Ras mRNA Research Articles

Discovery of an indolium-based, turn-on fluorescent ligand that represses the Neuroblastoma RAS expression by targeting the 5′-UTR G-quadruplex structure

The RNA G-quadruplex (RG4) at 5′-UTRs of Neuroblastoma RAS (NRAS) mRNA is considered to modulate the NRAS translation, providing new opportunities for interfering with NRAS hyperactivation. Therefore, targeting this RG4 structure by small molecules seems to be an alternative strategy for treating cancers with NRAS mutations or overexpression. In this study, we have developed a promising fluorescent ligand, which exhibited an extremely high fluorescence quantum yield as well as a relatively strong binding affinity with NRAS RG4. Then, this ligand was demonstrated to be able to illuminate the cellular RG4 structures. Furthermore, this ligand remarkably suppressed the translation level of NRAS mRNA, leading to the inhibition of downstream MAPK and AKT signaling pathways in NRAS-amplified MCF-7 cells. Increase of cell apoptosis as well as decrease of cell growth and migration were also detected in tumor cells treated with the ligand. Therefore, a novel and promising fluorescent ligand targeting NRAS RG4 was presented in this study, providing a potential strategy for the "undruggable" protein target.

α-Mangostin Suppresses Melanoma Growth, Migration, and Invasion and Potentiates the Anti-tumor Effect of Chemotherapy.

Purpose: Melanoma is a highly malignant tumor, which metastasizes and has poor prognosis in late-stage cancer patients. α-Mangostin possesses pharmacological properties, including antioxidant, anti-infective, and anticarcinogenic activities. We investigated α-Mangostin effect on melanoma growth, migration, and invasion and its possible molecular mechanism. Methods: Melanoma cells growth inhibition was determined by the colorimetric 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. Morphological changes of α-Mangostin-treated melanoma cells were evaluated by transmission electron microscopy and JC-1 staining. Cell apoptosis and cell cycle arrest were assessed by flow cytometry. The effect of α-Mangostin on tumor cells migration and invasion was observed by migration and invasion in vitro assay. Furthermore, the nude and C57BL/6 mouse subcutaneous melanoma models were used to evaluate the in vivo anti-tumor effect of α-Mangostin. Western blot and real time-PCR were performed to analyze the influence of α-Mangostin on some of the common signaling pathways in melanoma cell lines. Signaling pathways were further verified in dissected tumor tissues. Results: α-Mangostin inhibited in vitro melanoma cells proliferation, migration, and invasion of melanoma cells, induced cell cycle arrest in G0/G1 phase, and caused mitochondrial swelling and membrane depolarization, whereas it effectively suppressed melanoma growth in xenografted mice. In addition, α-Mangostin potentiated the in vitro and in vivo anti-tumor effects of cisplatin both in vitro and in vivo. Mechanistically, α-Mangostin down-regulated expression of RAS protein and mRNA, as well as phosphorylation of PI3K in A375, B16F10, M14 and SK-MEL-2 cells. MITF protein and mRNA were inhibited only in M14 cells. Conclusion: α-Mangostin suppresses melanoma cells growth, migration and invasion, and synergistically enhances the anti-tumor effect of chemotherapy, whose mechanism may be mediated through inhibiting Ras, PI3K and MITF.

Targeted Long-Read Sequencing Decodes the Transcriptional Atlas of the Founding RAS Gene Family Members.

The complicity of human RAS proteins in cancer is a well-documented fact, both due to the mutational hyperactivation of these GTPases and the overexpression of the genes encoding these proteins. Thus, it can be easily assumed that the study of RAS genes at the transcriptional and post-transcriptional level is of the utmost importance. Although previous research has shed some light on the basic mechanisms by which GTPases are involved in tumorigenesis, limited information is known regarding the transcriptional profile of the genes encoding these proteins. The present study highlights for the first time the wide spectrum of the mRNAs generated by the three most significant RAS genes (KRAS, NRAS and HRAS), providing an in-depth analysis of the splicing events and exon/intron boundaries. The implementation of a versatile, targeted nanopore-sequencing approach led to the identification of 39 novel RAS mRNA transcript variants and to the elucidation of their expression profiles in a broad panel of human cell lines. Although the present work unveiled multiple hidden aspects of the RAS gene family, further study is required to unravel the biological function of all the novel alternative transcript variants, as well as the putative protein isoforms.

