agar and Prostatic-Neoplasms

To develop an off-resonant frequency filtered method to selectively differentiate between implanted gold fiducial markers and platinum coated brachytherapy seeds.. The magnetic susceptibilities for gold fiducial markers and brachytherapy seeds differ in magnitude and also in their signs, resulting in B. Dual-plane 2D co-RASOR sequences were reconstructed off-resonance with applied frequency filters to create two projections displaying each seed, which were then colour-coded to negative and positive frequencies. Phantom validation showed that each seed contains its maximal CNR in opposing frequency regions as predicted. Local maxima can also appear in both negative and positive frequency regions. The relative difference between the signal of each seed and these local maxima ranged from 1.19 to 3.73, and an image threshold was determined in all cases. Tissue validation showed the technique differentiates seeds correctly and is limited by the hyperintensity patterns observed in the co-RASOR method.. Dual-plane co-RASOR offers sub-millimetre positive contrast from implanted seeds that contain unique off-resonant frequency maxima, which frequency filters can selectively differentiate.

Topics: Agar; Brachytherapy; Color; Computer Simulation; Contrast Media; Fiducial Markers; Gold; Humans; Magnetic Resonance Imaging; Male; Models, Theoretical; Phantoms, Imaging; Platinum; Prostatic Neoplasms; Prostheses and Implants; Reproducibility of Results; Tomography, X-Ray Computed

Ultrasound imaging has improved the treatment of prostate cancer by producing increasingly higher quality images and influencing sophisticated targeting procedures for the insertion of radioactive seeds during brachytherapy. However, it is critical that the needles be placed accurately within the prostate to deliver the therapy to the planned location and avoid complications of damaging surrounding tissues.. The authors have developed a compact mechatronic system, as well as an effective method for guiding and controlling the insertion of transperineal needles into the prostate. This system has been designed to allow guidance of a needle obliquely in 3D space into the prostate, thereby reducing pubic arch interference. The choice of needle trajectory and location in the prostate can be adjusted manually or with computer control.. To validate the system, a series of experiments were performed on phantoms. The 3D scan of the string phantom produced minimal geometric error, which was less than 0.4 mm. Needle guidance accuracy tests in agar prostate phantoms showed that the mean error of bead placement was less then 1.6 mm along parallel needle paths that were within 1.2 mm of the intended target and 1 degree from the preplanned trajectory. At oblique angles of up to 15 degrees relative to the probe axis, beads were placed to within 3.0 mm along a trajectory that were within 2.0 mm of the target with an angular error less than 2 degrees.. By combining 3D TRUS imaging system to a needle tracking linkage, this system should improve the physician's ability to target and accurately guide a needle to selected targets without the need for the computer to directly manipulate and insert the needle. This would be beneficial as the physician has complete control of the system and can safely maneuver the needle guide around obstacles such as previously placed needles.

Topics: Agar; Calibration; Humans; Imaging, Three-Dimensional; Male; Phantoms, Imaging; Prostatic Neoplasms; Radiotherapy, Computer-Assisted; Software; Ultrasonography

There are currently limitations associated with the prostate biopsy procedure, which is the most commonly used method for a definitive diagnosis of prostate cancer. With the use of two-dimensional (2D) transrectal ultrasound (TRUS) for needle-guidance in this procedure, the physician has restricted anatomical reference points for guiding the needle to target sites. Further, any motion of the physician's hand during the procedure may cause the prostate to move or deform to a prohibitive extent. These variations make it difficult to establish a consistent reference frame for guiding a needle. We have developed a 3D navigation system for prostate biopsy, which addresses these shortcomings. This system is composed of a 3D US imaging subsystem and a passive mechanical arm to minimize prostate motion. To validate our prototype, a series of experiments were performed on prostate phantoms. The 3D scan of the string phantom produced minimal geometric distortions, and the geometric error of the 3D imaging subsystem was 0.37 mm. The accuracy of 3D prostate segmentation was determined by comparing the known volume in a certified phantom to a reconstructed volume generated by our system and was shown to estimate the volume with less then 5% error. Biopsy needle guidance accuracy tests in agar prostate phantoms showed that the mean error was 2.1 mm and the 3D location of the biopsy core was recorded with a mean error of 1.8 mm. In this paper, we describe the mechanical design and validation of the prototype system using an in vitro prostate phantom. Preliminary results from an ongoing clinical trial show that prostate motion is small with an in-plane displacement of less than 1 mm during the biopsy procedure.

Topics: Agar; Biomechanical Phenomena; Biopsy, Needle; Equipment Design; Humans; Imaging, Three-Dimensional; In Vitro Techniques; Male; Needles; Phantoms, Imaging; Prostate; Prostatic Neoplasms; Reproducibility of Results; Stress, Mechanical; Ultrasonography; Urinary Bladder

Docetaxel-based chemotherapy is the only treatment that demonstrated an overall survival benefit in men with hormone refractory prostate cancer. 2-CADO inhibits the growth of PC3 cells by inducing apoptosis and cell cycle arrest through a mechanism that involves cellular uptake.. Androgen-independent and -sensitive (PC3 and LNCaP) prostate cancer cells and non-neoplastic HECV cells were used in the study. Proliferation and cell cycle progression were analyzed in the presence of 2-CADO and Docetaxel. Invasive potential was assessed by soft agar assay and metastatic ability by adhesion assay. IL-23 and PAR-1 expression were determined by real time PCR.. 2-CADO pre-treatment followed by Docetaxel at subclinical dosage reduced the viability of either PC3 or LNCaP while it did not enhance Docetaxel-induced cytotoxicity in adherent non-neoplastic HECV. The drugs reduced the invasive potential of PC3 cells by inducing apoptosis and blocking cell cycle progression in the S-phase. Down-regulation of PAR-1 gene expression resulted in a slightly lower metastatic potential, whereas up-regulation of IL-23 induced the activation of the immune system.. Pretreatment of PC3 cells with 2-CADO decreased the effective concentration of Docetaxel, lowered the metastatic potential, and induced the production of cytokines known to stimulate the immune response against cancer. The treatment was effective for prostate cancer cells independently on their androgen sensitiveness.

