Methionine Deprivation Research Articles

Abstract P4-07-04: Methioninase cell-cycle synchronization potentiates chemotherapy for breast cancer

Abstract Deprivation of methionine selectively arrests cancer cells during late S-phase (Proc. Natl. Acad. Sci. USA 77, 7306–7310, 1980; Biochim. Biophys. Acta, Reviews on Cancer 738, 49–87, 1984), where they are highly sensitive to chemotherapy drugs which damage DNA (J. Natl. Cancer Inst. 76, 629–639, 1986). Cancer cells, transformed to express different color fluorescent reporters during specific phases of the cell cycle (Cell 132, 487–498, 2008), were used to monitor the onset of the S/G2-phase block due to methionine deprivation effected by recombinant methioninase (rMETase). The S/G2-phase blocked cancer cells fluoresced yellow or green in contrast to cancer cells in G1 which fluoresced red. Cancer cells, including MCF-7 breast cancer, synchronously blocked in S/G2-phase by rMETase, were identified by their yellow-green fluorescence and allowed to accumulate to the maximum extent. At the point of maximum yellow/green cells in the culture, the cells were administered chemotherapy drugs which interact with DNA or block DNA synthesis such as doxorubicin. We termed this procedure color-coded chemotherapy (CCC). CCC was highly effective against the cancer cells (90% cell kill). In contrast, treatment of cancer cells with drugs only, and without rMETase-effected S/G2-phase synchrony, led to the majority of the cancer cell population being blocked in G1 phase (red fluorescent) where they were resistant to the drugs (40% cell kill). CCC, which identifies, by fluorescent color, when cancer cells are blocked in S/G2-phase by a unique cell-cycle-blocking agent, rMETase, demonstrates the potential of cell-synchronization-based chemotherapy for breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-07-04.

Chemotherapy of methioninase-synchronized S/G2 phase-blocked cancer cells identified by cell cycle-specific fluorescent reporters.

1063 Background: Cancer cells of all types have a generally elevated requirement for methionine compared to normal cells. This phenomenon is termed methionine-dependence and may be due to excessive methylation reactions in cancer cells, since methionine is the global source of cellular methyl groups (Biochim. Biophys. Acta, Reviews on Cancer 738, 49-87, 1984). Deprivation of methionine selectively arrests cancer cells during late S-phase (Proc. Natl. Acad. Sci. USA 77, 7306-7310, 1980), where they are highly sensitive to chemotherapy drugs which damage DNA (J. Natl. Cancer Inst. 76, 629-639, 1986). Methods: Cancer cells, transformed to express different color fluorescent reporters during specific phases of the cell cycle (Cell 132, 487-498, 2008), were used to monitor the onset of the S/G2-phase block due to methionine deprivation effected by recombinant methioninase (rMETase). The S/G2-phase blocked cancer cells fluoresced yellow or green in contrast to cancer cells in G1 which fluoresced red. Cancer cells, including MKN45 stromal cancer and MCF-7 breast cancer, synchronously blocked in S/G2-phase by rMETase, were identified by their yellow-green fluorescence and allowed to accumulate to the maximum extent. At the point of maximum yellow/green cells in the culture, the cells were administered chemotherapy drugs which interact with DNA or block DNA synthesis such as doxorubicin, cisplatin or 5-fluorouracil. We termed this procedure color-coded chemotherapy (CCC). Results: CCC was highly effective against the cancer cells (90% cell kill). In contrast, treatment of cancer cells with drugs only, and without rMETase-effected S/G2-phase synchrony, led to the majority of the cancer cell population being blocked in G1 phase (red fluorescent) where they were resistant to the drugs (40% cell kill). Conclusions: CCC, which identifies, by fluorescent color, when cancer cells are blocked in S/G2-phase by a unique cell-cycle-blocking agent, rMETase, demonstrates the potential of cell-synchronization-based chemotherapy.

