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[Cancer Research 59, 2318-2323, May 1, 1999]
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[Cancer Research 59, 2318-2323, May 15, 1999]
© 1999 American Association for Cancer Research

Advances in Brief

Adenovirus-mediated Gene Transfer of MMAC1/PTEN to Glioblastoma Cells Inhibits S Phase Entry by the Recruitment of p27Kip1 into Cyclin E/CDK2 Complexes

I. Wayne Cheney1, Saskia T. C. Neuteboom1, Mei-Ting Vaillancourt, Muralidhara Ramachandra and Robert Bookstein2

Canji, Inc., San Diego, California 92121


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Genetic alterations in the MMAC1 tumor suppressor gene (also referred to as PTEN or TEP1) occur in several types of human cancers including glioblastoma. Growth suppression induced by overexpression of MMAC1 in cells with mutant MMAC1 alleles is thought to be mediated by the inhibition of signaling through the phosphatidylinositol 3-kinase pathway. However, the exact biochemical mechanisms by which MMAC1 exerts its growth-inhibitory effects are still unknown. Here we report that recombinant adenovirus-mediated overexpression of MMAC1 in three different MMAC1-mutant glioblastoma cell lines blocked progression from G0/G1 to S phase of the cell cycle. Cell cycle arrest correlated with the recruitment of the cyclin-dependent kinase (CDK) inhibitor, p27Kip1, to cyclin E immunocomplexes, which resulted in a reduction in CDK2 kinase activities and a decrease in levels of endogenous phosphorylated retinoblastoma protein. CDK4 kinase activities were unaffected, as were the levels of the CDK inhibitor p21Cip1 present in cyclin E immunocomplexes. Therefore, overexpression of MMAC1 via adenovirus-mediated gene transfer suppresses tumor cell growth through cell cycle inhibitory mechanisms, and as such, represents a potential therapeutic approach to treating glioblastomas.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
The tumor suppressor gene MMAC1 was originally identified by homozygous deletion mapping of chromosome 10q, and deletions or inactivating mutations of this gene have been detected in a variety of tumor tissues and cell lines (1 , 2) . Germ-line mutations of the MMAC1 gene have also been described in Cowden disease and Bannayan-Zonana syndrome, in which affected individuals suffer increased susceptibility to cancer and developmental abnormalities (3 , 4) . The identification of MMAC1 as a tumor suppressor gene has been supported by various reports demonstrating that ectopic expression of MMAC1 in glioblastoma cell lines harboring MMAC1 mutations resulted in inhibition of cellular proliferation and suppression of both soft agar colony formation and tumorigenicity in nude mice (5, 6, 7) . Consistent with a distinct primary sequence motif, phosphatase activity of MMAC1 protein has been detected toward several types of phosphorylated substrates including poly-glutamine/phosphotyrosine peptides (8) , the cellular phosphoprotein focal adhesion kinase (9) , and the lipid second messenger PtdIns(3,4,5)P3 (10) ,3 which is generated by activated PI3-kinase. It is presently unclear which of these activities is most important for MMAC1 function. A role for MMAC1 in cell invasion and migration was suggested by the finding that MMAC1 gene transfer was associated with the dephosphorylation of focal adhesion kinase, p130Cas, and Shc (9 , 11 , 12) . On the other hand, exogenous expression of MMAC1 in various cells reduced levels of endogenous PtdIns(3,4,5)P3 and resulted in dephosphorylation of Akt/PKB, which is one of the main downstream targets of PI3-kinase (13, 14, 15, 16) . Furthermore, MMAC1-mutant cells possess constitutively elevated levels of PtdIns(3,4,5)P3 and activated Akt/PKB (17 , 18) . The phosphatidylinositol 3-phosphatase activity may be most critical in tumor suppression because the G129E mutation (isolated from two independent Cowden disease kindreds) specifically inhibited the ability of MMAC1 to dephosphorylate phosphoinositides but did not affect its protein phosphatase activity (13 , 19) . Nevertheless, the exact mechanism(s) by which MMAC1 functions in tumor suppression or growth inhibition remains unknown.