Topics: 2-Chloroadenosine; Agar; Antineoplastic Agents; Apoptosis; Cell Division; Cell Line, Tumor; Docetaxel; Drug Synergism; Gene Expression Regulation, Neoplastic; Humans; Interleukin-23 Subunit p19; Male; Prostatic Neoplasms; Receptor, PAR-1; S Phase; Stem Cells; Taxoids

PTEN mutations are among the most frequent genetic alterations found in human prostate cancers. Our previous works suggest that although precancerous lesions were found in Pten heterozygous mice, cancer progression and metastasis only happened when both alleles of Pten were deleted. To understand the molecular mechanisms underlying the role of PTEN in prostate cancer control, we generated two pairs of isogenic, androgen receptor (AR)-positive prostate epithelial lines from intact conditional Pten knock-out mice that are either heterozygous (PTEN-P2 and -P8) or homozygous (PTEN-CaP2 and PTEN-CaP8) for Pten deletion. Further characterization of these cells showed that loss of the second allele of Pten leads to increased anchorage-independent growth in vitro and tumorigenesis in vivo without obvious structural or numerical chromosome changes based on SKY karyotyping analysis. Despite no prior exposure to hormone ablation therapy, Pten null cells are tumorigenic in both male and female severe combined immunodeficiency mice. Furthermore, knocking down PTEN can convert the androgen-dependent Myc-CaP cell into androgen independence, suggesting that PTEN intrinsically controls androgen responsiveness, a critical step in the development of hormone refractory prostate cancer. Importantly, knocking down AR by shRNA in Pten null cells reverses androgen-independent growth in vitro and partially inhibited tumorigenesis in vivo, indicating that PTEN-controlled prostate tumorigenesis is AR dependent. These cell lines will serve as useful tools for understanding signaling pathways controlled by PTEN and elucidating the molecular mechanisms involved in hormone refractory prostate cancer formation.

Topics: Agar; Alleles; Animals; Cell Line, Tumor; Cell Proliferation; Chromosomes; Female; Gene Expression Regulation, Neoplastic; Karyotyping; Male; Mice; Mice, Knockout; Mutation; Prostatic Neoplasms; PTEN Phosphohydrolase

The HMGA architectural nuclear factors are involved in chromatin dynamics and their overexpression has been strongly linked to the neoplastic transformation process. Here we investigate the expression and post-translational modifications (PTMs) of HMGA proteins (HMGA1a, HMGA1b and HMGA2) in the rat prostatic cancer Dunning model (G, AT-1, and MAT-Ly-Lu cell lines). We demonstrate the expression of HMGA2, in addition to HMGA1a and HMGA1b, in both the anaplastic cell lines AT-1 and MAT-Ly-Lu and an extremely specific HMGA1a mono-methylation only in the most metastatic cell line MAT-Ly-Lu. The HMGA ectopic expression in HMGA-negative Dunning G cells does not significantly alter their growth ability, suggesting that, although HMGA expression is necessary for the progression of neoplastic transformation in several cellular models, in these cells it is not sufficient. These data suggest exploring HMGA2 as a potential marker in human prostate tumor and moreover indicate PTMs as an additional tool in the staging of tumor progression.

Topics: Agar; Animals; Blotting, Northern; Blotting, Western; Cell Line, Tumor; Chromatin; Chromatography, Liquid; Disease Progression; DNA, Complementary; Gene Expression Regulation, Neoplastic; HMGA Proteins; Humans; Male; Mass Spectrometry; Phenotype; Plasmids; Prostatic Neoplasms; Protein Processing, Post-Translational; Rats; Transfection

In prostate brachytherapy, an 18-gauge needle is used to implant radioactive seeds. This thin needle can be deflected from the preplanned trajectory in the prostate, potentially resulting in a suboptimum dose pattern and at times requiring repeated needle insertion to achieve optimal dosimetry. In this paper, we report on the evaluation of brachytherapy needle deflection and bending in test phantoms and two approaches to overcome the problem. First we tested the relationship between needle deflection and insertion depth as well as whether needle bending occurred. Targeting accuracy was tested by inserting a brachytherapy needle to target 16 points in chicken tissue phantoms. By implanting dummy seeds into chicken tissue phantoms under 3D ultrasound guidance, the overall accuracy of seed implantation was determined. We evaluated methods to overcome brachytherapy needle deflection with three different insertion methods: constant orientation, constant rotation, and orientation reversal at half of the insertion depth. Our results showed that needle deflection is linear with needle insertion depth, and that no noticeable bending occurs with needle insertion into the tissue and agar phantoms. A 3D principal component analysis was performed to obtain the population distribution of needle tip and seed position relative to the target positions. Our results showed that with the constant orientation insertion method, the mean needle targeting error was 2.8 mm and the mean seed implantation error was 2.9 mm. Using the constant rotation and orientation reversal at half insertion depth methods, the deflection error was reduced. The mean needle targeting errors were 0.8 and 1.2 mm for the constant rotation and orientation reversal methods, respectively, and the seed implantation errors were 0.9 and 1.5 mm for constant rotation insertion and orientation reversal methods, respectively.

