Adenoviral-mediated PTEN transgene expression sensitizes ... - Nature

Cancer Gene Therapy (2004) 11, 273–279 All rights reserved 0929-1903/04 $25.00

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Adenoviral-mediated PTEN transgene expression sensitizes Bcl-2-expressing prostate cancer cells to radiation Charles J Rosser1,a,c,d, Motoyoshi Tanaka1,b, Louis L Pisters1, Noriyoshi Tanaka1, Lawrence B Levy2, David C Hoover1, H Barton Grossman1, Timothy J McDonnell3, Deborah A Kuban4 and Raymond E Meyn5 1

Department of Urology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA; Department of Biomathematics, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA; 3Department of Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA; 4Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA; and 5Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA. 2

Bcl-2 is associated with resistance to radiotherapy in prostate cancer. It was recently demonstrated that transduction of LNCaP prostate cells with the PTEN gene resulted in Bcl-2 downregulation. We hypothesized that forced expression of PTEN in prostate cancer cells would sensitize cells to radiation, downregulate Bcl-2 expression, and potentiate the G2M block induced by radiation. Four cell lines — PC-3-Bcl-2 (Bcl-2 overexpression, deleted PTEN), PC-3-Neo (wild-type Bcl-2, deleted PTEN), LNCaP (Bcl-2 overexpression, deleted PTEN), and DU-145 (wild-type Bcl-2 and PTEN) — were transduced with a recombinant adenovirus-5 vector expressing the human wild-type PTEN cDNA under the control of a human cytomegalovirus promoter (Ad-MMAC). After correction for the effect of Ad-MMAC on plating efficiency, Ad-MMAC treatment reduced the surviving fractions after 2 Gy as follows: PC-3-Bcl-2, from 60.5 to 3.6%; PC-3-Neo, no reduction; LNCaP, from 29.6 to 16.3%; and DU-145, from 32.7 to 25.7%. PTEN expression was associated with the downregulation of Bcl-2 expression in PC-3-Bcl-2 and LNCaP cell lines. Ad-MMAC plus radiotherapy potentiated the G2M block seen with radiotherapy alone only in PC-3-Bcl-2 cells. These findings suggest that overexpression of Bcl-2 result in radioresistance and inability of radiation to cause its typical G2M cell-cycle arrest. Cancer Gene Therapy (2004) 11, 273–279. doi:10.1038/sj.cgt.7700673 Published online 6 February 2004 Keywords: prostate cancer; radiation; PTEN; Bcl-2; clonogenic assay

ecurrence after radiotherapy is a common phenomenon in prostate cancer treatment. Owing to the R increased risk of complications in nearby critical structures, the amount of radiation that can be delivered is limited and dose escalation is likely not the total solution to radiation resistance. Instead, investigators have turned to strategies for sensitizing prostate tumors to radiation. All of the strategies tested over the past 20 years have involved systemic administration of agents with side Received May 14, 2003.

Address correspondence and reprint requests to: Louis L Pisters, Department of Urology, Box 446, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA. E-mail: [email protected] a

CJR and MT contributed equally to this study. Current address: Department of Urology, Nara Medical School, Kashihara, Nara 634-8521, Japan. c Current address: Division of Urology, University of Florida Shands, 555 West 8th St., Jacksoulle, FL 32207 d Vukar-Lopez F, Rosser CJ, DeLacerda J, Grossman HB, Pisters L, Reyes A. unpublished data. b

effects of their own, which almost always limit pharmacologic doses to levels below what is needed to sensitize tumors. None of the sensitizing strategies tested to date are available for widespread use. Multiple studies have consistently implicated two genes, p53 and Bcl-2, as being important in postradiotherapy prostate cancer recurrence.1–7 Recently, Huang et al8 demonstrated that transient transfection of the PTEN gene into PTEN-null cells resulted in decreased levels of Bcl-2 mRNA and protein. PTEN is a tumor suppressor gene located on chromosome 10q23 and is known to be mutated or deleted in a variety of neoplasms, including prostate cancer.9,10 We demonstrated in a case–control study that patients in whom radiotherapy alone fails to control localized prostate cancer have decreased expression of PTEN and overexpression of phosphor-Akt in their cancers. Previously, researchers have demonstrated PTEN expression to potentiate the cytotoxicity effects of chemotherapeutic agents8 or irradiation.11 The loss of PTEN function and overexpression of Bcl-2 occur frequently in prostate cancer, and both events are associated with the development of prostate cancer

