Structural and biochemical characterization of the mitomycin C repair exonuclease MrfB.

Mitomycin C (MMC) repair factor A (mrfA) and factor B (mrfB), encode a conserved helicase and exonuclease that repair DNA damage in the soil-dwelling bacterium Bacillus subtilis. Here we have focused on the characterization of MrfB, a DEDDh exonuclease in the DnaQ superfamily. We solved the structure of the exonuclease core of MrfB to a resolution of 2.1 Å, in what appears to be an inactive state. In this conformation, a predicted α-helix containing the catalytic DEDDh residue Asp172 adopts a random coil, which moves Asp172 away from the active site and results in the occupancy of only one of the two catalytic Mg2+ ions. We propose that MrfB resides in this inactive state until it interacts with DNA to become activated. By comparing our structure to an AlphaFold prediction as well as other DnaQ-family structures, we located residues hypothesized to be important for exonuclease function. Using exonuclease assays we show that MrfB is a Mg2+-dependent 3'-5' DNA exonuclease. We show that Leu113 aids in coordinating the 3' end of the DNA substrate, and that a basic loop is important for substrate binding. This work provides insight into the function of a recently discovered bacterial exonuclease important for the repair of MMC-induced DNA adducts.

Structural Basis of Substrate Recognition by the Postmitosane Modification Enzyme MitM in Mitomycin Biosynthesis.

Mitomycins make up a class of natural molecules produced by Streptomyces with strong antibacterial and antitumor activities. MitM is a key postmitosane modification enzyme involved in mitomycin biosynthesis in Streptomyces caespitosus. This protein was previously suggested to catalyze the aziridinium methylation of mitomycin A and the mitomycin intermediate 9a-demethyl-mitomycin A as an N-methyltransferase. The structural basis for MitM to recognize cofactor S-adenosyl-l-methionine (SAM) and substrate mitomycin A is unknown. Here, we determined the crystal structures of apo-MitM and MitM-mitomycin A-S-adenosylhomocysteine (SAH) ternary complexes with resolutions of 2.23 and 2.80 Å, respectively. We found that MitM adopts a class I SAM-dependent methyltransferase fold and forms a homodimer in solution. Conformational changes in a series of residues involved in the formation of active pockets assist MitM in binding SAH and mitomycin A. In particular, the 28ALGAASLGE36 loop changes most significantly. When mitomycin A binds, the bending direction of this loop is reversed, changing the entrance of the active site from open to closed. This study provides structural insights into MitM's involvement in the postmitosane stage of mitomycin biosynthesis and provides a template for the engineering of methyltransferases.

Insight Into Factors Governing Formation, Synthesis and Stereochemical Configuration of DNA Adducts Formed by Mitomycins.

Mitomycin C, (MC), an antitumor drug used in the clinics, is a DNA alkylating agent. Inert in its native form, MC is reduced to reactive mitosenes in cellulo which undergo nucleophilic attack by DNA bases to form monoadducts as well as interstrand crosslinks (ICLs). These properties constitute the molecular basis for the cytotoxic effects of the drug. The mechanism of DNA alkylation by mitomycins has been studied for the past 30 years and, until recently, the consensus was that drugs of the mitomycins family mainly target CpG sequences in DNA. However, that paradigm was recently challenged. Here, we relate the latest research on both MC and dicarbamoylmitomycin C (DMC), a synthetic derivative of MC which has been used to investigate the regioselectivity of mitomycins DNA alkylation as well as the relationship between mitomycins reductive activation pathways and DNA adducts stereochemical configuration. We also review the different synthetic routes to access mitomycins nucleoside adducts and oligonucleotides containing MC/DMC DNA adducts located at a single position. Finally, we briefly describe the DNA structural modifications induced by MC and DMC adducts and how site specifically modified oligonucleotides have been used to elucidate the role each adduct plays in the drugs cytotoxicity.

Tracing of Acyl Carrier Protein-channeled Mitomycin Intermediates in Streptomyces caespitosus Facilitates Characterization of the Biosynthetic Steps for AHBA-GlcN Formation and Processing.

