Clinical Cancer Research The Future of Cancer Research: Science and Patient Impact Infection and Cancer: Biology, Therapeutics, and Prevention
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, F.
Right arrow Articles by Dong, Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, F.
Right arrow Articles by Dong, Z.
Clinical Cancer Research Vol. 11, 4512-4520, June 15, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

Blockade of Transforming Growth Factor-ß Signaling Suppresses Progression of Androgen-Independent Human Prostate Cancer in Nude Mice

Fahao Zhang2, Juwon Lee1, Shan Lu1, Curtis A. Pettaway2 and Zhongyun Dong1

Authors' Affiliations: 1 Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio and 2 Department of Cancer Biology, University of Texas M.D. Anderson Cancer Center, Houston, Texas

Requests for reprints: Zhongyun Dong, Department of Internal Medicine, Hematology-Oncology Division, Box 0508, University of Cincinnati College of Medicine, Room 1308, 3125 Eden Avenue, Cincinnati, OH 45267. Phone: 513-558-2176; Fax: 513-558-6703; E-mail: dongzu{at}ucmail.uc.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
We investigated the role of transforming growth factor-ß (TGF-ß) signaling in the growth and metastasis of PC-3MM2 human prostate cancer cells. Highly metastatic PC-3MM2 human prostate cancer cells were engineered to constitutively overexpress a dominant-negative type II TGF-ß receptor (DNR). Transfection of DNR had minimal direct effects on cell growth and attenuated TGF-ß-induced cell growth inhibition and TGF-ß1 production. There were no discernable differences in tumorigenicity (tumor incidence) among PC-3MM2 variants when the cells were implanted into the prostates of nude mice. Growth rate and metastatic incidence of DNR-engineered PC-3MM2 cells, however, were significantly reduced. Most cells in the control tumors were positively stained by an antibody to proliferation cell nuclear antigen and very few cells were stained by terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL). In sharp contrast, tumors formed by PC-3MM2-DNR cells contained fewer proliferation cell nuclear antigen–positive cells and many more TUNEL-positive cells. Staining with antibody against CD31 showed that control tumors contained more blood vessels than PC-3MM2-DNR tumors. Expression of interleukin-8 (IL-8) in tumors formed by PC-3MM2 cells was significantly reduced as revealed by both Northern blotting and ELISA. Finally, transfection of antisense IL-8 cDNA significantly reduced IL-8 production by PC-3MM2 cells and antisense IL-8-transfected PC-3MM2 cells grew slower in comparison with parental and control vector-transfected cells. Taken together, our data suggest that TGF-ß signaling, by regulating IL-8 expression in tumor cells and hence tumor angiogenesis, is critical for progressive growth of PC-3MM2 cells in the prostate of nude mice.

Key Words: TGF-ß • Receptor • prostate cancer • angiogenesis • IL-8

The progressive growth of primary tumors and metastases of prostate cancer depends on an adequate blood supply (i.e., angiogenesis; refs. 1, 2). Microvessel density in invasive tumors directly correlates with their metastatic potential (3, 4). Angiogenesis is a multistep process emanating from microvascular endothelial cells. The process begins with local degradation of the basement membrane surrounding capillaries followed by invasion of the surrounding stroma by the underlying endothelial cells in the direction toward the source of angiogenic molecules (5). Human prostate cancer cells and the stroma in the tumor environment produce a number of proangiogenic factors. Among them are transforming growth factor-ß1 (TGF-ß1; refs. 6–8) and interleukin-8 (IL-8; refs. 9–11).

TGF-ß1 is overexpressed in prostate cancer, especially in advanced disease. It has been shown that up-regulation of TGF-ß1 in prostate cancer tissues and high urinary and serum levels of TGF-ß1 are associated with enhanced tumor angiogenesis and tumor metastasis and with poor clinical outcome (12–16). Cells engineered to overexpress TGF-ß1 produce larger, less necrotic, and more metastatic tumors than control cells in mice (16, 17). Paradoxically, most prostate cancer cells do express functional TGF-ß receptors and their growth can be inhibited by TGF-ß1 in vitro (18). Compelling evidence indicates that TGF-ß1 promotes tumor progression by altering the tumor microenvironment, such as inhibiting immune responses (19, 20) and stimulating tumor angiogenesis (7, 8, 12, 17).

IL-8, originally discovered as a chemokine (21), is a proangiogenic factor (22) by either inducing the expression of matrix metalloproteinases (23) or recruiting inflammatory cells (24–26). In human prostate cancer, serum levels of IL-8 have been shown to correlate with increasing prostate cancer stages and can differentiate benign prostatic hyperplasia from prostate cancer (10, 27). Prostate cancer cells but not benign prostatic hyperplasia and normal prostate cells express IL-8. Furthermore, the level of IL-8 correlates with the microvessel density in tumors (11, 28). Our recent study further showed that increased mRNA expression of IL-8 is associated with both the Gleason score and pathologic stage of tumors and can distinguish organ-confined from nonconfined tumors (29). Preclinical studies showed that overexpression of IL-8 in prostate cancer cells correlates with metastatic potential (28), and IL-8 gene-transfected cells produce higher levels of matrix metalloproteinases, are more invasive in Matrigel, and are more tumorigenic in mice. In contrast, antisense IL-8-transfected cells express lower levels of matrix metalloproteinases and became less invasive in vitro and less tumorigenic in mice (23). Moreover, IL-8-neutralizing antibody inhibited the angiogenic activity of prostate cancer homogenates and reduced progression of prostate cancer cells in mice (30).

Our previous studies showed that forced expression of IFN-ß in orthotopic tumors of PC-3 human prostate cancer cells in nude mice led to down-regulation of TGF-ß1 and IL-8, inhibition of tumor angiogenesis, suppression of tumor growth, and prolongation of the survival of tumor-bearing mice (31, 32). Because IL-8 in our PC-3 model can be derived from tumor cells only and TGF-ß is capable of inducing IL-8 expression (33–35), we hypothesized that TGF-ß-stimulated IL-8 expression contributes to the tumor blood vessel formation in this model. The purpose of this study is to test this hypothesis. We report here that TGF-ß1 inhibits the growth of PC-3 cells and induces IL-8 expression in the cells in vitro. Disruption of TGF-ß signaling by overexpression of a dominant-negative type II TGF-ß receptor (DNR) does not alter tumor incidence but inhibits growth and metastasis of orthotopic PC-3 tumors, which correlates with poor angiogenesis and reduced IL-8 expression in the tumor lesions. These data show that TGF-ß-regulated IL-8 expression is involved in angiogenesis and the progression of orthotopic PC-3 tumors in nude mice.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Mice. Specific pathogen-free male athymic BALB/c nude mice were purchased from the Animal Production Area of the National Cancer Institute, Frederick Cancer Research Facility (Frederick, MD). The mice were maintained in a facility approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the U.S. Department of Agriculture, U.S. Department of Health and Human Services, and NIH. The mice were used according to institutional guidelines when they were 8 to 10 weeks of age.

