Pichia pastoris as a host for secretion of toxic saporin chimeras.

Most of the targeting moieties, such as antibody fragments or growth factor domains, used to construct targeted toxins for anticancer therapy derive from secretory proteins. These normally fold in the oxidative environment of the endoplasmic reticulum, and hence their folding in bacterial cells can be quite inefficient. For instance, only low amounts of properly folded antimetastatic chimera constituted by the amino-terminal fragment of human urokinase (ATF) fused to the plant ribosome-inactivating protein saporin could be recovered. ATF-saporin was instead secreted efficiently when expressed in eukaryotic cells protected from autointoxication with neutralizing anti-saporin antibodies. Pichia pastoris is a microbial eukaryotic host where these domains can fold into a transport-competent conformation and reach the extracellular medium. We show here that despite some host toxicity codon-usage optimization greatly increased the expression levels of active saporin but not those of an active-site mutant SAP-KQ in GS115 (his4) strain. The lack of any toxicity associated with expression of the latter confirmed that toxicity is due to saporin catalytic activity. Nevertheless, GS115 (his4) cells in flask culture secreted 3.5 mg/L of a histidine-tagged ATF-saporin chimera showing an IC(50) of 6 x 10(-11) M against U937 cells, thus demonstrating the suitability of this expression platform for secretion of toxic saporin-based chimeras.

Escherichia coli pfs transcription: regulation and proposed roles in autoinducer-2 synthesis and purine excretion.

Pfs expression is required for several metabolic pathways and limits the production of autoinducer-2, a molecule proposed to play a central role in interspecies quorum sensing. The present study reveals physiological conditions and promoter DNA elements that regulate Escherichia coli pfs transcription. Pfs transcription is shown to rely on both sigma 70 and sigma 38 (rpoS), and the latter is subject to induction that increases pfs expression. Transcription is maximal as the cells approach stationary phase, and this level can be increased by salt stress through induction of sigma 38-dependent expression. The pfs promoter is shown to contain both positive and negative elements, which can be used by both forms of RNA polymerase. The negative element is contained within the overlapping dgt promoter, which is involved in purine metabolism. Consideration of the physiological roles of sigma 38 and dgt leads to a model for how autoinducer production is controlled under changing physiological conditions.

Bacterial expression of biologically active recombinant musarmin 1 from bulbs of Muscari armeniacum L. and Miller.

Musarmins are type 1 ribosome-inactivating proteins with N-glycosidase activity on the 28 S rRNA that are present in bulbs of Muscari armeniacum L. and Miller at rather low concentrations. In the present work, a cDNA fragment coding for musarmin 1 was sub-cloned and expressed in Escherichia coli. The recombinant protein (rMU1) was synthesised as a polypeptide of 295 amino acids that was delivered to the periplasm and processed. Recombinant musarmin 1 present in the periplam has two forms: insoluble with a molecular mass of 29,423 and soluble with a molecular mass of 29,117 because of a small proteolytic shortening with respect to the insoluble one, presumably in the C-terminal. The yield of protein homogeneous by polyacrylamide gel electrophoresis was 23mgl-1 of bacterial culture. The recombinant musarmin 1 forms isolated from both the soluble and the insoluble (upon refolding) fractions retained full translational inhibitory and 28 S rRNA N-glycosidase activities as compared with the native protein. The recombinant protein displayed great stability towards trypsin, collagenase, rat plasma and rat liver protein extract, but was sensitive to the action of papain and proteinase K. The easy availability and full activity of the recombinant musarmin 1 makes it a good candidate for the preparation of immunotoxins for targeted therapy and for the construction of transgenic plants expressing it as antipathogenic agent.

Analysis of the in planta antiviral activity of elderberry ribosome-inactivating proteins.

