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Molecular Biology and Genetics |
Departments of Biochemistry [A. S., K. H.], Dermatology [T. I., M. M.], and Anatomy [H. S.], Akita University School of Medicine, Akita 010-8543, Japan; Department of Molecular Cell Biology, Research Institute for Microbial Disease [M. O., T. N.], Department of Dermatology [S. I.], and Department of Social and Environmental Medicine [N. K., J. T.], Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan; Department of Pharmacology, Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan [T. S.]; and Advanced Medical Discovery Institute, University of Toronto, Toronto, Ontario, M5G 2C1 Canada [T. W. M.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PTEN is a dual protein and lipid phosphatase (7 , 8) . PTEN's major substrate is PIP36 , a second messenger molecule generated by PI3'K activated in response to numerous growth factors such as EGF (9) , hepatocyte growth factor (10) , fibroblast growth factors (11) , and IGF-1 (12) . PIP3 in turn activates the serine-threonine kinase Akt/PKB, which is involved in antiapoptosis, proliferation and oncogenesis. Thus, by dephosphorylating PIP3, PTEN negatively regulates cell survival.
The epidermis of the skin is a keratinized and stratified squamous epithelium composed mainly of keratinocytes, cells whose proliferation and differentiation must be tightly regulated and coordinated (13, 14, 15) . Keratinocytes first attach to the basal membrane of the epidermis as undifferentiated precursor cells. These precursors migrate toward the surface of the epidermis and subsequently form its outermost layer (16) . Both the normal development of keratinocytes and the onset of tumors involving these cells require complex proliferative/apoptotic events. The biochemical pathways underlying many of these events have been delineated through the use of transgenic and knockout mice (17) . Hair follicle morphogenesis and hair remodeling also depend on a balance of proliferative and apoptotic events. However, the molecular mechanisms underlying hair morphogenesis are not well understood. A normal hair follicle cyclically traverses three phases of organ growth: hair shaft formation (anagen); organ involution (catagen); and relative quiescence (telogen). Hair follicle morphogenesis continues until the first hair cycle after birth (18) , after which periodical hair remodeling occurs throughout the life of the animal.
The importance of EGFR, IGF-1, and Ha-ras signaling in morphogenesis and carcinogenesis is clearly evident in skin (19, 20, 21)
. Activation of the transforming growth factor 
/EGFR pathway (22, 23, 24)
, IGF-1 (20
, 25)
, or v-Ha-ras (26)
in the epidermis of transgenic mice leads to skin hyperplasia, hyperkeratosis, and tumor formation. Transgenic animals overexpressing IGF-1 also show accelerated hair growth (27)
and mice lacking IGF-1R exhibit hypoplastic skin (28)
. Because each of EGFR, IGF-R, and Ras triggers PI3'K signaling and PTEN regulates this pathway, we felt it likely that the PTEN gene would play an important role in skin development and oncogenesis.
In previous work, we used gene targeting to create a complete null mutation of Pten in mice (Pten-/- mice; Ref. 29 ). However, total deficiency for Pten proved to be embryonic lethal in the very early stages of development, precluding the functional analysis of Pten in various organs. We subsequently showed in Pten-/- MEFs that Akt/PKB is a key player downstream of Pten and that Akt/PKB is hyperactivated in the absence of Pten (30) . Mice heterozygous for the Pten null mutation (Pten+/- mice) were frequently found to develop lymphoid hyperplasia and endometrial, prostatic, and breast cancers. With respect to skin anomalies, focal hyperkeratosis was reported in a proportion of Pten+/- mice (29 , 31 , 32) . Because Pten is expressed in keratinocytes and decreased Pten activity contributes to the malignant conversion stage in chemically induced mouse skin cancers (33) , we decided to investigate the role of Pten in skin development and oncogenesis using the Cre-loxP system.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PCR Analysis of Pten Genotypes.
