The prevention of thymic lymphomas in transgenic mice by human O6-alkylguanine-DNA alkyltransferase.

Nitrosoureas form O6-alkylguanine-DNA adducts that are converted to G to A transitions, the mutation found in the activated ras oncogenes of nitrosourea-induced mouse lymphomas and rat mammary tumors. These adducts are removed by the DNA repair protein O6-alkylguanine-DNA alkyltransferase. Transgenic mice that express the human homolog of this protein in the thymus were found to be protected from developing thymic lymphomas after exposure to N-methyl-N-nitrosourea. Thus, transgenic expression of a single human DNA repair gene is sufficient to block chemical carcinogenesis. The transduction of DNA repair genes in vivo may unravel mechanisms of carcinogenesis and provide therapeutic protection from known carcinogens.

Rapid repair of O6-methylguanine-DNA adducts protects transgenic mice from N-methylnitrosourea-induced thymic lymphomas.

The methylating agent N-methylnitrosourea (MNU) is biased, surprisingly, in its carcinogenic potential toward the mouse thymus. Previous studies have shown that single doses of MNU administered to adult mice induced thymic lymphomas in over 80% of mice, while transgenic mice expressing high levels of the human O6-alkylguanine-DNA alkyltransferase gene in the thymus (MGMT-CD2 transgenics) were protected from developing MNU-induced lymphomas. The mechanism of this protection was examined in this report. In nontransgenic mice given a lymphomagenic dose of 80 mg/kg MNU, depletion of thymic alkyltransferase activity occurred within 3 h and remained undetectable for the subsequent 192 h; whereas in MGMT-CD2-transgenic mice, this dose of MNU did not deplete thymic alkyltransferase, and the lowest level of alkyltransferase was still 10-fold higher than the constitutive level of thymic alkyltransferase in nontransgenic mice. Likewise, the level of O6-methylguanine adducts detected in the thymus of nontransgenic mice was 96 pg/micrograms guanine 3 h after MNU compared to only 8 pg/micrograms guanine in transgenic mice. By 18 h, the level of O6-methylguanine in MGMT-CD2-transgenic mice was below 2 pg/micrograms guanine, compared to over 70 pg/micrograms guanine in nontransgenic mice. In contrast, no differences were noted in the liver between groups because the MGMT transgene is not expressed in the liver of this strain of mouse. Our data establish that rapid O6-methylguanine-DNA adduct repair due to enhanced levels of alkyltransferase in MGMT-CD2-transgenic mice blocks the initiation of MNU-induced carcinogenesis.

Overexpression of human O6-alkylguanine DNA alkyltransferase (AGT) prevents MNU induced lymphomas in heterozygous p53 deficient mice.

O6-alkylguanine DNA alkyltransferase (AGT) is a key mechanism in the prevention against MNU induced malignant transformation by removal of O6 methyl guanine (O6mG) adducts. We asked whether heterozygous p53 deficient mice (p53+/-) would be more susceptible to MNU induced lymphomas than wild type mice, and whether O6mG adducts were responsible for this susceptibility. To determine whether MGMT overexpression would be protective, p53+/- mice were bred to human MGMT transgenic mice (MGMT+) and treated with 50 mg/kg MNU. MNU increased the incidence of thymic lymphomas in non-transgenic p53+/- mice from 23% (n=13) to 68% (n=22) and decreased the mean latency from 433 to 106 days (P=0.01 compared to untreated mice). Wild type mice had an incidence of 30% (n=38) and a mean latency of 135 days after MNU. Overexpression of MGMT in the thymus of p53+/- mice significantly reduced the lymphoma incidence from 68 to 28% (n=17) and increased the latency from 106 to 167 days (P=0.003). Similarly, the lymphoma incidence in MGMT+/wild type mice decreased from 30 to 8% (n=12) and the latency increased to 297 days (P=0.2). Loss of the wild type allele was found in only 2/17 lymphomas occurring in p53+/- mice and there were no significant point mutations in exons 5-8 of p53. Furthermore, there was no loss of p53 function in these mice. These data demonstrate that unrepaired O6mG lesions act cooperatively with the reduced p53 dose and lead to lymphomagenesis in p53+/- mice, but AGT overexpression and rapid removal of O6mG adducts is protective.

