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Abstract BORIS, or CTCFL, the so called Brother of the Regulator of Imprinted Sites because of the extensive homology in the central DNA binding region of the protein to the related regulator, CTCF, is expressed in early gametogenesis and in multiple cancers but not in differentiated somatic cells. Thus it is a member of the cancer testes antigen group (CTAs). Since BORIS and CTCF target common DNA binding sites, these proteins function on two levels, the first level is their regulation via the methylation context of the DNA target site and the second level is their distinct and different epigenetic associations due to differences in the non-homologous termini of the proteins. The regulation on both of these levels is extensive and complex and the sphere of influence of each of these proteins is associated with vastly different cellular signaling processes. On the level of gene expression, BORIS has three known promoters and multiple spliced mRNAs which adds another level of complexity to this intriguing regulator. BORIS expression is observed in the majority of cancer tissues and cell lines analyzed up to today. The expression profile and essential role of BORIS in cancer make this molecule very attractive target for cancer immunotherapy. This review summarizes what is known about BORIS regarding its expression, structure, and function and then presents some theoretical considerations with respect to its genome wide influence and its potential for use as a vaccine for cancer immunotherapy.

Objectives: Dental pulp stem cells (DPSCs) were shown to play an important role in regenerative medicine including reconstruction of various bone lesions. This study determined the impact of acemannan, an extracted product from Aloe vera , on in vitro proliferation of DPSCs and in vivo healing of mandibular defects in rabbits., Methods: DPSCs were isolated and characterized. The growth kinetics of cells exposed to acemannan (8 mg/mL) and Hank's balanced salt solution (HBSS) were compared in vitro . Fifteen male rabbits were divided into 3 groups. Five animals were left as control group without any therapeutic intervention. Five rabbits were considered as experimental group 1 and received 20 µL of a cell suspension containing 10 6 DPSCs in the bone defect. Another 5 rabbits were regarded as experimental group 2 and were injected in the bone defect with 20 µL of a cell suspension containing 10 6 DPSCs treated with acemannan for 24 h. After 60 days, the animals were assessed by radiography and histologically., Results: The mesenchymal properties of DPSCs were confirmed. Population doubling time (PDT) of DPSCs treated with acemannan (29.8 h) was significantly shorter than cells were just exposed to HBSS (45.9 h). DPSCs together with acemannan could significantly accelerate the healing process and osteogenesis in mandibular defects., Conclusions: As DPSCS showed an increased proliferation when treated with acemannan and accelerated the healing process in mandibular defects, these findings can open a new avenue in dentistry regenerative medicine when remedies of bone defects are targeted., Competing Interests: None., (AJSC Copyright © 2024.)

Infertility is a common clinical condition and about half of the major causes are due to male-related infertility. Pathogenesis of this abnormality is generally undefined; so establishing a proper treatment option is relatively uncertain. In recent years, several evidences demonstrated that mesenchymal stem cells (MSCs) can be a hope for innovative and efficient treatment of male infertility. This study reviews possible applications of MSCs in the restoration of spermatogenesis in male infertility of both humans and animals to suggest new avenues for future clinical practices. Articles published in "PubMed" and "Google Scholar" from January 1, 2000, to August 1, 2023, were investigated by searching items of "mesenchymal stem cells", "cell therapy", "cell transplantation", and, "regenerative medicine" keywords, in addition to the "urology", "andrology", "reproductive medicine", "male infertility", "azoospermia", and "spermatogenesis". The results obtained from the transplantation of MSCs in the treatment of male infertility seemed encouraging and they revealed the safety and efficacy of these cells to recover spermatogenesis; eventhough further stem cell research is still required before recruiting clinical application of MSCs in the treatment of human male infertility. Undertaking more well-defined, standardized, and reproducible protocols and enrolling larger sample sizes during a longer follow-up period can benefit the relevance of MSC transplantation in the restoration of spermatogenesis and treatment of male infertility. It seems that developing and utilizing stem cell transplantations, exosomes, scaffold delivery systems, and three dimensional (3D) culture methods may open a new window to getting more benefits from cell therapy in the treatment of men infertility.

Background: To stop the spread of the COVID-19 disease, it is crucial to create molecular tools to investigate and diagnose COVID-19. Current efforts focus on developing specific neutralizing monoclonal antibodies (NmAbs) elicited against the receptor-binding domain (RBD)., Methods: In the present study, recombinant RBD (rRBD) protein was produced in E. coli, followed by immunizing mice with purified rRBD. ELISA was applied to screen the hybridomas for positive reactivity with rRBD protein. The linear and conformational epitopes of the mAbs were subsequently identified using western blot. Finally, the reactivity, affinity, and neutralization activity of the purified mAbs were evaluated using ELISA., Results: All mAbs exhibited similar reactivity trends towards both eukaryotic RBD and prokaryotic rRBD in ELISA. Among them, 2E7-D2 and 2B4-G8 mAbs demonstrated higher reactivity than other mAbs. Additionally, in western blot assays, these two mAbs could detect reducing and non-reducing rRBD, indicating recognition of linear epitopes. Notably, five mAbs effectively blocked rRBD- angiotensin-converting enzyme 2 (ACE2) interaction, while two high-affinity mAbs exhibited potent neutralizing activity against eukaryotic RBD., Conclusion: In the current study, we generated and characterized new RBD-specific mAbs using the hybridoma technique that recognized linear and conformational epitopes in RBD with neutralization potency. Our mAbs are novel candidates for diagnosing and treating SARS-CoV-2., (© 2024. The Author(s).)

Background: Cancer stem cells (CSCs), a rare sub-fraction of tumor cells, with the capability of self-renewal and strong oncogenicity are tightly responsible for chemo and radio resistance and tumor metastasis in colorectal cancer. Hence, CSCs targeting would improve the efficacy of therapeutic strategies and clinical outcomes., Methods: Here, using three-dimensional CSC spheroids and syngeneic mice model, we evaluated the cancer preventive impact of CSCs-based vaccination. CSCs enrichment was performed via colonosphere formation from CT-26 cell line and CT-26-derived tumor biopsy and characterized by confirming high expression of key stemness genes (OCT4, SOX2, and NANOG) and CSC-related surface biomarkers (CD166, DCLK1, and CD133) via real-time PCR and flow cytometry, respectively. Then, the stemness phenotype and self-renewal in CSC-enriched spheroids were further confirmed by showing serial sphere formation capacity, clonogenicity potential, and enhanced in vivo tumorigenic capacity compared to their parental counterparts. CSCs lysates were used as vaccines in prophylactic settings compared to the parental cell lysate and PBS groups., Result: Immunization of syngeneic mice with CSCs lysates was effective in the prevention of tumor establishment and significantly decreased tumor growth rate accompanied by an improvement in survival rate in tumor-bearing mice compared to groups subjected to parental cells lysate and PBS. These results, for the first time, showed that mice immunized with cell lysate from tumor biopsy-derived spheroids are resistant to tumor induction. Immunofluorescence staining indicated that only the serum antibodies from CSC-vaccinated mice reacted with colonospheres., Conclusions: These findings represent CSCs lysate-based vaccination as a potential approach to hampering immunotherapy failure of colorectal cancer which along with other traditional therapies may effectively apply to prevent the establishment of aggressive tumors harboring stemness features., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

To assess in vitro and in vivo tracking of iron oxide labeled stem cells transfected by lipofectamine using magnetic resonance imaging (MRI), rat dental pulp stem cells (DPSCs) were characterized, labeled with iron oxide nanoparticles, and then transfected with lipofectamine to facilitate the internalization of these nanoparticles. Cell proliferation, viability, differentiation, and apoptosis were investigated. Prussian blue staining and MRI were used to trace transfected labeled cells. DPSCs were a morphologically spindle shape, adherent to culture plates, and positive for adipogenic and osteogenic inductions. They expressed CD73 and CD90 markers and lacked CD34 and CD45. Iron oxide labeling and transfection with lipofectamine in DPSCs had no toxic impact on viability, proliferation, and differentiation, and did not induce any apoptosis. In vitro and in vivo internalization of iron oxide nanoparticles within DPSCs were confirmed by Prussian blue staining and MRI tracking. Prussian blue staining and MRI tracking in the absence of any toxic effects on cell viability, proliferation, differentiation, and apoptosis were safe and accurate to track DPSCs labeled with iron oxide and transfected with lipofectamine. MRI can be a useful imaging modality when treatment outcome is targeted.