Abstract 2679: Color-coded selective chemotherapy of methioninase-synchronized S-phase cancer cells expressing cell-cycle-specific fluorescent reporters: A general approach to the treatment of cancer

Abstract Cancer cells of all types have a generally elevated requirement for methionine compared to normal cells. This phenomenon is termed methionine-dependence and may be due to excessive methylation reactions in cancer cells, since methionine is the global source of cellular methyl groups. Deprivation of methionine α,β lyase (methioninase or METase) selectively arrests cancer cells during late S-phase, where they are highly sensitive to chemotherapy drugs which damage DNA. Cancer cells, transformed to express different color fluorescent reporters during specific phases of the cell cycle (fluorescent ubiquitination-based cell cycle indicator [FUCCI]), were used to monitor the onset of the S-phase block due to methionine deprivation effected by METase. The S-phase-blocked cancer cells fluoresced yellow or green, in contrast to cancer cells in G1 which fluoresced red. Cancer cells, synchronously blocked in S-phase by METase and identified by their yellow-green fluorescence, were administered chemotherapy drugs which interact with DNA or block DNA synthesis such as doxorubicin, cisplatin or 5-fluorouracil. We have termed this strategy color-coded chemotherapy. The procedure was highly effective against the cancer cells. In contrast, treatment of cancer cells with drugs only, and without methioninase-effected S-phase synchrony, led to the majority of the cancer cell population being blocked in G phase (red fluorescent) where they were resistant to the drugs. The selective anticancer strategy and technology described in this report, whereby cancer cells are selectively and synchronously blocked in S-phase in the most drug-sensitive phase of the cell cycle where they are identified by fluorescent reporters and then treated with S-phase-specific-drugs, should be a general approach to the treatment of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2679. doi:1538-7445.AM2012-2679

Abstract 2686: Methioninase S-phase block of cancer cells imaged in real time by cell-cycle-specific fluorescence reporters

Abstract A general feature of cancer cells is their elevated requirement for methionine compared to normal cells, which may be due to excessive methylation reactions in cancer cells since methionine is the global source of cellular methyl groups. When deprived of methionine, cancer cells undergo a cell-cycle arrest in late S-phase. The recombinant bacterial enzyme, methionine β, α-lyase (methioninase or METase) degrades methionine very efficiently and effects an S-phase block selectively in cancer cells. In order to image the kinetics of cell cycle arrest of cancer cells due to deprivation of methionine by METase, cancer cells were engineered to express cell cycle-dependent fluorescent proteins using fluorescent ubiquitination-based cell cycle indicator (FUCCI) whereby cancer cells express Azami-green only in S-phase and Kusabira-orange only in G1. Thus, each cancer could be followed in real time as it becomes blocked in S-phase after METase treatment. FUCCI imaging demonstrated that cancer cells could remain in S-phase for 5 days before starting to die which indicates a new type of cancer cell dormancy. The use of METase treatment and cell-cycle specific fluorescent reporters provide a unique opportunity to image and study the cancer cell cycle and the consequences and advantages of an S-phase block in cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2686. doi:1538-7445.AM2012-2686

Methionine Deprivation Regulates the S6K1 Pathway and Protein Synthesis in Avian QM7 Myoblasts without Activating the GCN2/eIF2 alpha Cascade1–3

Amino acids modulate mRNA translation through the 70 kDa ribosomal protein S6 kinase (S6K1) and the general control nondepressible 2 protein kinase (GCN2)/eukaryotic initiation factor 2 alpha eIF2 alpha pathways. The aim of the present study was therefore to explore the signaling cascades potentially modulated by methionine availability in quail muscle QM7 myoblasts using media providing all other amino acids. Methionine deprivation caused a lower S6K1 phosphorylation compared with control (Ctl) cells. Supplying the methionine-deprived media with L- and DL-methionine isomers restored S6K1 phosphorylation to the levels observed in Ctl cells. Methionine also regulated downstream S6K1 targets (i.e. ribosomal protein S6 and eukaryotic elongation factor 2), modulated translation preinitiation complex (PIC) assembly, and stimulated protein synthesis. Replacing the lacking methionine with D-methionine or its hydroxyanalog [2-hydroxy-(4-methylthio) butanoic acid] did not restore S6K1 activation or protein synthesis. Conversely, the S6K1 pathway was activated by a methionine precursor, the ketoanalog of methionine. Methionine availability regulated the GCN2/eIF2 alpha pathway. However, our results indicate that methionine deprivation led to lower protein synthesis without activating eIF2 alpha phosphorylation, a process known to limit the formation of the 43S PIC. Using the amino acid alcohol methioninol did not decrease S6K1 phosphorylation or activity and did not alter the regulation of protein synthesis by methionine. These findings suggest that methionine exerts an effect on S6K1 signaling and protein synthesis in avian QM7 myoblasts through a mechanism partly independent of the global regulation via tRNA charging.

