E-cadherin negatively regulates neoplastic growth in non-small cell lung cancer: role of Rho GTPases.

Non-small cell lung cancers (NSCLC) that express the cell surface adhesion protein E-cadherin may carry a better prognosis than E-cadherin-negative tumors. Here, we found substantial inhibition of anchorage-independent growth in soft agar and cell migration in each of four NSCLC lines stably transfected with E-cadherin. The inhibitory effects were independent of the EGFR and beta-catenin/Wnt-signaling pathways. However, E-cadherin expression was associated with an adhesion-dependent reduction in the activity of Rho family proteins, RhoA in two lines and Cdc42 in the other two. The reduction of RhoA activity was dependent on DLC-1 Rho-GAP and p190 Rho-GAP and associated with an increase in a membrane-associated p190 Rho-GAP/p120 Ras-GAP complex. In parental cells with high levels of RhoA-GTP, siRNA-mediated knock-down of RhoA reduced cell migration and agar growth in a manner analogous to E-cadherin. In parental cells with high levels of Cdc42-GTP, transfection of a Cdc42 dominant-negative mutant reduced cell growth and migration similarly to cells expressing E-cadherin. Thus, E-cadherin can negatively regulate cell proliferation and migration in NSCLC by reducing the level of the predominant active form of Rho family protein, RhoA or Cdc42. These proteins can be considered downstream effectors of E-cadherin and might represent therapeutic targets in some NSCLC.

NHPV16 VLP vaccine induces human antibodies that neutralize divergent variants of HPV16.

Genital HPV genotypes are generally distinct serotypes, but whether variants within a genotype can represent serologic subtypes is unclear. In this study we used serum from human volunteers vaccinated with HPV16 L1 VLPs from variant 114K, to examine cross-neutralization of variants from each of the five major phylogenetic branches of HPV16. Recombinant Semliki Forest virus-derived pseudovirions for each variant were generated and combined with serum from vaccines, and the mixture was monitored for infectivity in a standard C127 cell focal transformation assay. Sera from all 10 VLP-immunized individuals had neutralizing activity against each of the variant pseudovirions. For each of the sera, variant titers differed by only fourfold or less from the median titer. Therefore, from a vaccine perspective, HPV16 variants belong to a single serotype. Vaccination with HPV16 114K L1 VLPs generates antibodies that should confer a similar degree of protection against all known phylogenetic branches of HPV16.

Efficiency alleles of the Pctr1 modifier locus for plasmacytoma susceptibility.

The susceptibility of BALB/c mice to pristane-induced plasmacytomas is a complex genetic trait involving multiple loci, while DBA/2 and C57BL/6 strains are genetically resistant to the plasmacytomagenic effects of pristane. In this model system for human B-cell neoplasia, one of the BALB/c susceptibility and modifier loci, Pctr1, was mapped to a 5.7-centimorgan (cM) chromosomal region that included Cdkn2a, which encodes p16(INK4a) and p19(ARF), and the coding sequences for the BALB/c p16(INK4a) and p19(ARF) alleles were found to be polymorphic with respect to their resistant Pctr1 counterparts in DBA/2 and C57BL/6 mice (45). In the present study, alleles of Pctr1, Cdkn2a, and D4Mit15 from a resistant strain (BALB/cDAG) carrying DBA/2 chromatin were introgressively backcrossed to the susceptible BALB/c strain. The resultant C.DAG-Pctr1 Cdkn2a D4Mit15 congenic was more resistant to plasmacytomagenesis than BALB/c, thus narrowing Pctr1 to a 1.5-cM interval. Concomitantly, resistant C57BL/6 mice, from which both gene products of the Cdkn2a gene have been eliminated, developed pristane-induced plasma cell tumors over a shorter latency period than the traditionally susceptible BALB/cAn strain. Biological assays of the p16(INK4a) and p19(ARF) alleles from BALB/c and DBA/2 indicated that the BALB/c p16(INK4a) allele was less active than its DBA/2 counterpart in inducing growth arrest of mouse plasmacytoma cell lines and preventing ras-induced transformation of NIH 3T3 cells, while the two p19(ARF) alleles displayed similar potencies in both assays. We propose that the BALB/c susceptibility/modifier locus, Pctr1, is an "efficiency" allele of the p16(INK4a) gene.

Epidermal growth factor receptor expression in neurofibromatosis type 1-related tumors and NF1 animal models.

