Raf-1 Expression Research Articles

Surprises from Raf-1 Knockout

Ehrenreiter et al. explored the signaling roles of the Raf-1 protein kinase by creating mice that lacked Raf-1 expression in the epidermis. Although Raf-1 can function in activation of mitogen-activated protein kinases, phosphorylation of ERK was not altered in the keratinocytes lacking Raf-1. Instead, a critical role of Raf-1 in wound healing and cell migration was revealed. The development of the epidermis was normal in the knockout animals, but wound healing in vivo was strongly inhibited, as was keratinocyte migration in Boyden chamber assays. The defects were similar to those observed in cells lacking Rok-α (Rho-associated kinase, also called ROCK-II). Indeed Rok-α activity was increased in the knockout cells, and treatment of knockout fibroblasts with a Rok inhibitor or transfection with a catalytically inactive mutant of Rok-α restored normal morphology and migration of the cells. Rok-α was also mislocalized to the plasma membrane in the cells lacking Raf-1. Endogenous Raf-1 and Rok-α were shown to interact by immunoprecipitation of the proteins together from migrating wild-type cells. A catalytically inactive form of Raf-1 could also rescue the knockout cells, indicating that the physical presence of Raf-1, rather than its kinase activity, was required. K. Ehrenreiter, D. Piazzolla, V. Velamoor, I. Sobczak, J. V. Small, J. Takeda, T. Leung, M. Baccarini, Raf-1 regulates Rho signaling and cell migration. J. Cell Biol. 168 , 955-964 (2005). [Abstract] [Full Text]

Electromagnetic fields at mobile phone frequency induce apoptosis and inactivation of the multi‐chaperone complex in human epidermoid cancer cells

The exposure to non-thermal microwave electromagnetic field (MW-EMF) at 1.95 MHz, a frequency used in mobile communication, affects the refolding kinetics of eukaryotic proteins (Mancinelli et al., 2004). On these basis we have evaluated the in vivo effect of MW-EMF in human epidermoid cancer KB cells. We have found that MW-EMF induces time-dependent apoptosis (45% after 3 h) that is paralleled by an about 2.5-fold decrease of the expression of ras and Raf-1 and of the activity of ras and Erk-1/2. Although also the expression of Akt was reduced its activity was unchanged likely as a consequence of the increased expression of its upstream activator PI3K. In the same experimental conditions an about 2.5-fold increase of the ubiquitination of ras and Raf-1 was also found and the addition for 12 h of proteasome inhibitor lactacystin at 10 microM caused an accumulation of the ubiquitinated isoforms of ras and Raf-1 and counteracted the effects of MW-EMF on ras and Raf-1 expression suggesting an increased proteasome-dependent degradation induced by MW-EMF. The exposure of KB cells to MW-EMF induced a differential activation of stress-dependent pathway with an increase of JNK-1 activity and HSP70 and 27 expression and with a reduction of p38 kinase activity and HSP90 expression. The overexpression of HSP90 induced by transfection of KB cells with a plasmid encoding for the factor completely antagonized the apoptosis and the inactivation of the ras --> Erk-dependent survival signal induced by MW-EMF. Conversely, the inhibition of Erk activity induced by 12 h exposure to 10 mM Mek-1 inhibitor U0126 antagonized the effects induced by HSP90 transfection on apoptosis caused by MW-EMF. In conclusion, these results demonstrate for the first time that MW-EMF induces apoptosis through the inactivation of the ras --> Erk survival signaling due to enhanced degradation of ras and Raf-1 determined by decreased expression of HSP90 and the consequent increase of proteasome dependent degradation.

Raf‐1 expression may influence progression to androgen insensitive prostate cancer

Recent evidence has implicated the MAP kinase pathway with the development of androgen insensitive prostate cancer (AIPC). We have previously reported gene amplification of critical members of this pathway with the development of androgen insensitive disease. Protein expression of Raf-1 was analyzed using immunohistochemistry (IHC) in a database of 65 paired tumor specimens obtained before and after the development of AIPC and correlated with other members of the pathway. Patients whose Raf-1 expression rose with development of AIPC had a significantly shorter median time to biochemical relapse compared to those whose expression fell or remained unchanged (1.16 vs. 2.62 years, P = 0.0005). In AIPC tumors, expression of Raf-1 correlated significantly with expression of HER2 and with expression of c-fos. We conclude that the HER2/Raf-1/AP-1 axis may promote the development of AIPC, leading to early relapse. Members of the pathway may act as novel therapeutic targets for patients.