Although the type-2 ribosome-inactivating proteins (SNA-I, SNA-V, SNLRP) from elderberry (Sambucus nigra L.) are all devoid of rRNA N-glycosylase activity towards plant ribosomes, some of them clearly show polynucleotide-adenosine glycosylase activity towards tobacco mosaic virus RNA. This particular substrate specificity was exploited to further unravel the mechanism underlying the in planta antiviral activity of ribosome-inactivating proteins. Transgenic tobacco (Nicotiana tabacum L. cv Samsun NN) plants expressing the elderberry ribosome-inactivating proteins were generated and challenged with tobacco mosaic virus in order to analyze their antiviral properties. Although some transgenic plants clearly showed antiviral activity, no clear correlation was observed between in planta antiviral activity of transgenic tobacco lines expressing the different ribosome-inactivating proteins and the in vitro polynucleotide-adenosine glycosylase activity of the respective proteins towards tobacco mosaic virus genomic RNA. However, our results suggest that the in planta antiviral activity of some ribosome-inactivating proteins may rely on a direct mechanism on the virus. In addition, it is evident that the working mechanism proposed for pokeweed antiviral protein cannot be extrapolated to elderberry ribosome-inactivating proteins because the expression of SNA-V is not accompanied by induction of pathogenesis-related proteins.

Purification and characterization of Moschatin, a novel type I ribosome-inactivating protein from the mature seeds of pumpkin (Cucurbita moschata), and preparation of its immunotoxin against human melanoma cells.

A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin (Cucurbita moschata) has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of approximately 29 kD. It is a rRNA N-glycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a IC50 of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M21 with a IC50 of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.

Normal somatic hypermutation of Ig genes in the absence of 8-hydroxyguanine-DNA glycosylase.

The hypermutation cascade in Ig V genes can be initiated by deamination of cytosine in DNA to uracil by activation-induced cytosine deaminase and its removal by uracil-DNA glycosylase. To determine whether damage to guanine also contributes to hypermutation, we examined the glycosylase that removes oxidized guanine from DNA, 8-hydroxyguanine-DNA glycosylase (OGG1). OGG1 has been reported to be overexpressed in human B cells from germinal centers, where mutation occurs, and could be involved in initiating Ab diversity by removing modified guanines. In this study, mice deficient in Ogg1 were immunized, and V genes from the H and kappa L chain loci were sequenced. Both the frequency of mutation and the spectra of nucleotide substitutions were similar in ogg1(-/-) and Ogg1(+/+) clones. More importantly, there was no significant increase in G:C to T:A transversions in the ogg1(-/-) clones, which would be expected if 8-hydroxyguanine remained in the DNA. Furthermore, Ogg1 was not up-regulated in murine B cells from germinal centers. These findings show that hypermutation is unaffected in the absence of Ogg1 activity and indicate that 8-hydroxyguanine lesions most likely do not cause V gene mutations.

DNA damage and repair system in spinal cord ischemia.

BACKGROUND AND PURPOSE: Spinal cord ischemia-reperfusion injury may be initiated by a number of mediators, including reactive oxygen species. Recent studies have shown that human MutY homologue (hMYH), human 8-oxo-7,8-dihydrodeoxyguanine (8-oxoG) glycosylase (hOGG1), and human MutS homologue 2 (hMSH2) are important DNA mismatch repair genes. We hypothesized that ischemia-reperfusion injury in spinal cord causes DNA damage manifested by 8-oxoG production and activates the DNA repair system involving hMYH, hOGG1, and hMSH2. METHODS: Spinal cords of rabbits were removed at 1, 3, 6, 24, and 48 hours after 30 minutes of infrarenal aortic occlusion. DNA damage was determined with 8-oxoG staining. The expression and localization of DNA repair enzymes, such as hMYH, hOGG1, and hMSH2, were studied with Western blot analysis and immunohistochemical staining. The level of apoptosis was determined with TUNEL study. Activation of caspase-3, an enzyme induced by cellular injury that leads to apoptosis by degrading cellular structural proteins, was also studied. RESULTS: DNA damage monitored with 8-oxoG level was significantly present from 1 hour to 6 hours after reperfusion in gray matter neurons of ischemic spinal cord. The levels of hMYH, hOGG1, and hMSH2 were markedly increased in gray matter neurons at 6 hours after reperfusion. Caspase-3 was also induced at 6 hours to 24 hours after reperfusion in ischemic spinal cord. However, the peak level of TUNEL reactivity was found at 48 hours after reperfusion in spinal cord neurons. CONCLUSION: This study has shown, for the first time, the rapid expression of DNA damage-repair processes associated with spinal cord ischemia and subsequent reperfusion.