Furnari et al. (19) reported recently that MMAC1 controls growth by regulating the cell cycle. Overexpression of MMAC1 with plasmid-based transfection systems conferred an observable G0/G1 cell cycle arrest that was dependent on the presence of reduced serum concentrations and a catalytically active MMAC1 phosphatase domain (19) . In this study, we report that adenovirus-mediated expression of MMAC1 induced G0/G1 arrest in glioblastoma cells possessing mutant but not wild-type MMAC1 alleles. Furthermore, MMAC1-induced cell cycle arrest correlated with the recruitment of the CDK inhibitor, p27Kip1, into cyclin E immunocomplexes, a concomitant decrease in CDK2 activity, and a reduction in the levels of phosphorylated endogenous pRb.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Cells and Culture Conditions.
The glioblastoma cell lines U87MG and U373MG were obtained from American Type Culture Collection. LN229 glioblastoma cells were a kind gift of Dr. N. de Tribolet (University Hospital, Lausanne, Switzerland; Ref. 20 ). The U251MG glioblastoma cell line was obtained from Dr. Peter Steck (M. D. Anderson Cancer Center, Houston, TX). U87MG cells contain a splice site mutation that results in an in-frame deletion of MMAC1 exon 3, whereas U373MG and U251MG harbor insertional frame-shifting mutations at codon 241 (1 , 2) . LN229 glioblastoma cells possess wild-type sequence for MMAC14 (5) . U87MG, U251MG, and LN229 cells were grown in DMEM, whereas U373MG cells were propagated in MEM. All cells were maintained in the presence of 10% fetal bovine serum, without antibiotics, at 37°C in a humidified atmosphere containing 7% CO2.

Viruses.
Replication-defective recombinant adenoviruses were constructed, propagated, and purified as described previously (6) . The rAd termed MMCB contains the constitutively active cytomegalovirus promoter driving expression of the wild-type MMAC1 cDNA sequence. As a control, an empty cassette rAd, which retains the cytomegalovirus promoter in the deleted E1 region, was constructed and is referred to as ZZCB. Replication-competent adenoviruses were undetectable in these virus preparations.

Cell Cycle Analysis.
Exponentially growing, asynchronous U87MG, U373MG, U251MG, and LN229 cells were infected with either ZZCB or MMCB at a dose of 2 x 108 virus particles/ml for 2 h, after which the virus was washed off and replaced with fresh medium. Infected cells were incubated an additional 48 h before a 4-h pulse with 0.01 mM BrdUrd and were then harvested by trypsinization. Detection of DNA-incorporated BrdUrd and PI DNA staining was accomplished by methods described by Demers et al. (21) . FACS analysis on 10,000 gated cells was performed on a Beckton Dickinson FACS station using CellQuest software. Results from three independent experiments were used to determine the average percentage of cells in each phase of the cell cycle.

Immune Complex Kinase Assays.
Subconfluent monolayers of U87MG, U373MG, U251MG, and LN229 glioblastoma cells were left untreated or infected with either MMCB or ZZCB at doses of 2 x 108 or 2 x 109 particles/ml for 2 h, washed with fresh medium, and then incubated for a total of 48 h after infection. Cells were harvested by scraping into cold PBS and were then pelleted and lysed in immunoprecipitation buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, and 0.1% Tween 20] containing 10% glycerol, 10 mM ß-glycerophosphate, 1 mM NaF, 0.1 mM sodium orthovanadate, plus Complete Protease Inhibitor Cocktail (Boehringer Mannheim; Ref. 22 ). Cell lysates were clarified by centrifugation at 10,000 x g for 10 min. Protein concentrations were determined by the Bradford assay (Bio-Rad), and equal quantities (72–100 µg) of each lysate were immunoprecipitated with 2 µg of anti-CDK2 goat polyclonal antibody (Santa Cruz 163-G) or 2 µg of anti-CDK4 rabbit polyclonal antibody (Santa Cruz 260-G) for 2 h at 4°C. Protein A-agarose beads (Upstate Biotechnology) were then added for an additional hour. Immunocomplexes were washed three times with immunoprecipitation buffer and once with 50 mM HEPES (pH 7.5) containing 1 mM DTT. Kinase assays were performed exactly as described by Matsushime et al. (22) , except that the substrate was 1 µg of recombinant retinoblastoma protein (pRb) from Escherichia coli lysates as described previously (23) . Relative intensities of 32P-phosphorylated pRb were quantitated with a Storm phosphorimager (Molecular Dynamics).