Topics: Agar; Animals; Brachytherapy; Chickens; Humans; Image Processing, Computer-Assisted; Imaging, Three-Dimensional; Male; Models, Statistical; Phantoms, Imaging; Prostatic Neoplasms; Radiometry; Radiotherapy Planning, Computer-Assisted; Radiotherapy, Computer-Assisted; Ultrasonics

An algorithm was developed in order to segment and track brachytherapy needles inserted along oblique trajectories. Three-dimensional (3D) transrectal ultrasound (TRUS) images of the rigid rod simulating the needle inserted into the tissue-mimicking agar and chicken breast phantoms were obtained to test the accuracy of the algorithm under ideal conditions. Because the robot possesses high positioning and angulation accuracies, we used the robot as a "gold standard," and compared the results of algorithm segmentation to the values measured by the robot. Our testing results showed that the accuracy of the needle segmentation algorithm depends on the needle insertion distance into the 3D TRUS image and the angulations with respect to the TRUS transducer, e.g., at a 10 degrees insertion anglulation in agar phantoms, the error of the algorithm in determining the needle tip position was less than 1 mm when the insertion distance was greater than 15 mm. Near real-time needle tracking was achieved by scanning a small volume containing the needle. Our tests also showed that, the segmentation time was less than 60 ms, and the scanning time was less than 1.2 s, when the insertion distance into the 3D TRUS image was less than 55 mm. In our needle tracking tests in chicken breast phantoms, the errors in determining the needle orientation were less than 2 degrees in robot yaw and 0.7 degrees in robot pitch orientations, for up to 20 degrees needle insertion angles with the TRUS transducer in the horizontal plane when the needle insertion distance was greater than 15 mm.

Topics: Agar; Algorithms; Animals; Brachytherapy; Chickens; Humans; Imaging, Three-Dimensional; Male; Needles; Phantoms, Imaging; Prostatic Neoplasms; Radiotherapy Planning, Computer-Assisted; Robotics; Ultrasonics

RWJ-241947 (MCC-555) is a novel peroxisome proliferator-activated receptor-gamma ligand of the thiazolidinedione class that was recently developed as an antidiabetic drug with unique properties. Some thiazolidinediones have anticancer activity against solid and hematological malignancies; the anticancer potency of RWJ-241947 has not been examined. We, therefore, investigated these effects in vitro and in vivo either alone or in combination with other compounds.. Tumor growth was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, soft agar colony assay in vitro, and xenografts in nude mice. Its effects on cell cycle, differentiation, and apoptosis were examined.. In vitro studies using various solid and hematological tumor cell lines showed that RWJ-241947 had antiproliferative activity against prostate cancer cells, with the strongest effect against the androgen-independent PC-3 prostate cancer cells. It increased expression of cyclin-dependent kinase inhibitor p21(WAF1), deceased cyclin E, and induced apoptosis in PC-3 cells. It increased E-cadherin and lowered protein expression of prostate-specific antigen without down-regulating the androgen receptor in androgen-dependent LNCaP prostate cancer cells. Reporter gene assays showed that this peroxisome proliferator-activated receptor-gamma ligand inhibited androgen activation of the androgen receptor response elements of the prostate-specific antigen gene. Remarkably, in vivo treatment of male beige/nude/X-linked immunodeficient (BNX) mice with RWJ-241947 profoundly suppressed growth of PC-3 prostate cancer xenografts with prominent apoptosis, as well as fibrosis, including inflammatory and giant cell reaction in the remaining tumor tissue. Notably, the experimented mice had a significantly decreased cholesterol. In addition, we studied the combination of arsenic trioxide (As2O3), which is used in the treatment of multiple myeloma, and RWJ-241947; these two reagents together prominently inhibited proliferation and caused apoptosis of multiple myeloma cells.. RWJ-241947 has surprisingly potent antiproliferative effects against prostate cancer cells in vivo, and it enhances the antitumor activity of As2O3 against myeloma cells. Small, well-defined clinical studies using RWJ-241947 are in order for these cancers.

Topics: Agar; Animals; Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Blotting, Western; Caspases; CD36 Antigens; Cell Cycle; Cell Differentiation; Cell Division; Cell Line, Tumor; Cell Survival; Genetic Linkage; Humans; Immunologic Deficiency Syndromes; Ligands; Luciferases; Male; Mice; Multiple Myeloma; Oxides; Prostatic Neoplasms; Receptors, Cytoplasmic and Nuclear; Tetrazolium Salts; Thiazoles; Thiazolidinediones; Time Factors; Transcription Factors; Transfection; U937 Cells; X Chromosome

Myopodin was previously reported as a gene that was frequently deleted in prostate cancer. This gene shares significant homology with a cell shape-regulating gene, synaptopodin. Myopodin was shown to bind actin and to induce actin bundling when cells were stimulated. To clarify the functional role of myopodin in prostate cancer, several assays were performed to evaluate the tumor suppression activity of myopodin. Our results indicate that myopodin inhibits tumor growth and invasion both in vitro and in vivo. The activity of tumor suppression of myopodin is located at the C-terminus region. To further evaluate the role of myopodin in suppressing the invasiveness of prostate cancer, an expression analysis of myopodin protein was performed in prostate tissues. The results indicate that down-regulation of myopodin expression occurs mostly in invasive stages of prostate cancer, implying a potential invasion suppression role for myopodin in prostate cancer. In addition, hemizygous deletion and down-regulation of myopodin expression occur in three aggressive prostate cancer cell lines. All these results support the hypothesis that myopodin functions as a tumor suppressor gene to limit the growth and to inhibit the metastasis of cancer cells.

