Genetic polymorphisms in androgen receptor-binding sites predict survival in prostate cancer patients receiving androgen-deprivation therapy.

BACKGROUND: Activated androgen receptor binds to androgen-responsive elements (AREs) in genome to regulate target gene transcription and, consequently, mediates physiological or tumorigenic processes of the prostate. Our aim was to determine whether genetic variants in AREs are associated with clinical outcomes after androgen-deprivation therapy (ADT) in prostate cancer patients. PATIENTS AND METHODS: We systematically investigated 55 common single-nucleotide polymorphisms (SNPs) in the genome-wide insilico-predicted AREs in a cohort of 601 men with advanced prostate cancer treated with ADT. The prognostic significance of these SNPs on disease progression, prostate cancer-specific mortality (PCSM) and all-cause mortality (ACM) after ADT was assessed by Kaplan-Meier analysis and Cox regression model. RESULTS: In univariate analysis, two, five, and four SNPs were associated with disease progression, PCSM, and ACM, respectively. After adjusting for known prognostic factors, ARRDC3 rs2939244, FLT1 rs9508016, and SKAP1 rs6504145 remained as significant predictors for PCSM and FBXO32 rs7830622 and FLT1 rs9508016 remained as significant predictors for ACM in multivariate analysis. Moreover, strong combined genotype effects on PCSM and ACM were also observed (P(trend) < 0.001). CONCLUSION: Our results suggest that SNPs in AREs influence prostate cancer survival and may further advance our understanding of the disease progression.

Genetic polymorphisms in oestrogen receptor-binding sites affect clinical outcomes in patients with prostate cancer receiving androgen-deprivation therapy.

BACKGROUND: Accumulating evidence indicates that oestrogens have significant direct effects on normal prostate development and carcinogenesis. The majority of the biological activities of oestrogens are mediated through the oestrogen receptor (ER), which functions as a hormone-inducible transcription factor to regulate target gene expression by binding to oestrogen response elements (EREs) in the regulatory regions of target genes. Sequence variants in EREs might affect the ER-ERE interaction and subsequent physiological activities. Therefore, we tested whether common single-nucleotide polymorphisms (SNPs) inside EREs are related to the clinical outcomes of androgen-deprivation therapy (ADT) in men with prostate cancer. METHODS: We systematically evaluated 49 ERE SNPs predicted using a genome-wide database in a cohort of 601 men with advanced prostate cancer treated with ADT. The prognostic significance of these SNPs on disease progression, prostate cancer-specific mortality (PCSM) and all-cause mortality (ACM) after ADT was assessed using Kaplan-Meier analysis and a Cox regression model. RESULTS: Based on multiple hypothesis testing, BNC2 rs16934641 was found to be associated with disease progression; in addition, TACC2 rs3763763 was associated with PCSM, and ALPK1 rs2051778 and TACC2 rs3763763 were associated with ACM. These SNPs remained significant in multivariate analyses that included known clinicopathological predictors. Moreover, a combined genotype effect on ACM was observed when ALPK1 rs2051778 and TACC2 rs3763763 were analysed in combination. Patients with a greater number of unfavourable genotypes had a shorter time to ACM during ADT (P for trend <0.001). CONCLUSION: The incorporation of ERE SNPs into models with known predictors might improve outcome prediction in patients with prostate cancer receiving ADT.

alpha-fetoprotein response predicts survival benefits of thalidomide in advanced hepatocellular carcinoma.

