Biomedical subjects
Publications and source records attributed to F McCormick.
Ras proteins activate a signaling cascade through direct binding of the serine/threonine kinase Raf. They also activate additional signaling pathways that are essential for full biological activity. Candidate effectors for these pathways include RalGDS and phosphatidyl inositol 3' kinase, as well as several other Ras binding proteins the biochemical and biological properties of which are poorly understood.
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The 55-kilodalton (kDa) protein from the E1B-region of adenovirus binds to and inactivates the p53 gene, which is mutated in half of human cancers. We have previously shown that the replication and cytopathogenicity of an E1B, 55-kDa gene-attenuated adenovirus, ONYX-015, is blocked by functional p53 in RKO and U20S carcinoma lines. We now report that normal human cells were highly resistant to ONYX-015-mediated, replication-dependent cytolysis. In contrast, a wide range of human tumor cells, including numerous carcinoma lines with either mutant or normal p53 gene sequences (exons 5-9), were efficiently destroyed. Antitumoral efficacy was documented following intratumoral or intravenous administration of ONYX-015 to nude mouse-human tumor xenografts; efficacy with ONYX-015 plus chemotherapy (cisplatin, 5-fluorouracil) was significantly greater than with either agent alone.
The Rho family members Cdc42, Rac, and Rho play a central role in the organization of the actin cytoskeleton and regulate transcription. Whereas Rac and Rho have been implicated in transformation by oncogenic Ras, the role of Cdc42 in this process remains unknown. In this study, we found that Rat1 fibroblasts expressing constitutively active V12-Cdc42 were anchorage independent and proliferated in nude mice but failed to show enhanced growth in low serum. Similar to V12-Rac1-expressing Rat1 fibroblasts, V12-Cdc42 lines displayed a high frequency of multinucleated cells. Interestingly, coexpression of dominant negative N17-Rac1 blocked the V12-Cdc42-induced multinucleated phenotype but not growth in soft agar, indicating that Cdc42 controls anchorage independence in a Rac-independent fashion. We also showed that dominant negative N17-Cdc42 inhibited Ras focus formation and anchorage-independent growth and caused reversion of the transformed morphology, indicating that Cdc42 is necessary for Ras transformation. N17-Cdc42 caused only partial inhibition of Ras-induced low-serum growth, however. In contrast, whereas N17-Rac1 also effectively inhibited Ras-induced anchorage independence, it did not revert the morphology of Ras-transformed cells. N17-Rac1 strongly inhibited low-serum growth of Ras-transformed cells, however. Together, these data provide a novel function for Cdc42 in cell proliferation and indicate that Cdc42 and Rac play distinct roles in growth control and Ras transformation.
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A novel alteration in exon 1 of KRAS was detected by single strand conformational polymorphism analysis of DNA amplified from the bone marrow of a 4-year-old child with myeloid leukemia. Sequencing of this mutant allele revealed an insertion of three nucleotides between codons 10 and 11 resulting in an in-frame insertion of glycine. Expression of the mutant protein in NIH 3T3 cells caused cellular transformation, and expression in COS cells activated the Ras-mitogen-activated protein kinase signaling pathway. Surprisingly, Ras.GTP levels measured in COS cells established that this novel mutant accumulates to 90% in the GTP state, considerably higher than a residue 12 mutant. Biochemical analysis confirmed that the higher Ras.GTP levels correspond to a dramatic decrease in intrinsic GTP hydrolysis as well as resistance to GTPase-activating proteins. This mutation is the first dominant Ras mutation found in human cancer that does not involve residues 12, 13, or 61, and its biochemical properties should help elucidate the mechanism of oncogenic activation.
