PTEN/MMAC1/TEP1 in signal transduction and tumorigenesis.

The level of phosphorylation within cells is tightly regulated by the concerted action of protein kinases and protein phosphatases [Hunter, T. (1995) Cell 80, 225-236]. Disregulation in the activity of either of these players can lead to cellular transformation. Many protein tyrosine kinases are proto-oncogenes and it has been postulated that some protein phosphatases may act as tumor suppressors. Herein we will review the recent findings addressing the roles the candidate tumor suppressor PTEN/MMAC1/TEP1 (PTEN, phosphatase and tensin homologue deleted from chromosome 10; MMAC 1, mutated in multiple advanced cancers 1; TEP1, TGF beta regulated and epithelial cell enriched phosphatase 1) plays in signal transduction and tumorigenesis. PTEN is a dual specificity protein phosphatase (towards phospho-Ser/Thr and phospho-Tyr) and, unexpectedly, also has a phosphoinositide 3-phosphatase activity. PTEN plays an important role in the modulation of the 1-phosphatidylinositol 3-kinase (PtdIns 3-kinase) pathway, by catalyzing the degradation of the PtdIns(3,4,5)P3 generated by PtdIns 3-kinase; this inhibits the downstream functions mediated by the PtdIns 3-kinase pathway, such as activation of protein kinase B (PKB, also known as Akt), cell survival and cell proliferation. Furthermore, PTEN modulates cell migration and invasion by negatively regulating the signals generated at the focal adhesions, through the direct dephosphorylation and inhibition of focal adhesion kinase (FAK). Growth factor receptor signaling is also negatively regulated by PTEN, through the inhibition of the adaptor protein Shc. While some of the functions of PTEN have been elucidated, it is clear that there is much more to discover about the roles of this unique protein.     

Alternative splicing of the human PTEN/MMAC1/TEP1 gene

The human tumour suppressor gene PTEN/MMAC1/TEP1 encodes a lipid and protein phosphatase. Using RT-PCR, alternatively spliced forms of PTEN mRNA, encoding full-length PTEN and two forms of the protein truncated at the C-terminal end, were detected in normal human tissue. Cultured tumour and non-tumour cell lines show similar splicing patterns.     

抑癌基因PTEN／MMAC1的cDNA克隆及其表达质粒的构建

为了获得了PTEN/MMAC1的cDNA并构建其酵母双杂交系统中的诱饵质粒和逆转录病毒表达质粒。利用RTPCR方法,从293细胞中扩增出一约12.kb的DNA片段,与pGEMT Easy连接,作全自动测序确证,重组入pLexA载体,构建成pLexA-PTEN/MMAC1,并用醋酸锂法转化酶母菌EGY48（p8op-LacZ）,在选择性培养基上观察pLexA-PTEN/MMAC1在EGY48(p8op-LacZ）中的表达情况;同时,PTEN/MMAC1的cDNA也重组入pLXSN构建pLXSN-PTEN/MMAC1。结果PCR获得1.2kb的DNA序列与献报道的PTEN/MMAC1的cDNA一致,转化的酵母菌在选择性培养上培养3d后,长出约1mm大小的白色菌落;pLXSN-PTEN/MMAC1可酶切出1.2kb的PTEN/MMAC1片段。结果表明获得了PTEN/MMAC1的cDNA,pLexA-PTEN/MMAC1可作为酵母双杂交系统中的诱饵质粒,而pLXSN-PTEN/MMAC1的构建为进一步研究其抑癌作用打下了基础。     

人肝细胞癌中抑癌基因PTEN／MMAC1／TEP1的突变分析

PTEN/MMAC1/TEP1 is a tumor suppressor gene. Its mutation has been found in several different types of human cancers. 34 primary human hepatocellular carcinomas have been examined for mutations in exon 5 and exon 8 of the PTEN gene. Exon 5 and exon 8 were amplified by polymerase chain reaction (PCR) with intronic primers and subjected to single strand conformation polymorphism (SSCP) analysis. SSCPs were found in 4 of the 34 hepatocellular carcinomas analyzed. Direct sequencing of the PCR products identified single base-pair substitutions in the four tumor DNA samples, two in intron 4 and two in exon 8. One of the base-pair substitution in exon 8 is a missense mutation, which changed codon 304 of PTEN protein from Cys to Gly. These data suggest that PTEN may be involved in the carcinogenesis and development of hepatocellular carcinoma.     

