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The PTEN tumour suppressor is a phosphatase that dephosphorylates phosphatidylinositol 3, 4, 5 triphosphate (PIP3) and protein substrates. PTEN function is modulated by its carboxy-terminal region, which contains several clustered phosphorylation sites and a PDZ-binding motif (PDZbm). Although PTEN growth suppression effect is well demonstrated, its additional biological roles are less well understood. DAF-18, a Caenorhabditis elegans homologue PTEN, is a component of the insulin/IGF-I signalling pathway that controls entry to the dauer larval stage and adult longevity. To further explore the role of PTEN in the insulin signalling cascade and its possible involvement in the mechanisms of ageing, we undertook a study of PTEN function in C. elegans. We now report that human PTEN can substitute for DAF-18 and restores the dauer and longevity phenotypes in worms devoid of DAF-18. Furthermore, we provide genetic and biochemical evidence that dauer and lifespan control depends on PTEN-mediated regulation of PIP3 levels. Finally, we established that phosphorylation sites in the C-terminus of PTEN and its PDZbm are necessary for PTEN control of the insulin/IGF-I pathway. These results demonstrate that PTEN negatively regulates the insulin/IGF pathway in a whole organism and raise the hypothesis that PTEN may be involved in mammalian ageing.

The Kruppel-like transcription factor KLF6 is a novel tumor-suppressor gene mutated in a significant fraction of human prostate cancer. It is localized to human chromosome 10p14-15, a region that displays frequent loss of heterozygosity in glioblastoma multiforme (GBM). Indeed, mutations of the KLF6 gene have recently been reported in this tumor type. In this study, we report that the expression of KLF6 is attenuated in human GBM when compared with primary astrocytes. Expression of KLF6 in GBM cells reverts their tumorigenicity both in vitro and in vivo, which is correlated with its transactivation of the p21/CIP1/WAF1 promoter. Additionally, KLF6 inhibits cellular transformation induced by several oncogenes (c-sis/PDGF-B, v-src, H-Ras, and EGFR) that are components of signaling cascades implicated in GBM. Our results provide the first evidence of functional tumor suppression by KFL6, and its loss may contribute to glial tumor progression.

The GTP-binding protein, R-Ras3/M-Ras, is a novel member of the Ras subfamily of GTPases which shows highest sequence similarity to the TC21 gene. R-Ras3 is highly expressed in both human and mouse brain and ectopic expression of a constitutively active mutant of R-Ras3 induces cellular transformation in NIH3T3 cells. To gain further insight into the normal cellular function of R-Ras3, we examined the ability of R-Ras3 in activating several known intracellular signaling cascades. We observed that R-Ras3 is a relatively weak activator of the mitogen-activated protein kinase/extracellular-signal-regulated kinases (MAPK/ERKs) when compared to the H-Ras oncogene. On the contrary, both R-Ras3 and H-Ras activated the Jun N-terminal kinase (JNK) to a similar extent. Under similar experimental conditions, R-Ras3 significantly stimulated one of the phosphatidylinositol 3-kinase (PI3-K) downstream substrates, Akt/PKB/RAC (Akt), which has been extensively implicated in mediating cell survival signaling. The activation of Akt by R-Ras3 was most likely to be PI3-K-dependent since this biochemical event was blocked by the pharmacological inhibitors, Wortmannin and LY294002, as well as by a dominant negative mutant of PI3-K. More importantly, R-Ras3 affinity-precipitated PI3-K from cell extracts in a GTP-dependent manner, and associated lipid kinase activity was readily detectable in R-Ras3 immune complexes. The biological significance of R-Ras3 in inducing Akt kinase activity is evidenced by the ability of an activated R-Ras3 to confer cell survival in the rat pheochromocytoma cell line, PC12. As expected, this biological activity of R-Ras3 was also abrogated by the addition of LY294002. Thus, R-Ras3 represents a novel G-protein which may play a role in cell survival of neural-derived cells.