Imbalancing the DNA base excision repair pathway in the mitochondria; targeting and overexpressing N-methylpurine DNA glycosylase in mitochondria leads to enhanced cell killing.

The DNA base excision repair (BER) pathway is responsible for the repair of alkylation and oxidative DNA damage. The short-patch BER pathway, beginning with the simple glycosylase N-methylpurine DNA glycosylase (MPG), is responsible for the removal of damaged bases such as 3-methyladenine and 1,N(6)-ethenoadenine from the DNA after alkylation or oxidative DNA damage. The resulting apurinic site is further processed by the other members in the pathway, resulting in the insertion of the correct nucleotide. If apurinic sites accumulate, they are mutagenic and cytotoxic to the cell. To evaluate its efficacy in sensitizing breast cancer cells to chemotherapy, MPG has been overexpressed in the breast cancer cell line, MDA-MB231. With MPG overexpression, an increase in DNA damage and increased cytotoxicity to methyl methanesulfonate as well as increased apoptosis levels was observed in these cells. Because mitochondrial DNA has been shown to be more sensitive to DNA damage than nuclear DNA, a construct containing mitochondrial-targeted MPG using the human manganese superoxide dismutase mitochondrial-targeting sequence was made. Overexpression of the mitochondrially targeted MPG dramatically increased the breast cancer cells' sensitivity to methyl methanesulfonate. In conclusion, we believe that the increase in sensitivity to DNA damage by overexpression of nuclear MPG is because of an imbalance in the BER pathway, and an even greater increase in cell sensitivity is observed when mitochondrial DNA is targeted.

8-Hydroxyguanosine repair is defective in some microsatellite stable colorectal cancer cells.

Mutator phenotypes are involved in the carcinogenesis of some cancers, e.g., defects in mismatch repair produce a mutator phenotype that drives carcinogenesis and causes microsatellite instability in hereditary nonpolyposis colon cancers and some sporadic colorectal cancers (CRC). Less understood, however, is the potential role of mutator phenotypes in microsatellite stable (MSS) CRC carcinogenesis. A novel transversion mutator phenotype was reported recently in an MSS CRC cell line. We hypothesized that 8-hydroxyguanosine could be involved and found elevations in 5 of 15 (33%) MSS CRC cell lines analyzed. Repair of an adenine*8-hydroxyguanosine mispair was functionally defective in the same five cell lines. The human MutY homologue transcript and MutY homologue protein levels were also decreased. These findings may reflect a MSS mutator phenotype contributing to the development of CRC.

dUTPase and uracil-DNA glycosylase are central modulators of antifolate toxicity in Saccharomyces cerevisiae.