Background: In vivo cell tracking after transplantation in regenerative medicine remains an unmet challenge and limits current understanding of the wound healing mechanism through cell-based therapies. This study investigated tracking of human Wharton's jelly stem cells (hWJSCs) seeded onto an acellular dermal matrix (ADM) and labeled with superparamagnetic iron oxide nanoparticles (SPIONs) by magnetic resonance imaging (MRI) in burn injury., Method: The hWJSCs were characterized and assessed for growth kinetics. A total of 30 rats were enrolled in three equal groups. Group 1 underwent scald burn injury left without treatment, the group 2 was treated by an ADM that was prepared from cosmetic surgery skin samples and the group 3 received hWJSCs labeled with SPIONs seeded onto an ADM. Tensile strength was evaluated before and after interventions, real time PCR assessed apoptosis, and Prussian blue staining, scanning electron microscopy (SEM) and MRI were used for the tracking of labeled cells., Results: The hWJSCs exhibited mesenchymal stem cell properties. Population doubling time was 40.1 hours. SPIONs did not show any toxic effect. The hWJSCs seeded onto an ADM decreased Bax and increased Bcl-2 gene expression. Internalization of SPIONs within hWJSCs was confirmed by Prussian blue staining, SEM and MRI until day 21. There was a significant difference between the Young's moduli of normal skin and the group receiving hWJSCs seeded onto an ADM. Histological observations and SEM imaging confirmed that MRI is an accurate method to track SPION-labeled hWJSCs in vivo ., Conclusions: This study showed that SPION labeling coupled with MRI can be used to further understand the fate of stem cells after transplantation in a burn model., (© The Author(s) 2022. Published by Oxford University Press.)

Aims: the main purpose of this study was to identify new selective antitumor agents., Main Methods: several hydrazonoyl chlorides (HCs) were synthesized and human tumor cell line viability was determined using the MTT assay. Tumor development was assessed using Ehrlich ascites carcinoma (EAC)-bearing mice., Key Findings: our results showed that 2-oxo-N-phenyl-2-(phenylamino)acetohydrazonoyl chloride (compound 4; CPD 4) and 2-oxo-2-(phenylamino)-N-(p-tolyl)acetohydrazonoyl chloride (CPD 5) were the most cytotoxic HCs to human cervical tumor HeLa (IC 50 : 20 and 25 μM for CPD 4 and 5 respectively), breast MCF7 (IC 50 : 29 and 34 μM for CPD 4 and 5 respectively) and colon HCT116 cancer cells (IC 50 : 26 and 25 μM for CPD 4 and 5 respectively) with the least cytotoxicity to human non-tumor CCD-18Co colon fibroblasts as well as murine splenocytes. The active compounds significantly inhibited colony formation as well as tumor development in EAC-bearing mice. We also observed that PTEN-deficient cells displayed greater sensitivity than cells expressing wild type PTEN. At the molecular level, comet and cell cycle analyses indicated that the active compounds generate DNA damage. In light of the PTEN-dependent sensitivity and genomic instability we examined the influence of HCs on the DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) and the PI3K/AKT/mTOR pathway, which are each known to be synthetic lethal with PTEN. We found that both PNKP and the PI3K/AKT/mTOR pathway to be adversely affected by the HCs, which may partially account for their toxicity., Significance: hydrazonoyl chlorides can be considered as hit compounds for the development of new antitumor agents., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Inhibition of DNA repair enzymes is an attractive target for increasing the efficacy of DNA damaging chemotherapies. The ERCC1-XPF heterodimer is a key endonuclease in numerous single and double strand break repair processes, and inhibition of the heterodimerization has previously been shown to sensitize cancer cells to DNA damage. In this work, the previously reported ERCC1-XPF inhibitor 4 was used as the starting point for an in silico study of further modifications of the piperazine side-chain. A selection of the best scoring hits from the in silico screen were synthesized using a late stage functionalization strategy which should allow for further iterations of this class of inhibitors to be readily synthesized. Of the synthesized compounds, compound 6 performed the best in the in vitro fluorescence based endonuclease assay. The success of compound 6 in inhibiting ERCC1-XPF endonuclease activity in vitro translated well to cell-based assays investigating the inhibition of nucleotide excision repair and disruption of heterodimerization. Subsequently compound 6 was shown to sensitize HCT-116 cancer cells to treatment with UVC, cyclophosphamide, and ionizing radiation. This work serves as an important step towards the synergistic use of DNA repair inhibitors with chemotherapeutic drugs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Weilbeer, Jay, Donnelly, Gentile, Karimi-Busheri, Yang, Mani, Yu, Elmenoufy, Barakat, Tuszynski, Weinfeld and West.)

Inhibition of the DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) increases the sensitivity of cancer cells to DNA damage by ionizing radiation (IR). We have developed a novel inhibitor of PNKP, i.e., A83B4C63, as a potential radio-sensitizer for the treatment of solid tumors. Systemic delivery of A83B4C63, however, may sensitize both cancer and normal cells to DNA damaging therapeutics. Preferential delivery of A83B4C63 to solid tumors by nanoparticles (NP) was proposed to reduce potential side effects of this PNKP inhibitor to normal tissue, particularly when combined with DNA damaging therapies. Here, we investigated the radio-sensitizing activity of A83B4C63 encapsulated in NPs (NP/A83) based on methoxy poly(ethylene oxide)- b -poly(α-benzyl carboxylate-ε-caprolactone) (mPEO- b -PBCL) or solubilized with the aid of Cremophor EL: Ethanol (CE/A83) in human HCT116 colorectal cancer (CRC) models. Levels of γ-H2AX were measured and the biodistribution of CE/A83 and NP/A83 administered intravenously was determined in subcutaneous HCT116 CRC xenografts. The radio-sensitization effect of A83B4C63 was measured following fractionated tumor irradiation using an image-guided Small Animal Radiation Research Platform (SARRP), with 24 h pre-administration of CE/A83 and NP/A83 to Luc + /HCT116 bearing mice. Therapeutic effects were analyzed by monitoring tumor growth and functional imaging using Positron Emission Tomography (PET) and [ 18 F]-fluoro-3 ' -deoxy-3 ' -L:-fluorothymidine ([ 18 F]FLT) as a radiotracer for cell proliferation. The results showed an increased persistence of DNA damage in cells treated with a combination of CE/A83 or NP/A83 and IR compared to those only exposed to IR. Significantly higher tumor growth delay in mice treated with a combination of IR and NP/A83 than those treated with IR plus CE/A83 was observed. [ 18 F]FLT PET displayed significant functional changes for tumor proliferation for the drug-loaded NP. This observation was attributed to the higher A83B4C63 levels in the tumors for NP/A83-treated mice compared to those treated with CE/A83. Overall, the results demonstrated a potential for A83B4C63-loaded NP as a novel radio-sensitizer for the treatment of CRC., Competing Interests: The authors declare the following competing financial interest(s): Material in this manuscript has been included in recent US patent applications. AL is Vice-President of Meros Polymers which has the license to mPEO-b-PBCL polymer used in this manuscript., (Copyright © 2021 Sadat, Wuest, Paiva, Munira, Sarrami, Sanaee, Yang, Paladino, Binkhathlan, Karimi-Busheri, Martin, Jirik, Murray, Gamper, Hall, Weinfeld and Lavasanifar.)

