Mitomycin C Exposure Research Articles

Treating colorectal peritoneal metastases with an injectable cytostatic loaded supramolecular hydrogel in a rodent animal model

Patients with peritoneal metastases (PM) of colorectal cancer have a very poor outcome. Intraperitoneal delivery of chemotherapy is the preferred route for PM treatment. The main limitation of the treatment options is the short residence time of the cytostatic, with subsequent short exposure of the cancer cells. To address this, a supramolecular hydrogel has been developed that allows both local and slow release of its encapsulated drug, mitomycin C (MMC) or cholesterol-conjugated MMC (cMMC), respectively. This experimental study investigates if drug delivery using this hydrogel improves the therapeutic efficacy against PM. PM was induced in WAG/Rij rats (n = 72) by intraperitoneally injecting syngeneic colon carcinoma cells (CC531) expressing luciferase. After seven days, animals received a single intraperitoneal injection with saline (n = 8), unloaded hydrogel (n = 12), free MMC (n = 13), free cMMC (n = 13), MMC-loaded hydrogel (n = 13), or cMMC-loaded hydrogel (n = 13). Primary outcome was overall survival with a maximum follow-up of 120 days. Intraperitoneal tumor development was non-invasive monitored via bioluminescence imaging. Sixty-one rats successfully underwent all study procedures and were included to assess therapeutic efficacy. After 120 days, the overall survival in the MMC-loaded hydrogel and free MMC group was 78% and 38%, respectively. A trend toward significance was found when comparing the survival curves of the MMC-loaded hydrogel and free MMC (p = 0.087). No survival benefit was found for the cMMC-loaded hydrogel compared to free cMMC. Treating PM with our MMC-loaded hydrogel, exhibiting prolonged MMC exposure, seems effective in improving survival compared to treatment with free MMC.

Oxidative stress in the endothelial cell culture exposed to mitomycin C

Background. Atherosclerosis is one of the leading cardiovascular pathologies. Evidence suggests that DNA damage caused by endothelial cell exposure to mitomycin C (MMC) leads to endothelial dysfunction and is the risk factor for this disease. MMC is an alkylating mutagen involved in the development of oxidative stress, which is also a risk factor for atherosclerosis. Aim. To access the levels of oxidative stress markers in the primary human endothelial cell culture exposed to alkylating mutagen MMC.Material and Methods. Commercially available primary cultures of endothelial cells obtained from human coronary artery (HCAEC) and human internal thoracic artery (HITAEC) were used in the study. The cells were cultivated in the presence of 500 ng/mL MMC (experimental group) and without mutagenic load (control group). The levels of reactive oxygen species, reactive nitrogen species, and 8-OH-deoxyguanosine (8-OHdG) in cell growth media were assessed by enzyme-linked immunosorbent assay. The relative telomere length and expression of TERT and POT1 genes were accessed in endothelial cells by quantitative polymerase chain reaction. Statistical analysis of data was performed using GraphPad Prism 9 software.Results. There were no differences in the concentrations of reactive oxygen species, reactive nitrogen species (NO2 -, NO3 -, NO2 -/NO3 -), and 8-OHdG in HCAEC and HITAEC cultures exposed to MMC compared to the corresponding parameters in the non-exposed controls. At the same time, HCAEC and HITAEC exposed to MMC were characterized by a decrease in the relative telomere length compared to control (10.97 vs. 27.03 in HCAEC, p = 0.002 and 9.12 vs. 25.64 in HITAEC, p = 0.001). Moreover, we discovered 1.75-fold increase in the expression of POT1 gene in the experimental HCAEC compared to control (p = 0.019). No expression of TERT gene was observed in study groups.Conclusions. Alkylating mutagen MMC did not induce any pronounced oxidative stress in the primary human endothelial cells in vitro. The development of endothelial dysfunction caused by MMC exposure was triggered mainly by DNA alkylation resulting in the genotoxic stress in the endothelial cells.

