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Departments of Neuro-Oncology [M. A. D., D. K., H. D., W. K. A. Y., P. A. S.], Molecular Pathology [T. J. M.], and Cell Biology [R. B., D. M.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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ABSTRACT |
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Introduction |
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One candidate tumor suppressor gene on 10q, MMAC/PTEN, initially identified due to homozygous deletions in gliomas and breast cancer cell lines, was shown to be altered in a number of cancers including prostate carcinoma cells (8 , 9) . Furthermore, interest in MMAC/PTEN was heightened due to the presence of a structural motif for a dual specificity protein phosphatase in the protein product that strongly suggested its involvement in the regulation of signal transduction. Recently, MMAC/PTEN has been shown to have phosphatidylinositol phosphatase activity, specifically for the 3' position (10) , implying its involvement in PI3'K3 -mediated pathways. More recently, the lipid phosphatase activity has been shown to be essential for its ability to inhibit tumorigenesis and growth inhibition (11 , 12) . In this regard, our own studies and those of others have shown that reintroduction of MMAC/PTEN into cells deficient of a functional gene product modulates the activity of Akt/PKB (13 , 14) . Akt/PKB, a serine-threonine kinase, has been shown to be involved in a number of proliferative, metabolic, and antiapoptotic pathways that are dependent upon PI3'K signaling to be activated (15) .
To examine the effects of MMAC/PTEN expression, an adenovirus was developed to express MMAC/PTEN under the control of a cytomegalovirus promoter (14) . This adenovirus, Ad-MMAC, was used to assess the biological and biochemical response of prostate carcinomas cells to functional MMAC/PTEN expression. The LNCaP cells were originally derived from a metastatic prostate cancer specimen and represent a model for androgen-sensitive prostate cancer. Furthermore, the effects of MMAC/PTEN expression on LNCaP cells engineered to overexpress Bcl-2, a negative regulator of apoptosis that has been implicated in prostate cancer progression (16) , was also examined. In conjunction, the biological effects of MMAC/PTEN expression were compared to the effects of p53 expression via adenoviral vectors in the same cells.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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E1) has been described previously (18)
, along with the virus expressing the p53 transgene (19)
. Viruses were amplified in 293 cells, isolated by cesium chloride gradient or purified as reported previously (14)
, and titer was determined by absorbance.
Protein Analysis.
Subconfluent monolayers of cells were infected with Ad-MMAC, Ad-DE1 at the indicated MOI, or mock infected with culture media alone. Twenty-four h after infection, cells were changed to serum-free medium. Twenty-four h later, cells were harvested, either with or without stimulation with EGF (50 ng/ml), FCS (10%), or insulin-like growth factor 1 (10 ng/ml) for 10 min. Cells were harvested in lysis solution containing 50 mM HEPES (pH 7.0), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM sodium PPi, 10% glycerol, 1% Triton X-100, 1 mM Na3VO4, 10 µM pepstatin, 10 µg/ml aprotinin, 5 mM iodoacetic acid, and 2 µg/ml leupeptin. Western analysis was performed as described previously (14)
. Immunoblotting was done using antibodies against total and phospho-specific Akt and MAPK (New England Biolabs, Boston, MA), Bcl-2 (Santa Cruz Biotechnology, Santa Cruz, CA), and MMAC1/PTEN (Santa Cruz Biotechnology), followed by horseradish peroxidase-conjugated secondary antibody and detection by chemiluminescence (Amersham, Arlington Heights, IL).
Cell Proliferation Assays.
For all assays, cells were grown in media supplemented with 10% FCS. To assay growth, cells (1 x 103) were plated on 96-well plates and infected 18 h later. Cells were then processed using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation assay (Promega Corp., Madison, WI) at the indicated time points thereafter. Statistical comparison of samples was by unpaired Student’s t test. For cell count analysis, cells (1 x 104) were plated on six-well dishes and infected 18 h later, and viable cell numbers were determined at indicated times thereafter.
Apoptosis Analysis.
