Raf mRNA Research Articles

Griffipavixanthone, a dimeric xanthone extracted from edible plants, inhibits tumor metastasis and proliferation via downregulation of the RAF pathway in esophageal cancer

Metastasis causes a large number of deaths among esophageal cancer patients. The activation of RAF family proteins elevates tumor metastasis and proliferation. In screen targeting the RAF protein, we identified that Griffipavixanthone (GPX), a dimeric xanthone isolated from Garcinia esculenta, is a B-RAF and C-RAF inhibitor against esophageal cancer cells. Using wound healing, transwell migration and matrigel invasion assays, we confirmed that GPX significantly inhibited cell migration and invasion. Furthermore, exposure to GPX rendered cell proliferation and induced G2/M cell cycle arrest. Our mechanistic study showed that GPX suppressed cancer metastasis and proliferation through downregulation of RAF-MEK-ERK cascades proteins as well as RAF mRNA levels. In a pulmonary metastasis model, the intraperitoneal injection of GPX significantly suppressed esophageal tumor metastasis and ERK protein level in vivo. In conclusion, our present study suggested that GPX could inhibit tumor migration, invasion and proliferation in vitro and in vivo, which indicated the potential of GPX for preventing and treating esophageal cancer.

Antisense oligonucleotide targeting of Raf-1: importance of raf-1 mRNA expression levels and raf-1-dependent signaling in determining growth response in ovarian cancer.

We sought to identify determinants of growth response to the Raf-1-targeted antisense oligonucleotide (ASO; ISIS 5132) using a large panel of ovarian cancer cell lines. First-(ISIS 5132) and second-generation (ISIS 13650) anti-Raf 1 ASOs were compared with control oligonucleotides. Growth was assessed by cell counts; apoptosis was assessed by poly(ADP-ribose) polymerase cleavage; and cell cycle analysis was assessed by flow cytometry. Protein expression was detected by Western blot analysis, and mRNA expression was detected by quantitative reverse transcription-PCR. Raf-1 kinase activity was detected by anti-Raf-1 immunoprecipitation, followed by myelin basic protein phosphorylation. A panel of 15 ovarian cancer cell lines was used to model a range of growth responses to ASOs targeting Raf-1 mRNA. Growth inhibition varied from 10% to >90% inhibition. Growth inhibition was associated with increased apoptosis and accumulation of cells in the G(2)-M and S phases of the cell cycle. Growth response was not related to level of Raf-1 protein expression, Raf-1 kinase activity, intracellular ASO uptake, or degree of Raf-1 protein inhibition. However, ASO growth response was associated with a high proportion of Raf-1 mRNA [relative to total (i.e., Raf-1 + A-Raf + B-Raf) Raf mRNA] and significantly higher Raf-1 kinase activity induction following growth factor (transforming growth factor alpha) stimulation in the cell lines consistent with dependency of these cell lines on Raf-1. These data indicate that ovarian cancers demonstrate differential sensitivity to ASOs targeted against Raf-1, and target expression levels and degree of utilization of Raf-1 signaling are implicated. Clinically sensitive tumors could feasibly be identified.

Biophysical and antisense properties of oligodeoxynucleotides containing 7-propynyl-, 7-iodo- and 7-cyano-7-deaza-2-amino-2'-deoxyadenosines

The synthesis of 7-propynyl-, 7-iodo- and 7-cyano-7-deaza-2-amino-2'-deoxyadenosines is described. The nucleosides were synthesized, functionalized into the phosphoramidites and incorporated into oligodeoxynucleotides. Spectroscopic melting experiments against complementary RNA showed increases of 3-4 degreesC per modification for single substitutions and smaller increases per incorporation for multiple substitutions relative to unmodified control sequences. The 7-propyne and 7-iodo nucleosides were incorporated into antisense sequences targeting the 3'-UTR of murine C- raf mRNA. Both nucleosides demonstrated substitution-dependent potency. The sequences with three and four substitutions of the 7-propyne-7-deaza-2-amino-2'-deoxyadenosine exhibited a 2-3-fold increase in potency over unmodifed controls.

