Is uracil misincorporation into DNA mutagenic?
Explain why Methotrexate treatment of cells can cause misincorporation of uracil into DNA? Even though methotrexate inhibits dihydrofolate reductase it has broad reaching effects on other metabolic pathways. Explain why methotrexate would also inhibit purine nucleotide metabolism, methionine metabolism and potentially interfere with all reactions involving single carbon …
Does dietary folate affect uracil misincorporation into DNA?
Usually, the drugs work by damaging the RNA or DNA that tells the cell how to copy itself in division. If the cells are unable to divide, they die. The faster the cells are dividing, the more likely it is that chemotherapy will kill the cells, causing the tumor to shrink.
Is methotrexate a chemotherapy drug?
Explain why Methotrexate treatment of cells can cause misincorporation of uracil into DNA. In the absence of the nucleotide thymidine, the pyrimidine nucleotide dUMP, which actually is a substrate for dTMP may be mistakenly utilized by the cell in the synthesis of DNA.
Why do old mice have high uracil content in liver DNA?
Jun 21, 2019 · explain why methotrexate treatment of cells can cause misincorporation of uracil into dna. even though methotrexate inhibits dihydrofolate reductase it has broad reaching effects on other metabolic pathways. explain why methotrexate would also inhibit purine nucleotide metabolism, methionine metabolism and potentially interfere with all reactions …
Precautions
Before starting methotrexate treatment, make sure you tell your doctor about any other medications you are taking (including prescription, over-the-counter, vitamins, herbal remedies, etc.). Do not take aspirin, or products containing aspirin unless your doctor specifically permits this.
Self-Care Tips
Drink at least two to three quarts of fluid every 24 hours, unless you are instructed otherwise.
Monitoring and Testing
You will be checked regularly by your health care provider while you are taking methotrexate, to monitor side effects and check your response to therapy. Periodic blood work to monitor your complete blood count (CBC) as well as the function of other organs (such as your kidneys and liver) will also be ordered by your doctor.
How Methotrexate Works
Cancerous tumors are characterized by cell division, which is no longer controlled as it is in normal tissue. "Normal" cells stop dividing when they come into contact with like cells, a mechanism known as contact inhibition. Cancerous cells lose this ability.
What are the two systems of cellular uptake of folates?
Two functionally different systems exist for cellular uptake of folates: ( i) membrane-bound FR (FRα and FRβ), which is linked to cell surfaces via a glycosylphosphatydilinositol ( GPI) anchor 20 and internalizes folates by receptor-mediated endocytosis (described in detail below) and ( ii) RFC, which uses a bidirectional anion-exchange mechanism to transport folates into the cytoplasm. 21, 22 The remaining discussion is restricted to FRα, the most widely studied FR isoform. The transport kinetics and affinity for folates and antifolates differ significantly between systems (Table I ), in part, because FRα does not bind and carry folate into the cell as does RFC, but binds and internalizes folate via endocytosis. Therefore, the dissociation constant, Kd, describes the binding affinity of folates by FRα, whereas the Michaelis constant, Km, describes the binding affinity plus transport of folates by RFC. 23 FRα binds oxidized folate – folic acid – with high affinity at low, physiologic concentrations ( Kd: <1 nM), 21 has high affinity for reduced folates such as 5-mTHF ( Kd: 1–10 nM), and relatively low affinity for methotrexate ( Kd: >100 nM). 21 Although folic acid is normally found in low concentrations in human serum, folic acid consumed in quantities generally found in multivitamin supplements can be transported in serum in the oxidized form. 24, 25 FRα can bind folic acid with ∼10 times greater affinity than any of the reduced forms of the vitamin 26 or methotrexate. 27 The significance of increased serum folic acid in the presence of FOLR1 -expressing tumors is unclear.
What is the role of FR in cancer?
