Combination Chemotherapy of BCNU and Didox Acts Synergystically in 9L Glioma Cells

Compounds inhibiting DNA repair and synthesis are expected to act synergistically with BCNU, a standard agent in the therapy of glioblastoma multiforme, and improve survival of patients with malignant gliomas. Ribonucleotide reductase (EC1.17.4.1; RR) catalyzes the rate‐limiting step in DNA synthesis and plays a critical role in maintaining crucial substrates for DNA repair. We have studied the effects of Didox, an inhibitor of RR on 9L glioma cells in combination with BCNU². We analyzed intracellular dNTP pools and found that Didox significantly depleted the intracellular dNTP concentrations. Experiments using cytotoxicity, growth inhibition and clonogenic assays showed significant synergism of Didox and BCNU. Combination regimens using synchronous administration demonstrated highest cytotoxicity. We have also identified altered gene expression in a number of DNA repair related enzymes after BCNU treatment using large‐scale cDNA arrays. The coadministration with Didox could reverse the expression of some of the overexpressed repair gene suggesting possible pathways to circumvent the developing resistance in 9L glioma cells against BCNU. These results introduce the combination of Didox and BCNU as a viable alternative for the treatment of malignant gliomas.     

In vitro repair synthesis of BCNU-induced DNA damage

The nitrosoureas including BCNU are potent chemotherapeutic drugs and have been used extensively for treatment of brain tumors and other neoplasias but the mechanisms of action for the DNA lesions created and their repair are still unclear. We have recently determined the in vitro repair of BCNU-treated DNA with cellular extracts and with DNA modifying enzymes. BCNU not only caused an increase in breaks in plasmid DNA, but an increase in cross-linked DNA was also observed after restriction enzyme digestion followed by gel electrophoresis. When HeLa cell-extracts were incubated with BCNU-treated DNA, 5-10 fold increases in DNA repair synthesis were observed as compared with untreated control. Substantial increases in 5'OH and 3'OH sites of the breaks were also found in BCNU-treated DNA as determined by the 10-20 fold increases in labeling with T4-DNA kinase and by endogenous polymerases, while the amount of ligatable sites were at a minimal. When the repair capacity of two glioma cell lines (UWR1 and UWR3) with differential BCNU sensitivity, and cells from a chromosomal breakage disease, Bloom's syndrome (BS), were assessed, the activities of the two glioma cells were about 20-30% of the normal lymphoblastoid cells and HeLa cells, whereas no difference was observed in BS cells. However, differential patterns of DNA bands were observed in the glioma samples suggesting cell-type specific capacities of repair synthesis. These data are in accordance with the concept that BCNU creates multiple DNA lesions and suggests different cell types may develop a variety of repair capabilities.     

O6-methylguanine DNA methyltransferase and p53 status predict temozolomide sensitivity in human malignant glioma cells

Temozolomide (TMZ) is a methylating agent which prolongs survival when administered during and after radiotherapy in the first-line treatment of glioblastoma and which also has significant activity in recurrent disease. O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair enzyme attributed a role in cancer cell resistance to O6-alkylating agent-based chemotherapy. Using a panel of 12 human glioma cell lines, we here defined the sensitivity to TMZ in acute cytotoxicity and clonogenic survival assays in relation to MGMT, mismatch repair and p53 status and its modulation by dexamethasone, irradiation and BCL-X(L). We found that the levels of MGMT expression were a major predictor of TMZ sensitivity in human glioma cells. MGMT activity and clonogenic survival after TMZ exposure are highly correlated (p < 0.0001, r2 = 0.92). In contrast, clonogenic survival after TMZ exposure does not correlate with the expression levels of the mismatch repair proteins mutS homologue 2, mutS homologue 6 or post-meiotic segregation increased 2. The MGMT inhibitor O6-benzylguanine sensitizes MGMT-positive glioma cells to TMZ whereas MGMT gene transfer into MGMT-negative cells confers protection. The antiapoptotic BCL-X(L) protein attenuates TMZ cytotoxicity in MGMT-negative LNT-229 but not in MGMT-positive LN-18 cells. Neither ionizing radiation (4 Gy) nor clinically relevant concentrations of dexamethasone modulate MGMT activity or TMZ sensitivity. Abrogation of p53 wild-type function strongly attenuates TMZ cytotoxicity. Conversely, p53 mimetic agents designed to stabilize the wild-type conformation of p53 sensitize glioma cells for TMZ cytotoxicity. Collectively, these results suggest that the determination of MGMT expression and p53 status will help to identify glioma patients who will or will not respond to TMZ.     