Reagents. EMEM, Ca2+, Mg2+-free HBSS, and fetal bovine serum (FBS) were purchased from M.A. Bioproducts (Walkersville, MD). An mRNA isolation kit was purchased from Invitrogen (San Diego, CA). Human TGF-ß1, human IL-8, and mouse tumor necrosis factor-{alpha} ELISA kits were purchased from BioSource International (Camarillo CA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was purchased from Sigma Chemical Co. (St. Louis, MO).

Tumor cells and culture. The well-characterized androgen-independent PC-3MM2 human prostate carcinoma cells (28, 36–38) were used. Cells were maintained as a monolayer culture in EMEM supplemented with 5% FBS, nonessential amino acids, sodium pyruvate, vitamin A, and glutamine. Cells in exponential growth phase were harvested by a 1-minute treatment with a 0.25% trypsin/0.02% EDTA solution. The flasks were tapped to detach the cells, MEM/10% FBS was added, and the cell suspension was gently agitated to produce a single-cell suspension. The cells were washed and resuspended in HBSS. Only suspensions of single cells with viability exceeding 95% (ascertained by trypan blue exclusion) were used.

Plasmids and transfection. The human TGF-ß type II receptor sequence between nucleotides 1 and 687, encoding the entire extracellular and transmembrane domains together with seven amino acids of the intracellular domain, was amplified by reverse-transcriptase PCR. Forward and reverse primers used were 5'-GCCATGGGTCGGGGGCTGCT-3' and 5'-TCAGGAGCTGATGTCAGAGCGGTC-3', respectively. The fragment amplified was inserted into a TA cloning vector (Invitrogen) according to the manufacturer's protocol. PC-3MM2 cells were transfected with the plasmid using FuGENE 6 transfection reagent (Roche Applied Science, Indianapolis, IN) at 37°C for 24 hours. The plasmid pcDNA3 (Invitrogen) was used as a control vector. Transfected cells were selected in medium containing 800 µg/mL G418. After 2 weeks of continuous culture, drug-resistant clones were isolated and expanded to derive PC-3MM2-DNR and PC-3MM2-Neo cells. Full coding region of antisense IL-8 cDNA was amplified by PCR using a plasmid containing IL-8 cDNA as a template and inserted into the TA cloning vector (Invitrogen). A plasmid containing the cDNA in antisense orientation was selected to transfect PC-3MM2 cells and the pcDNA3 vector was transfected as a control. After selection in G418, cells resistant to G418 were expanded to derive PC-3MM2-neo and PC-3MM2-IL8AS.

Doubling time. Tumor cells were plated at 5 x 103 cells per well of 24-well plates. After incubation for various lengths of time (1-5 days), the cells were harvested by trypsinization and counted. Doubling time was calculated from the growth curve of the cultures.

Effects of transforming growth factor-ß1 on cell growth in vitro. Effects of TGF-ß1 on cell growth in vitro were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as detailed in our previous studies (39, 40). PC-3MM2 parental, PC-3MM2-DNR, or PC-3MM2-Neo cells in 0.1 mL EMEM-5% FBS were plated at a density of 1,000 cells per 38-mm2 well of a 96-well plate. After an overnight incubation culture period, the cells were cultured in EMEM/1% FBS containing different concentrations of human TGF-ß1 at 37°C in 5% CO2 for 96 hours. During this period, the cells grew exponentially without change of medium. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (2 mg/mL in PBS) was added to the cultures at 0.05 mL per well during the final 2 hours of incubation. The medium was then carefully removed, and the dark blue formazan was dissolved in 100 µL per well of DMSO. The absorbance of each well was measured with a FluoStar Optima multi-detection microplate reader (BMG Labtechnologies, Durham, NC) at 570 nm. The percentages of growth inhibition were calculated according to the following formula: growth inhibition (%) = (1 – A570 nm of treated group / A570 nm of control group) x 100.

Tumor cell inoculation. Mice were anesthetized with Nembutal and placed in the supine position. The surgical procedure was done as detailed in our previous study (31, 38). Briefly, a lower midline incision was created and the prostate exposed. A tumor cell suspension (105 cells in 20 µL HBSS) was injected into the dorsal prostatic lobes using a 30-gauge needle, a 1-mL disposable syringe, and a calibrated push button-controlled dispensing device (Hamilton Syringe Company, Reno, NV). The abdominal wound was closed in one layer with wound clips (Autoclip, Clay Adama, Parsippany, NJ). Primary tumors (including the prostate) were excised and weighed after the mice were killed by cervical dislocation at days 21 to 28 after tumor inoculation. Regional lymph node metastasis was assessed by microscopic examination of H&E-stained serial paraffin sections. The tumor samples were collected for H&E staining, mRNA extraction, and immunohistochemical analysis.

Immunohistochemical staining. Immunohistochemical analyses were done as described previously (31, 38). Briefly, the tumor tissues were placed in optimal cutting temperature compounds and snap frozen in liquid nitrogen. Frozen sections (8-10 µm) were fixed in cold acetone and treated with 3% hydrogen peroxide (H2O2) in methanol (v/v). The treated slides were incubated in blocking solution and then reacted with an antibody to CD31 (41), also called platelet/endothelial cell adhesion molecule 1, a 130-kDa membrane protein constitutively expressed in microvessel endothelial cells (PharMingen, San Diego, CA) for 18 hours at 4°C in a humidified chamber. The sections were rinsed and incubated with peroxidase-conjugated secondary antibodies. A positive reaction was visualized by incubating the slides with stable 3,3'-diaminobenzidine and counterstaining with Mayer's hematoxylin. The slides were dried and mounted with Universal mount, and images were digitized using a Sony 3CD color video camera (Sony Corp., Tokyo, Japan) and a personal computer equipped with Optimas Image Analysis Software (Optimas Corp., Bothell, WA). For immunohistochemical staining of proliferative cell nuclear antigen, a 36-kDa DNA polymerase {delta} subunit whose expression levels peak during the S phase of the cell cycle (42), paraffin sections (3-5 µm) of the tumor samples were dewaxed and stained with a mouse monoclonal antibody (PharMingen) as described for the frozen sections.