The thymidylate synthase reaction remains an important target for widely used anticancer agents; however, the clinical utility of these drugs is limited by the occurrence of cellular resistance. Despite the considerable amount of information available regarding mechanisms of drug action, the relative significance of downstream events that result in lethality remains unclear. In this study, we have developed a model system using the budding yeast Saccharomyces cerevisiae to dissect the influence of dUMP misincorporation into DNA as a contributing mechanism of cytotoxicity induced by antifolate agents. The activities of dUTPase and uracil-DNA glycosylase, key enzymes in uracil-DNA metabolism, were diminished or augmented, and the manipulated strains were analyzed for biochemical endpoints of toxicity. Cells overexpressing dUTPase were protected from cytotoxicity by their ability to prevent dUTP pool expansion and were able to recover from an early S-phase checkpoint arrest. In contrast, depletion of dUTPase activity leads to the accumulation of dUTP pools and enhanced sensitivity to antifolates. These cells were also arrested in early S-phase and were unable to complete DNA replication after drug withdrawal, resulting in lethality. Inactivation of uracil base excision repair induced partial resistance to early cytotoxicity (within 10 h); however, lethality ultimately resulted at later time points (12-24 h), presumably because of the detrimental effects of stable uracil misincorporation. Although these cells were able to complete replication with uracil-substituted DNA, they arrested at the G(2)-M phase. This finding may represent a novel mechanism by which the G(2)-M checkpoint is signaled by the presence of uracil-substituted DNA. Together these data provide both genetic and biochemical evidence demonstrating that lethality from antifolates in yeast is primarily dependent on uracil misincorporation into DNA, and that uracil-independent mechanisms associated with dTTP depletion play a minor role. Our findings indicate that the relative expression levels of both dUTPase and uracil-DNA glycosylase can have great influence over the efficacy of thymidylate synthase-directed chemotherapy, thereby enhancing the candidacy of these proteins as prognostic markers and alternative targets for therapeutic development.

A single nucleotide polymorphism at the splice donor site of the human MYH base excision repair genes results in reduced translation efficiency of its transcripts.

BACKGROUND: Adenine paired with 8-hydroxyguanine, a major oxidatively damaged DNA lesion, is excised by mutY homologue (MYH) base excision repair protein in human cells. Since genetic polymorphisms of DNA repair genes associated with the activities and the expression levels of their products may modulate cancer susceptibility of individuals, we investigated the effect of a single nucleotide polymorphism (SNP) in the MYH gene on the difference in the expression levels of its products. RESULTS: An aberrant size of the beta type nuclear form transcript was detected in a lung cancer cell line, VMRC-LCD, by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The transcript contained the intron 1 sequence, and it was due to alternative splicing resulting from IVS1+5G/C SNP. The presence of the upstream open reading frame (ORF) on the 5'-side of the native ORF in the beta type transcript from the IVS1+5C allele could reduce the translation efficiency of the transcript into the nuclear form protein. Thus, expression vectors bearing the 5'-untranslated region sequence of either the IVS1+5G or 5C allele were constructed. In vitro translation analysis, as well as Western blot and quantitative RT-PCR analyses of the H1299 lung cancer cell line transfected with these vectors, revealed that the translation efficiency of the IVS1+5C transcript into MYH protein was much lower (approximately 30) than that of the IVS1+5G transcript. CONCLUSIONS: The SNP at the splice donor site of the MYH gene resulted in reduced translation efficiency of its transcripts. This is the fourth case of single nucleotide variations that cause alterations in translation initiation sites and translation efficiencies in human cells.

Altered expression of the DNA repair protein, N-methylpurine-DNA glycosylase (MPG) in human gonads.

BACKGROUND: The multifunctional mammalian MPG is responsible for a damaged DNA base in the nucleus. The DNA repair enzyme is transported from the cytoplasm to nucleus to repair the DNA base when it is damaged. If the enzyme does not work properly, the damaged DNA may lead to carcinogenesis, cell death, aging or infertility. MATERIALS AND METHODS: This study was performed to determine mRNA expression and intracellular localization of the DNA repair protein, N-methylpurine-DNA glycosylase (MPG), in human ovary and testicular tissues, particularly in epithelial ovarian tumor and spermatogenic (maturation) arrest infertile patients, by RT-PCR and immunohistochemical staining using human MPG monoclonal antibody. RESULTS: MPG mRNA expression in epithelial ovarian tumor and spermatogenic arrest testis tissues was slightly higher than in normal ovarian and testicular tissues, respectively. The present study demonstrated new and unexpected patterns of cellular and subcellular localization of this enzyme. In a normal ovary, immunostaining for MPG was observed in the nucleus of oocyte, granulosa and stromal cells. MPG was stained mostly in the nucleus and faintly-stained in the cytoplasm of normal coelomic epithelium as well as in benign epithelial ovarian tumors. However, the MPG expression of the nucleus in malignant epithelial tumors, both serous and mucinous type, disappeared. The spermatocyte and Leydig cells in normal testis were immunostained only in the cytoplasm. The spermatocyte and Leydig cells in spermatogenic arrest testis tissues showed up both in the nucleus and cytoplasm. The subcellular localization of MPG in the tissues tested was heterogeneous, while the altered MPG expression was found in ovarian tumor and spermatogenic arrest testis. CONCLUSION: These results suggest MPG's role in human gonadal tissues and raise the possibility that the altered mRNA level and intracellular localization could be associated with ovarian tumorigenesis and male infertility.

