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Endocrinology |
The Urologic Laboratory, Department of Urology, Children’s Hospital and the Department of Surgery, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, or heparin-binding EGF-like growth factor]. In response to ErbB1 activation by ligand, the p85 regulatory subunit of PI3K associated specifically with ErbB3 but not detectably with ErbB1. The anti-apoptotic effect of ErbB1 activation was significantly reduced when cells were treated simultaneously with wortmannin and PD98059. These data indicate that survival signals can be evoked in LNCaP cells by several distinct pathways and can be triggered by nuclear and cell-surface receptors. Constitutive signaling through the PI3K pathway is required to prevent cell death in LNCaP, whereas activation of the Erk/MAPK and androgen response pathways is not obligatory for cell survival. These results also show that survival signals, as distinguished from mitogenic signals, can be evoked in PCa cells by ErbB1 ligands known to be synthesized within the human prostate. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell survival signals can be transmitted by other intercellular signaling molecules, such as diffusible growth factors (4, 5, 6, 7) . It is possible that the androgen-response pathway in PCa cells intersects with other survival pathways that operate in non-androgen-dependent cell types. This cross-talk may play a role in mechanisms of normal prostate growth control and in apoptosis regulation as well as in the development of the resistance of prostate cancer to androgen ablation therapy. The emergence of androgen-independent PCa, in which mechanisms of androgen-dependent cell survival must no longer play a role, is a common outcome of endocrine therapy and generally results in progression to end-stage disease.
The PI3K pathway is an important intracellular mediator of survival signals originating from trophic factors (4 , 7 , 8 , 13) . PI3K is recruited and activated by cell surface receptors for peptide growth factors and cytokines, providing a mechanism whereby exogenous peptide growth factors can directly activate intracellular cell survival pathways. PI3K phosphorylates inositol lipids that act as second messengers for several targets, including the serine-threonine Akt kinase and the ribosomal S6 kinase, pp70S6K. The cell survival function of the PI3K pathway is mediated in part by Akt-dependent inactivation of pro-apoptotic proteins, such as the Bcl-2-related phosphoprotein, BAD, by site-specific phosphorylation (9) .
In this report, we demonstrate that, in LNCaP human PCa cells, constitutive signaling through the PI3K pathway is required to avoid spontaneous activation of an apoptotic signal. LNCaP cells were not similarly dependent on the androgen-response pathway or on the Erk/MAPK pathway, an established mediator of growth factor-activated survival signals. The LNCaP cell line resembles clinical PCa in many important respects. Consequently, these data support a critical role for PI3K-dependent survival signals in human PCa.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cultured Cells.
The human PCa cell lines LNCaP, PC3, and DU145 were purchased from the American Type Culture Collection (Rockville, MD). They were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc., Grand Island, NY). These supplements were used in all of the media unless otherwise indicated. Normal human prostate epithelial cells (PrEC, strain 4428) were obtained at first passage (Clonetics, San Diego, CA) and expanded in serum-free growth medium (PrEBM, Clonetics) supplemented with bovine pituitary extract, recombinant human EGF, hydrocortisone, gentamicin-amphotericin (GA-1000), tri-iodothyronine, insulin, transferrin, retinoic acid, and epinephrine. Experiments were performed on cells between passages 4 and 6. All of the cells were cultured in a humidified atmosphere at 37°C, 5% CO2.
Cell Transfection and Determination of Luciferase Activity.
LNCaP cells were transiently transfected using the calcium phosphate method (11)
. The pMMTV-luciferase and pSV-ß-gal plasmids were cotransfected for 24 h in DMEM and 10% FBS. Cells were harvested and replated in 24-well plates in RPMI 1640 and 5% FBS for 24 h. Cells were then treated with Casodex for 36 h in RPMI medium containing 5% charcoal dextran-treated FBS. Cell lysates were prepared in reporter lysis buffer (Promega). Luciferase activity was determined using a commercially available substrate (Promega). Chemiluminescence was measured with a liquid scintillation counter (LKB, Wallac, Gaithersburg, MD) for 30 s at 25°C immediately after the addition of luciferase substrate (luciferin). ß-galactosidase activities were determined using the ß-galactosidase Enzyme Assay System (Promega).