Rat-1 fibroblasts were used to study the role of the sustained activation of extracellular signal-regulated kinase 1 (ERK1) in lysophosphatidic acid (LPA)-stimulated mitogenic signalling. Mitogenic doses of LPA, like serum, stimulated biphasic, sustained, ERK activation that persisted towards the G1/S boundary. The EC50 for LPA-stimulated ERK activation after 10 min, the time of peak response, was 2 orders of magnitude to the left of that for the sustained response after 3 h or that for DNA synthesis after 22 h, with the result that non-mitogenic doses stimulated a maximal peak response but no second phase. To complement these studies, we examined the role of different signal pathways in regulating the sustained and acute phases of ERK activation using defined biochemical inhibitors and mimetics. Activation of protein kinase C and Ca2+ fluxes played a minor and transient role in regulation of ERK1 activity by LPA in Rat-1 cells. Sustained ERK1 activation stimulated by LPA was completely inhibited by pertussis toxin, whereas the early peak response was only partly affected; this is correlated with the specific inhibition of LPA-stimulated DNA synthesis by pertussis toxin. The selective tyrosine kinase inhibitor herbimycin A completely inhibited sustained ERK1 activation by LPA but, again, the early phase of the response was only partially inhibited. In addition, low doses of staurosporine inhibited ERK1 activation by LPA. The effects of herbimycin A and staurosporine were selective for the response to LPA but did not affect that to epidermal growth factor. The results suggest a strong correlation between sustained ERK1 activation and DNA synthesis in LPA-stimulated Rat-1 cells. Furthermore, the two discrete phases of ERK activation by LPA are regulated by a combination of at least two different signalling pathways; the sustained activation of ERK1 in Rat-1 cells proceeds via a G1- or Gzero-mediated pathway which may also involve a tyrosine kinase.
The role of protein kinase C (PKC) in inflammation, mitogenesis, and differentiation has been deduced in part through the use of a variety of PKC inhibitors. Two widely used inhibitors are the structurally related compounds GF109203X and Ro-31-8220, both of which potently inhibit PKC activity and are believed to be highly selective. While using GF109203X and Ro-31-8220 to address the role of PKC in immediate early gene expression, we observed striking differential effects by each of these two compounds. Growth factors induce the expression of the immediate early gene products MAP kinase phosphatase-1 (MKP-1), c-Fos and c-Jun. Ro-31-8220 inhibits growth factor-stimulated expression of MKP-1 and c-Fos but strongly stimulated c-Jun expression, even in the absence of growth factors. GF109203X displays none of these properties. These data suggest that Ro-31-8220 may have other pharmacological actions in addition to PKC inhibition. Indeed, Ro-31-8220 strongly stimulates the stress-activated protein kinase, JNK1. Furthermore, Ro-31-8220 apparently activates JNK in a PKC-independent manner. Neither the down-regulation of PKC by phorbol esters nor the inhibition of PKC by GF109203X affected the ability of Ro-31-8220 to activate JNK1. These data suggest that, in addition to potently inhibiting PKC, Ro-31-8220 exhibits novel pharmacological properties which are independent of its ability to inhibit PKC.
The human adenovirus E1B gene encodes a 55-kilodalton protein that inactivates the cellular tumor suppressor protein p53. Here it is shown that a mutant adenovirus that does not express this viral protein can replicate in and lyse p53-deficient human tumor cells but not cells with functional p53. Ectopic expression of the 55-kilodalton EIB protein in the latter cells rendered them sensitive to infection with the mutant virus. Injection of the mutant virus into p53-deficient human cervical carcinomas grown in nude mice caused a significant reduction in tumor size and caused complete regression of 60 percent of the tumors. These data raise the possibility that mutant adenoviruses can be used to treat certain human tumors.
PEA3, a member of the Ets family of transcriptional regulatory proteins, binds to the PEA3 promoter element and stimulates transcription through this site. The activity of the PEA3 element is regulated by mitogens, activated receptor tyrosine kinases, and oncogenic members of the Ras signal transduction pathway. However, it is not clear whether PEA3 mediates transcriptional regulation by these agents because a number of different Ets proteins can functionally interact with the PEA3 element. To specifically learn whether the activity of PEA3 is regulated, we investigated the ability of constitutively-activated Ras (Ha-RasV12) and signaling proteins downstream of Ras to alter PEA3-dependent reporter gene expression in COS cells. Ha-RasV12 and activated proteins in both the extra-cellular regulated kinase (ERK) and the stress-activated protein kinase (SAPK) or Jun N-terminal kinase (JNK) cascades independently stimulated PEA3-mediated gene expression. Ha-RasV12 stimulation of PEA3 activity was reduced by dominant-negative mutants in each of these protein kinase cascades, suggesting that Ras activates PEA3 through both pathways. Furthermore, the ability of unique activators of each kinase cascade to stimulate PEA3-dependent gene expression was selectively reduced by dominant-negative mutants within the homologous but not the heterologous pathway. Hence two distinct mitogen-activated protein kinase (MAPK) cascades regulate PEA3 activity. PEA3 was phosphorylated in vivo at serine residues consistent with the possibility that it may be a direct target of MAPKs.