Novel Strategy of Photodynamic Therapy Targeting RAS mRNA with G-Quadruplex Ligands for Cancer Treatment

The constitutive activation of RAS proteins is closely linked with the aggressiveness of cancer cells. Although RAS proteins are effective targets for cancer therapy, the development of drugs targeting RAS proteins has been unsuccessful so far due to the absence of a suitable active site for small molecule drugs. RAS mRNA that forms a Gquadruplex (G4) in the 5′-untranslated region (UTR) has been deemed as a druggable target. In this study, we demonstrated the advantages of photodynamic therapy (PDT) for the treatment of cancer using G4 ligands targeting RAS mRNA, including our previously reported ligand anionic phthalocyanine ZnAPC.

The effect and mechanism of microRNA-83a targeting on differentiation of bone marrow mesenchymal stem cells in vitro

Objective To study the anti-proliferation and inducing apoptosis effects of microRNA (miRNA, miR)-83a on bone marrow mesenchymal stem cells. Methods The experiment was divided into 3 groups: group A (PEF miR-83a and PSV2neo co-transfection group), group B (non-transfection group), and group C (PEF and PSV2neo co-transfection group). Group A served as the experimental group, and groups B and C group as the control groups. Transfection according to instructions, effects of application of expression detected miR-83a transcription of bone marrow mesenchymal stem cells lines in vitro antiproliferative effect of and biological function; by detecting the expression level of miR-83a immunohistochemical method and real-time quantitative polymerase chain reaction (Real-time PCR) technology in cells, Real-time PCR was measured after transfection, bone marrow mesenchymal stem cells lines Ras, Raf, protein kinase B (Akt) and extracellular signal-regulated kinase (ERK): mRNA expression changes, and through the methyl thiazol tetrazolium (MTT) method, the scratch test, hematoxylin and eosin (HE) staining, microscope observation and comparison of miR-83a transfection on the migration ability of cell proliferation, apoptosis and morphological changes. To analyse by using SPSS 14.0 statistical software. Results Real-time PCR and immunohistochemistry confirmed in miR-83a transfected cells with stable expression; Assingland staining results: after seventh days culture, the cells of the induction differentiation group were full of brown specific staining granules, and no specific staining granules were found in the undifferentiated differentiation group. Real-time PCR results: The expression of Ras, Raf and Akt mRNA in group A were 0.97±0.27, 0.54±0.18 and 0.99±0.31, in group B were 1.68±0.30, 1.58±0.25 and 2.06±0.42, in group C were 1.68±0.30, 1.58±0.25 and 1.89±0.31. Compared with group B and C, the levels of Ras, Raf and Akt mRNA in group A were significantly lower (t=1.531 and 1.838, P=0.016 and 0.011). MTT assay showed that the apoptotic rates of the cells were (23.49±4.64)%, (45.73±8.72)% and (59.25±13.16)% after 24, 48 and 72 h, respectively. There was significant difference between the two groups (χ2= 5.203 and 2.146, P =0.013 and 0.045). The scratch test showed that miR-83a transfected cell migration ability is lower than the normal cells. The effect of miR-83a on lung cancer cell 24 h, morphological observation of lung cancer cell apoptosis or necrosis. Conclusion Transfection of miR-83a can inhibit proliferation and promote apoptosis effect on pituitary adenoma cells in vitro cells, which is likely to play a role through inhibition of epidermal growth factor receptor (EGFR) and EGFRvⅢ signal transduction pathway in the RAS-RAF-mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PI3K)-Akt of two important signaling pathways activated. Key words: Bone marrow mesenchymal stem cells; MicroRNA-83a; Proliferation; Apoptosis; Biological function

Traditional Chinese medicine suppresses left ventricular hypertrophy by targeting extracellular signal-regulated kinases signaling pathway in spontaneously hypertensive rats