Genomic DNA Fragmentation Assay.
Cells (1.2–1.5 x 106) were seeded in 6-cm dishes for 24 h and treated with the various agents in RPMI medium for different times with or without 5% FBS. For androgen depletion experiments, LNCaP cells were cultured in 5% charcoal-dextran-treated FBS for various times (at least 3 days). Cells were harvested by scraping, and low molecular weight DNA was isolated according to Herrmann et al. (12)
with only slight modifications. The precipitated DNAs were separated in 1.8% agarose gels and visualized by ethidium bromide staining.
Quantitative Assay of DNA Fragmentation.
A quantitative sandwich ELISA was used to measure mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates according to the manufacturer’s protocol (Cell Death Detection ELISA, Boehringer Mannheim, Indianapolis, IN). Briefly, 1.5–2.0 x 104 cells/well were seeded in poly-L-lysine-coated 96-well plates for 2 days and cell lysates were collected after stimulation with growth factors and inhibitors under serum-free conditions as indicated. The amount of histone-associated DNA fragments was quantified by spectrophotometric measurement of peroxidase activity retained in the immunocomplex (415 nm). The apoptosis enrichment factor was calculated as follows:
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Immunoprecipitation and Western Blot Analysis.
To determine whether PI3K associates specifically with ErbB1 or ErbB3 after cell activation by EGF, 95%-confluent LNCaP cells were serum-starved for 24 h and stimulated with 10 ng/ml EGF for 10 min on ice, followed by incubation at 37°C for 5 min. Cell lysis was performed in NP40 lysis buffer [1% NP40, 10 mM Tris (pH 7.4), 10 mM NaF, 10 mM NaPP, 1 mM Na2VO3, and protease inhibitor cocktail (Boehringer Mannheim)]. Cell lysates (500 µg of protein) were incubated with 5 µl of monoclonal anti-p85 antibody (UBI) at 4°C overnight with gentle shaking, and immunocomplexes were captured on Protein A/G Plus (Santa Cruz). Immunoprecipitates were electrophoresed through 6% SDS-PAGE, and associated proteins were identified in Western blots using monospecific primary antibodies; proteins were visualized with the ECL Detection System (Amersham) according to the manufacturer’s specifications.
PI3K Assays.
The effects of PI3K inhibitors on PI3K and Akt1 kinase activities were tested under the same conditions as those used for cell death determinations. We chose one time point to assess kinase activity. Briefly, LNCaP cells were plated on poly-L-lysine-coated 60-mm dishes in RPMI 1640 and 10% FBS medium for 2 days. The cells were then treated with 50 nM wortmannin for 45 min followed by 10-ng/ml EGF stimulation for 10 min on ice and at 37°C for 5 min under serum-free conditions. Cell lysis was performed in NP40 lysis buffer as above. Cleared lysates were immunoprecipitated by monoclonal anti-p85 antibody (UBI). The kinase activity was measured by TLC using L--phosphatidylinositol (Avandi Polar Lipids, Inc., Alabaster, AL) as substrate.
Akt Kinase Assay.
Akt kinase activity was measured by the method of Crowder and Freeman (8)
. Briefly, the cell lysates were immunoprecipitated by anti-Akt1 polyclonal antibody (Santa Cruz). The immune complexes were washed three times with lysis buffer, once with ice-cold H2O, and twice with kinase buffer [20 mM HEPES (pH 7.4), 10 mM MgCl2, 10 mM MnCl2, 1 mM DTT, and 0.2 mM EGTA with 5 µM of ATP]; washed complexes were incubated for 30 min at 30°C in 30 µl of kinase buffer containing 1 µM protein kinase A inhibitor H89 (Biomol), 0.1 mg/ml histone H2B (Boehringer Mannheim), and 10 µCi/reaction [
-P32]ATP (specific activity = 3000 Ci/mmol; Amersham). Phosphorylation of histone H2B was analyzed by 15% SDS-PAGE, followed by autoradiography.