Deoxynucleotide pools and DNA synthesis in resting and PHA-stimulated human lymphocytes treated with mutagens.

In resting and PHA-stimulated PBL treated with uv light or MMS we measured the sizes of the dTTP and dATP pools and the variation of ATP content taken as an indicator of cytotoxicity. The effects on DNA synthesis were examined by measuring DNA repair (unscheduled DNA synthesis) in resting PBL and the inhibition of DNA replication in stimulated PBL. While both treatments affected DNA synthesis, only MMS perturbed dNTP pools and decreased the intracellular concentration of ATP. All the effects were more evident in cycling than in resting lymphocytes.     

Enhanced Anti-Tumor Effect of Zoledronic Acid Combined with Temozolomide against Human Malignant Glioma Cell Expressing O6-Methylguanine DNA Methyltransferase

Temozolomide (TMZ), a DNA methylating agent, is widely used in the adjuvant treatment of malignant gliomas. O⁶-methylguanine-DNA methyltranferase (MGMT), a DNA repair enzyme, is frequently discussed as the main factor that limits the efficacy of TMZ. Zoledronic acid (ZOL), which is clinically applied to treat cancer-induced bone diseases, appears to possess direct anti-tumor activity through apoptosis induction by inhibiting mevalonate pathway and prenylation of intracellular small G proteins. In this study, we evaluated whether ZOL can be effectively used as an adjuvant to TMZ in human malignant glioma cells that express MGMT. Malignant glioma cell lines, in which the expression of MGMT was detected, did not exhibit growth inhibition by TMZ even at a longer exposure. However, combination experiment of TMZ plus ZOL revealed that a supra-additive effect resulted in a significant decrease in cell growth. In combined TMZ/ZOL treatment, an increased apoptotic rate was apparent and significant activation of caspase-3 and cleavage of poly-(ADP-ribose) polymerase were observed compared with each single drug exposure. There were decreased amounts of Ras-GTP, MAPK and Akt phosphorylation and MGMT expression in the ZOL-treated cells. Subcutanous xenograft models showed significant decrease of tumor growth with combined TMZ/ZOL treatment. These results suggest that ZOL efficaciously inhibits activity of Ras in malignant glioma cells and potentiates TMZ-mediated cytotoxicity, inducing growth inhibition and apoptosis of malignant glioma cells that express MGMT and resistant to TMZ. Based on this work, combination of TMZ with ZOL might be a potential therapy in malignant gliomas that receive less therapeutic effects of TMZ due to cell resistance.     

Influence of the Expression Level of O6-Alkylguanine-DNA Alkyltransferase on the Formation of DNA Interstrand Crosslinks Induced by Chloroethylnitrosoureas in Cells: A Quantitation Using High-Performance Liquid Chromatography-Mass Spectrometry

