Tritiated Thymidine Incorporation Does Not Measure DNA Synthesis in Ribavirin-Treated Human Cells

The eukaryotic translation initiation factor eIF4E is deregulated in many human cancers, and its overexpression in cells leads to malignant transformation. Oncogenic properties of eIF4E are directly linked to its ability to bind 7-methyl guanosine of the 5 mRNA. Here, we observe that the antiviral guanosine analogue ribavirin binds to eIF4E with micromolar affinity at the functional site used by 7-methyl guanosine mRNA cap, competes with eIF4E:mRNA binding, and, at low micromolar concentrations, selectively disrupts eIF4E subcellular organization and transport and translation of mRNAs posttranscriptionally regulated by eIF4E, thereby reducing levels of oncogenes such as cyclin D1. Ribavirin potently suppresses eIF4E-mediated oncogenic transformation of murine cells in vitro, of tumor growth of a mouse model of eIF4E-dependent human squamous cell carcinoma in vivo, and of colony formation of eIF4E-dependent acute myelogenous leukemia cells derived from human patients. These findings describe a specific, potent, and unforeseen mechanism of action of ribavirin. Quantum mechanical and NMR structural studies offer directions for the development of derivatives with improved cytostatic and antiviral properties. In all, ribavirin's association with eIF4E may provide a pharmacologic means for the interruption of posttranscriptional networks of oncogenes that maintain and enhance neoplasia and malignancy in human cancer.cancer ͉ drug ͉ virus ͉ oncogenic network W hereas the precise causes of neoplasia and malignancy are known only for a few human cancers, deregulated tumor suppressors and oncogenes that maintain and enhance the malignant phenotype are becoming relatively well described (1, 2). Among these molecules are tumor suppressors like p53, Rb, and APC and oncogenes such as myc, cyclin D1, and eIF4E. Their interactions constitute a network of self-reinforcing feedback loops, whereby inactivation of principal elements can lead to reversal and at times even sustained loss of neoplastic phenotype (3, 4). eIF4E is overexpressed in a wide variety of malignant cell lines and primary human tumors such as carcinomas of the breast (5), colon (6), and head and neck (7), non-Hodgkin's lymphomas (8), and chronic and acute M4͞M5 myelogenous leukemias (9). Consistently, even moderate overexpression of eIF4E in rodent cells leads to deregulated proliferation and malignant transformation (10). eIF4E is essential for growth and survival of eukaryotes by acting at a critical step of cap-dependent translation and recruiting transcripts to the ribosome as a result of a specific interaction with the 5Ј 7-methyl guanosine (m 7 G) mRNA cap (11). It is important to stress that, although the interaction of eIF4E with the 5Ј mRNA cap is required for initiation of translation of cap-dependent mRNAs, up-regulation of eIF4E does not increase translation of all cap-dependent transcripts, but only of a specific subset of eIF4E-sensitive transcripts, as described below.As much as 70% of eIF4E is present in the nuclei of mammalian cells, where it i...

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“…7c), which is consistent with ribavirin's down-regulation of cyclin D1 (Fig. 2b) and with earlier studies (25).…”

Section: Resultssupporting

confidence: 80%

Ribavirin suppresses eIF4E-mediated oncogenic transformation by physical mimicry of the 7-methyl guanosine mRNA cap

Kentsis

Topisirović

Čuljković

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

272

357

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“…Our studies, as well as those of others (35) (Fig. 6), diminished the capacity of lectin-stimulated PBMC to progress through the cell cycle (Figs.…”

Section: Discussionmentioning

confidence: 61%

“…1-6). We have employed highly sensitive methods to assess the effects ofribavirin on the PBMC response to lectin and differences between the methodologies used by Drach et al (35) and us most likely account for the disparities between these studies.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of immune functions by antiviral drugs.

Heagy

Crumpacker²,

Lopez³

et al. 1991

J. Clin. Invest.

131

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Immune functions were evaluated in vitro for PBMC isolated from healthy donors and cultured with the antiviral agents, 3'-azido-3'-deoxythymidine (AZT), ribavirin, ganciclovir, 2'3'-dideoxyinosine (ddl), or acyclovir. To identify methods for assessing the effects of antiviral drugs on immune cells, the PBMC response to mitogens, Con A, or phytohemagglutinin was evaluated from measurements of [Hjthymidine and 11[Cleucine incorporation, cell growth, cellular RNA, DNA, and protein levels, and the PBMC proliferative cycle (i.e., progression from GO --G1 --S -G2 + M).At clinically relevant concentrations, AZT, ribavirin, or ganciclovir diminished PBMC responsiveness to mitogen. The numbers of proliferating cells in G1, S., and G2 + M phases of the cell cycle, DNA content, and VHjthymidine uptake were decreased in cultures treated with AZT, ribavirin, or ganciclovir. AZT or ribavirin but not ganciclovir reduced RNA and protein in the cultures and inhibited cell growth. Whereas AZT, ribavirin, or ganciclovir were antiproliferative, ddI or acyclovir had little, if any, effect on PBMC mitogenesis. The inhibitory effects of antivirals on immune cells may contribute to the immune deterioration observed in patients following prolonged use of the drugs. (J. Clin.