Genomic DNA from mouse tails was isolated and amplified by PCR following a published protocol (29)
. Sense primer [5'-GTCACCAGGATGCTTCTGAC-3'] and antisense primer [5'-GAAACGGCCTTAACGACGTAG-3'] were used to detect the floxed Pten allele; sense primer [5'-GTCACCAGGATGCTTCTGAC-3'] and antisense primer [5'-GTGACATCAACATGCAACACTG-3'] were used to detect the wild-type Pten allele; and sense primer [5'-ATGCCAATGCCCCCTCAGTTCCT-3'] and antisense primer [5'-TGCCCCTTTTTATCCCTTCCAGA-3'] were used to detect the Keratin5Cre transgene. Amplified fragments of 512 bp, 413 bp and 
300 bp, respectively, were obtained.
Preparation and in Vitro Culture of Keratinocytes.
Full-thickness skin taken from newborn mice was treated with 250 units/ml dispase (Godoshusei, Tokyo, Japan) overnight at 4°C. The epidermis was peeled off from the dermis and trypsinized to prepare single cells that were suspended in Defined Keratinocyte-SFM medium (Life Technologies, Inc.) with supplements and 2% FCS. Cells were seeded at 1 x 107 cells/10-cm dish (for proliferation assays) or 5 x 105 cells/well of a 6-well plate (for apoptosis assays), which had been precoated with collagen type I (Iwaki Glass, Tokyo, Japan). Cells were cultured at 37°C in 5% CO2 for 5 h until cells had attached and spread. Unattached cells were removed by washing with PBS, and the attached cells were additionally cultured in fresh medium without FCS for 24 h before proliferation and apoptosis assays (see below).
Southern and Western Blots.
Genomic Southern blots of DNA obtained from keratinocytes were performed using a previously described probe and protocol (29)
. For Western blots, keratinocytes (5 x 106/10 ml) were either left untreated or stimulated for 10 min or 16 h with 30 µg/ml EGF (Biomedical Technologies). Total cell lysates were prepared, and 15 µg of lysate aliquots were analyzed by Western blotting as described previously (30)
. Antibodies directed against the NH2 terminus of Pten and antiactin were from Santa Cruz, whereas antiphospho-Akt/PKB (Ser473), antitotal Akt/PKB, antiphospho-MAPK (p42/p44), and antitotal MAPK(p42/p44; Thr202/Tyr204) antibodies were from New England Biolabs.
Histological Analysis and Immunohistochemistry.
For histological analysis, dorsal skin samples and tumors were fixed in formalin and embedded in paraffin before sectioning according to standard protocols. Sections of 5 µm were cut and stained with H&E. For immunohistochemical staining, freshly dissected skin samples were covered with Tissue-Tek OCT compound (Miles, Inc.) and quickly frozen in liquid nitrogen. Frozen sections (5-µm thick) were fixed in ice-cold acetone. Immunofluorescent staining was performed using anti-Keratin5, anti-Keratin6, anti-Keratin10 (all from BabCo) and anti-Ki-67 (Novocastra) as primary antibodies, and fluorescein-conjugated donkey antirabbit or antimouse IgG (Jackson ImmunoResearch) as secondary antibodies.
Proliferation Assay.
Keratinocytes cultured in a 10 cm dish were replated in round-bottomed 96-well plates (1 x 104/well) in Defined Keratinocyte-SFM medium without supplements and cultured in the presence of EGF (30 µg/ml) for 72 h. Cells were pulsed with 1µCi [3H]thymidine (Amersham) for another 16 h before harvesting by trypsinization. Incorporated radioactivity was measured using a ß-scintillation counter.
Apoptosis Assay.
Keratinocytes cultured in 6-well plates coated with type I collagen were treated with either UV- or 
-irradiation at the doses indicated in Fig. 4
. One day after treatment, cell viability was determined by staining with 7-amino-actinomycin D (Sigma) as described previously (35)
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Tumor Induction.
