Over-expression of p53 mutants in LNCaP cells alters tumor growth and angiogenesis in vivo.

This study has investigated the impact of three specific dominant-negative p53 mutants (F134L, M237L, and R273H) on tumorigenesis by LNCaP prostate cancer cells. Mutant p53 proteins were associated with an increased subcutaneous "take rate" in NOD-SCID mice, and increased production of PSA. Tumors expressing F134L and R273H grew slower than controls, and were associated with decreased necrosis and apoptosis, but not hypoxia. Interestingly, hypoxia levels were increased in tumors expressing M237L. There was less proliferation in F134L-bearing tumors compared to control, but this was not statistically significant. Angiogenesis was decreased in tumors expressing F134L and R273H compared with M237L, or controls. Conditioned medium from F134L tumors inhibited growth of normal human umbilical-vein endothelial cells but not telomerase-immortalized bone marrow endothelial cells. F134L tumor supernatants showed lower levels of VEGF and endostatin compared with supernatants from tumors expressing other mutants. Our results support the possibility that decreased angiogenesis might account for reduced growth rate of tumor cells expressing the F134L p53 mutation.

Immunohistochemical characterisation of the monoclonal antibody BLCA-38 for the detection of prostate cancer.

BACKGROUND: Monoclonal antibodies (MAbs) can be used to detect, image and treat cancers. This study aimed to characterise the binding of BLCA-38 MAbs to human prostate cancer cell lines, human prostate cancer biopsy samples and normal tissues to enable future targeted studies. METHODS: BLCA-38 antigen expression on cancer lines was determined by flow cytometry; that on patient specimens from normal tissues and cancers was tested by immunohistochemistry using fresh frozen tissues or paraffin-embedded tissues that had undergone antigen retrieval. RESULTS: Cell surface BLCA-38 antigen expression was seen on DU-145, PC-3, PC-3 M and PC-3 M-MM2 prostate cancer lines, but LNCaP, MDA PCa 2a or MDA PCa 2b lines were negative. Other human lines, including 8/12 bladder cancer and A431 vulval epidermoid cells, but not breast cancer lines, expressed BLCA-38 antigen. Staining occurred in glandular epithelial cells in the majority of frozen, and paraffin-embedded prostate cancer tissues and was occasionally seen in prostatic intraepithelial neoplasia (PIN). No staining was observed in normal cadaver tissues or in benign areas from various other cancer tissues. CONCLUSIONS: The BLCA-38 antibody binds to the majority of human prostate cancers but not to normal cells, and has potential for targeting novel therapies in patients with this disease.

Inverse correlation between KAI1 mRNA levels and invasive behaviour in bladder cancer cell lines.

We have previously shown that levels of KAI1 mRNA are dramatically reduced in invasive human bladder cancers. To further investigate the role of KAI1 in bladder cancer, we have examined the relationship between KAI1 mRNA levels and cell behaviour in 18 bladder cancer cell lines and a virus-transformed uro-epithelial cell line. We found that low KAI1 mRNA levels correlated with increased in vitro invasive ability, reduced Ca(2+)-dependent and -independent cell-cell adhesion and reduced adhesion to fibronectin. These data support the idea that loss of KAI1 expression is an important factor in tumour cell invasive behaviour.

Elevated expression of FGF-2 does not cause prostate cancer progression in LNCaP cells.

BACKGROUND: Androgen-independent (AI) prostate cancer (CaP) resulting from progression of disease is untreatable. Such progression may relate to upregulation and autocrinicity of growth factor expression. We studied one candidate growth factor, basic fibroblast growth factor (FGF-2). METHODS: LNCaP cells made autocrine for FGF-2 by stable transfection with FGF-2 were examined for cancer progression, measured by 1) altered response to androgen, 2) ability to grow more quickly when cocultured with bone cells in vitro or to form tumors when coinoculated with bone cells in vivo, or 3) increase in metastatic ability. RESULTS: Stably transfected lines differed in FGF-2 protein expression. LNCaP-HF (high production of FGF-2) expressed more FGF-2 than LNCaP-LF (low production of FGF-2); controls were negative. In vitro, compared with LNCaPs, LNCaP-HF cells showed a slightly increased growth rate, reduced proliferation in response to androgen but not to estrogen or progesterone, and a decreased proliferative response to epidermal growth factor (EGF) and FGF-2. Although giving a slightly faster take rate, LNCaP-HF cells without Matrigel only formed small, fast-regressing tumors in male nude mice, and with Matrigel, did not differ from LNCaPs in growth rate or tumor size. No metastases occurred. No tumors grew in females. Mixed growth of FGF-2 transfectants with human fetal osteoblasts failed to cross-stimulate in vitro, or to allow tumor formation in vivo. CONCLUSIONS: Although FGF-2 is overexpressed in AI CaPs, our experiments show that upregulation of FGF-2 expression is not sufficient to cause androgen independence, tumorigenicity, or metastases production (i.e., prostate cancer progression) in LNCaP cells.