Chloroethylnitrosoureas (CENUs), which are bifunctional alkylating agents widely used in the clinical treatment of cancer, exert anticancer activity by inducing crosslink within a guanine-cytosine DNA base pair. However, the formation of dG-dC crosslinks can be prevented by O⁶-alkylguanine-DNA alkyltransferase (AGT), ultimately leading to drug resistance. Therefore, the level of AGT expression is related to the formation of dG-dC crosslinks and the sensitivity of cells to CENUs. In this work, we determined the CENU-induced dG-dC crosslink in mouse L1210 leukemia cells and in human glioblastoma cells (SF-763, SF-767 and SF-126) containing different levels of AGT using high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. The results indicate that nimustine (ACNU) induced more dG-dC crosslinks in L1210 leukemia cells than those induced by carmustine (BCNU), lomustine (CCNU) and fotemustine (FTMS). This result was consistent with a previously reported cohort study, which demonstrated that ACNU had a better survival gain than BCNU, CCNU and FTMS for patients with high-grade glioma. Moreover, we compared the crosslinking levels and the cytotoxicity in SF-763, SF-767 and SF-126 cells with different AGT expression levels after exposure to ACNU. The levels of dG-dC crosslink in SF-126 cells (low AGT expression) were significantly higher than those in SF-767 (medium AGT expression) and SF-763 (high AGT expression) cells at each time point. Correspondingly, the cytotoxicity of SF-126 was the highest followed by SF-767 and SF-763. The results obtained in this work provided unequivocal evidence for drug resistance to CENUs induced by AGT-mediated repair of DNA ICLs. We postulate that the level of dG-dC crosslink has the potential to be employed as a biomarker for estimating drug resistance and anticancer efficiencies of novel CENU chemotherapies.     

The Efficacy of the Ribonucleotide Reductase Inhibitor Didox in Preclinical Models of AML

Acute Myeloid Leukemia (AML) is an aggressive malignancy which leads to marrow failure, and ultimately death. There is a desperate need for new therapeutics for these patients. Ribonucleotide reductase (RR) is the rate limiting enzyme in DNA synthesis. Didox (3,4-Dihydroxybenzohydroxamic acid) is a novel RR inhibitor noted to be more potent than hydroxyurea. In this report we detail the activity and toxicity of Didox in preclinical models of AML. RR was present in all AML cell lines and primary patient samples tested. Didox was active against all human and murine AML lines tested with IC₅₀ values in the low micromolar range (mean IC₅₀ 37 µM [range 25.89–52.70 µM]). It was active against primary patient samples at concentrations that did not affect normal hematopoietic stem cells (HSCs). Didox exposure resulted in DNA damage and p53 induction culminating in apoptosis. In syngeneic, therapy-resistant AML models, single agent Didox treatment resulted in a significant reduction in leukemia burden and a survival benefit. Didox was well tolerated, as marrow from treated animals was morphologically indistinguishable from controls. Didox exposure at levels that impaired leukemia growth did not inhibit normal HSC engraftment. In summary, Didox was well tolerated and effective against preclinical models of AML.     

组蛋白H2B单泛素化参与DNA损伤修复的调控机制

作为一种重要的组蛋白修饰形式,H2B的单泛素化(uH2B)广泛地参与DNA复制、基因的表达与转录、DNA损伤修复及异染色质维持等生物学事件.在裂殖酵母中,H2B的单泛素化发生在其羧基端的119位赖氨酸(K119),并依赖于Rhp6/Bre1泛素连接酶复合体.研究表明,uH2B通过破坏H2A/H2B二聚体的结构促进mRNA在转录过程中的延伸,同时促进H3K4的三甲基化激活基因的表达及参与DNA损伤修复.本研究发现,Rhp6能够对核糖核苷酸还原酶抑制基因(Spd1)位点进行活跃的染色质修饰,促进H2B的单泛素化并抑制基因表达,从而促进dNTP的合成并调控DNA复制及损伤修复.重要的是,本研究发现,该过程不依赖于H3K4而决定于H3K9的三甲基化.同时uH2B直接在DNA双链断裂位点富集,通过改变染色质的结构参与DNA损伤修复,该过程中可能存在其他更为复杂的分子机制.     