We have found that EGF-R expression is associated with the development of the Schwann cell-derived tumors characteristic of neurofibromatosis type 1 (NF1) and in animal models of this disease. This is surprising, because Schwann cells normally lack EGF-R and respond to ligands other than EGF. Nevertheless, immunoblotting, Northern analysis, and immunohistochemistry revealed that each of 3 malignant peripheral nerve sheath tumor (MPNST) cell lines from NF1 patients expressed the EGF-R, as did 7 of 7 other primary MPNSTs, a non-NF1 MPNST cell line, and the S100(+) cells from each of 9 benign neurofibromas. Furthermore, transformed derivatives of Schwann cells from NF1(-/-) mouse embryos also expressed the EGF-R. All of the cells or cell lines expressing EGF-R responded to EGF by activation of downstream signaling pathways. Thus, EGF-R expression may play an important role in NF1 tumorigenesis and Schwann cell transformation. Consistent with this hypothesis, growth of NF1 MPNST lines and the transformed NF1(-/-) mouse embryo Schwann cells was greatly stimulated by EGF in vitro and could be blocked by agents that antagonize EGF-R function.

The Sos1 and Sos2 Ras-specific exchange factors: differences in placental expression and signaling properties.

Targeted disruption of both alleles of mouse sos1, which encodes a Ras-specific exchange factor, conferred mid-gestational embryonic lethality that was secondary to impaired placental development and was associated with very low placental ERK activity. The trophoblastic layers of sos1(-/-) embryos were poorly developed, correlating with high sos1 expression in wild-type trophoblasts. A sos1(-/-) cell line, which expressed readily detectable levels of the closely related Sos2 protein, formed complexes between Sos2, epidermal growth factor receptor (EGFR) and Shc efficiently, gave normal Ras.GTP and ERK responses when treated with EGF for < or =10 min and was transformed readily by activated Ras. However, the sos1(-/-) cells were resistant to transformation by v-Src or by overexpressed EGFR and continuous EGF treatment, unlike sos1(+/-) or wild-type cells. This correlated with Sos2 binding less efficiently than Sos1 to EGFR and Shc in cells treated with EGF for > or =90 min or to v-Src and Shc in v-Src-expressing cells, and with less ERK activity. We conclude that Sos1 participates in both short- and long-term signaling, while Sos2-dependent signals are predominantly short-term.

The tuberous sclerosis-1 (TSC1) gene product hamartin suppresses cell growth and augments the expression of the TSC2 product tuberin by inhibiting its ubiquitination.

We report here that overexpression of the tuberous sclerosis-1 (TSC1) gene product hamartin results in the inhibition of growth, as well as changes in cell morphology. Growth inhibition was associated with an increase in the endogenous level of the product of the tuberous sclerosis-2 (TSC2) gene, tuberin. As overexpression of tuberin inhibits cell growth, and hamartin is known to bind tuberin, these results suggested that hamartin stabilizes tuberin and this contributes to the inhibition of cell growth. Indeed, transient transfection of TSC1 increased the endogenous level of tuberin, and transient co-transfection of TSC1 with TSC2 resulted in higher tuberin levels. The stabilization was explained by the finding that tuberin is highly ubiquitinated in cells, while the fraction of tuberin that is bound to hamartin is not ubiquitinated. Co-expression of tuberin stabilized hamartin, which is weakly ubiquitinated, in transiently transfected cells. The amino-terminal two-thirds of tuberin was responsible for its ubiquitination and for stabilization of hamartin. A mutant of tuberin from a patient missense mutation of TSC2 was also highly ubiquitinated, and was unable to stabilize hamartin. We conclude that hamartin is a growth inhibitory protein whose biological effect is likely dependent on its interaction with tuberin.

Ras-specific exchange factor GRF: oligomerization through its Dbl homology domain and calcium-dependent activation of Raf.