Chinese herbal medicine Bu-Shen-Jiang-Ya decoction (BSJYD) is reported to be beneficial for hypertension. Over expression of extracellular signal regulated kinases (ERK) pathway plays an important role in left ventricular hypertrophy (LVH). This study aimed to observe effects of BSJYD on LVH in spontaneously hypertensive rats (SHRs) and explore its possible mechanism on regulation of ERK pathway. Sixty 12-week-old SHRs were randomly allocated into 5 groups: BSJYD high dose group, middle dose group, low dose group, captopril group, and control group. Besides, a control group of Wistar-Kyoto rats was established. All rats were treated for 8 weeks. Systolic blood pressure (SBP), heart rate (HR), pathology, and left ventricular mass index (LVMI) were measured. Western blotting and Real-time PCR were used to assess the expressions of BDNF, Ras, ERK1/2, and c-fox levels. SBP and HR were significantly decreased compared with the control group and LVMI was markedly improved by BSJYD treatment in a dose-dependent manner. BSJYD inhibited the expression of BDNF, Ras, ERK1/2, and c-fox mRNA in LVH. In conclusion, BSJYD suppressed hypertension-induced cardiac hypertrophy by inhibiting the expression of ERK pathway. These changes in gene expression may be a possible mechanism by which BSJYD provides myocardial protection from hypertension.

The Prognostic Value of HRAS mRNA Expression in Cutaneous Melanoma.

This study aimed to investigate the prognostic value of HRAS mRNA expression in cutaneous melanoma. Cutaneous melanoma is an aggressive cancer with an increasing incidence. Few studies have focused on the transcriptional level of RAS isoforms (KRAS, NRAS, and HRAS) in cutaneous melanoma. To gain further insight into RAS isoforms at transcriptional level, we obtained the cutaneous melanoma data from cBioPortal and investigated the RAS mRNA expression levels in different stages of melanoma and evaluated their correlation with clinical characteristics and patients' survival. Furthermore, we retrieved and analyzed the coexpression data and performed pathway enrichment analysis. Totally, 452 cutaneous melanoma cases were included in this study. We found that lower HRAS expression level was associated with longer patient survival. 206 genes that negatively correlated with HRAS expression were positively correlated with KRAS and NRAS expression. In contrast, no gene that positively correlated with HRAS expression was positively correlated with KRAS and NRAS expression. In conclusion, our data showed that transcriptional regulation was different for the three RAS isoforms in cutaneous melanoma. This study highlighted the prognostic value of HRAS mRNA expression and revealed that HRAS greatly differs from KRAS and NRAS at the transcriptional level.

Functional RNAs: combined assembly and packaging in VLPs.

We describe here a one pot RNA production, packaging and delivery system based on bacteriophage Qβ. We demonstrate a method for production of a novel RNAi scaffold, packaged within Qβ virus-like particles (VLPs). The RNAi scaffold is a general utility chimera that contains a functional RNA duplex with paired silencing and carrier sequences stabilized by a miR-30 stem-loop. The Qβ hairpin on the 5΄ end confers affinity for the Qβ coat protein (CP). Silencing sequences can include mature miRNAs and siRNAs, and can target essentially any desired mRNA. The VLP–RNAi assembles upon co-expression of CP and the RNAi scaffold in E. coli. The annealing of the scaffold to form functional RNAs is intramolecular and is therefore robust and concentration independent. We demonstrate dose- and time-dependent inhibition of GFP expression in human cells with VLP–RNAi. In addition, we target the 3΄UTR of oncogenic Ras mRNA and suppress Pan-Ras expression, which attenuates cell proliferation and promotes mortality of brain tumor cells. This combination of RNAi scaffold design with Qβ VLP packaging is demonstrated to be target-specific and efficient.

Lower Ras expression as an independent predictor of patient outcomes in lung cancer treated with bevacizumab plus chemotherapy