Terminal deoxynucleotidyl transferase–mediated nick-end labeling assay. Cell death in tumor lesions was determined by the terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) method (43) using the DeadEnd Fluorometric TUNEL system (Promega, Madison, WI). Paraffin sections were dewaxed in xylene and rehydrated by sequentially immersing in graded ethanol. The slides were washed in saline and PBS, fixed in 4% methanol-free formaldehyde-PBS, treated with 20 µg/mL of proteinase K in distilled H2O for 15 minutes at room temperature, rinsed with distilled H2O, and incubated in 3% H2O2 in methanol for 5 minutes. The treated slides were incubated in terminal deoxynucleotidyl transferase buffer [30 mmol/L Trizma base (pH 7.2), 140 mmol/L sodium cacolydate, and 1 mmol/L CoC12] containing fluorescein-12-dUTP and recombinant terminal deoxynucleotidyl transferase for 1 hour at 37°C. The reaction was stopped with a buffer containing 300 mmol/L NaCl and 30 mmol/L sodium citrate and washed in PBS. Samples were immediately analyzed under a fluorescence microscope to view the green fluorescence of fluorescein.

mRNA isolation and Northern blot analyses. The mRNA was isolated from cells or tumor tissue using a FastTrack kit (Invitrogen) and analyzed by Northern blotting as described in our previous studies (31, 38, 44). Two to 3 µg of mRNA were fractionated on 1% denaturing formaldehyde/agarose gels, transferred to Gene Screen nylon membrane (DuPont Co., Boston, MA), and UV cross-linked with 120,000 µJ/cm2 using a UV Stratalinker 1800 (Stratagene, La Jolla, CA). Hybridization with cDNA probes was done as described. Filters were washed two to three times at 50°C to 60°C with 30 mmol/L NaCl/3 mmol/L sodium citrate (pH 7.2)/0.1% SDS. The DNA probes used were cDNA fragments corresponding to human IL-8 and ß-actin.

ELISA. PC-3MM2 tumors were collected and homogenized in PBS containing a proteinase inhibitor cocktail (Roche Applied Science). Protein concentrations were determined using a protein assay kit (Bio-Rad Laboratory, Hercules, CA) and IL-8, TGF-ß1, and tumor necrosis factor-ß in the lysates were measured by using the ELISA kits following the manufacturer's protocols.

Statistical analysis. The tumor growth was done with five mice per group and repeated at least once. Differences in tumor incidence among groups were analyzed with the {chi}2 test. Differences in tumor weight, effects of TGF-ß1 on cell growth and TGF-ß1 production in vitro, IL-8 and TGF-ß1 production in tumors among groups were compared by ANOVA.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Establishment of cell lines that overexpress dominant-negative type II transforming growth factor-ß receptor. To directly examine the role of TGF-ß signaling in regulating growth and progression of prostate cancer, we stably transfected the highly metastatic PC-3MM2 human prostate cancer cells with a dominant-negative mutant form of DNR. This construct of dominant-negative receptor lacks the intracellular kinase domain and hence cannot transduce TGF-ß signals to downstream targets in the cells (45). Twenty clones of cells that express high or low levels of DNR were pooled to derive PC-3MM2-DNR-PC-3MM2-DNR-H1, PC-3MM2-DNR-H2, PC-3MM2-DNR-L1, and PC-3MM2-DNR-L2. Parental cells and control vector-transfected cells serve as controls. DNR expression was confirmed by Northern blotting (Fig. 1A).



View larger version (26K):
[in this window]
[in a new window]
  		Fig. 1. Characterization of DNR-transfected cells. DNR-transfected cells were pooled to derive PC-3MM2-DNR-H1, PC-3MM2-DNR-H2, PC-3MM2-DNR-L1, and PC-3MM2-DNR-L2. Expression of DNR, a fragment of type II TGF-ß receptor, in parental, vector-transfected, or DNR-transfected cells was determined by Northern blot analysis.

 
To determine whether transfection of DNR or the control vector has any direct effect on the growth of PC-3MM2 cells, we measured the doubling times of the variants in their exponential phase of growth in culture and found that there were no discernable differences in the doubling time among parental, vector-transfected, and DNR-transfected cells (Table 1). To determine whether the transfection procedure or the transfected gene alters growth of cells under suboptimal conditions, the cells were cultured in medium supplemented with different concentrations of FBS (0.5-5%). We again found that there was no significant difference in growth rates among the variants (data not shown).


View this table:
[in this window]
[in a new window]
  		Table 1. Doubling times

 
Previous studies showed that PC-3 cells are susceptible to TGF-ß-induced growth inhibition (46–48). To test whether overexpression of the DNR construct in PC-3MM2 cells could compromise functions of TGF-ß1 in the cells, we evaluated effects of TGF-ß1 on growth of PC-3MM2 cells in vitro (Fig. 2A). Because TGF-ß1 secreted naturally by most types of cells are present in latent forms (49), recombinant active TGF-ß1 was added into cultures of PC-3MM2 cells. Growth of parental and control vector-transfected PC-3MM2 cells was suppressed by TGF-ß1 in a dose-dependent manner and the growth was inhibited up to 40% when the cells were exposed to 10 ng/mL TGF-ß1. In contrast, PC-3MM2-DNR-L1, PC-3MM2-DNR-L2, PC-3MM2-DNR-H1, and PC-3MM2-DNR-H2 cells became relatively resistant or refractory to the inhibitory effects of TGF-ß1. At 10 ng/mL, TGF-ß1 inhibited growth of PC-3MM2-DNR-L1 and PC-3MM2-DNR-L2 cells by 25% and of PC-3MM2-DNR-H1 and PC-3MM2-DNR-H2 cells by <5%, respectively. Taking advantage of the fact that TGF-ß1 can induce TGF-ß1 expression in an autocrine manner, we further determined whether the overexpression of DNR could affect TGF-ß-regulated gene expression. Cells of PC-3MM2 variants were cultured in serum-free medium with and without 3 ng/mL of TGF-ß1 for 48 hours, and TGF-ß1 in the culture supernatants was measured by ELISA. Data in Fig. 2B (left) showed that control and DNR-engineered cells constitutively produced 2 to 3 ng/106 cells/24 hours and there were no significant differences in the constitutive production of TGF-ß1 among the variants. Production of TGF-ß1 by parental and vector-transfected PC-3MM2 cells was increased by >4-fold after treatment with TGF-ß1 (Fig. 2B, right). In contrast, TGF-ß1-induced TGF-ß1 production was significantly reduced in PC-3MM2-DNR-L1 and PC-3MM2-DNR-L2 cells and was abolished in PC-3MM2-DNR-H1 and PC-3MM2-DNR-H2 cells (Fig. 2B, right). In control experiments, we verified that TGF-ß1 used in the treatment was not detectable after incubation in the same vessels (without cells) at 37°C for 24 hours in serum-free medium. These data clearly showed that overexpression of DNR compromised responses of the cells to TGF-ß1.



View larger version (28K):
[in this window]
[in a new window]
  		Fig. 2. Effects of TGF-ß1 on PC-3MM2 cells. A, parental, control vector, or DNR-transfected cells were plated into 96-well plates at 1,000 cells per well. After an overnight attachment period, the cells were treated with various concentrations of TGF-ß1 in medium supplemented with 0.5% FBS for 5 days. Viable cells in the culture were then stained by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and effects of TGF-ß1 on cell growth were determined. B, variants of PC-3MM2 cells were plated into 24-well plates at 2 x 105 cells per well. After an overnight incubation, the cells were incubated in serum-free medium supplemented with 3 ng/mL of TGF-ß1 for 48 hours. TGF-ß1 in culture supernatants of PC-3MM2 cell variants were determined by ELISA. Columns, mean of three independent experiments; bars, ±SE. *, P < 0.05 in comparison with PC-3MM2.