The full-length versions of the Ras-specific exchange factors Ras-GRF1 (GRF1) and Ras-GRF2 (GRF2), which are expressed in brain and a restricted number of other organs, possess an ionomycin-dependent activation of Erk mitogen-activated protein kinase activity in 293T cells (C. L. Farnsworth et al., Nature 376:524-527, 1995; N. P. Fam et al., Mol. Cell. Biol. 17:1396-1406, 1996). Each GRF protein contains a Dbl homology (DH) domain. A yeast two-hybrid screen was used to identify polypeptides that associate with the DH domain of GRF1. In this screen, a positive cDNA clone from a human brain cDNA library was isolated which consisted of the GRF2 DH domain and its adjacent ilimaquinone domain. Deletion analysis verified that the two-hybrid interaction required only the DH domains, and mutation of Leu-263 to Gln (L263Q) in the N terminus of the GRF1 DH domain abolished the two-hybrid interaction, while a cluster of more C-terminally located mutations in the DH domain did not eliminate the interaction. Oligomers between GRF1 and GRF2 were detected in a rat brain extract, and forced expression of GRF1 and GRF2 in cultured mammalian cells formed homo- and hetero-oligomers. Introduction of the L263Q mutation in GRF1 led to a protein that was deficient in oligomer formation, while GRF1 containing the DH cluster mutations formed homo-oligomers with an efficiency similar to that of wild type. Compared to wild-type GRF1, the focus-forming activity on NIH 3T3 cells of the GRF1 DH cluster mutant was reduced, while the L263Q mutant was inactive. Both mutants were impaired in their ability to mediate ionomycin-dependent Erk activity in 293T cells. In the absence of ionomycin, 293T cells expressing wild-type GRF1 contained much higher levels of Ras-GTP than control cells; the increase in Erk activity induced by ionomycin in the GRF1-expressing cells also induced a concomitant increase in Raf kinase activity, but without a further increase in the level Ras-GTP. We conclude that GRF1 and GRF2 can form homo- and hetero-oligomers via their DH domains, that mutational inactivation of oligomer formation by GRF1 is associated with impaired biological and signaling activities, and that in 293T cells GRF1 mediates at least two pathways for Raf activation: one a constitutive signal that is mainly Ras-dependent, and one an ionomycin-induced signal that cooperates with the constitutive signal without further augmenting the level of GTP-Ras.

N terminus of Sos1 Ras exchange factor: critical roles for the Dbl and pleckstrin homology domains.

We have studied the functional importance of the N terminus of mouse Sos1 (mSos1), a ubiquitously expressed Ras-specific guanine nucleotide exchange factor whose C-terminal sequences bind Grb-2. Consistent with previous reports, addition of a myristoylation signal to mSos1 (MyrSos1) rendered it transforming for NIH 3T3 cells and deletion of the mSos C terminus (MyrSos1-deltaC) did not interfere with this activity. However, an N-terminally deleted myristoylated mSos1 protein (MyrSos1-deltaN) was transformation defective, although the protein was stable and localized to the membrane. Site-directed mutagenesis was used to examine the role of the Dbl and pleckstrin homology (PH) domains located in the N terminus. When mutations in the PH domain were introduced into two conserved amino acids either singly or together in MyrSos1 or MyrSos1-deltaC, the transforming activity was severely impaired. An analogous reduction in biological activity was seen when a cluster of point mutations was engineered into the Dbl domain. The mitogen-activation protein (MAP) kinase activities induced by the various Dbl and PH mutants of MyrSos1 correlated with their biological activities. When NIH 3T3 cells were transfected with a myristoylated Sos N terminus, their growth response to epidermal growth factor (EGF), platelet-derived growth factor, lysophosphatidic acid or serum was greatly impaired. The dominant inhibitory biological activity of the N terminus correlated with its ability to impair EGF-dependent activation of GTP-Ras and of MAP kinase, as well with the ability of endogenous Sos to form a stable complex with activated EGF receptors. The N terminus with mutations in the Dbl and PH domains was much less inhibitory in these biological and biochemical assays. In contrast to wild-type Sos1, nonmyristoylated versions of Sos1-deltaN and Sos1-deltaC did not form a stable complex with activated EGF receptors. We conclude that the Dbl and PH domains are critical for Sos function and that stable association of Sos with activated EGF receptors requires both the Sos N and C termini.

The Ras-specific exchange factors mouse Sos1 (mSos1) and mSos2 are regulated differently: mSos2 contains ubiquitination signals absent in mSos1.

