Search Results (37)

Increased expression of branched-chain amino acid (BCAA) transaminase 1 (BCAT1) often correlates with tumor aggressiveness and drug resistance in cancer. We have recently reported that BCAT1 was overexpressed in a subgroup of T-cell acute lymphoblastic (T-ALL) samples, especially those with NOTCH1 activating mutations. Interestingly, BCAT1-depleted cells showed pronounced sensitivity to DNA-damaging agents such as etoposide; however, how BCAT1 regulates this sensitivity remains uncertain. Here, we provide further clues on its chemo-sensitizing effect. Indeed, BCAT1 protein regulates the non-homologous end joining (c-NHEJ) DNA repair pathway by physically associating with the KU70/KU80 heterodimer. BCAT1 inhibition during active repair of DNA double-strand breaks (DSBs) led to increased KU70/KU80 acetylation and impaired c-NHEJ repair, a dramatic increase in DSBs, and ultimately cell death. Our results suggest that, in T-ALL, BCAT1 possesses non-metabolic functions that confer a drug resistance mechanism and that targeting BCAT1 activity presents a novel strategy to improve chemotherapy response in T-ALL patients. Full article

Targeting CDC20 can enhance the radiosensitivity of tumor cells, but the function and mechanism of CDC20 on DNA damage repair response remains vague. To examine that issue, tumor cell lines, including KYSE200, KYSE450, and HCT116, were utilized to detect the expression, function, and underlying mechanism of CDC20 in radio-chemoresistance. Western blot and immunofluorescence staining were employed to confirm CDC20 expression and location, and radiation could upregulate the expression of CDC20 in the cell nucleus. The homologous recombination (HR) and non-homologous end joining (NHEJ) reporter gene systems were utilized to explore the impact of CDC20 on DNA damage repair, indicating that CDC20 could promote HR repair and radio/chemo-resistance. In the early stages of DNA damage, CDC20 stabilizes the RPA1 protein through protein-protein interactions, activating the ATR-mediated signaling cascade, thereby aiding in genomic repair. In the later stages, CDC20 assists in the subsequent steps of damage repair by the ubiquitin-mediated degradation of RPA1. CCK-8 and colony formation assay were used to detect the function of CDC20 in cell vitality and proliferation, and targeting CDC20 can exacerbate the increase in DNA damage levels caused by cisplatin or etoposide. A tumor xenograft model was conducted in BALB/c-nu/nu mice to confirm the function of CDC20 in vivo, confirming the in vitro results. In conclusion, this study provides further validation of the potential clinical significance of CDC20 as a strategy to overcome radio-chemoresistance via uncovering a novel role of CDC20 in regulating RPA1 during DNA damage repair. Full article

Carotenoids are valuable pigments naturally occurring in all photosynthetic plants and microalgae as well as in selected fungi, bacteria, and archaea. Green microalgae developed a complex carotenoid profile suitable for efficient light harvesting and light protection and harbor great capacity for carotenoid production through the substantial power of the endogenous 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Previous works established successful genome editing and induced significant changes in the cellular carotenoid content in Chlamydomonas reinhardtii. This study employs a tailored carotenoid pathway for engineered bioproduction of the valuable ketocarotenoid astaxanthin. Functional knockout of lycopene ε-cyclase (LCYE) and non-homologous end joining (NHEJ)-based integration of donor DNA at the target site inhibit the accumulation of α-carotene and consequently lutein and loroxanthin, abundant carotenoids in C. reinhardtii without changes in cellular fitness. PCR-based screening indicated that 4 of 96 regenerated candidate lines carried (partial) integrations of donor DNA and increased ß-carotene as well as derived carotenoid contents. Iterative overexpression of CrBKT, PacrtB, and CrCHYB resulted in a 2.3-fold increase in astaxanthin accumulation in mutant ΔLCYE#3 (1.8 mg/L) compared to the parental strain UVM4, which demonstrates the potential of genome editing for the design of a green cell factory for astaxanthin bioproduction. Full article

