in athymic mice than in humans; other than this shorter half-life, PSA elimination in the murine model appears similar to that in humans, with both following first-order kinetics characteristic of a two-compartment model. Interestingly, following prolonged growth (greater than 21 days) in castrate hosts, LNCaP tumors are capable of adapting to an androgen-deprived environment whereby LNCaP tumors regain the ability to secrete PSA in amounts similar to the precastrate state. In LNCaP cells, androgens increase PSA mRNA levels 4-fold in vivo and in vitro. PSA mRNA expression is also altered by various growth factors. Changes in PSA production induced by androgens and growth factors do not always parallel changes in LNCaP cell growth rate induced by these factors, suggesting that PSA production occurs independently of cell growth rate and may be influenced by various interrelated factors, including hormonal and stromal milieu. Observations from this murine model suggest that androgens and tumor volume are independent determinants of serum PSA levels and imply that decreases in circulating PSA following antiandrogen therapy may not always reflect a corresponding reduction in tumor volume.

Animals↗

PubMed1992

Human prostate cancer model: roles of growth factors and extracellular matrices.

A human prostate cancer model was established by inoculating a prostate specific antigen (PSA)-producing LNCaP cell line with either prostate or bone fibroblasts. Alternatively, this human prostate cancer model can also be established by inoculating LNCaP cells with growth factor(s) (GFs) and extracellular matrix (ECM) immobilized on Gelfoam. The resulting LNCaP tumors were used to evaluate PSA production and excretion in athymic hosts. This model was also employed to examine the biochemical nature of mesenchymal cell-derived growth-promoting protein(s) and to assess the efficacy of potential chemotherapeutic agents. Because of the propensity of human prostate cancer to metastasize to the bone, this study defined a 1.0 M NaCl-eluted fraction, MS1, from the conditioned medium of a bone stromal cell line (MS) by heparin-affinity column chromatography. The growth-promoting activity was assayed both in vivo (e.g., tumor formation) and in vitro (e.g., soft agar colony formation). We found that the growth-promoting activity was trypsin- and heat-sensitive, and partially degraded by acid and dithiothreitol. Immunochemical studies indicated that the polyclonal antibody raised against MS1 blocked the growth-promoting effect elicited by the bone-conditioned media. This growth-promoting factor was found to be immunochemically dissimilar to KGF, HGF, and bFGF. However, addition of bFGF, HGF and NGF, but not aFGF, TGF beta, IGF1, IGF2, PDGF, EGF, TGF alpha and KGF, stimulated anchorage-independent growth of prostate cells, a condition closely parallel to tumor formation in vivo. We found that the MS1 fraction also contained fibronectin and tenascin but not laminin or collagen IV.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Prostate and bone fibroblasts induce human prostate cancer growth in vivo: implications for bidirectional tumor-stromal cell interaction in prostate carcinoma growth and metastasis.

Prostate cancer selectively metastasizes to the axial skeleton to produce osteoblastic lesions, which suggests that bidirectional paracrine interactions exist between prostate cancer and bone cells. To evaluate the role of tumor-stromal cell interaction and stromal-specific growth factors in prostate cancer growth and dissemination, we coinoculated nontumorigenic human prostate cancer cells (LNCaP) and various tissue-specific fibroblasts subcutaneously in athymic mice. LNCaP tumors were induced most consistently by human bone fibroblasts (62%), followed by two prostate fibroblast cell lines (31% and 17%), but not by lung, kidney, or embryonic 3T3 fibroblasts. Carcinomas formed preferentially in male hosts, demonstrating in vivo androgen sensitivity. Immunohistochemical and biochemical techniques confirmed the human prostate component of these tumors and were paralleled by elevations in serum prostate specific antigen. In vitro mitogenic assays revealed a two-to three-fold bidirectional stimulation between LNCaP and bone or prostate fibroblast conditioned media, but not lung, kidney, or 3T3 fibroblast conditioned media. A novel method developed to deliver concentrated bone or prostate fibroblast conditioned media in vivo using a slowly absorbed matrix (gelfoam) also induced tumor formation, emphasizing the importance of fibroblast growth factors in LNCaP tumor formation. Northern analysis identified the stromal compartment as the primary source of extracellular matrix (collagen, fibronectin), while only LNCaP cells expressed transforming growth factor alpha. Although LNCaP and stromal cells express basic fibroblast growth factor (bFGF), the bidirectional paracrine-mediated mitogenic activity between these cells is not inhibited by anti-bFGF antibodies, suggesting that other undefined growth factors may be involved in stimulating LNCaP growth. These observations illustrate the importance of stromal-epithelial interaction in prostate tumor growth and suggest that extracellular matrix and paracrine-mediated growth factors play a role in prostate cancer growth and metastasis.