We have previously demonstrated that growth inhibition of untransformed intestinal epithelial cells by transforming growth factor beta1 (TGFbeta) and TGFbeta2 was associated with a rapid activation of both Ras and extracellular signal-regulated kinase 1 (Erk1) (Mulder, K. M., and Morris, S. L. (1992) J. Biol. Chem. 267, 5029-5031; Hartsough, M. T., and Mulder, K. M. (1995) J. Biol. Chem. 270, 7117-7124). In order to determine whether Ras was required for TGFbeta regulation of both Erk1 and downstream components associated with TGFbeta-mediated growth inhibition, the intestinal epithelial cell (IEC) line IEC 4-1 was transfected with a vector containing a dominant-negative mutant of Ras (RasN17) under the control of an inducible metallothionein promoter. Using two different RasN17-transfected clones treated with ZnCl2, we demonstrate here that induction of Ras expression by at least 4-fold completely abrogated the TGFbeta-mediated activation of Erk1. Moreover, the RasN17-mediated reversal of the TGFbeta effect on Erk1 was dependent upon the level of expression of the dominant-negative protein. ZnCl2 treatment of control cells transfected with the empty vector did not alter Ras expression or the activation of Erk1 by TGFbeta. In order to determine whether the activation of Ras by TGFbeta was required for the growth inhibitory effect of TGFbeta, we examined TGFbeta2 effects on Cdk2-associated histone H1 kinase activity, cyclin A protein expression levels, and DNA synthesis in two intestinal epithelial cell clones transfected with RasN17. In cells expressing RasN17, we observed a 50% reversal of the inhibition of Cdk2 activity, a 78% reversal of the down-regulation of cyclin A protein expression, and a 21% reversal of the inhibition of DNA synthesis by TGFbeta. Collectively, these results indicate that Ras activation is obligatory for TGFbeta-mediated activation of Erk1, whereas it is partially required for the growth inhibitory effect of TGFbeta.
In response to stimulation with epidermal growth factor (EGF), the guanine nucleotide exchange factor human SOS1 (hSOS1) promotes the activation of Ras by forming a complex with Grb2 and the human EGF receptor (hEGFR). hSOS1 was phosphorylated in cells stimulated with EGF or phorbol 12-myristate 13-acetate or following co-transfection with activated Ras or Raf. Co-transfection with dominant negative Ras resulted in a decrease of EGF-induced hSOS1 phosphorylation. The mitogen-activated protein kinase (MAPK) phosphorylated hSOS1 in vitro within the carboxyl-terminal proline-rich domain. The same region of hSOS1 was phosphorylated in vivo, in cells stimulated with EGF. Tryptic phosphopeptide mapping showed that MAPK phosphorylated hSOS1 in vitro on sites which were also phosphorylated in vivo. Phosphorylation by MAPK did not affect hSOS1 binding to Grb2 in vitro. However, reconstitution of the hSOS1-Grb2-hEGFR complex showed that phosphorylation by MAPK markedly reduced the ability of hSOS1 to associate with the hEGFR through Grb2. Similarly, phosphorylated hSOS1 was unable to form a complex with Shc through Grb2. Thus phosphorylation of hSOS1, by affecting its interaction with the hEGFR or Shc, down-regulates signal transduction from the hEGFR to the Ras pathway.
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Replication-competent viruses are used as selective cancer therapeutics and the mechanisms leading to tumor-specific replication and antitumoral efficacy are now becoming apparent. The specific viruses in development include tumor-targeting herpes simplex viruses, autonomous parvoviruses, Newcastle disease viruses and adenovirus. Information is also available on antiviral immunology and viral defenses against host-mediated immunity. This approach has many potential attributes, in addition to potential hurdles that must be overcome.
Individuals with neurofibromatosis type 1 (NF1) are predisposed to certain cancers including juvenile chronic myelogenous leukaemia (JCML). The NF1 tumour-suppressor gene encodes a protein (neurofibromin) that accelerates GTP hydrolysis on Ras proteins. Here we show that primary leukaemic cells from children with NF1 show a selective decrease in NF1-like GTPase activating protein (GAP) activity for Ras but retain normal cellular GAP activity. Leukaemic cells also show an elevated percentage of Ras in the GTP-bound conformation. JCML cells are hypersensitive to granulocyte-macrophage colony stimulating factor (GM-CSF), and we observed a similar pattern of aberrant growth in haematopoietic cells from Nf1-/- mouse embryos. These data define a specific role for neurofibromin in negatively regulating GM-CSF signaling through Ras in haematopoietic cells and they suggest that hypersensitivity to GM-CSF may be a primary event in the development of JCML.
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