Mitomycins are a family of naturally occurring, potent alkylating agents in which the C member has been clinically used for cancer chemotherapy for over 5 decades. In Streptomyces caespitosus, mitomycins are derived from an N-glycoside composed of a 3-amino-5-hydroxybenzoic acid (AHBA) unit and a d-glucosamine (GlcN) unit; however, how this N-glycoside is formed and rearranged to a mitosane, for example, the compact polycyclic ring system of mitomycin C, remains elusive. Benefiting from the development of a method used to trace the mitomycin intermediates that accumulate on an acyl carrier protein (ACP), we here dissect the enzymatic steps for AHBA-GlcN formation and processing to underlie the mitosane structure. Following the N-glycosylation of AHBA with activated N-acetyl-GlcN, deacetylation occurs on ACP to provide AHBA-GlcN. Then, the sugar portion of this N-glycoside is transformed into a linear aminodiol that terminates with an epoxyethane, yielding an ACP-channeled intermediate that is ready for mitosane formation through crosslinking between the AHBA and linearized sugar units. This transformation is unusual and relies on the functional association of a dihydronicotinamide adenine dinucleotide (phosphate)-dependent protein with a radical S-adenosyl-l-methionine protein. Characterization of these ACP-based enzymatic steps for AHBA-GlcN formation and processing sheds light on the poorly understood biosynthetic pathway of mitomycins.

Cytotoxicity, crosslinking and biological activity of three mitomycins.

While interstrand crosslinks (ICLs) have been considered as one type of DNA damage in the past, there is mounting evidence suggesting that these highly cytotoxic lesions are processed differently by the cellular machinery depending upon the ICL structure. In this study, we examined the crosslinking ability of three mitomycins, the structure of the ICLs they produce and the cytotoxicity of the drugs toward three different cell lines. The drugs are: mitomycin C (1), decarbamoylmitomycin C (2), and a mitomycin-conjugate (3) whose mitosane moiety is linked to a N-methylpyrrole carboxamide. We found that, overall, both MC and compound 3 show strong similarities regarding their alkylation of DNA, while DMC alkylating behavior is markedly different. To gain further insight into the mode of action of these drugs, we performed high throughput gene expression and gene ontology analysis to identify gene expression and cellular pathways most impacted by each drug treatment in MCF-7 cell lines. We observed that the novel mitomycin derivative (3) specifically causes changes in the expression of genes encoding proteins involved in cell integrity and tissue structure. Further analysis using bioinformatics (IPA) indicated that the new derivative (3) displays a stronger downregulation of major signaling networks that regulate the cell cycle, DNA damage response and cell proliferation when compared to MC and DMC. Collectively, these findings demonstrate that cytotoxic mechanisms of all three drugs are complex and are not solely related to their crosslinking abilities or the structure of the ICLs they produce.

Advances in the Synthesis of Ring-Fused Benzimidazoles and Imidazobenzimidazoles.

This review article provides a perspective on the synthesis of alicyclic and heterocyclic ring-fused benzimidazoles, imidazo[4,5-f]benzimidazoles, and imidazo[5,4-f]benzimidazoles. These heterocycles have a plethora of biological activities with the iminoquinone and quinone derivatives displaying potent bioreductive antitumor activity. Synthesis is categorized according to the cyclization reaction and mechanisms are detailed. Nitrobenzene reduction, cyclization of aryl amidines, lactams and isothiocyanates are described. Protocols include condensation, cross-dehydrogenative coupling with transition metal catalysis, annulation onto benzimidazole, often using CuI-catalysis, and radical cyclization with homolytic aromatic substitution. Many oxidative transformations are under metal-free conditions, including using thermal, photochemical, and electrochemical methods. Syntheses of diazole analogues of mitomycin C derivatives are described. Traditional oxidations of o-(cycloamino)anilines using peroxides in acid via the t-amino effect remain popular.

Determination of the Protein-Protein Interactions within Acyl Carrier Protein (MmcB)-Dependent Modifications in the Biosynthesis of Mitomycin.

