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The essential checkpoint kinase Chk1 is required for cell-cycle delays after DNA damage or blocked DNA replication 1 , 2 . However, it is unclear whether Chk1 is involved in the repair of damaged DNA. Here we establish that Chk1 is a key regulator of genome maintenance by the homologous recombination repair (HRR) system. Abrogation of Chk1 function with small interfering RNA or chemical antagonists inhibits HRR, leading to persistent unrepaired DNA double-strand breaks (DSBs) and cell death after replication inhibition with hydroxyurea or DNA-damage caused by camptothecin. After hydroxyurea treatment, the essential recombination repair protein RAD51 is recruited to DNA repair foci performing a vital role in correct HRR 3 , 4 . We demonstrate that Chk1 interacts with RAD51, and that RAD51 is phosphorylated on Thr 309 in a Chk1-dependent manner. Consistent with a functional interplay between Chk1 and RAD51, Chk1-depleted cells failed to form RAD51 nuclear foci after exposure to hydroxyurea, and cells expressing a phosphorylation-deficient mutant RAD51 T309A were hypersensitive to hydroxyurea. These results highlight a crucial role for the Chk1 signalling pathway in protecting cells against lethal DNA lesions through regulation of HRR.

The Multicenter Study of Hydroxyurea in Sickle Cell Anemia (MSH) was a randomized double-blind placebo-controlled trial to test whether hydroxyurea could reduce the rate of painful crises in adults who had at least 3 painful crises per year. Because hydroxyurea is known to be carcinogenic, mutagenic, and teratogenic in animals, a major inclusion criterion in MSH was the use of contraceptives both by females and males in order to avoid exposure of the fetus to hydroxyurea. Despite this precautionary measure, some women became pregnant while taking hydroxyurea or their male partners were on hydroxyurea. We followed surviving patients who were enrolled in the original MSH trial for up to 17 years postrandomization. Our findings suggest that exposure of the fetus to hydroxyurea does not cause teratogenic changes in those pregnancies that terminate in live birth whether full term or premature. This seems to be true whether the parent taking hydroxyurea was the mother or the father. The same argument seems to apply for exposure to opioids. However, it will take a much longer follow-up of many more hydroxyurea-exposed sickle cell disease subjects to establish the results conclusively.

Recent studies have demonstrated the role of adenosine (ADO) in sickle-cell anemia (SCA). ADO is produced by CD39 and CD73 and converted to inosine by adenosine deaminase (ADA). We evaluated the effects of hydroxycarbamide (HU) treatment on the modulation of adenosine levels in SCA patients. The expressions of CD39, CD73, and CD26 were evaluated by flow cytometry on blood cells in 15 HU-treated and 17 untreated patients and 10 healthy individuals. RNA was extracted from monocytes, and ADA gene expression was quantified by real-time PCR. ADA activity was also evaluated. We found that ADA transcripts were two times higher in monocytes of HU-treated patients, compared with untreated ( P  = 0.039). Monocytes of HU-treated patients expressed CD26, while monocytes of controls and untreated patients did not ( P  = 0.023). In treated patients, a lower percentage of T lymphocytes expressed CD39 compared with untreated ( P  = 0.003), and the percentage of T regulatory (Treg) cells was reduced in the treated group compared with untreated ( P  = 0.017) and controls ( P  = 0.0009). Besides, HU-treated patients displayed increased ADA activity, compared with untreated. Our results indicate a novel mechanism of action of HU mediated by the reduction of adenosine levels and its effects on pathophysiological processes in SCA.

Hydroxyurea (HU) is mostly referred to as an inhibitor of ribonucleotide reductase (RNR) and as the agent that is commonly used to arrest cells in the S-phase of the cycle by inducing replication stress. It is a well-known and widely used drug, one which has proved to be effective in treating chronic myeloproliferative disorders and which is considered a staple agent in sickle anemia therapy and—recently—a promising factor in preventing cognitive decline in Alzheimer’s disease. The reversibility of HU-induced replication inhibition also makes it a common laboratory ingredient used to synchronize cell cycles. On the other hand, prolonged treatment or higher dosage of hydroxyurea causes cell death due to accumulation of DNA damage and oxidative stress. Hydroxyurea treatments are also still far from perfect and it has been suggested that it facilitates skin cancer progression. Also, recent studies have shown that hydroxyurea may affect a larger number of enzymes due to its less specific interaction mechanism, which may contribute to further as-yet unspecified factors affecting cell response. In this review, we examine the actual state of knowledge about hydroxyurea and the mechanisms behind its cytotoxic effects. The practical applications of the recent findings may prove to enhance the already existing use of the drug in new and promising ways.