Cells (1 x 106) were seeded on a 10-cm tissue culture dish and incubated overnight, followed by infection with the indicated viruses or mock treated. Cells were maintained in media supplemented with 10% FCS. Cells were harvested at various times thereafter. For protein analysis, detached and attached cells were collected and lysed in lysis buffer as above. Immunoblotting was done using antibodies against caspase-3 (Transduction Laboratories, Lexington, KY) and caspase-8 (PharMingen, San Diego, CA). Cells for DNA analysis were fixed in 1% paraformaldehyde, stored in 70% ethanol, and analyzed for apoptosis by flow cytometry using the Apo-BrdU kit (Phoenix Flow Systems, San Diego, CA) according to the manufacturer’s instructions. For caspase inhibitor experiments, cells were treated with the general caspase inhibitor ZVAD (20 µM; Enzyme Systems Products, Livermore, CA) at 24 and 72 h after infection, followed by harvesting at 96 h after infection. Cells were washed and resuspended in a solution of propidium iodide (50 µg/ml) containing 0.1% sodium citrate and 0.1% Triton X-100 and then analyzed by flow cytometry.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. Treatment of the cells with 5 MOI of Ad-MMAC resulted in a decrease of phosphorylation of both activating residues that was >65%, whereas a 25 MOI infection resulted in a decrease of phosphorylation by >95%. The inhibition of Akt/PKB phosphorylation was not due to a decrease in the expression of Akt/PKB because Ad-MMAC-infected cells had similar levels of the protein when compared with control-treated cells (Fig. 1a)
. Furthermore, infection of the cells with control adenovirus (Ad-DE1) or adenovirus expressing p53 (Ad-p53) revealed a significant stimulation of Akt/PKB phosphorylation, further illustrating the ability of MMAC/PTEN to down-regulate the phosphorylation of Akt/PKB and its specific biochemical effects. The ability of Ad-MMAC to inhibit the phosphorylation of Akt/PKB was observed in LNCaP and LNCaP-Bcl-2 cells maintained in media supplemented with 10% FCS, as well as in cells acutely stimulated with EGF, insulin-like growth factor 1, or 10% FCS after a period of serum deprivation. MMAC/PTEN expression was as effective as a 30-min pretreatment with wortmannin, a PI3'K inhibitor, at blocking EGF-induced Akt/PKB phosphorylation in LNCaP cells (Fig. 1a)
. The specificity of substrates for MMAC/PTEN was illustrated by its inability to inhibit growth factor-mediated stimulation of phosphorylation of MAPK (ERK1/2). Furthermore, the in vitro Akt/PKB kinase activity was significantly decreased by MMAC/PTEN expression and, similar to that observed with glioma cells (14)
, MMAC/PTEN was more effective than wortmannin in inhibiting the Akt/PKB kinase activity.
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. At 24 and 48 h after infection, the expression of MMAC/PTEN via the adenoviral construct was significantly reduced in both LNCaP and LNCaP-Bcl-2 cells compared with the levels we observed in U251 glioma cells (Fig. 1b)
. However, higher levels of expression were observed at later time points. A similar increase in expression of p53 via Ad-p53 over the same time course suggests that this difference reflects a difference in the kinetics of expression for LNCaP cells versus U251.
Growth Inhibition and Apoptosis.
The ability of MMAC/PTEN expression to mediate proliferation and apoptosis of LNCaP cells was assessed by several independent methods. Six days after infection, Ad-MMAC-infected cells were profoundly growth inhibited versus mock-infected cells, as determined by a MTS assay that assesses metabolic activity (Fig. 2a)
. LNCaP cells infected with 5 MOI exhibited a 75% decrease in growth, whereas cells infected with 25 MOI showed a 98% inhibition of growth compared with cells mock infected. Ad-MMAC infection resulted in a significantly greater inhibition of growth than that observed with Ad-p53 under the control of the same promoter. Cells infected with Ad-p53 at 5 MOI under the same conditions exhibited a 44% inhibition of growth, whereas 25 MOI produced 78% of the growth of control cells (Fig. 2a)
. Both of these values for Ad-p53 treatment were statistically less effective than treatment with Ad-MMAC at the same MOI (5 MOI, P < 0.01; 25 MOI, P < 0.0001). Control adenovirus infection exerted a notable toxic effect that resulted in some growth inhibition, but it was significantly less than that observed for either Ad-MMAC or Ad-p53. Similar results were observed when proliferation was determined by counting of viable cells in parallel experiments (Fig. 2b)
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. At day 6, cells infected with Ad-MMAC at 5 and 25 MOI revealed a 67 and 92% growth inhibition, respectively, compared with mock-infected cells. The growth inhibition produced by Ad-MMAC was again significantly greater than that produced by Ad-p53 at both MOIs, and it appeared that Bcl-2 overexpression slightly decreased the growth-inhibitory effect of p53. As with the LNCaP cells, analysis of growth by cell counts showed a similar differential between the two viruses (Fig. 2d)
.