Formation of 8-hydroxydeoxyguanosine and cell-cycle arrest in the rat liver via generation of oxidative stress by phenobarbital: association with expression profiles of p21(WAF1/Cip1), cyclin D1 and Ogg1.

To evaluate the risk of exposure to so-called non-genotoxic chemicals and elucidate mechanisms underlying their promoting activity on rat liver carcinogenesis the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), cytochrome P-450 (P-450) and hydroxyl radicals induction, DNA repair and alteration to cellular proliferation and apoptosis in the rat liver were investigated during 2 weeks of phenobarbital (PB) administration at a dose of 0.05%. Significant increase of hydroxyl radical levels by day 4 of PB exposure accompanied the accumulation of 8-OHdG in the nucleus and P-450 isoenzymes CYP2B1/2 and CYP3A2 in the cytoplasm of hepatocytes. Conspicuous elevation of 8-OHdG and apoptosis in the liver tissue were associated with reduction of the proliferating cell nuclear antigen (PCNA) index after 8 days of PB application. Thereafter, 8-OHdG levels decreased with an increase in mRNA expression for the 8-OHdG repair enzyme, DNA glycosylase 1 (Ogg1). Analysis with LightCycler quantitative 2-step RT-PCR demonstrated induction of cyclin D1 (CD1) and p21(WAF1/Cip1) mRNA expression on days 4 and 6, respectively, preceding marked elevation of PCNA and apoptotic indices. These results suggest that similar to genotoxic, non-genotoxic chemicals might induce reversible alteration to nuclear 8-OHdG in the rat liver after several days of continuous application; however, by a different mechanism. Increased 8-OHdG formation is caused by developing oxidative stress or apoptotic degradation of DNA and coordinated with enhanced expression of CD1 mRNA and cell proliferation, subsequent increase of p21(WAF1/Cip1) mRNA expression, cell-cycle arrest and apoptosis, while activation of 8-OHdG repair mechanisms contributes to protection of tissue against reactive oxygen species-induced cell death.

Human 8-oxoguanine DNA glycosylase 1 mRNA expression as an oxidative stress exposure biomarker of cooking oil fumes.

Epidemiological studies have indicated that the exposure to carcinogenic components formed during the cooking of food might be associated with lung cancer risk of Chinese women. Previous studies have confirmed that cooking oil fumes from frying fish (COF) contained relatively high amount of benzo[a]pyrene, 2-methyl-3,8-dimethylimidazo[4,5-f] qunoxaline, benzene, and 1,3-butadiene, reported in fumes from heated soybean oil. Thus, we consider that oxidative stress induced by COF may play a role in lung cancer development among Chinese women. To verify whether the oxidative DNA damage was induced by COF, high-performance liquid chromatography (HPLC) analysis data showed that the levels of 8-hydroxydeoxyguanine (8-OH dG) were increased in a dose-dependent manner when calf thymus DNA reacted with various concentrations of COF. Since human 8-oxoguanine DNA glycosylase 1 (hOGG1) was a repair enzyme for removing 8- OH dG from damaged DNA, we hypothesized that hOGG1 mRNA may be used to assess the risk of oxidative damage induced by the exposure of COF. The results from reverse-transcription polymerase chain reaction showed that the hOGG1 mRNA expression was induced by hydrogen peroxide (H2O2) and COF in human lung adenocarcinoma CL-3 cells. To elucidate whether hOGG1 mRNA expression was an exposure biomarker of COF, a cross-sectional study of 238 subjects including 94 professional cooks, 43 housewives, and 101 COF-nonexposed control subjects was conducted. The hOGG1 mRNA expression frequencies of COF-exposed cooks (27 of 94, 28.7%) and housewives (6 of 43, 14%) were significantly higher than those of control subjects (4 of 101, 4%). After adjusting for age, sex, and smoking and drinking status, the odds risks (ORs) of housewives versus control and cooks versus control were 3.94 (95% confidence interval [CI] = 0.95-16.62) and 10.12 (95% CI = 2.83-36.15), respectively. These results indicated that hOGG1 may be adequate to act as an exposure biomarker to assess the oxidative DNA damage induced by COF. This also suggests that oxidative stress induced by COF may play a role in lung cancer development among Chinese women.