Quantification of raf antisense oligonucleotide (rafAON) in biological matrices by LC-MS/MS to support pharmacokinetics of a liposome-entrapped rafAON formulation

An LC-MS/MS method was developed to quantify an antisense oligonucleotide against Raf-1 expression (rafAON) in monkey and mouse plasma and in mouse tissue homogenates from animals dosed with a liposome-entrapped rafAON easy-to-use formulation (LErafAON-ETU) intended for use as an antineoplastic agent. RafAON was extracted from mouse and monkey plasma using solid-phase extraction. Tissues were homogenized and sample cleanup was achieved by protein precipitation. RafAON and the internal standard (IS) were separated on a Hamilton PRP-1 column and quantified by tandem mass spectrometry using an electrospray source in negative ion mode. The total run time was 4.0 min. The peak areas of two rafAON transitions were summed and plotted against the peak area of an IS transition to generate the standard curve. In monkey plasma the linear range was 50-10,000 ng/mL, and in mouse plasma it was 25-5000 ng/mL. The lower limit of quantification was 500 ng/mL (10 microg/g tissue) in heart, kidney, liver, lung and spleen homogenates, and the standard curve was linear up to 10,000 ng/mL. Accuracy, precision and stability were evaluated and found to be acceptable in all three matrices. The assay was used to support pharmacokinetics and tissue distribution studies of LErafAON-ETU in mice and monkeys.

The role of Raf-1 in radiation resistance of human hepatocellular carcinoma Hep G2 cells

Development of radiation resistance is one of the major reasons that cancer cells do not respond to radiotherapy and the mechanism for resistance is still not clear. Two sublines of human hepatocellular carcinoma Hep G2 cells were established from cells that survived two different irradiation regimes, 2 Gy for 10 days or 10 Gy for 2 days, respectively. Using MTT assay, the radiation conditioned cells were found to be more resistant to gamma-irradiation and have a greater extent of potentially lethal damage repair (PLDR) for radiation than the parent cells. By Western blot analysis, the radiation-conditioned cells were found to overexpress Raf-1 which is known to regulate the radiation resistance of cells. Inhibition of Raf-1 expression by antisense oligonucleotides increased the radiation sensitivity of the radiation-conditioned cells while inhibitors of Ras (L744,832), PI3K (LY294002) and p38 (SB203580) had no effect. Moreover, antisense Raf-1 oligonucleotides also decreased the radiation induced PLDR capacity of the radiation conditioned cells. It is therefore suggested that Raf-1 may induce radiation resistance through an increase in radiation induced PLDR capacity in Hep G2 cells.

Novel actions of tyrphostin AG 879: inhibition of RAF-1 and HER-2 expression combined with strong antitumoral effects on breast cancer cells

Binding of growth factors to cell surface receptors activates protein tyrosine kinases (PTKs) that initiate cascades of downstream signaling events including the mitogen-activated protein (MAP) kinase cascade. This study reports that the PTK inhibitor AG 879 inhibits proliferation of human breast cancer cells through an effect involving inhibition of MAP kinase activation, but which cannot be explained by effects of AG 879 on its known PTK targets. Instead, AG 879 markedly inhibits expression of the RAF-1 gene, which encodes an upstream MAP kinase kinase kinase. Additionally, expression of HER-2, but not of other genes tested, is inhibited by this compound. These novel effects have to be considered when using AG 879 as a TRK-A and HER-2 inhibitor but may have useful therapeutic implications.

Enhanced therapeutic effects of doxorubicin and paclitaxel in combination with liposome-entrapped ends-modified raf antisense oligonucleotide against human prostate, lung and breast tumor models

