DNA interstrand crosslinks (ICLs) formed by antitumor agents, such as cisplatin or mitomycin C, are highly cytotoxic DNA lesions. Their repair is believed to be triggered primarily by the stalling of ...replication forks at ICLs in S-phase. There is, however, increasing evidence that ICL repair can also occur independently of replication. Using a reporter assay, we describe a pathway for the repair of cisplatin ICLs that depends on transcription-coupled nucleotide excision repair protein CSB, the general nucleotide excision repair factors XPA, XPF and XPG, but not the global genome nucleotide excision repair factor XPC. In this pathway, Rev1 and Polζ are involved in the error-free bypass of cisplatin ICLs. The requirement for CSB, Rev1 or Polζ is specific for the repair of ICLs, as the repair of cisplatin intrastrand crosslinks does not require these genes under identical conditions. We directly show that this pathway contributes to the removal of ICLs outside of S-phase. Finally, our studies reveal that defects in replication- and transcription-dependent pathways are additive in terms of cellular sensitivity to treatment with cisplatin or mitomycin C. We conclude that transcription- and replication-dependent pathways contribute to cellular survival following treatment with crosslinking agents.
Fanconi anemia is a human cancer predisposition syndrome caused by mutations in 13 Fanc genes. The disorder is characterized by genomic instability and cellular hypersensitivity to chemicals that ...generate DNA interstrand cross-links (ICLs). A central event in the activation of the Fanconi anemia pathway is the mono-ubiquitylation of the FANCI-FANCD2 complex, but how this complex confers ICL resistance remains enigmatic. Using a cell-free system, we showed that FANCI-FANCD2 is required for replication-coupled ICL repair in S phase. Removal of FANCD2 from extracts inhibits both nucleolytic incisions near the ICL and translesion DNA synthesis past the lesion. Reversal of these defects requires ubiquitylated FANCI-FANCD2. Our results show that multiple steps of the essential S-phase ICL repair mechanism fail when the Fanconi anemia pathway is compromised.
DNA interstrand crosslinks (ICLs) are toxic DNA lesions whose repair occurs in the S phase of metazoans via an unknown mechanism. Here, we describe a cell-free system based on
Xenopus egg extracts ...that supports ICL repair. During DNA replication of a plasmid containing a site-specific ICL, two replication forks converge on the crosslink. Subsequent lesion bypass involves advance of a nascent leading strand to within one nucleotide of the ICL, followed by incisions, translesion DNA synthesis, and extension of the nascent strand beyond the lesion. Immunodepletion experiments suggest that extension requires DNA polymerase ζ. Ultimately, a significant portion of the input DNA is fully repaired, but not if DNA replication is blocked. Our experiments establish a mechanism for ICL repair that reveals how this process is coupled to DNA replication.
Background
Pediatric patients with advanced chronic kidney disease (CKD) are often prescribed oral phosphate binders (PBs) for the management of hyperphosphatemia. However, available PBs have ...limitations, including unfavorable tolerability and safety.
Methods
This phase 3, multicenter, randomized, open-label study investigated safety and efficacy of sucroferric oxyhydroxide (SFOH) in pediatric and adolescent subjects with CKD and hyperphosphatemia. Subjects were randomized to SFOH or calcium acetate (CaAc) for a 10-week dose titration (stage 1), followed by a 24-week safety extension (stage 2). Primary efficacy endpoint was change in serum phosphorus from baseline to the end of stage 1 in the SFOH group. Safety endpoints included treatment-emergent adverse events (TEAEs).
Results
Eighty-five subjects (2–18 years) were randomized and treated (SFOH,
n
= 66; CaAc,
n
= 19). Serum phosphorus reduction from baseline to the end of stage 1 in the overall SFOH group (least squares LS mean ± standard error SE) was − 0.488 ± 0.186 mg/dL;
p
= 0.011 (post hoc analysis). Significant reductions in serum phosphorus were observed in subjects aged ≥ 12 to ≤ 18 years (LS mean ± SE − 0.460 ± 0.195 mg/dL;
p
= 0.024) and subjects with serum phosphorus above age-related normal ranges at baseline (LS mean ± SE − 0.942 ± 0.246 mg/dL;
p
= 0.005). Similar proportions of subjects reported ≥ 1 TEAE in the SFOH (75.8%) and CaAc (73.7%) groups. Withdrawal due to TEAEs was more common with CaAc (31.6%) than with SFOH (18.2%).
Conclusions
SFOH effectively managed serum phosphorus in pediatric patients with a low pill burden and a safety profile consistent with that reported in adult patients.
: VPM1002BC is a modified mycobacterium Bacillus Calmette Guérin (BCG) for the treatment of non-muscle invasive bladder cancer (NMIBC). The genetic modifications are expected to result in better ...immunogenicity and less side effects. We report on patient safety and immunology of the first intravesical application of VPM1002BC in human.