Sulfur amino acids deficiency caused by grass pea diet plays an important role in the toxicity of l-β-ODAP by increasing the oxidative stress: Studies on a motor neuron cell line

Neurolathyrism is a motor neuron disease caused by the overconsumption of grass pea ( Lathyrus sativus L.) containing l-β-ODAP. The precise mechanism to cause motor neuron degeneration has yet to be elucidated, but should agree with the epidemiological backgrounds. Considering the amino acid content of the legume, and the epidemiological link with prolonged unbalanced nutrition, the shortage of sulfur amino acids methionine and cysteine could affect the toxicity of l-β-ODAP. We analyzed the effect of these amino acids in the media on the toxicity using primary motor neuron culture and a motor neuron cell line NSC-34. Deprivation of both methionine and cysteine exacerbated the toxicity of l-β-ODAP by 66% compared to the complete medium. The glutathione content of these cells was greatly decreased in sulfur amino acid-deprived medium. l-β-ODAP further lowered the content in the deprived media to be 32–44% of the controls compared to normal media being 62–74%. The increased motor neuron toxicity in this medium was neutralized by the addition of reduced glutathione ethyl ester or N-acetylcysteine suggesting the importance of the mitochondrial oxidative stress induced by l-β-ODAP under sulfur amino acid-deficient conditions.

Dietary Methionine Restriction with FOLFOX Regimen as First Line Therapy of Metastatic Colorectal Cancer: A Feasibility Study

The methionine (MET) dependency of tumor cells opens interesting perspectives for targeting tumor cells and potentiating chemotherapy treatment, like 5-fluorouracil (5-FU) and platinum compound. Since MET deprivation can individually potentiate the different chemotherapeutic agents used in the 48-hour combined regimen of 5-FU, leucovorin and oxaliplatin (FOLFOX) regimen, we initiated a feasibility study associating dietary MET restriction with the FOLFOX regimen in patients with metastatic colorectal cancer. Objectives: (i) To evaluate the depletion in the plasma MET concentration, and (ii) to assess the feasibility of this combination. Methods: Eleven patients were enrolled in this study. They received a median number of 3 two-week cycles of a MET-free diet (3 consecutive days) and FOLFOX6 regimen. Results: The plasma MET concentration was reduced by dietary MET restriction, with a depletion of 58% on the 1st day of MET-free diet. Indeed, we demonstrated the feasibility and good tolerance (nutritional status and toxicity) of the association of a MET-free diet with the FOLFOX regimen. Despite good compliance to the diet, this study revealed the difficulty of administering this combination during further months. Among the 4 patients evaluable for response, 3 experienced a partial response and 1 patient a disease stabilization.

