C-raf mRNA Research Articles

Expression of transcription, growth factors, steroid hormone receptors, LC3B in papillary thyroid cancer tissue, association with prognosis and risk of recurrence

Introduction. Biological characteristics of the tumor play a major role in it’s development and progression. Currently, using the molecular markers aimed at resolving the problems in clinical oncology is becoming more important, including thyroid carcinomas. Heterogeneous contradictory data had been accumulated to date showing the ability of tumors genetic and biological parameters to predict the diseases outcome.Aim. To investigate prognostic value of transcription, growth factors, components of AKT / mTOR signaling pathway and autophagy protein LC3B in patients with papillary thyroid cancer in relation to recurrences and overall survival.Materials and methods. The study included 65 patients with T1–4N0–1M0 papillary thyroid cancer. According to the criteria of the American Thyroid Association (ATA) (2015), patients were divided into groups of patients with high, low and intermediate risk. 30 patients were classified as low risk, 23 as intermediate risk, and 12 as high risk. The BRAFV600 mutation was identified in 18 samples. The expression of transcription factors (p65 and p50 subunits of nuclear factor kappa B (NF-κB p65, NF-κB p50), hypoxia-inducible factor 1 (HIF-1), hypoxia-inducible factor 2 (HIF-2), growth factors (vascular endothelial growth factor (VEGF), receptor VEGF (VEGF-2), carbonic anhydrases of type 9 (CAIX)), AKT, c-RAF, GSK- 3β, p70S6, mammalian target of rapamycin (m-TOR), PDK, PTEN, 4E-BP1 in the tumor was assessed by real-time polymerase chain reaction (PCR). The BRAFV600 mutation was investigated using real-time allele-specific PCR. The content of the LC3B protein was examined using the Western Blot method.Results. As a result of the study, there is an increase in c-RAF expression with an increase in risk from low to high, which was accompanied by a decrease in 4E-BP1 expression. c-RAF mRNA levels were increased 3.0- and 2.8‑fold in the intermediate and high-risk groups, respectively, compared to low risk patients. There is a change in the expression of Brn-3α depending on the relapse risk. The maximum mRNA levels were found in patients with intermediate risk, where the figure was 4.3 and 6.2 times higher than in patients with low and high risk, respectively. An increase in LC3B expression by 56.0 and 28.0 times was shown in the tumor tissue of patients with intermediate risk compared with patients with low and high risk. This fact corresponds with an increasing content of the protein itself, which was higher in patients with intermediate risk. Patients with a negative BRAF gene status had an intermediate and high risk of tumor recurrence. The prognostic significance of the estrogen receptor β (ER-β) and NF-κB p50 expression level had been revealed in relation with relapse-free and overall survival of patients with papillary thyroid cancer.Conclusion. As a result of the study, additional molecular markers were found in order to for predict the tumors recurrence risk. The study showed the significance of ERβ and NF-κB p50 expression levels for predicting disease outcomes.

Actionable Gene Expression-Based Patient Stratification for Molecular Targeted Therapy in Hepatocellular Carcinoma

BackgroundThe effectiveness of molecular targeted agents is modest in hepatocellular carcinoma (HCC). Efficacy of molecular targeted therapies has been better in cancer patients with high expression of actionable molecules defined as cognate target molecules. However, patient stratification based on the actionable molecules dictating the effectiveness of targeted drugs has remained understudied in HCC.Experimental Design & ResultsPaired tumor and non-tumoral tissues derived from a total of 130 HCC patients were studied. Real-time RT-PCR was used to analyze the mRNA expression of actionable molecules in the tissues. mRNA levels of EGFR, VEGFR2, PDGFRβ, FGFR1, and mTOR were up-regulated in tumors compared to non-tumors in 35.4, 42.3, 61.5, 24.6, and 50.0% of patients, respectively. Up-regulation of EGFR was observed at early stage and tended to gradually decrease toward late stages (BCLC stage A: 41.9%; B: 30.8%; C: 17.6%). Frequency of VEGFR2 expression in tumors at stage C was lower than that in the other stages (BCLC stage A: 45.9%; B: 41.0%; C: 29.4%). PDGFRβ and mTOR were observed to be up-regulated in more than 50% of tumors in all the stages whereas FGFR1 was up-regulated in only about 20% of HCC irrespective of stages. A cluster analysis of actionable gene expression revealed that HCC can be categorized into different subtypes that predict the effectiveness of molecular targeted agents and combination therapies in clinical trials. Analysis of in vitro sensitivity to sorafenib demonstrated that HCC cells with up-regulation of PDGFRβ and c-Raf mRNA are more susceptible to sorafenib treatment in a dose and time-dependent manner than cells with low expression of the genes.ConclusionsmRNA expression analysis of actionable molecules could provide the rationale for new companion diagnostics-based therapeutic strategies in the treatment of HCC.