The objective of this study was to analyze the predictive roles of VEGF/KDR/Ras/MAPK gene expression in patients with advanced non-small-cell lung cancer (NSCLC) treated with bevacizumab plus chemotherapy. Twenty-five patients participating in an open-label phase IV trial (SAiL, MO19390) with available tumor tissues were analyzed. The mRNA expression levels of VEGF, kinase insert domain receptor (KDR), Ras, and mitogen-activated protein kinase (MAPK) in tumor tissues were detected using real-time quantitative PCR methods. The relationships between gene expression and disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) were assessed. Patients with lower Ras expression had a longer PFS and OS than patients with higher expression (median PFS, 9.9 vs 5.5 months, χ(2)=3.944, P=0.047; OS, 19.3 vs 7.1 months, χ(2)=9.384, P=0.002). The PFS and OS of patients with lower and higher MAPK expression exhibited a marginal and significant difference (median PFS, 9.9 vs 5.5 months, χ(2)=3.464, P=0.063; OS, 19.3 vs 9.7 months, χ(2)=5.298, P=0.021), respectively. Multivariate analyses using Cox's proportional hazards model showed that Ras is an independent predictor of OS (χ(2)=9.384, P=0.002). No differences in DCR were found according to Ras expression level. The results indicate that Ras is an independent predictor of OS. Thus, patients with lower Ras expression are most likely to benefit from bevacizumab plus chemotherapy treatment regimen. Patients with higher levels of Ras should receive other inhibitors that target Ras. The results also suggest that gene therapies that decrease RAS expression combined with bevacizumab may improve lung cancer treatment. Although there is a very important implication to patient selection in the target therapy, the data in this study are very preliminary owing to the too small sample size. Therefore, further research involving large numbers of patients and a prospective assessment of low and high RAS mRNA expressions getting the same treatments need to be done before conclusions can be made.

Abstract P5-03-09: Ovarian Hormones Promote Mammary Carcinogenesis by Altering Growth Factor Receptor Signaling

Abstract Study of carcinogenesis in rat models plays an important role in understanding the nature of neoplastic transformation. Identification of biological, cellular and molecular alterations involved in the carcinogen induced preneoplastic transformation has vital importance in mammary cancer development. Copenhagen rats are resistant to the carcinogen, N-methyl-N-nitrosourea (MNU) induced mammary carcinogenesis. Our previous studies indicate that MNU induced preneoplastic lesions were promoted to mammary cancer by the hormones estrogen and progesterone. The purpose of this study is to delineate the molecular mechanisms of hormonal promotion of mammary carcinogenesis. Seven week old female Copenhagen rats were administered a single dose of 50 mg/kg body weight MNU intraperitoneally. Four weeks post carcinogen administration, rats were divided into 4 groups, 1) Control, 2) rats treated with 30 mg estradiol, 3) rats treated with 30 mg progesterone, 4) rats treated with 30 mg estradiol plus 30 mg of progesterone. Two weeks after the implantation rats were killed and mammary glands were excised and used for further experiments. Quantitative RT-PCR experiments showed up-regulation of growth factor receptors such as Igf-1r, Egfr and growth factor signaling members Grb2, Pi3k and Ras mRNA expression. Immuno histochemical studies showed strong staining to growth factor receptors and western blotting studies showed increased protein levels of growth factor receptors such as IGF-1R and EGFR and active form of proliferative signaling molecules Akt and SAPK/JNK were up-regulated in combination of estradiol and progesterone treated rats. Phosphorylated p38 was reduced in the combination of hormones treated rat preneoplastic lesions when compared to control. Combination of ovarian hormones treatment provided enhanced growth factor signaling environment and promoted the mammary lesions through activation of SAPK/JNK and growth factor signaling pathways. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-03-09.

Cycling exercise affects the expression of apoptosis-associated microRNAs after spinal cord injury in rats

There are two major aspects to a spinal cord injury (SCI): an acute, primary mechanical trauma and a progressive phase of secondary tissue damage provoked by inflammation, excitotoxicity, apoptosis, and demyelination. MicroRNAs (miRs) are small, ~ 22 nucleotide, non-protein-coding RNAs that function at the post-transcriptional level to regulate gene expression. They have important roles in homeostatic processes such as cell proliferation and programmed cell death. In the injured rat spinal cord we performed an expression analysis of miRs and their downstream targets involved in apoptotic pathways and used post-injury cycling exercise to test for activity-dependent plasticity of miR expression. We show that SCI results in increased expression of miR Let-7a and miR16 while exercise leads to elevated levels of miR21 and decreased levels of miR15b. These changes in miR expression are correlated with changes in expression of their target genes: pro-apoptotic (decreased PTEN, PDCD4, and RAS mRNA) and anti-apoptotic (increased Bcl-2 mRNA) target genes. This is accompanied by a down-regulation of mRNA for caspase-7 and caspase-9 and reduced levels of caspase-7 protein. These results indicate possible beneficial effects of exercise through action on multiple miRs and their targets that contribute to the functional regulation of apoptosis after SCI.