人肝细胞癌中抑癌基因PTEN/MMAC1/TEP1的突变分析

PTEN／MMAC1／TEP1(PTEN)是新近分离到的抑癌基因,在多种肿瘤中存在突变。我们检测了34例人肝细胞癌中PTEN基因第5外显子和第8外显子的突变。采用聚合酶链方法以内含子引物扩增第5和第8外显子,继之以单链构象多态性和测序技术分析PTEN基因突变。有4例肝细胞癌SSCP显示异常条带并经测序证实存在突变。2例发生于第4内含子,突变位点相同;另两例发生于第8外显子,其中1例碱基颠换导致PTEN蛋白产物304位半胱氨酸突变为甘氨酸。     

PTEN/MMAC1 overexpression decreases insulin-like growth factor-I-mediated protection from apoptosis in neuroblastoma cells.

Insulin-like growth factor I (IGF-I) protects cells from apoptosis primarily through the action of phosphatidylinositol-3 kinase and the downstream serine/threonine kinase Akt. The PTEN gene product, a protein which dephosphorylates phosphatidylinositol lipids, prevents activation of Akt and regulates several cellular functions, including cell cycle progression, cell migration, and survival from apoptosis. In this study, PTEN overexpression decreases IGF-I-induced Akt activity, enhances serum withdrawal-induced apoptosis, and decreases IGF-I protection and cell growth in SHEP cells. The PTEN lipid phosphatase mutant G129E fails to inhibit IGF-I-stimulated Akt activity and protection from apoptosis. The C124S mutation, which abolishes both lipid and protein phosphatase activity, fails to inhibit Akt activity and IGF-I protection against hyperosmotic-induced apoptosis but still inhibits growth and IGF-I protection against serum withdrawal-induced apoptosis. These data suggest a role for PTEN in modulating the effect of IGF-I on Akt activity, neuroblastoma cell growth, and protection against apoptotic stimuli.     

Downregulation of PTEN/MMAC/TEP1 expression in human prostate cancer cell line DU145 by growth stimuli

Genetic alterations and/or deletion of the tumor suppressor gene PTEN/MMAC/TEP1 occur in many types of human cancer including prostate cancer. We describe the production of monoclonal antibody against recombinant human PTEN and the study of PTEN gene and protein expression in three commercially available human prostate cancer cell lines, PC-3, LNCaP, and DU 145. Northern blotting analyses showed that LNCaP and DU145 but not PC-3 cells expressed PTEN mRNA. However, Western blotting analyses using a monoclonal antibody against PTEN demonstrated the expression of PTEN protein in DU145 but not LNCaP cells. In DU145 cells, PTEN expression at both the mRNA and protein levels inversely correlated with serum concentrations and levels of PKB/Akt phosphorylation. In addition, the basal activity of PKB/Akt as indicated by level of phosphorylation was higher in prostate cancer cells which do not express PTEN than that in the cells expressing wild type PTEN. Thus, PTEN may play a critical role in regulating cellular signaling in prostate cancer cells.     