Ribosome-inactivating and adenine polynucleotide glycosylase activities in Mirabilis jalapa L. tissues.

Several tissues of Mirabilis jalapa L. (Nyctaginaceae) were assayed for inhibition of translation by a rabbit reticulocyte lysate (as a signal of ribosome-inactivating activity) and for adenine DNA glycosylase activity, activities that are both due to the presence of a class of enzymes called ribosome-inactivating proteins (RIPs), currently classified as rRNA N-glycosylases (EC ). These activities were highest in seed; intermediate in flower bud, immature seed, sepal + gynoecium, leaf, and root; and very low in all other tissues. By cation-exchange chromatography, four protein peaks with inhibitory activity on cell-free translation were identified in extracts from seeds, and two proteins were isolated from peaks 1 and 4, all of which have the properties of single-chain type 1 RIP. One is Mirabilis antiviral protein (MAP), so far purified only from roots. The second is a new protein that we propose to call MAP-4. The distribution of MAP and MAP-4 in several tissues was determined with a novel experimental approach based on liquid chromatography/mass spectrometry. The direct enzymatic activity of MAP on several substrates is described here for the first time. MAP depurinated not only rRNA in intact ribosomes, thus inhibiting protein synthesis, but also other polynucleotides such as poly(A), DNA, and tobacco mosaic virus RNA. Autologous DNA was depurinated more extensively than other polynucleotides. Therefore, the enzymatic activity of this protein may be better described as adenine polynucleotide glycosylase activity rather than rRNA N-glycosylase activity. Finally, MAP does not cross-react immunologically with other commonly utilized RIPs.

Gene amplification and expression of the DNA repair enzyme, N-methylpurine-DNA glycosylase (MPG) in HPV-infected cervical neoplasias.

BACKGROUND: Lethal and mutagenic damages of DNA is caused by a variety of agents including viruses. It is known that HPV is one of the major causes of cervical carcinogenesis and that cells eliminate DNA lesions with DNA repair enzymes. However, the role of N-methylpurine-DNA glycosylase (MPG) is not known in the development of cervical cancer. MATERIALS AND METHODS: Multiplex polymerase chain reaction (PCR) was used for the detection and typing of HPV in the biopsy. Gene amplification of MPG was measured by a PCR-based assay. The mRNA levels of MPG were determined by reverse transcription-PCR using hypoxanthine-guanine phosphoribosyl transferase as the reference gene. An immunohistochemical technique was used to examine the distribution of MPG in the tissues. RESULTS: Of 68 Korean cervical neoplasia patients, 86.8% showed HPV infection. High-risk HPV 16/18 were the most prevalent but positive only in 47.3% of the invasive cancer patients. Gene amplification of MPG was significantly increased in high-risk HPV-infected tissues as compared to low-risk HPV-infected and normal tissues (p < 0.05). The mRNA levels of MPG were higher in HPV-infected invasive carcinoma than normal cervical tissues. Immunohistochemical staining revealed that the intracellular expression and distribution (localization) of MPG altered in the cervical neoplasia. Interestingly, MPG expression in CIN III and invasive carcinoma (IC) was much higher than normal and CIN I. Granular positivity of MPG was notable in the perinuclear regions of the cytoplasm in HPV-infected invasive cancer. CONCLUSION: This is the first report on MPG expression in cervical neoplasia. Our results indicate that the gene amplification and expression of MPG were increased in high-risk HPV-infected cervical neoplasias and the intracellular distribution of MPG protein was altered, suggesting a role of MPG in carcinogenesis.