Methionine-deficient diet induces post-transcriptional downregulation of cystathionine β-synthase

Objective Elevated plasma total homocysteine (tHcy) is a risk factor for a variety of human diseases. Homocysteine is formed from methionine and has two primary metabolic fates: remethylation to form methionine or commitment to the transsulfuration pathway by the action of cystathionine β-synthase (CBS). We have examined the metabolic response in mice of a shift from a methionine-replete to a methionine-free diet. Methods and results We found that shifting 3-mo-old C57BL6 mice to a methionine-free diet caused a transient increase in tHcy and an increase in the tHcy/methionine ratio. Because CBS is a key regulator of tHcy, we examined CBS protein levels and found that within 3 d on the methionine-deficient diet, animals had a 50% reduction in the levels of liver CBS protein and enzyme activity. Examination of CBS mRNA and studies of transgenic animals that express CBS from a heterologous promoter indicated that this reduction is occurring post-transcriptionally. Loss of CBS protein was unrelated to intracellular levels of S-adenosylmethionine, a known regulator of CBS activity and stability. Conclusion Our results imply that methionine deprivation induces a metabolic state in which methionine is effectively conserved in tissue by shutdown of the transsulfuration pathway by an S-adenosylmethionine–independent mechanism that signals a rapid downregulation of CBS protein.

Combined Methionine Deprivation and Chloroethylnitrosourea Have Time-Dependent Therapeutic Synergy on Melanoma Tumors That NMR Spectroscopy-Based Metabolomics Explains by Methionine and Phospholipid Metabolism Reprogramming

Methionine (Met) deprivation stress (MDS) is proposed in association with chemotherapy in the treatment of some cancers. A synergistic effect of this combination is generally acknowledged. However, little is known on the mechanism of the response to this therapeutic strategy. A model of B16 melanoma tumor in vivo was treated by MDS alone and in combination with chloroethylnitrosourea (CENU). It was applied recent developments in proton-NMR spectroscopy-based metabolomics for providing information on the metabolic response of tumors to MDS and combination with chemotherapy. MDS inhibited tumor growth during the deprivation period and growth resumption thereafter. The combination of MDS with CENU induced an effective time-dependent synergy on growth inhibition. Metabolite profiling during MDS showed a decreased Met content (P < 0.01) despite the preservation of the protein content, disorders in sulfur-containing amino acids, glutamine/proline, and phospholipid metabolism [increase of glycerophosphorylcholine (P < 0.01), decrease in phosphocholine (P < 0.05)]. The metabolic profile of MDS combined with CENU and ANOVA analysis revealed the implication of Met and phospholipid metabolism in the observed synergy, which may be interpreted as a Met–sparing metabolic reprogramming of tumors. It follows that combination therapy of MDS with CENU seems to intensify adaptive processes, which may set limitations to this therapeutic strategy.

Glutamine Deprivation Causes Enhanced Plating Efficiency of a Herpes Simplex Virus Type 1 ICP0-Null Mutant

Isoleucine deprivation of cellular monolayers prior to infection has been reported to result in partial complementation of a herpes simplex virus type 1 (HSV-1) ICP0 null (ICP0(-)) mutant. We now report that glutamine deprivation alone is able to enhance the plating efficiency of an ICP0(-) virus and that isoleucine deprivation has little or no effect. Because a low glutamine level is associated with stress and because stress is known to induce reactivation, low levels of glutamine may be relevant to the reactivation of HSV-1 from latency. Additionally, we demonstrate that arginine and methionine deprivation result in partial complementation of the ICP0(-) virus.

Effect of methionine and cysteine deprivation on growth of different natural isolates of Lactobacillus spp. in chemically defined media

The purpose of this study was to determine the ability of natural isolates of lactobacilli from different ecological niches to grow in a chemically defined medium in the presence or absence of sulphur-containing amino acids, methionine and/or cysteine. The obtained results indicate that cysteine is essential for growth of L. paracasei subsp. paracasei BGHN14 and BGSJ2-8, while methionine is essential for isolates BGHN40, BGCG31, and BGHV54T of the species L. plantarum. Methionine is also essential for growth of L. rhamnosus BGHV58T. Other analyzed strains, such as L. plantarum BGSJ3-18, BGZB19, BGHV52Ta, and BGHV43T, require the presence of both amino acids for their growth.

