Mitomycin C Resistance Research Articles

Lack of the Bacterial Phytochrome Protein Decreases Deinococcus radiodurans Resistance to Mitomycin C.

Deinococcus radiodurans known for its extraordinary resistance to ionizing radiation contains bacterial phytochrome (BphP), a member of the family of red/far-red light-sensing proteins. In this study, we constructed a bphP mutant strain (ΔbphP) to investigate the role of D. radiodurans BphP (DrBphP) in the DNA damage response. When cells were incubated under light and dark conditions following exposure to DNA damaging agents, such as γ- and UV-radiation and mitomycin C (MMC), no significant difference in cell survival was observed between the wild-type D. radiodurans strain (WT) and ΔbphP. However, when continuously exposed to MMC under light conditions, the WT strain notably exhibited increased survival compared to cells grown in the dark. The increased survival was not observed in the ΔbphP strain. These results are indicative of the protective role of light-activated DrBphP in the presence of MMC. Site-directed mutagenesis revealed that the conserved amino acids Cys-24 and His-532 involved in chromophore binding and signal transduction, respectively, were essential for the protective function of DrBphP. Inactivation of the cognate response regulator (RR; DrBphR) of DrBphP increased MMC resistance in the dark. In trans complementation of the bphP bphR double mutant strain (ΔbphPR) with DrBphR decreased MMC resistance. Considering that DrBphP acts as a light-activated phosphatase that dephosphorylates DrBphR, it appears that phosphorylated DrBphR exerts a negative effect on cell survival in the presence of MMC. DrBphP overexpression resulted in an increase in MMC resistance of ΔbphPR, implying that other RRs might be involved in the DrBphP-mediated signaling pathway. A mutant lacking the dr_0781 gene (Δdr_0781) demonstrated the same MMC phenotype as ΔbphR. Survival was further increased in the bphR dr_0781 double mutant strain compared to each single mutant ΔbphR or Δdr_0781, suggesting that DR_0781 is also involved in the DrBphP-dependent MMC sensitivity. This study uncovered a previously unknown phenomenon of red/far-red light-dependent DNA damage survival mediated by BphP by identifying the conditions under which DrBphP exhibits a fitness advantage.

Changing the Natural History of Fanconi Anemia Complementation Group-À with Gene Therapy: Early Results of U.S. Phase I Study of Lentiviral-Mediated Ex-Vivo FANCA Gene Insertion in Human Stem and Progenitor Cells

Background Fanconi anemia (FA) is a rare genetic disorder characterized by defective cellular DNA repair, associated developmental abnormalities, progressive bone marrow failure (BMF), and a predisposition to hematologic malignancies and solid tumors. 80% of FA patients develop BMF due to progressive depletion of their BM stem cells. Although allogeneic HSCT is a curative treatment for BMF, its utilization and efficacy is limited by availability of donors, risk of GVHD and transplant-related toxicities. Pre-clinical studies showed that ex-vivo insertion of a functional FANCA gene into autologous FA-A CD34+ HSPCs provides a survival advantage to the gene-modified stem cells, leading to correction of BMF. Feasibility of this approach was established in the FANCOLEN-1 clinical trial (Spain). Modifications to the collection and manufacturing processes were made in the Phase I study in the U.S. Design and Methods RP-L102-0418 (clinicaltrials.gov # NCT03814408) is a Phase I clinical trial evaluating the feasibility and safety of autologous CD34+ cells transduced with a lentiviral vector (LV) carrying the FANCA gene (PGK-FANCA-WPRE) in children with FA-A. Patients with early evidence of cytopenias, and with BM CD34+ count >30/µL were eligible for treatment. PBMCs were collected via leucocytapheresis after mobilization with G-CSF and Plerixafor. CD34+ HSPCs were enriched, placed in culture with cytokines, and transduced with PGK-FANCA-WPRE LV. The investigational drug product (DP) (RP-L102) was infused fresh in patients within 4 hours of release, without any conditioning regimen. Patients are being followed for safety assessments (replication competent lentivirus, insertion site analysis) and efficacy (increasing peripheral blood vector copy number and BM mitomycin-C resistance), along with monitoring of cytopenias. Results Two FA-A patients (aged 5 and 6 years) were consented and enrolled on the study at Stanford University. Mobilization and apheresis procedures were performed successfully without any serious adverse events. Both the patients needed RBC priming of the apheresis circuit because of their weight Conclusions DP was successfully manufactured and infused in the Phase I study and both FA-A patients are showing early signs of stabilization of cytopenias. Plans for opening the Phase II study are in progress. Preventing BMF by gene therapy without any exposure to chemo-radiotherapy has the potential to change the natural history of FA.