Mutations and differential expression of the ras family genes in human nasal polyposis

Although it is well established that ras genes contribute to tumourigenesis either through the accumulation of mutations or by aberrant expression in a wide range of human cancers, little is known regarding their involvement in human nasal polyps (NPs). In the present study, the occurrence of mutations in codons 11 and 12 of the ras family genes was examined by PCR/RFLP and direct sequencing in 23 human NPs and their adjacent turbinates, as well as in turbinates from 13 control subjects. Moreover, the expression pattern of ras mRNA levels was assessed in NP specimens and compared to adjacent and control tissues. K-ras codon 11 and 12 mutations were detected in 17 and 35% of NPs, respectively, and were found in the adjacent inferior turbinate (AIT) (22 and 16%, respectively) and adjacent middle turbinates (AMT) (16 and 26%, respectively). K- and H-ras expression levels were elevated, whereas N-ras mRNA levels were lower in NPs and adjacent turbinates as compared to the control tissues. K-ras mRNA levels were up-regulated in advanced-stage polyps (P=0.037), while N-ras levels were found elevated in small polyps (P=0.046). Statistically significant negative correlations between K- and N-ras expression profiles arose in NPs and AITs (P=0.009 and 0.003, respectively). This, to our knowledge, is the first report on ras mutations and expression analysis in NPs. Our findings suggest a potential key role for activated members of ras family genes in terms of their contribution to the development of NPs as well as to the hypertrophy of adjacent turbinates.

Impact of MAPK cascade pathway and P53 pathway upon liver transplant

The change and the role of MAPK cascade pathway and P53 pathway after liver transplantation were explored. Thirty-four punctured donor liver specimens and 10 normal liver specimens were classified as group A (no rejection, n = 10), group B (mild/moderate acute rejection, n = 10), group C (serious acute rejection, n = 8), group D (chronic rejection/fibrosis, n = 6) and group E (control, n = 10). By using immunohistochemistry, the expression levels of mitogen activated protein kinase (MAPK), Ras and P53 proteins, and by in situ hybridization, MAPK and ras mRNA expression levels were detected. The results showed that the expression levels of MAPK and Ras proteins were increased by turns in groups A, B and C, and decreased by turns in groups D and E. The protein expression of P53 was higher in the treated groups. The expression of Ras, HSP70 mRNA was identical as that of protein. It is suggested that the MAPK cascade pathway and P53 pathway can protect the hepatocytes by different mechanisms after liver transplantation. MAPKs cascade pathway repairs hepatocyte injury or accelerates hepatocytes into proliferation or differentiation. P53 pathway blocks cell cycle within G1 phase to make hepatocyte repair or apoptosis to reduce disorder differentiation.

Resveratrol modulates gene expression associated with apoptosis, proliferation and cell cycle in cells with mutated human c-Ha-Ras, but does not alter c-Ha-Ras mRNA or protein expression

An accumulating body of evidence suggests that resveratrol can inhibit carcinogenesis through antiproliferative and apoptotic effects. One proposed mechanism for this is the modulation of genes, for example, Ras and p53, frequently associated with human cancer. To test the effect of resveratrol on gene expression, we used the WR-21 cell line because it contains a mutated human c-Ha-ras gene. Cells at ≥70% confluency were incubated with media alone or with increasing concentrations of trans-resveratrol (0.1–1000 μM) for 24 h. Resveratrol (30–100 μM) decreased cellular proliferation by 80% (bromodeoxyuridine incorporation) and increased apoptosis by 60% (TUNEL). Cells were then treated with media alone or with 50-μM resveratrol for 24 h. RNA was isolated for nylon-based macroarray analyses and protein for immunoblotting. Resveratrol increased (+) and decreased (−) gene expression associated with apoptosis (Birc5+, Cash+, Mcl-1+, Mdm2+, Rpa-like+), cellular proliferation (Ctsd+, Mdm2+, Egr1+, ODC+) and cell cycle (cyclin D+, cyclin g+, Gadd45a−, Mad2l−, Mdm2+). Resveratrol consistently increased by ≥6-fold Mdm2 expression and other downstream p53 effectors, but not p53 itself at 24 h. Subsequent cell cycle analysis indicated a significant accumulation of cells in G2/M, and a decrease in G1/G0 suggesting a G2/M blockade. Further RT-PCR and Western blot analyses indicated no differential changes in Ras mRNA expression or p21 ras protein levels, respectively. These results suggest that resveratrol potently inhibits cellular proliferation, increases apoptosis, alters cell cycle dynamics and modulates associated gene expression. Furthermore, these effects appear mediated, in part, by p53 without direct modulation of mutant c-Ha-ras expression.