Topics: Agar; Animals; Cell Division; Cell Line, Tumor; Collagen; Disease Progression; Down-Regulation; Drug Combinations; Gene Deletion; Humans; Immunoblotting; Immunohistochemistry; Laminin; Male; Mice; Mice, SCID; Microfilament Proteins; Neoplasm Metastasis; Neoplasm Transplantation; Prostatic Neoplasms; Protein Structure, Tertiary; Proteoglycans; Time Factors

Carcinoma-associated fibroblasts (CAF) promote tumor progression of pre-neoplastic epithelial cells. To investigate the basis of this phenomenon, we compared the properties of fibroblasts cultured from normal human prostate (NHPF) to prostate CAF. NHPF and CAF were assayed for growth potential, cell death and proliferative capacity by measuring population doubling time, cell cycle distribution and capability to form colonies in soft agar. Resistance to genotoxic (UV radiation: 0-50 J/cm2) and chemotoxic (0-200 nM Taxol) agents were compared between CAF and NHPF by measuring cell viability and cell cycle analysis. Transforming growth factor beta1 (TGF-beta1) immunoreactivity was assessed in non-malignant and malignant prostatic tissue. No detectable differences were found when comparing CAF and NHPF with respect to population doubling time, cell cycle distribution and response to genotoxic and chemotoxic agents. The mean number of colonies in soft agar was 120.5 for CAF vs. 18.2 for NHPF (p < 0.05). Because TGF-beta1 and matrix metalloproteinase (MMP)-9 have been associated with growth of fibroblasts in soft agar and tumor promotion, we measured the expression of these factors in NHPF and CAF by ELISA. There was no difference in expression of MMP-9; however, TGF-beta1 was expressed in higher concentrations in CAF than in NHPF (p < 0.0014). Furthermore, TGF-beta1 expression was higher in the carcinoma-associated stroma of prostate cancer tissue than stroma of non-malignant prostatic tissue. Increased capability of CAF as compared to NHPF to form colonies in soft agar may be due to a higher expression of TGF-beta1 and correlates with the ability of CAF to promote malignant progression of prostate epithelial cells.

Topics: Agar; Antineoplastic Agents; Carcinoma; Cell Cycle; Cell Death; Cell Division; Culture Media; Drug Resistance, Neoplasm; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Gene Expression Profiling; Humans; Male; Prostate; Prostatic Neoplasms; Radiation Tolerance; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Individuals supplemented with selenium have reduced incidence of prostate cancer. This study determines whether selenomethionine specifically affects the secretion of prostate specific antigen (PSA) in vitro.. LNCaP cells were supplemented with selenomethionine for 7 days. PSA secretion was determined by ELISA. Cell proliferation was assessed by enumeration of trypan blue excluding cells. Colony formation was determined in soft agar. Cell cycle distribution was determined by FACS analysis of propidium iodide stained cells.. Selenomethionine at > or = 70 microM inhibited LNCaP cell growth and colony formation. 0-100 microM selenomethionine did not affect the secretion of PSA by LNCaP cells in cell culture supernatants when normalized to the number of cells in culture. At supra-nutritional concentrations of selenomethionine, LNCaP cells had longer G(0)/G(1) phase in agreement with the inhibitory effects on cell growth.. PSA secretion is not specifically inhibited by concentrations of selenomethionine corresponding to plasma selenium concentrations found in individuals supplemented with chemopreventive concentrations of selenized yeast. These data suggest that changes in serum PSA levels in individual patients during selenium supplementation is not an effect specific for PSA secretion, but rather may be a useful indicator for changes in disease progression in individual patients.

Topics: Agar; Cell Division; Humans; Male; Prostate-Specific Antigen; Prostatic Neoplasms; Selenium; Selenomethionine; Stem Cells; Tumor Cells, Cultured

The molecular mechanism leading to the cancer metastasis to bone is poorly understood but yet determines prognosis and therapy. Here, we define a new molecular pathway that may account for the extraordinarily high osteotropism of prostate cancer. By using SPARC (secreted protein, acidic and rich in cysteine)-deficient mice and recombinant SPARC, we demonstrated that SPARC selectively supports the migration of highly metastatic relative to less metastatic prostate cancer cell lines to bone. Increased migration to SPARC can be traced to the activation of integrins alphaVbeta3 and alphaVbeta5 on tumor cells. Such activation is induced by an autocrine vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR)-2 loop on the tumor cells, which also supports the growth and proliferation of prostate cancer cells. A consequence of SPARC recognition by alphaVbeta5 is enhanced VEGF production. Thus, prostate cancer cells expressing VEGF/VEGFR-2 will activate alphaVbeta3 and alphaVbeta5 on their surface and use these integrins to migrate toward SPARC in bone. Within the bone environment, SPARC engagement of these integrins will stimulate growth of the tumor and further production of VEGF to support neoangiogenesis, thereby favoring the development of the metastatic tumor. Supporting this model, activated integrins were found to colocalize with VEGFR-2 in tissue samples of metastatic prostate tumors from patients.

Topics: Agar; Animals; Bone and Bones; Bone Neoplasms; Cell Division; Cell Movement; Flow Cytometry; Humans; Immunohistochemistry; Integrin alphaVbeta3; Integrins; Male; Mice; Models, Biological; Neoplasm Metastasis; Neoplasm Transplantation; Osteonectin; Prostatic Neoplasms; Receptors, Vitronectin; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured; Vascular Endothelial Growth Factor Receptor-2

G3139 is an 18-mer phosphorothioate oligodeoxyribonucleotide, which is targeted to the initiation codon region of the bcl-2mRNA. Although treatment of PC3 prostate cancer cells with G3139, which contains two CpG motifs, causes a dramatic decrease in bcl-2 protein expression after 3 days, it did not result in significant cellular apoptosis, as it does in many other cell lines. The absence of apoptosis was demonstrated by the absence of pro-caspase 3 cleavage products and of Annexin V cell surface expression. In addition, ATP production and the mitochondrial membrane potential DeltaPsim were preserved. Despite this, G3139 significantly inhibited the rate of cellular proliferation in complete media and blocked cloning in soft agar. G4232, a variant of G3139 that down-regulates bcl-2 expression to the same extent but has both CpG cytidines C5 methylated, was only minimally antiproliferative. A series of mismatched G3139-related oligomers were synthesized that could also substantially down-regulate bcl-2 protein expression, but only if the CpG motifs were preserved, demonstrating the presence of additional non-antisense mechanisms. G3139 caused production of reactive oxygen species in growth-arrested cells and oxidation of nuclear guanosine to 8-hydroxy-2'-deoxyguanosine, as determined by 1F7 monoclonal antibody staining. Bromodeoxyuridine incorporation studies demonstrated that G3139 induced a G1-S entry block and an intra-S-phase block in PC3 cells that persisted as long as 3 days. This finding coincides with the observation that expression of several proteins encoded by S-phase genes, including c-myb and poly(ADP-ribose) polymerase, were significantly reduced. These results illustrate the complexity of the mechanism of action of G3139 in PC3 cells.