Mice over-expressing human O6 alkylguanine-DNA alkyltransferase selectively reduce O6 methylguanine mediated carcinogenic mutations to threshold levels after N-methyl-N-nitrosourea.

While it is well known that MNU induces thymic lymphomas in the mouse, it remains unclear which pre-mutagenic lesions are responsible for lymphomagenic transformation. One lesion thought to play a critical role is O6methylguanine[O6mG]which initiates G: C to A:T transition mutations in K-ras and other oncogenes. O6alkylguanine-DNA alkyltransferase (AGT), encoded by the methylguanine methyltransferase gene [MGMT], removes the methyl group thereby preventing the mutation from occurring. When overexpressed in the thymus, MGMT protects mice from MNU-induced thymic lymphomas. To determine whether MGMT overexpression reduced G: C to A: T mutation frequency after MNU, Big Blue lacI and MGMT+/Big Blue mice were treated with MNU and analysed for mutations in the lacI and K-ras genes. The incidence of MNU-induced lymphomas was 84% in Big Blue lacI mice compared to 14% in MGMT+Big Blue lacI mice. Sixty-two per cent of the lymphomas had a GGT to GAT activating mutation in codon 12 of K-ras consistent with O6mG adduct-mediated point mutagenesis. LacI mutation frequency in thymus of MNU treated Big Blue mice was 45-fold above background whereas it was 11-fold above background in MNU treated MGMT+/Big Blue mice. Most lacI mutations were G:C to A:T transitions, implicating O6mG even in the MGMT+mice. No mutations were attributable to chromosomal aberrations or rearrangements. Thus, O6mG adducts account for the carcinogenic effect of MNU and MGMT overexpression is selectively able to reduce O6methylguanine adducts below a carcinogenic threshold. Other adducts are mutagenic but appear to contribute much less to malignant transformation or oncogene activation.

Potentiation of lymphomagenesis by methylnitrosourea in mice transgenic for LMO1 is blocked by O6-alkylguanine DNA-alkyltransferase.

We evaluated induction of lymphomas by the methylating carcinogen, N-methylnitrosourea [MNU], in transgenic mice expressing both LMO1 and the DNA repair gene, MGMT, in the thymus. The goal was to determine whether environmental mutagens shorten the latency or increase the incidence of LMO1 + lymphomas and whether mice transgenic for both LMO1 and MGMT, and thereby able to repair O6-methylguanine DNA adducts induced by MNU, would be protected. Mice heterozygous for LMO1 or MGMT were crossed and offspring treated with MNU at 6 weeks of age. MNU induced lymphoma incidence was highest in the LMO1 mice, 91% and lowest in the hMGMT + mice, 15%. MNU induced K-ras mutations in codon 12 in non-MGMT transgenics resulted in a shorter latency of tumors and accounting for half of the early lymphomas in LMO1 mice. The effect of MNU was abrogated in the LMO1/hMGMT transgenic mice, indicating the ability of MGMT expression to block the carcinogenic effect of MNU even in cancer prone mice. Thus, methylating agents potentiate lymphomagenesis of LMO1, in part through activation of K-ras and the MAPK pathway, a process which appear to synergize with LMO1 mediated transcription activation. O6-alkylguanine DNA-alkyltransferase mediated DNA repair effectively blocks chemical carcinogenesis in mice carrying the LMO1 oncogene.

Determinants of O6-alkylguanine-DNA alkyltransferase activity in human colon cancer.