Increased p21 ras activity in human fibroblasts transduced with survivin enhances cell proliferation

Survivin is critically involved in mitosis and when overexpressed enhances the activity of the Aurora B kinase, a serine-threonine kinase belonging to the family of oncogenic Aurora/IpI1p-related kinases. Both proteins interact with Ras GTPase-activating protein suggesting an impact on the Ras pathway. This study aimed at defining the role of survivin in proliferation and potential transformation of cells. When survivin was overexpressed in normal human lung fibroblasts, the characteristic track lanes of fibroblasts were disturbed and the rate of cell proliferation was increased. An enhanced level of p21 ras mRNA and protein expression and concomitant rise in levels of activated p21 ras were observed. Despite increased proliferation cell survival remained dependent on serum and cells were not able to form colonies in soft agar assays. These data suggest that overexpression of survivin increases cell growth but, despite the increase in active p21 ras, is not sufficient to transform primary cells. Yet, in addition to its anti-apoptotic function it might contribute to the accelerated growth of tumour cells by increasing p21 ras activity.

Transcriptional activation of H- and N- ras oncogenes in human cervical cancer

Objective. Overexpression of p21 protein has been detected in human cervical cancer. However, to date, there are no data on the differential activation of the three ras genes at the transcriptional level in cervical lesions. The purpose of this study was to evaluate the quantitative and qualitative changes of expression of the ras family genes in the development of human cervical cancer. Methods. The expression of ras mRNA levels in 35 human cervical specimens [11 normal cervix, 15 cervical intraepithelial neoplasia (CIN), 9 cervical cancer] was examined using the RT-PCR technique. In addition, we studied the incidence of point mutations in codon 12 of each ras gene using RFLP analysis and human papilloma virus (HPV) status. Results. The transcript levels for H- ras and N- ras were significantly higher in cancer cases compared to normal cervical tissues ( P = 0.0002 and P = 0.001, respectively) and CIN lesions ( P < 0.0001 and P = 0.002, respectively). The transcript levels for K- ras were similar in normal cervical tissue, CIN and cervical cancer. A strong positive correlation was found between H- and N- ras expression ( P = 0.001) and no correlation between H- and K- or N- and K- ras expression. Point mutations were detected only in three samples, located in codon 12 of K- ras gene. No relationship was found between expression levels of each ras gene and the presence of HPV. Conclusion. Our findings indicate the expression pattern of the three ras genes in cervical tissue and the involvement of H- and N- ras up-regulation in the pathogenesis of cervical cancer independent of HPV infection.

Consequences of Mevalonate Depletion: DIFFERENTIAL TRANSCRIPTIONAL, TRANSLATIONAL, AND POST-TRANSLATIONAL UP-REGULATION OF Ras, Rap1a, RhoA, AND RhoB

Ras-related proteins are small GTPases that are post-translationally modified with mevalonate-derived isoprenoids. Although the effects of inhibition of isoprenylation on protein function have been examined, the consequences of depletion of isoprenoid pools on regulation of expression of isoprenylated proteins have yet to be investigated. In these studies we have shown that depletion of mevalonate results in increased total levels of Ras, Rap1a, RhoA, and RhoB in K562 cells. Cycloheximide and [(35)S]methionine pulse/pulse-chase experiments reveal that mevalonate depletion increases the de novo synthesis of Ras and RhoA and decreases the degradation of existing Ras and RhoA protein. Pretreatment with actinomycin D completely prevents the induced up-regulation of RhoB and only partially prevents the up-regulation of Ras, Rap1a, and RhoA. Although depletion of mevalonate does not alter steady state levels of Ras mRNA, there is an increase in RhoB mRNA. Our results are the first to demonstrate that mevalonate depletion induces up-regulation of Ras and Ras-related proteins by discrete mechanisms that include modulation of transcriptional, translational, and post-translational processes.