Cross-talk between Aryl Hydrocarbon Receptor and Mitogen-Activated Protein Kinase Signaling Pathway in Liver Cancer through c-raf Transcriptional Regulation

c-raf is a serine-threonine kinase and a downstream effector of ras signaling. This kinase plays an essential role in cell proliferation, differentiation, and apoptosis. In the past, we reported induction of c-raf gene expression in rat liver cancer on treatment with a mixture of aryl hydrocarbon receptor (AhR) agonists. This prompted our interest in investigating the role of AhR in the transcriptional regulation of c-raf. Initially, we cloned the rat c-raf promoter and sequenced the genomic DNA and cDNA by Southern blotting and capillary electrophoresis. Then, a genetic algorithm was applied to search for putative AhR-binding sites. DNA-binding activity of AhR was confirmed by electromobility shift assay. We also studied c-raf gene expression in rat hepatoma cell lines with functional and/or devoid AhR and in primary human and rat hepatocyte cultures. Overall, we identified five and three AhR-binding sites in the human and rat c-raf gene, respectively. Treatment of hepatocyte cultures with the AhR antagonist resveratrol reduced DNA binding of AhR. Only rat hepatoma cells with functional AhR responded to 1 nmol/L 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment with >10-fold c-raf mRNA induction. Treatment of human and rat hepatocyte cultures with various AhR-activating chemicals resulted in induction of c-raf gene expression, albeit at different levels. Taken collectively, we show AhR to be a master regulator of c-raf and propose cross-talk between AhR and the mitogen-activated protein kinase signaling pathway in chemically induced hepatocarcinogenesis.

First- and second-generation antisense oligonucleotide inhibitors targeted against human c-raf kinase.

Following extensive screening of more than 50 antisense-designed phosphorothioate oligodeoxynucleotides targeted to human c-raf mRNA, one oligodeoxynucleotide (ISIS 5132/CGP 69846A) was identified as being the most potent inhibitor of c-raf gene expression both in vitro and in vivo. ISIS 5132 is a highly sequence-specific and target-specific inhibitor of c-raf mRNA and protein levels. c-raf inhibition results in dramatic alteration of downstream signalling events within the MAP kinase signalling pathway. Moreover, this oligodeoxynucleotide displays potent antitumour activity against a broad spectrum of tumour types in mouse models and has progressed to Phase I clinical trails. In an effort to identify potential back-up compounds to ISIS 5132, a variety of second-generation 2' sugar modifications have been evaluated for activity against c-raf in cell culture. We have identified a number of second-generation oligonucleotides with improved biophysical characteristics that result in enhanced activity against c-raf in cell culture. Activity enhancement was most pronounced for 2'-O-methoxyethyl-modified oligonucleotides and this modification also resulted in significantly improved antitumour activity in vivo.

Phase I study of LErafAON-ETU, an easy-to-use formulation of liiposome entrapped c-raf antisense oligonucleotide, in advanced cancer patients