We have compared aspects of the mouse sos1 (msos1) and msos2 genes, which encode widely expressed, closely related Ras-specific exchange factors. Although an msos1 plasmid did not induce phenotypic changes in NIH 3T3 cells, addition of a 15-codon myristoylation signal to its 5' end enabled the resulting plasmid, myr-sos1, to induce approximately one-half as many foci of transformed cells as a v-H-ras control. By contrast, an isogenic myr-sos2 plasmid, which was made by fusing the first 102 codons from myr-sos1 at homologous sequences to an intact msos2 cDNA, did not induce focal transformation directly, although it could form foci in cooperation with c-H-ras. Pulse-chase experiments indicated that the half-life of Sos1 in NIH 3T3 cells was greater than 18 h, while that of Sos2 was less than 3 h. While in vitro-translated Sos1 was stable in a rabbit reticulocyte lysate, Sos2 was degraded in the lysate, as were each of two reciprocal chimeric Sos1-Sos2 proteins, albeit at a slower rate. In the lysate, Sos2 and the two chimeric proteins could be stabilized by ATPgammaS. Unlike Sos1, Sos2 was specifically immunoprecipitated by antiubiquitin antibodies. In a myristoylated version, the chimeric gene encoding Sos2 at its C terminus made a stable protein in NIH 3T3 cells and induced focal transformation almost as efficiently as myr-msos1, while the myristoylated protein encoded by the other chimera was unstable and defective in the transformation assay. We conclude that mSos2 is much less stable than mSos1 and is degraded by a ubiquitin-dependent process. A second mSos2 degradation signal, mapped to the C terminus in the reticulocyte lysate, does not seem to function under the growth conditions of the NIH 3T3 cells.

Co-localization of the TSC2 product tuberin with its target Rap1 in the Golgi apparatus.

Tuberin is the protein product of the tuberous sclerosis-2 (TSC2) gene, which is associated with tuberous sclerosis (TSC), a human genetic syndrome characterized by the development of tumors in a variety of tissues. We have previously shown that tuberin is a widely expressed 180 kDa protein which exhibits specific GTPase activating activity in vitro towards the Ras-related Rap1 protein. In this study we have used affinity-purified antibodies against tuberin to analyse its expression in human and rat tissues and to examine its subcellular localization. Tuberin expression was detected in all adult human tissues tested, with the highest levels found in brain, heart and kidney, organs that are commonly affected in TSC patients. By contrast, in adult rats the highest levels of tuberin were found in brain, liver and testis. Indirect immunofluorescence of tuberin in various cultured cell lines revealed a punctate, mostly perinuclear staining pattern. Double-indirect immunofluorescence analysis with anti-tuberin sera and antisera against known Golgi markers (mannosidase-II and furin) revealed that the staining of tuberin was consistent with its localization in the stacks of the Golgi apparatus. In support of this, treatment of cells with brefeldin A, a drug known to cause disassembly of the Golgi apparatus, abolished the perinuclear staining of tuberin. Moreover, conventional and confocal immunofluorescence demonstrated co-localization of tuberin with Rap1, which has previously been localized to the Golgi apparatus. The co-localization of tuberin and Rap1 in vivo strengthens the likelihood that the in vitro catalytic activity of tuberin toward Rap1 plays a physiologically relevant role in the tumor suppressor function of tuberin.

SHC and GRB-2 are constitutively by an epidermal growth factor receptor with a point mutation in the transmembrane domain.

A single point mutation, Glu627--> Val, equivalent to the activating mutation in the Neu oncogene, was inserted in the transmembrane domain of the human epidermal growth factor (EGF) receptor. Unlike the wild type, Glu627-EGF receptor, transfected in NIH3T3 cells, gave rise to focal transformation and growth in agar even in the absence EGF. Constitutive activity of mutant EGF receptor amounted to 20% of that of wild type receptor stimulated by EGF. In addition, the mutant receptor was more sensitive to EGF, reaching maximum transforming activity at 5 ng/ml EGF. NIH3T3 cells expressing Glu627-EGF receptor showed a transformed phenotype and were not arrested in G0 upon serum deprivation. The mutant receptor was constitutively autophosphorylated, and several other cellular proteins were phosphorylated on tyrosine in absence of the ligand. Among these, the SHC adaptor protein was phosphorylated in absence of EGF, the other adaptor, GRB-2 was constitutively associated with the Glu627-EGF receptor in vivo and in vitro, and mitogen-activated protein kinase was constitutively phosphorylated. In contrast, other EGF receptor substrates, like phospholipase C gamma, were not phosphorylated in absence of EGF. The mutant receptor showed a higher sensitivity to cleavage by calpain both in absence and presence of EGF, appeared as a 170- and 150-kDa doublet in cell extracts, and a specific calpain inhibitor blocked the appearance of the 150-kDa form. Since the calpain cleavage site is located in the receptor cytoplasmic tail, this finding suggests that the Glu627 mutation induces a slightly different conformation in the EGF receptor intracellular domain. In conclusion, our data show that a point mutation in the EGF receptor transmembrane domain was able to constitutively activate the receptor and to induce transformation via constitutive activation of the Ras pathway.