Because MMAC/PTEN and p53 have both been shown to mediate apoptosis, and because the cell number was decreased below the initial seeding number after infection with 25 MOI of either virus in the proliferation analysis, the ability of the different adenovirus constructs to induce apoptosis in LNCaP and LNCaP-Bcl-2 cells was assessed. TUNEL and propidium iodide staining was performed on cells 4 days after infection (Fig. 3a)
. LNCaP cells infected with 5 MOI of Ad-MMAC showed 
5% of the cells undergoing apoptosis, and about 20% of cells were apoptotic after 25 MOI treatment (Fig. 3b)
. In contrast, Ad-p53 infection resulted in 5% and 50% of the cells displaying evidence of apoptosis at 5 and 25 MOI, respectively. Ad-p53 induced a greater amount of apoptosis than Ad-MMAC at other time points after infection as well, implying that this discrepancy did not reflect simply a delay in timing of apoptosis induction. Infection of LNCaP cells with control adenovirus at 25 MOI induced a small apoptotic response, but again this was much less than was observed with either Ad-MMAC or Ad-p53. Bcl-2 overexpression largely abrogated the apoptotic effect of MMAC/PTEN or p53 expression in LNCaP cells (Fig. 3, a and c)
. However, the cells were nearly as sensitive to the growth-suppressive effects of MMAC/PTEN as parental LNCaP cells. Both LNCaP and LNCaP-Bcl-2 cells infected with Ad-MMAC demonstrated an accumulation of cells in G1 phase of the cell cycle when compared with mock-infected cells. However, cells infected with control adenovirus also exhibited some G1 accumulation, thus making it difficult to distinguish whether this arrest was due to MMAC/PTEN expression, adenovirus toxicity, or both.
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. Five MOI of Ad-MMAC failed to induce significant cleavage of either caspase. LNCaP-Bcl-2 cells did not exhibit evidence of caspase processing, consistent with the blockade of apoptosis seen by flow cytometric analysis (Fig. 3, a and c)
. To further examine the significance of caspase cleavage, the LNCaP and LNCaP-Bcl-2 cells infected with 25 MOI of Ad-MMAC or Ad-p53 were examined for cell death using propidium iodide staining at various times after treatment with or without ZVAD, a general caspase inhibitor. LNCaP cells infected with Ad-MMAC or Ad-p53 and treated with ZVAD revealed a decrease in the percentage of cells undergoing apoptosis. ZVAD had little effect on the LNCap-Bcl-2 cells, although a significant growth inhibition was still noted in the MMAC/PTEN-expressing cells. These observations imply that the induced MMAC/PTEN growth suppression was largely independent of its apoptosis-inducing ability, whereas for p53, a closer relationship was noted between the two processes. |
Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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The present study was designed to assess the biological consequences of expression of MMAC/PTEN in prostate cells that are devoid of a functional MMAC/PTEN gene product. Acute expression of MMAC/PTEN in LNCaP cells causes inhibition of activation of Akt/PKB, similar to that shown in other cell types and recently in LNCaP cells (13
, 14
, 25)
. Furthermore, MMAC/PTEN expression by an adenoviral construct inhibited growth and induced apoptosis in LNCaP cells. However, apoptosis does not appear to account for the majority of the observed growth inhibition and, therefore, suggests that additional growth-regulatory mechanisms are being modulated. This is supported by several observations: (a) growth inhibition by 5 MOI of Ad-MMAC is significant (60–70% of control), whereas minimal apoptosis is observed at this MOI; (b) also, LNCaP cells treated with 25 MOI of Ad-MMAC exhibited a decrease in cell growth that could not be adequately accounted for by the apoptotic population, as assessed by TUNEL. MMAC/PTEN may also induce nonapoptotic cell death at high expression levels, although viral toxicity in LNCaP cells may also play a significant role; (c) additionally, stable overexpression of Bcl-2 in LNCaP cells dramatically decreased MMAC/PTEN-induced apoptosis, but it did not substantially diminish the growth-inhibitory effects of MMAC/PTEN; and (d) LNCaP cells treated with