RAF expression in human astrocytic tumors

RAF proteins are well known oncoproteins. The B-RAF has been shown to be activated by mutations in a multitude of human cancers. Alterations of C-RAF expression are discussed to play a role in lung cancer. Only for A-RAF no link to tumorigenesis has been published so far. Malignant gliomas are the most prevalent primary brain tumors of adults. They are highly invasive and very difficult to treat, despite of surgery, gamma-irradiation and chemotherapy. Although a role of the mitogenic Ras-RAF-MEK-ERK signalling cascade in brain tumor development is well established, there are only few reports available addressing alterations in RAF sequence or protein expression and function in human gliomas. We analysed the mutational status of A-RAF and B-RAF in human glioblastomas (GBM) by sequencing. Then we checked for RAF gene amplification by dot blot hybridization and examined RAF mRNA and protein expression patterns in human astrocytic gliomas of WHO grade II (LGA) and IV (GBM) by semiquantitative RT-PCR and Western blotting, respectively. The results were correlated with patients prognosis. Finally, we performed functional assays to address a putative function of A-RAF in glioma cell proliferation and migration. We showed that RAF mutations are a rare event in glioblastoma multiforme. A-raf gene amplification was more often detected and overexpression of all three RAF proteins on mRNA and protein level was regularly found in human malignant gliomas. Whereas A-RAF and C-RAF expression was negatively correlated with the patients prognosis, B-RAF expression had a positive effect. Since neither A-RAF, nor C-RAF expression had any influence on proliferation and migration of GBM cells, putative functions of C-RAF in angiogenesis and of A-RAF in regulation of metabolism are discussed. Our data indicate that RAF proteins might be valuable targets for small molecule therapies. However, initially specific functions of RAF during tumorigenesis have to be elucidated.

A rapid in vitro method for obtaining RNA accessibility patterns for complementary DNA probes: correlation with an intracellular pattern and known RNA structures.

A technique is described to identify the rare sequences within an RNA molecule that are available for efficient interaction with complementary DNA probes: the target RNA is digested by RNase H in the presence of a random pool of complementary DNA fragments generated from the same DNA preparation that was used for target RNA synthesis. The DNA region was amplified by PCR, partially digested with DNase and denatured prior to RNA binding. In the presence of single-stranded DNA fragments the RNA was digested with RNase H such that, on average, each molecule was cut once. Cleavage sites were detected by gel electrophoresis either directly with end-labeled RNA or by primer extension. The pattern of accessible sites on c- raf mRNA was determined and compared with the known profile of activity of oligonucleotides found in cells, showing the merit of the method for predicting oligonucleotides which are efficient for in vivo antisense targeting. New susceptible sites in the 3'-untranslated region of c- raf mRNA were identified. Also, four RNAs were probed to ascertain to what extent structure predicts accessibility: the P4-P6 domain of the Tetrahymena group I intron, yeast tRNAAsp, Escherichia coli tmRNA and a part of rat 18S rRNA.

Alterations in the methylation status and expression of the raf oncogene in phenobarbital-induced and spontaneous B6C3F1 mouse liver tumors.

The liver tumor-prone B6C3F1 mouse (C57Bl/6 female x C3H/He male), in conjunction with the more susceptible C3H/He paternal strain and the resistant C57BL/6 maternal strain, is an excellent model for studying the mechanisms involved in carcinogenesis. The study reported here indicated that the B6C3F1 mouse inherited a maternal raf allele containing a methylated site not present in the paternal allele. Seven days after partial hepatectomy or after administration of a promoting dose of phenobarbital (PB) for 14 d; raf in B6C3F1 mouse liver was hypomethylated. The additional methylated site in the allele inherited from C57BL/6 was not maintained. The methylation status of raf in the liver of the C57BL/6 mouse was not affected by PB treatment. This indicates that the B6C3F1 mouse is less capable of maintaining methylation of raf than the C57BL/6 strain is. In both PB-induced and spontaneous B6C3F1 liver tumors, raf was hypomethylated in a nonrandom fashion. The level of raf mRNA increased in seven of 10 PB-induced tumors but in only one of five spontaneous tumors, whereas the level of Ha-ras mRNA increased in nine of 10 PB-induced tumors and in four of five spontaneous tumors. The results of our investigation (a) support the hypothesis that hypomethylation of DNA is a nongenotoxic mechanism involved in tumorigenesis, (b) support the notion that PB promotes liver tumors that develop along a pathway different from that leading to spontaneous tumors, and (c) indicate that differences in DNA methylation between C57BL/6 and B6C3F1 mice could, in part, account for the unusually high tendency of the latter strain to develop liver tumors.