Otorhinolaryngology enrolls head and neck surgery in various tissues such as ear, nose, and throat (ENT) that govern different activities such as hearing, breathing, smelling, production of vocal sounds, the balance, deglutition, facial animation, air filtration and humidification, and articulation during speech, while absence of these functions can lead to high morbidity and even mortality. Conventional therapies for head and neck damaged tissues include grafts, transplants, and artificial materials, but grafts have limited availability and cause morbidity in the donor site. To improve these limitations, regenerative medicine, as a novel and rapidly growing field, has opened a new therapeutic window in otorhinolaryngology by using cell transplantation to target the healing and replacement of injured tissues. There is a high risk of rejection and tumor formation for transplantation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs); mesenchymal stem cells (MSCs) lack these drawbacks. They have easy expansion and antiapoptotic properties with a wide range of healing and aesthetic functions that make them a novel candidate in otorhinolaryngology for craniofacial defects and diseases and hold immense promise for bone tissue healing; even the tissue sources and types of MSCs, the method of cell introduction and their preparation quality can influence the final outcome in the injured tissue. In this review, we demonstrated the anti-inflammatory and immunomodulatory properties of MSCs, from different sources, to be safely used for cell-based therapies in otorhinolaryngology, while their achievements and challenges have been described too.

Phosphatase and TENsin homolog deleted on chromosome 10 (PTEN) is a major tumor-suppressor protein that is lost in up to 75% of aggressive colorectal cancers (CRC). The co-depletion of PTEN and a DNA repair protein, polynucleotide kinase 3'-phosphatase (PNKP), has been shown to lead to synthetic lethality in several cancer types including CRC. This finding inspired the development of novel PNKP inhibitors as potential new drugs against PTEN-deficient CRC. Here, we report on the in vitro and in vivo evaluation of a nano-encapsulated potent, but poorly water-soluble lead PNKP inhibitor, A83B4C63, as a new targeted therapeutic for PTEN-deficient CRC. Our data confirmed the binding of A83B4C63, as free or nanoparticle (NP) formulation, to intracellular PNKP using the cellular thermal shift assay (CETSA), in vitro and in vivo. Dose escalating toxicity studies in healthy CD-1 mice, based on measurement of animal weight changes and biochemical blood analysis, revealed the safety of both free and nano-encapsulated A83B4C63, at assessed doses of ≤50 mg/kg. Nano-carriers of A83B4C63 effectively inhibited the growth of HCT116/PTEN -/- xenografts in NIH-III nude mice following intravenous (IV) administration, but not that of wild-type HCT116/PTEN +/+ xenografts. This was in contrast to IV administration of A83B4C63 solubilized with the aid of Cremophor EL: Ethanol (CE), which led to similar tumor growth to that of formulation excipients (NP or CE without drug) or 5% dextrose. This observation was attributed to the higher levels of A83B4C63 delivered to tumor tissue by its NP formulation. Our data provide evidence for the success of NPs of A83B4C63, as novel synthetically lethal nano-therapeutics in the treatment of PTEN-deficient CRC. This research also highlights the potential of successful application of nanomedicine in the drug development process., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Relapse and metastasis in colorectal cancer (CRC) are often attributed to cancer stem-like cells (CSCs), as small sub-population of tumor cells with ability of drug resistance. Accordingly, development of appropriate models to investigate CSCs biology and establishment of effective therapeutic strategies is warranted. Hence, we aimed to assess the capability of two widely used and important colorectal cancer cell lines, HT-29 and Caco-2, in generating spheroids and their detailed morphological and molecular characteristics., Methods: CRC spheroids were developed using hanging drop and forced floating in serum-free and non-attachment conditions and their morphological features were evaluated by scanning electron microscopy (SEM). Then, the potential of CSCs enrichment in spheroids was compared to their adherent counterparts by analysis of serial sphere formation capacity, real-time PCR of key stemness genes (KLF4, OCT4, SOX2, NANOG, C-MYC) and the expression of potential CRC-CSCs surface markers (CD166, CD44, and CD133) by flow cytometry. Finally, the expression level of some EMT-related (Vimentin, SNAIL1, TWIST1, N-cadherin, E-cadherin, ZEB1) and multi-drug resistant (ABCB1, ABCC1, ABCG2) genes was evaluated., Results: Although with different morphological features, both cell lines were formed CSCs-enriched spheroids, indicated by ability to serial sphere formation, significant up-regulation of stemness genes, SOX2, C-MYC, NANOG and OCT4 in HT-29 and SOX2, C-MYC and KLF4 in Caco-2 spheroids (p-value < 0.05) and increased expression of CRC-CSC markers compared to parental cells (p-value < 0.05). Additionally, HT-29 spheroids exhibited a significant higher expression of both ABCB1 and ABCG2 (p-value = 0.02). The significant up-regulation of promoting EMT genes, ZEB1, TWIST1, E-cadherin and SNAIL1 in HT-29 spheroids (p-value = 0.03), SNAIL1 and Vimentin in Caco-2 spheroids (p-value < 0.05) and N-cadherin down-regulation in both spheroids were observed., Conclusion: Enrichment of CSC-related features in HT-29 and Caco-2 (for the first time without applying special scaffold/biochemical) spheroids, suggests spheroid culture as robust, reproducible, simple and cost-effective model to imitate the complexity of in vivo tumors including self-renewal, drug resistance and invasion for in vitro research of CRC-CSCs.

Background: Methamphetamine (METH) may be administered for weight loss purposes and to understand the METH side-effects more in details, this study aimed at determining the effect of METH on changes in adipose tissue in experimental rats., Methods: Forty five male Wistar rats were randomly allocated to three equal groups. Group 1 was experimental receiving METH [0.4 mg/kg, subcutaneously (S/C), 0.6 mL/rat] for 3 weeks, group 2 was the sham group receiving normal saline (0.6 mL/rat, S/C) and the 3 rd group was the control receiving distilled water, identically. The elevated plus maze test was used to confirm cognitive impairment and distraction as anxiety and to verify addiction to METH by assessing the percent time spent in open arm (OAT), the percent time spent in closed arm (CAT), the percent time spent in central parts and head dipping over the side of the maze. Adipose tissue was assessed histologically 7, 14 and 21-days after interventions., Results: A significant increase in anxiety level, and histologically inflammation, degeneration and necrosis in adipose tissue were visible after METH use., Conclusion: METH use resulted in a significant inflammation and necrosis in adipose tissue denoting to the dangers of METH use, when recreationally targeted for weight loss purposes.

The worldwide epidemiology of inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), still shows an increasing trend in Asia and Iran. Despite an improvement in the treatment landscape focused on symptomatic control, long-term colectomies have not decreased over the last 10-year period. Thus, novel therapies are urgently needed in clinics to supplement the existing treatments. Mesenchymal stem cells (MSCs) are multipotent adult stem cells with immunosuppressive effects, targeting IBD as a new treatment strategy. They have recently received global attention for their use in cell transplantation due to their easy expansion and wide range of activities to be engrafted, and because they are home to the mucosa of the intestine. Moreover, MSCs are able to differentiate into epithelial and other cells that can directly promote repair in the mucosal damages in UC. It seems that there is a need to deepen our understanding to target MSCs as a promising treatment option for UC patients who are refractory to conventional therapies. Here, we overviewed the therapeutic effects of MSCs in UC and discussed the achievements and challenges in the cell transplantation of UC.