Heat shock proteins (HSPs) are highly expressed in cancer cells and represent a promising target in anti-cancer therapy. In this study, we investigated for the first time the expression of high-molecular-weight HSP110, belonging to the HSP70 family of proteins, in Primary Effusion Lymphoma (PEL) and explored its role in their survival. This is a rare lymphoma associated with KSHV, for which an effective therapy remains to be discovered. The results obtained from this study suggest that targeting HSP110 could be a very promising strategy against PEL, as its silencing induced lysosomal membrane permeabilization, the cleavage of BID, caspase 8 activation, downregulated c-Myc, and strongly impaired the HR and NHEJ DNA repair pathways, leading to apoptotic cell death. Since chemical inhibitors of this HSP are not commercially available yet, this study encourages a more intense search in this direction in order to discover a new potential treatment that is effective against this and likely other B cell lymphomas that are known to overexpress HSP110. Full article

Radiation therapy is an essential component of present-day cancer management, utilizing ionizing radiation (IR) of different modalities to mitigate cancer progression. IR functions by generating ionizations in cells that induce a plethora of DNA lesions. The most detrimental among them are the DNA double strand breaks (DSBs). In the course of evolution, cells of higher eukaryotes have evolved four major DSB repair pathways: classical non-homologous end joining (c-NHEJ), homologous recombination (HR), alternative end-joining (alt-EJ), and single strand annealing (SSA). These mechanistically distinct repair pathways have different cell cycle- and homology-dependencies but, surprisingly, they operate with widely different fidelity and kinetics and therefore contribute unequally to cell survival and genome maintenance. It is therefore reasonable to anticipate tight regulation and coordination in the engagement of these DSB repair pathway to achieve the maximum possible genomic stability. Here, we provide a state-of-the-art review of the accumulated knowledge on the molecular mechanisms underpinning these repair pathways, with emphasis on c-NHEJ and HR. We discuss factors and processes that have recently come to the fore. We outline mechanisms steering DSB repair pathway choice throughout the cell cycle, and highlight the critical role of DNA end resection in this process. Most importantly, however, we point out the strong preference for HR at low DSB loads, and thus low IR doses, for cells irradiated in the G₂-phase of the cell cycle. We further explore the molecular underpinnings of transitions from high fidelity to low fidelity error-prone repair pathways and analyze the coordination and consequences of this transition on cell viability and genomic stability. Finally, we elaborate on how these advances may help in the development of improved cancer treatment protocols in radiation therapy. Full article

Cisplatin (CDDP) is the cornerstone of standard treatment for ovarian cancer. However, the resistance of ovarian cancer cells to CDDP leads to an inevitable recurrence. One of the strategies to overcome resistance to CDDP is the combined treatment of ovarian cancer with CDDP and etoposide (VP-16), although this strategy is not always effective. This article presents a new approach to sensitize CDDP-resistant human ovarian carcinoma cells to combined treatment with CDDP and VP-16. To replicate the tumor conditions of cancers, we performed analysis under hypoxia conditions. Since CDDP and VP-16 induce DNA double-strand breaks (DSB), we introduce DSB repair inhibitors to the treatment scheme. We used novel HRR and NHEJ inhibitors: YU238259 inhibits the HRR pathway, and DDRI-18 and A12B4C3 act as NHEJ inhibitors. All inhibitors enhanced the therapeutic effect of the CDDP/VP-16 treatment scheme and allowed a decrease in the effective dose of CDDP/VP16. Inhibition of HRR or NHEJ decreased survival and increased DNA damage level, increased the amount of γ-H2AX foci, and caused an increase in apoptotic fraction after treatment with CDDP/VP16. Furthermore, delayed repair of DSBs was detected in HRR- or NHEJ-inhibited cells. This favorable outcome was altered under hypoxia, during which alternation at the transcriptome level of the transcriptome in cells cultured under hypoxia compared to aerobic conditions. These changes suggest that it is likely that other than classical DSB repair systems are activated in cancer cells during hypoxia. Our study suggests that the introduction of DSB inhibitors may improve the effectiveness of commonly used ovarian cancer treatment, and HRR, as well as NHEJ, is an attractive therapeutic target for overcoming the resistance to CDDP resistance of ovarian cancer cells. However, a hypoxia-mediated decrease in response to our scheme of treatment was observed. Full article