Neurofibromin can inhibit Ras-dependent growth by a mechanism independent of its GTPase-accelerating function.

The NF1 gene, which is altered in patients with type 1 neurofibromatosis, has been postulated to function as a tumor suppressor gene. The NF1 protein product neurofibromin stimulates the intrinsic GTPase activity of active GTP-bound Ras, thereby inactivating it. Consistent with a tumor suppressor function, we have found that the introduction of NF1 in melanoma cell lines that are deficient in neurofibromin inhibited their growth and induced their differentiation. In addition, overexpression of neurofibromin in NIH 3T3 cells was growth inhibitory but did not alter the level of GTP.Ras in the cells. Transformation by v-ras, whose protein product is resistant to GTPase stimulation by neurofibromin, was inhibited in a cell line overexpressing neurofibromin, while transformation by v-raf was not altered. The results demonstrate that NF1 is a tumor suppressor gene that can inhibit Ras-dependent growth by a regulatory mechanism that is independent of neurofibromin's ability to stimulate Ras GTPase.

Regulated and constitutive activity by CDC25Mm (GRF), a Ras-specific exchange factor.

Serum stimulates cells to increase their proportion of Ras protein in the active GTP-bound state. We have recently identified four types (I to IV) of apparently full-length cDNAs from a single mammalian gene, called CDC25Mm or GRF, which is homologous to the Ras-specific exchange factor CDC25 of S. cerevisiae. The largest cDNA (type IV) is brain specific, with the other three classes, although they have distinct 5' ends, essentially representing progressive N-terminal deletions of this cDNA. When placed in a retroviral expression vector, all four types of cDNAs induced morphologic transformation of NIH 3T3 cells and an increase in the basal level of GTP.Ras. Serum stimulation of these transformants lead to a further increase in GTP.Ras only in cells expressing the type IV cDNA. Each type of GRF protein was found in cytosolic and membrane fractions, and the protein in each fraction could stimulate guanine nucleotide release from GDP.Ras in vitro. When NIH 3T3 cells and cells expressing the type IV protein were transfected with two versions of a mutant ras gene, one encoding membrane-associated Ras protein and the other encoding a cytosolic Ras protein, the basal levels of GTP bound to both forms of the mutant Ras protein were significantly higher in the cells expressing the type IV protein. However, serum increased the level of GTP bound to the membrane-associated mutant Ras protein in NIH 3T3 cells and in cells expressing the type IV protein but not in cells expressing the cytosolic version of the Ras protein. We conclude that each type of CDC25Mm induces cell transformation via the ability of its C terminus to stimulate guanine nucleotide exchange on Ras, the presence of N-terminal sequences is associated with a serum-dependent change in GTP.Ras, and the serum-dependent increase in GTP.Ras by exogenous CDC25Mm or by endogenous exchange factors probably requires membrane association of both Ras and the exchange factor.

Functional role of GTPase-activating protein in cell transformation by pp60v-src.

Morphological transformation of NIH 3T3 cells was observed following coexpression of a portion of the ras GTPase-activating protein (GAP) comprising the amino terminus (GAP-N) and a mutant of v-src (MDSRC) lacking the membrane-localizing sequence. Cells expressing either of these genes alone remained nontransformed. Coexpression of GAP-N with MDSRC did not alter the subcellular localization, kinase activity, or pattern of cellular substrates phosphorylated by the MDSRC product. In contrast to SHC, phospholipase C-gamma 1, and the p85 alpha phosphatidylinositol 3'-kinase subunit, the endogenous GAP product (p120GAP) was highly tyrosine-phosphorylated only in cells transformed by wild-type v-src. Furthermore, for transformation induced by wild-type v-src as well as by coexpression of MDSRC and GAP-N, a strict correlation was observed between cell transformation, elevated tyrosine phosphorylation of p62, p190, and a novel protein of 150 kDa, and complex formation between these proteins and p120GAP. As with cells transformed by wild-type v-src, the MDSRC plus GAP-N transformants remained dependent on endogenous Ras. The results suggest that tyrosine phosphorylation and complex formation involving p120GAP represent critical elements of cell transformation by v-src and that complementation of the cytosolic v-src mutant by GAP-N results, at least in part, from the formation of these complexes.