FANCN Hypomorphic Mutation Retains BRCA1 Binding Domain

Introduction: Fanconi anemia (FA) is a recessive genetic disease that can result in bone marrow failure, morphological defects and cancer predisposition. Patients with mutations in the FA gene, FANCN/PALB2 (partner and localizer of BRCA2) typically have more severe disease with earlier onset of cancer and bone marrow failure. FANCN is known to promote homologous recombination (HR) and contains an N-terminal BRCA1 binding domain, and a C-terminal BRCA2 binding domain. To further investigate the function of FANCN, we used recombinant adeno-associated virus (rAAV)-mediated gene targeting techniques to make a conditional null FANCN cell line. We targeted early exons 2 and 3 and found that removal of the conditional allele resulted in spontaneous chromosomal breaks and rearrangements that ultimately lead to chromosomal catastrophe within 48 hours. Due to the essential function of FANCN in genome stability, we were curious how FANCN patients can survive with biallelic loss of function mutations in FANCN.Methods: A hypomorphic FANCN cell line was created using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated 9 (Cas9)-mediated gene targeting of FANCN in exon 5. In addition, we complemented the FANCN conditional null cell line with four different FANCN cDNAs containing patient mutations with premature stop codons that completely eliminate or disrupt the N-terminal BRCA2 binding domain. These FANCN mutant cell lines have been investigated for expression of truncated protein and will be tested in their ability to bind and form foci with BRCA1 and BRCA2. Finally, these FANCN mutant cell lines will be evaluated in their sensitivity to mitomycin C (MMC), PARP inhibitors, replication stress, and in their ability to repair DNA.Results: We have determined that FANCN is essential for maintaining genomic stability and cellular viability. However, contrary to the lethality of deleting exons 2 and 3, the biallelic frameshift mutations in exon 5 resulted in a viable FANCN mutant cell line. These hypomorphic FANCN exon 5 frameshift mutations truncate the C-terminal BRCA2 binding domain, and this cell line has a growth defect and MMC sensitivity. Furthermore, we have demonstrated that the patient mutation complemented cell lines are expressing truncated FANCN proteins that will be functionally characterized.Conclusions: We are beginning to unveil important and separate functional domains of FANCN. For example, the FANCN N-terminus with the BRCA1 binding domain serves an essential function in viability and genomic stability, whereas the FANCN C-terminus with the BRCA2 binding domain servers an essential role in cell proliferation and MMC resistance. This research is expected to continue to advance our mechanistic understanding of FANCN and how these FANCN patient mutations result in disease. In addition, these novel FANCN cell lines provide a means for screening potential new therapeutics for the treatment of Fanconi anemia. DisclosuresHendrickson:Horizon Discovery , Humanetics and Intellia Therapeutics: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties: licensing fees, Research Funding.

Mitomycin C treatment induces resistance and enhanced migration via phosphorylated Akt in aggressive lung cancer cells.