To repair ionizing radiation (IR)-induced double strand breaks (DSBs), mammalian cells primarily use canonical non-homologous end-joining (cNHEJ), the homologous recombination (HR) pathway, and the alternative non-homologous end-joining (aEJ) as a backup. These pathways function either compensatively or competitively. High linear energy transfer (LET) compared to low-LET IR kills more cells at the same doses by inhibiting only cNHEJ, but not HR or aEJ. The mechanism remains unclear. The activation of each repair pathway requires the binding of different proteins to DNA fragments of varying lengths. We previously observed an increased generation of small DNA fragments (≤40 bp) in cells following high-LET IR compared to low-LET IR, suggesting that short DNA fragments were one of the major factors interfering with cNHEJ. To provide direct evidence, here we compare the efficiencies of cNHEJ, HR, or aEJ in repairing DSBs containing 30- or 60-bp fragments in vitro and in cells. We show that only cNHEJ but not HR or a-EJ was inefficient for repairing DSBs with 30-bp fragments compared to 60-bp ones, which strongly supports our hypothesis. These results not only enhance our understanding of the DSB repair pathway choice but also hold potential benefits for protection against high-LET IR-induced damage or improving high-LET radiotherapy. Full article

Alt-EJ is an error-prone DNA double-strand break (DSBs) repair pathway coming to the fore when first-line repair pathways, c-NHEJ and HR, are defective or fail. It is thought to benefit from DNA end-resection—a process whereby 3′ single-stranded DNA-tails are generated—initiated by the CtIP/MRE11-RAD50-NBS1 (MRN) complex and extended by EXO1 or the BLM/DNA2 complex. The connection between alt-EJ and resection remains incompletely characterized. Alt-EJ depends on the cell cycle phase, is at maximum in G₂-phase, substantially reduced in G₁-phase and almost undetectable in quiescent, G₀-phase cells. The mechanism underpinning this regulation remains uncharacterized. Here, we compare alt-EJ in G₁- and G₀-phase cells exposed to ionizing radiation (IR) and identify CtIP-dependent resection as the key regulator. Low levels of CtIP in G₁-phase cells allow modest resection and alt-EJ, as compared to G₂-phase cells. Strikingly, CtIP is undetectable in G₀-phase cells owing to APC/C-mediated degradation. The suppression of CtIP degradation with bortezomib or CDH1-depletion rescues CtIP and alt-EJ in G₀-phase cells. CtIP activation in G₀-phase cells also requires CDK-dependent phosphorylation by any available CDK but is restricted to CDK4/6 at the early stages of the normal cell cycle. We suggest that suppression of mutagenic alt-EJ in G₀-phase is a mechanism by which cells of higher eukaryotes maintain genomic stability in a large fraction of non-cycling cells in their organisms. Full article

Extensive research for the last two decades has significantly contributed to understanding the roles of lamins in the maintenance of nuclear architecture and genome organization which is drastically modified in neoplasia. It must be emphasized that alteration in lamin A/C expression and distribution is a consistent event during tumorigenesis of almost all tissues of human bodies. One of the important signatures of a cancer cell is its inability to repair DNA damage which befalls several genomic events that transform the cells to be sensitive to chemotherapeutic agents. This genomic and chromosomal instability is the most common feature found in cases of high-grade ovarian serous carcinoma. Here, we report elevated levels of lamins in OVCAR3 cells (high-grade ovarian serous carcinoma cell line) in comparison to IOSE (immortalised ovarian surface epithelial cells) and, consequently, altered damage repair machinery in OVCAR3. We have analysed the changes in global gene expression as a sequel to DNA damage induced by etoposide in ovarian carcinoma where lamin A is particularly elevated in expression and reported some differentially expressed genes associated with pathways conferring cellular proliferation and chemoresistance. We hereby establish the role of elevated lamin A in neoplastic transformation in the context of high-grade ovarian serous cancer through a combination of HR and NHEJ mechanisms. Full article

