Cytidine deaminase (CDA) deficiency causes pyrimidine pool disequilibrium. We previously reported that the excess cellular dC and dCTP resulting from CDA deficiency jeopardizes genome stability, ...decreasing basal poly(ADP-ribose) polymerase 1 (PARP-1) activity and increasing ultrafine anaphase bridge (UFB) formation. Here, we investigated the mechanism underlying the decrease in PARP-1 activity in CDA-deficient cells. PARP-1 activity is dependent on intracellular NAD
concentration. We therefore hypothesized that defects of the NAD
salvage pathway might result in decreases in PARP-1 activity. We found that the inhibition or depletion of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD
salvage biosynthesis pathway, mimicked CDA deficiency, resulting in a decrease in basal PARP-1 activity, regardless of NAD
levels. Furthermore, the expression of exogenous wild-type NAMPT fully restored basal PARP-1 activity and prevented the increase in UFB frequency in CDA-deficient cells. No such effect was observed with the catalytic mutant. Our findings demonstrate that (1) the inhibition of NAMPT activity in CDA-proficient cells lowers basal PARP-1 activity, and (2) the expression of exogenous wild-type NAMPT, but not of the catalytic mutant, fully restores basal PARP-1 activity in CDA-deficient cells; these results strongly suggest that basal PARP-1 activity in CDA-deficient cells decreases due to a reduction of NAMPT activity.
Abstract
Cytidine deaminase (CDA), an enzyme of the pyrimidine salvage pathway, deaminates cytidine, deoxycytidine and analogs, such as gemcitabine. Constitutive low levels of CDA activity have been ...reported in the blood of patients with hematological malignancies or suffering from gemcitabine toxicity. We previously reported that cellular CDA deficiency leads to genetic instability. We therefore hypothesized that constitutive CDA deficiency might confer a predisposition to cancer. We analyzed CDA activity and expression in blood samples from breast cancer (BC) patients with a suspected predisposition to the disease, and in healthy controls. Contrary to our hypothesis, we found that both CDA activity and mRNA levels were higher in blood samples from BC patients than in those from controls, and that this difference was not due to excess neutrophils. CDA activity levels were significantly higher in the serum samples of BC patients treated by radiotherapy (RT) than in those of untreated healthy controls, and hormone therapy in RT-treated BC patients was associated with significantly lower levels of CDA activity. A preliminary analysis of CDA activity in the serum of the very few BC patients who had undergone no treatment other than surgery suggested that the increase in CDA activity might be due to the breast cancer itself. Our findings raise important questions, which should lead to studies to elucidate the origin and significance of the increase in CDA activity in the serum of BC patients, and the impact of hormone therapy.
Cells from Bloom's syndrome patients display genome instability due to a defective BLM and the downregulation of cytidine deaminase. Here, we use a genome-wide RNAi-synthetic lethal screen and ...transcriptomic profiling to identify genes enabling BLM-deficient and/or cytidine deaminase-deficient cells to tolerate constitutive DNA damage and replication stress. We found a synthetic lethal interaction between cytidine deaminase and microtubule-associated protein Tau deficiencies. Tau is overexpressed in cytidine deaminase-deficient cells, and its depletion worsens genome instability, compromising cell survival. Tau is recruited, along with upstream-binding factor, to ribosomal DNA loci. Tau downregulation decreases upstream binding factor recruitment, ribosomal RNA synthesis, ribonucleotide levels, and affects ribosomal DNA stability, leading to the formation of a new subclass of human ribosomal ultrafine anaphase bridges. We describe here Tau functions in maintaining survival of cytidine deaminase-deficient cells, and ribosomal DNA transcription and stability. Moreover, our findings for cancer tissues presenting concomitant cytidine deaminase underexpression and Tau upregulation open up new possibilities for anti-cancer treatment.Cytidine deaminase (CDA) deficiency leads to genome instability. Here the authors find a synthetic lethal interaction between CDA and the microtubule-associated protein Tau deficiencies, and report that Tau depletion affects rRNA synthesis, ribonucleotide pool balance, and rDNA stability.
Cytidine deaminase (CDA) catalyzes the deamination of cytidine (C) and deoxycytidine (dC) to uridine and deoxyuridine, respectively. We recently showed that CDA deficiency leads to genomic ...instability, a hallmark of cancers. We therefore investigated whether constitutive CDA inactivation conferred a predisposition to cancer development. We developed a novel mouse model of Cda deficiency by generating Cda-knockout mice. Cda+/+ and Cda−/− mice did not differ in lifetime phenotypic or behavioral characteristics, or in the frequency or type of spontaneous cancers. However, the frequency of chemically induced tumors in the colon was significantly lower in Cda−/− mice. An analysis of primary kidney cells from Cda−/− mice revealed an excess of C and dC associated with significantly higher frequencies of sister chromatid exchange and ultrafine anaphase bridges and lower Parp-1 activity than in Cda+/+ cells. Our results suggest that, despite inducing genetic instability, an absence of Cda limits the number of chemically induced tumors. These results raise questions about whether a decrease in basal Parp-1 activity can protect against inflammation-driven tumorigenesis; we discuss our findings in light of published data for the Parp-1-deficient mouse model.
•We developed a novel mouse model of cytidine deaminase (Cda) deficiency (Cda−/−).•Cda deficiency results in cellular genetic instability and lower PARP-1 activity.•Cda deficiency does not increase the risk of spontaneous cancers.•Cda deficiency decreases the risk of chemically-induced tumors.•Lower PARP-1 activity may reduce inflammation-driven tumorigenesis in Cda−/− mice.