Immunoprecipitations.
Cell monolayers of U87MG, U251MG, U373MG, and LN229 were prepared and infected as described for the immune complex kinase assays. Cells were lysed in buffer A containing 50 mM HEPES, 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 2.5 mM EDTA, 1% Triton X-100, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and Complete Protease Inhibitor Cocktail. Cell lysates (270–400 µg) were incubated with 2 µg of rabbit polyclonal anti-cyclin E antibody C-19 (Santa Cruz Biotechnology) for 2 h at 4°C. Subsequently, 40 µl of a 50% recombinant protein A-agarose slurry (Upstate Biotechnology) were added to all samples, and the incubation was continued for another 2 h. The immune complexes were recovered by centrifugation at 4°C, washed three times with buffer A, and subjected to immunoblotting using either the mouse monoclonal anti-p27Kip1 or anti-p21Cip1 antibody (Transduction Laboratories).

Immunoblotting.
Protein lysates (7–15 µg) or immunoprecipitates were separated by SDS-PAGE using 6, 8, or 4–20% gels (Novex). Samples were transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore Corp.), blocked in Blotto [5% non-fat dry milk in Tris-buffered saline with 0.05% Tween 20 (TBST, Sigma)], and then incubated for 2 h with primary antibody diluted in Blotto. Membranes were washed (3 x 15 min) in TBST and incubated for 1 h with horseradish peroxidase-conjugated secondary antibody (Amersham Life Science) diluted in Blotto. After three 15-min washes in TBST, the bands were visualized with the ECL detection system (Amersham Life Science). Primary antibodies used were: rabbit polyclonal anti-MMAC1 antibody BL74 (6) ; rabbit polyclonal anti-cyclin E antibody C-19 (Santa Cruz Biotechnology); mouse monoclonal anti-pRb antibody C38 (24) ; mouse monoclonal anti-p27Kip1 antibody (Transduction Laboratories); mouse monoclonal anti-p21Cip1 antibody (Transduction Laboratories); rabbit polyclonal anti-Akt antibody (New England Biolabs); and the rabbit polyclonal anti-Phospho-Akt (Ser 473) antibody (New England Biolabs).


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Adenovirus-mediated Expression of Biologically Active MMAC1.
Exogenous MMAC1 expression was detected by immunoblotting using the anti-MMAC1 antibody BL74. At viral doses of 2 x 108 and 2 x 109 particles/ml, the MMAC1 transgene was expressed at comparable levels in the rAd (MMCB)-infected U87MG, U251MG, and LN229 cells (Fig. 1)Citation . In U373MG cells, the transgene was expressed with greater efficiency. Endogenous MMAC1 protein was undetectable in all cell lysates using the BL74 antibody. Because overexpression of MMAC1 has been demonstrated to inhibit intracellular signaling through the PI3-kinase pathway, resulting in reduced phosphorylation of Akt (13 , 14 , 18) , we assessed the effect of MMCB infection on Akt. MMCB infection of all four glioblastoma cell lines described above reduced or eliminated phosphorylation of Akt at the serine 473 residue (Fig. 1)Citation , demonstrating that the adenovirus mediated expression of MMAC1 produced a biologically active protein. Consistent with the finding that adenovirus infections result in {alpha}v integrin-mediated activation of PI3-kinase (25) , an up-regulation of Akt phosphorylation in LN229 lysates was observed upon infection with the control rAd ZZCB compared with the uninfected sample (Fig. 1)Citation . Exogenous MMAC1 expression in LN229 cells was, nevertheless, sufficient to overcome this rAd-dependent enhancement of Akt phosphorylation. Adenovirus-induced Akt phosphorylation was inapparent in the three MMAC1-mutant cell lines because Akt phosphorylation in these cells is already at an elevated level due to mutational inactivation of MMAC1 (Fig. 1)Citation .



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  		Fig. 1. Exogenous MMAC1 expression and effect on Akt phosphorylation. Immunoblot analysis of cell lysates (7.5–15 µg), prepared from uninfected or ZZCB- or MMCB-infected glioblastoma cell lines as indicated, using anti-MMAC1 BL74, anti-phospho-Akt (Ser 473), and anti-Akt antibodies.