The structure-specific ERCC1-XPF endonuclease is essential for repairing bulky DNA lesions and helix distortions induced by UV radiation, which forms cyclobutane pyrimidine dimers (CPDs), or chemicals that crosslink DNA strands such as cyclophosphamide and platinum-based chemotherapeutic agents. Inhibition of the ERCC1-XPF endonuclease activity has been shown to sensitize cancer cells to these chemotherapeutic agents. In this study, we have conducted a structure activity relationship analysis based around the previously identified hit compound, 4-((6-chloro-2-methoxyacridin-9-yl)amino)-2-((4-methylpiperazin1-yl)methyl)phenol (F06), as a reference compound. Three different series of compounds have been rationally designed and successfully synthesized through various modifications on three different sites of F06 based on the corresponding suggestions of the previous pharmacophore model. The in vitro screening results revealed that 2-chloro-9-((3-((4-(2-(dimethylamino)ethyl)piperazin-1-yl)methyl)-4-hydroxyphenyl)amino)acridin-2-ol (B9) has a potent inhibitory effect on the ERCC1-XPF activity (IC 50  = 0.49 μM), showing 3-fold improvement in inhibition activity compared to F06. In addition, B9 not only displayed better binding affinity to the ERCC1-XPF complex but also had the capacity to potentiate the cytotoxicity effect of UV radiation and inhibiting the nucleotide excision repair, by the inhibition of removal of CPDs, and cyclophosphamide toxicity to colorectal cancer cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

The heterodimer of DNA excision repair protein ERCC-1 and DNA repair endonuclease XPF (ERCC1-XPF) is a 5'-3' structure-specific endonuclease essential for the nucleotide excision repair (NER) pathway, and it is also involved in other DNA repair pathways. In cancer cells, ERCC1-XPF plays a central role in repairing DNA damage induced by chemotherapeutics including platinum-based and cross-linking agents; thus, its inhibition is a promising strategy to enhance the effect of these therapies. In this study, we rationally modified the structure of F06, a small molecule inhibitor of the ERCC1-XPF interaction (Molecular Pharmacology, 84, 2013 and 12), to improve its binding to the target. We followed a multi-step computational approach to investigate potential modification sites of F06, rationally design and rank a library of analogues, and identify candidates for chemical synthesis and in vitro testing. Our top compound, B5, showed an improved half-maximum inhibitory concentration (IC 50 ) value of 0.49 µM for the inhibition of ERCC1-XPF endonuclease activit, and lays the foundation for further testing and optimization. Also, the computational approach reported here can be used to develop DNA repair inhibitors targeting the ERCC1-XPF complex., (© 2019 John Wiley & Sons A/S.)

The development of efficient and repeatable protocols for biobanking and prolonged storage of cancer stem cells (CSCs), with minimum alterations in biological function, is valuable and desired, particularly for retrospective analysis and clinical applications. In particular, data regarding the effect of cryopreservation on CSCs's functional features is scarce. In this regard, few studies have been shown that 3D spheroid structures, which enriched for CSCs, can keep their biological phenotype and genetic profiles. Here, for the first time, we present data on cryopreservation of CT-26 colonospheres, with the focus on essential stem cell-like properties after thawing. Tumor biopsy-derived colonospheres were frozen in standard freezing media (90% fetal bovine serum + 10% dimethyl sulfoxide) and stored in liquid nitrogen for 10 months. Then, cryopreservation effect on preservation of CSCs-related features was verified using real-time polymerase chain reaction for evaluation of stemness genes and flow cytometry for the putative colorectal CSC surface biomarkers. The self-renewal capacity of thawed spheres was also compared with their fresh counterparts using serial formation assay. Finally, tumorigenic capacity of both groups was evaluated in immunocompetence mouse model. Our data indicated that postthawed colonospheres had high viability without drastic alteration in biological and structural features and maintained self-renewal potential after sequential passages. Real-time analysis showed that both fresh and frozen colonospheres displayed similar expression pattern for key stemness genes: SOX2 and OCT4. Cryopreserved spheroids expressed CD133, CD166, and DCLK1 CSCs surface biomarkers at elevated levels when compared with parental as non-cryopreserved counterparts. Our electron scanning microscopy micrographs clearly demonstrated that postthawed colonospheres retain their integrity and cell surface morphology and characteristics. We also found that both fresh and frozen spheroids were equally tumorigenic. This study represented an effective strategy for reliable storage of intact CT-26 colonospheres; this can provide researchers with a functionally reliable repository of murine colorectal CSCs for their future CSCs projects., (© 2019 Wiley Periodicals, Inc.)

The ERCC1-XPF heterodimer is a 5'-3' structure-specific endonuclease, which plays an essential role in several DNA repair pathways in mammalian cells. ERCC1-XPF is primarily involved in the repair of chemically induced helix-distorting and bulky DNA lesions, such as cyclobutane pyrimidine dimers (CPDs), and DNA interstrand cross-links. Inhibition of ERCC1-XPF has been shown to potentiate cytotoxicity of platinum-based drugs and cyclophosphamide in cancer cells. In this study, the previously described ERCC1-XPF inhibitor 4-((6-chloro-2-methoxyacridin-9-yl)amino)-2-((4-methylpiperazin-1-yl)methyl)phenol (compound 1 ) was used as a reference compound. Following the outcome of docking-based virtual screening (VS), we synthesized seven novel derivatives of 1 that were identified in silico as being likely to have high binding affinity for the ERCC1-XPF heterodimerization interface by interacting with the XPF double helix-hairpin-helix (HhH2) domain. Two of the new compounds, 4-((6-chloro-2-methoxyacridin-9-yl)amino)-2-((4-cyclohexylpiperazin-1-yl)methyl)phenol (compound 3 ) and 4-((6-chloro-2-methoxyacridin-9-yl)amino)-2-((4-(2-(dimethylamino)ethyl) piperazin-1-yl) methyl) phenol (compound 4 ), were shown to be potent inhibitors of ERCC1-XPF activity in vitro. Compound 4 showed significant inhibition of the removal of CPDs in UV-irradiated cells and the capacity to sensitize colorectal cancer cells to UV radiation and cyclophosphamide.

Formation of atherosclerotic plaques is the major cause of coronary artery disease (CAD). Several lines of study have revealed the role of oxidative stress in CAD pathogenesis. In the present study the aim was to investigate the oxidative and antioxidative markers in CAD patients and a control population. The study sample comprised of acute coronary syndrome (ACS) patients, chronic CAD patients and healthy controls (n=30/group). Blood samples of patients and control subjects were collected to measure the concentrations of reduced glutathione (GSH), malondialdehyde (MDA) and the percentage of MDA release as well as the activity of erythrocyte glutathione peroxidase (GPx) and total antioxidant capacity (TAC) of plasma. All parameters were measured by spectrophotometric methods. Additionally, oxidant/antioxidant status was compared between CAD patients with single, double or triple-vessel stenosis and in comparison with controls. The results indicated a significant increase in MDA level and the percentage of MDA release (P<0.05), and a marked decrease in GSH concentration (P<0.0001), TAC (P<0.0001) and the activity of erythrocyte GPx (P<0.0001) in the patient groups compared controls. ACS patients exhibited a similar pattern of data when compared with the chronic CAD group. Similar results were also observed when chronic CAD patients with single, double or triple vessel stenosis and controls were compared. The present study indicates that the acute form of CAD is more susceptible to oxidative damage, suggesting that use of antioxidant therapy may be warranted to ameliorate oxidative stress in this condition.