The DNA repair protein O6-alkylguanine-DNA alkyltransferase (alkyltransferase) repairs cytotoxic DNA O6-alkylguanine adducts induced by the nitrosoureas, triazines, and tetrazines. In this study, we determined whether there was a relationship between alkyltransferase activity in colon cancer and that of adjacent normal mucosa, and whether there were demographic patient characteristics which correlated with alkyltransferase expression in either tissue. Alkyltransferase activity and expression of the alkyltransferase gene, MGMT, were measured in 49 paired primary colon cancer samples and adjacent normal appearing mucosa. Alkyltransferase activity was found in all samples. The mean activity was higher in the tumor than the mucosa (r = 0.374, P < 0. 01), although the low correlation coefficient suggested that multiple factors influence the alkyltransferase activity. MGMT mRNA could also be detected in all samples and was highly correlated with alkyltransferase activity (r = 0.64, P < 0.001). No correlation was found between alkyltransferase activity and age, or gender of the patient, or location of the tumor, although activity tended to be higher in patients with lower stage disease. Thus, alkyltransferase activity is present in most, if not all, colon cancer samples, suggesting that it could play an important role in chemotherapeutic resistance of human colon cancer. Patients with colon cancer would appear to be prime candidates for studies utilizing O6-benzylguanine to deplete alkyltransferase prior to therapy with a nitrosourea, triazine, or tetrazine.

Alkyltransferase transgenic mice: probes of chemical carcinogenesis.

Transgenic mice expressing DNA-repair genes are an instructive model with which to study the protective role of DNA-repair pathways in both spontaneous and chemical carcinogenesis. Of particular interest in chemical carcinogenesis is the DNA-repair protein O6-alkylguanine-DNA alkyltransferase (alkyltransferase) which repairs O6-alkylguanine-DNA adducts. Transgenic mice carrying expression constructs for the alkyltransferase gene--either the human MGMT cDNA or the bacterial ada gene--express increased levels of alkyltransferase and have increased capacity to remove O6-methylguanine-DNA adducts. Protection from the DNA damaging effects of N-nitroso compounds occurs specifically in the cells and tissues in which the alkyltransferase transgene is expressed. For instance, mice carrying the PEPCKada construct have increased alkyltransferase in the liver and more rapid removal of O6methylguanine-DNA adducts. The protective effect is noted in hepatocytes, which express PEPCK-linked genes, not in nonparenchymal cells of the liver, which do not. Other tissues that express the transgene in the various models include the thymus, spleen, testes, muscle, stomach and brain. Mice expressing the human alkyltransferase in the thymus have a reduced incidence of thymic lymphomas following exposure to methyl nitrosourea (MNU), evidence of a role for this DNA-repair protein in protection from carcinogenesis due to N-nitroso compounds. Protection has also been observed in the induction of hepatic tumors by N-nitroso-dimethylamine (NDMA). These models will be used to identify whether overexpression of a single DNA-repair gene can block the carcinogenic process of N-nitroso compounds in many different tissues.

Evolutionary divergence of the cytochrome b5 gene of Drosophila.

Cytochrome proteins perform a broad spectrum of biological functions ranging from oxidative metabolism to electron transport and are thus essential to all organisms. The b-type cytochrome proteins bind heme noncovalently, are expressed in many different forms and are localized to various cellular compartments. We report the characterization of the cytochrome b5 (Cyt-b) gene of Drosophila virilis and compare its structure to the Cyt-b gene of Drosophila melanogaster. As in D. melanogaster, the D. virilis gene is nuclear encoded and single copy. Although the intron/exon structures of these homologues differ, the Cyt-b proteins of D. melanogaster and D. virilis are approximately 75% identical and share the same size coding regions (1,242 nucleotides) and protein products (414 amino acids). The Drosophila Cyt-b proteins show sequence similarity to other b-type cytochromes, especially in the N-terminal heme-binding domain, and may be targeted to the mitochondrial membrane. The greatest levels of similarity are observed in areas of potential importance for protein structure and function. The exon sequences of the D. virilis Cyt-b gene differ by a total of 292 base changes. However, 62% of these changes are silent. The high degree of conservation between species separated by 60 million years of evolution in both the DNA and amino acid sequences suggests this nuclear cytochrome b5 locus encodes an essential product of the Drosophila system.

Transgenic expression of human MGMT protects against azoxymethane-induced aberrant crypt foci and G to A mutations in the K-ras oncogene of mouse colon.