 
Effects of overexpression of dominant-negative type II transforming growth factor-ß receptor on tumorigenicity and tumor progression. Next, we investigated whether overexpression of DNR could alter growth and metastasis of prostate cancer cells in nude mice. PC-3MM2-DNR-H1 and PC-3MM2-DNR-H2 and PC-3MM2-DNR-L1 and PC-3MM2-DNR-L2 cells were pooled to derive PC-3MM2-DNR-H and PC-3MM2-DNR-L cells, respectively. PC-3MM2, PC-3MM2-neo, PC-3MM2-DNR-L, or PC-3MM2-DNR-H cells (105 per mouse in 20 µL of HBSS) were inoculated into the prostate of nude mice. On day 28 after tumor cell inoculation, mice were sacrificed and tumor incidence and weight measured. Paraffin sections of aortic lymph node were stained by H&E to identify metastases. As shown in Table 2, all mice inoculated with PC-3MM2, PC-3MM2-neo, or PC-3MM2-DNR-L cells, and 8 of 10 mice injected with PC-3MM2-DNR-H cells developed orthotopic tumors. Therefore, overexpression of DNR did not significantly alter the tumorigenicity of PC-3MM2 cells. Tumors formed by PC-3MM2-DNR-L and PC-3MM2-DNR-H cells were, however, significantly smaller than those formed by parental and control vector-transfected cells (Fig. 3A). Paraaortic lymph node metastases were found in 8 of 10 and 9 of 10 mice inoculated with PC-3MM2 and PC-3MM2-neo cells, respectively. In contrast, only 6 of 10 and 1 of 10 mice injected with PC-3MM2-DNR-L and PC-3MM2-DNR-H cells, respectively, developed lymph node metastases (Table 2). These data indicate that a blockade of TGF-ß signaling significantly compromised tumor progression.


View this table:
[in this window]
[in a new window]
  		Table 2. Tumor and metastasis incidences

 


View larger version (12K):
[in this window]
[in a new window]
  		Fig. 3. Growth of PC-3MM2 cells in the prostate of nude mice. PC-3MM2 cell variants (parental, Neo, DNR-L, and DNR-H) were inoculated into the prostates of nude mice at 2 x 105 cells per mouse (10 mice per group). The experiments were terminated 4 to 5 weeks later. Primary tumors in the prostates were removed and weighed. Columns, mean of 10 mice; bars, ±SD. *, P < 0.05 in comparison with PC-3MM2 parental tumors.

 
Effects of overexpression of dominant-negative type II TGF-ß receptor on cell growth, apoptosis, and angiogenesis in orthotopic tumors of PC-3MM2 cells in nude mice. On day 14 after tumor cell inoculation, cell growth and apoptosis (the two variables that determine tumor growth rates) were evaluated by immunohistochemical staining using proliferative cell nuclear antigen–specific antibody and TUNEL staining, respectively. As shown in Fig. 4, most cells (over 95%) in tumors of PC-3MM2 parental and PC-3MM2 control vector-transfected cells were stained strongly by the proliferative cell nuclear antigen antibody and very few cells (<1%) stained positive in the TUNEL assay. A majority of cells in PC-3MM2-DNR-L (~80%) and PC-3MM2-DNR-H (~60%) tumors were also stained positively by the proliferative cell nuclear antigen–specific antibody. The intensity of the staining, however, seemed weaker than that in tumors from control cells (Fig. 4). In sharp contrast to control tumors, many cells in PC-3MM2-DNR tumors (~10%), especially the PC-3MM2-DNR-H tumors (over 40%), were stained strongly positive by the TUNEL method (Fig. 4). To elucidate mechanisms responsible for the differences in cell growth and apoptosis among tumors of control and DNR-transfected cells, microvessel densities in the tumors were measured. As illustrated in Fig. 4, tumors from parental and control vector-transfected cells contained significantly more CD31-positive cells than tumors from DNR-transfected cells. Quantitative analysis revealed that the microvessel densities in tumors of PC-3MM2, PC-3MM2-Neo, PC-3MM2-DNR-L, and PC-3MM2-DNR-H were 217 ± 67/mm2, 254 ± 85/mm2, 126 ± 32/mm2, and 53 ± 17/mm2, respectively.



View larger version (122K):
[in this window]
[in a new window]
  		Fig. 4. Immunohistochemical staining of prostatic tumors formed by PC-3MM2 cell variants. On day 14 after tumor cell inoculation, prostatic tumors were removed and fixed for in vitro staining. For PCNA and TUNEL staining, paraffin-embedded sections were used and for CD31 staining and frozen sections were used.

 
IL-8 is a potent angiogenic molecule in prostate cancer (11, 23, 30, 50) and growth of PC-3 tumors in nude mice can be inhibited by IL-8-neutralizing antibody (30). Our previous studies showed that IFN-ß gene therapy–induced growth inhibition of PC-3MM2 tumors in nude mice correlates down-regulation of TGF-ß1 and IL-8 expression (31, 32). We therefore examined the expression of these two cytokines in orthotopic PC-3MM2 tumors. IL-8 mRNA was detected in tumors formed by parental and control vector-transfected PC-3MM2 cells but not in tumors formed by PC-3MM2 cells that overexpress the DNR construct (Fig. 5A). ELISA analysis revealed IL-8 levels in tumors formed by PC-3MM2, PC-3MM2-neo, PC-3MM2-DNR-L, and PC-3MM2-DNR-H cells were 28.5, 65, 12.6, and 1.8 ng/100 mg tumor tissue protein, respectively (Table 3). TGF-ß1 levels in tumors formed by DNR-transfected cells, especially those from PC-3MM2-DNR-H cells, were also moderately reduced (Table 3). Tumor necrosis factor-{alpha} a classic IL-8-inducing proinflammatory cytokine that can stimulate IL-8 expression by PC-3MM2 cells (data not shown), was not detectable in all tumor samples. To investigate whether TGF-ß1 directly stimulated IL-8 expression, we determined effects of TGF-ß1 on IL-8 expression in PC-3MM2 cells in vitro. As shown in Fig. 5B, the three lines of PC-3MM2 cells (PC-3MM2, PC-3MM2-Neo, and PC-3MM2-DNR-H) constitutively produced IL-8. After incubation with 10 ng/mL of TGF-ß1 for 24 hours, the production of IL-8 in parental cells and control vector-transfected cells, but not DNR-transfected cells, was significantly enhanced.