Statistical Analysis.
Statistical evaluation of differences between values was determined by paired Student t test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. DNA laddering was observed at lower concentrations of inhibitors when serum-free medium was used in place of serum-containing medium (not shown), consistent with previous reports that showed PI3K levels are reduced in cells cultured under serum-free conditions (4)
. DNA laddering results were verified by DNA fragmentation ELISA, which quantifies mono- and oligonucleosomes in the cytosol (Fig. 2)
. In contrast, apoptosis was not observed when LNCaP cells were treated with rapamycin (Fig. 1
and Fig. 2A
), an inhibitor of the kinase pp70S6K, which acts downstream of PI3K (14)
; with PD98059, a specific inhibitor of the Erk/MAPK kinase, MEK (15)
; with culture in androgen-depleted medium for 7 days; or with Casodex, a specific inhibitor of the AR (Fig. 1)
. Casodex inhibits the AR—expressed by LNCaP cells—which contains a mutation in the ligand-binding domain that alters the ligand-binding properties of the receptor (10)
. Apoptosis was also not observed when the androgen-insensitive PC-3 or DU145 cells (Fig. 1)
, or when normal human prostate epithelial cells (Fig. 2B)
, were treated with the PI3K inhibitors. Taken together, these results identify PI3K as an essential mediator of cell survival in LNCaP cells.
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, which indicates that survival signals are mediated by AR activation. The protective effect of DHT was not observed when wortmannin and DHT were added simultaneously or when DHT pretreatment was for less than 12 h (not shown), which suggests that the survival function of DHT is dependent on increases in gene expression, consistent with AR’s role as a transcription factor. To verify AR function in LNCaP cells, cells were transfected with an androgen-responsive pMMTV-luciferase plasmid. DHT treatment (1 nM) activated promoter activity approximately 24-fold (not shown), confirming that the AR was activated by DHT in the experiment shown in Fig. 3
. Promoter activation by DHT was inhibited to basal levels by 
0.5 µM Casodex, demonstrating that in the Casodex experiment shown in Fig. 1
, AR function was suppressed. These results indicate that androgen exerts a protective effect downstream from the PI3K pathway; however, AR function is not essential for LNCaP cell survival.
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, which are expressed predominantly by ductal epithelial cells and PCa cells (16)
, and HB-EGF, which is expressed predominantly by smooth muscle cells of the fibromuscular stroma (17)
. To determine whether these growth factors can attenuate the apoptotic effects of PI3K inhibition, EGF, TGF-, and HB-EGF were each added simultaneously with LY294002, and apoptosis was measured quantitatively by DNA fragmentation ELISA, 24 h after treatment. Apoptosis was inhibited between 50 and 70% with all of the three ligands (Fig. 4A)
. A similar protective effect was observed when cells were treated with wortmannin, an irreversible PI3K inhibitor, followed by a treatment of the cells with EGF 1 h later (Fig. 4B)
. Results obtained by DNA fragmentation ELISA were confirmed independently in DNA laddering experiments (Fig. 4C)
. These data indicate that ErbB1 activation by ligand is capable of significantly attenuating the apoptotic effect of PI3K inhibition.
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. The serine/threonine kinase Akt is activated by phospholipid products of PI3K and is a prominent downstream target of PI3K in cell survival signaling (reviewed in Ref. 19
). Akt activation was observed in LNCaP cells in response to EGF, TGF-, or HB-EGF (Fig. 5C)
, which suggests that the PI3K survival pathway can be activated by EGF-like growth factors in this cell line.
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. However, differences in both the PI3K and Akt kinase activities between the wortmannin alone and the wortmannin + EGF conditions were reproducibly small or not evident (Fig. 6)
. These data suggest that, under conditions of irreversible PI3K inhibition, ErbB1 activation by ligand activates a PI3K-independent cell survival pathway.