Raf-1 protein kinase plays an important role in cell growth, proliferation and cell survival. We have previously described the use of liposome-entrapped antisense raf oligonucleotide (LErafAON) to inhibit Raf-1 expression resulting in tumor growth inhibition and radiosensitization. The present study was undertaken to evaluate the chemosensitization effects of LErafAON in combination with doxorubicin or paclitaxel on a panel of human tumor xenografts. LErafAON (25.0 mg/kg i.v. x 10) displayed significant antitumor activity (P<0.05) when administered as a single agent in prostate (PC-3), lung (A549) and breast (MDA-MB 231) carcinoma models. Doxorubicin (1.0-4.0 mg/kg i.v. per week x 3) and paclitaxel (1.0-4.0 mg/kg i.v. on alternate days x 3) were administered as single agents at non-toxic doses that led to only minimal to moderate antitumor activity. However, a combination of LErafAON with doxorubicin or paclitaxel led to significantly enhanced antitumor activity in all the tumor types tested (PC-3, P<0.03; A549, P<0.035; MDA-MB 231, P<0.045) as compared with LErafAON alone or chemotherapeutic agents alone treated groups. This effect of chemosensitization appeared to be sequence-specific because a mismatch control oligonucleotide continued to show significant tumor growth. Additionally, no inhibition in Raf-1 expression in MDA-MB 231 tumor tissue was observed with mismatch oligonucleotide treatment. On the other hand, LErafAON treatment led to >75% inhibition of Raf-1 expression in tumor tissue. These preclinical observations support the use of LErafAON in combination with chemotherapeutic agents to improve the treatment of human cancers.

Inhibition of gene expression by nucleic acid enzymes in rodent models of human disease.

Nucleic acid enzymes have emerged as a versatile technique for sequence-specific gene silencing in a wide range of cells. However, the question remains as to whether, for example, DNA enzymes and ribozymes are functional in animals. In this chapter, we describe two different rodent models of human diseases--namely, leukemia and chronic heart failure. We specifically reduced Raf-1 expression in leukemic mice using an anti-Raf-1 DNA enzyme. A continuous supply of this catalytic molecule led to a substantial reduction in leukemic-cell burden and survival. Rats with postinfarction heart failure were treated with a DNA enzyme targeting TNFa, and this led to a substantial improvement of cardiac function concomitant with a restoration of the hemodynamic status of the animals. The described protocols should facilitate the in vivo evaluation of other oligonucleotide-based therapy such as small interfering RNAs (siRNAs).

Identification of molecular targets for dietary energy restriction prevention of skin carcinogenesis: an idea cultivated by Edward Bresnick.

Dietary energy restriction (DER) has long been known to strikingly inhibit carcinogenesis in many animal models. The animal data has been corroborated by recent and ongoing epidemiological studies demonstrating the importance of energy balance, physical exercise and obesity in human cancer. Dr. Edward Bresnick provided key insights into this important area of research and pivotal direction for the author's research while he served as Director of the Eppley Institute for Research in Cancer, Omaha, NE. These insights moved this research toward demonstrating that DER reduced the expression of key protein kinase C isoforms in mouse skin. More recent studies have uncovered downstream events that are inhibited by DER including blockage of tumor promoter activation of Raf-1, ERK 1,2 and AP-1 expression. Parallel studies have demonstrated the DER inhibition of these key cellular signaling events in mouse skin carcinogenesis are dependent upon an intact adrenal gland because adrenalectomized mice fed DER diet did not have reduced tumor burden or inhibited signaling and blocked AP-1 activation as was observed in DER mice with intact adrenal glands. In addition, the DER inhibition of tumorigenesis and AP-1 signaling was restored in adrenalectomized mice that were given corticosterone in the drinking water. This showed that in mice in the chemical carcinogenesis protocol glucocorticoid hormone plays a major role in mediating DER prevention of cancer. Studies are ongoing to further assess the mechanism of DER modulation of skin cancer by assessing impacts on transcriptional regulation and expression of genes that are critical in skin carcinogenesis.

Potentiation of signal transduction mitogenesis and cellular proliferation upon binding of receptor-recognized forms of α 2-macroglobulin to 1-LN prostate cancer cells

The α 2-macroglobulin signalling receptor is upregulated in highly metastatic 1-LN prostate cancer cells. Stimulation of 1-LN cells with activated α 2-macroglobulin (α 2M*) caused a two- to threefold increase in [ 3H]thymidine uptake and cell number. These events require the Ras-dependent MAPK and PI 3-kinase/Akt signalling cascades. Incubation of 1-LN cells with α 2M* induced Grb2, shc, sos and Raf-1 expression, as well as phosphorylation of MEK 1/2, ERK 1/2, p38 MAPK and JNK. This treatment also increased PI 3-kinase activation, PDK1 expression, Akt phosphorylation and p70s6k phosphorylation. Levels of the early gene products c-fos protein and thymidylate synthase were comparably increased. Exposure of 1-LN cells to α 2M* significantly raised the levels of phosphorylated CREB by about 15–20 min and phosphorylated p53 by about 60–90 min of incubation. We conclude that the growth regulatory effects of ligating the α 2M* signalling receptor on 1-LN cells are exerted via the onset and crosstalk between the Ras-dependent MAPK and PI 3-kinase/Akt signalling cascades.