BACKGROUND: Radiographic measurements do not always reflect the biological response of hepatocellular carcinoma to drug therapy. AIMS: To evaluate the clinical implications of tumour marker (alpha-fetoprotein) response in advanced hepatocellular carcinoma patients with thalidomide treatment. PATIENTS AND METHODS: Forty-two advanced hepatocellular carcinoma patients with baseline alpha-fetoprotein levels above 200 ng/mL and thalidomide therapy were included. Serum alpha-fetoprotein levels were measured every 4 weeks. alpha-fetoprotein response was defined as a 50% or greater reduction of alpha-fetoprotein levels for 4 or more weeks during treatment. Radiographic response was assessed by World Health Organization criteria; survivals were estimated by Kaplan-Meier method and prognostic factors were assessed by Cox's proportional hazard model. RESULTS: With intention-to-treat analysis, radiographic response and alpha-fetoprotein response were obtained in 7% (three of 42, 95% confidence interval: 0-15) and 24% (10 of 42, 95% CI: 10-38) of patients, respectively. All radiographic response was observed in alpha-fetoprotein responders. Multivariate analyses showed alpha-fetoprotein response was independent prognostic factor for both progression-free survival (relative risk = 0.394, 95% CI: 0.189-0.820, P = 0.013) and overall survival (relative risk = 0.241, 95% CI: 0.096-0.606, P =0.003), whereas radiographic response was not. CONCLUSION: alpha-fetoprotein response can more accurately reflect the biological response of advanced hepatocellular carcinoma to thalidomide therapy than radiographic response.

Suppression of tumorigenicity and metastasis in murine UV-2237 fibrosarcoma cells by infection with a retroviral vector harboring the interferon-beta gene.

In this study, we endeavored to determine the effectiveness of interferon beta (IFNbeta) gene therapy against highly metastatic murine UV-2237m fibrosarcoma cells. UV-2237m cells were engineered to produce murine IFNbeta constitutively following infection by a retroviral vector harboring the murine IFNbeta gene. Parental (UV-2237m-P), control-vector-transduced (UV-2237m-Neo), and IFNbeta-transduced (UV-2237m-IFNbeta) cells were injected subcutaneously (s.c.) or intravenously (i.v.) into syngeneic mice. Parental and control-transduced cells produced rapidly growing tumors, whereas IFNbeta-transduced cells did not. The tumorigenicity of IFNbeta-sensitive or -resistant parental cells was significantly suppressed when they were injected s.c. together with IFNbeta-transduced cells. The IFNbeta-transduced cells did not inhibit growth of parental cells injected s.c. at a distant site. UV-2237m-IFNbeta cells produced s.c. tumors in nude, SCID/Beige, and natural killer(NK)-cell-compromised syngeneic mice. The IFNbeta-transduced cells were more sensitive to in vitro splenic cell-mediated lysis than were the parental or control-transduced cells. Pretreatment of C3H/HeN mice with the NK-cell-selective antiserum (anti-asialoGM1) partially abrogated the cytotoxic activity of the cells. Cytotoxic activity was not observed in mixed culture of UV-2237m-IFNbeta cells and splenic cells from SCID/Beige mice. Significant cytotoxicity against UV-2237m-IFNbeta cells was mediated by macrophages activated by either IFNgamma, lipopolysaccharide, or a combination of both. Our data led us to conclude that the constitutive expression of IFNbeta can suppress tumorigenicity and metastasis of UV-2237m cells, which is due, in part, to activation of host effector cells.

Suppression of tumorigenicity and metastasis of human renal carcinoma cells by infection with retroviral vectors harboring the murine inducible nitric oxide synthase gene.

The purpose of this study was to determine whether retrovirus-mediated transfer of the murine macrophage inducible nitric oxide synthase (iNOS) gene can inhibit tumorigenicity and metastasis of human renal cancer cells. Retroviral vectors encoding murine macrophage iNOS were constructed in the pLXSN retroviral vector with the iNOS gene under the control of a long terminal repeat promoter and a neomycin resistance gene under the control of an internal simian virus 40 promoter. Highly metastatic human renal carcinoma SN12PM6 cells were infected with control or iNOS retrovirus. Expression of iNOS was confirmed by Northern and Western blot analyses, and expression of the functional iNOS protein, i.e., production of nitric oxide (NO), was determined by measuring nitrite accumulation in culture supernatants. Noninfected or control cells produced large orthotopic tumors in the kidney of nude mice and a larger number of experimental lung metastases, whereas iNOS-infected cells produced small tumors in the kidneys and few to no lung metastases. The data indicate that the infection of human renal cancer cells by retroviruses harboring the murine iNOS gene can induce the production of high levels of NO, which is associated with autocytotoxicity, suppression of tumorigenicity, and abrogation of metastasis.

Use of retroviral vectors encoding murine inducible nitric oxide synthase gene to suppress tumorigenicity and cancer metastasis of murine melanoma.