Since 1984, mitomycin C (MMC) has been applied in the treatment of non-small-cell lung cancer (NSCLC). MMC-based chemotherapeutic regimens are still under consideration owing to the efficacy and low cost as compared with other second-line regimens in patients with advanced NSCLC. Hence, it is important to investigate whether MMC induces potential negative effects in NSCLC. Here, we found that the malignant lung cancer cells, CL1-2 and CL1-5, were more resistant to MMC than were the parental CL1-0 cells and pre-malignant CL1-1 cells. CL1-2 and CL1-5 cells consistently showed lower sub-G1 fractions post MMC treatment. DNA repair-related proteins were not induced more in CL1-5 than in CL1-0 cells, but the levels of endogenous and MMC-induced phosphorylated Akt (p-Akt) were higher in CL1-5 cells. Administering a p-Akt inhibitor reduced the MMC resistance, demonstrating that p-Akt is important in the MMC resistance of CL1-5 cells. Furthermore, we revealed that cell migration was enhanced by MMC but lowered by a p-Akt inhibitor in CL1-5 cells. This study suggests that in CL1-5 cells, the activity of p-Akt, rather than DNA repair mechanisms, may underlie the resistance to MMC and enhance the cells' migration abilities after MMC treatment.

Identification of Escherichia coli ygaQ and rpmG as novel mitomycin C resistance factors implicated in DNA repair

Using the ASKA (A Complete Set of Escherichia coli K-12 ORF Archive) library for genome-wide screening of E. coli proteins we identified that expression of ygaQ and rpmG promotes mitomycin C resistance (MMC(R)). YgaQ mediated MMC(R) was independent of homologous recombination involving RecA or RuvABC, but required UvrD. YgaQ is an uncharacterized protein homologous with α-amylases that we identified to have nuclease activity directed to ssDNA of 5' flaps. Nuclease activity was inactivated by mutation of two amino acid motifs, which also abolished MMC(R). RpmG is frequently annotated as a bacterial ribosomal protein, although forms an operon with MutM glycosylase and a putative deubiquitinating (DUB) enzyme, YicR. RpmG associated MMC(R) was dependent on MutM. MMC(R) from RpmG resembles DNA repair phenotypes reported for 'idiosyncratic ribosomal proteins' in eukaryotes.

Selective Alleviation of Mitomycin C Sensitivity in lexA3 Strains of Escherichia coli Demands Allele Specificity of rif-nal Mutations: A Pivotal Role for rpoB87-gyrA87 Mutations

Very recently, we have reported about an unconventional mode of elicitation of Mitomycin C (MMC) specific resistance in lexA3 (SOS repair deficient) mutants due to a combination of Rif-Nal mutations (rpoB87-gyrA87). We have clearly shown that UvrB is mandatory for this unconventional MMC resistance in rpoB87-gyrA87-lexA3 strains and uvrB is expressed more even without DNA damage induction from its LexA dependent promoter despite the uncleavable LexA3 repressor. The rpoB87 allele is same as the rpoB3595 which is known to give rise to a fast moving RNA Polymerase and gyrA87 is a hitherto unreported NalR allele. Thus, it is proposed that the RNA Polymerase with higher elongation rate with the mutant DNA Gyrase is able to overcome the repressional hurdle posed by LexA3 to express uvrB. In this study we have systematically analysed the effect of three other rpoB (rif) mutations-two known to give rise to fast moving RNAP (rpoB2 and rpoB111) and one to a slow moving RNAP (rpoB8) and four different alleles of gyrA NalR mutations (gyrA199, gyrA247, gyrA250, gyrA259) isolated spontaneously, on elicitation of MMC resistance in lexA3 strains. Our results indicate that in order to acquire resistance to 0.5 µg/ml MMC cells require both rpoB87 and gyrA87 but resistance to 0.25 µg/ml of MMC can be brought about by either rpoB87, gyrA87, fast moving rpoB mutations or other nal mutations also. We have also depicted increased constitutive uvrB expression in strains carrying fast moving RNAP (rpoB2 and rpoB111) with gyrA87 and another nal mutation with rpoB87 and expression level in these strains is lesser than rpoB87-gyrA87 strain. These results evidently suggest an allele specific role for the rif-nal mutations to acquire MMC resistance in lexA3 strains via increased constitutive uvrB expression and a pivotal role for rpoB87-gyrA87 combination to elicit higher levels of resistance.