Mice (6–7 weeks old) were shaved on their backs 2 days before tumor induction. To induce tumors, the shaved dorsal skin of mice was treated topically with 25 nmol of DMBA (Sigma) in acetone. After 1 week, each animal received subsequent topical treatments of 10 nmol of TPA (Sigma) in acetone twice weekly for 4 weeks. Control mice were treated with acetone only.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Briefly, Keratin5Cre transgenic (k5CreTg) mice (34)
were crossed to mice homozygous for the floxed Pten allele (Ptenflox/flox mice) to generate Keratin5CrePtenflox/flox mice (k5Ptenflox/flox mice). The Keratin5 promotor directs gene expression from day 13.5 postcoitum (d.p.c.) in the basal layer of epidermal and follicular keratinocytes so that expression of a floxed gene under the control of this promoter is disrupted throughout the epidermis and the outer root sheaths of hair follicles (36
, 37) . In mice bearing a floxed allele of the Pig-a gene, expression of Cre using the Keratin5 promotor induced the disruption of Pig-a not only in the vast majority of keratinocytes but also in the esophagus and stomach (34)
. These results are consistent with a previous report in which the Keratin5 gene was found to be expressed in esophagus and stomach as well as in epidermis (36
, 38)
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allele (Fig. 1B)
. The deletion of Pten was confirmed at the protein level by Western blotting using antibody recognizing the NH2 terminus of Pten (Fig. 1C)
. Extracts of keratinocytes from k5Ptenflox/flox mice showed a major reduction in Pten protein, whereas keratinocytes of k5Ptenflox/+ mice showed only a minor decrease.
Lethality in the Lactation Period and Gross Skin Abnormalities in k5Ptenflox/flox Mice.
We monitored the growth and health of k5Pten+/+, k5Ptenflox/+, and k5Ptenflox/flox mice for several months. All k5Ptenflox/flox mice could be identified without genotyping because the skin of mutant animals 3 days of age or older was wrinkled because of hyperplasia (Fig. 2A)
. In addition, most k5Ptenflox/flox mice were significantly smaller than their k5Pten+/+ littermates from 3–5 days of age on (Fig. 2B)
, and their hair coats were abnormally ruffled and shaggy (Fig. 2C)
. k5Ptenflox/flox mice had hyperplastic noses and lips (Fig. 2, D and E)
and slightly enlarged ears (data not shown), giving the mutants the appearance of a wrinkled bear (Fig. 2, D and E)
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6 days after birth because of malnutrition during the lactation period (Fig. 2F)
. However, animals that survived this period and made it to the 2-month mark had normal life spans without severe malnutrition. Overall, these mutant mice looked similar to k5Pten+/+ and k5Ptenflox/+ mice, although their skin and hair abnormalities were still obvious. Gross histological examination of k5Ptenflox/flox pups revealed marked hyperkeratosis in the esophagus (Fig. 2, G and H)
. Milk was easily identified in the stomachs of wild-type mice but only faintly visible in the stomachs of k5Ptenflox/flox pups. It is highly likely that esophageal dysfunction because of hyperkeratosis caused the dysphagia, perhaps accounting for the small body size of the mutant mice and their deaths because of malnutrition. Examination of the hair of k5Ptenflox/flox mice showed that the cuticles, which normally cover the hair shaft, were almost completely detached from the hair shafts (Fig. 2, I and J)
. In contrast, k5Ptenflox/+ mice were indistinguishable from k5Pten+/+ mice in both gross appearance and histological findings (data not shown).
Precocious Hair Follicle Morphogenesis in Pten-deficient Epidermis.
To determine the cause of the gross abnormalities of k5Ptenflox/flox skin, we analyzed the microscopic architecture of skin in tissue specimens taken at various time points after birth. Histological comparison with wild-type mice revealed that hyperkeratosis, hypergranulosis, and epidermal hyperplasia were present in Pten-deficient newborns (Fig. 3
; day 0, top and middle panels). Moreover, the high density of the hair follicles in the mutant caused a reduction of interfollicular epidermis (Fig. 3
; day 0, top panel and arrow in bottom panel; day 7, top panel), and sebaceous glands showed advanced development in k5Ptenflox/flox skin (Fig. 3
; day 7, arrows in top and bottom panels). At 10 days after birth, the epithelia of k5Ptenflox/flox mice became papillomatous. Immunostaining using antibodies specific for Keratin5 and Keratin10 showed no obvious difference between k5Pten+/+ and k5Ptenflox/flox mice, except that ectopic staining by anti-Keratin10 was observed in the infundibular epidermis of the mutants (Fig. 3
; day 10, middle panel).