Clinical Severity Correlates with in Vitro Residual Function of FANCB Missense Variants

Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome, characterized by congenital anomalies, bone marrow failure and cancer predisposition. FA patients with mutations in FANCB exhibit X-linked inheritance pattern. FANCB patients have been frequently associated with a severe phenotype, resembling VACTERL association with hydrocephalus, although heterogeneity in these patients still exists and is not well understood. To determine the underlying pathogenesis of clinical heterogeneity, we investigated phenotype-genotype correlations in 19 patients from 16 families with FANCB pathogenic variants. We also investigated functional properties of five FANCB missense variants to determine their possible contribution to clinical manifestations.Patients were accrued at the Rockefeller University Hospital in the International Fanconi Anemia Registry. Among the 16 families with FANCB pathogenic variants, three carry nonsense variants, five have indels, two with large deletions, one has an intragenic duplication, two with canonical splice site variants that should result in aberrant splicing, and five display missense variants. One patient carried both an indel and a missense variant. Patients with variants leading to FANCB truncations (including nonsense variants, indels, large deletions/duplications, and splice variants) tend to show shorter survival and earlier hematologic onset, than patients with missense variants. However, the differences did not reach statistical significance due to small sample size.Functional properties of five missense variants were examined by overexpression of variant cDNA (HA-tagged) in FANCB-null fibroblasts, followed by assessment of FANCD2 ubiquitination, mitomycin C (MMC) sensitivity, FANCD2 foci formation and HA-FANCB localization. We found that expression of p.W479G and p.L676P FANCB variants leads to near-normal FANCD2 ubiquitination upon MMC exposure, and MMC sensitivity. Conversely, p.L43S-expressing cells behaved like FANCB-null fibroblasts. Expression of p.L329P and p.G750V resulted in low levels of FANCD2 ubiquitination upon MMC exposure and intermediate MMC sensitivity. Expression of p.L43S resulted in smaller-sized and lowest quantity of FANCD2 foci with predominant cytoplasmic localization of HA-FANCB, while other variants showed intermediate phenotype. We quantified the nuclear expression of HA-FANCB, which confirmed the lowest relative nuclear expression of p.L43S variant, suggesting a defect in nuclear localization necessary for the interstrand crosslink repair. We also assessed splicing defects in the patient primary cells by reverse transcription PCR. Majority of the transcripts in the patient carrying c.1435T>G (predicted to express p.W479G) variant showed aberrant splicing, exon 7 skipping, which should result in an out-of-frame truncated protein. Although the expression level of c.1435T>G encoding the missense variant p.W479G is lower than the splicing variant, its functional characteristics were near-normal, thus reflecting relatively milder phenotype in this patient. Overall patient survival correlated with residual function of their FANCB variants as assessed by in vitro assays described above.In summary, we report clinical course and mutation types of 19 patients with FANCB pathogenic variants. We also report that FANCB missense variants can have varying degree of residual function, which correlates with patient survival. Our data suggest that variant-level analysis may be a useful prognostic marker of patient outcome. In vitro assays evaluating the residual function of proteins with missense variants may help to predict patient outcome. DisclosuresNo relevant conflicts of interest to declare.

Assessment of the efficacy of repeated instillations of mitomycin C mixed with a thermosensitive hydrogel in an orthotopic rat bladder cancer model.

Background:We investigated a thermoreversible hydrogel that is highly viscous at body temperature, while fluid-like at a low temperature, thus aiming for a slow and prolonged intravesical drug release. Our study purposed to assess antitumor efficacy of mitomycin C (MMC) mixed with hydrogel in an orthotopic rat bladder cancer model.Methods:Bladders of female Fischer F344 rats were grafted with 1.5 × 106 AY-27 urothelial carcinoma cells. On day 5, tumor presence was assessed by cystoscopy and rats were divided into six groups (five treatment, one control, n = 10/group). Intravesical treatments (0.5 mg or 1 mg MMC-H2O or MMC-hydrogel, or 2 mg MMC-hydrogel) were administered on days 5, 8 and 11. Rats were sacrificed at day 14 and bladders were evaluated.Results:Rats with tumor at cystoscopy (47/60) were evaluated for efficacy. At necropsy, all control animals (8/8) had tumors. No microscopic tumors were present in the 0.5 mg and 1 mg MMC-hydrogel groups compared with 2/8 and 1/8 rats in the 0.5 mg and 1 mg MMC-H2O groups (p = 0.47 and p = 1.00, respectively).Greater toxicity was seen in animals treated with MMC-hydrogel compared with MMC-H2O, as demonstrated by lower body weights at necropsy (p = 0.000) and a tendency for more severe clinical signs in the 1 and 2 mg MMC-hydrogel groups. Rats that died prematurely received 1 mg (4/10) or 2 mg (9/10) of MMC-hydrogel.Conclusions:Under the current model conditions it is unclear whether instillation of MMC-hydrogel is more effective than MMC-H2O. Nonetheless, the observed difference in toxicity, acting as a surrogate marker for systemic MMC exposure in the MMC-hydrogel-treated rats, supports the prolonged drug release mechanism of the hydrogel.