Topics: Agar; Amino Acid Motifs; Annexin A5; Blotting, Northern; Blotting, Western; Bromodeoxyuridine; Cell Cycle; Cell Division; Cell Line, Tumor; Cell Membrane; Coloring Agents; CpG Islands; DNA; DNA Methylation; Down-Regulation; Enzyme Inhibitors; Flow Cytometry; G1 Phase; Guanosine; Humans; Male; Membrane Potentials; Mitochondria; Mutation; Oligonucleotides; Oligonucleotides, Antisense; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Reactive Oxygen Species; S Phase; Thionucleotides; Time Factors; Transfection

We have derived a panel of p53-null prostatic "basal" and "luminal" epithelial cell lines and their ras transformed counterparts to study stromal/epithelial interactions and the properties of tumors arising from "basal" and "luminal" cells.. Previously derived normal murine prostatic "basal" epithelial (PE-B-1) and "luminal" epithelial (PE-L-1) cell lines were transformed with N-Ras. These lines and a spontaneously transformed "luminal" cell line were inoculated subcutaneously or orthotopically into athymic mice, alone or in combination with normal prostatic smooth muscle cells (SMC).. All transformed lines formed subcutaneous tumors. SMC significantly enhanced the growth rate of the tumors arising from the "basal" and one of the "luminal" cell lines. The transformed "basal" line gave rise to tumors expressing both "basal" and "luminal" cytokeratins.. Prostatic SMC promote the growth of transformed epithelial cells, suggesting that prostatic stroma may promote tumor development. Furthermore, transformed "basal" cells give rise to tumors containing "luminal" cells, suggesting that although most human tumors have a "luminal" phenotype, they may originate from transformed "basal" cells.

Topics: Agar; Animals; Cell Differentiation; Cell Division; Cell Line, Transformed; Cell Transplantation; Culture Media; Dihydrotestosterone; Male; Mice; Mice, Inbred C57BL; Muscle, Smooth; Neoplasm Transplantation; Phenotype; Prostate; Prostatic Neoplasms; Stromal Cells; Transforming Growth Factor beta; Tumor Cells, Cultured

Microcell-mediated chromosome transfer allows for the introduction of normal chromosomes into tumor cells in an effort to identify putative tumor suppressor genes. We have used this approach to introduce an intact copy of chromosome 18 into the prostate cancer cell line DU145, and independently to introduce human chromosomes 8 and 18 into the prostate cancer cell line TSU-PR1. Introduction of an extra copy of human chromosome 8 had no effect on the growth properties in vitro or the tumorigenicity in vivo of TSU-PR1 cells. However, microcell hybrids containing an introduced copy of human chromosome 18 exhibited a longer population doubling time, retarded growth in soft agar, and slowed tumor growth in athymic nude mice. These experiments provide functional evidence for the presence of one or more tumor suppressor genes on human chromosome 18 that are involved in prostate cancer.

Topics: Agar; Animals; Cell Culture Techniques; Cell Division; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 18; Gene Transfer Techniques; Genes, Tumor Suppressor; Humans; Hybrid Cells; Male; Mice; Mice, Nude; Neoplasm Transplantation; Prostatic Neoplasms; Tumor Cells, Cultured

15-Lipoxygenase (15-LOX)-2 is expressed in benign prostate secretory cells and benign prostate produces 15S-hydroxyeicosatetraenoic acid (15S-HETE) from exogenous arachidonic acid (AA). In contrast, 15S-LOX-2 and 15S-HETE formation are reduced in prostate carcinoma (Pca). The mechanisms whereby reduced 15-LOX-2 may contribute to Pca development or progression are not known. We investigated the expression of peroxisome proliferator-activated receptor (PPAR) gamma in benign and malignant prostate tissues and the ability of 15S-HETE to activate PPARgamma-dependent transcription and modulate proliferation of the Pca cell line PC3. In contrast to benign prostate and similar to most Pca tissues, 15-LOX-2 mRNA was not detected in PC3 cells, and they did not produce detectable 15-HETE from [14C]AA. By reverse transcription-PCR, PPARgamma mRNA was present in 18 of 18 benign and 9 of 9 tumor specimens. The PPARgamma ligand BRL 49653 and 15S-HETE caused a dose-dependent inhibition of PC3 proliferation in a 14-day soft agar colony-forming assay (IC50 of 3 and 30 microM, respectively). 15S-HETE (10 microM) caused greater inhibition than 10 microM 15R-HETE. At 3 days, BRL 49653 and 15S-HETE caused a slight increase in cells in G0-G1 and a corresponding decrease in cells in S phase. In PC3 cells transiently transfected with a luciferase reporter linked to a PPAR response element, 1 microM BRL 49653 and 10 microM 15S-HETE caused approximately threefold and greater than twofold induction of PPAR-dependent transcription, respectively. By quantitative real-time reverse transcription-PCR and Northern analysis, 3-day treatment with BRL 49653 and 15S-HETE caused a reduction of PPARgamma expression but a marked up-regulation of the PPAR response element containing adipocyte type fatty acid binding protein. These results support the hypothesis that 15-LOX-2-derived 15S-HETE may constitute an endogenous ligand for PPARgamma in the prostate and that loss of this pathway by reduced expression of 15-LOX-2 may contribute to increased proliferation and reduced differentiation in prostate carcinoma.

