RASless MEFs as a Tool to Study RAS-Dependent and RAS-Independent Functions.

RAS proteins are key players in multiple cellular processes. To study the role of RAS proteins individually or in combination, we have developed MEFs that can be rendered RASless, i.e., devoid of all endogenous RAS isoforms. These cells have significantly contributed to our understanding of the requirements for RAS functions in cell proliferation as well as their implications in diverse cellular processes. Here, we describe methods using RASless MEFs to study RAS-dependent cellular activities with special emphasis on proliferation. We provide the details to identify inducers of RAS-independent proliferation in colony assays. We recommend following these stringent guidelines to avoid false-positive results. Moreover, this protocol can be adapted to generate RASless MEFs ectopically expressing RAS variants to interrogate their function in the absence of endogenous RAS isoforms or to perform experiments in the absence of RAS. Finally, we also describe protocols to generate and use RASless MEFs for cell cycle analyses using the FUCCI cell cycle indicator.

Review article: panitumumab--a fully human anti-EGFR monoclonal antibody for treatment of metastatic colorectal cancer.

BACKGROUND: Panitumumab is a fully human monoclonal IgG2 antibody targeting the epidermal growth factor receptor (EGFR). AIM: To review the efficacy of panitumumab in the treatment of metastatic colorectal cancer (mCRC). METHODS: Available literature identified from PubMed and conference websites was reviewed. RESULTS: In phase 2-3 studies, panitumumab monotherapy achieved objective response rates (ORRs) of 8-13% in relapsed/refractory EGFR-expressing mCRC. In a randomized phase 3 study (463 patients), panitumumab almost halved the risk of disease progression/death vs. a control group receiving only best supportive care (hazard ratio 0.54; 95% CI: 0.44-0.66; P < 0.0001). Objective response was achieved in 22/231 (10%) patients randomized to panitumumab--and also in 20/176 (11%) patients assigned to the control group who received panitumumab in a separate crossover protocol after disease progression. Response was confined to patients with tumours harbouring wild-type KRAS (ORR approximately equal to 20%). Panitumumab is also being evaluated in earlier lines of treatment. Panitumumab monotherapy is generally well tolerated; the most common toxicities are skin toxicity (approximately equal to 90%) and diarrhoea (<30%). Development of anti-panitumumab antibodies (0.3% by ELISA) and grade 3-4 infusion reactions (<1%) are rare. CONCLUSION: Panitumumab is an effective monotherapy option for patients with relapsed/refractory EGFR-expressing mCRC harbouring wild-type KRAS.

Future treatment to lessen exacerbations of chronic obstructive pulmonary disease.

Therapies currently used to reduce exacerbations of chronic obstructive pulmonary disease (COPD) are compounds used almost entirely for asthma therapy. A notable exception is tiotropium, a long-acting parasympatholytic agent. This compound and its precursor, iprotropium, are only occasionally used for asthma therapy. Likewise, leukotriene-modifying drugs are used occasionally for the treatment of COPD. In neither circumstance is there agency-approved indication for these particular cross-over therapies, but the use of long-acting beta(2)-adrenergic compounds and high-solubility inhaled steroids is a mainstay for therapy in both asthma and COPD. Similarly, theophylline, although less often used for either process, is therapeutically applicable to both asthma and COPD. Although overlap syndromes point to the occurrence of a common pathway in some cases, the inflammatory process for asthma and chronic obstructive pulmonary disease (COPD) differs substantially in most cases. Hence, the application of therapies designed to relax airway smooth muscle and ameliorate asthmatic inflammation lacks a therapeutic rationale for a disease characterized by predominant neutrophilic inflammation occurring in the small airways and alveoli. By definition, COPD is poorly reversible airflow obstruction; hence, the use of drugs designed to relax airway smooth muscle is somewhat counterintuitive and does not address the pathophysiological process of the disease.

Blockade of airway inflammation and hyperresponsiveness by HIV-TAT-dominant negative Ras.

We have reported previously that HIV-TAT-dominant negative (dn) Ras inhibits eosinophil adhesion to ICAM-1 after activation by IL-5 and eotaxin. In this study, we evaluated the role of Ras in Ag-induced airway inflammation and hyperresponsiveness by i.p. administration into mice of dnRas, which was fused to an HIV-TAT protein transduction domain (TAT-dnRas). Uptake of TAT-dnRas (t(1/2) = 12 h) was demonstrated in leukocytes after i.p. administration. OVA-sensitization significantly increased eosinophil and lymphocyte numbers in bronchoalveolar lavage fluid 24 h after final challenge. Treatment of animals with 3-10 mg/kg TAT-dnRas blocked the migration of eosinophils from 464 +/- 91 x 10(3)/ml to 288 +/- 79 x 10(3)/ml with 3 mg/kg of TAT-dnRas (p < 0.05), and further decreased to 116 +/- 63 x 10(3)/ml after 10 mg/kg TAT-dnRas (p < 0.01). Histological examination demonstrated that inflammatory cell infiltration (largely eosinophils and mononuclear cells) and mucin production around the airways caused by OVA were blocked by TAT-dnRas. OVA challenge also caused airway hyperresponsiveness to methacholine, which was dose dependently blocked by treatment with TAT-dnRas. TAT-dnRas also blocked Ag-induced IL-4 and IL-5, but not IFN-gamma, production in lung tissue. Intranasal administration of IL-5 caused eosinophil migration into the airway lumen, which was attenuated by pretreatment with TAT-dnRas. By contrast, TAT-green fluorescent protein or dnRas lacking the TAT protein transduction domain did not block airway inflammation, cytokine production, or airway hyperresponsiveness. We conclude that Ras mediates Th2 cytokine production, airway inflammation, and airway hyperresponsiveness in immune-sensitized mice.