New MCM8 mutation associated with premature ovarian insufficiency and chromosomal instability in a highly consanguineous Tunisian family

To identify the gene(s) involved in the etiology of premature ovarian insufficiency in a highly consanguineous Tunisian family. Genetic analysis of a large consanguineous family with several affected siblings. University hospital-based cytogenetics and molecular genetics laboratories. A highly consanguineous Tunisian family with several affected siblings born to healthy second-degree cousins. None. Targeted exome sequencing was performed by next-generation sequencing for affected family members. Mutations were validated by Sanger sequencing. Functional experiments were performed to explore the deleterious effects of the identified mutation. DNA damage was induced by increasing mitomycin C (MMC) concentrations on cultured peripheral lymphocytes. Analysis of the next-generation sequencing data revealed a new homozygous missense mutation in the minichromosome maintenance 8 gene (MCM8).This homozygous mutation (c. 482A>C; p.His161Pro) was predicted to be deleterious and segregated with the disease in the family. MCM8 participates in homologous recombination during meiosis and DNA double-stranded break repair by dimerizing with MCM9. Mcm8 knock out results in an early block in follicle development and small gonads. Given this, we tested the chromosomal breakage repair capacity of homozygous and heterozygous MCM8 p.His161Pro mutation on cultured peripheral lymphocytes exposed to increasing MMC concentrations. We found that chromosomal breakage after MMC exposure was significantly higher in cells from homozygously affected individuals than in those from a healthy control. Our findings provide additional support to the view that MCM8 mutations are involved in the primary ovarian insufficiency phenotype.

Sex-specific effects of cytotoxic chemotherapy agents cyclophosphamide and mitomycin C on gene expression, oxidative DNA damage, and epigenetic alterations in the prefrontal cortex and hippocampus - an aging connection.

Recent research shows that chemotherapy agents can be more toxic to healthy brain cells than to the target cancer cells. They cause a range of side effects, including memory loss and cognitive dysfunction that can persist long after the completion of treatment. This condition is known as chemo brain. The molecular and cellular mechanisms of chemo brain remain obscure. Here, we analyzed the effects of two cytotoxic chemotherapy drugs—cyclophosphamide (CPP) and mitomycin C (MMC) - on transcriptomic and epigenetic changes in the murine prefrontal cortex (PFC) and hippocampal regions. We for the first time showed that CPP and MMC treatments led to profound sex- and brain region-specific alterations in gene expression profiles. Gene expression changes were most prominent in the PFC tissues of female mice 3 weeks after MMC treatment, and the gene expression response was much greater for MCC than CPP exposure. MMC exposure resulted in oxidative DNA damage, evidenced by accumulation of 8-oxo-2′-deoxyguanosine (8-oxodG) and a decrease in the level of 8-oxodG repair protein OGG1 in the PFC of female animals 3 weeks after treatment. MMC treatment decreased global DNA methylation and increased DNA hydroxymethylation in the PFC tissues of female mice. The majority of the changes induced by chemotherapy in the PFC tissues of female mice resembled those that occur during the brain's aging processes. Therefore, our study suggests a link between chemotherapy-induced chemo brain and brain aging, and provides an important roadmap for future analysis.

HUS1 regulates in vivo responses to genotoxic chemotherapies.

Cells are under constant attack from genotoxins and rely on a multifaceted DNA damage response (DDR) network to maintain genomic integrity. Central to the DDR are the ATM and ATR kinases, which respond primarily to double-strand DNA breaks (DSBs) and replication stress, respectively. Optimal ATR signaling requires the RAD9A-RAD1-HUS1 (9-1-1) complex, a toroidal clamp that is loaded at damage sites and scaffolds signaling and repair factors. Whereas complete ATR pathway inactivation causes embryonic lethality, partial Hus1 impairment has been accomplished in adult mice using hypomorphic (Hus1(neo)) and null (Hus1(Δ1)) Hus1 alleles, and here we use this system to define the tissue- and cell type-specific actions of the HUS1-mediated DDR in vivo. Hus1(neo/Δ1) mice showed hypersensitivity to agents that cause replication stress, including the crosslinking agent mitomycin C (MMC) and the replication inhibitor hydroxyurea, but not the DSB inducer ionizing radiation. Analysis of tissue morphology, genomic instability, cell proliferation and apoptosis revealed that MMC treatment caused severe damage in highly replicating tissues of mice with partial Hus1 inactivation. The role of the 9-1-1 complex in responding to MMC was partially ATR-independent, as a HUS1 mutant that was proficient for ATR-induced checkpoint kinase 1 phosphorylation nevertheless conferred MMC hypersensitivity. To assess the interplay between the ATM and ATR pathways in responding to replication stress in vivo, we used Hus1/Atm double mutant mice. Whereas Hus1(neo/neo) and Atm(-/-) single mutant mice survived low-dose MMC similar to wild-type controls, Hus1(neo/neo)Atm(-/-) double mutants showed striking MMC hypersensitivity, consistent with a model in which MMC exposure in the context of Hus1 dysfunction results in DSBs to which the ATM pathway normally responds. This improved understanding of the inter-dependency between two major DDR mechanisms during the response to a conventional chemotherapeutic illustrates how inhibition of checkpoint factors such as HUS1 may be effective for the treatment of ATM-deficient and other cancers.