抑癌基因PTEN及其假基因PTENP1在子宫内膜癌中的研究进展

子宫内膜癌的发病率在逐年上升,引起了人们的广泛关注,但其发病的分子遗传学机制仍不十分清楚。近年来基因改变致癌的研究成为热点。国内外研究报道发现：PTEN（与张力蛋白同源第10染色体丢失的磷酸酶基因）是目前已知的子宫内膜癌中突变率最高的基因,常发生在子宫内膜癌的早期,对其突变的检测有助于子宫内膜癌的早期诊断、治疗及预后评价,并为子宫内膜癌的基因治疗提供了新的靶点。另外,研究发现,PTENP1（PTEN的假基因）转录调控PTEN的表达,被认为与一些肿瘤的发生有关。本文就PTEN基因的结构、功能及在子宫内膜癌中的突变情况、临床意义及PTENP1的研究现状进行综述。     

Absence of nigral degeneration in aged parkin/DJ-1/PINK1 triple knockout mice

Recessively inherited loss-of-function mutations in the parkin , DJ-1 , or PINK1 gene are linked to familial cases of early-onset Parkinson's diseases (PD), and heterozygous mutations are associated with increased incidence of late-onset PD. We previously reported that single knockout mice lacking Parkin, DJ-1, or PINK1 exhibited no nigral degeneration, even though evoked dopamine release from nigrostriatal terminals was reduced and striatal synaptic plasticity was impaired. In this study, we tested whether inactivation of all three recessive PD genes, each of which was required for nigral neuron survival in the aging human brain, resulted in nigral degeneration during the lifespan of mice. Surprisingly, we found that triple knockout mice lacking Parkin, DJ-1, and PINK1 have normal morphology and numbers of dopaminergic and noradrenergic neurons in the substantia nigra and locus coeruleus, respectively, at the ages of 3, 16, and 24 months. Interestingly, levels of striatal dopamine in triple knockout mice were normal at 16 months of age but increased at 24 months. These results demonstrate that inactivation of all three recessive PD genes is insufficient to cause significant nigral degeneration within the lifespan of mice, suggesting that these genes may be protective rather than essential for the survival of dopaminergic neurons during the aging process. These findings also support the notion that mammalian Parkin and PINK1 may function in the same genetic pathway as in Drosophila .     

Absence of diabetic hyperalgesia in bradykinin B1 receptor-knockout mice

Experimental evidence has shown that the inducible bradykinin (BK) B1 receptor (BKB1-R) subtype is involved in the development of hyperalgesia associated with type 1 diabetes. Selective BKB1-R antagonists inhibited, whereas selective agonists increased the hyperalgesic activity in diabetic mice in thermal nociceptive tests. Here we evaluate the development of diabetic hyperalgesia in a BKB1-R-knockout (KO) genotype compared to wild-type (WT) mice. The BKB1-R-KO mice were backcrossed for 10 generations to C57BL/6 genetic background before use in the experiments. Diabetes was induced by streptozotocin (STZ) and thermal nociception was assessed by the hot plate and tail immersion tests. The hyperalgesia observed in wild type mice was totally absent in the BKB1-R-KO mice. Furthermore, the selective BKB1-R agonist, desArg9BK, significantly increased the hyperalgesic activity in diabetic WT mice but had no effect on nociceptive responses in diabetic BKB1-R-KO mice. Taken together, the results confirm the crucial role of the BKB1-R, upregulated alongside inflammatory diabetes, in the development of diabetes-induced hyperalgesia.     

False phylogenies on wood mice due to cryptic cytochrome-b pseudogene

The phylogeny and phylogeography of the Old World wood mice (subgenus Sylvaemus, genus Apodemus, Muridae) are well-documented. Nevertheless, the distributions of species, such as A. fulvipectus and A. ponticus remain dubious, as well as their phylogenetic relationships with A. sylvaticus. We analysed samples of Apodemus spp. across Europe using the mitochondrial cytochrome-b gene (cyt-b) and compared the DNA and amino-acid compositions of previously published sequences. The main result stemming from this study is the presence of a well-differentiated lineage of Sylvaemus including samples of various species (A. sylvaticus, A. fulvipectus, A. ponticus) from distant locations, which were revealed to be nuclear copies of the mitochondrial cyt-b. The presence of this cryptic pseudogene in published sequences is supported by different pathways. This has led to important errors in previous molecular trees and hence to partial misinterpretations in the phylogeny of Apodemus.     