The design and construction of synthetic therapeutic protocells capable of establishing cognate chemical communication channels with living cells is an important challenge for synthetic biology and bio-engineering. Here we develop a step towards protocell-mediated nitric-oxide-induced vasodilation by constructing a new synthetic cell model based on bio-derived coacervate vesicles with high haemocompatibility and increased blood circulation times. The hybrid protocells are prepared by the spontaneous self-assembly of haemoglobin-containing erythrocyte membrane fragments on the surface of preformed polysaccharide–polynucleotide coacervate micro-droplets containing glucose oxidase. We use the sequestered enzymes to program a spatially coupled glucose oxidase/haemoglobin reaction cascade, which in the presence of glucose and hydroxyurea generates a protocell-mediated flux of nitric oxide that we exploit for in vitro and in vivo blood vessel vasodilation. Taken together, our results provide new opportunities for the development of endogenously organized cell-like entities (biocompatible micro-bots) geared specifically towards active interfacing with individual living cells and cell communities.The self-assembly of haemoglobin-containing erythrocyte membrane fragments onto the surface of preformed coacervates has been used to make hybrid synthetic cells that can initiate nitric-oxide-induced vasodilation. These synthetic cells encapsulate enzymes that generate a flux of nitric oxide, as well as exhibiting high haemocompatibility and increased blood circulation times.

Elevated levels of reactive oxygen species (ROS) reduce replication fork velocity by causing dissociation of the TIMELESS-TIPIN complex from the replisome. Here, we show that ROS generated by exposure of human cells to the ribonucleotide reductase inhibitor hydroxyurea (HU) promote replication fork reversal in a manner dependent on active transcription and formation of co-transcriptional RNA:DNA hybrids (R-loops). The frequency of R-loop-dependent fork stalling events is also increased after TIMELESS depletion or a partial inhibition of replicative DNA polymerases by aphidicolin, suggesting that this phenomenon is due to a global replication slowdown. In contrast, replication arrest caused by HU-induced depletion of deoxynucleotides does not induce fork reversal but, if allowed to persist, leads to extensive R-loop-independent DNA breakage during S-phase. Our work reveals a link between oxidative stress and transcription-replication interference that causes genomic alterations recurrently found in human cancer. Excessive oxidative stress is widely perceived as a key factor in cancer progression. Here, the authors reveal that oxidative stress induces transcription-dependent replication fork stalling that appears to be a major source of chromosomal rearrangements found in human cancers.

We previously reported initial clinical results of post-transcriptional gene silencing of BCL11A expression (NCT 03282656) reversing the fetal to adult hemoglobin switch. A goal of this approach is to increase fetal hemoglobin (HbF) expression while coordinately reducing sickle hemoglobin (HbS) expression. The resulting combinatorial effect should prove effective in inhibiting HbS polymerization at lower physiologic oxygen values thereby mitigating disease complications. Here we report results of exploratory single-cell analysis of patients in which BCL11A is targeted molecularly and compare results with cells of patients treated with hydroxyurea (HU), the current standard of care. We use single-cell assays to assess HbF, HbS, oxygen saturation, and hemoglobin polymer content in RBCs for nine gene therapy trial subjects (BCL shmiR , median HbF% = 27.9) and compare them to 10 HU-treated subjects demonstrating high and comparable levels of HbF (HU High Responders, median HbF% = 27.0). All BCL11A patients achieved the primary endpoint for NCT 03282656, which was defined by an absolute neutrophil count greater than or equal to 0.5 × 10 9 cells/L for three consecutive days, achieved within 7 weeks following infusion. Flow cytometric assessment of single-RBC HbF and HbS shows fewer RBCs with high HbS% that would be most susceptible to sickling in BCL shmiR vs. HU High Responders: median 42% of RBCs with HbS%>70% in BCL shmiR vs. 61% in HU High Responders ( p  = 0.004). BCL shmiR subjects also demonstrate more RBCs resistant to HbS polymerization at lower physiologic oxygen tension: median 32% vs. 25% in HU High Responders ( p  = 0.006). Gene therapy-induced BCL11A down-regulation reverses the fetal-to-adult hemoglobin switch and induces RBCs with higher HbF%, lower HbS%, and greater resistance to deoxygenation-induced polymerization in clinical trial subjects compared with a cohort of highly responsive hydroxyurea-treated subjects. The authors report in full the primary endpoint data of a pilot clinical trial (NCT 03282656) that used post-transcriptional gene silencing of BCL11A expression to reverse the fetal to adult hemoglobin switch in sickle cell disease. They develop new single-cell flow cytometry and microfluidic techniques to predict the efficacy of HbF induction and show that red blood cells from these patients exhibit greater resistance to deoxygenation-induced polymerization than red blood cells from hydroxyurea-responsive patients.

