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[Cancer Research 60, 1457-1462, March 1, 2000]
© 2000 American Association for Cancer Research

Virology

Overexpression of PTEN/MMAC1 and Decreased Activation of Akt in Human Papillomavirus-infected Laryngeal Papillomas1

Ping Zhang and Bettie M. Steinberg2

Department of Otolaryngology, Long Island Jewish Medical Center, The Long Island Campus of the Albert Einstein College of Medicine, New Hyde Park, New York 11040


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Laryngeal papillomas are benign, human papillomavirus-induced hyperplastic tumors of the respiratory tract. They are characterized by overexpression of the epidermal growth factor receptor, constitutive activation of mitogen-activated protein kinase, a low proliferative rate, and defects in differentiation. We have now found that phosphoinositol 3-kinase (PI 3-K) activity is significantly increased in papilloma tissue. However, phosphorylated Akt (also known as protein kinase B), a downstream effector of PI 3-K, is reduced when compared with normal tissue. The ratio of activated Akt to total Akt is much lower in papillomas than in normal laryngeal tissue, suggesting decreased Akt activation. PTEN/MMAC1 is a tumor suppressor that dephosphorylates phosphatidylinositol 3,4,5-triphosphate, an intermediate in the PI 3-K/Akt signaling pathway. We have found that PTEN protein is overexpressed in laryngeal papillomas when compared with normal laryngeal tissues. On the basis of reverse transcription-PCR analysis, PTEN mRNA is more abundant in papillomas, suggesting transcriptional up-regulation. We postulate that negative regulation of the PI 3-K/Akt pathway by PTEN may modulate the effects of the hyperactive epidermal growth factor receptor/mitogen-activated protein kinase pathway, contributing to the low proliferation and dysfunctional differentiation of laryngeal papillomas.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Laryngeal papillomas are benign squamous epithelial tumors caused by low-risk HPVs3 types 6 or 11 (1) . They are characterized by a hyperplastic suprabasal epithelium surrounding cords of connective tissues, with the increase in thickness of the spinous layer attributable to disruption of normal differentiation rather than high rates of basal or suprabasal cell proliferation (2) . Immunohistochemical (3) and Western blot analysis have shown that the EGF receptor is overexpressed, activated MAP kinase and phosphotyrosine levels are increased, and EGF thresholds for MAP kinase activation are lower in laryngeal papillomas and cells cultured from the papillomas (4) . Moreover, the block to differentiation can be relieved by removing EGF from the medium of organotypic cultures of papilloma cells (3) . Taken together, these data suggest that alterations in the EGF receptor/MAP kinase signal transduction pathway contribute significantly to the phenotype of laryngeal papillomas.

The PI 3-K/Akt pathway is a second signal transduction pathway that serves as a mediator of effects of insulin and several growth factors, participating in the regulation of proliferation and differentiation by the MAP kinase pathway (5 , 6) . PI 3-K is activated in response to a variety of growth stimuli that activate receptor tyrosine kinases, including the insulin receptor and the EGF receptor. Activation of PI 3-K induces the production of PtdIns (3,4)P2 and PtdIns (3,4,5)P3. Both of these lipid second messengers bind to Akt, presumably altering confirmation and enhancing phosphorylation on threonine at residue 308 and on serine at residue 473 by other cellular kinases (7) . Maximal activation requires phosphorylation at both of these sites. A critical modulator of the PI 3-K/Akt pathway, PtdIns (3,4,5)P3, also directly activates the kinase that phosphorylates Akt on threonine 308 (8) . Activation of Akt through the PI 3-K pathway is required for cell survival and proliferation when the MAPK pathway is activated (9 , 10) . Therefore, we postulated that changes in this pathway might also occur in papilloma tissues.