Absence of erythrogenesis and vasculogenesis in Plcg1-deficient mice.

Mice nullizygous for Plcg1 cease growing at early to mid-gestation. An examination of carefully preserved wild-type embryos shows clear evidence of erythropoiesis, but erythropoiesis is not evident in Plcg1 nullizygous embryos at the same stage. The analyses of embryonic materials demonstrate that in the absence of Plcg1, erythroid progenitors cannot be detected in the yolk sac or embryo body by three different assays, burst-forming units, colony-forming units, and analysis for the developmental marker Ter119. However, non-erythroid granulocyte/macrophage colonies are produced by Plcg1 null embryos. Further analysis of these embryos demonstrates significantly diminished vasculogenesis in Plcg1 nullizygous embryos based on the lack of expression of the endothelial marker platelet endothelial cell adhesion molecule-1. In addition, Plcg1 nullizygous embryos express a greatly reduced level of vascular endothelial growth factor receptor-2/Flk-1, consistent with significantly impaired vasculogenesis and erythropoiesis. Interestingly, these early embryos do express phospholipase C-gamma2, however, it is unable to substitute for the absence of phospholipase C-gamma1, which can be detected in its tyrosine-phosphorylated state.     

Interaction of the tumor suppressor PTEN/MMAC with a PDZ domain of MAGI3, a novel membrane-associated guanylate kinase

PTEN/MMAC is a phosphatase that is mutated in multiple human tumors. PTEN/MMAC dephosphorylates 3-phosphorylated phosphatidylinositol phosphates that activate AKT/protein kinase B (PKB) kinase activity. AKT/PKB is implicated in the inhibition of apoptosis, and cell lines and tumors with mutated PTEN/MMAC show increased AKT/PKB kinase activity and resistance to apoptosis. PTEN/MMAC contains a PDZ domain-binding site, and we show here that the phosphatase binds to a PDZ domain of membrane-associated guanylate kinase with inverted orientation (MAGI) 3, a novel inverted membrane-associated guanylate kinase that localizes to epithelial cell tight junctions. Importantly, MAGI3 and PTEN/MMAC cooperate to modulate the kinase activity of AKT/PKB. These data suggest that MAGI3 allows for the juxtaposition of PTEN/MMAC to phospholipid signaling pathways involved with cell survival.     

Hypomorphic mutation of PDK1 suppresses tumorigenesis in PTEN(+/-) mice

Many cancers possess elevated levels of PtdIns(3,4,5)P(3), the second messenger that induces activation of the protein kinases PKB/Akt and S6K and thereby stimulates cell proliferation, growth, and survival. The importance of this pathway in tumorigenesis has been highlighted by the finding that PTEN, the lipid phosphatase that breaks down PtdIns(3,4,5)P(3) to PtdIns(4,5)P(2), is frequently mutated in human cancer. Cells lacking PTEN possess elevated levels of PtdIns(3,4,5)P(3), PKB, and S6K activity and heterozygous PTEN(+/-) mice develop a variety of tumors. Knockout of PKBalpha in PTEN-deficient cells reduces aggressive growth and promotes apoptosis, whereas treatment of PTEN(+/-) mice with rapamycin, an inhibitor of the activation of S6K, reduces neoplasia. We explored the importance of PDK1, the protein kinase that activates PKB and S6K, in mediating tumorigenesis caused by the deletion of PTEN. We demonstrate that reducing the expression of PDK1 in PTEN(+/-) mice, markedly protects these animals from developing a wide range of tumors. Our findings provide genetic evidence that PDK1 is a key effector in mediating neoplasia resulting from loss of PTEN and also validate PDK1 as a promising anticancer target for the prevention of tumors that possess elevated PKB and S6K activity.