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. Consistent with the results shown in Fig. 1
, treatment with PD98059 alone did not stimulate apoptosis significantly. These results indicate that, although signaling through the Erk/MAPK pathway is not obligatory for cell survival in LNCaP, cytoprotective signals can be transmitted through this pathway in response to ErbB1 activation.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and HB-EGF—have all been shown to be present endogenously in the human prostate, as synthetic products either of ductal epithelial cells and carcinoma cells or of smooth muscle cells within the interstitial fibromuscular stroma (16
, 17)
. These studies have demonstrated that these diffusible growth factors are PCa survival factors in addition to their role as epithelial and carcinoma cell mitogens. Importantly, however, analysis of PI3K activity and Akt kinase activity—after wortmannin and EGF treatment—indicated that survival signals stimulated by ErbB1 ligands are also likely to be transmitted through a PI3K-independent pathway. This was demonstrated by experiments in which the protective effect of ErbB1 activation by ligand was abolished when wortmannin and PD98059 were used in combination. This finding indicates that ErbB1-activated survival signals can also be transmitted through the Erk/MAPK pathway, despite the fact that the MEK inhibitor, when used alone, did not induce apoptosis. Our findings indicate that multiple cell survival pathways, distinct from the well-known androgen-mediated survival pathway, coexist in human PCa cells and can be activated by endogenous autocrine and paracrine prostatic growth factors as well as by steroid hormones.
The PI3K pathway has been shown previously to be an essential survival pathway for a number of cell types. PI3K and downstream effectors can be activated by ligands for multiple receptor tyrosine kinases, including receptors for insulin-like growth factors, PDGF, and EGF-like factors (6 , 7 , 23, 24, 25) . The mechanism of cytoprotection in this pathway is not fully understood; however, it seems to involve phosphorylation of the pro-apoptotic protein, BAD, by the serine-threonine protein kinase, Akt (9 , 26) . Phosphorylation prevents BAD from heterodimerizing with cytoprotective proteins, such as Bcl-2 and Bcl-xL, thereby inhibiting its pro-apoptotic activity. However, as shown previously by others (4) as well as by data presented in this report, a dependence on PI3K for cell survival is a cell-specific characteristic. In fact, PI3K activation mediates ligand-dependent apoptotic signals in some cells (5 , 27 , 28) . In our study, the androgen-independent prostate cell lines, PC-3 and DU145, and normal prostate epithelial cells obtained from a young donor, were insensitive to the apoptotic trigger. These observations suggest that the sensitivity of LNCaP cells to the PI3K inhibitors is an intrinsic property of this cell line and that normal prostate epithelial cells and some androgen-independent PCa cells are resistant to this particular apoptotic signal.
The LNCaP cell line has been of interest to prostate cancer researchers for many years. It remains the only continuous human PCa cell line that expresses a variety of properties characteristic of the human disease (reviewed in Ref. 29 ). PC-3 and DU145 cells do not express definitive prostatic markers, and their relevance to PCa seen clinically has been questioned by some investigators. LNCaP cells can be grown and studied as androgen-dependent, prostate-specific antigen-secreting xenografts in immune-deficient animals (30, 31, 32, 33) . Recently, several groups have reported the development of novel LNCaP variants that show more aggressive in vivo behavior than the parent line, including alterations in androgen-responsiveness, metastasis to the skeleton, and formation of osteoblastic lesions at bony sites (31 , 33) . These recent reports have greatly expanded the capability of the LNCaP system to model the human disease. Several groups have also noted that LNCaP cells can be induced to differentiate along a neural or neuroendocrine pathway in response to a variety of signals (34, 35, 36) . This is significant because there is some evidence that neuroendocrine differentiation is a marker of a more aggressive phenotype in PCa, particularly in the case of androgen-independent tumors (37) . Hence, a tendency toward neural/neuroendocrine differentiation by LNCaP cells may be a reflection of in vivo properties exhibited by some clinically aggressive PCa. Interestingly, the apoptotic sensitivity of LNCaP cells to PI3K inhibitors is similar to that reported for some neural cells. Neurons, oligodendrocytes, and oligodendrocyte precursors rapidly undergo programmed cell death in response to the inhibition of PI3K using pharmacological approaches similar to the ones we used in this study (8 , 38 , 39) . This similarity suggests the possibility that a dependence on constitutive activation of the PI3K pathway is a reflection of the neuroendocrine properties of LNCaP cells. In oligodendrocytes, neurotrophic factors were found to be incapable of inhibiting the apoptotic response to PI3K inhibition (39) . In contrast, our results indicate that such a reversal is possible in LNCaP by ligand-mediated receptor tyrosine kinase activation. It is interesting to speculate that LNCaP cells, which originate from a PCa lymph node metastasis, have acquired atypical means of bypassing apoptotic signals as a result of malignant progression. Resistance to apoptosis has been observed to correlate with increasing metastatic potential in some LNCaP variants (40) .