The purpose of this study was to determine whether retrovirus-mediated transfer of murine macrophage inducible nitric oxide synthase (iNOS) can produce inhibition of tumorigenicity and metastasis. Retroviral vectors encoding macrophage iNOS constructed in pLXSN, a retroviral vector with the iNOS gene under the control of a long terminal repeat promoter, were stably transfected into PA317 cells. Medium harvested from confluent monolayers of the virus-producing cell lines was used for infection of the murine K-1735 melanoma cells. Expression of iNOS was confirmed by northern and Western blot analyses. Functional iNOS protein expression was confirmed by bioassay of nitrite accumulation in the culture supernatant. Cells infected by a control iNOS-negative retrovirus produced fast-growing subcutaneous tumors and many lung metastases in nude mice, whereas iNOS-transduced cells produced slow-growing tumors and few lung metastases, showing that the infection of murine tumor cells by retroviruses harboring the iNOS gene can suppress tumorigenicity and metastasis.

Destruction of bystander cells by tumor cells transfected with inducible nitric oxide (NO) synthase gene.

BACKGROUND: The activation of an enzyme, inducible nitric oxide synthase (iNOS), catalyzes the production of endogenous nitric oxide (NO). NO, in turn, is associated with cell death, suppression of tumor development, and inhibition of metastasis of murine melanoma cells. Moreover, the in vivo induction of iNOS is associated with regression of established hepatic metastases. Whether this regression required the activation of the iNOS gene in every tumor cell or whether NO-producing tumor cells can also kill bystander (neighboring) cells has been previously unknown. PURPOSE: The goal of this study was to determine whether cells producing NO and then undergoing autolysis can also kill bystander cells in vitro and in vivo. METHODS: Murine K-1735 C4.parental (C4.P) melanoma cells were transfected with the functional iNOS gene (transfectant denoted as C4.L8) or with a control truncated-nonfunctional iNOS gene (transfectant denoted as C4.S2). NO-mediated cytolysis and bystander cell killing were determined in vitro and in vivo. RESULTS: Only the functional iNOS-transfected C4.L8 cells produced NO and underwent autolysis. C4.L8 cells also produced statistically significant lysis of iNOS-negative C4.P cells. This lysis was suppressed by the specific iNOS inhibitor N(G)-methyl-L-arginine. NO-producing C4.L8 cells and control C4.P or C4.S2 cells were injected subcutaneously into syngeneic C3H/HeN mice. Control C4.P and C4.S2 cells produced rapidly growing subcutaneous tumors, whereas C4.L8 cells did not. The mixture of C4.P and C4.S2 cells (1:5 ratio) produced rapidly growing subcutaneous tumors, whereas the mixture of C4.P and C4.L8.5 cells (1:5 ratio) produced slow-growing tumors. The subcutaneous growth of C4.P cells was not affected by C4.L8.5 cells injected subcutaneously at a distant site. Mixtures of C4.P cells labeled with [(125)I]iododeoxyuridine and C4.L8 cells (NO producing) or C4.S2 cells (control) were injected subcutaneously. The survival rate of the radiolabeled cells indicated that the NO-producing C4.L8.5 cells lysed the bystander C4.P cells. CONCLUSION: The production of high-level endogenous NO causes autolysis in tumor cells and lysis of bystander cells under in vitro and in vivo conditions. IMPLICATIONS: NO-mediated cell killing does not require transfection of genes into every cell in a neoplasm.

Abrogation of tumorigenicity and metastasis of murine and human tumor cells by transfection with the murine IFN-beta gene: possible role of nitric oxide.