Objectives: The aim of this work was to compare and analyze the diagnostic value of serum prostasin, cancer antigen 125 (CA125), lactate dehydrogenase (LDH), alpha-fetoprotein (AFP), and human chorionic gonadotropin (hCG+β) in epithelial ovarian cancer (EOC) and evaluate if their serum levels could be used as a potential diagnostic markers of EOC from benign tumors and healthy women., Materials and Methods: Preoperative serum samples of 110 women (24 healthy controls, 66 ovarian benign tumors, and 20 EOC) were tested for prostasin, CA125, AFP, and hCG+β. The level of CA125, AFP, and hCG+β serum tumor markers were determined by electro-chemiluminescence immunoassay (ECLIA) and the serum level of prostasin was measured using enzyme-linked immunosorbent assay (ELISA) and LDH activity was measured by spectrophotometer and analyzed using SPSS version., Results: The Area Under the Curve (AUC) values of prostasin, CA125, LDH, AFP, and hCG+β for the discrimination of EOC from benign and healthy controls were, respectively, 0.89, 0.91, 0.77, 0.54, and 0.65, and significant increase in serum levels of prostasin, CA125, and LDH were observed for EOC compared with benign and control groups., Conclusion: The present study showed that CA 125 and LDH levels of serum increased in high stages, while prostasin level was decreased in high stages. The present results indicate that prostasin, CA125, and LDH are differentially expressed in EOC than in benign and healthy control population, that may be an indicative of a better diagnostic value, with higher sen- sitivity and specificity. Here the authors used a multimarker approach, consisting of CA125, AFP, beta hCG, prostasin, and LDH that could provide a more accurate tool for a differential diagnosis of patients with EOC.

Cryopreservation and biobanking of stem cells are becoming increasingly important as stem cell technology and application attract the interest of industry, academic research, healthcare and patient organisations. Stem cell are already being used in the treatment of some diseases and it is anticipated that stem cell therapy will play a central role in future medicine. Similarly, the discovery of both hematopoietic and solid tumor stem cells and their clinical relevance have profoundly altered paradigms for cancer research as the cancer stem cells are considered promising new targets against cancer. Consequently, long-term cryopreservation and banking of normal and malignant stem cells is crucial and will inevitably become a routine procedure that requires highly regulated and safe methods of specimen storage. There is, however, an increasing amount of evidence showing contradictory results on the impact of cryopreservation and thawing of stem cells, including extensive physical and biological stresses, apoptosis and necrosis, mitochondrial injuries, changes to basal respiration and ATP production, cellular structural damage, telomere shortening and cellular senescence, and DNA damage and oxidative stress. Notably, cell surface proteins that play a major role in stem cell fate and are used as the biomarkers of stem cells are more vulnerable to cold stress than other proteins. There are also data supporting the alteration in some biological features and genetic integrity at the molecular level of the post-thawed stem cells. This article reviews the current and future challenges of cryopreservation of stem cells and stresses the need for further rigorous research on the methodologies for freezing and utilizing cancer stem cells following long-term storage.

Scientific revolution is changing the world forever. Many new disciplines and fields have emerged with unlimited possibilities and opportunities. Biobanking is one of many that is benefiting from revolutionary milestones in human genome, post-genomic, and computer and bioinformatics discoveries. The storage, management, and analysis of massive clinical and biological data sets cannot be achieved without a global collaboration and networking. At the same time, biobanking is facing many significant challenges that need to be addressed and solved including dealing with an ever increasing complexity of sample storage and retrieval, data management and integration, and establishing common platforms in a global context. The overall picture of the biobanking of the future, however, is promising. Many population-based biobanks have been formed, and more are under development. It is certain that amazing discoveries will emerge from this large-scale method of preserving and accessing human samples. Signs of a healthy collaboration between industry, academy, and government are encouraging.

Background: Lung cancer is the leading cause of death among cancers in the world. The annual death toll due to this disease exceeds the combined deaths caused by colon, breast, prostate, and pancreatic cancers. As a result, there has been a tremendous effort to identify new biomarkers for early detection and diagnosis of lung cancer., Methods: In this study we report the results of screening a panel of eight non-small cell lung cancer (NSCLC) cell lines originating from different subtypes of lung cancer in an attempt to identify potential biomarkers unique to this disease. We used real-time polymerase chain reaction and flow cytometry techniques to analyze the expression of ALDHA1, EpCAM, CD133, CD24, and CD38 in this panel., Results: We demonstrate for the first time that the majority of NSCLC cells do not express levels of CD38 that would qualify it as a new biomarker for the disease. In contrast, we found that CD24 is over-expressed in 6 out of 8 of the cell lines. The combined CD24+/CD38-/low phenotype was detected in 50% of the cell lines that are also positive for CD133 and EpCAM., Conclusions: We report that CD24+/CD38-/low signature could potentially be used as a new biomarker for the early detection of NSCLC.

Cryopreservation is a common procedure widely used in biological and clinical sciences. Similar protocols are also applied in preserving cancer stem cells, a field with high promises and challenges. Specific cell surface membrane proteins are considered to be biomarkers of cancer stem cells and they may play a critical role in differentiating stem cells from non stem cells. We have looked at the possible effect of long-term cryopreservation on the molecular integrity of breast MCF7 and lung, A549 and H460, cancer stem cells and to assess if these cells are more sensitive to long-term storage process. We analyzed the expression of CD24 and CD38 as two potent biomarkers of lung cancer stem cells and EpCAM and ALDH that are used as biomarkers of a wide range of cancer stem cells. We also selected three genes essential for the normal functioning of the cells, Fos, MUC1, and HLA. Our results indicate a pattern of down-regulation in the expression of the genes following freezing, in particular among cell surface marker proteins. Global gene expression of the post-thaw breast and lung cancer stem cells also reveals a significant down-regulation in freeze-thaw cells independent from each other. Analyzing the canonical pathways between two populations reveals a significant alteration in the gene expression of the pathways involved in cell cycle, mitosis, and ataxia telangiectasia mutated pathways. Overall, our results indicate that current protocols for long-term storage of lung and breast cancer stem cells may substantially influence the activity and function of genes.

Polynucleotide kinase/phosphatase (PNKP) is a bifunctional enzyme that can phosphorylate the 5'-OH termini and dephosphorylate the 3'-phosphate termini of DNA. It is a DNA repair enzyme involved in the processing of strand break termini, which permits subsequent repair proteins to replace missing nucleotides and rejoin broken strands. Little is known about DNA repair in Plasmodium falciparum, including the roles of PNKP in repairing parasite DNA. We identified a P. falciparum gene encoding a protein with 24% homology to human PNKP and thus suggestive of a putative PNKP. In this study, the PNKP gene of P. falciparum strain K1 (PfPNKP) was successfully cloned and expressed in E. coli as a GST-PfPNKP recombinant protein. MALDI-TOF/TOF analysis of the protein confirmed the identity of PfPNKP. Assays for enzymatic activity were carried out with a variety of single- and double-stranded substrates. Although 3'-phosphatase activity was detected, PfPNKP was observed to dephosphorylate single-stranded substrates or double-stranded substrates with a short 3'-single-stranded overhang, but not double-stranded substrates that mimicked single-strand breaks. We hypothesize that unlike human PNKP, PfPNKP may not be involved in single-strand break repair, since alternative terminal processing mechanisms can substitute for PfPNKP, and that PfPNKP DNA repair actions may be confined to overhanging termini of double-strand breaks., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Lung cancer is the number one killer among all cancers and is estimated to kill over 170,000 individual in 2010 in the United States. However, little is understood about the role of tumor initiating cells in the lung cancer and whether these cells play a major role in initiation, drug resistance, and metastases of this disease. We have isolated lungospheres from tumors grown in mice and have critically examined proposed biomarkers of lung cancer stem cells such as ALDH, EpCAM, CD133/1, CD133/2, CD24, and CD38, using global gene expression, flow cytometric analysis, and quantitative real time PCR. We present evidences that the pattern of overexpression of ALDH and EpCAM, two widely discussed biomarkers of cancer stem cells, in the tumor generated by lung cancer stem cells in mice are different that could be an indicative of tumor aggressiveness. We propose, for the first time, that CD38 in combination with CD24 is a biomarkers for H460 derived lung cancer stem cells and could be used to elucidate the characteristics of these sub-population of cells. Our results demonstrate that the combination of CD24(Low/-)/CD38+ and overexpression of ALDH1 and EpCAM is the signature of enriched tumor initiating cells in H460 non-small cell lung cancer cell line. Our results propose H460-derived cancer stem cells as a well defined cell for future comprehensive analysis of putative lung cancer stem cells-like cells.