Raf-1 expression is related to early hormone-relapse in prostate cancer

Raf-1 expression is related to early hormone-relapse in prostate cancer

BCL-2 and BCL-XL Restrict Lineage Choice during Hematopoietic Differentiation

Differentiation of hematopoietic cells from multipotential progenitors is regulated by multiple growth factors and cytokines. A prominent feature of these soluble factors is promotion of cell survival, in part mediated by expression of either of the anti-apoptotic proteins, BCL-2 and BCL-XL. The complex expression pattern of these frequently redundant survival factors during hematopoiesis may indicate a role in lineage determination. To investigate the latter possibility, we analyzed factor-dependent cell-Patersen (FDCP)-Mix multipotent progenitor cells in which we stably expressed BCL-2 or BCL-XL. Each factor maintained complete survival of interleukin-3 (IL-3)-deprived FDCP-Mix cells but, unexpectedly, directed FDCP-Mix cells along restricted and divergent differentiation pathways. Thus, IL-3-deprived FDCP-Mix BCL-2 cells differentiated exclusively to granulocytes and monocytes/macrophages, whereas FDCP-Mix BCL-XL cells became erythroid. FDCP-Mix BCL-2 cells grown in IL-3 were distinguished from FDCP-Mix and FDCP-Mix BCL-XL cells by a striking reduction in cellular levels of Raf-1 protein. Replacement of the BCL-2 BH4 domain with the related BCL-XL BH4 sequence resulted in a switch of FDCP-Mix BCL-2 cells to erythroid fate accompanied by persistence of Raf-1 protein expression. Moreover, enforced expression of Raf-1 redirected FDCP-Mix BCL-2 cells to an erythroid fate, and prohibited generation of myeloid cells. These results identify novel roles for BCL-2 and BCL-XL in cell fate decisions beyond cell survival. These effects are associated with differential regulation of Raf-1 expression, perhaps involving the previously identified interaction between BCL-2-BH4 and the catalytic domain of Raf-1.

Raf-1 antagonizes erythroid differentiation by restraining caspase activation.

The Raf kinases are key signal transducers activated by mitogens or oncogenes. The best studied Raf isoform, Raf-1, was identified as an inhibitor of apoptosis by conventional and conditional gene ablation in mice. c-raf-1 − / − embryos are growth retarded and anemic, and die at midgestation with anomalies in the placenta and fetal liver. Here, we show that Raf-1–deficient primary erythroblasts cannot be expanded in culture due to their accelerated differentiation into mature erythrocytes. In addition, Raf-1 expression is down-regulated in differentiating wild-type cells, whereas overexpression of activated Raf-1 delays differentiation. As recently described for human erythroid precursors, we find that caspase activation is necessary for the differentiation of murine fetal liver erythroblasts. Differentiation-associated caspase activation is accelerated in erythroid progenitors lacking Raf-1 and delayed by overexpression of the activated kinase. These results reveal an essential function of Raf-1 in erythropoiesis and demonstrate that the ability of Raf-1 to restrict caspase activation is biologically relevant in a context distinct from apoptosis.

High Glucose Stimulates Synthesis of Fibronectin via a Novel Protein Kinase C, Rap1b, and B-Raf Signaling Pathway