The purpose of this study was to determine whether sustained local production of murine IFN-beta (mIFN-beta) could inhibit the tumorigenicity and metastasis of human and murine tumor cells implanted into nude mice. Human melanoma cells (A375SM), renal carcinoma cells (SN12PM6), and colon carcinoma cells (KM12SM) were transfected with mIFN-beta or a control neomycin resistance vector. All cell lines grew well in culture. Tumor cells were injected into the subcutis, kidney, spleen, or lateral tail vein of nude mice. Parental or control transfected cells produced local tumors and experimental or spontaneous lung metastases, whereas mIFN-beta-transfected cells did not. In vivo survival experiments using [125I]IdUdR-labeled cells showed that by day 7 after s.c. implantation, all IFN-beta-transfected cells died. IFN-beta transfection prevented the outgrowth of parental or control-transfected cells only when they were injected together with transfected cells into one site, suggesting that IFN-beta promoted a local lysis of the bystander cells. Similar indirect antitumor activity was demonstrated in various human (KM12SM and SN12PM6) and murine (CT-26 colon carcinoma, RENCA renal cell carcinoma, and 3LL Lewis lung carcinoma) tumors. The IFN-beta-transfected tumor cells stimulated a high level of nitric oxide production by murine macrophages under in vitro and in vivo conditions, which correlated with the vigorous nonspecific antitumor activity. Collectively, these results demonstrate that local production of IFN-beta can eradicate tumor cells of different histology by inducing inducible nitric oxide synthase expression in infiltrating cells.

Bcl-2 protects cells from cytokine-induced nitric-oxide-dependent apoptosis.

Cytokine-mediated cell death in tumor cells can be achieved through endogenous nitric oxide (NO) from within tumor cells or exogenous NO from either activated macrophages or endothelial cells. The purpose of this study was to determine the role of Bcl-2 in NO-mediated apoptosis. The incubation of murine L929 and NIH3T3 cells with interleukin-1 alpha (IL-1 alpha) and interferon gamma (IFN gamma) induced high endogenous NO production only in the L929 cells that also underwent apoptosis. NIH3T3 cells were not resistant to NO-mediated apoptosis. In fact, the incubation of L929 and NIH3T3 cells with exogenous NO derived from NO donors, sodium nitroprusside, or S-nitroso-N-acetyl-DL-penicillamine (SNAP) induced death, characterized by typical apoptotic morphology and DNA fragmentation, in both cell types, but to a higher degree in NIH3T3 cells than in the L929 cells. We then measured the effect of Bcl-2 expression on exogenous NO-induced apoptosis. At both the mRNA and protein levels, L929 fibroblasts expressed higher levels of endogenous mouse Bcl-2 than did NIH3T3 cells. At the same time, L929 cells were much more resistant to exogenous NO-induced cell death than were NIH3T3 cells. The inverse correlation between mouse Bcl-2 expression and sensitivity to exogenous NO-mediated cell death was also found in the murine K-1735 melanoma C-23 and X-21 clonal populations. Transfection of both NIH3T3 cells and L929 cells with the human bcl-2 gene led to resistance to both exogenous and endogenous NO-mediated apoptosis. These data demonstrate that NO-mediated apoptosis can be suppressed by expression of Bcl-2, suggesting that abnormal expression of Bcl-2 may influence the efficacy of tumor immunotherapy.

Tyrosine phosphorylation at the membrane-microfilament interface: a p185neu-associated signal transduction particle containing Src, Abl and phosphorylated p58, a membrane- and microfilament-associated retroviral gag-like protein.

Microfilaments are associated with the microvillar membrane in the 13762 ascites rat mammary carcinoma cells by stable interaction with a large, multimeric signal transduction particle (STP) containing the (proto)oncogene receptor p185(neu). In vitro kinase assays on isolated microvilli and microvillar fractions enriched in the putative signal transduction particle showed a high specific activity of tyrosine kinase activity compared to that of membranes from EGF receptor-overexpressing A431 cells maximally activated by EGF. Assays of velocity sedimentation fractions from microvillar lysates in the presence and absence of the exogenous tyrosine kinase substrate poly-glu-tyr polypeptide (poly-E(4)Y) suggested association of the tyrosine kinase activity with STP-enriched microvillar fractions. The particulate fractions also contained discrete endogenous tyrosine-phosphorylated proteins, including prominent bands of approximately 42 and 58 kDa. Addition of ATP to these fractions resulted in a rapid increase in tyrosine phosphorylation of these and several other proteins, as detected by anti-phosphotyrosine blots. Immunoprecipitation and immunoblotting with anti-phosphotyrosine antibody of SDS-solubilized ascites cells and microfilament core fractions showed nine major bands; the electrophoretic mobilities of most of these in the cell immunoprecipitate and microfilament core were the same. In vivo and in situ phosphorylation, phosphoamino acid analysis, immunoprecipitation, 2-dimensional isoelectric focusing/SDS PAGE and immunoblot analysis showed that one of the prominent substrates is p58(gag), a retroviral Gag-like cytoplasmic STP component implicated in stabilizing microfilament-membrane interactions. Immunoblotting identified two peripheral membrane tyrosine kinases, p6O(src) and p120(abl), stably associated with the p185(neu)-containing signal transduction particle. These results provide further evidence for the constitutive activation of this aggressive mammary tumor and suggest a rote for phosphorylation of p58(gag) in organization of the STP at the membrane-microfilament interface in these cells.