Recent reports suggested a role for microtubules in double-strand-DNA break repair. We herein investigated the role of the microtubule-associated protein Tau in radio- and chemotherapy. Noticeably, a lowered expression of Tau in breast cancer cell lines resulted in a significant decrease in mouse-xenograft breast tumor volume after doxorubicin or X-ray treatments. Furthermore, the knockdown of Tau impaired the classical nonhomologous end-joining pathway and led to an improved cellular response to both bleomycin and X-rays. Investigating the mechanism of Tau’s protective effect, we found that one of the main mediators of response to double-stranded breaks in DNA, the tumor suppressor p53-binding protein 1 (53BP1), is sequestered in the cytoplasm as a consequence of Tau downregulation. We demonstrated that Tau allows 53BP1 to translocate to the nucleus in response to DNA damage by chaperoning microtubule protein trafficking. Moreover, Tau knockdown chemo-sensitized cancer cells to drugs forming DNA adducts, such as cisplatin and oxaliplatin, and further suggested a general role of Tau in regulating the nuclear trafficking of DNA repair proteins. Altogether, these results suggest that Tau expression in cancer cells may be of interest as a molecular marker for response to DNA-damaging anti-cancer agents. Clinically targeting Tau could sensitize tumors to DNA-damaging treatments. Full article

DNA double-strand breaks (DSBs) caused by platinum drugs are dangerous lesions that kill cancer cells in chemotherapy. Repair of DSB by homologous recombination (HR) and nonhomologous end joining (NHEJ) is frequently associated with platinum resistance in ovarian cancer. While the role of the HR pathway and HR-targeting strategy in platinum resistance is well studied, dissecting and targeting NHEJ machinery to overcome platinum resistance in ovarian cancer remain largely unexplored. Here, through an NHEJ pathway-focused gene RNAi screen, we found that the knockdown of XRCC4 significantly sensitized cisplatin treatment in the platinum-resistant ovarian cancer cell lines. Moreover, upregulation of XRCC4 is observed in a panel of platinum-resistant cell lines relative to the parental cell lines, as well as in ovarian cancer patients with poor progression-free survival. Mechanistically, the increased sensitivity to cisplatin upon XRCC4 knockdown was caused by accumulated DNA damage. In cisplatin-resistant ovarian cancer, the JNK-cJUN complex, activated by cisplatin, translocated into the nucleus and promoted the transcription of XRCC4 to confer cisplatin resistance. Knockdown of XRCC4 or treatment of the JNK inhibitor led to the attenuation of cisplatin-resistant tumor growth in the xenograft mouse models. These data suggest targeting XRCC4 is a potential strategy for ovarian cisplatin resistance in ovarian cancer. Full article

Trichophyton rubrum is the most prevalent causative agent responsible for 80–90% of all known superficial fungal infections in humans, worldwide. Limited available methods for genetic manipulations have been one of the major bottlenecks in understanding relevant molecular mechanisms of disease pathogenesis in T. rubrum. Here, a dual-plasmid-based CRISPR/Cas9 strategy to edit pH regulatory transcription factor, pacC, of a clinical isolate of T. rubrum by non-homologous end joining (NHEJ) repair is presented. A cas9–eGFP fusion that aids pre-screening of primary transformants through detection of GFP fluorescence is expressed from one plasmid while target-specific sgRNA from the other brings about mutagenesis of pacC with an overall efficiency of 33.8–37.3%. The mutants had reduced transcript levels of pacC at both acidic and alkaline pH with several morphological abnormalities. We believe this dual-plasmid-based CRISPR/Cas9 strategy will aid functional genomics studies, especially in non-lab-adapted clinical strains of T. rubrum. Full article