View larger version (26K):
[in this window]
[in a new window]
  		Fig. 5. Expression of IL-8 and TGF-ß1 in PC-3MM2 tumors. Tumor cells were inoculated into the prostate of nude mice (10 mice per group). A, 21 days later, the tumors were removed for in vitro analyses to determine DNR and IL-8 mRNA expression by Northern blotting. B, TGF-ß1-induced IL-8 expression in PC-3MM2, PC-3MM2-neo, and PC-3MM2-DNR-H cells in culture. Columns, mean of three to five tumors.

 

View this table:
[in this window]
[in a new window]
  		Table 3. Expression of IL-8, TGF-ß1, and TNF-{alpha} in tumors

 
Effects of down-regulation of interleukin-8 expression on growth of PC-3MM2 tumors. In the last set of experiments, we investigated the role of IL-8 in the growth of PC-3MM2 tumors. PC-3MM2 cells were transfected with antisense IL-8 cDNA or its pcDNA3 control vector and selected in G418 to establish a stable cell line PC-3MM2-IL8-AS or PC-3MM2-vect. ELISA analysis showed that the transfection of antisense IL-8 cDNA abolished constitutive production of IL-8 (5.25 ng/106 cells/24 hours versus 0 ng/106 cells/24 hours in PC-3MM2 cells and PC-3MM2-IL8-AS cells, respectively). TGF-ß1-induced IL-8 production was also significantly compromised (16.8 ng/106 cells/24 hours in PC-3MM2 versus 0.25 ng/106 cells/24 hours in PC-3MM2-IL8-AS cells). Transfection of the control vector had no significant effects on either constitutive or TGF-ß1-induced IL-8 production (data not shown). After verifying that the transfection of neither the antisense IL-8 cDNA nor its control vector affected in vitro cell growth, including doubling time and serum dependency (data not shown), we determined effects of down-regulation of IL-8 on growth and metastasis of PC-3MM2 cells in mice. Data in Fig. 6A showed that PC-3MM2-IL8AS cells produced significantly smaller tumors in the prostate of nude mice compared with PC-3MM2 parental and control vector-transfected cells. Figure 6B revealed that IL-8 expression levels in PC-3MM2-IL8AS tumors was significantly reduced in comparison with those in tumors formed by PC-3MM2 parental and PC-3MM2-vect cells.



View larger version (14K):
[in this window]
[in a new window]
  		Fig. 6. Effects of antisense IL-8 on growth of PC-3MM2 cells in the prostate of nude mice. PC-3MM2 cells were transfected with control vector pCR3.1 or the vector containing IL-8 cDNA in antisense orientation. After selection in G418, parental, control vector-transfected, or the antisense IL-8 cDNA-transfected cells were implanted into the prostates of nude mice (10 mice per group). Mice were sacrificed 4 weeks after tumor cell inoculation. Tumor weight (A) and IL-8 protein levels (B) were determined by ELISA. A, columns, mean of 10 mice; bars, ±SD. B, columns, mean of three to five tumors; bars, ±SD. *, P < 0.05 in comparison with PC-3MM2 parental tumors.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
We investigated the role of TGF-ß1 signaling in growth and metastasis of the highly metastatic PC-3MM2 human prostate cancer cells. The DNR was overexpressed to interrupt TGF-ß signaling. We found that although transfection of the mutant gene had no detectable direct effect on growth of PC-3MM2 cells (the doubling time and the serum dependence) in vitro, it reduced or abrogated responses of the cells to TGF-ß1 (growth inhibition and gene expression). The loss or attenuation of TGF-ß signaling in the transfected cells had minimal, if any, effects on tumorigenicity (tumor incidence) but significantly retarded the growth of primary tumors and reduced metastases of PC-3MM2 cells. IL-8 expression and angiogenesis were significantly reduced in tumors formed by the DNR-transfected cells. These results showed that TGF-ß signaling, in paracrine and/or autocrine fashions, is critical for the progression of orthotopic PC-3MM2 tumors in nude mice.

TGF-ß signaling plays complex roles in prostate cancer carcinogenesis and progression. In normal prostate tissue, activation of TGF-ß1 signaling inhibits cell growth and induces apoptosis in epithelial cells (51, 52) and thus serves as a tumor suppressor. Indeed, overexpression of a DNR induces malignant transformation of nontumorigenic rat prostate epithelial cells (45, 53). It is noteworthy, however, that a significant fraction of prostate cancer induced by overexpression of the dominant-negative type II receptor is squamous carcinoma, which only accounts for 0.5% to 1% of primary tumors of human prostate cancer (54). In addition, it has been shown that overexpression of type II TGF-ß receptor in LNCaP human prostate cancer cells can suppresses tumorigenicity via induction of caspase-1–mediated apoptosis (55, 56). Paradoxically, TGF-ß1 is overexpressed in prostate cancer, especially in advanced disease (6, 57). It has been suggested that prostate cancer cells are relatively more resistant to TGF-ß-mediated growth inhibition or apoptosis due to the down-regulation of TGF-ß receptors or an alteration of TGF-ß signaling pathways (6, 57). Most prostate cancer cells, however, do express functional receptors and their growth can be inhibited by TGF-ß1 in vitro (18). Cells forced to overexpress TGF-ß1 grow slower in vitro, due to the inhibitory effects of TGF-ß1, but produce larger, less necrotic, and more metastatic tumors than control cells in mice (16, 17). These findings, as well as the data presented in this article, support the hypothesis that TGF-ß signaling serve as tumor suppressors in tumorigenesis or carcinogenesis but promote tumor progression in advanced tumors (58). This hypothesis is also strongly supported by findings in breast cancer showing that overexpression of the dominant-negative type II receptor promotes mammary tumor formation in transgenic mice (59), but suppresses progression of the highly metastatic 4T1 murine mammary tumors (60). This TGF-ß switch is further documented in a recent study using a series of genetically related human breast-derived cell lines overexpressed with the dominant-negative type II receptor (61).