The human PTEN/MMAC1/TEP1 (PTEN) tumor suppressor gene encodes a phosphatase with specificity towards the D3 phosphate of phosphatidylinositides. PTEN mutations have been reported in the endometrioid type of uterine tumors which are associated with frequent activations of the Ras oncogenes. In this study, we report the ability of PTEN to potently inhibit H-Ras induced morphological transformation and anchorage-independent growth in NIH3T3 cells. This novel activity of PTEN was correlated more with its ability to suppress the phosphatidylinositol 3-kinase (PI3-K)-dependent signaling cascade, but not the mitogen-activated protein kinase (MAPK) pathway. To define the minimal region in PTEN protein that is responsible for this anti-oncogenic activity, a panel of carboxyl-terminal truncation mutants was generated. While deletions of 4 and 33 amino acids do not have marked effects, removal of up to 68 amino acids drastically reduced the ability of PTEN to inhibit Ras transformation. The propensity of these mutants to suppress Ras transformation is correlated with their relative ability to dephosphorylate inositol (1,3,4,5)-tetrakisphosphate in vitro, and to suppress Akt kinase activity in cultured cells. In addition, we have evidence to suggest that the C-terminal region of PTEN contributes to the stability of the encoded gene product.

Members of the Ras subfamily of GTP-binding proteins, including Ras (H-, K-, and N-), TC21, and R-ras have been shown to display transforming activity, and activating lesions have been detected in human tumors. We have identified an additional member of the Ras gene family which shows significant sequence similarity to the human TC21 gene. This novel human ras-related gene, R-ras3, encodes for a protein of 209 amino acids, and shows approximately 60-75% sequence identity in the N-terminal catalytic domain with members of the Ras subfamily of GTP-binding proteins. An activating mutation corresponding to the leucine 61 oncogenic lesion of the ras oncogenes when introduced into R-ras3, activates its transforming potential. R-ras3 weakly stimulates the mitogen-activated protein kinase (MAPK) activity, but this effect is greatly potentiated by the co-expression of c-raf-1. By the yeast two-hybrid system, R-ras3 interacts only weakly with known Ras effectors, such as Raf and RalGDS, but not with RglII. In addition, R-ras3 displays modest stimulatory effects on trans-activation from different nuclear response elements which bind transcription factors, such as SRF, ETS/TCF, Jun/Fos, and NF-kappaB/Rel. Interestingly, Northern blot analysis of total RNA isolated from various tissues revealed that the 3.8 kilobasepair (kb) transcript of R-ras3 is highly restricted to the brain and heart. The close evolutionary conservation between R-ras3 and Ras family members, in contrast to the significant differences in its biological activities and the pattern of tissue expression, raise the possibility that R-ras3 may control novel cellular functions previously not described for other GTP-binding proteins.

The heterotrimeric G-protein, G alpha12, together with the closely-related G alpha13, are members of the G12 class of alpha-subunits important in mediating the signaling from seven transmembrane domain-spanning receptors. Recent evidence implicating both G alpha12 and G alpha13 in the activation of signaling pathways involving members of the RHO gene family led us to examine the role of Rac1, RhoA and Cdc42Hs in the transforming properties of G alpha12. Asparagine 17 (Asn 17) dominant inhibitory mutants of Rac1, and to a lesser extent RhoA, block focus forming ability of the GTPase-deficient mutant of G alpha12 (G alpha12 Leu 229) in NIH3T3 cells. In turn, wild-type G alpha12 cooperates well with Rac1 Val 12 but not with RhoA Leu 63 mutant in transforming NIH3T3 cells. Interestingly, the morphology of foci induced by G alpha12 and RhoA mutants are strikingly similar and is distinct from those displayed by Rac1 Val 12 mutant. The fact that G alpha12's ability to induce mitogenesis in NIH3T3 cells is not significantly perturbed by C3 ribosyltransferase suggested that RhoA does not play a major role in G alpha12-induced mitogenic events. Activated mutant of Rac1 has previously been demonstrated to stimulate the activity of the stress-induced c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs). Transient co-transfection of Rac1 Val 12 mutant with the wild-type G alpha12 in COS7 cells leads to the further activation of an exogenously expressed hemagglutinin(HA)-tagged JNK. Furthermore, the cooperation between G alpha12 and Rac1 in cellular transformation is correlated with their ability to stimulate transcription from c-fos serum response element (SRE).