BMN673 is a relatively new PARP inhibitor (PARPi) that exhibits superior efficacy in vitro compared to olaparib and other clinically relevant PARPi. BMN673, similar to most clinical PARPi, inhibits the catalytic activities of PARP-1 and PARP-2 and shows impressive anticancer potential as monotherapy in several pre-clinical and clinical studies. Tumor resistance to PARPi poses a significant challenge in the clinic. Thus, combining PARPi with other treatment modalities, such as radiotherapy (RT), is being actively pursued to overcome such resistance. However, the modest to intermediate radiosensitization exerted by olaparib, rucaparib, and veliparib, limits the rationale and the scope of such combinations. The recently reported strong radiosensitizing potential of BMN673 forecasts a paradigm shift on this front. Evidence accumulates that BMN673 may radiosensitize via unique mechanisms causing profound shifts in the balance among DNA double-strand break (DSB) repair pathways. According to one of the emerging models, BMN673 strongly inhibits classical non-homologous end-joining (c-NHEJ) and increases reciprocally and profoundly DSB end-resection, enhancing error-prone DSB processing that robustly potentiates cell killing. In this review, we outline and summarize the work that helped to formulate this model of BMN673 action on DSB repair, analyze the causes of radiosensitization and discuss its potential as a radiosensitizer in the clinic. Finally, we highlight strategies for combining BMN673 with other inhibitors of DNA damage response for further improvements. Full article

Microhomology-mediated end joining (MMEJ) is a highly mutagenic pathway to repair double-strand breaks (DSBs). MMEJ was thought to be a backup pathway of homologous recombination (HR) and canonical nonhomologous end joining (C-NHEJ). However, it attracts more attention in cancer research due to its special function of microhomology in many different aspects of cancer. In particular, it is initiated with DNA end resection and upregulated in homologous recombination-deficient cancers. In this review, I summarize the following: (1) the recent findings and contributions of MMEJ to genome instability, including phenotypes relevant to MMEJ; (2) the interaction between MMEJ and other DNA repair pathways; (3) the proposed mechanistic model of MMEJ in DNA DSB repair and a new connection with microhomology-mediated break-induced replication (MMBIR); and (4) the potential clinical application by targeting MMEJ based on synthetic lethality for cancer therapy. Full article

PTEN has been implicated in the repair of DNA double-strand breaks (DSBs), particularly through homologous recombination (HR). However, other data fail to demonstrate a direct role of PTEN in DSB repair. Therefore, here, we report experiments designed to further investigate the role of PTEN in DSB repair. We emphasize the consequences of PTEN loss in the engagement of the four DSB repair pathways—classical non-homologous end-joining (c-NHEJ), HR, alternative end-joining (alt-EJ) and single strand annealing (SSA)—and analyze the resulting dynamic changes in their utilization. We quantitate the effect of PTEN knockdown on cell radiosensitivity to killing, as well as checkpoint responses in normal and tumor cell lines. We find that disruption of PTEN sensitizes cells to ionizing radiation (IR). This radiosensitization is associated with a reduction in RAD51 expression that compromises HR and causes a marked increase in SSA engagement, an error-prone DSB repair pathway, while alt-EJ and c-NHEJ remain unchanged after PTEN knockdown. The G₂-checkpoint is partially suppressed after PTEN knockdown, corroborating the associated HR suppression. Notably, PTEN deficiency radiosensitizes cells to PARP inhibitors, Olaparib and BMN673. The results show the crucial role of PTEN in DSB repair and show a molecular link between PTEN and HR through the regulation of RAD51 expression. The expected benefit from combination treatment with Olaparib or BMN673 and IR shows that PTEN status may also be useful for patient stratification in clinical treatment protocols combining IR with PARP inhibitors. Full article