Mitotic Arrest, Apoptosis, and Sensitization to Chemotherapy of Melanomas by Methionine Deprivation Stress

Methionine deprivation stress (MDS) eliminates mitotic activity in melanoma cells regardless of stage, grade, or TP53 status, whereas it has a negligible effect on normal skin fibroblasts. In most cases, apoptosis accounts for the elimination of up to 90% of tumor cells from the culture within 72 hours after MDS, leaving a scattered population of multinucleated resistant cells. Loss of mitosis in tumor cells is associated with marked reduction of cyclin-dependent kinase (CDK) 1 transcription and/or loss of its active form (CDK1-P-Thr(161)), which is coincident with up-regulation of CDKN1A, CDKN1B, and CDKN1C (p21, p27, and p57). Expression of the proapoptotic LITAF, IFNGR, EREG, TNFSF/TNFRSF10 and TNFRSF12, FAS, and RNASEL is primarily up-regulated/induced in cells destined to undergo apoptosis. Loss of Aurora kinase B and BIRC5, which are required for histone H3 phosphorylation, is associated with the accumulation of surviving multinucleated cells. Nevertheless, noncycling survivors of MDS are sensitized to temozolomide, carmustin, and cisplatin to a much greater extent than normal skin fibroblasts possibly because of the suppression of MGMT/TOP1/POLB, MGMT/RAD52/RAD54, and cMET/RADD52, respectively. Sensitivity to these and additional genotoxic agents and radiation may also be acquired due to loss of cMET/OGG1, reduced glutathione reductase levels, and a G(2)-phase block that is a crucial step in the damage response associated with enhancement of drug toxicity. Although the genes controlling mitotic arrest and/or apoptosis in response to low extracellular methionine levels are unknown, it is likely that such control is exerted via the induction/up-regulation of tumor suppressors/growth inhibitor genes, such as TGFB, PTEN, GAS1, EGR3, BTG3, MDA7, and the proteoglycans (LUM, BGN, and DCN), as well as the down-regulation/loss of function of prosurvival genes, such as NFkappaB, MYC, and ERBB2. Although MDS targets several common genes in tumors, mutational variability among melanomas may decide which metabolic and signal transduction pathways will be activated or shutdown.

Modulation of cell cycle and gene expression in pancreatic tumor cell lines by methionine deprivation (methionine stress): implications to the therapy of pancreatic adenocarcinoma

The effect of methionine deprivation (methionine stress) on the proliferation, survival, resistance to chemotherapy, and regulation of gene and protein expression in pancreatic tumor lines is examined. Methionine stress prevents successful mitosis and promotes cell cycle arrest and accumulation of cells with multiple micronuclei with decondensed chromatin. Inhibition of mitosis correlates with CDK1 down-regulation and/or inhibition of its function by Tyr(15) phosphorylation or Thr(161) dephosphorylation. Inhibition of cell cycle progression correlates with loss of hyperphosphorylated Rb and up-regulation of p21 via p53 and/or transforming growth factor-beta (TGF-beta) activation depending on p53 status. Although methionine stress-induced toxicity is not solely dependent on p53, the gain in p21 and loss in CDK1 transcription are more enhanced in wild-type p53 tumors. Up-regulation of SMAD7, a TGF-beta signaling inhibitor, suggests that SMAD7 does not restrict the TGF-beta-mediated induction of p21, although it may prevent up-regulation of p27. cDNA oligoarray analysis indicated a pleiotropic response to methionine stress. Cell cycle and mitotic arrest is in agreement with up-regulation of NF2, ETS2, CLU, GADD45alpha, GADD45beta, and GADD45gamma and down-regulation of AURKB, TOP2A, CCNA, CCNB, PRC1, BUB1, NuSAP, IFI16, and BRCA1. Down-regulation of AREG, AGTR1, M-CSF, and EGF, IGF, and VEGF receptors and up-regulation of GNA11 and IGFBP4 signify loss of growth factor support. PIN1, FEN1, and cABL up-regulation and LMNB1, AREG, RhoB, CCNG, TYMS, F3, and MGMT down-regulation suggest that methionine stress sensitizes the tumor cells to DNA-alkylating drugs, 5-fluorouracil, and radiation. Increased sensitivity of pancreatic tumor cell lines to temozolomide is shown under methionine stress conditions and is attributed in part to diminished O(6)-methylguanine-DNA methyltransferase and possibly to inhibition of the cell cycle progression.