Evidence for involvement of UvrB in elicitation of ‘SIR’ phenotype by rpoB87-gyrA87 mutations in lexA3 mutant of Escherichia coli

An unconventional DNA repair termed SIR (SOS Independent Repair), specific to mitomycin C (MMC) damage elicited by a combination of specific RifR (rpoB87) and NalR (gyrA87) mutations in SOS un-inducible strains of Escherichia coli was reported by Kumaresan and Jayaraman (1988). We report here that the rpoB87 mutation defines a C1565→T1565 transition changing S522→F522 and gyrA87 defines a G244→A244 transition changing D82→N82. The reconstructed lexA3 rpoB87 gyrA87 strain (DM49RN) exhibited resistance to MMC but not to UV as expected. When mutations in several genes implicated in SOS/NER were introduced into DM49RN strain, uvrB mutation alone decreased the MMC resistance and suppressed SIR phenotype. This was alleviated about two fold by a plasmid clone bearing the uvrB+ allele. Neither SulA activity as measured based on filamentation and sulA::gfp fluorescence analyses nor the transcript levels of sulA as seen based on RT-PCR analyses indicate a change in sulA expression in DM49RN strain. However, uvrB transcript levels are increased with or without MMC treatment in the same strain. While the presence of lexA3 allele in a plasmid clone was found to markedly decrease the MMC resistance of the DM49RN strain, the additional presence of uvrB+ allele in the same clone alleviated the suppression of MMC resistance by lexA3 allele to a considerable extent. These results indicate the increased expression of uvrB in the DM49RN strain is probably from the LexA dependent promoter of uvrB. The sequence analyses of various uvrB mutants including those isolated in this study using localized mutagenesis indicate the involvement of the nucleotide phosphate binding domain (ATPase domain) and the ATP binding domain and/or the DNA binding domain of the UvrB protein in the MMC repair in DM49RN. The possible involvement of UvrB protein in the MMC damage repair in DM49RN strain in relation to DNA repair is discussed.

Cyclophosphamide promotes engraftment of gene-modified cells in a mouse model of Fanconi anemia without causing cytogenetic abnormalities

A major hurdle for hematopoietic stem cell (HSC) gene therapy for inherited bone marrow disorders, including Fanconi anemia (FA), is adequate engraftment of gene-modified cells. A phenotypic defect in DNA repair renders FA patients sensitive to alkylating agents such as cyclophosphamide (Cy); however, at lower doses, Cy is well tolerated in the FA transplant setting. We tested whether non-alkylating agents could replace Cy for pretransplant conditioning to enhance engraftment of FANCA gene-modified hematopoietic cells. We compared Cy preconditioning with fludarabine (Flu) or cytarabine (AraC) or no conditioning as a control in fanca ( -/- ) mutant mice receiving gene-modified bone marrow cells. Only mice conditioned with Cy exhibited appreciable engraftment of gene-modified cells by PCR and resistance to mitomycin C (MMC). Cy administration following transplantation increased gene marking levels in all animals treated, but highest gene marking and corresponding MMC resistance were observed in mice receiving Cy pre- and posttransplantation. Importantly, no cytogenetic abnormalities were observed in Cy-treated mice. We conclude that Cy is an effective and superior preparative regimen with respect to engraftment of lentivirus-transduced cells and functional correction in fanca ( -/- ) mice. Thus, appropriately dosed Cy may provide a suitable conditioning regimen for FA patients undergoing HSC gene therapy.