Treatment of LNCaP cells with IL-6 induces a neuroendocrine phenotype by a PI3K-dependent mechanism (36) . This finding is consistent with the conclusion that the PI3K pathway plays a prominent role in the mechanisms of cell growth and differentiation in these cells. IL-6 has also been shown to activate ErbB2 and ErbB3 in LNCaP cells by a novel mechanism involving heterodimerization of the IL-6 receptor and the ErbB2 tyrosine kinase (41) . Taken together with the results of our study, these findings suggest the possibility that anti-apoptotic signals may be triggered in PCa cells by inflammatory cytokines through ErbB receptor-activatable survival mechanisms. Prostate tumors are often infiltrated with inflammatory cells capable of secreting potent bioactive factors into the local environment (42) .
In summary, our results have shown that several cell survival mechanisms operate in LNCaP human prostate cancer cells, a physiologically relevant cell line that resembles the human disease in important respects. These pathways can be activated by several diffusible biological mediators, including growth factors and androgenic hormones, which are present in hormonally intact and in androgen-depleted individuals. An important finding is that cell survival signals mediated by PI3K in these cells are obligatory for cell survival. It is uncertain as yet how broadly these findings can be applied to prostate cancers seen clinically; however, they suggest the possibility that targeting the PI3K pathway may prove to be an effective means of chemotherapeutic intervention in vivo.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Enders Research Laboratories, 1151, Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115. Phone: (617) 355-6054; Fax: (617) 355-8336; E-mail: freeman_m{at}a1.tch.harvard.edu 
2 The abbreviations used are: PCa, prostate carcinoma; PI3K, phosphatidylinositol 3' kinase; MAPK, mitogen-activated protein kinase; AR, androgen receptor; Erk, extracellular signal-regulated kinase; MEK, MAPK/Erk kinase; EGF, epidermal growth factor; TGF-, transforming growth factor ; HB-EGF, heparin-binding EGF-like growth factor; IL-6, interleukin 6; PDGF, platelet-derived growth factor; DHT, dihydrotestosterone; FBS, fetal bovine serum; pMMTV, mouse mammary tumor virus promoter plasmid.
Received 12/16/98. Accepted 4/15/99.