Mitomycin has a unique chemical structure and contains densely assembled functionalities with extraordinary antitumor activity. The previously proposed mitomycin C biosynthetic pathway has caused great attention to decipher the enzymatic mechanisms for assembling the pharmaceutically unprecedented chemical scaffold. Herein, we focused on the determination of acyl carrier protein (ACP)-dependent modification steps and identification of the protein-protein interactions between MmcB (ACP) with the partners in the early-stage biosynthesis of mitomycin C. Based on the initial genetic manipulation consisting of gene disruption and complementation experiments, genes mitE, mmcB, mitB, and mitF were identified as the essential functional genes in the mitomycin C biosynthesis, respectively. Further integration of biochemical analysis elucidated that MitE catalyzed CoA ligation of 3-amino-5-hydroxy-bezonic acid (AHBA), MmcB-tethered AHBA triggered the biosynthesis of mitomycin C, and both MitB and MitF were MmcB-dependent tailoring enzymes involved in the assembly of mitosane. Aiming at understanding the poorly characterized protein-protein interactions, the in vitro pull-down assay was carried out by monitoring MmcB individually with MitB and MitF. The observed results displayed the clear interactions between MmcB and MitB and MitF. The surface plasmon resonance (SPR) biosensor analysis further confirmed the protein-protein interactions of MmcB with MitB and MitF, respectively. Taken together, the current genetic and biochemical analysis will facilitate the investigations of the unusual enzymatic mechanisms for the structurally unique compound assembly and inspire attempts to modify the chemical scaffold of mitomycin family antibiotics.

Conjugating Aptamer and Mitomycin C with Reductant-Responsive Linker Leading to Synergistically Enhanced Anticancer Effect.

Mitomycin C (MMC) has been using for the treatment of a variety of digestive tract cancers. However, its nonspecific DNA-alkylating ability usually causes severe side effects, thus largely limiting its clinical applications. The utilization of an efficient active targeted drug delivery technique would address this issue. Accordingly, we report the design and development of aptamer-mitomycin C conjugates that use different cross-linking chemistry. The targeted delivery ability and cytotoxicity of these conjugates were carefully studied. It is worth noting that a linker-dependent cytotoxicity effect was observed for these conjugates. The use of a reductant-sensitive disulfide bond cross-linking strategy resulted in significantly enhanced cytotoxicity of MMC against the target cancer cell lines. Importantly, this cytotoxicity enhancement was suited to different types of aptamers, demonstrating the success of our design. Mechanistic studies of the enhanced cytotoxicity effect indicated that the target recognition, specific binding, and receptor-mediated internalization of aptamer were also critical for the observed effect.

Engineered Fn3 protein has targeted therapeutic effect on mesothelin-expressing cancer cells and increases tumor cell sensitivity to chemotherapy.

Mesothelin is a protein expressed at high levels on the cell surface in a variety of cancers, with limited expression in healthy tissues. The presence of mesothelin on tumor tissue correlates with increased invasion and metastasis, and resistance to traditional chemotherapies, through mechanisms that remain poorly understood. Molecules that specifically recognize mesothelin and interrupt its contribution to tumor progression have significant potential for targeted therapy and targeted drug delivery applications. A number of mesothelin-targeting therapies are in preclinical and clinical development, although none are currently approved for routine clinical use. In this work, we report the development of a mesothelin-targeting protein based on the fibronectin type-III non-antibody protein scaffold, which offers opportunities for applications where antibodies have limitations. We engineered protein variants that bind mesothelin with high affinity and selectively initiate apoptosis in tumor cells expressing mesothelin. Interestingly, apoptosis does not occur through a caspase-mediated pathway and does not require downregulation of cell-surface mesothelin, suggesting a currently unknown pathway through which mesothelin contributes to cancer progression. Importantly, simultaneous treatment with mesothelin-binding protein and chemotherapeutic mitomycin C had a greater cytotoxic effect on mesothelin-positive cells compared to either molecule alone, underscoring the potential for combination therapy including biologics targeting mesothelin.

Replication-independent repair of DNA interstrand crosslinks.

DNA interstrand crosslinks (ICLs) are cytotoxic lesions that covalently link opposite strands of the DNA helix and block DNA unwinding. ICLs are repaired during and outside S phase, and replication-independent ICL repair (RIR) is critical to maintain genomic integrity and to allow transcription in nondividing or slowly dividing cells. Here, we show that the Y family DNA polymerase kappa (Pol κ) is essential for RIR of a site-specific ICL lesion in Xenopus egg extracts, and that both its catalytic activity and UBZ domains are required for this function. We also demonstrate a requirement for PCNA and its modification on lysine 164. Finally, we show that Pol κ participates in ICL repair in mammalian cells, particularly in G0. Our results identify key components of the RIR pathway and begin to unravel its mechanism.