Cancer stem cell studies may improve understanding of tumor pathophysiology and identify more effective strategies for cancer treatment. In a variety of organisms, Piwil2 has been implicated in multiple roles including stem cell self-renewal, RNA silencing, and translational control. In this study, we documented specific expression of the stem cell protein Piwil2 in breast cancer with predominant expression in breast cancer stem cells. In patients who were evaluated, we determined that 90% of invasive carcinomas and 81% of carcinomas in situ exhibited highest expression of Piwil2. In breast cancer cells, Piwil2 silencing suppressed the expression of signal transducer and activator of transcription 3, a pivotal regulator of Bcl-X(L) and cyclin D1, whose downregulation paralleled a reduction in cell proliferation and survival. Our findings define Piwil2 and its effector signaling pathways as key factors in the proliferation and survival of breast cancer stem cells., (Copyright 2010 AACR.)

Cancer stem cells are believed to be the driving force behind tumor progression and development. Despite extensive studies on the effects of cryopreservation on embryonic and hematopoietic stem cells there is only limited data that directly deals with in the cryopreservation of cancer stem cells. In this study, we looked at the effect of cryopreservation on breast cancer progenitor cells known as mammospheres, which are derived from the MCF7 breast carcinoma cell line. We focused on the effect of cryopreservation on the cell biology and function of tumor-initiating cells using a standard method of cryopreservation with 15% dimethyl sulfoxide (Me(2)SO). Cell proliferation and survival was analyzed by alamarBlue solution on cryopreserved cells stored for 1-12 weeks and also by the expression of Ki-67. To assess self-renewal, single cells were harvested by limiting dilution procedure and wells were scored once a week. In order to investigate senescence, the activity of beta-galactosidase was detected by histochemical staining. Our results indicate that cryopreservation of breast cancer initiating cells will not reduce the ability of the cells to proliferate following cryopreservation storage for up to 12 months. Similarly, self-renewal, a unique property of stem cells, was shown to be maintained during cryopreservation. In contrast, cryopreservation of the mammospheres significantly increases the rate of senescence-mediated pathways. These data suggest that although cryopreservation of tumor-initiating cells is feasible but further studies are necessary to achieve a trustable repository of tumor-initiating cells and the design of new therapeutic measures to specifically target these cells., (Copyright 2010 Elsevier Inc. All rights reserved.)

XRCC1 plays a central role in mammalian single-strand break repair. Although it has no enzymatic activity of its own, it stimulates the activities of polynucleotide kinase/phosphatase (PNKP), and this function is enhanced by protein kinase CK2 mediated phosphorylation of XRCC1. We have previously shown that non-phosphorylated XRCC1 stimulates the kinase activity of PNKP by increasing the turnover of PNKP. Here we extend our analysis of the XRCC1-PNKP interaction taking into account the phosphorylation of XRCC1. We demonstrate that phosphorylated and non-phosphorylated XRCC1 interact with different regions of PNKP. Phosphorylated XRCC1 binds with high affinity (K(d) = 3.5 nM and 1 : 1 stoichiometry) to the forkhead associated (FHA) domain, while non-phosphorylated XRCC1 binds to the catalytic domain of PNKP with lower affinity (K(d) = 43.0 nM and 1 : 1 stoichiometry). Under conditions of limited enzyme concentration both forms of XRCC1 enhance the activities of PNKP, but the effect is more pronounced with phosphorylated XRCC1, particularly for the kinase activity of PNKP. The stimulatory effect of phosphorylated XRCC1 on PNKP can be totally inhibited by the presence of excess FHA domain polypeptide, but non-phosphorylated XRCC1 is not susceptible to competition by the FHA domain. Thus, XRCC1 can stimulate PNKP by two independent mechanisms.

Introduction: A subpopulation of cancer cells, tumor-initiating cells, is believed to be the driving force behind tumorigenesis and resistance to radiation and chemotherapy. The persistence of tumor-initiating cells may depend on altered regulation of DNA damage and checkpoint proteins, as well as a reduced propensity to undergo apoptosis or senescence., Methods: To test this hypothesis, we isolated CD24-/low/CD44+ tumor-initiating cells (as mammospheres) from MCF-7 breast cancer cells grown in adherent monolayer culture, and carried out a comprehensive comparison of cell death and DNA damage response pathways prior to and after exposure to ionizing radiation in mammospheres and monolayer MCF-7 cells. Single and double-strand break repair was measured by single-cell gel electrophoresis. The latter was also examined by phosphorylation of histone H2AX and formation of 53BP1 and Rad51 foci. Apoptosis was quantified by flow-cytometric analysis of annexin V-binding and senescence was analyzed on the basis of cellular beta-galactosidase activity. We employed the telomeric repeat amplification protocol to quantify telomerase activity. Expression of key DNA repair and cell cycle regulatory proteins was detected and quantified by western blot analysis., Results: Our data demonstrate that in comparison to the bulk population of MCF-7 cells (predominantly CD24+/CD44+), the MCF-7 mammosphere cells benefit from a multifaceted approach to cellular protection relative to that seen in monolayer cells, including a reduced level of reactive oxygen species, a more active DNA single-strand break repair (SSBR) pathway, possibly due to a higher level of expression of the key SSBR protein, human AP endonuclease 1 (Ape1), and a significantly reduced propensity to undergo senescence as a result of increased telomerase activity and a low level of p21 protein expression. No significant difference was seen in the rates of double-strand break repair (DSBR) between the two cell types, but DSBR in mammospheres appears to by-pass the need for H2AX phosphorylation., Conclusions: Enhanced survival of MCF-7 tumor-initiating cells in response to ionizing radiation is primarily dependent on an inherent down-regulation of the senescence pathway. Since MCF-7 cells are representative of cancer cells that do not readily undergo apoptosis, consideration of senescence pathways may play a role in targeting stem cells from such tumors.

Human polynucleotide kinase/phosphatase (hPNKP) is a 57.1-kDa enzyme that phosphorylates DNA 5'-termini and dephosphorylates DNA 3'-termini. hPNKP is involved in both single- and double-strand break repair, and cells depleted of hPNKP show a marked sensitivity to ionizing radiation. Therefore, small molecule inhibitors of hPNKP should potentially increase the sensitivity of human tumors to gamma-radiation. To identify small molecule inhibitors of hPNKP, we modified a novel fluorescence-based assay to measure the phosphatase activity of the protein, and screened a diverse library of over 200 polysubstituted piperidines. We identified five compounds that significantly inhibited hPNKP phosphatase activity. Further analysis revealed that one of these compounds, 2-(1-hydroxyundecyl)-1-(4-nitrophenylamino)-6-phenyl-6,7a-dihydro-1H-pyrrolo[3,4-b]pyridine-5,7(2H,4aH)-dione (A12B4C3), was the most effective, with an IC50 of 0.06 micromol/L. When tested for its specificity, A12B4C3 displayed no inhibition of two well-known eukaryotic protein phosphatases, calcineurin and protein phosphatase-1, or APTX, another human DNA 3'-phosphatase, and only limited inhibition of the related PNKP from Schizosaccharomyces pombe. At a nontoxic dose (1 micromol/L), A12B4C3 enhanced the radiosensitivity of human A549 lung carcinoma and MDA-MB-231 breast adenocarcinoma cells by a factor of two, which was almost identical to the increased sensitivity resulting from shRNA-mediated depletion of hPNKP. Importantly, A12B4C3 failed to increase the radiosensitivity of the hPNKP-depleted cells, implicating hPNKP as the principal cellular target of A12B4C3 responsible for increasing the response to radiation. A12B4C3 is thus a useful reagent for probing hPNKP cellular function and will serve as the lead compound for further development of PNKP-targeting drugs.