Activation of inducible nitric oxide synthase gene in murine macrophages requires protein phosphatases 1 and 2A activities.

The purpose of these studies was to identify phosphatase activities required for the production of nitric oxide in murine macrophages exposed to lipopolysaccharide (LPS), synthetic lipopeptide (LPP), and mouse interferon-gamma (IFN-gamma). The in vitro treatment of macrophages with IFN-gamma and LPS or IFN-gamma and LPP resulted in production of NO, which was inhibited by addition of the specific phosphatase 1 and 2A (PP1/2A) inhibitors okadaic acid (OA), calyculin A, and cantharidin (but not the nonactive analogues okadaic acid tetraacetate and 1,4-dimethylendothall). OA suppressed the accumulation of steady-state inducible NO synthase (iNOS) mRNA and iNOS protein (without alteration of their stability). The cytosol and nuclei of control macrophages contained large amounts of PP1/2A activities that were inhibited by OA in a dose-dependent manner. Taken together, these data indicate that PP1/2A activities are involved in the regulation of iNOS gene expression in murine macrophages.

Transfection with the inducible nitric oxide synthase gene suppresses tumorigenicity and abrogates metastasis by K-1735 murine melanoma cells.

Previous studies from our laboratory demonstrated an inverse relationship between the expression level of inducible nitric oxide synthase (iNOS) and the metastatic potential of murine K-1735 melanoma cells. The purpose of this study was to provide direct evidence that the expression of iNOS suppresses metastatic potential of melanoma cells. Highly metastatic K-1735 clone 4 cells (C4.P), which express low levels of iNOS, were transfected with a functional iNOS (C4.L8), inactive-mutated iNOS (C4.S2), or neomycin-resistance (C4.Neo) genes in medium containing 3 mM NG-methyl-L-arginine (NMA). Positive transfectants were identified by Southern and Northern blot analyses and homogeneous staining with a specific anti-iNOS monoclonal antibody. Semiconfluent cultures of C4.P (parental), C4.Neo.3 (control transfection), C4.S2.3 (inactive iNOS), and C4.L8.5 (functional iNOS) were harvested, and viable cells were injected intravenously into syngeneic C3H/HeN mice and allogeneic BALB/c nude mice. C4.P, C4.Neo.3, and C4.S2.3 cells were highly metastatic whereas C4.L8.5 cells were not metastatic. Experiments with [125I]dUrd-labeled tumor cells demonstrated that the initial arrest in the lung microvasculature did not differ among the lines, but that C4.L8.5 cells died by 48-72 h after injection. Enhanced survival of all K-1735 C4 cells (including C4.L8.5) was found in mice given twice daily injections of 20 mg NMA. The C4.L8.5 cells produced slow growing subcutaneous tumors in nude mice, whereas the other three lines produced fast growing tumors. In vitro studies confirmed that in the absence of NMA the expression of iNOS in C4.L8.5 cells induced apoptosis. Collectively, these data demonstrate that the expression of recombinant iNOS in melanoma cells is associated with apoptosis, suppression of tumorigenicity, and abrogation of metastasis.

Molecular cloning and sequencing of a 58-kDa membrane- and microfilament-associated protein from ascites tumor cell microvilli with sequence similarities to retroviral Gag proteins.