TGF-ß may promote progression of advanced tumors by several paracrine and autocrine mechanisms (58). TGF-ß1, one of most potent immune suppressors, inhibits the proliferation and/or activation of cytotoxic T cells, natural killer cells, neutrophils, and macrophages (62) and facilitates tumor progression by inhibiting functions of host defense systems (19, 20). In the highly metastatic 4T1 murine mammary tumor model, overexpression of a DNR is shown to significantly reduce tumor cell invasion (60). In addition, many clinical studies showed that up-regulation of TGF-ß1 in prostate cancer tissues and high urinary and serum levels of TGF-ß1 are associated with enhanced tumor angiogenesis and with poor clinical outcome (12–16). Effects of TGF-ßs on tumor angiogenesis are also confirmed in animal models in mice (7, 8, 12, 17). The mechanisms by which TGF-ß1 promotes angiogenesis remain obscure. It was noted, however, that TGF-ß1-induced angiogenesis is often associated with inflammatory infiltrations and blockade of TGF-ß function simultaneously inhibits the inflammation and angiogenesis (63). Data presented in this article suggest that TGF-ß may promote tumor angiogenesis by enhancing angiogenic molecule IL-8 expression in tumor cells. We based this conclusion on several lines of evidence. First, TGF-ß1 is capable of inducing IL-8 expression in PC-3MM2 cells. Second, overexpression of the DNR in PC-3MM2 cells does not affect their tumorigenicity (tumor incidence) but significantly reduces tumor progression (tumor volume and metastatic incidence). Third, tumors formed by PC-3MM2 cells engineered with the DNR are poorly vascularized and contain much lower levels of IL-8. Fourth, forced expression of anti-sense IL-8 does not affect in vitro growth of PC-3MM2 cells but significantly compromises growth of PC-3MM2 cells in the prostates of nude mice. Our data do not exclude, however, the possibility that TGF-ß1 may affect tumor angiogenesis through regulating expression of other angiogenesis-related genes, such as metalloproteinases as well as other cytokines (64). Moreover, it should be noted that tumor cells are heterogenic in multiple properties and, hence, effects of TGF-ß1 on angiogenic molecule expression and cell growth may vary with tumors and with tumor models. For example, it has been documented that overexpression of TGF-ß receptor in LNCaP cells enhances TGF-ß-induced apoptosis in vitro and reduces their tumorigenicity in mice (55, 56).

One striking observation in this study is that tumors formed by PC-3MM2 cells that overexpress the dominant-negative type II receptor of TGF-ß contain significantly higher levels of apoptotic cells. The direct and logical explanation for this massive apoptosis is the lack of angiogenesis due to the reduced or diminished IL-8 production stimulated by TGF-ß1. However, a recent study indicates that IL-8 promotes androgen-independent growth of prostate cancer cells (65) suggesting that the massive apoptosis may also be caused directly by the lack of the growth-stimulating effects of IL-8 or the combination effects of the two. Together with the finding that IL-8 promotes androgen-independent growth in LNCaP cells, our data also suggest the TGF-ß1-induced IL-8 expression may play an important role in the transition of androgen-dependent to androgen-independent growth.

In summary, we showed that overexpression of a DNR in PC-3MM2 cells did not alter tumorigenicity of the cells in the prostate of mice but compromised progression of orthotopic tumors formed by the cells and that IL-8 expression and angiogenesis in tumors formed by the dominant-negative receptor–engineered cells are significantly reduced. These data indicate that the TGF-ß-regulated IL-8 expression cascade is critical for the progression of orthotopic tumors formed by PC-3MM2 cells in nude mice. IL-8 is overexpressed in prostate cancer (10, 27) and our recent data further revealed that IL-8 overexpression is associated with both the Gleason's score and pathologic stage of tumors and distinguish organ-confined from nonconfined tumors. The mechanisms responsible for IL-8 overexpression in prostate cancer, especially in primary tumors, however, remain unclear. Data presented in this report, derived from a well-documented orthotopic model of human prostate cancer, suggest a potential mechanism by which IL-8 is up-regulated prostate cancer.


   Acknowledgments
 
We thank Dr. Isaiah J. Fidler (University of Texas M.D. Anderson Cancer Center, Houston, TX) for providing us with plasmid containing IL-8 expression cassette and for advice on our research, Donna Reynolds (University of Texas M.D. Anderson Cancer Center) for technical assistance with immunohistochemical staining, and Dr. Robert Franco and Kathryn Lund for critical reading of this article.