Folate is a basic component of cell metabolism and DNA synthesis and repair, and rapidly dividing cancer cells have an increased requirement for folate to maintain DNA synthesis, an observation supported by the widespread use of antifolates in cancer chemotherapy. FRα levels are high in specific malignant tumors of epithelial origin compared to normal cells, and are positively associated with tumor stage and grade, raising questions of its role in tumor etiology and progression. It has been suggested that FRα might confer a growth advantage to the tumor by modulating folate uptake from serum or by generating regulatory signals. Indeed, cell culture studies show that expression of the FRα gene, FOLR1, is regulated by extracellular folate depletion, increased homocysteine accumulation, steroid hormone concentrations, interaction with specific transcription factors and cytosolic proteins, and possibly genetic mutations. Whether FRα in tumors decreases in vivo among individuals who are folate sufficient, or whether the tumor's machinery sustains FRα levels to meet the increased folate demands of the tumor, has not been studied. Consequently, the significance of carrying a FRα-positive tumor in the era of folic acid fortification and widespread vitamin supplement use in countries such as Canada and the United States is unknown. Epidemiologic and clinical studies using human tumor specimens are lacking and increasingly needed to understand the role of environmental and genetic influences on FOLR1 expression in tumor etiology and progression. This review summarizes the literature on the complex nature of FOLR1 gene regulation and expression, and suggests future research directions. © 2006 Wiley-Liss, Inc.
How many exons are in the FOLR1 gene?
The FOLR1 gene is composed of 7 exons and 6 introns, spans 6.8 kb, and is flanked by consensus splice site sequences 20, 47 (the nucleotide sequence is available from Genbank, accession number U20391). Both the organization and transcription of FOLR1 are complex. Tissue-specific expression of the receptor is due to multiple transcripts arising from at least 2 promoter regions, one located upstream and within exon 1 (named P1) and the second upstream of exon 4 (named P4), and alternative splicing of exons 1 to 4. 20, 47 The 2 promoters encode FOLR1 transcripts with different 5′ ends but identical amino acid sequences and 3′ ends. 47 The identification of a steroid receptor-binding element upstream of P4 suggests steroid hormones may regulate gene expression (discussed below). 47 In contrast, the FOLR2 and FOLR3 genes each consist of 5 exons, 4 introns and 1 promoter that encodes a single transcript. 20, 51, 67, 68
What is the role of folic acid in cancer?
Folate, a basic component of cell metabolism and DNA synthesis and repair, 2 is an essential vitamin required by both normal and tumor cells, an observation supported by the widespread use of antifolates in cancer chemotherapy .
Is FR high in tumors?
FRα levels are high in specific malignant tumors of epithelial origin compared to normal cells, and are positively associated with tumor stage and grade, raising questions of its role in tumor etiology and progression.
What are the most common mutations in cancer?
The most common mutations in cancer are C to T transitions, but their origin has remained elusive. Recently, mutational signatures of APOBEC-family cytosine deaminases were identified in many common cancers, suggesting off-target deamination of cytosine to uracil as a common mutagenic mechanism. Here we present evidence from mass spectrometric quantitation of deoxyuridine in DNA that shows significantly higher genomic uracil content in B-cell lymphoma cell lines compared to non-lymphoma cancer cell lines and normal circulating lymphocytes. The genomic uracil levels were highly correlated with AID mRNA and protein expression, but not with expression of other APOBECs. Accordingly, AID knockdown significantly reduced genomic uracil content. B-cells stimulated to express endogenous AID and undergo class switch recombination displayed a several-fold increase in total genomic uracil, indicating that B cells may undergo widespread cytosine deamination after stimulation. In line with this, we found that clustered mutations (kataegis) in lymphoma and chronic lymphocytic leukemia predominantly carry AID-hotspot mutational signatures. Moreover, we observed an inverse correlation of genomic uracil with uracil excision activity and expression of the uracil-DNA glycosylases UNG and SMUG1. In conclusion, AID-induced mutagenic U:G mismatches in DNA may be a fundamental and common cause of mutations in B-cell malignancies.