Ad-p53 showed much greater apoptosis of the cellular population (50–60%) at day 4 but significantly less growth inhibition compared with cells treated with Ad-MMAC. Thus, the expression of MMAC/PTEN in LNCaP prostate cancer cells is capable of producing a marked growth inhibition that appears to be only partially due to its ability to induce apoptosis, although the constitutive expression of MMAC/PTEN appears to be incompatible with the viability and proliferation of LNCaP cells. This conclusion is supported by transfection studies where various forms of MMAC/PTEN were inserted into LNCaP cells. Colonies were observed only in cells transfected with mutant forms of MMAC/PTEN that affected the phosphatase domain, whereas no colonies were found in cells transfected with a wild-type construct in several independent experiments.
The growth inhibition demonstrated here is intriguing and is under further investigation. There are several possible alternative mechanisms that may be operational. MMAC/PTEN has been shown to modulate the activity of PI3'K-mediated signaling pathways (13, 14 , 25 , 26) ; therefore, regulation of nonapoptosis-related substrates such as GSK, p70 S6 kinase, or 4E-BP1 may account for the growth inhibition (15) . In support of this possibility, the observed inhibition of cell growth as assessed by the MTS assay was greater than that observed when cell number was determined, although both were significant. Interestingly, the expression of MMAC/PTEN from the adenoviral construct at early time points was significantly less in LNCaP cells than that we have observed in U251 glioma cells, although the cell lines demonstrate similar uptake when infected with reporter adenoviruses. This may simply represent a difference in the kinetics of protein metabolism between the two cell lines. However, Wu et al. (25) also observed a decrease in expression of several exogenous transient expression constructs in LNCaP cells expressing MMAC/PTEN. We have not observed any decrease in expression of endogenous or stably transfected gene products in the cells (Bcl-2, Akt/PKB, and MAPK). Furthermore, they observed that this effect of expression is also linked to the phosphatidylinositol phosphatase activity of MMAC/PTEN, because activated Akt/PKB appears to rescue cells from repression of gene expression. Therefore, although the mechanism(s) of down-regulation of transient gene expression constructs in LNCaP cells with a functional MMAC/PTEN is unknown, these observations suggest that the presence of MMAC/PTEN may influence the metabolic and/or transcriptional activities of the cells.
Previously, we have shown that adenoviral transfection of MMAC/PTEN into U251 human glioblastoma cells results in a similar inhibition of Akt/PKB activation. However, we did not observe apoptosis in U251 cells unless the cells received an additional apoptosis-inducing stimuli (14) . Ad-MMAC also produced a notable, but less pronounced, growth inhibition in U251 cells, which was dramatically less that that observed with adenoviral infection with p53 (19) . Growth inhibition without inducing apoptosis was also shown for U87 cells (12 , 26) . These observations are similar to those shown for fibroblasts that were generated from MMAC/PTEN-deficient mice (13) . However, breast cancer cells devoid of a functional MMAC/PTEN have been shown recently to respond to the expression of functional MMAC/PTEN by undergoing apoptosis (27) . The biological differences between the responses of these different cancer cell types does not appear to be due to differential expression of MMAC/PTEN because a number of different expression systems and doses were used. Furthermore, for the breast carcinoma cells, a number of different cell lines exhibited similar biological effects. Thus, MMAC/PTEN appears to have disparate effects on different types of cells, although the responses to the expression of MMAC/PTEN in cells devoid of a functional gene product within a particular type of cancer appears to be relatively similar. As such, it will be worthwhile to examine the effect of MMAC/PTEN expression in a variety of cell types to explore its different biological functions, as well as to evaluate its range of therapeutic uses.
