The majority of thyroid carcinomas maintain the expression of the cell growth suppressor p27, an inhibitor of cyclin-dependent kinase-2 (Cdk2). However, we find that 80% of p27-expressing tumors show an uncommon cytoplasmic localization of p27 protein, associated with high Cdk2 activity. To reproduce such a situation, a mutant p27 devoid of its COOH-terminal nuclear-localization signal was generated (p27-NLS). p27-NLS accumulates in the cytoplasm and fails to induce growth arrest in 2 different cell lines, indicating that cytoplasm-residing p27 is inactive as a growth inhibitor, presumably because it does not interact with nuclear Cdk2. Overexpression of cyclin D3 may account in part for p27 cytoplasmic localization. In thyroid tumors and cell lines, cyclin D3 expression was associated with cytoplasmic localization of p27. Moreover, expression of cyclin D3 in thyroid carcinoma cells induced cytoplasmic retention of cotransfected p27 and rescued p27-imposed growth arrest. Endogenous p27 also localized prevalently to the cytoplasm in normal thyrocytes engineered to stably overexpress cyclin D3 (PC-D3 cells). In these cells, cyclin D3 induced the formation of cytoplasmic p27-cyclin D3-Cdk complexes, which titrated p27 away from intranuclear complexes that contain cyclins A-E and Cdk2. Our results demonstrate a novel mechanism that may contribute to overcoming the p27 inhibitory threshold in transformed thyroid cells.J. Clin. Invest. 104:865-874 (1999). MethodsCell lines. The human cell lines used in this study are described in ref. 21. Bosc23 cells were a gift of M. Santoro (Consiglio Nazionale delle Ricerche, Naples, Italy). All cell lines were grown in DMEM containing 10% FCS. PC Cl 3 and PC-D3 cells (normal thyrocytes engineered to stably overexpress cyclin D3) were grown in Ham's F12 medium supplemented with 5% calf serum in the presence of 6 hormones (thyrotropin, hydrocortisone, insulin, transferrin, somatostatin, and glycyl-histidyllysine; Sigma Chemical Co., St. Louis, Missouri, USA).Immunoperoxidase staining. Immunohistochemistry was performed using anti-p27 monoclonal antibody k25020 (Transduction Laboratories, Lexington, Kentucky, USA) or anti-p27 polyclonal antibody C-19 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) as described previously (15,16). Antigen retrieval was performed by microwave irradiation. To define p27 expression we used cutoff values that have been defined in previous papers (15,16). Tumors were considered to be p27-positive when 50% or more of the tumor cells stained positive; if less than 50% of cells stained positive, a tumor was considered p27-negative. Counts were performed in 5 random high-power fields. At least 500 cells were counted.Western blotting, immunoprecipitation, and kinase assay. Cells were lysed in NP-40 buffer containing protease inhibitors. Proteins were separated on polyacrylamide gels and transferred to nitrocellulose membranes (Hybond-C; Amersham Pharmacia Biotech, Uppsala, Sweden). Membranes were incubated with primary and secondary antibodies and...

The r-PTPeta gene encodes a rat receptor-type protein tyrosine phosphatase whose expression is negatively regulated by neoplastic cell transformation. Here we first demonstrate a dramatic reduction in DEP-1/HPTPeta (the human homolog of r-PTPeta) expression in a panel of human thyroid carcinomas. Subsequently, we show that the reexpression of the r-PTPeta gene in highly malignant rat thyroid cells transformed by retroviruses carrying the v-mos and v-ras-Ki oncogenes suppresses their malignant phenotype. Cell cycle analysis demonstrated that r-PTPeta caused G(1) growth arrest and increased the cyclin-dependent kinase inhibitor p27(Kip1) protein level by reducing the proteasome-dependent degradation rate. We propose that the r-PTPeta tumor suppressor activity is mediated by p27(Kip1) protein stabilization, because suppression of p27(Kip1) protein synthesis using p27-specific antisense oligonucleotides blocked the growth-inhibitory effect induced by r-PTPeta. Furthermore, we provide evidence that in v-mos- or v-ras-Ki-transformed thyroid cells, the p27(Kip1) protein level was regulated by the mitogen-activated protein (MAP) kinase pathway and that r-PTPeta regulated p27(Kip1) stability by preventing v-mos- or v-ras-Ki-induced MAP kinase activation.