Rosiglitazone Reverses Mitomycin C Resistance in Human Gastric Cancer Cells

IntroductionTo explore the mechanisms of rosiglitazone (ROS), a selective peroxisome proliferator-activated receptor gamma ligand, in reversing mitomycin C (MMC) resistance in a human drug-resistant gastric cancer cell line. MethodsThe vincristine-resistant human gastric cancer cell line SGC7901/VCR and its parental cell line SGC7901 were treated with ROS, MMC (negative control), cyclosporine A+MMC (positive control) or ROS+MMC. A tetrazolium blue (methyl thiazolyl tetrazolium) assay was used to evaluate the sensitivity to these treatments. Flow cytometry analysis and acridine orange-ethidium bromide (AO-EB) fluorescent staining were used to determine the effects of ROS on MMC-induced apoptosis. Reverse transcription polymerase chain reaction and western blotting were used to measure the expression of multidrug resistant 1 (MDR1), Livin and P-glycoprotein (P-gp). ResultsROS administration dose dependently increased the reversal index in MMC-treated SCG7901/VCR cells. ROS increased apoptosis in SGC7901/VCR cells compared with the blank group and MMC group. ROS+MMC also increased apoptosis in SGC7901/VCR cells compared with other groups (P < 0.05 or P < 0.01). The mRNA expression of MDR1 and Livin and the protein expression of P-gp in SGC7901/VCR cells were significantly higher than those in SGC7901 cells (P < 0.01). However, ROS or ROS+MMC treatment markedly upregulated the mRNA expression of MDR1 and Livin and the protein expression of P-gp in SGC7901/VCR cells (P < 0.01). ConclusionsROS reverses MMC resistance in human gastric cancer SGC7901/VCR cells by reducing expression of MDR1, Livin and P-gp and increasing apoptosis.

Involvement of the aldo–keto reductase, AKR1B10, in mitomycin-c resistance through reactive oxygen species-dependent mechanisms

The human aldo-keto reductase (AKR) 1B10 is suggested as a tumor marker in various solid tumors. Using colon cancer cells, we found that AKR1B10 was induced with acquisition of resistance to the anticancer drug mitomycin-c (MMC). In the resistant cells, treatment with an AKR1B10 inhibitor decreased their MMC tolerance. In the nonresistant cells, overexpression and silencing of AKR1B10 decreased and increased, respectively, susceptibility to cytotoxic effects of MMC and 4-hydroxy-2-nonenal, which was formed as a product of lipid peroxidation by MMC treatment. These results suggest a role of AKR1B10 in the development of MMC resistance, which may be mediated by its ability to detoxify cytotoxic aldehydes including 4-hydroxy-2-nonenal.

Resistance of uveal melanoma to the interstrand cross-linking agent mitomycin C is associated with reduced expression of CYP450R.

Background:Uveal melanoma (UM) is the most common primary intraocular tumour of adults, frequently metastasising to the liver. Hepatic metastases are difficult to treat and are mainly unresponsive to chemotherapy. To investigate why UM are so chemo-resistant we explored the effect of interstrand cross-linking agents mitomycin C (MMC) and cisplatin in comparison with hydroxyurea (HU).Methods:Sensitivity to MMC, cisplatin and HU was tested in established UM cell lines using clonogenic assays. The response of UM to MMC was confirmed in MTT assays using short-term cultures of primary UM. The expression of cytochrome P450 reductase (CYP450R) was analysed by western blotting, and DNA cross-linking was assessed using COMET analysis supported by γ-H2AX foci formation.Results:Both established cell lines and primary cultures of UM were resistant to the cross-linking agent MMC (in each case P<0.001 in Student's t-test compared with controls). In two established UM cell lines, DNA cross-link damage was not induced by MMC (in both cases P<0.05 in Students's t-test compared with damage induced in controls). In all, 6 out of 6 UMs tested displayed reduced expression of the metabolising enzyme CYP450R and transient expression of CYP450R increased MMC sensitivity of UM.Conclusion:We suggest that reduced expression of CYP450R is responsible for MMC resistance of UM, through a lack of bioactivation, which can be reversed by complementing UM cell lines with CYP450R.