Expression and mutational analysis of DinB-like protein DR0053 in Deinococcus radiodurans.

In order to understand the mechanism governing radiation resistance in Deinococcus radiodurans, current efforts are aimed at identifying potential candidates from a large repertoire of unique Deinococcal genes and protein families. DR0053 belongs to the DinB/YfiT protein family, which is an over-represented protein family in D. radiodurans. We observed that dr0053 transcript levels were highly induced in response to gamma radiation (γ-radiation) and mitomycin C (MMC) exposure depending on PprI, RecA and the DrtR/S two-component signal transduction system. Protein profiles demonstrated that DR0053 is a highly induced protein in cultures exposed to 10 kGy γ-radiation. We were able to determine the transcriptional start site of dr0053, which was induced upon irradiation, and to assign the 133-bp promoter region of dr0053 as essential for radiation responsiveness through primer extension and promoter deletion analyses. A dr0053 mutant strain displayed sensitivity to γ-radiation and MMC exposure, but not hydrogen peroxide, suggesting that DR0053 helps cells recover from DNA damage. Bioinformatic analyses revealed that DR0053 is similar to the Bacillus subtilis protein YjoA, which is a substrate of bacterial protein-tyrosine kinases. Taken together, the DNA damage-inducible (din) gene dr0053 may be regulated at the transcriptional and post-translational levels.

Role of recombinant human erythropoietin against mitomycin C-induced cardiac, hepatic and renal dysfunction in Wistar rats.

Mitomycin C (MMC) is one of the most effective chemotherapeutic drugs. However, the dose of MMC is greatly limited by its toxicity in normal tissues. Recombinant human erythropoietin (rhEPO), an erythropoietic hormone, has also been shown to exert tissue protective effects. The purpose of this study was to explore the protective effect of rhEPO against MMC-induced heart, liver, and renal dysfunction. Adult male Wistar rats were divided into six groups (with six animals each), namely control, rhEPO alone group, MMC alone group, and rhEPO + MMC group (pre-, co-, and posttreatment conditions). The results showed that MMC induced a marked cardiac, renal, and liver failure characterized by a significant decrease in body weight, organs weight, and organs ratio and a significant increase in creatinine, blood urea nitrogen, alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase, and conjugated and total bilirubin levels in serum. Histological examination showed that MMC caused liver alterations. rhEPO treatment restored body weight, organs weight, and organs ratio as well as serum biochemical parameters and histological damage caused by MMC exposure.

The Hypothetical Protein ‘All4779’, and Not the Annotated ‘Alr0088’ and ‘Alr7579’ Proteins, Is the Major Typical Single-Stranded DNA Binding Protein of the Cyanobacterium, Anabaena sp. PCC7120

Single-stranded DNA binding (SSB) proteins are essential for all DNA-dependent cellular processes. Typical SSB proteins have an N-terminal Oligonucleotide-Binding (OB) fold, a Proline/Glycine rich region, followed by a C-terminal acidic tail. In the genome of the heterocystous nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC7120, alr0088 and alr7579 are annotated as coding for SSB, but are truncated and have only the OB-fold. In silico analysis of whole genome of Anabaena sp. strain PCC7120 revealed the presence of another ORF ‘all4779’, annotated as a hypothetical protein, but having an N-terminal OB-fold, a P/G-rich region and a C-terminal acidic tail. Biochemical characterisation of all three purified recombinant proteins revealed that they exist either as monomer or dimer and bind ssDNA, but differently. The All4779 bound ssDNA in two binding modes i.e. (All4779)35 and (All4779)66 depending on salt concentration and with a binding affinity similar to that of Escherichia coli SSB. On the other hand, Alr0088 bound in a single binding mode of 50-mer and Alr7579 only to large stretches of ssDNA, suggesting that All4779, in all likelihood, is the major typical bacterial SSB in Anabaena. Overexpression of All4779 in Anabaena sp. strain PCC7120 led to enhancement of tolerance to DNA-damaging stresses, such as γ-rays, UV-irradiation, desiccation and mitomycinC exposure. The tolerance appears to be a consequence of reduced DNA damage or efficient DNA repair due to increased availability of All4779. The ORF all4779 is proposed to be re-annotated as Anabaena ssb gene.