Androgens↗

PubMed1991

Reciprocal mesenchymal-epithelial interaction affecting prostate tumour growth and hormonal responsiveness.

A novel cell-cell recombination model was established to test the reciprocal mesenchymal (fibroblast)-epithelial interaction in the prostate gland. Both growth factors and ECM pathways were found to be actively engaged during cellular communications. The application of this cell-cell recombination concept to prostate cancer established a new human prostate cancer animal model in which the tumours actively secrete prostate specific antigen, a known human prostate cancer marker. This review explores the significance of mesenchymal-epithelial interaction in determining prostate hormonal responsiveness and prostate cell transformation and speculates on the potential roles of mesenchymal-epithelial interaction in prostate cancer growth and progression.

Adenocarcinoma↗

PubMedDate not supplied

Chemosensitization of human renal cell cancer using antisense oligonucleotides targeting the antiapoptotic gene clusterin.

BACKGROUND: Renal cell cancer (RCC) is a chemoresistant disease with no active chemotherapeutic agent achieving objective response rates higher than 15%. Clusterin is a cell survival gene that increases in human renal tubular epithelial cells after various states of injury and disease. Downregulation of clusterin, using antisense oligonucleotides (ASO), has recently been shown to increase chemosensitivity in several prostate cancer models. The objectives in this study were to evaluate clusterin expression levels in human RCC and normal kidney tissue, and to test whether clusterin ASO could also enhance chemosensitivity in human RCC Caki-2 cells both in vitro and in vivo. METHODS: Immunohistochemical staining was used to characterize clusterin expression in 67 RCC and normal kidney tissues obtained from radical nephrectomy specimens. Northern blot analysis was used to assess changes in clusterin mRNA expression after ASO and paclitaxel treatment. The effects of combined clusterin ASO and paclitaxel treatment on Caki-2 cell growth was examined using an MTT assay. Athymic mice bearing Caki-2 tumors were treated with clusterin ASO alone, clusterin ASO plus paclitaxel, and mismatch control oligonucleotides plus paclitaxel, over a period of 28 days with measurement of tumor volumes once weekly over 8 weeks. RESULTS: Immunohistochemistry of normal and malignant kidney tissue sections of 67 patients demonstrated positive clusterin staining for almost all RCC (98%) and an overexpression, compared to normal tissue, in a majority of RCC (69%). Clusterin ASO, but not mismatch control oligonucleotides, decreased clusterin mRNA expression in Caki-2 cells in a dose-dependent and sequence-specific manner. Pretreatment of Caki-2 cells with clusterin ASO significantly enhanced chemosensitivity to paclitaxel in vitro. Characteristic apoptotic DNA laddering was observed after combined treatment with ASO plus paclitaxel, but not with either agent alone. In vivo administration of clusterin ASO plus paclitaxel acted synergistically to increase apoptosis and significantly delay Caki-2 tumor growth, compared to mismatch control oligonucleotide plus paclitaxel. In addition, TUNEL staining revealed increased apoptotic cells in tumors treated with clusterin ASO plus paclitaxel compared to treatment with either clusterin ASO or paclitaxel alone. CONCLUSION: These findings confirm that the use of clusterin ASO may be a feasible strategy to enhance chemosensitivity for patients with advanced RCC.

Adenocarcinoma, Clear Cell↗