Human polynucleotide kinase (hPNK) is required for processing and rejoining DNA strand break termini. The 5'-DNA kinase and 3'-phosphatase activities of hPNK can be stimulated by the "scaffold" protein XRCC1, but the mechanism remains to be fully elucidated. Using a variety of fluorescence techniques, we examined the interaction of hPNK with XRCC1 and substrates that model DNA single-strand breaks. hPNK binding to substrates with 5'-OH termini was only approximately 5-fold tighter than that to identical DNA molecules with 5'-phosphate termini, suggesting that hPNK remains bound to the product of its enzymatic activity. The presence of XRCC1 did not influence the binding of hPNK to substrates with 5'-OH termini, but sharply reduced the interaction of hPNK with DNA bearing a 5'-phosphate terminus. These data, together with kinetic data obtained at limiting enzyme concentration, indicate a dual function for the interaction of XRCC1 with hPNK. First, XRCC1 enhances the capacity of hPNK to discriminate between strand breaks with 5'-OH termini and those with 5'-phosphate termini; and second, XRCC1 stimulates hPNK activity by displacing hPNK from the phosphorylated DNA product.

Human polynucleotide kinase (hPNK) is a bifunctional enzyme possessing a 5'-DNA kinase activity and a 3'-phosphatase activity. Studies based on cell extracts and purified proteins have indicated that hPNK can act on single-strand breaks and double-strand breaks (DSB) to restore the termini to the chemical form required for further action by DNA repair polymerases and ligases (i.e., 5'-phosphate and 3'-hydroxyl termini). These studies have revealed that hPNK can bind to XRCC4, and as a result, hPNK has been implicated as a participant in the nonhomologous end joining (NHEJ) pathway for DSB repair. We sought to confirm the role of hPNK in NHEJ in the cellular setting using a genetic approach. hPNK was stably down-regulated by RNA interference expression in M059K glioblastoma cells, which are NHEJ positive, and M059J cells, which are NHEJ deficient due to a lack of DNA-PK catalytic subunit (DNA-PKcs). Whereas depletion of hPNK significantly sensitized M059K cells to ionizing radiation, no additional sensitization was conferred to M059J cells, clearly implying that hPNK operates in the same DNA repair pathway as DNA-PKcs. On the other hand, depletion of hPNK did not increase the level of sister chromatid exchanges, indicating that hPNK is not involved in the homologous recombination DSB repair pathway. We also provide evidence that the action of hPNK in the repair of camptothecin-induced topoisomerase 1 "dead-end" complexes is independent of DNA-PKcs and that hPNK is not involved in the nucleotide excision repair pathway.

DNA glycosylases/AP lyases initiate repair of oxidized bases in the genomes of all organisms by excising these lesions and then cleaving the DNA strand at the resulting abasic (AP) sites and generate 3' phospho alpha,beta-unsaturated aldehyde (3' PUA) or 3' phosphate (3' P) terminus. In Escherichia coli, the AP-endonucleases (APEs) hydrolyze both 3' blocking groups (3' PUA and 3' P) to generate the 3'-OH termini needed for repair synthesis. In mammalian cells, the previously characterized DNA glycosylases, NTH1 and OGG1, produce 3' PUA, which is removed by the only AP-endonuclease, APE1. However, APE1 is barely active in removing 3' phosphate generated by the recently discovered mammalian DNA glycosylases NEIL1 and NEIL2. We showed earlier that the 3' phosphate generated by NEIL1 is efficiently removed by polynucleotide kinase (PNK) and not APE1. Here we show that the NEIL2-initiated repair of 5-hydroxyuracil (5-OHU) similarly requires PNK. We have also observed stable interaction between NEIL2 and other BER proteins DNA polymerase beta (Pol beta), DNA ligase IIIalpha (Lig IIIalpha) and XRCC1. In spite of their limited sequence homology, NEIL1 and NEIL2 interact with the same domains of Pol beta and Lig IIIalpha. Surprisingly, while the catalytically dispensable C-terminal region of NEIL1 is the common interacting domain, the essential N-terminal segment of NEIL2 is involved in analogous interaction. The BER proteins including NEIL2, PNK, Pol beta, Lig IIIalpha and XRCC1 (but not APE1) could be isolated as a complex from human cells, competent for repair of 5-OHU in plasmid DNA.

Mammalian polynucleotide kinase (PNK) is a key component of both the base excision repair (BER) and nonhomologous end-joining (NHEJ) DNA repair pathways. PNK acts as a 5'-kinase/3'-phosphatase to create 5'-phosphate/3'-hydroxyl termini, which are a necessary prerequisite for ligation during repair. PNK is recruited to repair complexes through interactions between its N-terminal FHA domain and phosphorylated components of either pathway. Here, we describe the crystal structure of intact mammalian PNK and a structure of the PNK FHA bound to a cognate phosphopeptide. The kinase domain has a broad substrate binding pocket, which preferentially recognizes double-stranded substrates with recessed 5' termini. In contrast, the phosphatase domain efficiently dephosphorylates single-stranded 3'-phospho termini as well as double-stranded substrates. The FHA domain is linked to the kinase/phosphatase catalytic domain by a flexible tether, and it exhibits a mode of target selection based on electrostatic complementarity between the binding surface and the phosphothreonine peptide.

Through the use of a keratinocyte/fibroblast co-culture system, we have recently identified a potent keratinocyte-derived anti-fibrogenic factor (KDAF) for dermal fibroblasts. A sequential chromatography of the active fractions of keratinocyte-conditioned medium (KCM) and peptide mapping of the candidate proteins identified KDAF as being the keratinocyte-releasable 14-3-3 sigma (14-3-3sigma) protein, which is also known as stratifin. In this study, we hypothesize that differentiated, but not proliferating, keratinocytes are the primary source of releasable 14-3-3sigma in conditioned medium. To address this hypothesis, in a longitudinal study, keratinocyte differentiation was induced by growing these cells in a medium consisting of 50% keratinocyte serum-free medium (KSFM) and 50% Dulbecco's modified eagle's medium without any additives for up to 20 d. When KCM was collected every other day and added to fibroblasts, the level of matrix metalloproteinase (MMP)-1 mRNA expression was markedly increased in fibroblasts receiving KCM and this increase was even greater in cells receiving conditioned media collected at later time points relative to that of controls. The results of a western blot analysis further showed a marked increase in the expression of 14-3-3sigma protein in keratinocytes grown in test medium from day 4 to day 10. This finding was consistent with the levels of 14-3-3sigma mRNA expression in differentiated keratinocytes. In contrast to a very high level of 14-3-3sigma mRNA expression seen in keratinocytes, fibroblasts that are highly responsive to14-3-3sigma were unable to express this factor. Interestingly, the level of 14-3-3sigma mRNA expression was markedly higher in keratinocytes co-cultured with fibroblasts relative to that of mono-cultured keratinocytes. In conclusion, this study provides evidence that keratinocytes express a high level of 14-3-3sigma at the levels of mRNA and protein. But the releasable form of 14-3-3sigma protein was only found in conditioned medium derived from differentiated keratinocytes. Further, our recently purified recombinant 14-3-3sigma protein mimics the collagenase stimulatory effect of KCM in dermal fibroblasts.