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resistant to radiotherapy. The activation of the protooncogene Akt, which encodes a serine–threonine kinase downstream of PTEN, promotes cell survival by inhibiting the function of proapoptotic proteins.12–15 These findings suggest that strategies designed to downregulate Bcl-2 through the expression of PTEN may represent promising new strategies for sensitizing of prostate cancer cells to radiation. We hypothesized that transducing prostate cancer cells with PTEN would sensitize them to the effects of radiation.

Materials and methods

Cell lines and recombinant adenovirus vectors PC-3-Bcl-2 cells (overexpression Bcl-2, deleted PTEN, and mutant p53) and PC-3-Neo cells (wild-type Bcl-2, deleted PTEN, and mutant p53) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, 4 mM glutamine, and 400 mg/ml G418. DU145 cells (wild-type Bcl-2, wild-type PTEN, and mutant p53) and LNCaP cells (overexpression Bcl-2, deleted PTEN, and wild-type p53) were purchased from the American Type Culture Collection (Manassas, VA). Cells were maintained in RPMI supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 4 mM glutamine. All cells were incubated at 371C in a humidified atmosphere of 5% CO2 in air. Recombinant adenovirus vector Ad-MMAC, which expresses the human wild-type PTEN cDNA under the control of the human cytomegalovirus immediate-early promoter/enhancer was provided by Introgen, Inc. (Houston, TX). Control adenovirus vector (hereafter designated Ad-CTL) was derived from the same vector without a transgene (Ad-DE1/Ad5-dE1). A recombinant GFP adenovirus vector was used to determine the transduction efficiency of infection in vitro. The titers of Ad-MMAC and Ad-GFP were 1.7 1011 and 1.5 1011 plaque-forming units/ml, respectively. The same MOIs were used for both Ad-MMAC and Ad-CTL and were as follows: PC-3-Bcl-2, MOI ¼ 50; PC-3-Neo, MOI ¼ 50; DU-145, MOI ¼ 25; and LNCaP, MOI ¼ 10. These MOIs were selected because preliminary studies performed with Ad-GFP revealed that they resulted in a transduction efficiency of approximately 75%.

Clonogenic survival For clonogenic survival assays, 5 105 cells were plated into sterile T25 flasks. Typically, after 48 h, 2 106 cells in each flask were available for gene transduction. Both AdMMAC and Ad-CTL viruses were maintained and diluted in serum-free DMEM/F12 until transduction. The cells in each flask were washed in PBS to remove any residual serum that might bind viral particles and affect the MOI. A measure of 1 ml of the Ad-MMAC or AdCTL viral solution was gently placed onto the monolayer of cells in each T25 flask. The flasks were returned to the incubator and left there for 1 h. Control flasks with and

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without control vector were exposed to identical manipulations. After incubation, the flasks were decanted and washed with PBS. Next, 4 ml of complete medium with serum was added to each flask, and flasks were returned to the incubator. At 48 h after viral exposure, some flasks were irradiated with a high-dose-rate cesium unit (4 Gy/ min) to a total of 2, 4, or 6 Gy. Immediately after irradiation, cells were trypsinized, serial diluted, and replated into 100-mm dishes. The plates were incubated for 12 days for PC-3-Bcl-2 and PC-3 Neo cells and 16 days for DU-145 and LNCaP cells. The colonies were stained with gentian violet and counted. The surviving fraction was calculated relative to the unirradiated cells. For each radiation dose and dilution, three experiments were performed, and an intraexperiment average was calculated.