Topics: Agar; Arachidonate 15-Lipoxygenase; Blotting, Northern; Catalysis; Cell Division; Culture Media; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Hydroxyeicosatetraenoic Acids; Isoenzymes; Luciferases; Male; Prostatic Neoplasms; Receptors, Cytoplasmic and Nuclear; Recombinant Fusion Proteins; RNA, Messenger; Rosiglitazone; Thiazoles; Thiazolidinediones; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

Research into molecular and genetic mechanisms underlying prostate carcinogenesis would be greatly advanced by in vitro models of prostate tumors representing primary tumors. We have successfully established an immortalized human prostate epithelial (HPE) cell culture derived from a primary tumor with telomerase. The actively proliferating early passaged RC-58T cells were transduced through infection with a retrovirus vector expressing the human telomerase catalytic subunit (hTERT). A high level of telomerase was detected in RC-58T/hTERT cells but not RC-58T cells. RC-58T/hTERT cells are currently growing well at passage 50, whereas RC-58T cells senesced at passage 7. RC-58T/hTERT cells exhibit transformed morphology. More importantly, these immortalized cells showed anchorage-independent growth as they formed colonies in soft agar and grew above the agar layer. Expression of androgen-regulated prostate specific gene NKX3.1 and epithelial specific cytokeratin 8 (CK8) but not prostate specific antigen (PSA) and androgen receptor was detected in RC-58T/hTERT cells. Prostate stem cell antigen (PSCA) and p16 were also expressed in this cell line. RC-58T/hTERT cells showed growth inhibition when exposed to retinoic acid and transforming growth factor (TGF)-beta1 known potent inhibitors of prostate epithelial cell growth. A number of chromosome alterations were observed including the loss of chromosomes Y, 3p, 10p, 17p, 18q and the gain of chromosomes 16 and 20. These results demonstrate that this primary tumor-derived HPE cell line retained its transformed phenotypes and should allow studies to elucidate molecular and genetic alterations involved in prostate cancer. This is the first documented case of an established human prostate cancer cell line from a primary tumor of a prostate cancer patient with telomerase.

Topics: Agar; Cell Culture Techniques; Cell Division; Cell Line, Transformed; Cell Size; Cell Transformation, Neoplastic; Chromosome Aberrations; DNA-Binding Proteins; Epithelial Cells; Humans; Karyotyping; Male; Models, Biological; Prostatic Neoplasms; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Telomerase; Transduction, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tretinoin; Tumor Cells, Cultured

To determine the thermal dose of a hyperthermia treatment, knowledge of the three-dimensional (3D) temperature distribution is mandatory. The aim of this paper is to validate an interstitial hyperthermia treatment planning system with which the full 3D temperature distribution can be obtained in individual patients. Within a phase I study, 12 patients with prostate cancer were treated with interstitial hyperthermia using our multi electrode current source interstitial hyperthermia treatment (MECS IHT) system. The temperature distribution was measured from within the heating devices and by additional thermometry. The perfusion level was estimated and the heating implant reconstructed. The steady-state temperature distribution was calculated using our interstitial hyperthermia treatment planning system. The simulated temperature distribution was validated by individually comparing the measured and simulated thermo-sensors, both for the thermometry integrated with the heating applicators and the additional thermometry. The entire procedure was also performed on a no-flow agar-agar phantom. It was shown that the calculated temperature distribution of an individual patient during MECS interstitial hyperthermia is very heterogeneous. The validation indicates that the calculated temperature elevations match the measurements within approximately 1 degrees C. Possible improvements are more precise reconstruction, incorporation of discrete vasculature and using a temperature-dependent, heterogeneous perfusion distribution. Further technical improvements of the MECS-IHT system may also result in better temperature calculations.

Topics: Agar; Calibration; Carcinoma; Electrodes; Humans; Hyperthermia, Induced; Male; Phantoms, Imaging; Prostatic Neoplasms; Reproducibility of Results; Temperature; Time Factors

This paper is a step in investigating whether three-dimensional (3D) ultrasound can be used intraoperatively to replace Computed Tomography (CT) for localization of brachytherapy seeds. In order to quantify the accuracy and variability of seed localization without introducing effects due to tissues, we first report our results with test phantoms. An inter- and intra-observer study was performed to assess the variability of 2 3D ultrasound scan acquisition methods: Tilt 3D scanning and pull-back 3D scanning. Seven observers measured the positions of gold seed markers in an agar phantom twice in each of the three orthogonal image planes. An analysis of variance (ANOVA) was performed to determine the intra- and inter-observer standard errors of measurement (SEM) and the minimum detectable changes in marker position (deltap). Average intra- and inter-observer SEMs for the tilt scan 3D image were 0.36 and 0.40 mm, respectively. Measurements of the pull-back scan 3D image yielded average intra- and inter-observer SEM of 0.46 and 0.49 mm, respectively. A paired difference analysis showed that the lower SEM for the tilt 3D scan image were statistically significant at a significance level of alpha= 0.05. The accuracy of the US measurements was tested by determining marker coordinates from CT images of the phantom in a stereotactic head frame. CT coordinates were matched to the ultrasound (US) coordinates by means of an affine transform. Average matching errors in x, y, and z were 0.02, 0.10, and -0.02 mm, respectively.

Topics: Agar; Analysis of Variance; Brachytherapy; Gold; Humans; Image Processing, Computer-Assisted; Male; Models, Statistical; Observer Variation; Phantoms, Imaging; Prostatic Neoplasms; Radiometry; Rectum; Reproducibility of Results; Tomography, X-Ray Computed; Ultrasonics

The contribution of the ETS2 transcription factor to the transformed state in prostate cancer cells has been assessed. Northern blot analysis easily detects ETS2 in DU145 and PC3, high grade human prostate cell lines, but ETS2 is not present in lower grade LNCaP cells. Stable transfection of PC3 and DU145 prostate cell lines with an antisense ETS2 vector or with a dominant negative ETS2 mutant significantly reduced the ability of DU145 and PC3 cells to form large colonies in soft agar. Thus, the presence of ETS2 is positively correlated with a more transformed phenotype and blockage of ETS2 function can reduce transformed properties of prostate cancer cells.