PTEN/MMAC1/TEP1 is a major new tumor suppressor gene located on human chromosome 10q23 (11) . Somatic mutations of PTEN have been identified in a number of malignancies, including carcinomas of the breast, prostate, lung, and head and neck (12, 13, 14, 15, 16) . Heterozygous disruption of PTEN in mice leads to tumor formation in multiple tissues, indicating that PTEN regulates fundamental cellular processes (17) . PTEN-knockout mouse embryos display regions of increased proliferation, and PTEN-deficient immortalized mouse embryo fibroblasts exhibit decreased sensitivity to cell death, providing in vivo evidence that PTEN negatively regulates a cell survival signaling pathway (18) .

PTEN has homology to tensin, an actin-binding protein localized to focal adhesion complexes (19) ; to auxilin, a protein involved in the uncoating of clatherin-coated vesicle (20) ; and to dual-specificity phosphatases (11 , 21) . Recombinant PTEN is capable of dephosphorylating both threonine-and tyrosine-phosphorylated substrates. It also dephosphorylates lipid second messengers in vitro, specifically removing the 3-phosphate from PtdIns (3,4)P2 and PtdIns (3,4,5)P3 (21 , 22) . Overexpression of PTEN suppresses growth and tumorigenicity and induces G1 cell cycle arrest in human glioblastoma cells, which is linked to inhibition of the PI 3-K/Akt pathway (23, 24, 25) . Overexpression of Akt can rescue cells from PTEN-dependent death (26) . Fig. 1Citation illustrates the EGF receptor/MAP kinase and PI 3-K/Akt pathways.



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  		Fig. 1. Diagram showing the PI 3-K/Akt pathway, the EGF receptor/MAP kinase pathway, and a known interaction between the two. PI-3 Kinase, PI 3-K; MAPK, MAP kinase.

 
Studies of PTEN in human tumors have focused on mutation or deletion of the PTEN gene, and changes in PTEN expression in HPV-induced benign tumors have not been described. We have analyzed PTEN expression in laryngeal papilloma tissues by Western blot, RT-PCR and immunohistochemistry, and analyzed the PTEN-regulated PI 3-K/Akt pathway. We found activated PI 3-K, overexpression of PTEN, and reduced activation of Akt. We propose that negative regulation of the PI 3-K/Akt pathway by PTEN may modulate the effects of the highly active EGF receptor/MAP kinase pathway in laryngeal papillomas, contributing to the low proliferation rate and dysfunctional differentiation of these benign tumors.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Samples
Biopsy specimens were removed during direct laryngoscopy. Normal tissues were surgical discards of stratified squamous epithelium from the epiglottis and false vocal fold from patients with no history of papilloma. A subset of biopsies was from clinically normal tissue adjacent to papillomas to rule out any systemic difference in signaling pathways in papilloma patients. Papilloma tissues were removed at the time of therapeutic laser excision from patients whose lesions had been assayed previously for HPV presence, typed as either HPV-6 or HPV-11, as described previously (27) . Use of these tissue discards was approved by the Institutional Review Board, and informed consent was obtained from each subject or subject’s guardian.

PI 3-K Assay
Functional kinase assays were done by a modification of the method of Carpenter et al. (28) . Tissue lysates containing 200 µg of protein were immunoprecipitated with anti-phosphotyrosine (Upstate Biotechnology, Inc., Waltham, MA) and protein G plus protein A agarose (CalBiochem Corp., San Diego, CA). Papilloma tissues were sufficiently large to analyze separately, whereas normal tissues were pooled to achieve protein concentrations equal to the papilloma extracts. Three normal samples (each a pool of four normal laryngeal tissues) and five separate papillomas were analyzed. The lipids L-æ phosphatidyl-L-serine and L-æ-phosphatidylinositol (Sigma Chemical Co., St. Louis, MO) were dried under nitrogen in a siliconized tube and then sonicated in 10 mM HEPES (pH 7.4), 0.1 mM EGTA, and 0.03 ml/100 ml NP40. Reaction mixtures containing 0.5 µCi of [32 P]ATP per nmol were incubated for 20 min at 30°C; reactions were stopped by the addition of HCl and extracted with chloroform-methanol (1:1). Lipids were separated by TLC using the solvent system n-propyl alcohol:2 M acetic acid (65:35, v/v). For later experiments, the faster chloroform, methanol, 2.2 M NH2OH (9:7:2, v/v/v) solvent system was used. Chromatography plates were visualized by autoradiography, and films were quantitated by image analysis (Alpha Innotech Corp., San Leandro, CA).