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; day10, top left panel). In contrast, the skin of k5Ptenflox/flox mice was much thicker than that of wild-type animals on day 7 but significantly thinner on day 10, suggesting that the mutants had already regressed into the catagen phase (Fig. 3
; day 7, top panel and day 10, top panel). In wild-type mice, relative quiescence (telogen) was observed at day 19 (Fig. 3
; day 19, top and middle left panels). However, on day 19 in k5Ptenflox/flox mice, the epithelia were papillomatous and thicker than in the wild-type, and well-developed sebaceous glands were present (Fig. 3
; day 19, top and middle right panels). Immunostaining using anti-Ki-67 antibody indicated that on day 19, basal cells in the mutant were proliferating more than those of wild-type littermates (Fig. 3
, day 19, bottom panels). Moreover, the proliferation of basal cells in the mutant on day 19 was greater than that of mutant basal cells on day 15 (data not shown), consistent with accelerated entrance into the next anagen phase. Although follicular remodeling was not impaired in the absence of Pten (data not shown), most epithelia in the mutant mice continued to show hyperkeratosis, hyperplasticity (especially the granular layer), and well-developed sebaceous glands even at 3 months of age (Fig. 3
, 3 months, right panels). The hair was consistently abnormal (data not shown). The presence of hyperplastic epithelia and the acceleration of skin morphogenesis in k5Ptenflox/flox mice indicate that Pten is indispensable for the normal development of skin.
Skin Tumor Formation in k5Ptenflox/+ and k5Ptenflox/flox Mice.
To further characterize the effects of Pten gene disruption on mouse epidermis, 32 wild-type, 31 heterozygous mutant, and 11 homozygous mutant mice that survived over 2 months after birth were monitored for spontaneous tumorigenesis. As shown in Fig. 4A
, no skin tumors were detected on k5Pten+/+ mice during the 8.5 month observation period. In contrast, spontaneous tumors developed in 23% of k5Ptenflox/+ mice and, most surprisingly, in 100% of k5Ptenflox/flox mice. Most of these spontaneous tumors were squamous papillomas that occurred on the face and palms of the front paws (Fig. 4, B and F)
. However, many of these papillomas went on to develop into squamous cell carcinomas with nuclear atypia and increased mitosis. These latter tumors were capable of invading the dermis (Fig. 4, C and G
). In addition to papillomas and squamous cell carcinomas, we observed sebaceous carcinomas characterized by an obvious sebaceous gland-like structure (in 11% of tumor-bearing mice; Fig. 4, D and H
) and adenocarcinomas of the sweat gland (in 11% of tumor-bearing mice; Fig. 4, E and I
). To confirm that the tumors arising in k5Ptenflox/+ mice arose because of a LOH, PCR assays on tumor DNA were carried out. The loss of the wild-type Pten allele was observed in 3 of 3 squamous carcinomas obtained from k5Ptenflox/+ mice (Fig. 4J)
.
To examine induced carcinogenesis in Pten-deficient skin, k5Pten+/+, k5Ptenflox/+ and k5Ptenflox/flox mice of 6–7 weeks of age (n = 8/group) were treated with either DMBA plus TPA or TPA alone. DMBA initiates skin tumorigenesis, and TPA promotes growth of an established skin tumor. Surprisingly, 100% of k5Ptenflox/flox mice treated with DMBA followed by TPA developed 5–15 skin papillomas in the treated area (only) within 5 weeks of the initial DMBA treatment (Fig. 4, K–M)
. In contrast, no tumors were present on the skin of either k5Ptenflox/+ or wild-type mice treated in the same fashion for the same duration. TPA treatment alone failed to induce tumorigenesis in any of the three groups during the 6-week observation period (data not shown). This result was not unexpected because spontaneous tumors did not appear in k5Ptenflox/flox mice until 3.5 months of age. These observations indicate that Pten functions as a tumor suppressor for both spontaneous and induced skin tumors in mice.
Hyperproliferation and Resistance to Apoptosis in k5Ptenflox/flox Keratinocytes.