Topics: Agar; Androgens; Blotting, Northern; Cell Division; Cell Transformation, Neoplastic; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Genes, Dominant; Humans; Male; Phenotype; Precipitin Tests; Prostatic Neoplasms; Proto-Oncogene Protein c-ets-2; Proto-Oncogene Proteins; Repressor Proteins; RNA, Antisense; Sequence Deletion; Trans-Activators; Transcription Factors; Transfection; Tumor Cells, Cultured

Most patients' prostate cancers respond to androgen deprivation but relapse after periods of several months to years. Only two prostate cancer xenografts, LNCaP and PC-346, have been reported to be responsive to androgen deprivation and to relapse subsequently. Both of these tumors shrink slightly, if at all, and relapse less than 5 weeks after androgen withdrawal. After androgen withdrawal, the human primary prostate cancer xenograft CWR22 regresses markedly, and prostate-specific antigen (PSA) falls up to 3000-fold in the blood of mice. PSA usually returns to normal. In some animals, the tumor relapses and is then designated CWR22R. In these animals, PSA starts to rise approximately 2-7 months, and tumor begins to grow 3-10 months after castration. Animals with CWR22 need to be euthanized because of large tumors 6-12 weeks after the transplantation of CWR22. Androgen withdrawal prolongs life approximately 3-4-fold.

Topics: Agar; Androgens; Animals; Humans; Male; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms, Hormone-Dependent; Orchiectomy; Prostate-Specific Antigen; Prostatic Neoplasms; Testosterone; Transplantation, Heterologous; Tumor Cells, Cultured

Thirty-one bone marrow aspirations were performed on patients with prostatic carcinoma metastatic to bone. After separation over a Ficoll-Hypaque gradient viable nucleated cells were cultured in semisolid agar. Colony formation occurred in 14 of 27 (52%) nonbacterially contaminated cultures. Characterization of cells from the colonies showed them to be consistent with malignant prostate cells. After staining, these cells were periodic acid-Schiff positive, prostatic acid phosphatase positive, and prostatic specific antigen positive. Other studies demonstrated the cells to be karyotypically abnormal, ultrastructurally similar to epithelial cells, and capable of secondary colony formation. Three bone marrow aspirate specimens did not have metastatic prostatic carcinoma detected by standard methods but did demonstrate colony formation. However, colony formation was most frequently seen when a radionuclide scan was positive at the aspiration site and when tumor cells were microscopically detectable by Wright staining of a smeared aspirate. The potential utility of colony forming cultures in prostate cancer is discussed. In working with bone marrow aspirates, additional cell separation procedures may be required to calculate and maximize plating efficiencies.

Topics: Agar; Biopsy; Bone Marrow; Bone Neoplasms; Carcinoma; Cells, Cultured; Humans; Karyotyping; Male; Microscopy, Electron; Neoplasm Metastasis; Prostatic Neoplasms

Soft agar cultures of human prostatic carcinoma cells obtained from prostates with needle biopsy and metastatic lymph nodes were examined. Colony formation were observed in 2 of the 4 needle biopsy specimens and in 7 of the 10 lymphatic nodes specimens. The plating efficiency was 0.04 to 0.43%. The effect of testosterone on the clonal growth of these cells in soft agar culture was studied. In half of the cases, colony formation was increased by the addition of testosterone. Whereas in the other cases, colony formation was not influenced by the testosterone. Clinically, most of the former cases responded well to the anti-androgen therapy. Neither EGF nor hydrocortisone showed any effect on the colony formation of the prostatic cancer cells in soft agar. These results suggest that primary culture of human prostatic cancer is possible in soft agar medium, and that androgen dependency of these cells can be examined in vitro.

Topics: Adenocarcinoma; Agar; Aged; Cells, Cultured; Colony-Forming Units Assay; Culture Media; Humans; Male; Middle Aged; Prostatic Neoplasms; Testosterone; Tumor Stem Cell Assay

Human tumors were cultured by the two-layer soft-agar technique and the time course of tumor colony development was evaluated during periods of up to 6 weeks in culture. All colony counting was performed with an automated tumor colony counter (Omnicon; Bausch and Lomb, Inc, Rochester, NY, USA). This instrument provided colony counts per culture plate in six size categories from greater than 60 microns diameter colonies to greater than 149 microns diameter colonies. Six to 24 culture plates were used for each "growth curve", generally 24. Control (non-drug-treated) cultures were obtained from 117 tumors, of which 25 also provided enough cells to allow evaluation of the time course of colony development after exposure to cytostatic agents. The development of colonies in non-drug-treated plates usually demonstrated a lag phase, a logarithmic growth phase to maximum colony development and a subsequent deterioration of colonies. In spite of clumps seeded into the agar, real colony growth could be recognized by frequent colony counting of culture dishes, although the temporal patterns of growth were sometimes different if pure single-cell suspensions were compared with suspensions containing clumps from the same tumor. Drug pre-incubation caused changes in the temporal pattern of colony growth as well as in the total number of colonies. Some cultures showed drug sensitivity when evaluated at certain time points while evaluation at later time points showed only borderline drug effect or none at all. The potential utility of tumor colony growth curves in the clinical applications of tumor colony cultures is discussed.