Reactions of deoxyribonucleotide synthesis system to irradiation and their modification by radioprotectors

The paper covers the problem on reactions of deoxyribonucleotide (dNTP) synthesis system in blood-forming organs of animals induced by irradiation. The synthesis of dNTP is a rate-limiting stage for DNA synthesis. Cellular requirements for dNTP pools during DNA synthesis are related with ensuring of the accuracy of DNA copying during replication and repair. It has been shown that organism defence mechanisms against irradiation include the following stages: 1. The prompt SOS-activation of dNTP synthesis 30 min later after irradiation, playing the important role in protecting of cell's genetic apparatus from damage. 2. The inhibition of dNTP synthesis within 3-24 h after irradiation resulting to the imbalance of four dNTP and the decrease of their pools. As result of that, the abnormal repair is observed due to depurinations, errors of base incorporations and "misrepair". 3. The restore of dNTP synthesis occurred 2 days later after irradiation. The increase of dNTP pools promotes the increase of DNA synthesis rate as well as proliferative activity of cells. Confirming the fact that the alterations in dNTP pools play essential role in the production of DNA lesions became an important step in understanding of the multistage process leading to radioprotection. To get high and balanced pools of dNTP needed for the increase in the volume of repair of DNA lesions the radioprotectors with high efficiency relative to the survival test were used in experiments. They induced the elevated dNTP synthesis in bone marrow and spleen during the time when the irradiation alone caused the essential prolonged suppression of dNTP synthesis as well as DNA and protein synthesis in organs of nonprotected animals. It has been shown that substances with antioxidant and antiradical activity induced the dNTP synthesis, too. In vivo regulatory factors of dNTP synthesis have been studied to elucidate the mechanisms of getting of high and balanced dNTP pools by using of different substances.     

RANTES-mediated control of excitatory amino acid release in mouse spinal cord

O⁶‐methylguanine‐DNA methyltransferase (MGMT) is a DNA‐repair protein promoting resistance of tumor cells to alkylating chemotherapeutic agents. Glioma cells are particularly resistant to this class of drugs which include temozolomide (TMZ) and carmustine (BCNU). A previous study using the RNA microarray technique showed that decrease of MGMT mRNA stands out among the alterations in gene expression caused by the cell growth‐depressing transfection of a T98G glioma cell line with liver‐type glutaminase (LGA) [Szeliga et al. (2009) Glia, 57, 1014]. Here, we show that stably LGA‐transfected cells (TLGA) exhibit decreased MGMT protein expression and activity as compared with non‐transfected or mock transfected cells (controls). However, the decrease of expression occurs in the absence of changes in the methylation of the promoter region, indicating that LGA circumvents, by an as yet unknown route, the most common mechanism of MGMT silencing. TLGA turned out to be significantly more sensitive to treatment with 100–1000 μM of TMZ and BCNU in the acute cell growth inhibition assay (MTT). In the clonogenic survival assay, TLGA cells displayed increased sensitivity even to 10 μM TMZ and BCNU. Our results indicate that enrichment with LGA, in addition to inhibiting glioma growth, may facilitate chemotherapeutic intervention.     

Possible Novel Therapy for Malignant Gliomas with Secretable Trimeric TRAIL

Malignant gliomas are the most common primary brain tumors. Despite intensive clinical investigation and many novel therapeutic approaches, average survival for the patients with malignant gliomas is only about 1 year. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown potent and cancer-selective killing activity and drawn considerable attention as a promising therapy for cancers, but concerns over delivery and toxicity have limited progress. We have developed a secretable trimeric TRAIL (stTRAIL) and here evaluated the therapeutic potential of this stTRAIL-based gene therapy in brain tumors. An adenovirus (Ad-stTRAIL) delivering stTRAIL was injected into intra-cranial human glioma tumors established in nude mice and tumor growth monitored using the magnetic resonance imaging (MRI). Ad-stTRAIL gene therapy showed potent tumor suppressor activity with no toxic side effects at therapeutically effective doses. When compared with 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a conventional therapy for malignant gliomas, Ad-stTRAIL suppressed tumor growth more potently. The combination of Ad-stTRAIL and BCNU significantly increased survival compared to the control mice or mice receiving Ad-stTRAIL alone. Our data indicate that Ad-stTRAIL, either alone or combined with BCNU, has promise as a novel therapy for malignant gliomas.     