3214 Background: Raf kinases play key roles in integrating the K-Ras and mitogen-activated protein kinase (MAPK) intracellular signaling cascades, which are important regulators of tumor cell proliferation. LErafAON-ETU is an easy-to-use, liquid, liposomal formulation of an antisense oligonucleotide complementary to the c-raf mRNA sequence. The lipid component consists of a novel, positively charged, synthetic cardiolipin (PCL-2). Formulation with liposomes eliminates the need for extensive phosphorothioate modification; thus, only the 3’- and 5’- termini of the LErafAON-ETU oligonucleotide are modified. Methods: This Phase I dose-escalation study is designed to determine the dose-limiting toxicity (DLT) and maximum-tolerated dose (MTD) of LErafAON-ETU in patients with advanced cancer. LErafAON-ETU is administered as a 60 minute intravenous infusion once weekly for 3 consecutive weeks (a treatment cycle). Patients are eligible to receive repeated treatment cycles until disease progression or unacceptable toxicity occurs. Dose levels of 7.5, 15, 30, 60, 120, 240, and 480 mg/m2 or more are planned, depending on the tolerability of LErafAON-ETU. An accelerated dosing strategy will be implemented after the safety and tolerability of the initial dose level has been established in 3 patients. In this design, a single patient will be treated at each subsequent dose level until an adverse event of Grade ≥2, a pre-infusion platelet count of <75,000/mm3, or an increase in the serum creatinine level to ≥1.5 x the upper limit of normal occurs. At such time this cohort will be expanded, and a standard dose-escalation scheme will be employed with 3–6 patients in each cohort dependent upon the occurrence of DLTs. Results: Patient enrollment has begun. Three patients have completed infusion at the initial dose of 7.5 mg/m2. Conclusions: This accelerated dose-escalation scheme should reach potentially therapeutic doses more rapidly than would a standard design. This study represents the initial clinical dosing and proof of concept for a novel, liquid, PCL-2 based liposomal formulation of an antisense oligonucleotide with minimal phosphorothioation. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration NeoPharm NeoPharm

2'-O-[2-[(N,N-dimethylamino)oxy]ethyl]-modified oligonucleotides inhibit expression of mRNA in vitro and in vivo.

Synthesis and antisense activity of oligonucleotides modified with 2'-O-[2-[(N,N-dimethylamino)oxy] ethyl] (2'-O-DMAOE) are described. The 2'-O-DMAOE-modified oligonucleotides showed superior metabolic stability in mice. The phosphorothioate oligonucleotide 'gapmers', with 2'-O-DMAOE- modified nucleoside residues at the ends and 2'-deoxy nucleosides residues in the central region, showed dose-dependent inhibition of mRNA expression in cell culture for two targets. 'Gapmer' oligonucleotides have one or two 2'-O-modified regions and a 2'-deoxyoligonucleotide phosphorothioate region that allows RNase H digestion of target mRNA. To determine the in vivo potency and efficacy, BalbC mice were treated with 2'-O-DMAOE gapmers and a dose-dependent reduction in the targeted C-raf mRNA expression was observed. Oligonucleotides with 2'-O-DMAOE modifications throughout the sequences reduced the intercellular adhesion molecule-1 (ICAM-1) protein expression very efficiently in HUVEC cells with an IC(50) of 1.8 nM. The inhibition of ICAM-1 protein expression by these uniformly modified 2'-O-DMAOE oligonucleotides may be due to selective interference with the formation of the translational initiation complex. These results demonstrate that 2'-O-DMAOE- modified oligonucleotides are useful for antisense-based therapeutics when either RNase H-dependent or RNase H-independent target reduction mechanisms are employed.

E-2. Inhibitors of MAP kinases

Mitogen activated protein (MAP) kinase cascades form a threekinase signaling module involved in transmitting membrane signals to the cell nucleus (Fig 1). In mammals, three major groups of MAPK have been identified the ERK, ~38 and JNK groups. A MAPK module consists of MAP kinase or extracellular signalgegulated &nase (ERK) activated by a MAP/ERK kinase (MEK, MAPKK) which, in turn, is activated by a MEK kinase (MEKK, MAPKKK). The most characterized MAP kinase pathway, involved in cellular differentiation and proliferation is the raf (MEKK) MEK (MAPKK) ERK pathway. Once activated, raf phosphorylates, MEK, and MEKa, which in mm phosphorylate ERKl and ERK2, @UMAPK and p42MAPK). MAPK activation results in phosphorylation and activation of ribosomal S6 kinase and transcription factors such as c-&z, c-Myc and c-Fos, resulting in the switching on of a number of genes associated with proliferation and carcinogenesis. Conditional MAP kinase activation is important in gene regulation, promoting Gl cell cycle progression before DNA replication, and spindle assembly during both meiotic and mitotic cell division, among other processes. Inappropriate activation of the MAP kinase pathway, through mutations introduced via oncogenes, is a feature of many neoplasms. In lung cancer cells, activation of the MAP kinase pathway has been described, and involves mutations in K-ras, constitutive activation of c-raf and B-raf mutations and baseline presence of phosphorylated MEK. Inhibitors of raf-kinase and MEK are currently undergoing clinical testing. ISIS 5 132 is an antisense oligonucleotide directed against c-raf mRNA. BAY 43-9006 is a small molecule inhibitor of raf-kinase. CI-1040 is a small molecule inhibitor of the dual specificity kinases MEKUMEK2. In addition, activated raf kinase has been shown to interact with the PI-3-Kinase/Akt pathway and inhibit apoptosis by phosphorylating BAD and upregulating Bcl-2. Thus, mTOR inhibitors such as CCI-779 and RADOOl, indirectly interfere with MAP kinase signaling pathways. Figure 1: Mammalian MAP Kinase Signaling Pathways

In vivo expression of single-stranded DNA in mammalian cells with DNA enzyme sequences targeted to C-raf.