The human DNA repair protein, hXRCC1, which is required for DNA single-strand break repair and genetic stability was produced as a histidine-tagged polypeptide in Escherichia coli, purified by affinity chromatography, and subjected to sedimentation and spectroscopic analyses. This study represents the first biophysical examination of full-length XRCC1. Sedimentation equilibrium measurements indicated that hXRCC1 exists as a monomer at lower protein concentrations but forms a dimer at higher protein concentrations with a K(d) of 5.7 x 10(-)(7) M. The size and shape of hXRCC1 in solution were determined by analytical ultracentrifugation studies. The protein exhibited an intrinsic sedimentation coefficient, s(0)(20,w), of 3.56 S and a Stokes radius, R(s), of 44.5 A, which together with the M(r) of 68000 suggested that hXRCC1 is a moderately asymmetric protein with an axial ratio of 7.2. Binding of model ligands, representing single-strand breaks with either a nick or a single nucleotide gap, quenched protein fluorescence, and binding affinities and stoichiometries were determined by carrying out fluorescence titrations as a function of ligand concentration. XRCC1 bound both nicked and 1 nucleotide-gapped DNA substrates tightly in a stoichiometric manner (1:1) with K(d) values of 65 and 34 nM, respectively. However, hXRCC1 exhibited lower affinities for a duplex with a 5 nucleotide gap, the intact duplex with no break, and a single-stranded oligonucleotide with K(d) values of 215, 230, and 260 nM, respectively. Our results suggest that hXRCC1 exhibits preferential binding to DNA with single-strand breaks with a gap size of <5 nucleotides.

The paradigm for repair of oxidized base lesions in genomes via the base excision repair (BER) pathway is based on studies in Escherichia coli, in which AP endonuclease (APE) removes all 3' blocking groups (including 3' phosphate) generated by DNA glycosylase/AP lyases after base excision. The recently discovered mammalian DNA glycosylase/AP lyases, NEIL1 and NEIL2, unlike the previously characterized OGG1 and NTH1, generate DNA strand breaks with 3' phosphate termini. Here we show that in mammalian cells, removal of the 3' phosphate is dependent on polynucleotide kinase (PNK), and not APE. NEIL1 stably interacts with other BER proteins, DNA polymerase beta (pol beta) and DNA ligase IIIalpha. The complex of NEIL1, pol beta, and DNA ligase IIIalpha together with PNK suggests coordination of NEIL1-initiated repair. That NEIL1/PNK could also repair the products of other DNA glycosylases suggests a broad role for this APE-independent BER pathway in mammals.

Human polynucleotide kinase (hPNK) is a 57.1-kDa monomeric protein with conserved motifs associated with phosphatase and kinase activities. hPNK catalyzes phosphorylation of 5'-DNA termini and dephosphorylation of 3'-DNA termini. Previous studies, employing cell-free systems, have suggested that hPNK participates in the repair of DNA strand breaks. To better define the cellular function of hPNK, a double-stranded small-interfering RNA molecule designed to stably target hPNK transcription was introduced into A549 human lung adenocarcinoma cells. The small-interfering RNA suppressed hPNK gene expression by at least 80-90%. These cells exhibited a 7-fold higher spontaneous mutation frequency based on the development of resistance to ouabain; elevated sensitivity to a broad range of genotoxic agents including gamma-radiation, UVC radiation, methyl methanesulfonate, hydrogen peroxide, and camptothecin; and slower repair of radiation-induced DNA strand breaks. These findings underscore the importance of hPNK in the maintenance of DNA integrity after damage induced by endogenous and exogenous agents.

Termination of wound healing requires a fine balance between collagen deposition and its hydrolysis. To dissect the underlying control mechanisms for this process, we established a keratinocyte/fibroblast co-culture system and subsequently demonstrated more than a 10-fold increase in collagenase expression in fibroblasts co-cultured with keratinocytes relative to that of control cells. This finding was further confirmed in fibroblasts grown in a keratinocyte/fibroblast collagen-GAG gel. The efficacy of keratinocyte-derived collagenase stimulatory factors on collagenase activity was evaluated, and the results showed that only conditioned medium derived from fibroblasts co-cultured with keratinocytes was able to break down markedly type I collagen to its one-quarter and three-quarter fragments of both alpha (alpha1 and alpha2) and beta (beta1.1 and beta1.2) chains. The results of a dose-response experiment showed that keratinocyte-conditioned medium (KCM) stimulates the expression of collagenase mRNA by dermal fibroblasts in a concentration-dependent fashion. In a similar experiment, the results of a time-response experiment revealed that KCM treatment increases the expression of collagenase mRNA in dermal fibroblasts as early as 6 h and reaches its maximum level within 24-48 h. Considering that this keratinocyte-releasable factor has a potent collagenase stimulatory effect on fibroblasts, which favors the resolution of accumulated type I and type III collagen found in fibrotic tissue, we referred to this protein as a keratinocyte-derived anti-fibrogenic factor (KDAF). In a series of chromatography experiments and a direct trypsin digestion of the proteins and subsequent peptide mapping, a keratinocyte-derived collagenase-stimulating factor turned out to be a releasable form of stratifin, also known as 14-3-3 sigma protein. To validate this finding, stratifin cDNA was cloned into a pGEX-6P-1 expressing vector and more than 50 mg of recombinant stratifin was generated and used to treat fibroblasts with various concentrations for 24 h. The results of northern analysis showed a remarkable dose-response increase in the expression of collagenase mRNA in stratifin-treated fibroblasts relative to that of the control. This finding was consistent with that obtained from collagenase activity assay. In conclusion, we identified a keratinocyte-releasable form of stratifin in KCM that mimics the collagenase stimulatory effect of KCM for dermal fibroblasts. This finding suggests that stratifin is likely to be, at least, one of the KDAFs found in KCM.

Human polynucleotide kinase (hPNK), which possesses both 5'-DNA kinase and 3'-DNA phosphatase activities, is a DNA repair enzyme required for processing and rejoining of single- and double-strand-break termini. Full-length hPNK was subjected to sedimentation and spectroscopic analyses in association with its ligands, a 20-mer oligonucleotide, ATP, and AMP-PNP (a nonhydrolyzable analogue of ATP). Sedimentation equilibrium measurements indicated that hPNK was a monomer in the presence and absence of the ligands. Circular dichroism measurements revealed that the ligands induced different conformational changes in hPNK, although AMP-PNP induced the same conformational changes as ATP. CD also indicated that the oligonucleotide could bind to the protein-AMP-PNP complex. Protein-ligand binding affinities and stoichiometries were determined by measuring changes in protein intrinsic fluorescence. Titrating hPNK with the oligonucleotide indicated tight binding with a K(d) value of 1.3 microM and with 1:1 stoichiometry. A 5'-phosphorylated oligonucleotide with the same sequence exhibited an almost 6-fold lower affinity (K(d) value, 7.2 microM). ATP and AMP-PNP bound with high affinity (K(d) values, respectively, of 1.4 and 1.6 microM), and the observed binding stoichiometries were 1:1. Furthermore, the nonphosphorylated oligonucleotide was able to bind to hPNK in the presence of AMP-PNP with a K(d) value of 2.5 microM, confirming the formation of a ternary complex. This study provides the first direct physical evidence for such a ternary complex involving a polynucleotide kinase, AMP-PNP, and an oligonucleotide, and supports a reaction mechanism in which ATP and DNA bind simultaneously to the enzyme.

The efficient repair of double-strand breaks (DSBs) in DNA is critical for the maintenance of genome stability. In mammalian cells, repair can occur by homologous recombination or by non-homologous end joining (NHEJ). DNA breaks caused by reactive oxygen or ionizing radiation often contain non- conventional end groups that must be processed to restore the ligatable 3'-OH and 5'-phosphate moieties which are necessary for efficient repair by NHEJ. Here, using cell-free extracts that efficiently catalyse NHEJ in vitro, we show that human polynucleotide kinase (PNK) promotes phosphate replacement at damaged termini, but only within the context of the NHEJ apparatus. Phosphorylation of terminal 5'-OH groups by PNK was blocked by depletion of the NHEJ factor XRCC4, or by an inactivating mutation in DNA-PK(cs), indicating that the DNA kinase activity in the extract is coupled with active NHEJ processes. Moreover, we find that end-joining activity can be restored to PNK-depleted extracts by addition of human PNK, but not bacteriophage T4 PNK. This work provides the first demonstration of a direct, specific role for human PNK in DSB repair.