Western Blotting
Tissue samples were minced and sonicated in lysis buffer [150 mM NaCl, 20 mM HEPES (pH 7.5), 1 mM MgCl2, 1 mM CaCl2, 10 g/100 ml glycerol, 1 g/100 ml NP40, with freshly added 1 µg/ml pepstatin, 1 µg/ml leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM NaVO4]. Protein concentrations were determined by the BCA assay procedure (Pierce), and 20 µg protein from each sample were loaded on 7.5% SDS-polyacrylamide gels. To achieve sufficient material, two or more normal tissue biopsies were pooled for each sample. Proteins were electroblotted to nylon transfer membranes, blocked with 5 g/100 ml nonfat milk in TTSB [10 mM Tris (pH 7.5), 100 mM NaCl, and 0.1 g/100 ml Tween 20], and probed with primary antibody. After incubation with the corresponding alkaline phosphatase-conjugated second antibody, bound immunoglobulins were detected using chemiluminescent film exposure. The membranes were stripped in 2 g/100 ml SDS, 62.5 mM Tris (pH 6.8), and 100 mM ß-mercaptoethanol for 30 min at 65°C, rinsed with TTSB, and reprobed with subsequent primary antibody.

Primary antibodies used in the study were: PTEN and actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), PI-3K (P85; Transduction Laboratories, Lexington, KY), and Akt, Akt-thr308p, and Akt-ser473p (New England BioLabs, Inc., Beverly, MA). All antibodies were used as described by the manufacturers.

Films were quantitated by densitometry. Results were normalized to actin intensity on the same blot and expressed as mean ± SE. Statistical significance was determined by a two-tailed Student t test, with significance set at P < 0.05.

PTEN mRNA Analysis
PTEN message levels were determined by RT-PCR. Total RNA was isolated from patient tissue samples with RNA STAT-60 (Tel-Test, Inc., Friendswood, TX) and treated with RNase-free DNase. First-strand cDNA was synthesized from 1.5 µg of total RNA with an oligo(dT)16 primer and AMV reverse transcriptase (Boehringer Mannheim Biochemicals, Indianapolis, IN). A total of eight normal biopsies were pooled into one sample to provide sufficient RNA, and three pools were analyzed. Papilloma RNAs were analyzed individually. Human PTEN-specific primers, which generate a 671-bp product, were described previously (29) . PTEN cDNA from 150 ng of total RNA was amplified with AmpliTaq Gold (PE Biosystems, Foster City, CA), starting at 95°C for 9 min, and then 43 cycles at 94°C for 1 min, 60°C for 1.5 min, followed by a final extension at 60°C for 10 min. The integrity of each RNA sample and equivalence of RNA amount were confirmed by using primers to 36B4 cDNA, a housekeeping gene encoding the human acidic ribosomal phosphoprotein PO (30) . The amount of template cDNA was titrated to permit relative quantitation of PTEN mRNA, with 1:10 and 1:25 dilutions of the cDNA used for 36B4 amplification. PCR products were detected by ethidium bromide staining of agarose gels. Negative controls included amplification of RNA that had not been reverse transcribed to rule out possible product from residual DNA and omission of template from the reaction.