   Footnotes
 
Grant support: University of Cincinnati College of Medicine Cancer Center and National Cancer Institute, NIH grant CA97099-01A1 (Z. Dong).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12/13/04; revised 3/ 9/05; accepted 3/21/05.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Folkman J. Clinical application of research on angiogenesis. N Engl J Med 1995;333:1753–63.
  2. Folkman J. Tumor angiogenesis. Advances in Cancer Research 1985;43:175–203.[Medline]
  3. Brawer MK. Quantitative microvessel density. A staging and prognostic marker for human prostatic carcinoma. Cancer 1996;78:345–9.[CrossRef][Medline]
  4. Strohmeyer D, Rossing C, Strauss F, Bauerfeind A, Kaufmann O, Loening S. Tumor angiogenesis is associated with progression after radical prostatectomy in pT2/pT3 prostate cancer. Prostate 2000;42:26–33.[CrossRef][Medline]
  5. Folkman J, Klagsburn M. Angiogenic factors. Science 1987;235:442–7.[Abstract/Free Full Text]
  6. Wikstrom P, Damber JE, Bergh A. Role of transforming growth factor-ß1 in prostate cancer. Microsc Res Tech 2001;52:411–9.[CrossRef][Medline]
  7. Yang EY, Moses HL. Transforming growth factor ß1-induced changes in cell migration, proliferation, and angiogenesis in the chicken chorioallantoic membrane. J Cell Biol 1990;111:731–41.[Abstract/Free Full Text]
  8. Roberts AB, Sporn MB. Regulation of endothelial cell growth, architecture, and matrix synthesis by TGF-ß. Am J Respir Crit Care Med 1989;140:1126–8.
  9. Balbay MD, Pettaway CA, Kuniyasu H, et al. Highly metastatic human prostate cancer growing within the prostate of athymic mice overexpresses vascular endothelial growth factor. Clin Cancer Res 1999;5:783–9.[Abstract/Free Full Text]
  10. Veltri RW, Miller MC, Zhao G, et al. Interleukin-8 serum levels in patients with benign prostatic hyperplasia and prostate cancer. Urology 1999;53:139–47.[CrossRef][Medline]
  11. Ferrer FA, Miller LJ, Andrawis RI, et al. Angiogenesis and prostate cancer: in vivo and in vitro expression of angiogenesis factors by prostate cancer cells. Urology 1998;51:161–7.[Medline]
  12. Wikstrom P, Stattin P, Franck-Lissbrant I, Damber JE, Bergh A. Transforming growth factor ß1 is associated with angiogenesis, metastasis, and poor clinical outcome in prostate cancer. Prostate 1998;37:19–29.[CrossRef][Medline]
  13. Perry KT, Anthony CT, Case T, Steiner MS. Transforming growth factor ß as a clinical biomarker for prostate cancer. Urology 1997;49:151–5.[CrossRef][Medline]
  14. Adler HL, McCurdy MA, Kattan MW, Timme TL, Scardino PT, Thompson TC. Elevated levels of circulating interleukin-6 and transforming growth factor-ß1 in patients with metastatic prostatic carcinoma. J Urol 1999;161:182–7.[CrossRef][Medline]
  15. Steiner MS, Zhou ZZ, Tonb DC, Barrack ER. Expression of transforming growth factor-ß1 in prostate cancer. Endocrinology 1994;135:2240–7.[Abstract]
  16. Steiner MS, Barrack ER. Transforming growth factor-ß1 overproduction in prostate cancer: effects on growth in vivo and in vitro. Mol Endocrinol 1992;6:15–25.[Abstract]
  17. Stearns ME, Garcia FU, Fudge K, Rhim J, Wang M. Role of interleukin 10 and transforming growth factor ß1 in the angiogenesis and metastasis of human prostate primary tumor lines from orthotopic implants in severe combined immunodeficiency mice. Clin Cancer Res 1999;5:711–20.[Abstract/Free Full Text]
  18. Barrack ER. TGF ß in prostate cancer: a growth inhibitor that can enhance tumorigenicity. Prostate 1997;31:61–70.[CrossRef][Medline]
  19. Fontana A, Constam DB, Frei K, Malipiero U, Pfister HW. Modulation of the immune response by transforming growth factor ß. Int Arch Allergy Immunol 1992;99:1–7.[CrossRef][Medline]
  20. Torre-Amione G, Beauchamp RD, Koeppen H, et al. A highly immunogenic tumor transfected with a murine transforming growth factor type ß1 cDNA escapes immune surveillance. Proc Natl Acad Sci U S A 1990;87:1486–90.[Abstract/Free Full Text]
  21. Walz A, Peveri P, Aschauer H, Baggiolini M. Purification and amino acid sequencing of NAF, a novel neutrophil-activating factor produced by monocytes. Biochem Biophys Res Commun 1987;149:755–61.[CrossRef][Medline]
  22. Koch AE, Polverini PJ, Kunkel SL, et al. Interleukin-8 as a macrophage-derived mediator of angiogenesis. Science 1992;258:1798–801.[Abstract/Free Full Text]
  23. Inoue K, Slaton JW, Eve BY, et al. Interleukin 8 expression regulates tumorigenicity and metastases in androgen-independent prostate cancer. Clin Cancer Res 2000;6:2104–19.[Abstract/Free Full Text]
  24. Sunderkotter C, Goebeler M, Schulze-Osthoff K, Bhardwaj R, Sorg C. Macrophage-derived angiogenesis factors. Pharmacol Ther 1991;51:195–216.[CrossRef][Medline]
  25. Arnold F, West D, Kumar S. Wound healing: the effect of macrophage and tumour derived angiogenesis factors on skin graft vascularization. Br J Exp Path 1987;68:569–74.[Medline]
  26. Chen HH, Chien CH, Liu HM. Correlation between angiogenesis and basic fibroblast growth factor expression in experimental brain infarct. Stroke 1994;25:1651–7.[Abstract]
  27. Lehrer S, Diamond EJ, Mamkine B, Stone NN, Stock RG. Serum interleukin-8 is elevated in men with prostate cancer and bone metastases. Technol Cancer Res Treat 2004;3:411.[Medline]
  28. Greene GF, Kitadai Y, Pettaway CA, von Eschenbach AC, Bucana CD, Fidler IJ. Correlation of metastasis-related gene expression with metastatic potential in human prostate carcinoma cells implanted in nude mice using an in situ messenger RNA hybridization technique. Am J Pathol 1997;150:1571–82.[Abstract]
  29. Uehara H, Troncoso P, Johnston D, et al. Expression of interleuken-8 gene in radical prostatectomy specimens is associated with advanced pathologic stage. Prostate. In press.
  30. Moore BB, Arenberg DA, Stoy K, et al. Distinct CXC chemokines mediate tumorigenicity of prostate cancer cells. Am J Pathol 1999;154:1503–12.[Abstract/Free Full Text]
  31. Cao G, Su J, Lu W, et al. Adenovirus-mediated interferon-ß gene therapy suppresses growth and metastasis of human prostate cancer in nude mice. Cancer Gene Ther 2001;8:497–505.[CrossRef][Medline]
  32. Zhang F, Lu W, Dong Z. Tumor-infiltrating macrophages are involved in suppressing growth and metastasis of human prostate cancer cells by INF-ß gene therapy in nude mice. Clin Cancer Res 2002;8:2942–51.[Abstract/Free Full Text]
  33. Kumar NM, Rabadi NH, Sigurdson LS, Schunemann HJ, Lwebuga-Mukasa JS. Induction of interleukin-1 and interleukin-8 mRNAs and proteins by TGF ß1 in rat lung alveolar epithelial cells. J Cell Physiol 1996;169:186–99.[CrossRef][Medline]
  34. Fong CY, Pang L, Holland E, Knox AJ. TGF-ß1 stimulates IL-8 release, COX-2 expression, and PGE(2) release in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2000;279:L201–7.[Abstract/Free Full Text]
  35. Gerritsma JS, van Kooten C, Gerritsen AF, van Es LA, Daha MR. Transforming growth factor-ß1 regulates chemokine and complement production by human proximal tubular epithelial cells. Kidney Int 1998;53:609–16.[CrossRef][Medline]
  36. Kozlowski JM, Fidler IJ, Campbell D, Xu ZL, Kaighn ME, Hart IR. Metastatic behavior of human tumor cell lines grown in the nude mouse. Cancer Res 1984;44:3522–9.[Abstract/Free Full Text]
  37. Stephenson RA, Dinney CP, Gohji K, Ordonez NG, Killion JJ, Fidler IJ. Metastatic model for human prostate cancer using orthotopic implantation in nude mice. J Natl Cancer Inst 1992;84:951–7.[Abstract/Free Full Text]
  38. Dong Z, Greene G, Pettaway C, et al. Suppression of angiogenesis, tumorigenicity, and metastasis by human prostate cancer cells engineered to produce interferon-ß. Cancer Res 1999;59:872–9.[Abstract/Free Full Text]
  39. Dong Z, Juang SH, Kumar R, et al. Suppression of tumorigenicity and metastasis in murine UV-2237 fibrosarcoma cells by infection with a retroviral vector harboring the interferon-ß gene. Cancer Immunol Immunother 1998;46:137–46.[CrossRef][Medline]
  40. Dong Z, Ward NE, Fan D, Gupta KP, O'Brian CA. In vitro model for intrinsic drug resistance: effects of protein kinase C activators on the chemosensitivity of cultured human colon cancer cells. Mol Pharmacol 1991;39:563–9.[Abstract]
  41. DeLisser HM, Newman PJ, Albelda SM. Molecular and functional aspects of PECAM-1/CD31. Immunol Today 1994;15:490–5.[CrossRef][Medline]
  42. Travali S, Ku DH, Rizzo MG, Ottavio L, Baserga R, Calabretta B. Structure of the human gene for the proliferating cell nuclear antigen. J Biol Chem 1989;264:7466–72.[Abstract/Free Full Text]
  43. Gavrieli Y, Shermen Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992;119:493–501.[Abstract/Free Full Text]
  44. Dong Z, Radinsky R, Fan D, et al. Organ-specific modulation of steady-state mdr gene expression and drug resistance in murine colon cancer cells. J Natl Cancer Inst 1994;86:913–20.[Abstract/Free Full Text]
  45. Tang B, de Castro K, Barnes HE, et al. Loss of responsiveness to transforming growth factor ß induces malignant transformation of nontumorigenic rat prostate epithelial cells. Cancer Res 1999;59:4834–42.[Abstract/Free Full Text]
  46. Bang YJ, Kim SJ, Danielpour D, et al. Cyclic amp induces transforming growth factor ß2 gene expression and growth arrest in the human androgen-independent prostate carcinoma cell line Pc-3. Proc Natl Acad Sci U S A 1992;89:3556–60.[Abstract/Free Full Text]
  47. Rowley DR, Dang TD, McBride L, Gerdes MJ, Lu B, Larsen M. ß-2 microglobulin is mitogenic to PC-3 prostatic carcinoma cells and antagonistic to transforming growth factor ß1 action. Cancer Res 1995;55:781–6.[Abstract/Free Full Text]
  48. Roberson KM, Penland SN, Padilla GM, et al. Fenretinide: induction of apoptosis and endogenous transforming growth factor ß in PC-3 prostate cancer cells. Cell Growth Differ 1997;8:101–11.[Abstract]
  49. Koli K, Saharinen J, Hyytiainen M, Penttinen C, Keski-Oja J. Latency, activation, and binding proteins of TGF-ß. Microsc Res Tech 2001;52:354–62.[CrossRef][Medline]
  50. Kim SJ, Uehara H, Karashima T, McCarty M, Shih N, Fidler IJ. Expression of interleukin-8 correlates with angiogenesis, tumorigenicity, and metastasis of human prostate cancer cells implanted orthotopically in nude mice. Neoplasia 2001;3:33–42.[CrossRef][Medline]
  51. Ilio KY, Sensibar JA, Lee C. Effect of TGF-ß1, TGF-{alpha}, and EGF on cell proliferation and cell death in rat ventral prostatic epithelial cells in culture. J Androl 1995;16:482–90.[Abstract/Free Full Text]
  52. Lee C, Sintich SM, Mathews EP, et al. Transforming growth factor-ß in benign and malignant prostate. Prostate 1999;39:285–90.[CrossRef][Medline]
  53. Song K, Cornelius SC, Danielpour D. Development and characterization of DP-153, a nontumorigenic prostatic cell line that undergoes malignant transformation by expression of dominant-negative transforming growth factor ß receptor type II. Cancer Res 2003;63:4358–67.[Abstract/Free Full Text]
  54. Little NA, Wiener JS, Walther PJ, Paulson DF, Anderson EE. Squamous cell carcinoma of the prostate: 2 cases of a rare malignancy and review of the literature. J Urol 1993;149:137–9.[Medline]
  55. Guo Y, Kyprianou N. Overexpression of transforming growth factor (TGF) ß1 type II receptor restores TGF-ß1 sensitivity and signaling in human prostate cancer cells. Cell Growth Differ 1998;9:185–93.[Abstract]
  56. Guo Y, Kyprianou N. Restoration of transforming growth factor ß signaling pathway in human prostate cancer cells suppresses tumorigenicity via induction of caspase-1-mediated apoptosis. Cancer Res 1999;59:1366–71.[Abstract/Free Full Text]
  57. Wikstrom P, Lindh G, Bergh A, Damber JE. Alterations of transforming growth factor ß1 (TGF-ß1) and TGFß receptor expressions with progression in Dunning rat prostatic adenocarcinoma sublines. Urol Res 1999;27:185–93.[CrossRef][Medline]
  58. Derynck R, Akhurst RJ, Balmain A. TGF-ß signaling in tumor suppression and cancer progression. Nat Genet 2001;29:117–29.[CrossRef][Medline]
  59. Gorska AE, Joseph H, Derynck R, Moses HL, Serra R. Dominant-negative interference of the transforming growth factor ß type II receptor in mammary gland epithelium results in alveolar hyperplasia and differentiation in virgin mice. Cell Growth Differ 1998;9:229–38.[Abstract]
  60. McEarchern JA, Kobie JJ, Mack V, et al. Invasion and metastasis of a mammary tumor involves TGF-ß signaling. Int J Cancer 2001;91:76–82.[CrossRef][Medline]
  61. Tang B, Vu M, Booker T, et al. TGF-ß switches from tumor suppressor to prometastatic factor in a model of breast cancer progression. J Clin Invest 2003;112:1116–24.[CrossRef][Medline]
  62. Letterio JJ, Roberts AB. Regulation of immune responses by TGF-ß. Annu Rev Immunol 1998;16:137–61.[CrossRef][Medline]
  63. Sakamoto T, Ueno H, Sonoda K, et al. Blockade of TGF-ß by in vivo gene transfer of a soluble TGF-ß type II receptor in the muscle inhibits corneal opacification, edema and angiogenesis. Gene Ther 2000;7:1915–24.[CrossRef][Medline]
  64. Siegel PM, Massague J. Cytostatic and apoptotic actions of TGF-ß in homeostasis and cancer. Nat Rev Cancer 2003;3:807–21.[CrossRef][Medline]
  65. Lee LF, Louie MC, Desai SJ, et al. Interleukin-8 confers androgen-independent growth and migration of LNCaP: differential effects of tyrosine kinases Src and FAK. Oncogene 2004;23:2197–205.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
M. Zhang, B. E. Berndt, J.-J. Chen, and J. Y. Kao
Expression of a Soluble TGF-{beta} Receptor by Tumor Cells Enhances Dendritic Cell/Tumor Fusion Vaccine Efficacy
J. Immunol., September 1, 2008; 181(5): 3690 - 3697.
[Abstract] [Full Text] [PDF]