Cell cycle analysis For the analysis of cell-cycle distribution, cells were seeded at 5 105 cells in 10-cm tissue culture dishes and incubated overnight. Cells were then infected with AdMMAC or Ad-CTL or mock infected. Cells were maintained in supplemented medium as described in the first paragraph of Materials and methods, and 48 h after gene transduction, some cells were irradiated with 2 Gy. After 48 h, cells were trypsinized, washed with 1 PBS, fixed in 1% paraformaldehyde, and stored at 41C in 70% ethanol. Following overnight incubation in 70% ethanol, cells were treated with RNase A and incubated in propidium iodide. Cell-cycle distribution was determined using FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ) measuring at least 10,000 gated cells. Cell-cycle analysis was performed in duplicate.

Cell lysis and Western blotting For protein analysis, 5 105 cells were plated on 10-cm dishes. Approximately 24 h later, cells were infected with various amounts of adenovirus in 4 ml of medium. At 48 h after gene transduction, cells were irradiated with 2 Gy. At various time points, cells were scraped, washed two times with cold 1 PBS, and harvested in lysis solution containing 50 mM HEPES (pH 7.0), 150 mM NaCl, 1 mM EDTA, 100 mM NaF, 10 mM NaPPi, 10% glycerol, 1% Triton X-100, 1 mM Na3VO4, 1 mM pepstatin, 10 mg/ml aprotinin, 5 mM iodoacetic acid, and 2 mg/ml leupeptin. Western blot was performed as described previously.11 Immunoblotting was performed using antibodies against Bcl-2, PTEN, and total and phospho-Akt (Santa Cruz Biotechnology, Santa Cruz, CA), followed by horseradish peroxidase-conjugated secondary antibody (Amersham, Chicago, IL). Protein–antibody complexes were detected using chemiluminescence (Amersham, Arlington Heights, IL).

Immunocytochemical staining For immunocytochemical analysis, PC-3-Bcl-2, PC-3Neo, DU-145, and LNCaP prostate cancer cells were grown on coverslips in six-well plates at a density of


5 105 cells per well. The cells were allowed to attach overnight. The cells were then infected with Ad-MMAC or Ad-CTL or mock infected for 1 h and then decanted and refed with fresh medium. After 48 h, some cells were irradiated with 2 Gy. At 48 h after irradiation, the cells were washed and fixed in 1% paraformaldehyde. Subsequently, cells were blocked with 1% goat serum in citric acid buffer for 3 h and then incubated overnight with the monoclonal mouse anti-PTEN (Zymed, San Francisco, CA) at room temperature. The following day, cells were washed, treated with a biotinylated horse anti-mouse antibody (secondary antibody), and incubated at room temperature for 1 h. This was followed by incubation with an avidin–biotin peroxidase complex. The peroxidase reaction was developed with diaminobenzidine and H2O2 substrate. Finally, the slides were counterstained with hematoxylin, dehydrated, and mounted with Permount.

In vitro cytotoxicity assay Briefly, all prostate cancer cell lines were seeded in 96-well plates at a density of 5 103 cells per well and infected with Ad-MMAC or Ad-CTL or mock infected. At 1 h after viral exposure, medium was decanted and fresh supplemented medium was added. After 1–6 days, the cells were fixed with 1% glutaraldehyde for 15 min, stained with 0.5% crystal violet for 15 min, washed with water for 10 min, and allowed to air-dry. Subsequently, Sorenson’s solution (30 mM trisodium citrate, 00.6% HCl, and 47.5% ethanol) was added to elute the dye, and the optical density was read on a microplate autoreader (Bio-Tek Instruments, Winooski, VT) at 540 nm. Absorbance values were normalized to the values

obtained for the vehicle-treated cells to determine the survival percentage. Each assay was performed in triplicate, and an intraexperiment average was calculated.

Statistical analysis Logistic regression was used to compare clonogenic curves. The comparison was based on the actual proportion that survived for each cell line, treatment, and dose. In addition, this was fit into the following model: [(1)-(2)][(3)-(4)], where (1), (2), (3), and (4) are the various therapeutic strategies. For example, to compare PC-3-Bcl2 treated with 2 Gy with mock or CTL, four expected proportions were generated: (1) dose ¼ 2, treatment ¼ CTL; (2) dose ¼ 0, treatment ¼ CTL; (3) dose ¼ 2, treatment ¼ mock; and (4) dose ¼ 0, treatment ¼ mock. A Po.05 was considered statistically significant. Statistical analyses were performed with commercially available computer software.