The alpha-subunit of the heterotrimeric G-protein, G12, has been shown to induce cellular transformation when overexpressed or oncogenically activated in rodent fibroblasts. To investigate the interaction between Galpha12 transforming pathway and the Ras-Raf-MAPK pathway, we examined the ability of mitogenic signaling molecules in cooperating with Galpha12 in transforming NIH3T3 fibroblasts. We observed a striking cooperative effect on focus-forming ability when Galpha12 and c-raf-1 cDNAs were co-transfected into NIH3T3 cells. NIH3T3 cells coexpressing both Galpha12 and c-raf-1 resulted in the constitutive activation of the mitogenic-activated protein kinase (MAPK). In addition, the levels of GTP-bound Ras were elevated in Galpha12 transformed NIH3T3 cells. Our results provide a model for studying the effects of simultaneous activation of two distinct growth regulatory pathways in cellular transformation.

We generated a cDNA expression library from a human neuroepithelioma cell line for detection of novel oncogenes by focus formation assay in NIH3T3 cells. A morphologically unique focus was identified upon transfection and the transforming plasmid was isolated. The transforming gene, designated NET1, encoded a predicted protein species of 54 kDa containing the Dbl-Homology (DH) motif. This motif is also present in other growth regulatory molecules including Bcr, Cdc24, Vav, Ras-Grf, Ect2, Ost, Tim and Tiam1, which have been implicated as regulators of small GTP-binding proteins. NIH3T3 cells transfected with NET1 expression plasmid showed altered growth properties in vitro and were tumorigenic when injected into nude mice. In addition, a 2.5 kb cDNA was isolated from a normal human cDNA library which represented the NET1 proto-oncogene contained a 5' extended open reading frame. The fact that the proto-oncogene failed to induce transformation in NIH3T3 cells suggested that the original NET1 oncogene was activated by 5'-truncation. The 3.0 and 2.4 kilobasepair (kb) transcripts of the NET1 gene was ubiquitously expressed in all tissues examined. Using fluorescence in situ hybridization, we localized the NET1 gene to the short arm of human chromosome 10 at band p15.

TC21 is the fourth member of the ras gene family to exhibit oncogenic activation in human tumor cells. To assess the prevalence of activated TC21 oncogenes in human tumors, we have developed sensitive single-strand conformational polymorphism (SSCP) conditions and immunological reagents for the detection of both single base alterations and/or overt overexpression in a wide spectrum of human tumor cell lines and surgical samples. In an initial examination of 33 human tumor specimens, we observed a novel nine basepair three amino acids insertion at TC21 codon 24 in one human uterine leiomyosarcoma cell line, SK-UT-1. This mutant allele when transfected into NIH3T3 cells, displayed high transforming activity comparable to that of the Leu72 oncogenic mutant identified by expression cDNA cloning from a human ovarian carcinoma cell line. Comparing the level of GTP-binding by the mutant and normal TC21 products revealed that this novel lesion increases the GTP-bound form of the TC21 molecule. These findings imply that the mechanism by which mutations activate the oncogenic properties of this ras-related molecule is analogous to that of previously known ras family members.

R-ras, K-rev-1/rap and TC21, are more closely related to prototype H-ras than any other known members of the ras superfamily. We recently isolated a mutationally activated TC21 oncogene from a human ovarian carcinoma cell line. Based upon these observations, we sought to re-examine the transforming potential of R-ras, which was reported earlier to lack transforming capacity. Mutations were introduced into the R-ras gene at codons 38 or 87, analogous to positions 12 and 61, respectively, responsible for H-ras oncogene activation. While both mutations resulted in acquisition of R-ras transforming capacity for NIH3T3 cells the position 61 was shown to be more active. Transfectants expressing either R-ras mutant formed colonies in soft agar and were tumorigenic in vivo. As has been reported for H-ras, R-ras cooperated with c-raf-1 in inducing transformation of NIH3T3 cells. These results imply interactions in R-ras and c-raf-1 signaling pathways. We observed R-ras transcripts of 4.6 and 1.2 kb ubiquitously expressed in each of a variety of tissues examined. All these findings raise the possibility that R-ras, like prototype ras genes, may be mutationally activated as an oncogene in some human malignancies.