We next investigated whether the accelerated skin morphogenesis and oncogenesis in k5Ptenflox/flox mice were associated with a defect in keratinocyte proliferation or apoptosis. Keratinocytes were stimulated in vitro with EGF to induce proliferation or subjected to UV- or -irradiation to induce apoptosis. As shown in Fig. 5A
, Pten-deficient keratinocytes showed enhanced proliferation in response to EGF. In addition, the mutant keratinocytes were more resistant than wild-type keratinocytes to apoptosis induced by either high dose UV- or -irradiation (Fig. 5B)
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and MAPK (Fig. 6B)
was significantly elevated in k5Ptenflox/flox keratinocytes compared with k5Pten+/+ or k5Ptenflox/+ keratinocytes. Thus, in keratinocytes, as in T cells, Akt/PKB and MAPK activation is subject to negative regulation by Pten.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hereditary heterozygous mutation of PTEN in humans is associated with Cowden’s disease (2) , a disorder characterized by the onset of multiple hamartoma in various tissues. These hamartoma frequently develop into malignancies such as breast and thyroid cancers (5) . The characteristic clinical features of Cowden’s disease include skin abnormalities such as trichilemmomas in the face papules, papillomatosis in the mucosal and cutaneous tissues, and hyperkeratosis in the acral region of the skin (4) . At the molecular level, skin lesions in Cowden’s disease patients have shown evidence of LOH for PTEN (42) . In this study, we observed significant similarities to Cowden’s disease symptoms in k5Ptenflox/flox mice. The mutant animals exhibited hyperkeratosis and spontaneous tumors such as papillomas and cutaneous squamous cell carcinomas. Although trichilemmomas, a type of tumor of the epidermal appendages, did not occur in k5Ptenflox/flox mice, the mutants frequently displayed other types of epidermal appendage tumors. We therefore believe our k5Ptenflox/flox mice represent not only a reasonable model of the skin lesions characteristic of Cowden’s disease but also a suitable model of skin carcinogenesis in general.
The phenotypes observed in k5Ptenflox/flox mice are reminiscent of those of Ha-ras (26)
, transforming growth factor (22)
, sons of sevenless (SOS) (24)
, and IGF-1 (25
, 27)
transgenic mice. The similarities between k5Ptenflox/flox mice and transgenic mice expressing IGF-1 under the control of the Keratin5 promotor (k5IGF-1 Tg) are particularly striking. k5IGF-1 Tg mice are small in size as neonates and have wrinkled, thick skin because of hyperkeratosis and epithelial hyperplasia. The hair of these mutants is ruffled and shaggy, and spontaneous tumors such as papillomas and squamous cell carcinomas develop with age. Cells of these mutants show evidence of apoptotic resistance and activation of PI3'K and its downstream mediators Akt/PKB and ERK (25)
. These observations, taken together with the results of our study, suggest that signal transduction downstream of many molecules affecting cell growth/death involves common Akt/PKB- and ERK-mediated pathways. Disruption of these pathways may account for the phenotypes of k5Ptenflox/flox mice.
One of the most striking phenotypes in k5Ptenflox/flox mice is the precocious morphogenesis of skin. The wnt/ß-catenin/Lef-1 pathway has been reported to be very important for the acceleration of developmental morphogenesis in the skin (43) . In the embryonic skin of 14.5 d.p.c mice, pilosebaceous units develop from epidermal down-growths under the influence of specific mesenchymal cell condensations. These condensations supply permissive and instructive signals that govern the position and type of hairs and other appendages developed (reviewed by Refs. 38 , 44 ). The expression of patterning genes such as those in the wnt/ß-catenin/Lef-1 signaling pathway are thought to regulate these signals (43) . Perhaps significantly, Pten has been shown to negatively regulate the ß-catenin/Lef-1 pathway by inhibiting the nuclear accumulation of ß-catenin and activation of Lef-1 in a prostatic cell line (45) . However, in our hands, no definite difference in the subcellular distribution of ß-catenin in k5Ptenflox/flox cells was observed (data not shown). Both k5Ptenflox/flox mice and k5IGF-1 Tg mice show accelerated hair growth at day 5 (27) , indicating that common molecules downstream of PKB/Akt in addition to ß-catenin, or molecules downstream of ERK, may account for the accelerated skin morphogenesis in these mice.