Synthesis of Mitomycin C and decarbamoylmitomycin C N6 deoxyadenosine-adducts.

Mitomycin C (MC), an anti-cancer drug, and its analog, decarbamoylmitomycin C (DMC), are DNA-alkylating agents. MC is currently used in the clinics and its cytotoxicity is mainly due to its ability to form Interstrand Crosslinks (ICLs) which impede DNA replication and, thereby, block cancer cells proliferation. However, both MC and DMC are also able to generate monoadducts with DNA. In particular, we recently discovered that DMC, like MC, can form deoxyadenosine (dA) monoadducts with DNA. The biological role played by these monoadducts is worthy of investigation. To probe the role of these adducts and to detect them in enzymatic digests of DNA extracted from culture cells treated by both drugs, we need access to reference compounds i.e. MC and DMC dA-mononucleoside adducts. Previous biomimetic methods used to generate MC and DMC mononucleoside adducts are cumbersome and very low yielding. Here, we describe the diastereospecific chemical synthesis of both C-1 epimers of MC and DMC deoxyadenosine adducts. The key step of the synthesis involves an aromatic substitution reaction between a 6-fluoropurine 2'-deoxyribonucleoside and appropriately protected stereoisomeric triaminomitosenes to form protected-MC-dA adducts with either an S or R stereochemical configuration at the adenine-mitosene linkage. Fluoride-based deprotection methods generated the final four reference compounds: the two stereoisomeric MC-dA adducts and the two stereoisomeric DMC-dA adducts. The MC and DMC-dA adducts synthesized here will serve as standards for the detection and identification of such adducts formed in the DNA of culture cells treated with both drugs.

The Use of TAT Peptide-Functionalized Graphene as a Highly Nuclear-Targeting Carrier System for Suppression of Choroidal Melanoma.

Tumorous metastasis is a difficult challenge to resolve for researchers and for clinicians. Targeted delivery of antitumor drugs towards tumor cells' nuclei can be a practical approach to resolving this issue. This work describes an efficient nuclear-targeting delivery system prepared from trans-activating transcriptional activator (TAT) peptide-functionalized graphene nanocarriers. The TAT peptide, originally observed in a human immunodeficiency virus 1 (HIV-1), was incorporated with graphene via an edge-functionalized ball-milling method developed by the author's research group. High tumor-targeting capability of the resulting nanocarrier was realized by the strong affinity between TAT and the nuclei of cancer cells, along with the enhanced permeability and retention (EPR) effect of two-dimensional graphene nanosheets. Subsequently, a common antitumor drug, mitomycin C (MMC), was covalently linked to the TAT-functionalized graphene (TG) to form a nuclear-targeted nanodrug MMC-TG. The presence of nanomaterials inside the nuclei of ocular choroidal melanoma (OCM-1) cells was shown using transmission electron microscopy (TEM) and confocal laser scanning microscopy. In vitro results from a Transwell co-culture system showed that most of the MMC-TG nanodrugs were delivered in a targeted manner to the tumorous OCM-1 cells, while a very small amount of MMC-TG was delivered in a non-targeted manner to normal human retinal pigment epithelial (ARPE-19) cells. TEM results further confirmed that apoptosis of OCM-1 cells was started from the lysis of nuclear substances, followed by the disappearance of nuclear membrane and cytoplasm. This suggests that the as-synthesized MMC-TG is a promising nuclear-target nanodrugfor resolution of tumorous metastasis issues at the headstream.

Interdependent Sequence Selectivity and Diastereoselectivity in the Alkylation of DNA by Decarbamoylmitomycin†C.