Transformation-specific interaction of the bovine papillomavirus E5 oncoprotein with the platelet-derived growth factor receptor transmembrane domain and the epidermal growth factor receptor cytoplasmic domain.

The bovine papillomavirus E5 transforming protein appears to activate both the epidermal growth factor receptor (EGF-R) and the platelet-derived growth factor receptor (PDGF-R) by a ligand-independent mechanism. To further investigate the ability of E5 to activate receptors of different classes and to determine whether this stimulation occurs through the extracellular domain required for ligand activation, we constructed chimeric genes encoding PDGF-R and EGF-R by interchanging the extracellular, membrane, and cytoplasmic coding domains. Chimeras were transfected into NIH 3T3 and CHO(LR73) cells. All chimeras expressed stable protein which, upon addition of the appropriate ligand, could be activated as assayed by tyrosine autophosphorylation and biological transformation. Cotransfection of E5 with the wild-type and chimeric receptors resulted in the ligand-independent activation of receptors, provided that a receptor contained either the transmembrane domain of the PDGF-R or the cytoplasmic domain of the EGF-R. Chimeric receptors that contained both of these domains exhibited the highest level of E5-induced biochemical and biological stimulation. These results imply that E5 activates the PDGF-R and EGR-R by two distinct mechanisms, neither of which specifically involves the extracellular domain of the receptor. Consistent with the biochemical and biological activation data, coimmunoprecipitation studies demonstrated that E5 formed a complex with any chimera that contained a PDGF-R transmembrane domain or an EGF-R cytoplasmic domain, with those chimeras containing both domains demonstrating the greatest efficiency of complex formation. These results suggest that although different domains of the PDGF-R and EGF-R are required for E5 activation, both receptors are activated directly by formation of an E5-containing complex.

Cell transformation by ras and regulation of its protein product.

We are studying the biological activity and regulation of mammalian Ras protein in tumours and in physiological signalling. We have shown that GAP (the GTPase-activating protein) is a potent negative regulator of normal Ras in cells. Reduction or loss of the NF1 gene product neurofibromin, in association with genetic abnormalities of the NF1 locus, has been identified in schwannoma cell lines from patients with neurofibromatosis and in melanoma and neuroblastoma lines from patients without neurofibromatosis. Although loss of neurofibromin in the schwannoma lines was associated with a high proportion of normal Ras protein in the active GTP-bound state, Ras-GTP appeared to be appropriately regulated in the melanoma and neuroblastoma lines, which contain normal levels of GAP. Therefore the GTPase-activating activity of neurofibromin is not essential for negative regulation of Ras in some cell types and the putative tumour suppressor function of neurofibromin in such cell types is independent of its GTPase-activating activity. Mitogen activation of Ras in fibroblasts is mediated primarily by exchange factors, which probably interact with a region on the Ras protein distinct from the region required for interaction with GAP. Multiple full-length cDNAs have identified a mouse gene whose products are related to yeast CDC25 guanine nucleotide exchange factor.

Mutant p53 can substitute for human papillomavirus type 16 E6 in immortalization of human keratinocytes but does not have E6-associated trans-activation or transforming activity.

Human papillomavirus type 16 (HPV16) E6 and E7 are selectively retained and expressed in HPV16-associated human genital tumors. E6 is active in several cell culture assays, including transformation of NIH 3T3 cells, trans activation of the adenovirus E2 promoter, and cooperation with E7 to immortalize normal human keratinocytes. Biochemically, the HPV16 E6 protein has been shown to bind to tumor suppressor protein p53 in vitro and induce its degradation in a rabbit reticulocyte lysate. To examine the relationship between the various biological activities of E6 and inactivation of p53, we tested the abilities of dominant negative mutants of p53 to substitute functionally for E6 in the three cell culture assays. While wild-type p53 inhibited keratinocyte proliferation, both mouse and human mutant p53s, in conjunction with E7, increased proliferation of the keratinocytes, resulting in generation of immortalized lines. However, in contrast to E6, mutant p53 was unable to induce transformation or trans activate the adenovirus E2 promoter in NIH 3T3 cells. These results suggest that inactivation of wild-type p53 is necessary for HPV-induced immortalization of human keratinocytes and that different or additional activities are required for E6-dependent transformation and trans activation of NIH 3T3 cells.