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“…However, when limited for thymidine, thyA mutants are known to undergo thymine-less death (1), a phenomenon during which chromosomal DNA suffers singlestrand breaks (24). (10,15,30).…”

Section: Phenol-chloroform Extraction Of [mentioning

confidence: 99%

“…However, when limited for thymidine, thyA mutants are known to undergo thymine-less death (1), a phenomenon during which chromosomal DNA suffers singlestrand breaks (24). (10,15,30).To avoid the possibility of thymine starvation in our experiments, we also attempted to visualize Okazaki fragments by using the [ 32 P]orthophosphate label which we routinely employ to label chromosomal DNA for pulsed-field gel electrophoresis (17, 36). Since we expected that the bulk of the 32 P label will be deposited into RNA, we removed RNA altogether by separating chromosomal DNA from replication intermediates in alkaline agarose gels.…”

mentioning

confidence: 99%

Polyphosphate Accumulation in Escherichia coli in Response to Defects in DNA Metabolism

Amado

Kuzminov

2009

J Bacteriol

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Phenol-chloroform extraction of [32 P]orthophosphate-labeled Escherichia coli cells followed by alkaline gel electrophoresis revealed, besides the expected chromosomal DNA, two non-DNA species that we have identified as lipopolysaccharides and polyphosphates by using a combination of biochemical and genetic techniques. We used this serendipitously found straightforward protocol for direct polyphosphate detection to quantify polyphosphate levels in E. coli mutants with diverse defects in the DNA metabolism. We detected increased polyphosphate accumulation in the ligA, ligA recBCD, dut ung, and thyA mutants. Polyphosphate accumulation may thus be an indicator of general DNA stress.DNA replication intermediates, also known as Okazaki fragments, have classically been detected by pulse labeling thymine-limited thyA mutant cells with [ 3 H]thymidine, a DNAspecific label (27). However, when limited for thymidine, thyA mutants are known to undergo thymine-less death (1), a phenomenon during which chromosomal DNA suffers singlestrand breaks (24). (10,15,30).To avoid the possibility of thymine starvation in our experiments, we also attempted to visualize Okazaki fragments by using the [ 32 P]orthophosphate label which we routinely employ to label chromosomal DNA for pulsed-field gel electrophoresis (17, 36). Since we expected that the bulk of the 32 P label will be deposited into RNA, we removed RNA altogether by separating chromosomal DNA from replication intermediates in alkaline agarose gels. We found, however, that Okazaki pieces cannot be detected using [ 32 P]orthophosphate even by alkaline agarose because there are other molecules in larger amounts in the cells that take in 32 P-label and mask the replication intermediates. We report on the identification and quantification of two of the "masking species" in wild-type Escherichia coli, as well as in several mutants. MATERIALS AND METHODSBacterial strains, growth conditions, labeling, and isolation of phosphatecontaining species. All strains are described in Table 1. Cells were grown with shaking at 30°C in MOPS (morpholinepropanesulfonic acid) low-phosphate medium (25) supplemented with 0.2% Casamino Acids to an optical density at 600 nm (OD 600 ) of 0.2 to 0.4. Cultures were further incubated at 30°C, 37°C, or 42°C and labeled with [ 32 P]orthophosphate (5 to 10 Ci/ml) for the amount of time indicated in the figures. Samples were processed by spinning down the cells and resuspending them in 50 l of 20% sucrose in Tris-EDTA. Three hundred fifty microliters of 2% sodium dodecyl sulfate (SDS) were added, and after thorough mixing, the cells were lysed by incubation at 70°C for 10 min. Isolation of nucleic acids was achieved by subsequent extraction with 400 l of phenol, followed by 400 l of phenol-chloroform, and finally, 400 l of chloroform, with two ethanol precipitations (18). Alternatively, the Wizard genomic DNA purification kit (Promega) was used.Enzymatic reactions. DNase I, exonuclease I, and calf intestinal phosphatase (CIP) were purchased from New England ...

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Tritiated Thymidine Incorporation Does Not Measure DNA Synthesis in Ribavirin-Treated Human Cells

Cited by 41 publications

References 19 publications

Ribavirin suppresses eIF4E-mediated oncogenic transformation by physical mimicry of the 7-methyl guanosine mRNA cap

Ribavirin suppresses eIF4E-mediated oncogenic transformation by physical mimicry of the 7-methyl guanosine mRNA cap

Inhibition of immune functions by antiviral drugs.

Polyphosphate Accumulation in Escherichia coli in Response to Defects in DNA Metabolism

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