Mitomycin C (MC), an antitumor drug, and decarbamoylmitomycin C (DMC), a derivative of MC, alkylate DNA and form deoxyguanosine monoadducts and interstrand crosslinks (ICLs). Interestingly, in mammalian culture cells, MC forms primarily deoxyguanosine adducts with a 1"-R stereochemistry at the guanine-mitosene bond (1"-α) whereas DMC forms mainly adducts with a 1"-S stereochemistry (1"-ß). The molecular basis for the stereochemical configuration exhibited by DMC has been investigated using biomimetic synthesis. Here, we present the results of our studies on the monoalkylation of DNA by DMC. We show that the formation of 1"-ß-deoxyguanosine adducts requires bifunctional reductive activation of DMC, and that monofunctional activation only produces 1"-α-adducts. The stereochemistry of the deoxyguanosine adducts formed is also dependent on the regioselectivity of DNA alkylation and on the overall DNA CG content. Additionally, we found that temperature plays a determinant role in the regioselectivity of duplex DNA alkylation by mitomycins: At 0 °C, both deoxyadenosine (dA) and deoxyguanosine (dG) alkylation occur whereas at 37 °C, mitomycins alkylate dG preferentially. The new reaction protocols developed in our laboratory to investigate DMC-DNA alkylation raise the possibility that oligonucleotides containing DMC 1"-ß-deoxyguanosine adducts at a specific site may be synthesized by a biomimetic approach.

Cholesterol Modification of an Anticancer Drug for Efficient Incorporation into a Supramolecular Hydrogel System.

Treatment of cancer in the peritoneal cavity may be improved with macroscale drug delivery systems that offer control over intraperitoneal concentration of chemotherapeutic agents. Currently, suitable drug carriers to facilitate a sustained release of small hydrophilic drugs such as mitomycin C are lacking. For this purpose, a pH-responsive supramolecular hydrogel based on ureido-pyrimidinone (UPy) chemistry is utilized here. In order to provide a sustained release profile, a lipophilicity-increasing cholesterol conjugation strategy is proposed that enhances affinity between the modified drug (mitomycin-PEG24 -cholesterol, MPC) and the hydrophobic compartments in the UPy gel. Additional advantages of cholesterol conjugation include improved chemical stability and potency of mitomycin C. In vitro the tunability of the system to obtain optimal effective concentrations over time is demonstrated with a combinatorial treatment of mitomycin C and MPC in one UPy hydrogel delivery system.

Targeting of folate-conjugated liposomes with co-entrapped drugs to prostate cancer cells via prostate-specific membrane antigen (PSMA).

Folate-targeted liposomes (FTL) were tested as drug delivery vehicles to PSMA-positive cancer cells. We used FL with co-entrapped mitomycin C lipophilic prodrug (MLP) and doxorubicin (DOX), and the LNCaP prostate cancer cell line which expresses PSMA but is negative for folate receptor. A major increase in cell drug levels was observed when LNCaP cells were incubated with FTL as compared to non-targeted liposomes (NTL). MLP was activated to mitomycin C, and intracellular and nuclear fluorescence of DOX was detected, indicating FTL processing and drug bioavailability. PMPA (2-(phosphonomethyl)-pentanedioic acid), a specific inhibitor of PSMA, blocked the uptake of FTL into LNCaP cells, but did not affect the uptake of FTL into PSMA-deficient and folate receptor-positive KB cells. The cytotoxic activity of drug-loaded FTL was found significantly enhanced when compared to NTL in LNCaP cells. FTL may provide a new tool for targeted therapy of cancers that over-express the PSMA receptor.

Characterization of Pegylated Liposomal Mitomycin C Lipid-Based Prodrug (Promitil) by High Sensitivity Differential Scanning Calorimetry and Cryogenic Transmission Electron Microscopy.