The molecular mechanism(s) by which high glucose induces fibronectin expression via G-protein activation in the kidney are largely unknown. This investigation describes the effect of high glucose (HG) on a small GTP-binding protein, Rap1b, expression and activation, and the relevance of protein kinase C (PKC) and Raf pathways in fibronectin synthesis in cultured renal glomerular mesangial cells (MCs). In vivo experiments revealed a dose-dependent increase in Rap1b expression in glomeruli of diabetic rat kidneys. Similarly, in vitro exposure of MCs to HG led to an up-regulation of Rap1b with concomitant increase in fibronectin (FN) mRNA and protein expression. The up-regulation of Rap1b mRNA was mitigated by the PKC inhibitors, calphostin C, and bisindolymaleimide, while also reducing HG- induced FN expression in non-transfected MCs. Overexpression of Rap1b by transfection with pcDNA 3.1/Rap1b in MCs resulted in the stimulation of FN synthesis; however, the PKC inhibitors had no significant effect in reducing FN expression in Rap1b-transfected MCs. Transfection of Rap1b mutants S17N (Ser --> Asn) or T61R (Thr --> Arg) in MCs inhibited the HG-induced increased FN synthesis. B-Raf and Raf-1 expression was investigated to assess whether Rap1b effects are mediated via the Raf pathway. B-Raf, and not Raf-1, expression was increased in MCs transfected with Rap1b. HG also caused activation of Rap1b, which was largely unaffected by anti-platelet-derived growth factor (PDGF) antibodies. HG-induced activation of Rap1b was specific, since Rap2b activation and expression of Rap2a and Rap2b were unaffected by HG. These findings indicate that hyperglycemia and HG cause an activation and up-regulation of Rap1b in renal glomeruli and in cultured MCs, which then stimulates FN synthesis. This effect appears to be PKC-dependent and PDGF-independent, but involves B-Raf, suggesting a novel PKC-Rap1b-B-Raf pathway responsible for HG-induced increased mesangial matrix synthesis, a hallmark of diabetic nephropathy.

Insulin-like Growth Factor-binding Protein-5 (IGFBP-5) Stimulates Growth and IGF-I Secretion in Human Intestinal Smooth Muscle by Ras-dependent Activation of p38 MAP Kinase and Erk1/2 Pathways

Insulin-like growth factor-binding protein-5 (IGFBP-5) and insulin-like growth factor-I (IGF-I) are produced by human intestinal smooth muscle cells. Endogenous IGF-I stimulates growth and increases IGFBP-5 secretion. IGFBP-5 augments the effects of IGF-I by facilitating interaction of IGF-I with the IGF-I receptor tyrosine kinase. Andress (Andress, D. L. (1998) Am. J. Physiol. 274, E744-E750) and Berfield et al. (Berfield, A. K., Andress, D. L., and Abrass, C. K. (2000) Kidney Int. 57, 1991-2003) have shown that in osteoblasts and kidney mesangial cells, IGFBP-5 stimulates proliferation and filopodia formation independently of IGF-I, presumably by activating a distinct IGFBP-5 receptor serine kinase. The present study determined whether IGFBP-5 exerts direct effects on growth in human intestinal smooth muscle cells and identified the intracellular signaling pathways involved. IGFBP-5 caused a concentration-dependent increase in [(3)H]thymidine incorporation and an increase in IGF-I secretion that occurred independently of IGF-I and the IGF-I receptor tyrosine kinase. IGFBP-5-induced phosphorylation of p38 MAP kinase, which was abolished by SB203580, or expression of a dominant negative Ras mutant, Ras(S17N), and phosphorylation of Erk1/2, which was abolished by a Raf1 kinase inhibitor, U1026, or expression of Ras(S17N). IGFBP-5-stimulated [(3)H]thymidine incorporation and IGF-I secretion were partly inhibited by SB203580 or U1026 and abolished by the combination of the two inhibitors or by expression of Ras(S17N). These data show that IGFBP-5 stimulates growth and IGF-I secretion in human intestinal smooth muscle cells by activation of p38 MAP kinase-dependent and Erk1/2-dependent pathways that are independent of IGF-I. A positive feedback mechanism therefore links IGFBP-5 and IGF-I secretion that reinforces their individual effects on growth.

Modulation of p53, ErbB1, ErbB2, and Raf-1 expression in lung cancer cells by depsipeptide FR901228.