Abnormal regulation of mammalian p21ras contributes to malignant tumor growth in von Recklinghausen (type 1) neurofibromatosis.

Tumor cell lines derived from malignant schwannomas removed from patients with neurofibromatosis type 1 (NF1) have been examined for the level of expression of NF1 protein. All three NF1 lines examined expressed lower levels of NF1 protein than control cells, and the level in one line was barely detectable. The tumor lines expressed normal levels of p120GAP and p21ras. Although the p21ras proteins isolated from the tumor cells had normal (nonmutant) biochemical properties in vitro, they displayed elevated levels of bound GTP in vivo. The level of total cellular GAP-like activity was reduced in extracts from the tumor line that expresses very little NF1 protein. Introduction of the catalytic region of GAP into this line resulted in morphological reversion and lower in vivo GTP binding by endogenous p21ras. These data implicate NF1 protein as a tumor suppressor gene product that negatively regulates p21ras and define a "positive" growth role for ras activity in NF1 malignancies.

The bovine papillomavirus E5 oncogene can cooperate with ras: identification of p21 amino acids critical for transformation by c-rasH but not v-rasH.

We have previously used a series of insertion-deletion mutants of the mutationally activated v-rasH gene to identify several regions of the encoded protein that are dispensable for cellular transformation (B. M. Willumsen, A. G. Papageorge, H.-F. Kung, E. Bekesi, T. Robins, M. Johnsen, W. C. Vass, and D. R. Lowy, Mol. Cell. Biol. 6:2646-2654, 1986). To determine if some of these amino acids are more important for the biological activity of c-rasH, we have now tested many of the same insertion-deletion mutants in the c-rasH form for their ability to transform NIH 3T3 cells. Since the transforming activity of c-rasH is low, we have used cotransfection with the bovine papillomavirus (BPV) genome to develop a more sensitive transformation assay for c-rasH mutants. The increased sensitivity of the assay, which is seen both in focal transformation and in anchorage-independent growth, is mediated by cooperation between the BPV E5 gene and ras. E5-dependent cooperation was seen for v-rasH as well as for c-rasH, which suggests that the major effect of E5 was to increase the susceptibility of the cell to transformation to a given level of ras activity. The cooperation assay was used to test the potential importance, in c-rasH, of codons 93 to 108, 123 to 130, and 166 to 183, which were nonessential for v-rasH transformation. Relative to the respective transforming activity of wild-type c-rasH and v-rasH, mutants with lesions in codons 102 and 103 were significantly less active in their c-rasH forms than in their v-rasH forms. We conclude that a region including amino acids 102 and 103 encodes a function that is more critical to c-rasH than to v-rasH. Guanine nucleotide exchange is one function that is compatible with such a phenotype.

Heterogeneous amino acids in Ras and Rap1A specifying sensitivity to GAP proteins.

Guanosine triphosphatase (GTPase) activity of Ras is increased by interaction with Ras-GAP (GTPase-activating protein) or with the GAP-related domain of the type 1 neurofibromatosis protein (NF1-GRD), but Ras is not affected by interaction with cytoplasmic and membrane forms of Rap-GAP; Rap1A, whose effector function can suppress transformation by Ras, is sensitive to both forms of Rap-GAP and resistant to Ras-GAP and NF1-GRD. A series of chimeric proteins composed of portions of Ras and Rap were constructed; some were sensitive to Ras-GAP but resistant to NF1-GRD, and others were sensitive to cytoplasmic Rap-GAP but resistant to membrane Rap-GAP. Sensitivity of chimeras to Ras-GAP and cytoplasmic Rap-GAP was mediated by amino acids that are carboxyl-terminal to the effector region. Residues 61 to 65 of Ras conferred Ras-GAP sensitivity, but a larger number of Rap1A residues were required for sensitivity to cytoplasmic Rap-GAP. Chimeras carrying the Ras effector region that were sensitive only to Ras-GAP or only to cytoplasmic Rap-GAP transformed NIH 3T3 cells poorly. Thus, distinct amino acids of Ras and Rap1A mediate sensitivity to each of the proteins with GAP activity, and transforming potential of Ras and sensitivity of Ras to Ras-GAP are at least partially independent properties.