The effect of a lipidated prodrug of mitomycin C (MLP) on the membrane of a pegylated liposome formulation (PL-MLP), also known as Promitil, was characterized through high-sensitivity differential scanning calorimetry (DSC) and cryo-TEM. The thermodynamic analysis demonstrated that MLP led to the formation of heterogeneous domains in the membrane plane of PL-MLP. MLP concentrated in prodrug-rich domains, arranged in high-ordered crystal-like structures, as suggested by the sharp and high enthalpy endotherm in the first heating scanning. After thiolytic cleavage of mitomycin C from MLP by dithiothreitol (DTT) treatment, the crystal-like prodrug domain disappears and a homogeneous membrane with stronger lipid interactions and higher phase transition temperature compared with the blank (MLP-free) liposomes is observed by DSC. In parallel, the rod-like discoid liposomes and the "kissing liposomes" seen by cryo-TEM in the PL-MLP formulation disappear, and liposome mean size and polydispersity increase after DTT treatment. Both MLP and the residual postcleavage lipophilic moiety of the prodrug increased the rigidity of the liposome membrane as indicated by DSC. These results confirm that MLP is inserted in the PL-MLP liposome membrane via its lipophilic anchor, and its mitomycin C moiety located mainly at the region of the phospholipid glycerol backbone and polar headgroup. We hypothesize that π-π stacking between the planar aromatic rings of the mitomycin C moieties leads to the formation of prodrug-rich domains with highly ordered structure on the PL-MLP liposome membrane. This thermodynamically stable conformation may explain the high stability of the PL-MLP formulation. These results also provide us with an interesting example of the application of high sensitivity DSC in understanding the composition-structure-behavior dynamics of liposomal nanocarriers having a lipid-based drug as pharmaceutical ingredient.

Dual-targeted hybrid nanoparticles of synergistic drugs for treating lung metastases of triple negative breast cancer in mice.

Lung metastasis is the major cause of death in patients with triple negative breast cancer (TNBC), an aggressive subtype of breast cancer with no effective therapy at present. It has been proposed that dual-targeted therapy, ie, targeting chemotherapeutic agents to both tumor vasculature and cancer cells, may offer some advantages. The present work was aimed to develop a dual-targeted synergistic drug combination nanomedicine for the treatment of lung metastases of TNBC. Thus, Arg-Gly-Asp peptide (RGD)-conjugated, doxorubicin (DOX) and mitomycin C (MMC) co-loaded polymer-lipid hybrid nanoparticles (RGD-DMPLN) were prepared and characterized. The synergism between DOX and MMC and the effect of RGD-DMPLN on cell morphology and cell viability were evaluated in human MDA-MB-231 cells in vitro. The optimal RGD density on nanoparticles (NPs) was identified based on the biodistribution and tumor accumulation of the NPs in a murine lung metastatic model of MDA-MB-231 cells. The microscopic distribution of RGD-conjugated NPs in lung metastases was examined using confocal microscopy. The anticancer efficacy of RGD-DMPLN was investigated in the lung metastatic model. A synergistic ratio of DOX and MMC was found in the MDA-MB-231 human TNBC cells. RGD-DMPLN induced morphological changes and enhanced cytotoxicity in vitro. NPs with a median RGD density showed the highest accumulation in lung metastases by targeting both tumor vasculature and cancer cells. Compared to free drugs, RGD-DMPLN exhibited significantly low toxicity to the host, liver and heart. Compared to non-targeted DMPLN or free drugs, administration of RGD-DMPLN (10 mg/kg, iv) resulted in a 4.7-fold and 31-fold reduction in the burden of lung metastases measured by bioluminescence imaging, a 2.4-fold and 4.0-fold reduction in the lung metastasis area index, and a 35% and 57% longer median survival time, respectively. Dual-targeted RGD-DMPLN, with optimal RGD density, significantly inhibited the progression of lung metastasis and extended host survival.

A three-dimensional skin equivalent reflecting some aspects of in vivo aged skin.

Human skin undergoes morphological, biochemical and functional modifications during the ageing process. This study was designed to produce a 3-dimensional (3D) skin equivalent in vitro reflecting some aspects of in vivo aged skin. Reconstructed skin was generated by co-culturing skin fibroblasts and keratinocytes on a collagen-glycosaminoglycan-chitosan scaffold, and ageing was induced by the exposition of fibroblasts to Mitomycin-C (MMC). Recently published data showed that MMC treatment resulted in a drug-induced accelerated senescence (DIAS) in human dermal fibroblast cultures. Next to established ageing markers, histological changes were analysed in comparison with in vivo aged skin. In aged epidermis, the filaggrin expression is reduced in vivo and in vitro. Furthermore, in dermal tissue, the amount of elastin and collagen is lowered in aged skin in vivo as well as after the treatment of 3D skin equivalents with MMC in vitro. Our results show histological signs and some aspects of ageing in a 3D skin equivalent in vitro, which mimics aged skin in vivo.