Enhancement of the efficacy of mitomycin C-mediated apoptosis in human colon cancer cells with RNAi-based thioredoxin reductase 1 deficiency

Thioredoxin reductase 1 (Trr1) is an antioxidant and redox regulator that functions in governing the cellular redox state and survival against oxidative insults in mammals. However, this selenoprotein is also overexpressed in various forms of malignant cancers, leading to the hypothesis that Trr1 may be a potential target for cancer therapy. A quinone anti-cancer drug, mitomycin C (MMC), has been clinically used in the treatment of several types of tumors, including those of the colon. MMC exerts its activity via ROS induction and further results in DNA cross-linkage. To evaluate the significant role of Trr1 in MMC resistance in human colon cancer (RKO) cells, specific reduction in the expression of Trr1 was achieved using short-hairpin RNA (shRNA)-based interference. Our results showed that stable Trr1 shRNA knockdown manifested higher cellular susceptibility to MMC in comparison to that in wild-type cells. In addition, increased intracellular ROS accumulation appeared in the Trr1 shRNA knockdown cells compared to the RKO wild-type cells, in proportion to a relatively higher fraction of the DNA damage reporter protein phosphorylated histone 'γ-H2AX'. Notably, a neutral comet assay demonstrated that DNA double-strand breaks were highly induced in the Trr1-deficient cancer cells in the presence of MMC, presumably stimulating cancer cell death. Our results also revealed that MMC-induced apoptosis was associated with enhancement of oxidative damage to DNA. These results suggest that the specific knockdown of Trr1 expression via shRNA vector interference technology may be a potent molecular strategy by which to enhance the effectiveness of MMC-mediated killing in human colon cancer cells, through acceleration of double-strand DNA damage-oxidative stress as a trigger for apoptosis. This implies that Trr1 may be a prime target for enhancing the effectiveness of MMC chemotherapy in combination with specific RNA interference.

Altered expression of FANCL confers mitomycin C sensitivity in Calu-6 lung cancer cells

Fanconi Anemia (FA) results from mutations in a group of genes whose products, including BRCA2 and BACH1/BRIP1, are known to function in one common pathway (the FA-BRCA pathway) to guard genome integrity, especially when challenged by DNA crosslinking agents, such as Cisplatin and mitomycin C (MMC). The extremely high incidence of cancer in FA patients reveals the essentialness of this pathway in tumor suppression. However, this pathway’s involvement in non-FA cancers is not well understood. To evaluate the contribution of the FA-BRCA pathway to cancer, we investigated the integrity of the FA-BRCA pathway in ten human cancer cell lines. We found that the Calu-6 lung cancer cell line carries a defective FA-BRCA pathway. In this cell line, the examination of six FA proteins, essential for the activation of the FA-BRCA pathway, detected substantially reduced expression of FANCL, a catalytic subunit of the ubiquitin ligase/E3-complex. Reconstitution of FANCL in these cells restored the activation of the FA-BRCA pathway, but MMC sensitivity of the cells with a complemented FA-BRCA pathway was decreased as compared to the cells with an impaired FA-BRCA pathway. Collectively, the abnormal FANCL expression is the cause leading to a defective FA-BRCA pathway, which confers the sensitivity of Calu-6 cells to MMC. This suggests that the correlation of an intact FA-BRCA pathway with MMC resistance may emerge as a common mechanism underlying resistance to DNA crosslinking agents in cancer patients.

Cytogenetic Instability in Ovarian Epithelial Cells from Women at Risk of Ovarian Cancer

Fanconi anemia is an inherited cancer predisposition disease characterized by cytogenetic and cellular hypersensitivity to cross-linking agents. Seeking evidence of Fanconi anemia protein dysfunction in women at risk of ovarian cancer, we screened ovarian surface epithelial cells from 25 primary cultures established from 22 patients using cross-linker hypersensitivity assays. Samples were obtained from (a) women at high risk for ovarian cancer with histologically normal ovaries, (b) ovarian cancer patients, and (c) a control group with no family history of breast or ovarian cancer. In chromosomal breakage assays, all control cells were mitomycin C (MMC) resistant, but eight samples (five of the six high-risk and three of the eight ovarian cancer) were hypersensitive. Lymphocytes from all eight patients were MMC resistant. Only one of the eight patients had a BRCA1 germ-line mutation and none had BRCA2 mutations, but FANCD2 was reduced in five of the eight. Ectopic expression of normal FANCD2 cDNA increased FANCD2 protein and induced MMC resistance in both hypersensitive lines tested. No FANCD2 coding region or promoter mutations were found, and there was no genomic loss or promoter methylation in any Fanconi anemia genes. Therefore, in high-risk women with no BRCA1 or BRCA2 mutations, tissue-restricted hypersensitivity to cross-linking agents is a frequent finding, and chromosomal breakage responses to MMC may be a sensitive screening strategy because cytogenetic instability identified in this way antedates the onset of carcinoma. Inherited mutations that result in tissue-specific FANCD2 gene suppression may represent a cause of familial ovarian cancer.