Results

Effect of Ad-MMAC on radiosensitivity of prostate cancer cells Eight control experiments were performed to characterize the clonogenic responses of PC-3-Bcl-2, PC-3-Neo, DU145, and LNCaP cells to irradiation without the presence of viral vector (Fig 1). After controlling for plating efficiencies, mock-infected PC-3-Bcl-2 cells proved to be the most radioresistant of the prostate cancer cell lines. Among these cells not treated with viral vector, the surviving fractions after 4 Gy were as follows: PC-3-Bcl-2,

Figure 1 Clonogenic surviving fraction of mock infected (1), Ad-CTL infected (2), and Ad-MMAC infected (3) cells. (a) PC-Bcl-2; (b) PC-3-Neo; (c) DU-145; and (d) LNCaP. The error bars represent one standard deviation above and below the mean. *Po.05.

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55.11%; DU-145, 19.75%, LNCaP, 7.27%; and PC-3Neo, 5.91%. After normalization of data in PC-3-Bcl-2 cells, the surviving fraction after 2 Gy was reduced from 0.6047 without Ad-MMAC to 0.0357 with Ad-MMAC (Po.0001). In PC-3-Neo cells, the addition of PTEN produced no reduction in the surviving fraction after irradiation (0.2336 compared to 0.3387 for 2 Gy, P4.05). The addition of PTEN did not produce a reduction in the surviving fraction in irradiated DU-145 cells; 2 Gy without Ad-MMAC (0.327), 2 Gy with Ad-MMAC 0.2573 (P ¼ .0596). In LNCaP cells, the surviving fraction after 2 Gy was 0.2962 without Ad-MMAC and 0.1631 with AdMMAC (P ¼ .0003). Exposure to Ad-CTL did not produce radiosensitization in any of the cell lines tested.

Effect of Ad-MMAC on the expression of PTEN and Bcl-2 Western blot analysis confirmed that only DU-145 cells expressed wild-type PTEN. In all four cell lines, AdMMAC treatment resulted in the overexpression of the PTEN protein. In all four cell lines, PTEN expression was not altered by the addition of radiation. Bcl-2 expression was evident in LNCaP cells and was overexpressed in PC-3-Bcl-2 cells. PC-3-Neo and DU-145 cells were confirmed to be wild type for Bcl-2 expression. Treatment with Ad-MMAC produced a pronounced decrease in Bcl-2 expression in PC-3-Bcl-2 cells and LNCaP cells. In the PC-3-Bcl-2 and LNCaP cell lines, expression of Bcl-2 was not altered by treatment with radiation; Ad-CTL; or Ad-CTL plus radiation (Fig 3).

Effect of Ad-MMAC on immunohistochemical detection of PTEN and cell morphology Effect of Ad-MMAC on cell-cycle distribution In PC-3-Bcl-2 and PC-3-Neo cells, expression of PTEN produced an 8–17% increase in the G1 population and a concurrent decrease in the S and G2M population compared to the population in mock-treated cells (Table 1). The changes in G1-phase population were not significant compared to either mock-treated cells or cells infected with Ad-CTL. In DU-145 and LNCaP cells, expression of PTEN produced no appreciable changes in cell-cycle distribution. Radiotherapy alone caused a modest G2M arrest in all cell lines, except in DU-145. Infection with Ad-MMAC followed by irradiation caused no significant change in the proportion of cells in G2M phase over that seen with irradiation alone except in PC3-Bcl-2 cells, where there was an increase in the number of cells in G2M phase (Po.05) (Fig 2). Table 1 Effect of various treatments on cell-cycle distribution in PC3-Bcl-2, PC-3-Neo, LNCaP, and DU-145 cells % of cells in indicated phase Cell line and cell-cycle phase PC-3-Bcl-2 G1 S G2+M PC-3-Neo G1 S G2+M LNCaP G1 S G2+M DU-145 G1 S G2+M