The MAT-C1 subline of the 13762 rat mammary adenocarcinoma has highly stable, branched microvilli and immobile cell surface receptors. A membrane- and microfilament-associated 58-kDa protein (p58) in the MAT-C1 microvilli has been implicated in the stabilization of the microvilli and microfilament-membrane interactions. This protein is associated with a high M(r) glycoprotein complex containing the (proto)oncogene p185neu and other signal transduction components in a putative microfilament-associated signal transduction particle. Amino acid sequences were obtained from two trypsin peptides of p58. Screening a MAT-C1 cDNA library with a degenerate oligonucleotide derived from the larger peptide and polymerase chain reaction amplification of cDNA ends permitted the isolation of overlapping cDNAs encoding the 427-amino acid open reading frame of p58. In vitro transcription and translation using a full-length cDNA gave a protein of approximately 55 kDa, which reacts with anti-p58 antiserum and reconstitutes into a complex with actin and glycoproteins from the membrane-microfilament interaction site. When COS-7 cells were transfected with the full-length cDNA, p58 was localized in a punctate distribution. In addition, the transfected cells exhibited fewer microfilament cables than untransfected neighboring cells. The amino acid sequence showed a surprising similarity to mammalian retroviral Gag proteins and included regions corresponding to p15, p12 and the N-terminal 80% of p30. Comparisons of p58 and the corresponding regions of the Gag proteins for Moloney murine leukemia virus indicated that about 60% of their amino acid residues were identical. These studies suggest that p58 is the product of an endogenous retroviral gene whose expression as a cellular protein alters the properties of the tumor cell to provide a selective advantage for tumor growth in the animal.

Membrane-microfilament interactions in ascites tumor cell microvilli. Identification and isolation of a large microfilament-associated membrane glycoprotein complex.

[14C]Glucosamine metabolic labeling and concanavalin A blots were used to identify four major glycoprotein species associated with ascites tumor cell microvillar microfilament cores and with a transmembrane complex containing actin. Phalloidin shift analysis of glucosamine-labeled microvilli showed that glycoproteins of 110-120, 80, 65, and 55 kDa are stably associated with the microfilament cores. Analysis of large (greater than 10(6) kDa) transmembrane complexes from microvillar membranes made under microfilament-depolymerizing conditions (Carraway, C. A. C., Jung, G., and Carraway, K. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 430-434) revealed glycoproteins of the same Mr values, showing the same relative staining or labeling patterns as those observed with the microfilament cores. Gel filtration of high salt, high pH extracts of intact microvilli, microfilament cores, or transmembrane complexes showed that in all of these fractions the glycoproteins are associated in a very large, stable complex. The glycoprotein multimer was isolated essentially free of actin and other components by Sephacryl S-1000 chromatography of microvilli, microvillar membranes prepared at pH 11, microfilament cores, or transmembrane complex fractions in Triton X-100, 1 M KCl, glycine, pH 9.5. Purified glycoprotein complex bound actin when incubated under polymerizing conditions. The presence of the glycoprotein heteromultimer in both microfilament cores and transmembrane complex from isolated membranes and the association of the purified glycoprotein complex with actin are consistent with our hypothesis that the glycoprotein-containing transmembrane complex is an association site for microfilaments at the plasma membrane.

Transcranial Doppler ultrasonography of the middle cerebral artery in the hemodynamic assessment of internal carotid artery stenosis.

We performed transcranial Doppler ultrasonography of the middle cerebral arteries in 43 patients who underwent cerebral angiography. Twelve patients had normal arteriograms, 18 had internal carotid artery stenosis, and 13 had complete occlusion. Parameters measured included: flow velocity (mean, maximum, and peak systolic), flow acceleration, systolic-to-diastolic ratio, and pulsatility index. Patients with 75% to 100% stenosis had lower average ipsilateral flow acceleration and mean velocity than did normal subjects. Correlation analysis revealed an inverse relationship between degree of stenosis and ipsilateral flow acceleration, as well as ipsilateral mean velocity. These correlations were no longer significant when we excluded normals, however. There was a linear relationship between right and left velocity values for both normal subjects and patients with occlusion but not for patients with stenosis. This technique has potential for the reliable assessment of the hemodynamic effect of carotid stenosis on intracranial circulation.