The dual-speci®city phosphatase PTEN/MMAC1/TEP1 has recently been identi®ed as the tumor suppressor gene most frequently mutated and/or deleted in human tumors. Germline mutations of PTEN give rise to Cowden Disease (CD), an autosomal dominantlyinherited cancer syndrome which predisposes to increased risk of developing breast and thyroid tumors. However, PTEN mutations have rarely been detected in sporadic thyroid carcinomas. In this study, we con®rm that PTEN mutations in sporadic thyroid cancer are infrequent as we found one point mutation and one heterozygous deletion of PTEN gene in 26 tumors and eight cell lines screened. However, we report that PTEN expression is reduced ± both at the mRNA and at the protein level ± in ®ve out of eight tumor-derived cell lines and in 24 out of 61 primary tumors. In most cases, decreased PTEN expression is correlated with increased phosphorylation of the PTEN-regulated protein kinase Akt/PKB. Moreover, we demonstrate that PTEN may act as a suppressor of thyroid cancerogenesis as the constitutive re-expression of PTEN into two di erent thyroid tumor cell lines markedly inhibits cell growth. PTEN-dependent inhibition of BrdU incorporation is accompanied by enhanced expression of the cyclin-dependent kinase inhibitor p27 kip1 and can be overcome by simultaneous co-transfection of an excess p27 kip1 antisense plasmid. Accordingly, in a subset of thyroid primary carcinomas and tumor-derived cell lines, a striking correlation between PTEN expression and the level of p27 kip1 protein was observed. In conclusion, our ®ndings demonstrate that inactivation of PTEN may play a role in the development of sporadic thyroid carcinomas and that one key target of PTEN suppressor activity is represented by the cyclin-dependent kinase inhibitor p27 kip1 . Oncogene (2000) 19, 3146 ± 3155.

PTEN/MMAC1/TEP1 (hereafter PTEN) is a tumor suppressor gene (located at 10q23) that is frequently mutated or deleted in sporadic human tumors. PTEN encodes a multifunctional phosphatase, which negatively regulates cell growth, migration and survival via the phosphatidylinositol 3 0 -kinase/AKT signalling pathway. Accordingly, Pten þ /À mice develop various types of tumors including teratocarcinomas and teratomas. We have investigated PTEN expression in 60 bioptic specimens of germ cell tumors (32 seminomas, 22 embryonal carcinomas and six teratomas) and 22 intratubular germ cell neoplasias (ITGCN) adjacent to the tumors for PTEN protein and mRNA expression. In total, 10 testicular biopsies were used as controls. In the testis, PTEN was abundantly expressed in germ cells whereas it was virtually absent from 56% of seminomas as well as from 86% of embryonal carcinomas and virtually all teratomas. On the contrary, ITGCN intensely expressed PTEN, indicating that loss of PTEN expression is not an early event in testicular tumor development. The loss of PTEN expression occurs mainly at the RNA level as determined by in situ hybridization of cellular mRNA (17/22) but also it may involve some kind of posttranscriptional mechanisms in the remaining 25% of cases. Analysis of microsatellites D10S551, D10S541 and D10S1765 in GCTs (n ¼ 22) showed LOH at the PTEN locus at 10q23 in at least 36% of GCTs (three embryonal carcinoma, three seminoma, two teratoma); one seminoma and one embryonal (9%) carcinoma presented an inactivating mutation in the PTEN gene (2/22). Finally, we demonstrated that the phosphatidylinositol 3 0 -kinase/ AKT pathway, which is regulated by the PTEN phosphatase, is crucial in regulating the proliferation of the NT2/D1 embryonal carcinoma cells, and that the cyclin-dependent kinase inhibitor p27 kip1 is a key downstream target of this pathway.

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