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 T. Ikezoe, S. Tanosaki, U. Krug, B. Liu, P. Cohen, H. Taguchi, and H. P. Koeffler Insulin-like growth factor binding protein-3 antagonizes the effects of retinoids in myeloid leukemia cells Blood, July 1, 2004; 104(1): 237 - 242. [Abstract] [Full Text] [PDF]
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 J. M. Kurie Role of Protein Kinase B-Dependent Signaling in Lung Tumorigenesis Chest, May 1, 2004; 125(5_suppl): 141S - 144S. [Abstract] [Full Text] [PDF]
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H. Huang, D. C. Muddiman, and D. J. Tindall Androgens Negatively Regulate Forkhead Transcription Factor FKHR (FOXO1) through a Proteolytic Mechanism in Prostate Cancer Cells J. Biol. Chem., April 2, 2004; 279(14): 13866 - 13877. [Abstract] [Full Text] [PDF]
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M. Holgado-Madruga and A. J. Wong Role of the Grb2-Associated Binder 1/SHP-2 Interaction in Cell Growth and Transformation Cancer Res., March 15, 2004; 64(6): 2007 - 2015. [Abstract] [Full Text] [PDF]
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H.-K. Lin, Y.-C. Hu, L. Yang, S. Altuwaijri, Y.-T. Chen, H.-Y. Kang, and C. Chang Suppression Versus Induction of Androgen Receptor Functions by the Phosphatidylinositol 3-Kinase/Akt Pathway in Prostate Cancer LNCaP Cells with Different Passage Numbers J. Biol. Chem., December 19, 2003; 278(51): 50902 - 50907. [Abstract] [Full Text] [PDF]
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H. Q. Wang, T. Quan, T. He, T. F. Franke, J. J. Voorhees, and G. J. Fisher Epidermal Growth Factor Receptor-dependent, NF-{kappa}B-independent Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway Inhibits Ultraviolet Irradiation-induced Caspases-3, -8, and -9 in Human Keratinocytes J. Biol. Chem., November 14, 2003; 278(46): 45737 - 45745. [Abstract] [Full Text] [PDF]
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C.-C. Yang, H.-P. Lin, C.-S. Chen, Y.-T. Yang, P.-H. Tseng, V. M. Rangnekar, and C.-S. Chen Bcl-xL Mediates a Survival Mechanism Independent of the Phosphoinositide 3-Kinase/Akt Pathway in Prostate Cancer Cells J. Biol. Chem., July 3, 2003; 278(28): 25872 - 25878. [Abstract] [Full Text] [PDF]
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H.-Y. Lee, H. Srinivas, D. Xia, Y. Lu, R. Superty, R. LaPushin, C. Gomez-Manzano, A. M. Gal, G. L. Walsh, T. Force, et al. Evidence That Phosphatidylinositol 3-Kinase- and Mitogen-activated Protein Kinase Kinase-4/c-Jun NH2-terminal Kinase-dependent Pathways Cooperate to Maintain Lung Cancer Cell Survival J. Biol. Chem., June 20, 2003; 278(26): 23630 - 23638. [Abstract] [Full Text] [PDF]
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L. Yang, H.-K. Lin, S. Altuwaijri, S. Xie, L. Wang, and C. Chang APPL Suppresses Androgen Receptor Transactivation via Potentiating Akt Activity J. Biol. Chem., May 2, 2003; 278(19): 16820 - 16827. [Abstract] [Full Text] [PDF]
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A. F. Taghiyev, N. V. Guseva, H. Harada, C. M. Knudson, O. W. Rokhlin, and M. B. Cohen Overexpression of BAD Potentiates Sensitivity to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Treatment in the Prostatic Carcinoma Cell Line LNCaP Mol. Cancer Res., May 1, 2003; 1(7): 500 - 507. [Abstract] [Full Text] [PDF]
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 A. E. Paradis, P. W. Kantoff, E. Giovannucci, M. J. Stampfer, and J. Ma Association between the Met326Ile Polymorphism of the p85{alpha} Regulatory Subunit of Phosphatidylinositol 3-Kinase and Prostate Cancer Risk: A Prospective Study Cancer Epidemiol. Biomarkers Prev., February 1, 2003; 12(2): 172 - 173. [Full Text] [PDF]