 
MMAC1 Expression Induces G0/G1 Cell Cycle Arrest.
To determine whether cell cycle regulation is one of the mechanisms by which MMAC1 exerts its growth-suppressive effects, as observed in glioblastoma and melanoma cells (5 , 6 , 26) , BrdUrd incorporation/proliferation assays were performed. In these assays BrdUrd was incorporated into asynchronous, exponentially growing rAd-infected U87MG, U251MG, U373MG, and LN229 glioblastoma cells. The total cellular DNA content (PI staining) and the proportion of cells in S phase (BrdUrd incorporation) were quantitated by FACS analysis. Scatter profiles of rAd-infected glioblastoma cells and the percentages of each in the various phases of the cell cycle (Fig. 2)Citation showed that MMCB infection of all three MMAC1-mutant cell lines resulted in a substantial increase in the G0/G1 phase (13–22% increase) and a reduction in the number of cells in S phase (14–21% decrease). Little or no effect was noted for the G2/M phase of the cell cycle. The G0/G1 cell cycle blockade was not observed in the MMAC1-wild-type cells LN229, suggesting that the suppressive effect induced by MMAC1 overexpression is MMAC1 genotype specific.



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  		Fig. 2. Flow cytometric analysis of exponentially growing uninfected or rAd-infected cells. Scatter profiles are presented as a two-parameter plot of anti-BrdUrd-FITC (DNA incorporation) versus PI (total DNA) staining. The percentage of cells in S phase was determined by assessing the fraction of cells staining positive for BrdUrd incorporation. A representative scatter profile from a single experiment is shown for each cell type analyzed, along with the cell cycle phase distribution percentages determined for each sample.

 
MMAC1 Expression Reduces the Activity of CDK2 but not of CDK4.
The kinase activities of G1 cyclin/cyclin-dependent kinase complexes are key determinants of progression from G1 to S phase. Cyclin D/CDK4 or CDK6 activity peaks in late G1, whereas cyclin E/CDK2 activity peaks just before S phase entry. To investigate whether the MMCB-induced G1 cell cycle arrest is due to a change in G1 cyclin/CDK activities, we examined the kinase activities of immune complexes precipitated by antibodies to CDK2 and CDK4 using exogenous retinoblastoma protein (pRb) as a substrate. CDK2 kinase activities were markedly suppressed (80–95%) in a dose-dependent fashion when U87MG, U251MG, and U373MG cells were infected with MMCB as compared with controls (Fig. 3A)Citation . Overexpression of MMAC1 in LN229 cells did not affect their CDK2 kinase activities. Unlike the effect on the CDK2 kinase activities in MMAC1-mutant cell types, MMCB infection had no effect on CDK4 kinase activities in U87MG and U373MG cells and only a small effect (25% decrease) in U251MG cells (Fig. 3A)Citation . MMCB-infected LN229 cells were similarly unaffected in the CDK4 kinase assay.



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  		Fig. 3. Influence of MMAC1 expression on CDK2 and CDK4 activities and pRb phosphorylation. Lysates prepared from either uninfected or ZZCB- or MMCB-infected glioblastoma cells were immunoprecipitated with anti-CDK2 (Lanes 1–5) or anti-CDK4 (Lanes 6–10) antibodies and then used for in vitro kinase assays with purified pRb as substrate. Autoradiographs show a single 32P-labeled band at Mr 110,000, which corresponds to phosphorylated retinoblastoma protein [pRB(P)]. B, phosphorylation status of endogenous pRb was determined in cell lysates (5 µg) of uninfected or ZZCB- or MMCB-infected glioblastoma cells by immunoblotting using the anti-pRb antibody C38. Endo pRB(P), endogenous phosphorylated pRB.

 
pRb Phosphorylation Status after MMCB Infection.
Because pRb phosphorylation at the G1 to S cell cycle transition can be regulated, in part, by cyclin E/CDK2 activities (27) and because our in vitro kinase assay showed a decrease in CDK2 kinase activity, we assessed the phosphorylation status of endogenous pRb. Protein lysates from uninfected or ZZCB- or MMCB-infected U87MG, U251MG, U373MG, and LN229 cells were immunoblotted and probed with the anti-pRb mouse monoclonal antibody C38. Levels of phosphorylated pRb as well as total pRb levels were significantly decreased in MMCB-infected U87MG, U251MG, and U373MG cells in a dose-dependent manner as compared with control (ZZCB)-infected or uninfected cells (Fig. 3B)Citation . The phosphorylation status of endogenous pRb in LN229 cells was unaffected by MMCB infection (Fig. 3B)Citation . Taken together, these data demonstrate that all the lysates that have a decrease in CDK2 activity, as determined in our in vitro kinase assays, also show a significant reduction in the levels of phosphorylated endogenous pRb.