Kimchi and an active component, beta-sitosterol, reduce oncogenic H-Ras(v12)-induced DNA synthesis.

The Korean fermented vegetable food, kimchi, has been demonstrated to have anticancer functional properties. This study examined the effect of kimchi samples, methanol extracts of commercially grown baechu cabbage kimchi (CK) and organically grown baechu cabbage kimchi (OK), as well as the dichloromethane fraction (DCM fr.) from CK, and the active compound (AC), which has been identified as largely beta-sitosterol, from DCM fr., on the Ras-dependent signaling pathway. CK, OK, and DCM fr. exhibited a greater inhibition against the proliferation of Rat2 fibroblasts transformed with Ras(v12) (HO6) than parental Rat2 fibroblasts. In addition, OK and DCM fr. showed a higher inhibitory effect than CK. Furthermore, we employed the single-cell microinjection technique, combined with 3-bromo-5'-deoxyuridine incorporation, to examine the effects of kimchi samples on DNA synthesis induced by microinjected oncogenic Ras(v12). When the DCM fr. and AC were used to treat Rat1 fibroblasts overexpressing human insulin receptors (HIRc-B) and microinjected with oncogenic H-Ras(v12), the DNA synthesis of injected cells was decreased, suggesting that kimchi might block the signaling pathway of oncogenic Ras(v12), thus preventing the proliferation of transformed cells. This study provides additional evidence that kimchi and its active components, including beta-sitosterol, have potential in both the prevention and treatment of cancer, and presents convincing evidence that the anticancer effects may be a result of an inhibition of Ras oncogene signaling.

Development of a murine mutant Ras CD8+ CTL peptide epitope variant that possesses enhanced MHC class I binding and immunogenic properties.

We recently identified a murine mutant Ras p21 CD8+ CTL epitope reflecting residues 4 to 12, containing the mutation of Gly to Val at codon 12, that bound weakly to H-2Kd in vitro and generated a weak primary CTL response in immunized BALB/c mice. Here, we explored the hypothesis that specific modifications to the Ras4-12 peptide sequence can improve MHC binding, leading to enhanced immunogenicity without altering immune specificity. We synthesized Ras4-12 peptides in which Val at residue 12 was replaced with the more dominant H-2Kd C-terminus anchor residue Leu or Ile. In functional H-2Kd binding assays, Ras4-12(L12 or I12) peptide variants competed more effectively than the Ras4-12(V12) peptide. Ras4-12(L12 or I12) peptide variants enhanced both in vitro cytotoxicity and proliferation responses of anti-Ras4-12 CTL compared with the mutant Ras4-12(V12) peptide. Additionally, the Ras4-12(L12) peptide variant induced a quantitatively greater T cell response in vivo compared with that produced by Ras4-12(V12) as determined by IFN-gamma production. Mice immunized with Ras4-12(L12) peptide elicited CD8+ CTL activity specific for target cells presenting the Ras4-12(V12) epitope exogenously and endogenously. Moreover, both anti-Ras4-12(V12)-derived and anti-Ras4-12(L12)-derived CTL lines were similar insofar as their TCR usage and amino acid contact residues in the Ras4-12(V12) peptide. These experiments demonstrate that modifications can be introduced in tumor-specific peptide epitopes to enhance both in vitro and in vivo immunogenicity. The design of oncogene-specific peptide epitope variants as immunogens may accelerate the generation of anti-tumor T cell responses for cancer immunotherapy.

Regulation of scatter factor/hepatocyte growth factor responses by Ras, Rac, and Rho in MDCK cells.

Scatter factor/hepatocyte growth factor (SF/HGF) stimulates the motility of epithelial cells, initially inducing centrifugal spreading of cell colonies followed by disruption of cell-cell junctions and subsequent cell scattering. These responses are accompanied by changes in the actin cytoskeleton, including increased membrane ruffling and lamellipodium extension, disappearance of peripheral actin bundles at the edges of colonies, and an overall decrease in stress fibers. The roles of the small GTP-binding proteins Ras, Rac, and Rho in regulating responses to SF/HGF were investigated by microinjection. Inhibition of endogenous Ras proteins prevented SF/HGF-induced actin reorganization, spreading, and scattering, whereas microinjection of activated H-Ras protein stimulated spreading and actin reorganization but not scattering. When a dominant inhibitor of Rac was injected, SF/HGF- and Ras-induced spreading and actin reorganization were prevented, although activated Rac alone did not stimulate either response. Microinjection of activated Rho inhibited spreading and scattering, while inhibition of Rho function led to the disappearance of stress fibers and peripheral bundles but did not prevent SF/HGF-induced motility. We conclude that Ras and Rac act downstream of the SF/HGF receptor p190Met to mediate cell spreading but that an additional signal is required to induce scattering.