Immunohistochemistry
Serial frozen sections were fixed in cold acetone and incubated with either anti-PTEN or anti-PTEN preincubated at 4°C overnight with PTEN peptide (Santa Cruz Biotechnology) to block the antigen recognition site, used as a negative control. Detection was performed with the Vectastain ABC kit (Vector Laboratories, Burlingame, CA), using diaminobenzidine as substrate. Sections were then lightly counterstained with hematoxylin.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PI 3-K Activity Is Elevated in Laryngeal Papillomas
We reported recently that laryngeal papilloma tissues contain high levels of tyrosine-phosphorylated proteins and increased activation of the EGF receptor/MAP kinase pathway (4) . We therefore asked whether PI 3-Kinase activity was also elevated in the papilloma tissues. Fig. 2ACitation shows one such assay. The levels of PI 3-K activity were elevated in the papilloma extracts (mean, 5.1 ± 2.1 fold higher than the normal tissue extracts). This difference was significant (P = 0.025), supporting the hypothesis that multiple signal transduction pathways may be elevated in laryngeal papillomas.



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  		Fig. 2. PI 3-K activity and protein abundance in extracts from laryngeal papillomas and normal laryngeal tissues. A, TLC of products from a lipid kinase assay. The position of PtdIns-3-phosphate (PIP) is shown. Ori, origin. B, Western blot showing protein levels in extracts. Actin levels on the same blot were used to correct for any difference in protein content in the extracts. PI3K, PI 3-K.

 
The PI 3-K assays were done using equal amounts of protein from the two types of tissues, not by adding equal amounts of total PI 3-K. To determine whether total PI 3-K levels were elevated in papilloma cells, Western blot analysis of both types of tissues was done (Fig. 2B)Citation . There was no elevation of total PI 3-K in the papilloma tissues. In fact, the levels were somewhat lower in papillomas relative to normal tissues (0.45 ± 0.13, compared with 1.0 ± 0.6), although this difference was not significant. Clearly, the fraction of PI 3-K that was activated was greater in papillomas than in uninfected tissues, consistent with the high level of tyrosine phosphorylation in these benign tumors.

Decreased Activation of Akt in Laryngeal Papillomas
Akt is a downstream mediator of PI 3-K, activated by PtdIns (3,4,5)P3 (8) . We therefore analyzed the relative levels of activated Akt by Western blot, using antibodies specific for either serine- or threonine-phosphorylated Akt and an antibody that recognized all Akt forms (Fig. 3)Citation . The amount of Ser-473-P Akt in papilloma tissue relative to normal tissue was 0.52 ± 0.06 to 1.0 ± 0.02. This difference was marginally not significant (P = 0.06). However, the relative amount of Thr-308-P Akt was significantly reduced (P = 0.01), with a papilloma:normal tissue ratio of 0.2 ± 0.06 to 1.0 ± 0.2. These results were clearly discordant with the enhanced PI 3-K activity in papilloma tissues.



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  		Fig. 3. Western blots of phosphorylated and total Akt in extracts from papillomas and normal tissues. Antibodies specific for Akt phosphorylated on serine 473, on threonine 308, and total Akt were used sequentially on the same blot, followed by anti-actin to correct for differences in protein content in the extracts.

 
The fraction of Akt that is active in any given tissue is a function of both the amount of phosphorylated Akt and the total Akt level. There was slightly more total Akt in papillomas (1.4 ± 0.2 fold higher than normal tissue). Although this difference was not significant, it further suggested that activation of Akt in papilloma tissues was suppressed. When the ratios of phosphorylated Akt to total Akt were calculated for papilloma tissues relative to normal tissues, there was a clear reduction in the fraction of Akt that was phosphorylated in papillomas (Fig. 4)Citation . The differences were highly significant for both phosphorylation sites (P = 0.02 for serine phosphorylation and P = 0.01 for threonine phosphorylation).



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  		Fig. 4. Fraction of total Akt that was phosphorylated on either serine 473 or threonine 308 in papilloma tissues, relative to levels in normal laryngeal epithelium. The low relative abundance of either phosphorylated form in papillomas was significantly different from normal tissue (P = 0.02 and 0.01, respectively). Bars, SE.