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FOOTNOTES |
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1 This work was supported by Grants R01 CA56041 and P01 CA55261 from the NIH, Grant RPG-96-036-04-CDD from the American Cancer Society, the State of Texas Advanced Technology Program (97-110), and grants from the Gilland Foundation and CaP CURE. 
2 To whom requests for reprints should be addressed, at Department of Neuro-Oncology, Box 316, The Brain Tumor Center, The University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-3002; Fax: (713) 745-1183; E-mail: steckpa{at}audumla.mdacc.tmc.edu
3 The abbreviations used are: PI3'K, phosphatidylinositol 3'-kinase; MAPK, mitogen-activated protein kinase; MOI, multiplicity of infection; EGF epidermal growth factor; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; ZVAD, Z-val-ala-Asp(ome)-CH2F; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling.
Received 1/26/99. Accepted 4/16/99.
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M. A. Davies, S. J. Kim, N. U. Parikh, Z. Dong, C. D. Bucana, and G. E. Gallick Adenoviral-mediated Expression of MMAC/PTEN Inhibits Proliferation and Metastasis of Human Prostate Cancer Cells Clin. Cancer Res., June 1, 2002; 8(6): 1904 - 1914. [Abstract] [Full Text] [PDF]
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G. O. Hellawell, G. D. H. Turner, D. R. Davies, R. Poulsom, S. F. Brewster, and V. M. Macaulay Expression of the Type 1 Insulin-like Growth Factor Receptor Is Up-Regulated in Primary Prostate Cancer and Commonly Persists in Metastatic Disease Cancer Res., May 1, 2002; 62(10): 2942 - 2950. [Abstract] [Full Text] [PDF]
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M. Ichinose, X.-H. Liu, N. Hagihara, and R. J. Youle Extracellular Bad Fused to Toxin Transport Domains Induces Apoptosis Cancer Res., March 1, 2002; 62(5): 1433 - 1438. [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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M. Nomura, A. Kaji, Z. He, W.-Y. Ma, K.-i. Miyamoto, C. S. Yang, and Z. Dong Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway J. Biol. Chem., November 30, 2001; 276(49): 46624 - 46631. [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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H. Thakkar, X. Chen, F. Tyan, S. Gim, H. Robinson, C. Lee, S. K. Pandey, C. Nwokorie, N. Onwudiwe, and R. K. Srivastava Pro-survival Function of Akt/Protein Kinase B in Prostate Cancer Cells. RELATIONSHIP WITH TRAIL RESISTANCE J. Biol. Chem., October 12, 2001; 276(42): 38361 - 38369. [Abstract] [Full Text] [PDF]
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H. Huang, J. C. Cheville, Y. Pan, P. C. Roche, L. J. Schmidt, and D. J. Tindall PTEN Induces Chemosensitivity in PTEN-mutated Prostate Cancer Cells by Suppression of Bcl-2 Expression J. Biol. Chem., October 12, 2001; 276(42): 38830 - 38836. [Abstract] [Full Text] [PDF]
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M. Chakraborty, S. G. Qiu, K. M. Vasudevan, and V. M. Rangnekar Par-4 Drives Trafficking and Activation of Fas and FasL to Induce Prostate Cancer Cell Apoptosis and Tumor Regression Cancer Res., October 1, 2001; 61(19): 7255 - 7263. [Abstract] [Full Text] [PDF]
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S. Gupta, S. Stuffrein, R. Plattner, M. Tencati, C. Gray, Y. E. Whang, and E. J. Stanbridge Role of Phosphoinositide 3-Kinase in the Aggressive Tumor Growth of HT1080 Human Fibrosarcoma Cells Mol. Cell. Biol., September 1, 2001; 21(17): 5846 - 5856. [Abstract] [Full Text] [PDF]