What are the consequences of exposure to TS inhibitors such as pemetrexed?
Misincorporation of genomic uracil and formation of DNA double strand breaks (DSBs) are known consequences of exposure to TS inhibitors such as pemetrexed. Uracil DNA glycosylase (UNG) catalyzes the excision of uracil from DNA and initiates DNA base excision repair (BER). To better define the relationship between UNG activity and pemetrexed anticancer activity, we have investigated DNA damage, DSB formation, DSB repair capacity, and replication fork stability in UNG (+/+) and UNG (-/-) cells. We report that despite identical growth rates and DSB repair capacities, UNG (-/-) cells accumulated significantly greater uracil and DSBs compared with UNG (+/+) cells when exposed to pemetrexed. ChIP-seq analysis of γ-H2AX enrichment confirmed fewer DSBs in UNG (+/+) cells. Furthermore, DSBs in UNG (+/+) and UNG (-/-) cells occur at distinct genomic loci, supporting differential mechanisms of DSB formation in UNG-competent and UNG-deficient cells. UNG (-/-) cells also showed increased evidence of replication fork instability (PCNA dispersal) when exposed to pemetrexed. Thymidine co-treatment rescues S-phase arrest in both UNG (+/+) and UNG (-/-) cells treated with IC50-level pemetrexed. However, following pemetrexed exposure, UNG (-/-) but not UNG (+/+) cells are refractory to thymidine rescue, suggesting that deficient uracil excision rather than dTTP depletion is the barrier to cell cycle progression in UNG (-/-) cells. Based on these findings we propose that pemetrexed-induced uracil misincorporation is genotoxic, contributing to replication fork instability, DSB formation and ultimately cell death.
What are the defects in DNA repair pathways?
This article reviews the available data on the influence of defects in proteins involved in the major DNA repair pathways (i.e., homologous recombination, non-homologous end joining, base excision repair, nucleotide excision repair, mismatch repair, Fanconi anemia repair, translesion synthesis and direct reversal repair) on the cytotoxicity of the FDA-approved anticancer drugs. It is shown that specific deficiencies in these DNA repair pathways alter the cytotoxicity of 60 anticancer drugs, including classical DNA-targeting drugs (e.g., alkylating agents, cytotoxic antibiotics, DNA topoisomerase inhibitors and antimetabolites) and other drugs whose primary pharmacological target is not the DNA (e.g., antimitotic agents, hormonal and targeted therapies). This information may help predict response to anticancer drugs in patients with tumors having specific DNA repair defects.
How does uracil occur in DNA?
Uracil arises in DNA from spontaneous deamination of cytosine and through incorporation of dUMP by DNA polymerase during DNA replication. Excision of uracil by the action of uracil-DNA glycosylase (Ung) initiates the base excision repair pathway to counter the promutagenic base modification. In this study, we cloned a cDNA-encoding Caenorhabditis elegans homologue (CeUng-1) of Escherichia coli Ung. There was 49% identity in amino acid sequence between E.coli Ung and CeUng-1. Purified CeUng-1 removed uracil from both U:G and U:A base pairs in DNA. It also removed uracil from single-stranded oligonucleotide substrate less efficiently than double-stranded oligonucleotide. The CeUng-1 activity was inhibited by Bacillus subtilis Ung inhibitor, indicating that CeUng-1 is a member of the family-1 Ung group. The mutation in the ung-1 gene did not affect development, fertility and lifespan in C.elegans, suggesting the existence of backup enzyme. However, we could not detect residual uracil excision activity in the extract derived from the ung-1 mutant. The present experiments also showed that the ung-1 mutant of C.elegans was more resistant to NaHSO (3)-inducing cytosine deamination than wild-type strain.
What is the difference between 5-FU and 5-FU?