S-Adenosylmethionine Decarboxylase Degradation by the 26 S Proteasome Is Accelerated by Substrate-mediated Transamination

The short-lived enzyme S-adenosylmethionine decarboxylase uses a covalently bound pyruvoyl cofactor to catalyze the formation of decarboxylated S-adenosylmethionine, which then donates an aminopropyl group for polyamine biosynthesis. Here we demonstrate that S-adenosylmethionine decarboxylase is ubiquitinated and degraded by the 26 S proteasome in vivo, a process that is accelerated by inactivation of S-adenosylmethionine decarboxylase by substrate-mediated transamination of its pyruvoyl cofactor. Proteasome inhibition in COS-7 cells prevents the degradation of S-adenosylmethionine decarboxylase antigen; however, even brief inhibition of the 26 S proteasome caused substantial losses of S-adenosylmethionine decarboxylase activity despite accumulation of S-adenosylmethionine decarboxylase antigen. Levels of the enzyme's substrate (S-adenosylmethionine) increased rapidly after 26 S proteasome inhibition, and this increase in substrate level is consistent with the observed loss of activity arising from an increased rate of inactivation by substrate-mediated transamination. Evidence is also presented that this substrate-mediated transamination accelerates normal degradation of S-adenosylmethionine decarboxylase, as the rate of degradation of the enzyme was increased in the presence of AbeAdo (5'-([(Z)-4-amino-2-butenyl]methylamino]-5'-deoxyadenosine) (a substrate analogue that transaminates the enzyme); conversely, when the intracellular substrate level was reduced by methionine deprivation, the rate of degradation of the enzyme was decreased. Ubiquitination of S-adenosylmethionine decarboxylase is demonstrated by isolation of His-tagged AdoMetDC (S-adenosylmethionine decarboxylase) from COS-7 cells co-transfected with hemagglutinin-tagged ubiquitin and showing bands that were immunoreactive to both anti-AdoMetDC antibody and anti-hemagglutinin antibody. This is the first study to demonstrate that AdoMetDC is ubiquitinated and degraded by the 26 S proteasome, and substrate-mediated acceleration of degradation is a unique finding.

Expression of spermidine/spermine N1-acetyltransferase in HeLa cells is regulated by amino acid sufficiency

The effect of amino acids on the regulation of the expression of spermidine/spermine N 1-acetyltransferase (SSAT), the key enzyme of polyamine catabolism, was studied in HeLa cells. When compared with similar exposure to complete medium, deprivation of arginine, methionine or leucine gave rise to a time-dependent, slowly reversible increase in the cellular level of SSAT mRNA that started to be significant after 8, 12 or 16 h and reached four-, five- and two-fold after 16 h, respectively. Experiments utilizing (i) constructs containing fragments of the SSAT promoter linked to a luciferase reporter gene or (ii) actinomycin D (Act-D)-treated cells indicated that the increase in the SSAT mRNA level was due to an augmentation in gene transcription and message stability after omission of one of the polyamine precursor amino acids. By contrast, SSAT mRNA stabilisation was only observed when leucine was the omitted amino acid. Amino acid deprivation was also found to cause increased intracellular activity of SSAT concurrent with changes in the cell polyamine content, namely increased putrescine but decreased spermine levels. Furthermore, stable expression of a dominant negative mutant of stress-activated protein kinase/extracellular signal-regulated protein kinase (SAPK/ERK) kinase 1 in HeLa cells was found to inhibit the increase in SSAT mRNA by amino acid deprivation. The data suggest that c-Jun N-terminal kinase/SAPK (JNK/SAPK) may be involved in the amino acid-dependent regulation of SSAT expression.