Several lines of evidence suggest that Akt/PKB may be a key molecule regulating the onset of skin carcinogenesis in mice. The transplantation of keratinocytes overexpressing Akt/PKB results in highly aggressive skin tumors characterized by increased invasiveness and altered differentiation (33) . In addition, Akt/PKB activation is one of the first events in the chemical induction of skin tumors (33) . Finally, the onset of skin tumor formation in mice requires EGFR and Akt/PKB signaling in addition to SOS/Ras/ERK signaling (24) . Indeed, MAPK/ERK has been reported to act in synergy with the PI3'K pathway to stimulate CycD1 transcription in NIH3T3 cells (46) . Thus, the onset of tumors in k5Ptenflox/flox mice may be caused primarily by cellular hyperproliferation and/or apoptotic resistance induced by PI3'K and Akt/PKB hyperactivation, with a contribution by deregulated ERK activation.
k5IGF-1 Tg mice are reportedly hypersensitive to both DMBA and TPA. However, analysis of papillomas observed in k5IGF-1 Tg mice treated with TPA alone revealed that essentially all of the tumors had Ha-ras mutations (25) . This finding suggests that enhanced IGF-1 signaling does not substitute for initiation but rather enhances the ability of TPA to select for already existing transformed cells (25) . Because both Pten deficiency and IGF-1 overexpression result in Akt/PKB and subsequent MAPK activation, it is conceivable that the same mechanism underlies the phenotypes in k5Ptenflox/flox mice. Prolonged observation of k5Ptenflox/flox mice subjected to treatment with either DMBA or TPA alone may help to define the role of Pten in chemically induced carcinogenesis.
In this study, both k5Ptenflox/flox and k5Ptenflox/+ mice developed spontaneous skin tumors at high frequency. Fully 100% of k5Ptenflox/flox mice and 23% of k5Ptenflox/+ mice acquired papillomas and/or squamous cell carcinomas during the 8.5-month observation period. The result for the k5Ptenflox/+ mice is particularly surprising in light of the fact that skin tumors are observed in Pten+/- mice at a frequency of <5%. We speculate that the tissue-specificity of the Pten mutation examined in this study may in some way account for this discrepancy. For example, an immunosurveillance mechanism able to block skin tumor formation may be triggered in response to global (only) inactivation of Pten in heterozygotes. Such a mechanism would be in line with our previous observations of mice with a T-cell-specific Pten deficiency. These mutants experience an accumulation of T cells that produce increased levels of Th1 and Th2 cytokines (35) .
Our study is the first reported in vivo analysis of Pten function in mouse skin. We clearly demonstrate that Pten is an essential regulator of normal homeostasis and oncogenesis in the organ. Our results suggest that inhibition of the PIP3-Akt/PKB pathway may be an attractive therapeutic target for the treatment of skin malignancies. Studies to examine PTEN expression in human keratinocyte malignancies are ongoing.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by grants from the Ministry of Education, Science, Sports and Culture, Japan; Japan Medical Association; the Naito Foundation; the ONO Medical Research Foundation; the Japanese Society for Promotion of Science (JSPS-RAT98L01101); the Sumitomo Foundation; and the Public Trust Haraguchi Memorial Cancer Research Foundation. 
2 These individuals contributed equally as second authors.
3 These individuals contributed equally as last authors.
4 To whom requests for reprints should be addressed, at Advanced Medical Discovery Institute, 620 University, Suite 706, Toronto, Ontario, M5G 2C1 Canada. Phone: (416) 204-2236; Fax: (416) 204-5300; E-mail: tmak{at}uhnres.utoronto.ca
5 To whom requests for reprints should be addressed, at Department of Molecular Cell Biology, Research Institute for Microbial Disease, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-8361; Fax: 81-6-6879-8362; E-mail: tnakano{at}biken.osaka-u.ac.jp
6 The abbreviations used are: PIP3, phosphatidylinositol 3,4,5-triphosphate; PI3'K, phosphoinositide-3-kinase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; IGF, insulin-like growth factor; MEF, murine embryonic fibroblast; SE, scanning electron microscopy; DMBA, 7,12-dimethylbenz(a)anthracene; TPA, 12-O-tetradecanoylphorbol-13-acetate; MAPK, mitogen-activated protein kinase; LOH, loss of heterozygosity; PKB, protein kinase B.