Topics: Agar; Animals; Antineoplastic Agents; Breast Neoplasms; Carcinoma, Small Cell; Colony-Forming Units Assay; Cystadenoma; Female; Humans; Male; Ovarian Neoplasms; Prostatic Neoplasms; Rats; Skin Neoplasms; Staining and Labeling; Time Factors; Tumor Stem Cell Assay

We have systematically analyzed the proliferative properties of the clonogenic cells of the R3327 transplantable rat prostate adenocarcinoma (colony forming units, prostatic adenocarcinoma, CFU-PA). Pronase was determined to be more effective than either trypsin or collagenase in obtaining the largest number of viable cells from a solid R3327 tumor. Digestion with 2.5 mg/ml yielded a maximum of 5.6 X 10(4) CFU-PA/g of tumor tissue, with higher concentrations resulting in s substantially lower fraction of CFU-PA while producing larger overall cell yields. Bacto-agar at 0.35% supported the growth of the largest number of CFU-PA and was sharply concentration dependent with concentrations greater than 0.4% completely suppressing CFU-PA growth. The addition of conditioned medium (CM) from R3327 liquid cell cultures to agar cultures resulted in a specific twofold increase in CFU-PA/10(4) cells, whereas CM from R3327A cells was less effective and CM from rat skin fibroblasts least stimulatory. The addition of washed rat red blood cells (RBC) either within the agar culture itself or in an overlayer was highly stimulatory, resulting in as much as a fivefold increase in CFU-PA to 6 to 8 x 10(4)/g.

Topics: Adenocarcinoma; Agar; Animals; Cell Count; Cell Division; Cell Line; Erythrocytes; Female; Male; Mercaptoethanol; Neoplasms, Experimental; Peptide Hydrolases; Pronase; Prostatic Neoplasms; Rats; Rats, Inbred Strains; Thrombin

Cellular growth interactions were studied between neonatal human lung fibroblasts (NLF-13) and human tumor lines derived from carcinomas of the prostate (PC-3, DU145), bladder (J82), and endometrium (HEC-1A) and from a glioma (Hs 683t). NLF-13 were interacted with tumor cells in soft agar or agarose media using two experimental protocols. In one system, NLF-13 cells were grown as anchored monolayers proliferating under the tumor cell layer. In the second, NLF-13 were embedded directly (nonanchored) into the agar or agarose layer with the tumor cells. The results from both interaction systems were similar for all five tumor lines. Anchored NLF-13 caused a dose-dependent inhibition of tumor growth, whereas nonanchored cells produced a dose-dependent growth stimulation. A time exposure experiment indicated that tumor stimulation and inhibition were biphasic responses to NLF-13. It was concluded that low concentrations of a diffusible NLF-13 product(s) accelerated tumor growth, whereas high concentrations were inhibitory. Further, the production of the active NLF-13 substance(s) was positively correlated with NLF-13 growth rate. Tumor cell inhibition was irreversible after a 5-day exposure to proliferating NLF-13 cells. Another line of normal neonatal human lung fibroblasts (NLF-147) showed inhibitory properties similar to those described for NLF-13. However, preliminary studies with fibroblasts from the skin of a Down's syndrome neonate (DS-172) and from a human kidney tumor (KTF-130) have shown both these fibroblast types to have a reduced ability to inhibit tumor cell cultures (J82) compared to the neonatal lung fibroblasts (NLF-13 and NLF-147).

Topics: Agar; Cell Communication; Cell Division; Cell Line; Female; Fibroblasts; Glioma; Humans; Infant, Newborn; Kinetics; Lung; Male; Prostatic Neoplasms; Urinary Bladder Neoplasms; Uterine Neoplasms

The influence of normal human lung fibroblasts (NLF) on the clonal growth of human prostatic carcinoma cells (PC-3) in soft agar was studied. PC-3 growth was assessed by both colony-forming efficiency and clonal growth rate as estimated from increase in colony diameter. Effects of anchored NLF (proliferating cell monolayer) and nonanchored NLF (nonproliferating cells embedded in agar) on PC-3 growth were compared. Marked differences were observed. Anchored NLF inhibited whereas nonanchored NLF stimulated PC-3. Under suboptimal growth conditions (7% fetal bovine serum), PC-3 growth was stimulated about three-fold when NLF was added to the agar at a NLF:PC-3 seeding ratio of 60:1. Similar seeding ratios using anchored NLF cells caused a 98% inhibition of PC-3 growth. Control experiments with NLF-seeded coverslips on agar cultures ruled out medium depletion as a cause of the PC-3 inhibition. PC-3 inhibition was independent of NLF cell contact, required the continued presence of NLF cells, and was thus attributed to a diffusible, labile factor derived from anchored NLF monolayers. Kinetic analysis was used to determine the nature of the nonanchored NLF-fetal bovine serum interaction on stimulated PC-3 growth. Although the NLF factor was not a component of fetal bovine serum, it acted synergistically with it to stimulate PC-3 growth. It was concluded that there were strong correlations between (a) nonanchored NLF cells and PC-3 stimulation and (b) anchored NLF cells and PC-3 inhibition.

Topics: Agar; Cell Adhesion; Cell Division; Cells, Cultured; Culture Media; Fibroblasts; Humans; Male; Neoplasm Metastasis; Prostatic Neoplasms

Topics: Abdominal Neoplasms; Agar; Aged; Antigens, Neoplasm; Breast Neoplasms; Cell Division; Cells, Cultured; Clone Cells; Colonic Neoplasms; Female; Humans; Immune Sera; Immunity, Cellular; Kidney Neoplasms; Liposarcoma; Lymphocytes; Male; Melanoma; Methods; Middle Aged; Neoplasms; Prostatic Neoplasms; Rectal Neoplasms; Thyroid Neoplasms

Topics: Adult; Agar; Blood Protein Electrophoresis; Breast Neoplasms; Contraceptives, Oral; Estrogens; Female; Gels; Humans; Male; Pregnancy; Pregnancy Complications; Progesterone; Prostatic Neoplasms; Thyroid Gland; Thyroxine-Binding Proteins

Topics: Acrylates; Agar; Aminocaproates; Animals; Blood Coagulation Disorders; Blood Coagulation Tests; Electrophoresis; Extracorporeal Circulation; Fibrinogen; Fibrinolysin; Fibrinolysis; Fibrinolytic Agents; Gels; Humans; Immune Sera; Immunoelectrophoresis; Liver Cirrhosis; Male; Molecular Weight; Nicotinic Acids; Plasma; Prostatic Neoplasms; Rabbits