5-fluorouracil (5-FU) and its metabolite 5-fluorodeoxyuridine (FdUrd; floxuridine) are chemotherapy agents that are converted to FdUMP and FdUTP. FdUMP inhibits thymidylate synthase and causes the accumulation of uracil in the genome, whereas FdUTP is incorporated by DNA polymerases as 5-FU in the genome; however, it remains unclear how either genomically incorporated U and 5-FU contribute to killing. We show that depletion of the uracil DNA glycosylase UNG sensitizes tumor cells to FdUrd. Furthermore, we show that UNG depletion does not sensitize cells to the thymidylate synthase inhibitor (raltitrexed), which induces uracil but not 5-FU accumulation, thus indicating that genomically incorporated 5-FU plays a major role in the anti-neoplastic effects of FdUrd. We also show that 5-FU metabolites do not block the first round of DNA synthesis but instead arrest cells at the G1/S border when cells again attempt replication and activate homologous recombination (HR). This arrest is not due to 5-FU lesions blocking DNA polymerase δ, but instead depends, in part, on the glycosylase TDG. Consistent with the activation of HR repair, disruption of HR sensitized cells to FdUrd, especially when UNG was disabled. These results show that 5-FU lesions that escape UNG repair activate HR, which promotes cell survival.
How many genes are mutated in colon cancer?
To this aim, we have analyzed 37 colon cancer samples by use of the Ion AmpliSeq™ Comprehensive Cancer Panel. Overall, we have found 307 mutated genes, most of which already implicated in the development of colon cancer. Among these, 15 genes were mutated in tumors originating in all six colon segments and were defined “common genes” (i.e. APC, PIK3CA, TP53) whereas 13 genes were preferentially mutated in tumors originating only in specific colon segments and were defined “site-associated genes” (i.e. BLNK, PTPRD). In addition, the presence of mutations in 10 of the 307 identified mutated genes (NBN, SMUG1, ERBB2, PTPRT, EPHB1, ALK, PTPRD, AURKB, KDR and GPR124) were found to be of clinical relevance. Among clinically relevant genes, NBN and SMUG1 were identified as independent prognostic factors that predicted poor survival in colon cancer patients. In conclusion, the findings reported here indicate that tumors arising in different colon segments present differences in the type and/or frequency of genetic variants, with two of them being independent prognostic factors that predict poor survival in colon cancer patients.
What is uracil misincorporation?
Uracil misincorporation into DNA is a consequence of pemetrexed inhibition of thymidylate synthetase. The base excision repair (BER) enzyme, uracil DNA glycosylase (UNG) is the major glycosylase responsible for removal of misincorporated uracil. We previously illustrated hypersensitivity to pemetrexed in UNG (-/-) human colon cancer cells. Here, we examined the relationship between UNG expression and pemetrexed sensitivity in human lung cancer. We observed a spectrum of UNG expression in human lung cancer cells. Higher levels of UNG are associated with pemetrexed resistance and are present in cell lines derived from pemetrexed-resistant histological subtypes (small cell and squamous cell carcinoma). Acute pemetrexed exposure induces UNG protein and mRNA, consistent with up-regulation of uracil-DNA repair machinery. Chronic exposure of H1299 adenocarcinoma cells to increasing pemetrexed concentrations established drug-resistant sublines. Significant induction of UNG protein confirmed up-regulation of BER as a feature of acquired pemetrexed resistance. Co-treatment with the BER inhibitor, methoxyamine (MX) overrides pemetrexed resistance in chronically exposed cells, underscoring the utility of BER directed therapeutics to offset acquired drug resistance. Expression of UNG-directed siRNA and shRNA enhanced sensitivity in A549 and H1975 cells, and in drug-resistant sublines, confirming that UNG up-regulation is protective. In human lung cancer, UNG deficiency is associated with pemetrexed-induced retention of uracil in DNA that destabilizes DNA replication forks resulting in DNA double strand breaks and cell death. Thus, in experimental models, UNG is a critical mediator of pemetrexed sensitivity that warrants evaluation to determine clinical value.