The use of antisense oligodeoxynucleotides (AS-ODN) remains a viable method to downregulate selected gene function. However, limitations to the antisense approach remain, such as (1) difficulties in delivery of the AS-ODN into target tissues, (2) instability of AS-ODN in vivo, (3) uncertanties about the precise mode of action, and (4) toxic effects in animal and human studies. To circumvent some of these difficulties, we designed a vector set that directs the in vivo production of single-stranded DNA (ssDNA) of a desired target sequence with limited extraneous vector nucleotide sequences. One plasmid was designed to express Moloney murine leukemia virus (MoMuLV) reverse transcriptase (RT). Another expression plasmid contains the MoMuLV primer binding site at the 3'-end of its RNA transcript so that an ssDNA would be synthesized by RT when both plasmids are cotransfected into cells. To test this expression system, we constructed a plasmid set, pssXA/pssXB that produces ssRNA-cleaving DNA 10-23 enzyme (Santoro, S.W., and Joyce, G.F. [1997]. Proc. Natl. Acad. Sci. USA 37, 13330-13342). The DNA enzyme sequence was placed between two oligonucleotide arms that are complementary and able to specifically target C-raf kinase mRNA. These plasmids were transfected into the A549 lung carcinoma cell line. Reduced C-raf mRNA levels by up to 34%-36%, as determined by Northern blot analysis, were observed in the transfected cells. Our results demonstrate the feasibility of using this novel ssDNA expression system to generate any sequence of interest in vivo for antisense, RNA-cleavage DNA enzyme, or triplex-forming strategies.

INHIBITION OF C-RAF EXPRESSION BY ANTISENSE OLIGONUCLEOTIDES EXTENDS HEART ALLOGRAFT SURVIVAL IN RATS1

C-raf is a well-characterized serine/ threonine (Ser/Thr) protein kinase that is involved in the transduction of multiple signals of T cells. We demonstrate that the inhibition of C-raf mRNA expression prolongs heart allograft survival. Three 20-mer C-raf antisense oligonucleotides, each with identical sequences, were synthesized with different chemical modifications: one as a uniform phosphorothioate oligodeoxynucleotide (PS oligo), a second with a PS backbone and 2'-methoxyethyl (ME) substitutions at the 2'-sugar positions in the first and last five nucleotides, and a third with a mixed PS and phosphodiester (PD) backbone and ME modifications on the first and last five nucleotides. Both ME-modified C-raf antisense oligos were at least 5-fold more effective than the PS C-raf antisense oligo in blocking C-raf mRNA expression in two cell lines. Similarly, each of the ME C-raf antisense oligos produced better heart allograft survival rates than did PS C-raf oligo. Furthermore, although the combination of PS C-raf antisense oligo with sirolimus (SRL) acted synergistically to extend heart allograft survival, the effect was potentiated by either of the ME-modified oligos. C-raf inhibition extends heart allograft survival, and ME-modification potentiates antisense activity.

Induction of oocyte maturation by jun-N-terminal kinase (JNK) on the oncogenic ras-p21 pathway is dependent on the raf-MEK signal transduction pathway.