SAM and the Anti-Antiterminator

Gene expression can be controlled by RNA structure, which can affect RNA translation or transcription. In bacteria, direct binding of cellular factors to the nascent RNA, which alters secondary structure, can regulate transcription termination. McDaniel et al. showed that transcripts from the S box genes, which are induced by methionine deprivation, bind S -adenosylmethionine (SAM) in vitro and that SAM, but not methionine or related compounds, represses in vitro transcription. Using an RNA-DNA hybrid cleavage assay with DNA oligonucleotides directed against different regions of one S box gene transcript, the authors showed that cleavage patterns were different in the presence and absence of SAM, consistent with SAM inducing a conformational change in the RNA structure. Under methionine replete conditions, the RNA assumes a terminator conformation. When methionine is depleted, an antiterminator structure is formed that permits readthrough of the terminator. The cleavage assays suggest that SAM promotes the formation of a third structure, the anti-antiterminator, which is necessary for the formation of the terminator structure. In Bacillus subtilis , overexpression of SAM synthetase delayed the stimulation of S box gene expression in response to methionine depletion. Regulation of RNA structure and control of RNA transcription termination are emerging as important regulatory mechanisms in prokaryotes. B. A. M. McDaniel, F. J. Grundy, I. Artsimovitch, T. M. Henkin, Transcription termination control of the S box system: Direct measurement of S-adenosylmethioinine by the leader RNA. Proc. Natl. Acad. Sci. U.S.A. 100 , 3083-3088 (2003). [Abstract] [Full Text]

Induction of caspase-dependent and -independent apoptosis in response to methionine restriction

Tumor cells are more sensitive to methionine restriction than normal tissues, a phenomenon known as methionine auxotrophy. Previous studies have demonstrated that methionine restriction causes tumor cell growth arrest and eventually apoptosis. The current studies were undertaken to elucidate the molecular pathways leading to apoptosis induced by methionine restriction. We found that methionine restriction induced formation of oligonucleosomal DNA fragment and cytochrome c release from mitochondria in methionine-dependent PC3 and Hela cells. Methionine restriction also led to cleavage and activation of initiator and effector caspases in Hela cells but not PC3 cells. Furthermore, methionine restriction resulted in cleavage of BID and reduction in Bcl-2 levels in both cell lines. These data suggest that apoptosis induced by methionine restriction is mitochondria-dependent. Methionine restriction induced caspase-independent cell death in PC3 cells, whereas it stimulated caspase-dependent cell death in Hela cells. Cleavage of BID and decreased expression of Bcl-2 upon methionine deprivation may be the underlying mechanism to stimulate release of cytochrome c from mitochondria.

Cytotoxic synergism of methioninase in combination with 5-fluorouracil and folinic acid

Potentiation of the cytotoxic activity of 5-fluorouracil (FUra) by folinic acid (5-HCO-H 4folate) is due to elevation of the methylene tetrahydrofolate (CH 2-H 4folate) level, which increases the stability of the ternary complex of thymidylate synthase (TS), fluorodeoxyuridine monophosphate, and CH 2-H 4folate that inactivates the TS. Methionine deprivation results in the production of tetrahydrofolate (H 4folate) and, subsequently, CH 2-H 4folate from methyl tetrahydrofolate, as a consequence of the induction of methionine synthesis. We hypothesized that the efficacy of FUra could be augmented by the combination of high-concentration 5-HCO-H 4folate and recombinant methioninase (rMETase), a methionine-cleaving enzyme. Studies in vitro were performed with the cell line CCRF-CEM. Cytotoxic synergism of FUra + rMETase and FUra + 5-HCO-H 4folate + rMETase was demonstrated with the combination index throughout a broad concentration range of FUra and rMETase. A subcytotoxic concentration of rMETase reduced the IC 50 of FUra by a factor of 3.6, and by a factor of 7.5, in the absence and in the presence of 5-HCO-H 4folate, respectively. 5-HCO-H 4folate increased the intracellular concentrations of CH 2-H 4folate and H 4folate from their baseline levels. Concentrations of folates were not changed by exposure to rMETase. Levels of free TS in cells treated with FUra + 5-HCO-H 4folate and with FUra + rMETase were lower than those in cells exposed to FUra alone. The decrease of TS was still more pronounced in cells treated with FUra + 5-HCO-H 4folate + rMETase. The synergism described in this study will be a basis for further exploration of combinations of fluoropyrimidines, folates, and rMETase.