 
PTEN Is Overexpressed in Laryngeal Papillomas
The inconsistency between elevated PI 3-K activity and reduced Akt phosphorylation suggested that signaling downstream of PI 3-K might be blocked. Ramaswamy et al. (25) reported recently that PTEN, the phosphatase that dephosphorylates PtdIns (3,4,5)P3, is linked to inhibition of the PI 3-K/Akt pathway. We therefore analyzed three normal samples (each a pool of two separate biopsies from different patients) and 10 papillomas from different patients for PTEN protein levels. Fig. 5ACitation shows one such assay. PTEN was consistently overexpressed in papillomas. When the PTEN signals were normalized to actin levels (Fig. 5B)Citation , the PTEN levels were 3.3 ± 0.6 fold higher in papilloma tissue than in normal tissues, which was statistically significant (P = 0.01). These results provided an explanation for the lower levels of phosphorylation of Akt in papilloma tissues.



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  		Fig. 5. Expression of PTEN in normal and papilloma tissues. A, extracts from both types of tissues were analyzed by Western blot. B, densitometric analysis of PTEN levels in papilloma tissues compared with normal laryngeal tissues, corrected for differences in protein loading with antibody to actin. Bars, SE.

 
PTEN RNA Levels Are Increased in Papilloma Cells
PTEN levels are down-regulated in several ways in malignancies, including reduction in either transcription or translation (31) . We therefore asked whether the elevation of PTEN protein in papillomas reflected differences in steady-state mRNA level. RT-PCR results were consistent with an increase in PTEN message in papilloma cells (Fig. 6)Citation . Each of the eight papilloma samples generated product that could be visualized on an ethidium bromide-stained gel, albeit at different levels. However, the PCR product from eight pooled normal tissue RNAs was below detectable levels. This did not reflect insufficient RNA, because the 36B4 product level was equal to the papillomas. Reamplification of the initial PCR products did demonstrate the presence of PTEN transcripts in normal tissue RNA, but at very low abundance (data not shown). Two additional pooled normal samples were equally low in abundance, only visible on reamplification (data not shown). The negative control, amplifying RNA without reverse transcription, showed no DNA contamination and confirmed that we were measuring differences in PTEN RNA abundance. Thus, we conclude that at least part of the increase in PTEN protein in laryngeal papillomas is attributable to an increase in PTEN mRNA levels.



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  		Fig. 6. RT-PCR analysis of PTEN mRNA in normal laryngeal epithelium and papillomas. M, molecular weight marker. N, amplified cDNA from pooled normal tissues. Papillomas, amplified cDNA from seven separate papilloma samples. Con, control amplification of RNA from papillomas, omitting reverse transcription. H2O, control amplification with no sample added. Amplification of 36B4 RNA was used to assure equivalent amounts of cDNA in each amplification reaction.

 
PTEN Distribution Is Different in Normal and Papilloma Tissues
We then asked whether all cells within a papilloma overexpressed PTEN, or whether the expression varied within the tissue (Fig. 7)Citation . Normal tissue showed faint staining for PTEN in the cytoplasm and slightly stronger in the suprabasal layers of the tissue (Fig. 7, A and B)Citation , suggesting that PTEN might be regulated coordinately with differentiation. In contrast, papilloma tissues showed strong staining throughout the epithelium (Fig. 7, D and E)Citation . The stronger staining was consistent with our Western blot results. Most striking was the presence of PTEN in the nuclei of many of the papilloma cells, as well as in the cytoplasm. Recently, Sano et al. (32) reported PTEN nuclear staining in a subset of neurons and endothelial cells, ruling out the likelihood that nuclear staining in papillomas was artifactual. These results suggest that PTEN is not only overexpressed in papillomas, but that it might also have additional effects on regulation of growth and differentiation beyond reducing Akt activation. We confirmed that the staining patterns were specific for PTEN, because preabsorption of antibody with PTEN peptide nearly abolished all staining (Fig. 7, C and F)Citation .