Histone deacetylases (HDACs) modulate chromatin structure by regulating acetylation of core histone proteins. HDAC inhibitors, such as depsipeptide FR901228 (FK228), induce growth arrest and apoptosis in a variety of human cancer cells by mechanisms that cannot be attributed solely to histone acetylation. This study evaluated the mechanisms by which FK228 mediates apoptosis in non-small-cell lung cancer (NSCLC) cells. Proliferation and apoptosis were assessed in a panel of NSCLC cell lines that vary in the expression of the growth-regulating proteins p53, pRb, and K-Ras treated with a clinically relevant dose of FK228 (25 ng/mL). Western blot and immunoprecipitation techniques were used to analyze expression of cell-cycle proteins (cyclin A, cyclin E, p53, and p21), signaling-related proteins (ErbB1, ErbB2, and Raf-1), activity of extracellular signal-regulated kinase 1 and 2 (ERK1/2), binding of mutant p53 and Raf-1 to heat shock protein (Hsp)90, and acetylation of Hsp90. FK228 treatment inhibited growth and induced apoptosis in NSCLC cells expressing wild-type or mutant p53. FK228 treatment led to altered expression of cyclin A, cyclin E, and p21, and to reduced expression of mutant, but not wild-type, p53. FK228-treated cells also were depleted of ErbB1, ErbB2, and Raf-1 proteins, and exhibited lower ERK1/2 activity. FK228 treatment also inhibited the binding of mutant p53 and Raf-1 to Hsp90; this inhibition was associated with acetylation of Hsp90. FK228 depletes the levels of several oncoproteins that are normally stabilized by binding to Hsp90 in cancer cells. The resulting ability of FK228 to diminish signal transduction via pathways involving Raf-1 and ERK may contribute to the potency and specificity of this novel antitumor agent.

Differential expression of ras signal transduction mediators in verrucous and squamous cell carcinomas of the upper aerodigestive tract.

ras gene mutations and expression of its gene product have been described in verrucous and squamous cell carcinomas. Other downstream signal-transduction mediators, extracellular signal-regulated kinases 1 and 2 (ERK-1 and ERK-2) and Raf-1, have not yet been as extensively studied. To determine patterns of expression of ERK-1, ERK-2, and Raf-1 in verrucous and squamous cell carcinomas of the upper aerodigestive tract. Seventeen verrucous carcinomas and 10 squamous cell carcinomas of the upper aerodigestive tract were examined for the immunohistochemical expression of ERK-1, ERK-2, and Raf-1 product. Raf-1 expression was intensely expressed in the most basal portions of the epithelium in verrucous carcinomas, but was minimally expressed in the suprabasalar areas. Anti-Raf-1 staining of the squamous cell carcinomas was diffuse and patchy throughout the tumor cells and was weak in intensity. There was no geographic preference of staining. The cytoplasmic expression of both ERK-1 and ERK-2 was predominantly negative in the most basal layers of the epithelium in the verrucous carcinomas, but was positive in the suprabasalar region of the epithelium. Immunohistochemical expression of ERK-1 and ERK-2 in the squamous carcinomas was diffuse throughout the tumor. There is strong correlation of the geographic expression of these mediators of ras signal transduction in verrucous and squamous carcinomas, but the cause of these differences remains unclear at present. The expression of these mediator proteins may have potential for diagnosis, as well as in understanding the biologic behavior of these lesions.

Independent control of cell survival by Raf-1 and Bcl-2 at the mitochondria.

Bcl-2 family proteins play a critical role in the regulation of cell survival by controlling the activation of the cell death executing caspase machinery. Recent work demonstrated that they also provide a link between growth factor signaling and cell survival control. Raf-1 has been identified initially as an essential component of the mitogenic Ras-Raf-MEK-ERK cascade. However, expression of oncogenic Raf-1 also efficiently suppresses apoptotic cell death. This process requires mitochondrial translocation of Raf-1 which can be achieved either by co-expression of the anti-apoptotic protein Bcl-2 or by fusion with the transmembrane domain of the yeast outer mitochondrial membrane protein Mas 70p. It is currently unclear how mitochondrial Raf-1 prevents apoptosis. One possible mechanism involves the phosphorylation of the pro-apoptotic protein Bad resulting in the restoration of Bcl-2 function. Alternatively, the role of Bcl-2 could be limited to the mitochondrial translocation of Raf-1 and survival signaling by Raf-1 is Bcl-2 independent. To test for the mutual requirement of Raf-1 and Bcl-2 in apoptosis suppression the individual proteins were singly tested for survival activity in a genetic background which precludes the expression of the other. The results obtained in these studies demonstrate that ablation of Raf-1 or Bcl-2 expression in fibroblast cells significantly increases the sensitivity towards doxorubicin induced cell death. Reversion of the mutant phenotype could be achieved in either case by introducing a functional bcl-2 gene or a mitochondria targeted version of oncogenic Raf-1, demonstrating that each protein by itself is sufficient to confer protection. Our data thus suggest the existence of two separate pathways of survival signaling at the mitochondria controlled either by Bcl-2 or by Raf-1.