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M. D. Ringel, N. Hayre, J. Saito, B. Saunier, F. Schuppert, H. Burch, V. Bernet, K. D. Burman, L. D. Kohn, and M. Saji Overexpression and Overactivation of Akt in Thyroid Carcinoma Cancer Res., August 1, 2001; 61(16): 6105 - 6111. [Abstract] [Full Text] [PDF]
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C. M. van Golen, T. S. Schwab, K. M. W. Ignatoski, S. P. Ethier, and E. L. Feldman PTEN/MMAC1 Overexpression Decreases Insulin-like Growth Factor-I-mediated Protection from Apoptosis in Neuroblastoma Cells Cell Growth Differ., July 1, 2001; 12(7): 371 - 378. [Abstract] [Full Text] [PDF]
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K. Kimura, M. Markowski, C. Bowen, and E. P. Gelmann Androgen Blocks Apoptosis of Hormone-dependent Prostate Cancer Cells Cancer Res., July 1, 2001; 61(14): 5611 - 5618. [Abstract] [Full Text] [PDF]
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X. Zhu, C.-H. Kwon, P. W. Schlosshauer, L. H. Ellenson, and S. J. Baker PTEN Induces G1 Cell Cycle Arrest and Decreases Cyclin D3 Levels in Endometrial Carcinoma Cells Cancer Res., June 1, 2001; 61(11): 4569 - 4575. [Abstract] [Full Text] [PDF]
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 J.-i. Hisatake, J. O'Kelly, M. R. Uskokovic, S. Tomoyasu, and H. P. Koeffler Novel vitamin D3 analog, 21-(3-methyl-3-hydroxy-butyl)-19-nor D3, that modulates cell growth, differentiation, apoptosis, cell cycle, and induction of PTEN in leukemic cells Blood, April 15, 2001; 97(8): 2427 - 2433. [Abstract] [Full Text] [PDF]
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 L.-P. Weng, J. L. Brown, and C. Eng PTEN induces apoptosis and cell cycle arrest through phosphoinositol-3-kinase/Akt-dependent and -independent pathways Hum. Mol. Genet., February 1, 2001; 10(3): 237 - 242. [Abstract] [Full Text] [PDF]
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J.-T. Dong, C.-L. Li, T. W. Sipe, and H. F. Frierson Jr. Mutations of PTEN/MMAC1 in Primary Prostate Cancers from Chinese Patients Clin. Cancer Res., February 1, 2001; 7(2): 304 - 308. [Abstract] [Full Text]
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 K. M. Yamada and M. Araki Tumor suppressor PTEN: modulator of cell signaling, growth, migration and apoptosis J. Cell Sci., January 7, 2001; 114(13): 2375 - 2382. [Abstract] [Full Text] [PDF]
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N. Nakamura, S. Ramaswamy, F. Vazquez, S. Signoretti, M. Loda, and W. R. Sellers Forkhead Transcription Factors Are Critical Effectors of Cell Death and Cell Cycle Arrest Downstream of PTEN Mol. Cell. Biol., December 1, 2000; 20(23): 8969 - 8982. [Abstract] [Full Text]
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M.-M. Georgescu, K. H. Kirsch, P. Kaloudis, H. Yang, N. P. Pavletich, and H. Hanafusa Stabilization and Productive Positioning Roles of the C2 Domain of PTEN Tumor Suppressor Cancer Res., December 1, 2000; 60(24): 7033 - 7038. [Abstract] [Full Text]
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L. L. Nielsen, M. Gurnani, B. Shi, G. Terracina, R. C. Johnson, J. Carroll, J. M. Mathis, and G. Hajian Derivation and Initial Characterization of a Mouse Mammary Tumor Cell Line Carrying the Polyomavirus Middle T Antigen: Utility in the Development of Novel Cancer Therapeutics Cancer Res., December 1, 2000; 60(24): 7066 - 7074. [Abstract] [Full Text]
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T. Hyun, A. Yam, S. Pece, X. Xie, J. Zhang, T. Miki, J. S. Gutkind, and W. Li Loss of PTEN expression leading to high Akt activation in human multiple myelomas Blood, November 15, 2000; 96(10): 3560 - 3568. [Abstract] [Full Text] [PDF]