Nitric oxide and BCNU chemoresistance in C6 glioma cells: role of S-nitrosoglutathione

Inducible nitric oxide synthase (iNOS or NOS2) is expressed in malignant glioma. Previously we noted that C6 glioma cells overexpressing NOS2 displayed chemoresistance against 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and other chloroethylnitrosourea derivatives with carbamoylating action. Herein we report experimental evidence supporting the contention that this NOS2 effect is mediated, at least in part, by S-nitrosoglutathione (GSNO), a potent antioxidant derived from interaction of NO and glutathione. Out of three NO donors tested, only GSNO was effective in protecting glioma cells against BCNU cytotoxicity. Furthermore, the protective effect of GSNO, similar to that of NOS2, was confined to carbamoylating, but not alkylating action. Experimental manipulations that were expected to increase or decrease cellular GSNO stores, as confirmed by immunocytochemical staining using a GSNO-specific antibody and HPLC analysis of GSNO contents in culture medium, led respectively to enhanced or reduced chemoresistance against carbamoylating cytotoxicity. Finally, neocuproine, a selective cuprous ion chelator known to neutralize GSNO actions, abolished NOS2-mediated chemoresistance against carbamoylating agents. Our results reveal a novel action of NOS2/GSNO that may potentially contribute to the development of chemoresistance against BCNU, which remains a mainstay in chemotherapy for glioblastoma multiforme.     

The role of N-nitrosourea-induced changes in the nucleoid structure and activity of repair enzymes in the development of drug resistance in mice with leukemia L1210

Using centrifugation of the nucleoid in a neutral sucrose gradient, the damages in the secondary structure of DNA and the activity of repair enzymes, such as DNA-polymerases alpha and beta and poly(ADP-riboso) polymerase, induced by 1-methyl-nitrosourea (MNU) and 1.3-bis (2-chloroethyl)-1-nitrosourea (BCNU) injected at maximal nonlethal single doses to mice bearing parent leukemia cells (L1210/0) and resistant to MNU and BCNU leukemia L1210 cells (L1210/MNU and L1210/BCNU), were studied. The MNU-induced production of single-strand breaks in L1210/0 and L1210/MNU cells was more conspicuous in newly replicated DNA than in those in preexisting DNA. A more fast repair of the damages in newly replicated DNA was detected in L1210/BCNU and especially in L1210/MNU leukemia cells as compared with L1210/0 cells. The data obtained suggest that there are prone errors in the repair of DNA template, since most of the single-strand breaks were revealed in the newly replicated DNA synthesized on the repaired DNA. The repair of DNA damages in L1210/BCNU and especially in L1210/MNU cells was accompanied by the activation of DNA-polymerases alpha and beta and poly(ADP-riboso)polymerase. Both DNA-polymerases--alpha and beta--were shown to be involved in repair of DNA damages induced by MNU and only DNA-polymerase beta was involved in the repair of damages induced by BCNU.     

The effects of X rays on BCNU-induced DNA crosslinking

We have used the technique of alkaline elution to study DNA interstrand crosslinking in 9L rat brain tumor cells treated with combinations of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and X rays. Irradiation with doses as low as 50 rad of X rays immediately or 6 hr after a 1-hr treatment with 60, 80, or 100 microM BCNU produced a significant increase in BCNU-induced DNA interstrand crosslinking. If cells were irradiated before BCNU treatment, the amount of crosslinking was not affected compared with BCNU alone. Cell survival experiments using 600 rad of X rays and 1-hr treatments with 0-30 microM BCNU were also performed. As found in the crosslinking studies, irradiation immediately or 6 hr after the BCNU treatment produced enhanced cell kill, but irradiation 6 hr before BCNU treatment did not produce enhanced cell kill. Therefore, the X-ray-mediated increase in BCNU-induced DNA interstrand crosslinking may be the mechanism through which cell kill is increased by combination treatment with the agents.     