Drug-Induced Trafficking of P-Glycoprotein in Human Brain Capillary Endothelial Cells as Demonstrated by Exposure to Mitomycin C

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality.

Characterization of the Burkholderia thailandensis SOS response by using whole-transcriptome shotgun sequencing.

The bacterial SOS response is a well-characterized regulatory network encoded by most prokaryotic bacterial species and is involved in DNA repair. In addition to nucleic acid repair, the SOS response is involved in pathogenicity, stress-induced mutagenesis, and the emergence and dissemination of antibiotic resistance. Using high-throughput sequencing technology (SOLiD RNA-Seq), we analyzed the Burkholderia thailandensis global SOS response to the fluoroquinolone antibiotic, ciprofloxacin (CIP), and the DNA-damaging chemical, mitomycin C (MMC). We demonstrate that a B. thailandensis recA mutant (RU0643) is ∼4-fold more sensitive to CIP in contrast to the parental strain B. thailandensis DW503. Our RNA-Seq results show that CIP and MMC treatment (P < 0.01) resulted in the differential expression of 344 genes in B. thailandensis and 210 genes in RU0643. Several genes associated with the SOS response were induced and include lexA, uvrA, dnaE, dinB, recX, and recA. At the genome-wide level, we found an overall decrease in gene expression, especially for genes involved in amino acid and carbohydrate transport and metabolism, following both CIP and MMC exposure. Interestingly, we observed the upregulation of several genes involved in bacterial motility and enhanced transcription of a B. thailandensis genomic island encoding a Siphoviridae bacteriophage designated E264. Using B. thailandensis plaque assays and PCR with B. mallei ATCC 23344 as the host, we demonstrate that CIP and MMC exposure in B. thailandensis DW503 induces the transcription and translation of viable bacteriophage in a RecA-dependent manner. This is the first report of the SOS response in Burkholderia spp. to DNA-damaging agents. We have identified both common and unique adaptive responses of B. thailandensis to chemical stress and DNA damage.

Ionizing radiation or mitomycin-induced micronuclei in lymphocytes of BRCA1 or BRCA2 mutation carriers

BRCA1 and BRCA2 genes are essential in preserving the integrity of genome, and it is not unambiguously clear whether the heterozygosity status may affect BRCA1 or BRCA2 functions. This may have implications for the clinical management of BRCA1 and BRCA2 mutation carriers both in breast cancer (BC) screening modality and in cancer treatment based on DNA-damaging or DNA-repair-inhibiting drugs. We investigated whether lymphocytes carrying BRCA1 or BRCA2 mutations displayed an increased sensitivity to radiation or mitomycin C (MMC) in vitro treatments. Peripheral blood from 21 BRCA1 mutation carriers (12 with BC and 9 healthy), 24 BRCA2 carriers (13 with BC and 11 healthy), 15 familial BC patients without detected mutation in BRCA1 or BRCA2 and 16 controls without familial history of cancer (5 with BC and 11 healthy) were irradiated or treated with MMC. Chromosomal damage was measured using the cytokinesis-block micronucleus assay. We evaluated micronuclei (MN) and nucleoplasmic bridges (NPBs). The BRCA2 mutation carriers and familial BC patients without detected mutation in BRCA1 or BRCA2 showed less basal NPB than BRCA1 carriers and controls. The BRCA1 (+/-) or BRCA2 (+/-) lymphocytes did not have increased frequencies of MN or NPB after irradiation. In contrast, BRCA2 (+/-) lymphocytes presented higher levels of MN after MMC exposure than BRCA1 carriers and controls. The monoallelic BRCA1 or BRCA2 pathogenic mutations seem not to be associated with an enhanced radiosensitivity. The mutation of one BRCA2 allele conferred an increased sensitivity to MMC, presumably because of the role of this gene in the repair of MMC-induced DNA damage. This finding indicates that the MMC-induced MN analysis could be useful in identifying functional deficiencies of BRCA2 or genes related to BRCA2. Since MMC can be used as an anti-cancer drug, these data may be relevant for the management and follow-up of BRCA2 mutation carriers.