The Ankyrin Repeat-containing Adaptor Protein Tvl-1 Is a Novel Substrate and Regulator of Raf-1

Tvl-1 is a 269-amino acid ankyrin repeat protein expressed primarily in thymus, lung, and testes that was identified by screening a murine T-cell two-hybrid cDNA library for proteins that associate with the serine-threonine kinase Raf-1. The interaction of Tvl-1 with Raf-1 was confirmed by co-immunoprecipitation of the two proteins from COS-1 cells transiently transfected with Tvl-1 and Raf-1 expression constructs as well as by co-immunoprecipitation of the endogenous proteins from CV-1 and NB2 cells. Tvl-1 interacts with Raf-1 via its carboxyl-terminal ankyrin repeat domain. The same domain also mediates Tvl-1 homodimerization. Tvl-1 was detected by immunofluorescence in both the cytoplasm and the nucleus suggesting that in addition to Raf-1 it may also interact with nuclear proteins. Activated Raf-1 phosphorylates Tvl-1 both in vitro and in vivo. In baculovirus-infected Sf9 insect cells, Tvl-1 potentiates the activation of Raf-1 by Src and Ras while in COS-1 cells it potentiates the activation of Raf-1 by EGF. These data suggest that Tvl-1 is both a target as well as a regulator of Raf-1. The human homologue of Tvl-1 maps to chromosome 19p12, upstream of MEF2B with the two genes in a head to head arrangement.

Antisense raf oligodeoxyribonucleotide is a radiosensitizer in vivo.

Raf-1, a cytosolic protein serine/threonine kinase, plays important roles in cell growth, proliferation, transformation, and cell survival. The aim of the present study was to evaluate the radiotherapeutic efficacy of a fully phosphorothioated and well-characterized antisense raf oligodeoxyribonucleotide (ODN) corresponding to the 3'-untranslated region of human c-raf-1 mRNA (ISIS 5132/5132). Using our recently developed liposome encapsulation of ODN approach, we first compared the pharmacokinetic parameters of a liposomal formulation of 5132 (LE-5132) and 5132. The peak plasma concentrations 5 minutes after ODN administrations (30 mg/kg i.v.) were 28.5 microg/ml and 13.5 microg/ml for LE-5132 and 5132, respectively. The decrease in plasma concentration of LE-5132 and 5132 followed a biexponential pattern, with initial distribution half-lives (t1/2alpha) of 34.8 minutes and 21.6 minutes, respectively. The terminal half-lives (t1/2beta) with LE-5132 and 5132 were 14.5 hours and 4.3 hours, respectively. The area under the plasma concentration-time curve (AUC) was 5.8 times higher with LE-5132 than with 5132. Significantly higher intact ODN levels could be measured in most organs within 48 hours of administration of LE-5132 compared with 5132 (liver 18.4-fold, spleen, 31-fold, heart 3-fold, lungs 1.5-fold). In kidneys, the level was lower with LE-5132 (0.77-fold). LE-5132 composition, unlike 5132, did not affect clotting time in vitro. Significant decline in the level of Raf-1 protein was observed in vitro in relatively radioresistant human laryngeal squamous cell carcinoma cells (SQ-20B) treated with LE-5132 compared with SQ-20B cells treated with equimolar concentration of 5132 or liposome-encapsulated mismatched 5132 (0.5 microM LE-5132, 71.3%+/-22.5%; 1.0 microM LE-5132, 79.6%+/-16.7%). In addition, LE-5132 appeared to be a more potent antitumor compound than 5132 (p < 0.001). These data established the suitability of LE-5132 for in vivo radiotherapeutic efficacy studies. Intravenous administration of LE-5132 into SQ-20B tumor-bearing athymic mice inhibited Raf-1 expression in tumor tissue compared with blank liposome-treated or untreated control groups. LE-5132 or ionizing radiation (IR) treatment alone caused significant but transient inhibition of SQ-20B tumor growth but not tumor regression. Remarkably, a combination of LE-5132 and IR treatments led to significant and sustained tumor regression for at least 27 days after the last treatment (< 0.001). Histopathologic examination of tumor samples revealed a significant proportion of cells containing fragmented chromatin in the LE-5132 + IR treatment group as compared with single agent and untreated control groups. These in vivo data support the notion that Raf-1 has proliferative and survival functions and advance the scientific and technologic bases for the use of antisense raf ODN in the management of radioresistant malignancies.