Received 8/ 5/02. Accepted 12/16/02.
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G. Li, Y. Hu, Y. Huo, M. Liu, D. Freeman, J. Gao, X. Liu, D.-C. Wu, and H. Wu PTEN Deletion Leads to Up-regulation of a Secreted Growth Factor Pleiotrophin J. Biol. Chem., April 21, 2006; 281(16): 10663 - 10668. [Abstract] [Full Text] [PDF]
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L. Li, K. Sampat, N. Hu, J. Zakari, and S. H. Yuspa Protein Kinase C Negatively Regulates Akt Activity and Modifies UVC-induced Apoptosis in Mouse Keratinocytes J. Biol. Chem., February 10, 2006; 281(6): 3237 - 3243. [Abstract] [Full Text] [PDF]
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D. Yao, C. L. Alexander, J. A. Quinn, M. J. Porter, H. Wu, and D. A. Greenhalgh PTEN Loss Promotes rasHa-Mediated Papillomatogenesis via Dual Up-Regulation of AKT Activity and Cell Cycle Deregulation but Malignant Conversion Proceeds via PTEN-Associated Pathways Cancer Res., February 1, 2006; 66(3): 1302 - 1312. [Abstract] [Full Text] [PDF]
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S. Ruiz, M. Santos, M. F. Lara, C. Segrelles, C. Ballestin, and J. M. Paramio Unexpected Roles for pRb in Mouse Skin Carcinogenesis Cancer Res., November 1, 2005; 65(21): 9678 - 9686. [Abstract] [Full Text] [PDF]
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L. Yang, C. Mao, Y. Teng, W. Li, J. Zhang, X. Cheng, X. Li, X. Han, Z. Xia, H. Deng, et al. Targeted Disruption of Smad4 in Mouse Epidermis Results in Failure of Hair Follicle Cycling and Formation of Skin Tumors Cancer Res., October 1, 2005; 65(19): 8671 - 8678. [Abstract] [Full Text] [PDF]
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 L. Alonso, H. Okada, H. A. Pasolli, A. Wakeham, A. I. You-Ten, T. W. Mak, and E. Fuchs Sgk3 links growth factor signaling to maintenance of progenitor cells in the hair follicle J. Cell Biol., August 15, 2005; 170(4): 559 - 570. [Abstract] [Full Text] [PDF]
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 L. Ma, J. Teruya-Feldstein, N. Behrendt, Z. Chen, T. Noda, O. Hino, C. Cordon-Cardo, and P. P. Pandolfi Genetic analysis of Pten and Tsc2 functional interactions in the mouse reveals asymmetrical haploinsufficiency in tumor suppression Genes & Dev., August 1, 2005; 19(15): 1779 - 1786. [Abstract] [Full Text] [PDF]
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Q. Yue, M. Groszer, J. S. Gil, A. J. Berk, A. Messing, H. Wu, and X. Liu PTEN deletion in Bergmann glia leads to premature differentiation and affects laminar organization Development, July 15, 2005; 132(14): 3281 - 3291. [Abstract] [Full Text] [PDF]
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T. B. El-Abaseri, J. Fuhrman, C. Trempus, I. Shendrik, R. W. Tennant, and L. A. Hansen Chemoprevention of UV Light-Induced Skin Tumorigenesis by Inhibition of the Epidermal Growth Factor Receptor Cancer Res., May 1, 2005; 65(9): 3958 - 3965. [Abstract] [Full Text] [PDF]
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 C. Kurlawalla-Martinez, B. Stiles, Y. Wang, S. U. Devaskar, B. B. Kahn, and H. Wu Insulin Hypersensitivity and Resistance to Streptozotocin-Induced Diabetes in Mice Lacking PTEN in Adipose Tissue Mol. Cell. Biol., March 15, 2005; 25(6): 2498 - 2510. [Abstract] [Full Text] [PDF]