Targeted nitric oxide delivery preferentially induces glioma cell chemosensitivity via altered p53 and O6‐Methylguanine‐DNA Methyltransferase activity

Glioblastoma multiforme is the most common malignant central nervous system tumor, and also among the most difficult to treat due to a lack of response to chemotherapeutics. New methods of countering the mechanisms that confer chemoresistance to malignant gliomas could lead to significant advances in the quest to identify novel drug combinations or targeted drug delivery systems for cancer therapy. In this study, we investigate the use of a targeted nitric oxide (NO) donor as a pretreatment to sensitize glioma cells to chemotherapy. The protein chlorotoxin (CTX) has been shown to preferentially target glioma cells, and we have developed CTX–NO, a glioma‐specific, NO‐donating CTX derivative. Pretreatment of cells with CTX–NO followed by 48‐h exposure to either carmustine (BCNU) or temozolomide (TMZ), both common chemotherapeutics used in glioma treatment, resulted in increased efficacy of both therapeutics. After CTX–NO exposure, both T98G and U‐87MG human malignant glioma cells show increased sensitivity to BCNU and TMZ. Further investigation revealed that the consequences of this combination therapy was a reduction in active levels of the cytoprotective enzyme MGMT and altered p53 activity, both of which are essential in DNA repair and tumor cell resistance to chemotherapy. The combination of CTX–NO and chemotherapeutics also led to decreased cell invasion. These studies indicate that this targeted NO donor could be an invaluable tool in the development of novel approaches to treat cancer. Biotechnol. Bioeng. 2013; 110: 1211–1220. © 2012 Wiley Periodicals, Inc.     

Ribonucleotide reductase subunit p53R2 regulates mitochondria homeostasis and function in KB and PC-3 cancer cells

Ribonucleotide reductase (RR) is a rate-limiting enzyme that catalyzes de novo conversion of ribonucleotide 5′-diphosphates to the corresponding 2′-deoxynucleotide, essential for DNA synthesis and replication. The mutations or knockout of RR small subunit, p53R2, results in the depletion of mitochondrial DNA (mtDNA) in human, implying that p53R2 might play a critical role for maintaining mitochondrial homeostasis. In this study, siRNA against p53R2 knockdown approach is utilized to examine the impact of p53R2 depletion on mitochondria and to derive underlying mechanism in KB and PC-3 cancer cells. Our results reveal that the p53R2 expression not only positively correlates with mtDNA content, but also partakes in the proper mitochondria function, such as ATP synthesis, cytochrome c oxidase activity and membrane potential maintenance. Furthermore, overexpression of p53R2 reduces intracellular ROS and protects the mitochondrial membrane potential against oxidative stress. Unexpectedly, knockdown of p53R2 has a modest, if any, effect on mitochondrial and total cellular dNTP pools. Taken together, our study provides functional evidence that mitochondria is one of p53R2-targeted organelles and suggests an unexpected function of p53R2, which is beyond known RR function on dNTP synthesis, in mitochondrial homeostatic control.     

APRIL, a new member of the tumor necrosis factor family, modulates death ligand-induced apoptosis

APRIL (a proliferation-inducing ligand) is a newly identified member of the tumor necrosis factor (TNF) family. Tumor growth-promoting as well as apoptosis-inducing effects of APRIL have been described. Here, we report that five of 12 human malignant glioma cell lines express APRIL. APRIL gene transfer experiments revealed that malignant glioma cells are refractory to growth-promoting activity of APRIL in vitro and in vivo. Interestingly, ectopic expression of APRIL confers minor protection from apoptotic cell death induced by the death ligands, CD95 ligand (CD95L) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2 ligand (Apo2L). This antiapoptotic activity is specific for death ligand/receptor-mediated apoptosis since APRIL does not protect glioma cells from the cytotoxicity of the drugs, teniposide, vincristine, lomustine or cisplatin. Ectopic expression of APRIL is associated with the upregulation of X-linked inhibitor of apoptosis protein (XIAP), providing a possible explanation for the antiapoptotic activity observed here. In contrast, APRIL does not regulate the expression levels of the antiapoptotic proteins FLICE-inhibitory protein (FLIP), Bcl-2 or Bcl-X(L). These findings suggest that APRIL is involved in the regulation of death ligand-induced apoptotic signaling in malignant glioma cells.     