One of the main challenges in cancer therapy is the identification of molecular mechanisms mediating resistance or sensitivity to treatment. Cytidine deaminase (CDA) was reported to be downregulated ...in cells derived from patients with Bloom syndrome, a genetic disease associated with a strong predisposition to a wide range of cancers. The purpose of this study was to determine whether CDA deficiency could be associated with tumors from the general population and could constitute a predictive marker of susceptibility to antitumor drugs.
We analyzed
expression
, in large datasets for cancer cell lines and tumors and in various cancer cell lines and primary tumor tissues using IHC, PDXs, qRT-PCR, and Western blotting. We also studied the mechanism underlying CDA silencing and searched for molecules that might target specifically CDA-deficient tumor cells using
analysis coupled to classical cellular experimental approaches.
We found that CDA expression is downregulated in about 60% of cancer cells and tissues. We demonstrate that DNA methylation is a prevalent mechanism of CDA silencing in tumors. Finally, we show that CDA-deficient tumor cells can be specifically targeted with epigenetic treatments and with the anticancer drug aminoflavone.
CDA expression status identifies new subgroups of cancers, and CDA deficiency appears to be a novel and relevant predictive marker of susceptibility to antitumor drugs, opening up new possibilities for treating cancer.
.
Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA) deficiency leads to an excess of dCTP, ...slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation: the intracellular accumulation of dCTP inhibits PARP-1 activity. CDA deficiency results in incomplete DNA replication when cells enter mitosis, leading to the formation of ultrafine anaphase bridges between sister-chromatids at "difficult-to-replicate" sites such as centromeres and fragile sites. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. The stimulation of PARP-1 activity in CDA-deficient cells restores replication and, thus, chromosome segregation. Moreover, increasing intracellular dCTP levels generates under-replication-induced sister-chromatid bridges as efficiently as PARP-1 knockdown. These results have direct implications for Bloom syndrome (BS), a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3'-5' DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our study describes previously unknown pathological consequences of the distortion of dNTP pools and reveals an unexpected role for PARP-1 in preventing DNA under-replication and chromosome segregation defects.
Defects in DNA replication are associated with genetic instability and cancer development, as illustrated in Bloom syndrome. Features of this syndrome include a slowdown in replication speed, ...defective fork reactivation and high rates of sister chromatid exchange, with a general predisposition to cancer. Bloom syndrome is caused by mutations in the BLM gene encoding a RecQ helicase. Here we report that BLM deficiency is associated with a strong cytidine deaminase defect, leading to pyrimidine pool disequilibrium. In BLM-deficient cells, pyrimidine pool normalization leads to reduction of sister chromatid exchange frequency and is sufficient for full restoration of replication fork velocity but not the fork restart defect, thus identifying the part of the Bloom syndrome phenotype because of pyrimidine pool imbalance. This study provides new insights into the molecular basis of control of replication speed and the genetic instability associated with Bloom syndrome. Nucleotide pool disequilibrium could be a general phenomenon in a large spectrum of precancerous and cancer cells.
Identifying new molecular targets for novel anticancer treatments is a major challenge in clinical cancer research. We have shown that cytidine deaminase (CDA) expression is downregulated in about ...60% of cancer cells and tissues. In this study, we aimed to develop a new anticancer treatment specifically inhibiting the growth of CDA-deficient tumor cells. High-throughput screening of a chemical library led to the identification of a naphthol derivative, X55, targeting CDA-deficient tumor cells preferentially, without affecting the growth of non-tumoral cells regardless of CDA expression status. Metabolomic profiling revealed that CDA-deficient HeLa cells differed markedly from control HeLa cells. X55 treatment had a moderate effect on control cells, but greatly disturbed the metabolome of CDA-deficient HeLa cells, worsening the deregulation of many metabolites. In particular, the levels of the three oncometabolites, fumarate, succinate and 2-hydroxyglutarate, were significantly lower in CDA-depleted cells, and this decrease in levels was exacerbated by X55 treatment, revealing an unexpected link between CDA deficiency, mitochondrial function and X55 response. Finally, we identified strong downregulation of
MAPT
(encoding Tau, a microtubule associated protein) expression as a reliable predictive marker for tumor cell X55 sensitivity.
Bloom Syndrome (BS) is a rare genetic disease characterized by high levels of chromosomal instability and an increase in cancer risk. Cytidine deaminase (CDA) expression is downregulated in BS cells, ...leading to an excess of cellular dC and dCTP that reduces basal PARP-1 activity, compromising optimal Chk1 activation and reducing the efficiency of downstream checkpoints. This process leads to the accumulation of unreplicated DNA during mitosis and, ultimately, ultrafine anaphase bridge (UFB) formation. BS cells also display incomplete sister chromatid disjunction when depleted of cohesin. Using a combination of fluorescence in situ hybridization and chromosome spreads, we investigated the possible role of CDA deficiency in the incomplete sister chromatid disjunction in cohesin-depleted BS cells. The decrease in basal PARP-1 activity in CDA-deficient cells compromised sister chromatid disjunction in cohesin-depleted cells, regardless of BLM expression status. The observed incomplete sister chromatid disjunction may be due to the accumulation of unreplicated DNA during mitosis in CDA-deficient cells, as reflected in the changes in centromeric DNA structure associated with the decrease in basal PARP-1 activity. Our findings reveal a new function of PARP-1 in sister chromatid disjunction during mitosis.
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