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  		Fig. 7. Immunohistochemical detection of PTEN in tissues. A and B, normal laryngeal tissue, showing low levels of cytoplasmic PTEN. D and E, papilloma tissue, showing both cytoplasmic and nuclear PTEN staining throughout the epithelium. Higher magnification of papilloma tissue (E) clearly shows nuclear PTEN in a fraction of cells in the basal layer. C and F, negative controls for normal and papilloma tissue, with antibody preadsorbed with PTEN peptide prior to staining papilloma tissue. Tissues were lightly counterstained with hematoxylin after immunohistochemical detection of PTEN. Bar, 100 µm.

 
Correlation between PTEN and PI 3-K Levels
There was variation in PTEN levels in the different tissue samples, with papilloma tissues ranging from 1.9 to 5.8 fold higher than the normal tissue. We had previously noted some variation in PI 3-K levels. We therefore analyzed the relationship between PI 3-K levels and PTEN levels. There was a correlation (r = 0.98) between PI 3-K and PTEN protein level in papillomas (Fig. 8)Citation . We were also able to analyze four samples for correlation between relative PI 3-K activity and PTEN level. This analysis, even with the small sample size, showed a similar correlation, with r = 0.93 (data not shown). Because of the need to pool multiple normal tissues for analysis, we were not able to do the same correlation analysis for the normal tissues. We do not yet know the mechanism for the coordinate increase in expression of both proteins or the apparent relationship between PI 3-K activity and PTEN abundance.



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  		Fig. 8. Correlation of abundance of PI 3-K and PTEN in papilloma tissues. Six separate papilloma biopsies were analyzed by Western blots, and protein levels were quantitated by densitometry and normalized to actin on the same blots. There was a clear positive correlation (r = 0.98) between PI 3-K and PTEN levels in these tissues.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study analyzed the abundance and distribution of the tumor suppressor PTEN in laryngeal papilloma tissues and the PI 3-K/Akt pathway it regulates. We found that both PTEN and the signaling pathway were altered when compared with normal laryngeal epithelium. PTEN protein was consistently overexpressed in papillomas, with differences in both abundance and distribution.

The suprabasal distribution of PTEN in normal tissue is consistent with the role of PTEN as a negative regulator of the PI 3-K/Akt pathway. Activation of that pathway is important in induction of cell proliferation, and the upper layers of the epithelium are terminally differentiated and not proliferating. We reported previously that papilloma tissues show a lower percentage of proliferating basal cells than normal tissue (2) . Similarly, cultured papilloma cells proliferate more slowly than uninfected cells (3) . The presence of elevated levels of PTEN in basal cells could explain the lower proliferative rate of these benign tumors.

The identification of endogenous PTEN substrates has been essential for a better understanding of its intracellular role in papilloma tissues. PTEN is a phosphatase, capable of dephosphorylating both threonine- and tyrosine-phosphorylated substrates and also targeting the lipid second messenger PtdIns (3,4,5)P3 in vitro (21 , 22) and in vivo (17) . PtdIns (3,4,5)P3 is the product of PI 3-K. PI 3-K can be activated by many extracellular stimuli, including EGF, platelet-derived growth factor, and insulin, phosphorylating the hydroxyl group at position 3 on the inositol ring of phosphoinositides. Its lipid products bind to multiple signaling molecules that initiate secondary signaling cascades that regulate many diverse physiological functions, such as trafficking, adhesion, actin arrangement, cell growth, and cell survival (6) . Our data show that PI 3-K activity was 2.5–7.8 times higher in papillomas. This elevation in activity could be attributable to the overexpression of the EGF receptor and increased activity of the EGF receptor/MAP kinase pathway seen in papillomas (3 , 4) . Although the small size of the biopsy samples precluded assaying both pathways in the same samples, we have consistently seen over overexpression of the EGF receptor and constitutive activation of MAP kinase in papilloma tissues.