Oxidative DNA damage and 8-hydroxy-2-deoxyguanosine DNA glycosylase/apurinic lyase in human breast cancer.

To test the hypothesis that oxidative stress is involved in breast cancer, we compared the levels of 8-hydroxy-2-deoxyguanosine (8-oxo-dG), an oxidized DNA base common in cells undergoing oxidative stress, in normal breast tissues from women with or without breast cancer. We found that breast cancer patients (N = 76) had a significantly higher level of 8-oxo-dG than control subjects (N = 49). The mean ( +/- SD) values of 8-oxo-dG/10(5) dG, as measured by high-performance liquid chromatography electrochemical detection, were 10.7 +/- 15.5 and 6.3 +/- 6.8 for cases and controls, respectively (P = 0.035). This difference also was found by immunohistochemistry with double-fluorescence labeling and laser-scanning cytometry. The average ratios (x10(6)) of the signal intensity of antibody staining to that of DNA content were 3.9 +/- 7.2 and 1.1 +/- 1.4 for cases (N = 57) and controls (N = 34), respectively (P = 0.008). There was no correlation between the ages of the study subjects and the levels of 8-oxo-dG. Cases also had a significantly higher level of 8-hydroxy-2-deoxyguanosine DNA glycosylase/apurinic lyase (hOGG1) protein expression in normal breast tissues than controls (P = 0.008). There was no significant correlation between hOGG1 expression and 8-oxo-dG. Polymorphism of the hOGG1 gene was very rare in this study population. The previously reported exon 1 polymorphism and two novel mutations of the hOGG1 gene were found in three of 168 cases and two of 55 controls. In conclusion, normal breast tissues from cancer patients had a significantly higher level of oxidative DNA damage. The elevated level of 8-oxo-dG in cancer patients was not related to age or to deficiency of the hOGG1 repair gene.

Retrovirus-mediated expression of the base excision repair proteins, formamidopyrimidine DNA glycosylase or human oxoguanine DNA glycosylase, protects hematopoietic cells from N,N',N"-triethylenethiophosphoramide (thioTEPA)-induced toxicity in vitro and in vivo.

Modulation of DNA damage repair activity could lead to new approaches to reduce cytotoxic side effects of chemotherapy. N,N',N"-Triethylenethiophosphoramide (thioTEPA) induces the formation of amino-ethyl adducts of guanine, resulting in imidazole ring opening [formamidopyrimidine (Fapy)] and is associated with significant myelosuppression in dose-intensive therapies. In Escherichia coli, Fapy lesions are repaired by the Fapy-DNA glycosylase (Fpg) protein. We hypothesized that the expression of the Fpg could increase resistance of hematopoietic cells to thioTEPA-induced cytotoxicity. Expression of Fpg in bone marrow (BM) cells via a retrovirus vector was associated with demonstrable 8-oxodeoxyguanosine DNA glycosylase activity. BM cells were infected with a recombinant retrovirus, SF91, containing the Fpg gene and expressing the enhanced green fluorescence protein (EGFP) via an internal ribosomal entry site element. Control mice received BM transduced with the backbone containing IRES-EGFP alone. Fpg-transduced and GFP+ BM hematopoietic cells were resistant in vitro to thioTEPA at multiple concentrations. Mice transplanted with transduced cells were treated with four doses of thioTEPA (10 mg/kg) given over 7 weeks. Despite low transduction efficiency, peripheral blood leukocytes, hemoglobin, and platelet counts of thioTEPA-treated Fpg mice were significantly higher than treated control mice (P < 0.05). In addition, after treatment, the BM, spleen, and thymic cellularity as well as the number of GFP+ progenitor cells in the BM of treated mice were significantly higher than those of control group. Selection of Fpg-transduced cells in vivo was demonstrated by an increase in the mean fluorescence intensity of peripheral mononuclear cells of Fpg mice compared with pretreatment value. In addition, a significant increase in the EGFP-bright cells was demonstrated, suggesting preferential survival of high-expressing hematopoietic cells. Similar results were demonstrated in vitro with primary BM expressing the human functional counterpart of Fpg, OGG1. These results show that expression of the Fpg or hOGG1 protein protects hematopoietic cells from thioTEPA-induced DNA damage and suggest that a high level of expression of these repair proteins is required to establish resistance to this drug. Expression of Fpg and/or OGG1 may provide an novel approach to preventing thioTEPA-induced toxicity of primary hematopoietic cells.