Enhancement of mitomycin C-induced apoptosis in Nrf2-deficient human colon cancer cells

The nuclear erythroid factor 2-related factor 2 (Nrf2) is widely known as an important redox factor to protect cells from oxidative stress. However, recently it is known that Nrf2 in cancer cells provides advantages for cell viability from the exposure to anticancer drugs. Mitomycin C (MMC) is an anticancer drug for treatment of several types of cancers including colon and breast cancers. It is potent DNA crosslinker. MMC is able to induce ROS as part of its mechanism of action, which exerts detrimental effects to cancer cells. In this study, we investigated the role of Nrf2 in resistance to MMC in human colon cancer cells. Stable Nrf2 shRNA-expressing RKO showed higher sensitivity to MMC exposure than Nrf2 wild type cells. Furthermore, significantly increased apoptosis was associated with high level of oxidative stress and expression of DNA damage sensitive protein, phosphorylated H2AX (gamma-H2AX) in Nrf2 deficient human colon RKO cells. In conclusion, we demonstrated for the first time that Nrf2 enhanced MMCresistance via inhibition of oxidative stress and DNA damage in human colon cancer cells. Therefore, we suggested that Nrf2 might be potential therapeutic target in the MMC treatment of colon cancer.

Comparison of Standard (0.02%) and Low Dose (0.002%) Mitomycin C in the Prevention of Corneal Haze Following Surface Ablation for Myopia

To retrospectively compare the safety and efficacy of lower dose mitomycin C (MMC) (0.002%) to that of the standard dose (0.02%) in eyes treated with photorefractive keratectomy (PRK) for myopia. The clinical efficacy of 95 eyes receiving myopic PRK with a standard concentration of MMC (0.02%) is sequentially compared to 126 eyes receiving low dose MMC (0.002%). The topical exposure times for MMC varied between 30 seconds and 2 minutes in both groups, and direct contralateral eye comparison of these two exposure times was analyzed in a prospective subset of 39 patients from among the low dose group. Patients were examined preoperatively and postoperatively at 1, 3, 6-9, and 9-12 months. Haze, visual acuity, and efficacy ratio outcomes were analyzed. The preoperative findings were overall statistically similar, except for higher spherical equivalent refractive error (P = .007) and best spectacle-corrected visual acuity (P = .007) in the standard MMC group. Postoperatively, haze levels ranged from 0 to 4+. With multivariable analysis, significantly less haze was noted among the standard dose MMC eyes for high myopia and higher ablation depth at all postoperative time points. In contrast, the haze levels were statistically similar for moderate myopia and lower ablation depths at the latter postoperative time points. The subset of contralateral eyes randomly receiving low dose MMC (0.002%) at either 30 seconds or 2 minutes exposure showed no significant difference in haze between these exposure times. The standard concentration of topical MMC (0.02%) is more effective than low dose MMC (0.002%) in preventing postoperative haze following surface ablation for myopia > or = -6.00 D and deeper ablation depth > or = 75 microm. However, for moderate myopia and shallow depth, low dosing appears to be equally effective. The duration of MMC exposure appears to be less important than its concentration.

Expression of 1-Cys Peroxiredoxin in the Corneal Wound-Healing Process

To study the regulation by mitomycin C (MMC) of 1-cys peroxiredoxin (Prx) expression during the corneal wound-healing process and its induction pathway in cultured bovine keratocytes (BKs). Rat corneas were excised at 4 hours, 12 hours, 1 day, 3 days, and 7 days after photorefractive keratectomy (PRK). Expression of 1-cys Prx in the corneas was examined by Northern blot and immunoblot analyses. Cultured BKs were exposed to 0.02% MMC for 5 minutes and maintained under normal culture conditions for different time periods. Subsequently, levels of 1-cys Prx and extracellular signal-regulated kinase (ERK)1/2 expression were measured by immunoblot analysis using polyclonal 1-cys Prx, ERK1/2, or phospho-ERK1/2 antibodies. To inhibit ERK1/2 activation, the BKs were pretreated with 50 micromol/mL PD98059 for 1 hour before MMC exposure and incubated in complete medium with or without PD98059 for 24 hours. MMC-induced cytotoxicity was determined by colorimetric cell-counting kit-8 assay. Increased levels of 1-cys Prx expression were seen in wounded rat corneas at 12 hours after injury and reached the highest level between 1 and 3 days, during which time active proliferation occurred. Induction of 1-cys Prx expression was obvious in proliferating BKs stimulated by growth factors. MMC treatment in cultured BKs resulted in increased expression of 1-cys Prx and phospho-ERK1/2 in a time-dependent manner. Treatment with 50 micromol/mL PD98059 significantly inhibited the active ERK1/2 and 1-cys Prx expression induced by MMC, leading to synergistic cytotoxicity in the BKs. 1-Cys Prx may function as an important enzyme in cell proliferation during the postinjury corneal wound-healing process. Furthermore, the induction of 1-cys Prx expression through the ERK1/2 signaling pathway may contribute to cellular defense against cytotoxic agents, thus playing an important role in cell survival.