We have previously found that microinjection of activated MEK (mitogen activated kinase kinase) and ERK (mitogen-activated protein; MAP kinase) fails to induce oocyte maturation, but that maturation, induced by oncogenic ras-p21 and insulin-activated cell ras-p21, is blocked by peptides from the ras-binding domain of raf. We also found that jun kinase (JNK), on the stress-activated protein (SAP) pathway, which is critical to the oncogenic ras-p21 signal transduction pathway, is a strong inducer of oocyte maturation. Our purpose in this study was to determine the role of the raf-MEK-MAP kinase pathway in oocyte maturation and how it interacts with JNK from the SAP pathway. We microinjected raf dominant negative mutant mRNA (DN-raf) and the MEK-specific phosphatase, MKP-T4, either together with oncogenic p21 or c-raf mRNA, into oocytes or into oocytes incubated with insulin to determine the effects of these raf-MEK-MAP kinase pathway inhibitors. We found that oocyte maturation induced by both oncogenic and activated normal p21 is inhibited by both DN-raf and by MKP-T4. The latter more strongly blocks the oncogenic pathway. Also an mRNA encoding a constitutively activated MEK strongly induces oocyte maturation that is not inhibited by DN-raf or by MKP-T4. Surprisingly, we found that oocyte maturation induced by JNK is blocked both by DN-raf and MKP-T4. Furthermore, we discovered that c-raf induces oocyte maturation that is inhibited by glutathione-S-transferase (GST), which we have found to be a potent and selective inhibitor of JNK. We conclude that there is a strong reciprocal interaction between the SAP pathway involving JNK and the raf-MEK-MAP kinase pathway and that oncogenic ras-p21 can be preferentially inhibited by MEK inhibitors. The results imply that blockade of both MEK and JNK-oncogenic ras-p21 interactions may constitute selective synergistic combination chemotherapy against oncogenic ras-induced tumors.

Anti-proliferative effects of unmodified antisense oligodeoxynucleotides targeted against c-raf mRNA: use of poly (lysine/serine) copolymers or cationic lipopolyamines.

1. It is now known that nuclease-resistant phosphorothioate antisense oligodeoxynucleotides (ODN) have some actions that are unrelated to antisense mechanisms. In the present study we assessed the anti-proliferative effects of phosphorothioate (PS) and phosphodiester (PO; unmodified) antisense ODN targeted against c-raf mRNA on pancreatic cancer cells in vitro, using poly (lysine/serine) copolymers conjugated with polyethylene glycol (PLSP) or cationic lipopolyamines (Transfectam) as carriers. 2. The anti-proliferative effect of the PO antisense ODN was significantly (P < 0.05) greater than that of the PS ODN, either complexed with PLSP (2 mumol/L ODN) or the Transfectam (0.5 mumol/L ODN). However, the effect of the PS or PO antisense ODN was not dependent on the antisense sequence. The c-raf mRNA levels, assessed by reverse transcription-polymerase chain reaction, were obviously reduced by both PO and PS antisense ODN compared with mismatched ODN when complexed with the Transfectam (1 mumol/L ODN). 3. Although the anti-proliferative effects were mainly unrelated to antisense mechanisms, unmodified antisense ODN complexed with some carriers could be used as anti-tumour agents considering that synthetic carriers can be modified to improve functions, such as delivery.

Abrogation of c-Raf expression induces apoptosis in tumor cells.

Signal transduction pathways involving the c-Raf protein kinase are frequently activated in tumor cells. We have addressed the relevance of this activation by a loss-of-function approach. An anti-sense phosphorothioate oligonucleotide (ODN) specifically targeted against c-raf mRNA (Monia et al., 1996a) was used to block c-Raf protein expression in four different cell lines derived from lung, cervical, prostate and colon carcinomas. Concomitant with the abrogation of c-Raf expression we observed the occurrence of classical apoptotic markers, including chromatin condensation, inter-nucleosomal DNA cleavage, annexin V binding and cleavage of PARP, which was followed by cell death, affecting most of the cell population. This induction of apoptosis occurred independent of the p53 status of the cell. These findings demonstrate that c-Raf can protect tumor cells from undergoing programmed cell death, and suggest that the interference with c-Raf expression or function by ODNs or specific drugs could represent a powerful means for improving the efficacy of anti-cancer therapy.

G-CSF receptor-mediated up-regulation of c-fos but not c-raf mRNA expression in myeloid cells.

Granulocyte colony stimulating factor (G-CSF) regulates survival, proliferation, and differentiation of myeloid precursor cells. One of the signaling mechanisms for the G-CSF receptor (G-CSF-R) involves the activation of Ras/MAP kinase (MEK) pathway. Since Raf is an important, common link between Ras and MEK in the Ras-Raf-MEK cascade, we studied the expression of c-raf mRNA in G-CSF-treated myeloid precursor cell lines--NFS-60 and HL-60. G-CSF did not alter c-raf mRNA expression in these cells up to 24 h, but induced a transient up-regulation of c-fos mRNA expression between 15-60 min post-treatment. Our results show that G-CSF triggers a de novo induction of c-fos but not c-raf mRNA, and suggests that G-CSF-R-mediated activation of Ras/MEK pathway may involve post-transcriptional mechanisms of Raf regulation.