We generated a cDNA expression library from a human mammary epithelial cell line for detection of novel oncogenes by focus formation assay in NIH3T3 cells. A morphologically unique focus was identified and the transforming plasmid was isolated. The transforming gene, designated TIM, encoded a predicted protein species of 60 kDa containing a Dbl-Homology (DH) motif. This motif is also present in other growth regulatory molecules including Bcr, Cdc24, Vav, Ras-grf, and Ect2 which have been implicated as regulators of small GTP-binding proteins. NIH3T3 cells transfected with TIM expression plasmid showed altered growth properties in vitro and were tumorigenic when injected into nude mice. The 6.5 kilobasepair (kb) transcript of the TIM gene was mainly expressed in kidney, liver, pancreas, lung, and placenta. By analysing a panel of human-hamster somatic cell hybrids, we localized the TIM gene to human chromosome 7.

By ectopic expression of cDNAs derived from a Ewing sarcoma cell line in NIH3T3 cells, we isolated a transforming gene (est). Sequence analysis revealed homology to the cot oncogene, which encodes a novel serine kinase. Whereas the cot product was truncated at its carboxy-terminal end as a result of gene rearrangement during transfection, est encodes the normal cot product. Thus, this gene can be activated as an oncogene by overexpression as well as by gene rearrangement. NIH3T3 cells transfected with est formed progressively growing colonies in soft agar and were tumorigenic in nude mice. The 3.2-kb est transcript was expressed at low level in both human fibroblasts and epithelial cells. Addition of the tumor promoter, okadaic acid (OA), or cytokine, interleukin 1 (IL-1), but not serum or platelet-derived growth factor (PDGF), induced increased expression of the est transcript. Using fluorescence in situ hybridization, we localized the est gene to the short arm of human chromosome 10 at band p11.2.

The DNA sequence of cDNA clones prepared from transcripts of the mouse met proto-oncogene reveals that the mouse met gene encodes a 1380 amino acid protein with the characteristics of a growth factor receptor. This protein can be divided into several putative domains, including an intracellular protein tyrosine kinase domain, a transmembrane domain and a 929 amino acid extracellular domain, possessing a potential proteolytic cleavage site with the sequence Lys-Arg-Arg-Lys-Arg-Ser. To gain additional insights into the function of the met protein we have examined the level of met transcripts in tissues of the late-gestation mouse conceptus. Transcription of met was observed in most of the tissues analysed, but the highest levels of met mRNA were detected in the yolk sac, amnion and kidney; no transcripts were detectable in the calvaria. Chromosomal localization using a series of mouse-hamster hybrid cell lines has demonstrated that met is located on mouse chromosome 6.

The primary structure of the protein tyrosine kinase domain of the human met gene has been determined from cDNA clones prepared from transcripts of the activated human met gene. These analyses reveal that the met kinase domain (located on human chromosome 7) possesses unique features that distinguish met from other members of the src family of protein tyrosine kinases. The results also demonstrate that the product of the activated met gene is a fusion protein and that the amino terminal end of this fusion protein, which is encoded by human chromosome 1, exhibits homology to laminin B1.

Recent studies have identified a specific chromosomal translocation, t(X;18)(p11.2;q11.2), in a high proportion of human synovial sarcomas. As a first step towards characterizing the X;18 translocation we have established a synovial sarcoma cell line. Fusion of this cell line to mouse RAG cells gave rise to somatic cell hybrids that contain the derivative (X) marker chromosome in the absence of other genetic material from chromosomes 18 and X. Southern analysis of DNA from these somatic cell hybrids demonstrated that the human X chromosome markers DXS94, DXS14, DXZ1 and DXS62 were retained. In contrast DXS7, GAPDP1, ARAF1, DXS146 were not consistently present in the hybrids indicating that these markers were on the region of the X chromosome replaced by part of the long arm of chromosome 18 during the generation of the X;18 translocation. The predicted position of the translocation relative to X chromosome markers is DXS7-DXS146-X; 18-DXS14-DXZ1-DXS94.