Development of Chemopreventive Strategies for Radiation-Induced Cancer: Targeting Radiation-Induced Genetic Alterations

Carcinogenesis is a multistage process involving dysregulation of signal transduction and cell cycle pathways. This dysregulation results in specific molecular and genetic alterations, including gene amplification, mutations, and chromosomal rearrangements. These aberrations can be measured to provide a novel means to assess carcinogenic risk or as targets for chemointervention. Recent human and in vivo studies have demonstrated that genetic alterations, such as oncogenes and oncoproteins, were observed in preneoplastic tissues or serum following exposure to chemical carcinogens or low-level radiation (LLR). Identification of preneoplastic changes following radiation exposure may provide information that will allow development of LLR chemopreventive strategies. Radiation carcinogenesis studies in vivo with a lung tumor model showed that a low-dose cobalt-60 radiation exposure induced persistent time-dependent genetic alterations, such as elevated ras expression. This radiation exposure also resulted in lung tumor formation in 26% of the irradiated animals at 232 days after irradiation. A significant and progressive increase in ras oncogene expression was measured using Northern blot analysis in 80% of the irradiated animals over the duration of the experiment. Pharmacological intervention strategies are being tested using buthionine-[S,R]-sulfoximine (BSO). BSO has been previously shown to down-regulate ras expression. Administration of BSO prevented radiation-induced changes in ras mRNA levels in this lung tumor model. Further studies are being conducted with an LLR-induced leukemia model in which detection of circulating levels of oncoproteins will be more feasible. Based on these preliminary results and on its clinical efficacy and low clinical toxicity, BSO warrants further study as an LLR chemopreventive agent. Furthermore, this strategy to target LLR-induced preneoplastic alterations may be an effective means of developing modulators of LLR-induced cancers.

An unnatural base pair for incorporating amino acid analogs into proteins.

An unnatural base pair of 2-amino-6-(2-thienyl)purine (denoted by s) and pyridin-2-one (denoted by y) was developed to expand the genetic code. The ribonucleoside triphosphate of y was site-specifically incorporated into RNA, opposite s in a template, by T7 RNA polymerase. This transcription was coupled with translation in an Escherichia coli cell-free system. The yAG codon in the transcribed ras mRNA was recognized by the CUs anticodon of a yeast tyrosine transfer RNA (tRNA) variant, which had been enzymatically aminoacylated with an unnatural amino acid, 3-chlorotyrosine. Site-specific incorporation of 3-chlorotyrosine into the Ras protein was demonstrated by liquid chromatography-mass spectrometry (LC-MS) analysis of the products. This coupled transcription-translation system will permit the efficient synthesis of proteins with a tyrosine analog at the desired position.

Chromosomal rearrangements involving telomeric DNA sequences in Balb/3T3 cells transfected with the Ha-ras oncogene.

Chromosomal instability involving telomeric DNA sequences was studied in mouse Balb/3T3 fibroblasts transfected with a mutated human c-Ha-ras-1 gene (B61 cells) and spontaneously immortalized normal parental cells (A31 cells), using fluorescence in situ hybridization (FISH). FISH analysis with a telomeric probe revealed high frequencies of chromosome alterations involving telomeric regions, mainly stable and unstable Robertsonian fusion-like configurations (RLC) (0.25 and 1.95/cell in A31 and B61 cells, respectively) and chromosome ends lacking telomeric signals in one (LTS') or both chromatids (LTS") (5.9 and 17.5/cell for A31 and B61 cells, respectively). Interstitial telomeric sequences (ITS) were also detected at both non-telomeric sites and in the centromeres of RLC. The frequencies of RLCs with ITS located in the centromeres were 3-fold higher in B61 compared with A31 cells. We demonstrated a high level of chromosome instability involving telomeric DNA sequences in ras-transfected cells overexpressing ras mRNA, which could be a consequence of rapid cell cycle progression associated with a deficient telomere capping mechanism.