CHOP/GADD153 Gene Expression Response to Cellular Stresses Inhibited by Prior Exposure to Ultraviolet Light Wavelength Band C (UVC): INHIBITORY SEQUENCE MEDIATING THE UVC RESPONSE LOCALIZED TO EXON 1

CHOP/GADD153 is both an activating and repressing transcription factor that is markedly induced in response to a variety of cellular stresses. The CHOP/GADD153 gene was originally cloned because of its inducibility by ultraviolet light wavelength band C (UVC) and has since been found to be activated in response to many different cellular stresses. Some of the recent studies have questioned the UVC responsiveness of the CHOP gene. Contradiction in our own data led us to reexamine the UVC effects on CHOP expression. UVC is capable of strongly activating the mouse CHOP promoter in stably transfected NIH 3T3 cells but has only a modest and transient effect on the level of the CHOP messenger RNA. In addition to its positive effect on CHOP promoter activity, we show that UVC negatively affects CHOP mRNA and protein expression. Pretreatment of NIH 3T3 cells with UVC markedly attenuates the subsequent induction of CHOP mRNA by the cellular stress activators methylmethane sulfate, tunicamycin, glucose deprivation, and methionine deprivation for as long as at least 16 h. This inhibitory effect of UVC on CHOP expression in response to stress is independent of the presence or absence of p53 and does not involve mRNA degradation as opposed to the UVC effect that inhibits p21 expression seen only in the absence of p53. The target of the inhibitory effect of UVC on CHOP expression is located in the first exon of the gene, a 5'-untranslated region that is unusually conserved between different species. These findings suggest that an unknown function encoded by the 5'-untranslated region somehow modifies the response of CHOP gene transcription to UVC.

The essential role of phosphatidylinositol 3-kinase and of p38 mitogen-activated protein kinase activation in the antioxidant response element-mediated rGSTA2 induction by decreased glutathione in H4IIE hepatoma cells.

The protective adaptive response to electrophiles and reactive oxygen species is mediated by the enhanced expression of the phase II detoxifying genes through antioxidant response elements (AREs). The current study was designed to identify the signaling pathways responsible for the expression of rGSTA2 in response to cellular oxidative stress and to establish the molecular mechanistic basis. Deprivation of cystine and methionine caused oxidative stress in H4IIE hepatoma cells as evidenced by a marked decrease in the reduced glutathione (first order rate constant = 0.056 h(-1); t(1/2) = 12.6 h) and an increase in pro-oxidant production. Electrophoretic mobility shift assay revealed that the ARE complex, consisting of Nrf-1/2 and Maf proteins, was activated 12 to 48 h after sulfur amino acid deprivation (SAAD). The rGSTA2 mRNA level was elevated by SAAD beginning at 24 h, whereas the rGSTA2 subunit was maximally induced at 48 h. Nuclear ARE activation and rGSTA2 mRNA increase were both completely inhibited by wortmannin or LY294002, the phosphatidylinositol 3-kinase (PI3-kinase) inhibitors. The p38 mitogen-activated protein (MAP) kinase was activated at 0.5 to 3 h after SAAD, followed by sustained diminished activation up to 12 h. Inhibition of p38 MAP kinase by SB203580 prevented the ARE-mediated rGSTA2 induction. The activation of p38 MAP kinase, however, failed to be inhibited by wortmannin or LY294002, showing that PI3-kinase is not involved in the activation of p38 MAP kinase. Data showed that PI3-kinase plays an essential role in the ARE-mediated rGSTA2 induction by oxidative stress after SAAD, which activates the p38 MAP kinase and leads to rGSTA2 induction.