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K. N. Thimmaiah, J. Easton, S. Huang, K. A. Veverka, G. S. Germain, F. C. Harwood, and P. J. Houghton Insulin-like Growth Factor I-mediated Protection from Rapamycin-induced Apoptosis Is Independent of Ras-Erk1-Erk2 and Phosphatidylinositol 3'-Kinase-Akt Signaling Pathways Cancer Res., January 15, 2003; 63(2): 364 - 374. [Abstract] [Full Text] [PDF]
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R. M. Adam, J. Kim, J. Lin, A. Orsola, L. Zhuang, D. C. Rice, and M. R. Freeman* Heparin-Binding Epidermal Growth Factor-Like Growth Factor Stimulates Androgen-Independent Prostate Tumor Growth and Antagonizes Androgen Receptor Function Endocrinology, December 1, 2002; 143(12): 4599 - 4608. [Abstract] [Full Text] [PDF]
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M. A. Eid, R. W. Lewis, and M. V. Kumar Mifepristone Pretreatment Overcomes Resistance of Prostate Cancer Cells to Tumor Necrosis Factor {alpha}-related Apoptosis-inducing Ligand (TRAIL) Mol. Cancer Ther., August 1, 2002; 1(10): 831 - 840. [Abstract] [Full Text] [PDF]
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R. P. Singh, S. Dhanalakshmi, A. K. Tyagi, D. C. F. Chan, C. Agarwal, and R. Agarwal Dietary Feeding of Silibinin Inhibits Advance Human Prostate Carcinoma Growth in Athymic Nude Mice and Increases Plasma Insulin-like Growth Factor-binding Protein-3 Levels Cancer Res., June 1, 2002; 62(11): 3063 - 3069. [Abstract] [Full Text] [PDF]
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H.-Y. Lee, K.-H. Chun, B. Liu, S. A. Wiehle, R. J. Cristiano, W. K. Hong, P. Cohen, and J. M. Kurie Insulin-like Growth Factor Binding Protein-3 Inhibits the Growth of Non-Small Cell Lung Cancer Cancer Res., June 1, 2002; 62(12): 3530 - 3537. [Abstract] [Full Text] [PDF]
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L. Zhuang, J. Lin, M. L. Lu, K. R. Solomon, and M. R. Freeman Cholesterol-rich Lipid Rafts Mediate Akt-regulated Survival in Prostate Cancer Cells Cancer Res., April 1, 2002; 62(8): 2227 - 2231. [Abstract] [Full Text] [PDF]
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J. Kim, R. M. Adam, and M. R. Freeman Activation of the Erk Mitogen-activated Protein Kinase Pathway Stimulates Neuroendocrine Differentiation in LNCaP Cells Independently of Cell Cycle Withdrawal and STAT3 Phosphorylation Cancer Res., March 1, 2002; 62(5): 1549 - 1554. [Abstract] [Full Text] [PDF]
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T. Ueda, N. Bruchovsky, and M. D. Sadar Activation of the Androgen Receptor N-terminal Domain by Interleukin-6 via MAPK and STAT3 Signal Transduction Pathways J. Biol. Chem., February 22, 2002; 277(9): 7076 - 7085. [Abstract] [Full Text] [PDF]
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 Y.-S. Bae, Y. Kim, J. C. Park, P.-G. Suh, and S. H. Ryu The synthetic chemoattractant peptide, Trp-Lys-Tyr-Met-Val-D-Met, enhances monocyte survival via PKC-dependent Akt activation J. Leukoc. Biol., February 1, 2002; 71(2): 329 - 338. [Abstract] [Full Text] [PDF]
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J. P. Carson, M. Behnam, J. N. Sutton, C. Du, X. Wang, D. F. Hunt, M. J. Weber, and G. Kulik Smac Is Required for Cytochrome c-induced Apoptosis in Prostate Cancer LNCaP Cells Cancer Res., January 1, 2002; 62(1): 18 - 23. [Abstract] [Full Text] [PDF]
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H. Murillo, H. Huang, L. J. Schmidt, D. I. Smith, and D. J. Tindall Role of PI3K Signaling in Survival and Progression of LNCaP Prostate Cancer Cells to the Androgen Refractory State Endocrinology, November 1, 2001; 142(11): 4795 - 4805. [Abstract] [Full Text] [PDF]
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 B. A. Ballif and J. Blenis Molecular Mechanisms Mediating Mammalian Mitogen-activated Protein Kinase (MAPK) Kinase (MEK)-MAPK Cell Survival Signals Cell Growth Differ., August 1, 2001; 12(8): 397 - 408. [Full Text] [PDF]