Changes in DNA 8-hydroxyguanine levels, 8-hydroxyguanine repair activity, and hOGG1 and hMTH1 mRNA expression in human lung alveolar epithelial cells induced by crocidolite asbestos.

We examined 8-hydroxyguanine (8-OH-Gua) formation and 8-OH-Gua repair enzyme activity in pulmonary type-II-like epithelial cells to determine whether oxidative stress induced by asbestos plays a role in its carcinogenic mechanism. A549 cells were incubated with crocidolite asbestos at concentrations of 0, 10, 50 and 100 microg/ml over 27 h. We then evaluated 8-OH-Gua formation, 8-OH-Gua repair enzyme activity and gene expression of 8-oxoguanine-DNA glycosylase 1 (hOGG1) and human MUtT homologue (hMTH1). This was done using a high-performance liquid chromatography system equipped with an electrochemical detector, endonuclease nicking assay and reverse transcription polymerase chain reaction, respectively. Crocidolite induced the formation of 8-OH-Gua in DNA at concentrations of 50 and 100 microg/ml. 8-OH-Gua levels increased at 9 h and had declined to near baseline at 27 h, whereas 8-OH-Gua repair enzyme activity peaked at 18 h post-crocidolite exposure. hOGG1 and hMTH1 mRNA levels were also increased by crocidolite exposure. These data suggest that crocidolite asbestos is associated with epithelial cell injury in the process of carcinogenesis through oxidative stress.

RAG1 and RAG2 expression by B cell subsets from human tonsil and peripheral blood.

It has been suggested that B cells acquire the capacity for secondary V(D)J recombination during germinal center (GC) reactions. The nature of these B cells remains controversial. Subsets of tonsil and blood B cells and also individual B cells were examined for the expression of recombination-activating gene (RAG) mRNA. Semiquantitative analysis indicated that RAG1 mRNA was present in all tonsil B cell subsets, with the largest amount found in naive B cells. RAG2 mRNA was only found in tonsil naive B cells, centrocytes, and to a lesser extent in centroblasts. Neither RAG1 nor RAG2 mRNA was routinely found in normal peripheral blood B cells. In individual tonsil B cells, RAG1 and RAG2 mRNAs were found in 18% of naive B cells, 22% of GC founder cells, 0% of centroblasts, 13% of centrocytes, and 9% of memory B cells. Individual naive tonsil B cells containing both RAG1 and RAG2 mRNA were activated (CD69(+)). In normal peripheral blood approximately 5% of B cells expressed both RAG1 and RAG2. These cells were uniformly postswitch memory B cells as documented by the coexpression of IgG mRNA. These results indicate that coordinate RAG expression is not found in normal peripheral naive B cells but is up-regulated in naive B cells which are activated in the tonsil. With the exception of centroblasts, RAG1 and RAG2 expression can be found in all components of the GC, including postswitch memory B cells, some of which may circulate in the blood of normal subjects.