We do not know whether the elevated PTEN levels in papillomas are directly caused by HPV infection. HPV proteins can alter transcription of multiple cellular proteins (33, 34, 35) and could possibly enhance PTEN transcription by elevating amounts of a limiting transcription factor. Conversely, increased PTEN could reflect the hyperplastic state of the papilloma tissues. The fact that PTEN and PI 3-K levels correlated in papillomas suggests a potential coordinated regulation of these proteins, thus maintaining a type of homeostatic balance of proteins within the PI 3-K/Akt pathway. Alternatively, PTEN levels could actually be regulated by PI 3-K activity, not simply protein level, because the limited number of papilloma samples we were able to analyze suggested a correlation between PI 3-K activity and PTEN abundance. Because the PI 3-K in papillomas was highly activated, increase in protein abundance would result in increased active protein. These possibilities must be studied further to determine whether one or more are correct.

Akt is a crucial downstream target for PI 3-K activity. Both PtdIns (3,4,5)P3 and PtdIns (3,4)P2 take part in Akt activation, with PtdIns (3,4,5)P3 playing a dual role by binding to the PH domain at the NH2-terminal end of Akt to allow phosphorylation by the upstream kinase and also directly activating the upstream kinase (8 , 36) . Phosphorylation of Akt at Thr-308 and Ser-473 has been proposed to be essential for its activation (37 , 38) . Our results with phospho-specific Akt antibodies showed that both of these sites were underphosphorylated in papillomas compared with normal tissue. Akt is a serine/threonine kinase that phosphorylates multiple substrates, resulting in a variety of biological effects, including glycogen synthesis, GLUT-4 translocation, cell cycle regulation, differentiation, and suppression of apoptosis (reviewed in Ref. 39 ). Thus, reduction in active Akt by PTEN could have multiple effects in papilloma cells.

Overexpression of PTEN could affect regulation of papilloma cells by other pathways as well as by reducing activated AKT. The NH2 terminus of PTEN is homologous to the cytoskeletal protein tensin. A role for PTEN as a molecule regulating the connection between the cytoskeleton and intracellular signaling pathways has been proposed (11 , 12 , 21) . Studies with glioblastoma cells reported that PTEN inhibited cell migration, spreading, and focal adhesions by inhibiting integrin and growth factor-mediated MAP kinase signaling pathways by targeting focal adhesion kinase, Shc, and its interaction with the adapter protein growth factor receptor binding protein 2, without affecting c-Jun NH2-terminal kinase or Akt signaling, (40 , 41) . We reported previously (42) that the actin cytoskeleton structure was altered in papilloma cells, suggesting that multiple different signaling processes regulated by PTEN are altered in these cells. The presence of PTEN in the nuclei of papilloma cells (Fig. 7)Citation and endothelial cells and neurons (32) suggests that PTEN may also have a nuclear function, at least in some tissues. We are now beginning to investigate the interactive role of PTEN with these various pathways and the regulation of PTEN expression in HPV-infected cells, using cells derived from laryngeal papillomas.


   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 Supported by Grant P50DC 00203 from the National Institute on Deafness and Other Communication Disorders and a grant from the Helen and Irving Schneider Family Foundation. Back

2 To whom requests for reprints should be addressed, at Department of Otolaryngology, Long Island Jewish Medical Center, 270–05 76th Avenue, New Hyde Park, NY 11040. Phone: (718) 470-7553; Fax: (718) 347-2320; E-mail: bsteinbe{at}lij.edu Back

3 The abbreviations used are: HPV, human papillomavirus; EGF, epidermal growth factor; PI 3-K, phosphatidylinositol 3-kinase; PtdIns (3,4,5)P3, phosphoinositol 3,4,5-trisphosphate; PtdIns (3,4)P2, phosphoinositol 3,4-bisphosphate; MAP, mitogen-activated protein; RT-PCR, reverse transcription-PCR. Back

Received 6/29/99. Accepted 12/30/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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