Alterations in the cyclic AMP signal transduction pathway regulating ribonucleotide reductase gene expression in malignant H-ras transformed cell lines

Ribonucleotide reductase is a highly regulated activity responsible for reducing ribonucleotides to deoxyribonucleotides, which are required for DNA synthesis and DNA repair. We have tested the hypothesis that malignant cell populations contain alterations in signal pathways important in controlling the expression of the two genes that code for ribonucleotide reductase, R1 and R2. A series of radiation and H-ras transformed mouse 10T1/2 cell lines with increasing malignant potential were exposed to stimulators of cAMP synthesis (forskolin and cholera toxin), an inhibitor of cAMP degradation (3-isobutyl-1-methylxanthine) and a biologically stable analogue of cAMP (8-bromo-cAMP). Dramatic elevations in the expression of the R1 and R2 genes at the message and protein levels were observed in malignant metastatic populations, which were not detected in the normal parental cell line or in cells capable of benign tumor formation. These changes in ribonucleotide reductase gene expression occurred without any detectable modifications in the rates of DNA synthesis, showing that they were regulated by a novel mechanism independent of the S phase of the cell cycle. Furthermore, studies with forskolin (a stimulator of the protein kinase A signal pathway) and the tumor promoter 12–0-tetradecanoylphorbol-13-acetate (a stimulator of the protein kinase C signal pathway), alone or in combination, indicated that their effects on R1 and R2 gene expression in a highly malignant cell line were greater than when they were tested individually, suggesting that the two pathways modulating R1 and R2 gene expression can cooperate to regulate ribonucleotide reduction, and interestingly this can occur in a synergistic fashion. Also, a direct relationship between H-ras expression and ribonucleotide reductase gene expression was observed; analysis of forskolin mediated elevations in R1 and R2 message levels closely correlated with the levels of H-ras expression in the various cell lines. In total, these studies demonstrate that ribonucleotide reductase expression is controlled by a complex process, and malignant ras transformed cells contain alterations in the regulation of signal transduction pathways that lead to novel modifications in ribonucleotide reductase gene expression. This signal mechanism, which is aberrantly regulated in malignant cells, may be related to regulatory pathways involved in determining ribonucleotide reductase expression in a S phase independent manner during periods of DNA repair. © 1994 Wiley-Liss, Inc.     

The role of N3-ethyldeoxythymidine in mutagenesis and cytotoxicity by ethylating agents

The significance of DNA ethylation at the central hydrogen-bonding site (N3) of thymine was investigated using an in vitro DNA replication system. The system utilized a primed template in which the 3'-end of the primer is eight nucleotides away from N3-ethyldeoxythymidine (N3-Et-dT), present at template position 26 from the 3'-end. The 34-nucleotide template corresponds to a specific DNA sequence at gene G of bacteriophage phi X174. DNA synthesis products were quantitated by electrophoretic separation and autoradiography. At 10 microM dNTP and 0.5 mM Mn2+, N3-Et-dT blocked DNA synthesis by Escherichia coli polymerase I (Klenow fragment): 60% after incorporating a nucleotide opposite N3-Et-dT (incorporation-dependent blocked product) and 39% 3' to N3-Et-dT. DNA replication past the lesion (post-lesion synthesis) was negligible. Post-lesion synthesis increased using higher concentrations of dNTP, reaching 68% at 200 microM dNTP. DNA sequencing revealed that dA was incorporated opposite N3-Et-dT in the incorporation-dependent blocked product. In the post-lesion synthesis product, dT was exclusively incorporated opposite N3-Et-dT. Formation of the N3-Et-dT.dA base pair at the replication fork terminated DNA synthesis, while the N3-Et-dT.dT base pair formed at the 3'-end of the growing chain was extended, leading to an A.T----T.A transversion mutation. The results suggest a dual role for the N3-Et-dT lesion, contributing in part to the cytotoxicity and mutagenicity of ethylating agents. These studies provide a basis for understanding the activation of oncogene neu by A.T----T.A transversion mutation in rat neuroblastomas induced by N-ethyl-N-nitrosourea.