Transgenic mice over-expressing MGMT, which codes for the human protein O6-alkylguanine-DNA alkyltransferase, are protected from methylating agent-induced thymic lymphomas. In this study we evaluated the ability of transgenic overexpression of MGMT in the colon to protect mice from the development of azoxymethane(AOM)-induced aberrant crypt foci (ACF) and mutations in K-ras. Colonic alkyltransferase in MGMT+ transgenic mice was > 5-fold higher than in nontransgenics: 10.5 +/- 1.1 vs 2.2 +/- 1.1 fmol/micrograms DNA, P = < 0.0001. Mice received 20 mg AOM/kg i.p. at 6 weeks or 15 mg AOM/kg at 6 and 7 weeks of age, and 8 wks later colons were examined for ACF. A significant protective effect of MGMT was seen in mice given single dose of 20 mg AOM/kg. The incidence of ACF/colon was lower in MGMT+ mice (2.0 +/- 1.2) than in nontransgenic mice (3.9 +/- 1.8, P = 0.02). G to A mutations in codon 12 of K-ras were detected by PCR-RFLP in ACF and in random samples of normal appearing mucosa. The incidence of ACF with mutant K-ras in MGMT transgenic mice (0.6 +/- 0.7/colon) was significantly reduced compared to nontransgenic mice (2.3 +/- 1.7/colon, P = 0.02). We propose that AOM induces at least two overlapping but not identical premalignant lesions (aberrant crypt foci and K-ras mutations) which can be prevented by over-expression of MGMT. Thus, MGMT may protect colonic mucosa from carcinogenesis involving methylating agents such as AOM.

Functional requirements for phenotypic correction of murine beta-thalassemia: implications for human gene therapy.

As initial human gene therapy trials for beta-thalassemia are contemplated, 2 critical questions important to trial design and planning have emerged. First, what proportion of genetically corrected hematopoietic stem cells (HSCs) will be needed to achieve a therapeutic benefit? Second, what level of expression of a transferred globin gene will be required to improve beta-thalassemic erythropoiesis? These questions were directly addressed by means of a murine model of severe beta-thalassemia. Generation of beta-thalassemic mice chimeric for a minority proportion of genetically normal HSCs demonstrated that normal HSC chimerism levels as low as 10% to 20% resulted in significant increases in hemoglobin (Hb) level and diminished extramedullary erythropoiesis. A large majority of the peripheral red cells in these mice were derived from the small minority of normal HSCs. In a separate set of independent experiments, beta-thalassemic mice were bred with transgenic mice that expressed different levels of human globins. Human gamma-globin messenger RNA (mRNA) expression at 7% of the level of total endogenous alpha-globin mRNA in thalassemic erythroid cells resulted in improved red cell morphology, a greater than 2-g/dL increase in Hb, and diminished reticulocytosis and extramedullary erythropoiesis. Furthermore, gamma-globin mRNA expression at 13% resulted in a 3-g/dL increase in Hb and nearly complete correction of red cell morphology and other indices of inefficient erythropoiesis. These data indicate that a significant therapeutic benefit could be achieved with expression of a transferred globin gene at about 15% of the level of total alpha-globin mRNA in patients with severe beta-thalassemia in whom 20% of erythroid precursors express the vector genome.

Enforced expression of the GATA-2 transcription factor blocks normal hematopoiesis.