Oncogenes in X-ray-transformed C3H 10T1/2 mouse cells and in X-ray-induced mouse fibrosarcoma (RIF-1) cells.

In order to better understand the molecular basis of X-ray induced carcinogenesis we have investigated RNA levels of oncogenes in an X-ray transformed C3H 10T1/2 fibroblast line (XTD) and RIF-1 cells isolated from an X-ray-induced fibrosarcoma in a C3H mouse. Steady-state levels of K-ras, H-ras, N-ras, abl, sis, src, and fos were unchanged in the X-ray-transformed cells compared with non-transformed C3H 10T1/2 cells. However, myc and raf mRNA levels were increased dramatically in the transformed cells. Data further suggests a possible alteration in processing of raf RNA in the XTD cells. Southern blot analysis of secondary transfectants induced with XTD DNA indicated that the oncogenic phenotype did not segregate with the myc or raf loci; nor with nine other oncogenes analysed.

Xenopus c‐raf proto‐oncogene: cloning and expression during oogenesis and early development

We have isolated and characterized a cDNA which contains the entire coding sequence of Xenopus laevis raf protein. raf mRNA is identified as a member of the class of maternal RNAs. It is already relatively abundant at the beginning of oogenesis and is stable at least until the midblastula transition. The RNA is also detected later during embryogenesis in particular in gastrula, neurula, tailbud and feeding tadpole. We have also found the RNA in several adult tissues (skin, testis, stomach, intestine) at different levels.

Changes in the polyadenylation of specific stable RNA during the early development of Xenopus laevis

The distribution of four specific RNA species between the poly(A) + and poly(A) − fractions has been studied during the first hours of Xenopus laevis development, before the mid-blastula transition (MBT). Two of these specific RNA species correspond to clones selected by differential hybridization from a Xenopus egg cDNA library. Another corresponds to Xenopus c- raf mRNA and the last one to RNA revealed by a mouse omithine decarboxylase probe. We show that two of these RNAs are adenylated after fertilization and remain in the poly(A) + population. During the same period, the other two RNAs are deadenylated and these new poly(A) − RNAs remain stable at least until the MBT. These results show (i) that polyadenylation of specific RNA species occurs after fertilization in Xenopus and (ii) that, in the absence of transcription, adenylation and deadenylation can occur simultaneously in the fertilized egg.

Activation of human raf transforming genes by deletion of normal amino-terminal coding sequences.

Three activated cellular raf genes have been detected by transfection of NIH 3T3 cells with human tumor DNAs. Blot hybridization analysis indicated that all three transforming raf genes had recombined with non-raf sequences in the vicinity of raf exon 7-intron 7, resulting in the deletion of about 40% of the normal coding sequence from the raf amino terminus. By cloning sequences upstream of the truncated raf loci we have shown that the rearrangements involve the fusion of three different 5' non-raf human sequences to the human raf gene. No rearrangements could be detected in the raf loci of the three original human tumor DNAs, suggesting that the raf genes were activated by DNA rearrangements occurring during transfection. Significant overexpression of raf mRNA was not evident in two of the three transformant lines, indicating that raf overexpression is not necessary and 5' truncation alone may be sufficient to activate the transforming potential of cellular raf genes.

Activation of human raf transforming genes by deletion of normal amino-terminal coding sequences.

Three activated cellular raf genes have been detected by transfection of NIH 3T3 cells with human tumor DNAs. Blot hybridization analysis indicated that all three transforming raf genes had recombined with non-raf sequences in the vicinity of raf exon 7-intron 7, resulting in the deletion of about 40% of the normal coding sequence from the raf amino terminus. By cloning sequences upstream of the truncated raf loci we have shown that the rearrangements involve the fusion of three different 5' non-raf human sequences to the human raf gene. No rearrangements could be detected in the raf loci of the three original human tumor DNAs, suggesting that the raf genes were activated by DNA rearrangements occurring during transfection. Significant overexpression of raf mRNA was not evident in two of the three transformant lines, indicating that raf overexpression is not necessary and 5' truncation alone may be sufficient to activate the transforming potential of cellular raf genes.