Mock infection

PTEN

XRT

PTEN+XRT

Effect of Ad-MMAC on cell proliferation in vitro 45.93 32.27 21.8

53.78 27.75 18.47

42.09 29.82 28.09

44.18 20.83 34.99

38.37 27.34 34.29

55.79 22.7 21.52

30.51 23.01 46.47

37.58 20.11 42.31

65.82 10.88 23.3

68.67 10.26 21.07

53.64 17.48 28.87

66.73 6.41 26.85

53.13 19.15 27.72

50.8 25.55 23.65

48.52 25.85 25.63

47.31 23.06 29.63

Results are representative of four replicates of two experiments. Cancer Gene Therapy

Immunohistochemical analysis using specific antibody to human PTEN showed strong protein expression in approximately 75% of cells in all four cell lines assayed 92 h after Ad-MMAC exposure. Cells of all four cell lines were seeded at the same passage and same initial cell density. Slight staining was present in DU-145 cells (wildtype PTEN). In all four cell lines, no abnormal cellular morphology was noted in mock-infected or Ad-CTL-treated groups, and only a few morphologic changes were noted in the radiation-treated groups. In contrast, in all four cell lines, treatment with Ad-MMAC plus radiation produced numerous morphologic aberrations (multinucleated cells, apoptotic bodies, and perinuclear vacuolation). These changes were especially common in the PC-3-Bcl-2 cell line (Fig 4). Similarly, in all four cell lines, treatment with Ad-MMAC resulted in some of these same morphologic changes, but to a dramatically lesser degree than did treatment with Ad-MMAC combined with radiation. Furthermore, in all four cell lines cell density was lower in Ad-MMAC plus radiation-treated group than in the other treatment groups.

To determine the effect of PTEN expression on cell proliferation, prostate cancer cells were infected with AdMMAC or Ad-CTL or mock infected. In both PC-3-Neo and PC-3-Bcl-2 cells, the maximum growth inhibition was 35%, and this was observed 72 h after infection with AdMMAC (MOI ¼ 50). In LNCaP cells, the maximum growth inhibition was 63% and this occurred 48 h after infection with Ad-MMAC (MOI ¼ 10). Similarly, the greatest effect in DU-145 cells (11% growth inhibition) was seen 48 h after infection with Ad-MMAC (MOI ¼ 25). The growth inhibitory effects of PTEN expression were transient. In prior work, cellular regrowth at 4 days after viral transfection was demonstrated in DU-145. However, PC-3 and LNCaP cells continued to show inhibition out to 7 days after viral transfection (Data not shown).


277 PC-3-Bcl-2 Ad-PTEN

400

Phase G1

:% :45.93

600

S G2+M

:32.27 :21.80

400

300

Number

Number

Mock

200

Phase G1

:% :53.78

S G2+M

:27.75 :18.47

300

200 100

100

0

0 0

30

60 90 Channels

XRT

180

0

Phase G1

:% :42.09

S G2+M

:29.82 :28.09

120

60

30

60 90 Channels

120

280

150

:% :44.18 : 20.83 :34.99

Phase G1

Ad-PTEN/XRT

Number

Number

240

120

S G2+M

210

140

70

0

0 0

30

60

90

120

150

Channels

0

30

60

90

120

150

Channels

Figure 2 Assessment of flow cytometric analysis of PC-3-Bcl-2 cells stained for DNA content. Cells were mock infected (a), infected with Ad-CTL (data not shown; statistically no different from mock), infected with Ad-MMAC (b), irradiated (c), or infected with Ad-MMAC and then irradiated (d). Experiments were performed in duplicate. Infection with Ad-MMAC followed by irradiation caused no increase in the number of cells in G2M phase (Po.05).

Figure 3 Western analysis of PTEN and Bcl-2 expression in PC-3Bcl-2 cells 48 h after treated with Ad-CTL, Ad-MMAC, or radiotherapy (2 Gy). Total Akt was used as the loading control.