Biallelic inactivation of BRCA2 in Fanconi anemia.

Fanconi anemia (FA) is a rare autosomal recessive cancer susceptibility disorder characterized by cellular hypersensitivity to mitomycin C (MMC). Six FA genes have been cloned, but the gene or genes corresponding to FA subtypes B and D1 remain unidentified. Here we show that cell lines derived from FA-B and FA-D1 patients have biallelic mutations in BRCA2 and express truncated BRCA2 proteins. Functional complementation of FA-D1 fibroblasts with wild-type BRCA2 complementary DNA restores MMC resistance. Our results link the six cloned FA genes with BRCA1 and BRCA2 in a common pathway. Germ-line mutation of genes in this pathway may result in cancer risks similar to those observed in families with BRCA1 or BRCA2 mutations.

Overexpression of Metallothionein-II Sensitizes Rodent Cells to Apoptosis Induced by DNA Cross-linking Agent through Inhibition of NF-κB Activation

DNA cross-linking agents such as mitomycin C (MMC) and cisplatin are used as chemotherapeutic agents in cancer treatment. However, the molecular mechanism underlying their antitumor activity is not entirely clear. Critical steps in cytotoxicity toward cross-linking agents can involve DNA repair efficiency, inhibition of replication, cell-cycle checkpoints, regulation, and induction of apoptosis. The complexity of the mechanisms of the mammalian cell defense against cross-linking agents is reflected by the existence of many complementation groups identified in rodent cells that are specifically sensitive to MMC. We recently showed that increased induction of apoptosis contributes to the MMC sensitivity of the group represented by the V-H4 hamster mutant cell line. In this study, through the analyses of a substractive library, we discovered that sensitive V-H4 cells display a 40-fold increase of steady-state expression of metallothionein II (MT-II) mRNA compared with resistant parental V79 cells. Down-regulation of MT-II by antisense oligonucleotides partially restores MMC resistance in V-H4 cells, indicating that MT-II overexpression is directly involved in MMC hypersensitivity of these cells. MTs have been reported to regulate the activation of NF-kappaB, one of the key proteins that modulates the apoptotic response. Here we found that NF-kappaB activation by MMC is impaired in V-H4 cells and is partially restored following down-regulation of MT-II by antisense oligonucleotides. All these data suggest that the overexpression of MT-II in V-H4 cells impairs NF-kappaB activation by MMC, resulting in decreased cell survival and enhanced induction of apoptosis.

In vivo titration of mitomycin C action by four Escherichia coli genomic regions on multicopy plasmids.

Mitomycin C (MMC), a DNA-damaging agent, is a potent inducer of the bacterial SOS response; surprisingly, it has not been used to select resistant mutants from wild-type Escherichia coli. MMC resistance is caused by the presence of any of four distinct E. coli genes (mdfA, gyrl, rob, and sdiA) on high-copy-number vectors. mdfA encodes a membrane efflux pump whose overexpression results in broad-spectrum chemical resistance. The gyrI (also called sbmC) gene product inhibits DNA gyrase activity in vitro, while the rob protein appears to function in transcriptional activation of efflux pumps. SdiA is a transcriptional activator of ftsQAZ genes involved in cell division.