Characterisation of the anti-bladder-cancer monoclonal antibody BLCA-8: identification of its antigen as a neutral glycolipid.

A monoclonal antibody, BLCA-8, was raised against the human bladder cancer cell line, UCRU-BL-17CL. By flow cytometry and immunoperoxidase staining, this antibody was found to possess high specificity for bladder tumours, some reactivity with fetal tissues, and no reactivity with normal bladder, or any normal or malignant tissue. This high specificity and the stability of the antigen to the urinary environment suggest that BLCA-8 may have potential for use as an anti-bladder-cancer therapeutic agent. By thin-layer chromatography and autoradiography, BLCA-8 was found to bind four components within the neutral lipid fraction of a bladder cancer cell line, UCRU-BL-17/23 alpha. These components had RF values of 0.22, 0.16/0.15 (doublet), 0.12 and 0.08, and migrated below globoside, indicating the presence of more than four sugars. By enzyme-linked immunosorbent assay and thin-layer chromatography it was found that the binding of BLCA-8 to the lipid extract was increased by both mild alkaline hydrolysis and enzymatic treatments, indicating that adjacent phospholipids and glycolipids interfere with the accessibility of the antibody-binding site. Full biochemical characterisation of the BLCA-8 antigen is currently underway.

Detection of malignant cells in voided urine from patients with bladder cancer, a novel monoclonal assay.

A simple assay is described for detecting malignant cells in the voided urine from patients with transitional cell carcinoma of the bladder. Agarose-embedded urothelial cells from 24 biopsy-proven cancer patients and 10 controls were stained for surface immunofluorescence with four monoclonal antibodies reactive with human bladder cancer and three monoclonals reactive with blood group A. Reactivity was assessed by fluorescence microscopy. One antibody, BLCA-8 appeared to have particular diagnostic utility. Thus, 24.3 +/- 5.8 percent of outer layer and 27.0 +/- 4.6 percent of inner layer urothelial cells reacted with BLCA-8 in patient samples, compared to 2.9 +/- 1.0 and 0.8 +/- 0.5 percent of similar cells from control urines. BLCA-8 antigen expression was found to be relatively stable even after prolonged exposure to urine. In a comparison with conventional cytology, samples from 4/8 patients were considered positive by standard methods, whereas, 8/8 were BLCA-8 positive. This new technique may thus be a useful adjunct to conventional methods.

Purification and characterization of a secondary alcohol dehydrogenase from a pseudomonad.

Growth of Pseudomonas sp. NRRL B3266 in the presence of oleic acid resulted in the induction of two enzymes: oleate hydratase, which produced 10(R)hydroxyoctadecanoate, and hydroxyoctadecanoate dehydrogenase, which catalyzed the oxidized nicotinamide adenine dinucleotide-dependent production of 10-oxooctadecanoate. This latter enzyme was purified to homogeneity and shown to consist of two polypeptide chains of about 29,000 daltons each. The enzyme had a broad substrate specificity, catalyzing the dehydrogenation of a number of 18-carbon hydroxy fatty acids. The kinetic parameters for various 10- and 12-hydroxy fatty acids were similar (Km ca. 5 micron and Vmax ca. 50 to 200 mumol/min per mg of protein). The enzyme also catalyzed the dehydrogenation of unsubstituted secondary alcohols. The effectiveness of these alcohols as substrates was highly dependent on their hydrophobicity, the Km decreasing from 9 mM for 4-heptanol to 7 micron for 6-dodecanol. Inhibition of the enzyme by primary alcohols also showed a dependence on hydrophobicity, the Ki decreasing from 350 mM for methanol to 90 micron for decanol.