MMCB Infection Does Not Affect Cyclin E Levels but Results in Recruitment of CDK Inhibitor p27Kip to Cyclin E Complexes.
The kinase activity of cyclin E/CDK2 complexes is determined by a delicate balance between cyclin E, CDK2, and CDK inhibitors. Because cyclin E is a rate-limiting regulator in the formation of catalytically active cyclin E/CDK2 complexes (28 , 29) , we examined whether endogenous cyclin E levels were changed upon overexpression of MMAC1 in MMCB-infected cells. Protein lysates made from uninfected or ZZCB- or MMCB-infected U87MG, U251MG, U373MG, and LN229 cells were immunoblotted and probed with anti-cyclin E antibodies. rAd infection (ZZCB control and MMCB) of U251MG and U373MG did result in a slight reduction in the total levels of cyclin E; however, no MMCB-specific effect was observed in any of the four glioma cell lines tested (Fig. 4A)Citation .



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  		Fig. 4. Effect of MMAC1 expression on cyclin E levels and on the level of CDK inhibitors bound to cyclin E immunocomplexes. A, total levels of cyclin E were determined in cell lysates (15 µg) from uninfected or ZZCB- or MMCB-infected glioblastoma cells by immunoblotting using the anti-cyclin E antibody C-19. Detection of p27Kip1 or p21Cip1 in cyclin E immunocomplexes was performed by immunoprecipitating cyclin E complexes from cell lysates of uninfected or ZZCB- or MMCB-infected glioblastoma cells using the anti-cyclin E antibody C-19, followed by immunoblotting with either the anti-p27Kip1 (B) or anti-p21Cip1 (C) antibodies.

 
Because cyclin E levels were unaltered by MMAC1 overexpression, we next addressed the question of whether the activities of the cyclin E/CDK2 inhibitors, p27Kip1 and p21Cip1, were affected upon MMCB infection. p27Kip1 and p21Cip1 exert their inhibitory functions by forming a tight ternary complex with cyclin E/CDK2, thereby inactivating the kinase activity (30 , 31) . We therefore analyzed by Western blotting the level of endogenous p27Kip1 present in cyclin E immunocomplexes isolated from uninfected or ZZCB- or MMCB-infected glioblastoma cells. Cyclin E immunocomplexes from MMCB-infected U87MG, U373MG, U251MG, but not from LN229 cells, had p27Kip1 levels that were 2–4-fold higher than those found in ZZCB-treated or uninfected controls (Fig. 4B)Citation . Background levels of p27Kip1 were slightly elevated in all control U251MG samples, probably because these cells tended to grow in islands, the centers of which were likely to be contact inhibited and p27Kip1 up-regulated (30) . When cyclin E immunocomplexes were analyzed for the presence of endogenous p21Cip1, no substantial difference could be detected between uninfected or ZZCB- or MMCB-infected cells (Fig. 4C)Citation .


   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
In this study, we report a biochemical mechanism to account for the growth-suppressive effects associated with reintroduction of MMAC1 into MMAC1-mutant tumor cells. The data presented here indicate that the CDK inhibitor, p27Kip1, is recruited into cyclin E/CDK2 immunocomplexes when MMAC1 is overexpressed in MMAC1-mutant, but not MMAC1 wild-type, glioblastoma cells. Moreover, the recruitment of p27Kip1 to this complex leads to a reduction in cyclin E/CDK2 kinase activities by 80–95% and an overall diminution of phosphorylation levels in endogenous pRb. These effects culminate in a substantial reduction in the number of cells reaching S phase of the cell cycle; hence, MMCB-transduced cells fail to divide further. Our results suggest that the MMAC1 effect targets specifically the kinase activity of CDK2-associated complexes because CDK4-associated kinase activities were unaffected. Furthermore, the CDK2 inhibitor p27Kip1 seems to be the key inhibitory component, because overexpression of MMAC1 leads to the recruitment of p27Kip1, but not p21Cip1, into cyclin E immunocomplexes. Levels of p21Cip1 in these complexes were unaffected.