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 C. Abate-Shen and M. M. Shen Molecular genetics of prostate cancer Genes & Dev., October 1, 2000; 14(19): 2410 - 2434. [Full Text]
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X. Shan, M. J. Czar, S. C. Bunnell, P. Liu, Y. Liu, P. L. Schwartzberg, and R. L. Wange Deficiency of PTEN in Jurkat T Cells Causes Constitutive Localization of Itk to the Plasma Membrane and Hyperresponsiveness to CD3 Stimulation Mol. Cell. Biol., September 15, 2000; 20(18): 6945 - 6957. [Abstract] [Full Text]
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F. Vazquez, S. Ramaswamy, N. Nakamura, and W. R. Sellers Phosphorylation of the PTEN Tail Regulates Protein Stability and Function Mol. Cell. Biol., July 15, 2000; 20(14): 5010 - 5018. [Abstract] [Full Text]
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V. Stambolic, M.-S. Tsao, D. Macpherson, A. Suzuki, W. B. Chapman, and T. W. Mak High Incidence of Breast and Endometrial Neoplasia Resembling Human Cowden Syndrome in pten+/- Mice Cancer Res., July 1, 2000; 60(13): 3605 - 3611. [Abstract] [Full Text]
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H. Miyake, C. Nelson, P. S. Rennie, and M. E. Gleave Overexpression of Insulin-Like Growth Factor Binding Protein-5 Helps Accelerate Progression to Androgen-Independence in the Human Prostate LNCaP Tumor Model through Activation of Phosphatidylinositol 3'-Kinase Pathway Endocrinology, June 1, 2000; 141(6): 2257 - 2265. [Abstract] [Full Text] [PDF]
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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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R. E. Bachelder, M. J. Ribick, A. Marchetti, R. Falcioni, S. Soddu, K. R. Davis, and A. M. Mercurio P53 Inhibits {alpha}6{beta}4 Integrin Survival Signaling by Promoting the Caspase 3-Dependent Cleavage of Akt/PKB J. Cell Biol., November 29, 1999; 147(5): 1063 - 1072. [Abstract] [Full Text] [PDF]
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 I. U. Ali, L. M. Schriml, and M. Dean Mutational Spectra of PTEN/MMAC1 Gene: a Tumor Suppressor With Lipid Phosphatase Activity J Natl Cancer Inst, November 17, 1999; 91(22): 1922 - 1932. [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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P. Li, S. V. Nicosia, and W. Bai Antagonism between PTEN/MMAC1/TEP-1 and Androgen Receptor in Growth and Apoptosis of Prostatic Cancer Cells J. Biol. Chem., June 1, 2001; 276(23): 20444 - 20450. [Abstract] [Full Text] [PDF]
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B. Belletti, M. Prisco, A. Morrione, B. Valentinis, M. Navarro, and R. Baserga Regulation of Id2 Gene Expression by the Insulin-like Growth Factor I Receptor Requires Signaling by Phosphatidylinositol 3-Kinase J. Biol. Chem., April 20, 2001; 276(17): 13867 - 13874. [Abstract] [Full Text] [PDF]
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M. Nomura, A. Kaji, W.-Y. Ma, S. Zhong, G. Liu, G. T. Bowden, K.-i. Miyamoto, and Z. Dong Mitogen- and Stress-activated Protein Kinase 1 Mediates Activation of Akt by Ultraviolet B Irradiation J. Biol. Chem., June 29, 2001; 276(27): 25558 - 25567. [Abstract] [Full Text] [PDF]
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, 5 MOI; 
, 25 MOI), Ad-p53 (
, 5 MOI; 
, 25 MOI), or Ad-MMAC (
, 5 MOI; 
, 25 MOI). Data are presented as a percentage of MTS value of mock-infected LNCaP cells. Columns, means of five replicate samples; bars, SD. B, cell number of LNCaP cells mock infected (line with Xs) or infected with Ad-DE1 (
,25 MOI), or Ad-MMAC (
, 5 MOI; 
, 25 MOI). C, MTS assay of LNCaP-Bcl-2 cells 4 days and 6 days after infection; labels same as in A. D, cell number of LNCaP-Bcl-2 cells; labels same as in B.