Molecular and cellular characterization of baboon C-Raf as a target for antiproliferative effects of antisense oligonucleotides.

C-Raf is a an essential member of the growth factor-ras pathway and a target for intervention strategies aimed at blocking cell proliferative responses. Excessive smooth muscle proliferation is considered one cause of the arterial closure in restenosis. Because of the similarity to the human cardiovascular system, a useful current animal model of the disease is a baboon model. As a foundation for animal studies employing antisense oligonucleotides, efforts were made to characterize the molecular and cellular biology of the baboon system. The nucleotide sequence of baboon c-raf cDNA was determined. Antisense phosphorothioate oligonucleotides specific to the 3'-UTR of c-raf mRNA from human and baboon were compared using primary baboon smooth muscle cells in culture. A particular human antisense oligonucleotide, referred to as ISIS 5132, was different by only 2 of 20 bases from the baboon sequence. The corresponding baboon antisense oligonucleotide ISIS 12959, however, was markedly more effective to inhibit c-raf mRNA, protein production, and DNA synthesis, and the results attest to the species specificity of the approach. After antisense treatment, c-raf mRNA levels dropped rapidly, whereas protein levels decreased with a half-life of roughly 24-48 hours, consistent with the antiproliferative effects. The data are discussed with regard to the profile of protein-protein interactions made by C-Raf and with the view that the baboon system closely parallels the human one at the signal transduction level. As this work progressed, a baboon cDNA homolog of a human c-raf-2 pseudogene was isolated, sequenced, and shown to be transcribed into mRNA.

Involvement of Ras/Raf/AP-1 in BMP-4 signaling during Xenopus embryonic development.

Previously, we elucidated the role of bone morphogenetic protein 4 (BMP-4) in the dorsal-ventral patterning of the Xenopus embryo by using a dominant negative mutant of the BMP-4 receptor (DN-BR). The present paper describes the involvement of Ras, Raf, and activator protein 1 (AP-1) in BMP-4 signaling during Xenopus embryonic development. The AP-1 activity was determined by injecting an AP-1-dependent luciferase reporter gene into two-cell-stage Xenopus embryos and measuring the luciferase activity at various developmental stages. We found that injection of BMP-4 mRNA increased AP-1 activity, whereas injection of DN-BR mRNA inhibited AP-1 activity. Similar inhibitory effects were seen with injection of mRNAs encoding dominant negative mutants of c-Ha-Ras, c-Raf, or c-Jun. These results suggest that the endogenous AP-1 activity is regulated by BMP-4/Ras/Raf/Jun signals. We next investigated the effects of Ras/Raf/AP-1 signals on the biological functions of BMP-4. DN-BR-induced dorsalization of the embryo, revealed by the formation of a secondary body axis or dorsalization of the ventral mesoderm explant analyzed by histological and molecular criteria, was significantly reversed by coinjection of [Val12]Ha-Ras, c-Raf, or c-Jun mRNA. Furthermore, the BMP-4-stimulated erythroid differentiation in the ventral mesoderm was substantially inhibited by coinjection with the dominant negative c-Ha-Ras, c-Raf, or c-Jun mutant. Our results suggest the involvement of Ras/Raf/AP-1 in the BMP-4 signaling pathway.

2‘-O-Aminopropyl Ribonucleotides: A Zwitterionic Modification That Enhances the Exonuclease Resistance and Biological Activity of Antisense Oligonucleotides