The plenteous resistance to and undesirable consequences of the existing antipiroplasmic therapies have emphasized the urgent need for new chemotherapeutics and drug targets for both prophylaxis and chemotherapy. Hydroxyurea (HYD) is an antineoplastic agent with antitrypanosomal activity. Eflornithine (α-difluoro-methyl ornithine, DFMO) is the best choice therapy for the treatment of late-stage Gambian human African trypanosomiasis. In this study, the inhibitory and combination efficacy of HYD and DFMO with existing babesicidal drugs (diminazene aceturate (DA), atovaquone (ATV), and clofazimine (CLF)) deoxyribonucleotide in vitro against the multiplication of Babesia and Theileria. As well as, their chemotherapeutic effects were assessed on B. microti strain that infects rodents. The Cell Counting Kits-8 (CCK-8) test was used to examine their cytotoxicity on human foreskin fibroblast (HFF), mouse embryonic fibroblast (NIH/3T3), and Madin-Darby bovine kidney (MDBK) cells. HYD and DFMO suppressed the multiplication of all tested species (B. bigemina, B. bovis, B. caballi, B. divergens, and T. equi) in a dose-related manner. HFF, NIH/3T3, or MDBK cell viability was not influenced by DFMO at 1000 μM, while HYD affected the MDBK cell viability at EC50 value of 887.5±14.4 μM. The in vitro combination treatments of DFMO and HYD with CLF, DA, and ATV exhibited synergistic and additive efficacy toward all tested species. The in vivo experiment revealed that HYD and DFMO oral administration at 100 and 50 mg/kg inhibited B. microti multiplication in mice by 60.1% and 78.2%, respectively. HYD-DA and DFMO-DA combined treatments showed higher chemotherapeutic efficacy than their monotherapies. These results indicate the prospects of HYD and DFMO as drug candidates for piroplasmosis treatment, when combined mainly with DA, ATV, and CLF. Therefore, further studies are needed to combine HYD or DFMO with either ATV or CLF and examine their impact on B. microti infection in mice.

The aim of the present study was to investigate the potential of human plasma derived exosomes for the delivery of hydroxyurea to enhance its therapeutic efficacy in breast cancer. Plasma derived exosomes were isolated using differential centrifugation along with ultrafiltration method. Hydroxyurea was encapsulated in exosomes using a freeze–thaw method. The exosomes and Exo-HU were characterized for their size distribution, drug entrapment efficiency, in-vitro drug release profile, morphological analysis and cytotoxic effects on MCF-7 cell line. The results showed a mean size of 178.8 nm and a zeta potential of -18.3 mV, indicating good stability and 70% encapsulation effectiveness for HU. Exo-HU produced sustained drug release action with a considerable percentage released within 72 h. The morphological analysis indicated that the plasma derived exosomes were spherical, and cup shaped. In cytotoxicity studies on MCF-7 cells, Exo-HU has reduced cell viability compared to HU and blank exosomes. Findings of this study showed that human plasma-derived exosomes have been considered as effective delivery vehicle for hydroxyurea, potentially improving breast cancer treatment outcomes. Graphical Abstract

Stem cell transplantation and genetic therapies offer potential cures for patients with sickle cell disease (SCD), but these options require advanced medical facilities and are expensive. Consequently, these treatments will not be available for many years to the majority of patients suffering from this disease.What is urgently needed now is an inexpensive oral drug in addition to hydroxyurea, the only drug approved by the FDA that inhibits sickle-hemoglobin polymerization. Here, we report the results of the first phase of our phenotypic screen of the 12,657 compounds of the Scripps ReFRAME drug repurposing library using a recently developed high-throughput assay to measure sickling times following deoxygenation to 0% oxygen of red cells from sickle trait individuals. The ReFRAME library is a very important collection because the compounds are either FDA-approved drugs or have been tested in clinical trials. From dose-response measurements, 106 of the 12,657 compounds exhibit statistically significant antisickling at concentrations ranging from 31 nM to 10 μM. Compounds that inhibit sickling of trait cells are also effective with SCD cells. As many as 21 of the 106 antisickling compounds emerge as potential drugs. This estimate is based on a comparison of inhibitory concentrations with free concentrations of oral drugs in human serum. Moreover, the expected therapeutic potential for each level of inhibition can be predicted from measurements of sickling times for cells from individuals with sickle syndromes of varying severity. Our results should motivate others to develop one or more of these 106 compounds into drugs for treating SCD.