Home page
 Protein Eng Des SelHome page
E. V. Verona, Y. Tang, T. K. Millstead, A. P. Hinck, J. K. Agyin, and L.-Z. Sun
Expression, purification and characterization of BGERII: a novel pan-TGF{beta} inhibitor
Protein Eng. Des. Sel., July 1, 2008; 21(7): 463 - 473.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
S. Lu, J. Lee, M. Revelo, X. Wang, S. Lu, and Z. Dong
Smad3 Is Overexpressed in Advanced Human Prostate Cancer and Necessary for Progressive Growth of Prostate Cancer Cells in Nude Mice
Clin. Cancer Res., October 1, 2007; 13(19): 5692 - 5702.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
S. Lu, A. Wang, S. Lu, and Z. Dong
A novel synthetic compound that interrupts androgen receptor signaling in human prostate cancer cells
Mol. Cancer Ther., July 1, 2007; 6(7): 2057 - 2064.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
R. Ge, V. Rajeev, P. Ray, E. Lattime, S. Rittling, S. Medicherla, A. Protter, A. Murphy, J. Chakravarty, S. Dugar, et al.
Inhibition of Growth and Metastasis of Mouse Mammary Carcinoma by Selective Inhibitor of Transforming Growth Factor-{beta} Type I Receptor Kinase In vivo.
Clin. Cancer Res., July 15, 2006; 12(14): 4315 - 4330.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, F.
Right arrow Articles by Dong, Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, F.
Right arrow Articles by Dong, Z.