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D.-L. Lin, M. C. Whitney, Z. Yao, and E. T. Keller Interleukin-6 Induces Androgen Responsiveness in Prostate Cancer Cells through Up-Regulation of Androgen Receptor Expression Clin. Cancer Res., June 1, 2001; 7(6): 1773 - 1781. [Abstract] [Full Text] [PDF]
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G. Kulik, J. P. Carson, T. Vomastek, K. Overman, B. D. Gooch, S. Srinivasula, E. Alnemri, G. Nunez, and M. J. Weber Tumor Necrosis Factor {{alpha}} Induces BID Cleavage and Bypasses Antiapoptotic Signals in Prostate Cancer LNCaP Cells Cancer Res., March 1, 2001; 61(6): 2713 - 2719. [Abstract] [Full Text]
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 K. W. Kang, J. H. Ryu, and S. G. Kim The Essential Role of Phosphatidylinositol 3-Kinase and of p38 Mitogen-Activated Protein Kinase Activation in the Antioxidant Response Element-Mediated rGSTA2 Induction by Decreased Glutathione in H4IIE Hepatoma Cells Mol. Pharmacol., November 1, 2000; 58(5): 1017 - 1025. [Abstract] [Full Text]
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H. Zhong, K. Chiles, D. Feldser, E. Laughner, C. Hanrahan, M.-M. Georgescu, J. W. Simons, and G. L. Semenza Modulation of Hypoxia-inducible Factor 1{{alpha}} Expression by the Epidermal Growth Factor/Phosphatidylinositol 3-Kinase/PTEN/AKT/FRAP Pathway in Human Prostate Cancer Cells: Implications for Tumor Angiogenesis and Therapeutics Cancer Res., March 1, 2000; 60(6): 1541 - 1545. [Abstract] [Full Text]
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 H. T. Nguyen, S. H. Bride, A.-B. Badawy, R. M. Adam, J. Lin, A. Orsola, P. D. Guthrie, M. R. Freeman, and C. A. Peters Heparin-Binding EGF-Like Growth Factor Is Up-Regulated in the Obstructed Kidney in a Cell- and Region-Specific Manner and Acts to Inhibit Apoptosis Am. J. Pathol., March 1, 2000; 156(3): 889 - 898. [Abstract] [Full Text] [PDF]
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R. M. Adam, J. G. Borer, B. J. Williams, J. A. Eastham, K. R. Loughlin, and M. R. Freeman Amphiregulin Is Coordinately Expressed with Heparin-Binding Epidermal Growth Factor-Like Growth Factor in the Interstitial Smooth Muscle of the Human Prostate Endocrinology, December 1, 1999; 140(12): 5866 - 5875. [Abstract] [Full Text]
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A. D. Ebert, C. Wechselberger, S. Frank, B. Wallace-Jones, M. Seno, I. Martinez-Lacaci, C. Bianco, M. De Santis, H. K. Weitzel, and D. S. Salomon Cripto-1 Induces Phosphatidylinositol 3'-Kinase-dependent Phosphorylation of AKT and Glycogen Synthase Kinase 3{beta} in Human Cervical Carcinoma Cells Cancer Res., September 1, 1999; 59(18): 4502 - 4505. [Abstract] [Full Text] [PDF]
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D.-Q. Zheng, A. S. Woodard, G. Tallini, and L. R. Languino Substrate Specificity of alpha vbeta 3 Integrin-mediated Cell Migration and Phosphatidylinositol 3-Kinase/AKT Pathway Activation J. Biol. Chem., August 4, 2000; 275(32): 24565 - 24574. [Abstract] [Full Text] [PDF]
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J.-i. Okano, I. Gaslightwala, M. J. Birnbaum, A. K. Rustgi, and H. Nakagawa Akt/Protein Kinase B Isoforms Are Differentially Regulated by Epidermal Growth Factor Stimulation J. Biol. Chem., September 29, 2000; 275(40): 30934 - 30942. [Abstract] [Full Text] [PDF]
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J. Lin, L. Hutchinson, S. M. Gaston, G. Raab, and M. R. Freeman BAG-1 Is a Novel Cytoplasmic Binding Partner of the Membrane Form of Heparin-binding EGF-like Growth Factor. A UNIQUE ROLE FOR proHB-EGF IN CELL SURVIVAL REGULATION J. Biol. Chem., August 3, 2001; 276(32): 30127 - 30132. [Abstract] [Full Text] [PDF]
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, normal prostate epithelial cells; 
, LNCaP.