The mechanism of MMCB-induced G1 arrest in MMAC1-mutant cells resembles mechanisms described for the cell cycle arrest resulting from transforming growth factor-ß treatment of mink Mv1Lu cells (32) and for suppression of cyclin E-CDK2 activities in suspended, untransformed, human diploid fibroblasts (33) . In both instances, total levels of the CDK inhibitor p27Kip1 were up-regulated 2–4-fold and were sufficient to produce a G1 block in the cell cycle. Our results are in agreement with data reported recently by Li and Sun (34) , showing that transient MMAC1 expression in U87MG cells, introduced by retroviral infection, induced a G1 cell cycle arrest that correlated with an increase in the total levels of p27Kip1. Total levels of p27Kip1 can, however, be elevated without having a growth-inhibitory effect, as was observed recently in esophageal adenocarcinomas where mislocalization of p27Kip1 in the cytoplasm appeared to result in its inactivation through sequestration from nuclear targets (35 , 36) . Therefore, our data extend Li and Sun’s observations (34) by demonstrating that upon MMCB infection of glioblastoma cells mutant for MMAC1, the CDK inhibitor p27Kip1 is specifically recruited into cyclin E immunocomplexes to exert its inhibitory function on the cyclin E/CDK2 activity, ultimately producing a G1 cell cycle arrest. One question for future examination is whether MMAC1- tumor cells with impaired p27Kipl function, either from reduced expression or mislocalization, are capable of G1 arrest in response to MMAC1 replacement.

Unlike the MMAC1-mediated G1 growth arrest observed by Furnari et al. (19) , the growth suppression imparted by MMCB transduction did not depend upon low serum concentrations. All rAd transductions were performed in normal growth medium conditions containing 10% fetal bovine serum; hence, it is unlikely that MMCB-induced growth suppression is dependent on reduced concentrations of inhibitory serum factors. In accordance with Furnari et al. (5) , we found that exogenous MMAC1 did not induce its growth-inhibitory effects in a glioblastoma cell line containing wild-type MMAC1-. These results differ from those of Li et al. (15) , who reported that various MMAC1 wild-type epithelial cell lines were responsive to exogenous MMAC1-induced growth suppression. This discrepancy may be explained, in part, by cell type-specific differences in signaling by MMAC1.

The effect of MMAC1 on p27Kip1 activity is consistent with previous reports that this gene acts as an opponent of PI3-kinase. Takuwa and Takuwa (37) showed that PI3-kinase activity is required for G1 to S phase cell cycle progression, because the addition of the PI3-kinase inhibitor wortmannin to mitogen-stimulated cells caused a G1 arrest by preventing proper down-regulation of p27Kip1. Furthermore, microinjected antibodies capable of neutralizing PI3-kinase activity correlated with a G1 cell cycle blockade of NIH 3T3 cells (38) . How many steps and which components comprise the link between PI3-kinase activity and p27Kip1 levels remain open questions. Further investigation into the biological function of MMAC1 will be required to completely understand the effects of reintroducing this tumor suppressor gene into MMAC1 mutant tumor cells and its potential application as a gene therapeutic for cancer.


   ACKNOWLEDGMENTS
 
We thank Drs. N. de Tribolet and P. Steck for their kind gifts of LN229 and U251MG glioma cell lines, respectively. We thank A. Levy for excellent technical support and Drs. G. W. Demers and E. Lees for helpful discussions.


   FOOTNOTES
 
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.

1 These authors contributed equally to this work. Back

2 To whom requests for reprints should be addressed, at Canji, Inc., 3525 John Hopkins Court, San Diego, CA 92121. Phone: (619) 597-0177; Fax: (619) 623-2032; E-mail: rob.bookstein{at}canji.com Back

3 The abbreviations used are: PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5 triphosphate; rAd, recombinant adenovirus; PI3-kinase, phosphatidylinositol 3-kinase; BrdUrd, 5-bromo-2'-deoxyuridine; PI, propidium iodide; pRb, retinoblastoma protein; FACS, fluorescence-activated cell sorter; CDK, cyclin-dependent kinase. Back

4 A. Levy and R. Bookstein, unpublished data. Back

Received 3/ 5/99. Accepted 3/31/99.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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