Discussion

We found that infection of PTEN-mutant, Bcl-2-overexpressing prostate cancer cells (PC-3-Bcl-2 and LNCaP) with a replication-deficient adenovirus expressing the

PTEN gene under the cytomegalovirus promoter resulted in the overexpression of PTEN, downregulation of Bcl-2, and synergistic sensitization of cells to radiotherapy. We also found that treatment with Ad-MMAC potentiated the G2M block caused by radiotherapy in PC-3-Bcl-2 cells. Finally, we found that treatment with Ad-MMAC and, to a much greater degree, treatment with Ad-MMAC followed by radiotherapy resulted in morphologic aberrations in cells. Our finding that Ad-MMAC reduced Bcl-2 levels in cells overexpressing this protein is of particular interest because overexpression of Bcl-2 is associated with resistance to the cytotoxic effects of radiation. This was reflected in our study by the fact that the surviving fraction after 4 Gy in cells not treated with adenovirus was 55.11% for PC-3-Bcl-2 cells, which overexpress Bcl-2, versus 5.91% in PC-3-Neo cells, which do not overexpress Bcl-2. Bcl-2 overexpression may delay the response to radiation via a number of mechanisms including p53. p53 is a critical regulator of the cellular response to radiationinduced apoptosis.16 We demonstrated an increased rate of apoptosis with Ad-MMAC plus radiotherapy compared to radiotherapy alone only in LNCaP cells, which have deleted PTEN, wild-type p53 (data not shown). However, the greatest effect of Ad-MMAC on cell

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Figure 4 Results of immunocytochemical analysis using anti-PTEN antibody in PC-3-Bcl-2 cells initially seeded on coverslips in six-well plates at 5 105 cells/well. After 24 h, the cells were subjected to mock or adenoviral transfection. After 48 h, the cultures were irradiated with 2 Gy. After 48 h, cells were washed with PBS and fixed as described in Materials and methods. No cytoplasmic staining for PTEN was evident in mock infected (a) or radiation-treated (c) cells. Staining of the cytoplasm for PTEN was strong in cells treated with Ad-MMAC (b) and Ad-MMAC plus radiation (d). All images at 100 magnification. In the Ad-MMAC-treated cells, approximately 75% of the cells stained positive for PTEN protein expression. A similar gene transduction rate was observed in the other cell lines (PC-3-Neo, LNCaP, and DU-145) (data not shown). Radiotherapy alone (c) produced a slight increase in apoptotic bodies (arrowhead). The addition of radiotherapy to cells previously treated with Ad-MMAC (d) produced an abundance of cells with abnormal morphologic features, including an increase in the number of apoptotic bodies, perinuclear vacuolation (arrowhead), multinucleated cells (thin arrows), and overall decrease in cell number.

proliferation was seen in the clonogenic assay in PC-3Bcl-2 cells, which have mutated p53. One of the cellular responses to radiation is a profound G2M cell-cycle block. After treatment with radiation, a modest G2M block was evident in PC-3-Neo, which has wild-type Bcl-2 and deleted PTEN, and PC-3-Bcl-2 and LNCaP cells, which overexpress Bcl-2 and have deleted PTEN (Table 2). These findings suggest that overexpression of Bcl-2 result in radioresistance and limit the ability of radiation to cause its typical G2M cell-cycle arrest. We are currently assessing the exact mechanism (i.e. transcriptional versus post-transcriptional) as to how forced overexpression of PTEN can: (1) downregulate Bcl-2 expression and (2) synergistically sensitize cells to the affects of radiation. Furthermore, we will examine the relationship between PTEN, Bcl-2, and p53. The expression of PTEN may counteract anomalies of p53 and/or Bcl-2 expression. Given the many genetic defects seen in prostate tumors, strategies designed to replace defective genes are inherently fraught with difficult problems, which may account for the lack of response in previous clinical trials that have employed this approach. We expect that future studies of gene therapy for prostate cancer will employ an approach

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Table 2 Prostate cancer cell lines phenotype p53

Bcl-2

PTEN

PC-3-Bcl-2 PC-3-Neo

Mutant Mutant

Overexpression Expression

Deleted Deleted

DU-145

Mutant

Wild type

Wild type

LNCaP

Wild type

Overexpression

Deleted

more like the one we used here combining gene therapy with traditional therapies (surgery, hormonal therapy, radiotherapy, and chemotherapy) in an effort to improve response. Acknowledgments

Stephanie Deming and Dr Kwai Wa Cheng for his careful review of the manuscript. This work was supported by American Foundation of Urologic Disease grant and National Cancer Institute Grants CA56973 and CA16672.


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