Knockdown of the Fanconi Anemia Gene FANCD2 Directly Affects Hematopoiesis in Human Embryonic Stem Cells.

Fanconi anemia (FA) is a rare autosomal recessive disease characterized by the progressive bone marrow failure, developmental anomalies and cancer susceptibility. Twelve distinct FA complementation groups have been identified (A–C, D1, D2, E–G, I, J, L, M) and 11 cDNAs cloned. A current working model proposes that eight FA proteins (A, B, C, E, F, G, L and M) assemble to form a multi-protein nuclear complex, involved the repair of damaged DNA. The FA complex facilitates the monoubiquitination of the FANCD2 protein following exposure to DNA damaging agents. Ubiquinated FANCD2 co-localizes with the key DNA repair proteins RAD51, BRCA2 (know as FANCD1), BRCA1, and FANCJ (BACH1) to promote homologous recombination. The hallmark of FA patients is marrow failure due to defective hematopoietic stem cells. The study of human FA stem cell biology is severely restricted due to the few CD34+ cells that can be isolated from FA patients compared with normal individuals. We generated human embryonic stem cells (hESC) exhibiting FA phenotype by introducing small double strand RNA species to ablate FANCD2 gene function in hESC. Human ES cells were then differentiated into hematopoietic cells to investigate FA hematopoiesis. FANCD2-specific small hairpin RNAs (shRNAs) were designed and cloned into a self-inactivated lentiviral vector with a GFP cDNA. High titer virus (2×108 iu/ml) was used for transduction of the hESC line H1. More than 90% of H1 cells were transduced with lentivirus that was observed by GFP expression. The FANCD2 protein expression was analyzed by Western blotting; FANCD2 shRNA targeted cells expressed 1–3% of the FANCD2 protein compared with control H1 cells. Functional assessment of the H1-FANCD2 was performed by incubation with DNA cross-linking agent, mitomycin C (MMC). The H1-FANCD2 cells were nearly 10 times more sensitive to MMC than untreated or scrambled shRNA ES cells. The effective dose 50 (ED50) of MMC required to induce apoptosis was only 4 ng/ml in H1-FANCD2 cells compared with 30 ng/ml in mock-infected of scrambled shRNA controls. Cytogentic abnormalities are the hallmark of Fanconi anemia. When H1-FANCD2 cells were exposed to MMC, 57% of cells had abnormal cytogenetics compared with 2% of scrambled shRNA transduced H1 cells. In addition, 75.34±0.69% of H1-FANCD2 cells were arrested at the G2/M phase of the cell cycle after MMC exposure compared with 60.63±2.5% of H1-scramble cells. MMC hypersensitivity, cell cycle defects and chromosomal abnormalities of H1-FANCD2 confirmed the FA phenotype. Differentiation of H1 and H1-scramble cells through a blastocyst intermediate produced a discrete number of KDR+/CD31+ hemangioblast cells that give rise to erythroid and myeloid hematopoietic colonies in methylcellulose culture. H1-FANCD2 ES cells produced blastocyst and hemangioblast cells without significant development of end-stage hematopoietic lineages. In conclusion, we successfully converted, using shRNAs, hESC H1 cells that exhibit the FA phenotype including abnormal hematopoiesis. Generation of mutant FA hES cells will be a valuable model to study FA pathophysiology and treatment.