: Six patients with BCG failure received a treatment of 6 weekly instillations with VPM1002BC. Patients were monitored for adverse events (AE), excretion of VPM1002BC and cytokines, respectively.
: No DLT (dose limiting toxicity) occurred during the DLT-period. No grade ≥3 AEs occurred. Excretion of VPM1002BC in the urine was limited to less than 24 hours. Plasma levels of TNFα significantly increased after treatment and blood-derived CD4+ T cells stimulated with PPD demonstrated significantly increased intracellular GM-CSF and IFN expression.
: The intravesical application of VPM1002BC is safe and well tolerated by patients and results in a potential Th1 weighted immune response.
The cytotoxicity of SN1-type alkylating agents such as N-methyl-N'-nitrosourea (MNU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), or the cancer chemotherapeutics temozolomide, dacarbazine and ...streptozotocin has been ascribed to the persistence of O 6-methylguanine (meG) in genomic DNA. One hypothesis posits that meG toxicity is caused by futile attempts of the mismatch repair (MMR) system to process meG/C or meG/T mispairs arising during replication, while an alternative proposal suggests that the latter lesions activate DNA damage signaling, cell cycle arrest and apoptosis directly. Attempts to elucidate the molecular mechanism of meG-induced cell killing in vivo have been hampered by the fact that the above reagents induce several types of modifications in genomic DNA, which are processed by different repair pathways. In contrast, defined substrates studied in vitro did not undergo replication. We set out to re-examine this phenomenon in replication-competent Xenopus laevis egg extracts, using either phagemid substrates containing a single meG residue, or methylated sperm chromatin. Our findings provide further support for the futile cycling hypothesis.
Plasmids containing a site-specific DNA interstrand cross-link (ICL) are invaluable tools for the investigation of ICL repair pathways at the biochemical and cellular level. We describe a procedure ...for preparation of plasmid DNA substrates containing a single ICL at a specific site. The procedure is versatile, leads to reliable yields of pure DNA substrate, and is suitable for the incorporation of virtually any type of DNA lesion into plasmids.
Introduction: This study aimed to investigate the efficacy and safety of sucroferric oxyhydroxide (SFOH) versus sevelamer carbonate in controlling serum phosphorus (sP) in adult Chinese dialysis ...patients with hyperphosphataemia (sP >1.78 mmol/L). Methods: Open-label, randomised (1:1), active-controlled, parallel group, multicentre, phase III study of SFOH and sevelamer at starting doses corresponding to 1,500 mg iron/day and 2.4 g/day, respectively, with 8-week dose titration and 4-week maintenance (NCT03644264). Primary endpoint was non-inferiority analysis of change in sP from baseline to week 12. Secondary endpoints included sP over time and safety. Results: 415 patients were screened; 286 were enrolled and randomised (142 and 144 to SFOH and sevelamer, respectively). Mean (SD) baseline sP: 2.38 (0.57) and 2.38 (0.52) mmol/L, respectively. Mean (SD) change in sP from baseline to week 12: – 0.71 (0.60) versus −0.63 (0.52) mmol/L, respectively; difference (sevelamer minus SFOH) in least squares means (95% CI): 0.08 mmol/L (−0.02, 0.18) with the lower limit of 95% CI above the non-inferiority margin of −0.34 mmol/L. The SFOH group achieved target sP (1.13–1.78 mmol/L) earlier than the sevelamer group (56.5% vs. 32.8% at week 4) and with a lower pill burden (mean 3.7 vs. 9.1 tablets/day over 4 weeks of maintenance, respectively). Safety and tolerability of SFOH was consistent with previous studies, and no new safety signals were observed. Conclusion: SFOH effectively reduced sP from baseline and was non-inferior to sevelamer after 12 weeks of treatment but had a lower pill burden in Chinese dialysis patients with hyperphosphataemia; SFOH benefit-risk profile is favourable in Chinese patients.
DNA interstrand crosslinks (ICLs), inhibit DNA metabolism by covalently linking two strands of DNA and are formed by antitumor agents such as cisplatin and nitrogen mustards. Multiple complex repair ...pathways of ICLs exist in humans that share translesion synthesis (TLS) past a partially processed ICL as a common step. We have generated site-specific major groove ICLs and studied the ability of Y-family polymerases and Pol ζ to bypass ICLs that induce different degrees of distortion in DNA. Two main factors influenced the efficiency of ICL bypass: the length of the dsDNA flanking the ICL and the length of the crosslink bridging two bases. Our study shows that ICLs can readily be bypassed by TLS polymerases if they are appropriately processed and that the structure of the ICL influences which polymerases are able to read through it.
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