Phenotypic correction of Fanconi anemia group C knockout mice

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, congenital anomalies, and a predisposition to malignancy. FA cells demonstrate hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC). Mice with a targeted disruption of the FANCC gene (fancc -/- nullizygous mice) exhibit many of the characteristic features of FA and provide a valuable tool for testing novel therapeutic strategies. We have exploited the inherent hypersensitivity of fancc -/- hematopoietic cells to assay for phenotypic correction following transfer of the FANCC complementary DNA (cDNA) into bone marrow cells. Murine fancc -/- bone marrow cells were transduced with the use of retrovirus carrying the human fancc cDNA and injected into lethally irradiated recipients. Mitomycin C (MMC) dosing, known to induce pancytopenia, was used to challenge the transplanted animals. Phenotypic correction was determined by assessment of peripheral blood counts. Mice that received cells transduced with virus carrying the wild-type gene maintained normal blood counts following MMC administration. All nullizygous control animals receiving MMC exhibited pancytopenia shortly before death. Clonogenic assay and polymerase chain reaction analysis confirmed gene transfer of progenitor cells. These results indicate that selective pressure promotes in vivo enrichment of fancc-transduced hematopoietic stem/progenitor cells. In addition, MMC resistance coupled with detection of the transgene in secondary recipients suggests transduction and phenotypic correction of long-term repopulating stem cells. (Blood. 2000;95:700-704)

Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA.

The mitomycin C-resistance gene, mcrA, of Streptomyces lavendulae produces MCRA, a protein that protects this microorganism from its own antibiotic, the antitumor drug mitomycin C. Expression of the bacterial mcrA gene in mammalian Chinese hamster ovary cells causes profound resistance to mitomycin C and to its structurally related analog porfiromycin under aerobic conditions but produces little change in drug sensitivity under hypoxia. The mitomycins are prodrugs that are enzymatically reduced and activated intracellularly, producing cytotoxic semiquinone anion radical and hydroquinone reduction intermediates. In vitro, MCRA protects DNA from cross-linking by the hydroquinone reduction intermediate of these mitomycins by oxidizing the hydroquinone back to the parent molecule; thus, MCRA acts as a hydroquinone oxidase. These findings suggest potential therapeutic applications for MCRA in the treatment of cancer with the mitomycins and imply that intrinsic or selected mitomycin C resistance in mammalian cells may not be due solely to decreased bioactivation, as has been hypothesized previously, but instead could involve an MCRA-like mechanism.

The XRCC2 and XRCC3 repair genes are required for chromosome stability in mammalian cells

The irs1 and irs1SF hamster cell lines are mutated for the XRCC2 and XRCC3 genes, respectively. Both show heightened sensitivity to ionizing radiation and particularly to the DNA cross-linking chemical mitomycin C (MMC). Frequencies of spontaneous chromosomal aberration have previously been reported to be higher in these two cell lines than in parental, wild-type cell lines. Microcell-mediated chromosome transfer was used to introduce complementing or non-complementing human chromosomes into each cell line. irs1 cells received human chromosome 7 (which contains the human XRCC2 gene) or, as a control, human chromosome 4. irs1SF cells received human chromosome 14 (which contains the XRCC3 gene) or human chromosome 7. For each set of hybrid cell lines, clones carrying the complementing human chromosome recovered MMC resistance to near-wild-type levels, while control clones carrying noncomplementing chromosomes remained sensitive to MMC. Fluorescence in situ hybridization with a human-specific probe revealed that the human chromosome in complemented clones remained intact in almost all cells even after extended passage. However, the human chromosome in noncomplemented clones frequently underwent chromosome rearrangements including breaks, deletions, and translocations. Chromosome aberrations accumulated slowly in the noncomplemented clones over subsequent passages, with some particular deletions and unbalanced translocations persistently transmitted throughout individual subclones. Our results indicate that the XRCC2 and XRCC3 genes, which are now considered members of the RAD51 gene family, play essential roles in maintaining chromosome stability during cell division. This may reflect roles in DNA repair, possibly via homologous recombination.