Comparative Error-Free and Error-Prone Translesion Synthesis of N(2)-2'-Deoxyguanosine Adducts Formed by Mitomycin C and Its Metabolite, 2,7-Diaminomitosene, in Human Cells.

Mitomycin C (MC) is a cytotoxic and mutagenic antitumor agent that alkylates DNA upon reductive activation. 2,7-Diaminomitosene (2,7-DAM) is a major metabolite of MC in tumor cells, which also alkylates DNA. MC forms seven DNA adducts, including monoadducts and inter- and intrastrand cross-links, whereas 2,7-DAM forms two monoadducts. Herein, the biological effects of the dG-N(2) adducts formed by MC and 2,7-DAM have been compared by constructing single-stranded plasmids containing these adducts and replicating them in human embryonic kidney 293T cells. Translesion synthesis (TLS) efficiencies of dG-N(2)-MC and dG-N(2)-2,7-DAM were 38 ± 3 and 27 ± 3%, respectively, compared to that of a control plasmid. This indicates that both adducts block DNA synthesis and that dG-N(2)-2,7-DAM is a stronger replication block than dG-N(2)-MC. TLS of each adducted construct was reduced upon siRNA knockdown of pol η, pol κ, or pol ζ. For both adducts, the most significant reduction occurred with knockdown of pol κ, which suggests that pol κ plays a major role in TLS of these dG-N(2) adducts. Analysis of the progeny showed that both adducts were mutagenic, and the mutation frequencies (MF) of dG-N(2)-MC and dG-N(2)-2,7-DAM were 18 ± 3 and 10 ± 1%, respectively. For both adducts, the major type of mutation was G → T transversions. Knockdown of pol η and pol ζ reduced the MF of dG-N(2)-MC and dG-N(2)-2,7-DAM, whereas knockdown of pol κ increased the MF of these adducts. This suggests that pol κ predominantly carries out error-free TLS, whereas pol η and pol ζ are involved in error-prone TLS. The largest reduction in MF by 78 and 80%, respectively, for dG-N(2)-MC and dG-N(2)-2,7-DAM constructs occurred when pol η, pol ζ, and Rev1 were simultaneously knocked down. This result strongly suggests that, unlike pol κ, these three TLS polymerases cooperatively perform the error-prone TLS of these adducts.

Intracrine sex steroid synthesis and signaling in human epidermal keratinocytes and dermal fibroblasts.

Peripheral intracrine sex steroid synthesis from adrenal precursors dehydroepiandrosterone (DHEA) and DHEA-sulfate has evolved in humans. We sought to establish if there are differences in intracrine, paracrine, and endocrine regulation of sex steroids by primary cultures of human skin epidermal keratinocytes and dermal fibroblasts. Microarray analysis identified multifunctional genes modulated by steroids, quantitative RT-PCR (qRT-PCR) mRNA expression, enzymatic assay aromatase activity, scratch assay cell migration, immunocytochemistry α-smooth muscle actin (α-SMA), and collagen gel fibroblast contraction. All steroidogenic components were present, although only keratinocytes expressed the organic anion organic anion transporter protein (OATP) 2B1 transporter. Both expressed the G-protein-coupled estrogen receptor (GPER1). Steroids modulated multifunctional genes, up-regulating genes important in repair and aging [angiopoietin-like 4 (ANGPTL4), chemokine (C-X-C motif) ligand 1 (CXCL1), lamin B1 (LMNB1), and thioredoxin interacting protein (TXNIP)]. DHEA-sulfate (DHEA-S), DHEA, and 17ß-estradiol stimulated keratinocyte and fibroblast migration at early (4 h) and late (24-48 h) time points, suggesting involvement of genomic and nongenomic signaling. Migration was blocked by aromatase and steroid sulfatase (STS) inhibitors confirming intracrine synthesis to estrogen. Testosterone had little effect, implying it is not an intermediate. Steroids stimulated fibroblast contraction but not α-SMA expression. Mechanical wounding reduced fibroblast aromatase activity but increased keratinocyte activity, amplifying the bioavailability of intracellular estrogen. Cultured fibroblasts and keratinocytes provide a biologically relevant model system to investigate the complex pathways of sex steroid intracrinology in human skin.