Laser Epithelial Keratomileusis With Mitomycin C: Indications and Limits

To assess the advantages and safety of using mitomycin C (MMC) with laser epithelial keratomileusis (LASEK) by measuring high order aberrations of the cornea. We analyzed the results of 86 eyes that had LASEK plus a brushstroke of MMC 0.01%. Another group of 100 eyes had LASEK only (control group). Mean follow-up was 312 days for the MMC group and 774 days for the control group. A Nidek EC-5000 laser was used in all eyes. We measured mean refractive error, best spectacle-corrected visual acuity (BSCVA), haze, and high order aberrations before and at 1 month and nearly 1 year after LASEK. Mean baseline spherical equivalent refraction was -6.80 +/- 3.00 D for the MMC group and -6.00 +/- 3.10 D for the control group (P=.34). In eyes with low myopia (-1.20 to -10.80 D), a single ablation zone of 6 mm with a transition zone of 8 mm was used, and in the higher myopia group (-5.60 to -15.50 D), smoothing and a cross sphere procedure (+0.75 D) were used. Mean spherical equivalent refraction after LASEK for low myopia in the MMC group at last examination (mean follow-up 312 days) was +0.50 +/- 0.93, and 0 +/- 0.34 D for the control group. Mean spherical equivalent refraction after LASEK for high myopia in the MMC group at last examination (mean follow-up 312 days) was +0.50 +/- 1.03 D, and +0.40 +/- 0.78 D for the control group. Mean haze in the low myopia MMC group was 0.06 and in the control group, 0.11; in the high myopia MMC group it was 0.1, and in the control group, 0.27 (all differences P<.05). No differences between baseline and postoperative BSCVA were noted. Early and late overcorrection were noted in the MMC group. Increased high order aberrations were evident at 1 month and 1 year in the MMC groups. The use of mitomycin C 0.01% with LASEK significantly decreased subepithelial haze. However, refractive outcome was less predictable. High order aberrations increased after LASEK with MMC. Additional studies with longer follow-up are necessary to evaluate long-term effects, and ideal MMC concentration and exposure time.

Topical exposure of mitomycin C reduces opacification of the residual anterior lens capsule and lenticular regeneration after vitrectomy and lensectomy in rabbits.

To examine the effect of topical administration of mitomycin C (MMC) on the opacification of residual anterior lens capsule in a vitrectomized/lensectomized eye. Adult Japanese albino rabbits ( n=8) received two-port vitrectomy and lensectomy with or without topical administration of MMC (0.4 mg/ml) to the residual anterior lens capsule in one eye under both general and topical anesthesia. Four eyes after vitrectomy and lensectomy were exposed to MMC for 1 min and washed out with balanced salt solution. After 1 month, the anterior segment of each globe was observed with EAS-1000 in vivo under general anesthesia and then the enucleated globes were observed through the Miyake view. Histological examination was performed. During healing intervals in eyes without MMC exposure, regenerated lens structure of Soemmering's ring or fibrous tissue was formed in peripheral or central areas of the residual capsule, respectively. Both the anterior lens capsule opacification and lens fiber regeneration were statistically significantly reduced by MMC topical exposure. Lens epithelial cells produce regenerated lenticular structure and fibrous tissue on the residual capsule following vitrectomy and lensectomy in rabbits. Exposure of the residual anterior capsule to MMC may be effective in reducing both its opacification and lens fiber regeneration after vitrectomy/lensectomy.

Outcomes of primary phakic trabeculectomies without versus with 0.5- to 1-minute versus 3- to 5-minute mitomycin C

PURPOSE: To compare the intraocular pressure and hypotony outcomes of primary phakic trabeculectomies with no mitomycin C (MMC), shorter MMC, and longer MMC exposure. METHODS: We evaluated primary phakic trabeculectomies with no MMC (36 eyes of 36 patients), 0.5- to 1-minute MMC (50 eyes of 50 patients), and 3- to 5-minute MMC (38 eyes of 38 patients) at the concentration of 0.5 mg/ml. Successful trabeculectomy was defined as an intraocular pressure of 21 mm Hg or less without development of a marked visual acuity loss associated with prolonged hypotony (intraocular pressure < 6 mm Hg over 3 months) and without the need for additional surgery to control intraocular pressure or treat postoperative complications. RESULTS: The three groups were similar in demographics, preoperative intraocular pressure, and medical dependency. However, the incidence of hypotony during the postoperative periods of 3 to 12 months was significantly higher in the 3- to 5-minute MMC group ( P < .05, chi-square test). Severe visual acuity loss associated with hypotony was also more frequently found in the 3- to 5-minute MMC group than in the 0.5- to 1-minute ( P = .009, chi-square test) group or the control group ( P = .014, chi-square test). In addition, the success probabilities were significantly different among the three groups ( P = .001, Kaplan-Meier survival analysis with log-rank test) and were the highest in the 0.5- to 1-minute MMC group and the lowest in the 3- to 5-minute MMC group. CONCLUSION: Shorter application (0.5 to 1 minute) of MMC appears to be optimal for the successful outcome of primary phakic trabeculectomy compared with no MMC or longer application of MMC at a concentration of 0.5 mg/ml.