Oligonucleotides containing 2'-O-aminopropyl-substituted RNA have been synthesized. The 2'-O-(aminopropyl)adenosine (APA), 2'-O-(aminopropyl)cytidine (APC), 2'-O-(aminopropyl)-guanosine (APG), and 2'-O-(aminopropyl)uridine (APU) have been prepared in high yield from the ribonucleoside, protected, and incorporated into an oligonucleotide using conventional phosphoramidite chemistry. Molecular dynamics studies of a dinucleotide in water demonstrates that a short alkylamine located off the 2'-oxygen of ribonucleotides alters the sugar pucker of the nucleoside but does not form a tight ion pair with the proximate phosphate. A 5-mer with the sequence ACTUC has been characterized using NMR. As predicted from the modeling results, the sugar pucker of the APU moiety is shifted toward a C3'-endo geometry. In addition, the primary amine rotates freely and is not bound electrostatically to any phosphate group, as evidenced by the different sign of the NOE between sugar proton resonances and the signals from the propylamine chain. Incorporation of aminopropyl nucleoside residues into point-substituted and fully modified oligomers does not decrease the affinity for complementary RNA compared to 2'-O-alkyl substituents of the same length. However, two APU residues placed at the 3'-terminus of an oligomer gives a 100-fold increase in resistance to exonuclease degradation, which is greater than observed for phosphorothioate oligomers. These structural and biophysical characteristics make the 2'-O-aminopropyl group a leading choice for incorporation into antisense therapeutics. A 20-mer phosphorothioate oligonucleotide capped with two phosphodiester aminopropyl nucleotides targeted against C-raf mRNA has been transfected into cells via electroporation. This oligonucleotide has 5-10-fold greater activity than the control phosphorothioate for reducing the abundance of C-raf mRNA and protein.

Uniform response of c-raf expression to differentiation induction and inhibition of proliferation in a rat rhabdomyosarcoma cell line

The clonal rat rhabdomyosarcoma cell line BA-HAN-1C is composed of proliferating mononuclear cells, some of which spontaneously fuse to terminally differentiated myotube-like giant cells. Both the induction of differentiation by retinoic acid (RA) and by sodium butyrate (NaBut), as well as the inhibition of proliferation by fetal calf serum (FCS)-depleted medium uniformly resulted in the same effects. There was a significant (p less than 0.001) inhibition of proliferation and induction of cellular differentiation, as evidenced by a significant (p less than 0.05) increase in creatine kinase activity. Furthermore, after exposure to RA-supplemented or FCS-depleted medium, a significant (p less than 0.001) increase in the number of myotube-like giant cells was observed. These effects were preceded by a uniform enhancement of c-raf mRNA expression, which became evident 6 h after exposure to RA, NaBut and FCS-depleted media. C-raf mRNA expression persisted at an elevated level throughout the observation period of 5 days after exposure to RA or NaBut, whereas the increased expression of c-raf mRNA observed after FCS-depletion declined near to the basal level after only 24 h. Furthermore, a transient c-fos mRNA expression was observed 15 and 30 min after exposure to RA-supplemented and FCS-depleted medium but not after exposure to NaBut. The present results suggest a possible role of c-raf in the regulation of differentiation and proliferation of this cell line. Since all our experiments with RA, NaBut and FCS-depletion resulted in an early peak of c-raf mRNA expression, it is suggested that this early peak may be sufficient to trigger events crucial for differentiation and proliferation of BA-HAN-1C tumor cells.

Expression of c-raf-1 and A-raf-1 during differentiation of 3T3-L1 preadipocyte fibroblasts into adipocytes

The objective of this study was to examine the expression of the c-raf-1 and A-raf-1 protooncogenes during differentiation of 3T3-L1 preadipocytes into adipocytes. At confluence, prior to initiation of differentiation c-raf and A-raf steady state mRNA levels were low. Expression of c-raf and A-raf began to increase 72 hours following initiation of differentiation by treatment with differentiation medium, reaching a maximum increase of 3 to 6-fold and 3 to 4-fold respectively by 190 hours. The increase of c-raf and A-raf steady state message levels occurred concomittent with the onset of differentiation as indicated by increased levels of glycerol-3-phosphate dehydrogenase mRNA. These changes were compared with those for several other protooncogene mRNAs including c-myc, c-fos, H-ras and histone H3. These results are the first to show increased expression of the raf protooncogenes during terminal differentiation rather than in association with proliferation.

Activation of rat c-raf during transfection of hepatocellular carcinoma DNA.

Rat c-raf was found to be activated in a transformant obtained with DNA of a hepatocellular carcinoma induced by 2-amino-3-methylimidazo[4,5-f]quinoline. This activated c-raf was cloned in a cosmid vector and actively transforming clones were obtained. Comparison of the restriction maps of this activated c-raf and cloned normal rat c-raf revealed a recombination in the 5'-terminal region of the activated form of this gene. The recombined DNA was shown to be actively transcribed and possibly to form a fused mRNA with c-raf, which is slightly smaller than normal c-raf mRNA. Since this recombination was not detected in the original tumor by Southern blot analysis, it presumably occurred during transfection.