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N. Di-Poi, C. Y. Ng, N. S. Tan, Z. Yang, B. A. Hemmings, B. Desvergne, L. Michalik, and W. Wahli Epithelium-Mesenchyme Interactions Control the Activity of Peroxisome Proliferator-Activated Receptor {beta}/{delta} during Hair Follicle Development Mol. Cell. Biol., March 1, 2005; 25(5): 1696 - 1712. [Abstract] [Full Text] [PDF]
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N. Wijesekara, D. Konrad, M. Eweida, C. Jefferies, N. Liadis, A. Giacca, M. Crackower, A. Suzuki, T. W. Mak, C. R. Kahn, et al. Muscle-Specific Pten Deletion Protects against Insulin Resistance and Diabetes Mol. Cell. Biol., February 1, 2005; 25(3): 1135 - 1145. [Abstract] [Full Text] [PDF]
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M. M. Fraser, X. Zhu, C.-H. Kwon, E. J. Uhlmann, D. H. Gutmann, and S. J. Baker Pten Loss Causes Hypertrophy and Increased Proliferation of Astrocytes In vivo Cancer Res., November 1, 2004; 64(21): 7773 - 7779. [Abstract] [Full Text] [PDF]
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 M. Cully, A. Elia, S.-H. Ong, V. Stambolic, T. Pawson, M.-S. Tsao, and T. W. Mak grb2 heterozygosity rescues embryonic lethality but not tumorigenesis in pten+/- mice PNAS, October 26, 2004; 101(43): 15358 - 15363. [Abstract] [Full Text] [PDF]
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J. A. McCormick, Y. Feng, K. Dawson, M. J. Behne, B. Yu, J. Wang, A. W. Wyatt, G. Henke, F. Grahammer, T. M. Mauro, et al. Targeted Disruption of the Protein Kinase SGK3/CISK Impairs Postnatal Hair Follicle Development Mol. Biol. Cell, September 1, 2004; 15(9): 4278 - 4288. [Abstract] [Full Text] [PDF]
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J.-H. Mao, M. D. To, J. Perez-Losada, D. Wu, R. Del Rosario, and A. Balmain Mutually exclusive mutations of the Pten and ras pathways in skin tumor progression Genes & Dev., August 1, 2004; 18(15): 1800 - 1805. [Abstract] [Full Text] [PDF]
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 C. Segrelles, S. Ruiz, M. Santos, J. Martinez-Palacio, M. F. Lara, and J. M. Paramio Akt mediates an angiogenic switch in transformed keratinocytes Carcinogenesis, July 1, 2004; 25(7): 1137 - 1147. [Abstract] [Full Text] [PDF]
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S. A. Backman, D. Ghazarian, K. So, O. Sanchez, K.-U. Wagner, L. Hennighausen, A. Suzuki, M.-S. Tsao, W. B. Chapman, V. Stambolic, et al. Early onset of neoplasia in the prostate and skin of mice with tissue-specific deletion of Pten PNAS, February 10, 2004; 101(6): 1725 - 1730. [Abstract] [Full Text] [PDF]
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 J. M. Ward and D. E. Devor-Henneman Mouse Models of Human Familial Cancer Syndromes Toxicol Pathol, January 1, 2004; 32(1_suppl): 90 - 98. [Abstract] [PDF]
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C.-H. Kwon, X. Zhu, J. Zhang, and S. J. Baker mTor is required for hypertrophy of Pten-deficient neuronal soma in vivo PNAS, October 28, 2003; 100(22): 12923 - 12928. [Abstract] [Full Text] [PDF]
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, loxP sites by black arrowheads. The probe used to analyze Southern blots is indicated and has been described previously 
) are plotted. G and H, severe hyperkeratosis (arrow) in the lower esophagus. Comparative histology of lower esophagus of k5CrePten+/+ (G) and k5CrePtenflox/flox (H) mice. I and J, cuticle detachment. SE analysis of cuticles (arrow) of k5CrePten+/+ (I) and k5CrePtenflox/flox (J) mice. The cuticle of the k5CrePtenflox/flox hair has almost completely separated from the hair shaft.