The zinc finger transcription factor GATA-2 is highly expressed in immature hematopoietic cells and declines with blood cell maturation. To investigate its role in normal adult hematopoiesis, a bicistronic retroviral vector encoding GATA-2 and the green fluorescent protein (GFP) was used to maintain the high levels of GATA-2 that are normally present in primitive hematopoietic cells. Coexpression of the GFP marker facilitated identification and quantitation of vector-expressing cells. Bone marrow cells transduced with the GATA-2 vector expressed GFP as judged by flow cytometry and GATA-2 as assessed by immunoblot analysis. A 50% to 80% reduction in hematopoietic progenitor-derived colony formation was observed with GATA-2/GFP-transduced marrow, compared with marrow transduced with a GFP-containing vector lacking the GATA-2 cDNA. Culture of purified populations of GATA-2/GFP-expressing and nonexpressing cells confirmed a specific ablation of the colony-forming ability of GATA-2/GFP-expressing progenitor cells. Similarly, loss of spleen colony-forming ability was observed for GATA-2/GFP-expressing bone marrow cells. Despite enforced GATA-2 expression, marrow cells remained viable and were negative in assays to evaluate apoptosis. Although efficient transduction of primitive Sca-1(+) Lin- cells was observed with the GATA-2/GFP vector, GATA-2/GFP-expressing stem cells failed to substantially contribute to the multilineage hematopoietic reconstitution of transplanted mice. Additionally, mice transplanted with purified, GATA-2/GFP-expressing cells showed post-transplant cytopenias and decreased numbers of total and gene-modified bone marrow Sca-1(+) Lin- cells. Although Sca-1(+) Lin- bone marrow cells expressing the GATA-2/GFP vector were detected after transplantation, no appreciable expansion in their numbers occurred. In contrast, control GFP-expressing Sca-1(+) Lin- cells expanded at least 40-fold after transplantation. Thus, enforced expression of GATA-2 in pluripotent hematopoietic cells blocked both their amplification and differentiation. There appears to be a critical dose-dependent effect of GATA-2 on blood cell differentiation in that downregulation of GATA-2 expression is necessary for stem cells to contribute to hematopoiesis in vivo.

The immature thymocyte is protected from N-methylnitrosourea-induced lymphoma by the human MGMT-CD2 transgene.

N-methylnitrosourea (MNU) induces thymic lymphoma in a high proportion of susceptible C57BL/6xSJL (C57/SJL) mice. Expression of the human DNA repair gene, MGMT cDNA, which encodes O6-methylguanine-DNA-methyltransferase, in transgenic mice effectively prevents MNU-induced thymic lymphomas. In this study, we determined the phenotype of thymocytes expressing the transgene and defined whether the target cell population for MNU induced lymphomas were actually those that expressed the transgene. Transgene expression was characterized by in situ hybridization for MGMT mRNA and immunohistochemistry for the human alkyltransferase protein and was compared to the phenotype of the MNU induced lymphomas. The MGMT transgene was expressed uniformly in immature cortical thymocytes that were CD4+CD8+J11d+ and to a lesser extent in the medullary thymocyte. Lymphomas were induced by single [50 or 80 mg/kg] or multiple doses [30 mg/kg x 5] of MNU to evaluate the dose response of tumor induction and protection by the MGMT-CD2 transgene. Forty-seven of the 108 treated mice developed lymphomas: 38 of 58 nontransgenic and 9 of 50 MGMT+ mice. The T-cell phenotype of thymic lymphomas was established by immunohistochemistry and FACS analysis. Most of the lymphomas were J11d+ (98%), 70% of the tumors were CD4+CD8+, 21% were CD4-CD8+, 9% were CD4-CD8-, and none were CD4-CD8-. All lymphomas in MGMT+ transgenic mice were CD4+CD8+. Since the main phenotype of MNU induced lymphomas in these mice, CD4+CD8+J11d+, is also the cell phenotype which expresses the MGMT-CD2 transgene at high levels, it appears that MGMT-induced protection has occurred in the cell target for MNU induced transformation.

Retroviral-mediated transfer of the green fluorescent protein gene into murine hematopoietic cells facilitates scoring and selection of transduced progenitors in vitro and identification of genetically modified cells in vivo.

We have investigated the utility of the green fluorescent protein (GFP) to serve as a marker to assess retroviral gene transfer into hematopoietic cells and as a tool to identify and enrich for cells expressing high levels of the vector-encoded transcript. GFP, by virtue of a naturally occurring chromophore encoded in its primary sequence, displays autonomous fluorescence, thus eliminating the need for antibody or cytochemical staining to detect its expression. A bicistronic murine stem cell virus (MSCV)-based retroviral vector was constructed containing the GFP cDNA and a mutant, human dihydrofolate reductase gene. High-titer, ecotropic retroviral producer cells free of replication competent virus were generated and used to transduce murine bone marrow cells by cocultivation. Within 24 hours after completion of the transduction procedure, a high proportion (40% to 70%) of the marrow cells were intensely fluorescent compared to mock-transduced cells or cells transduced with a control retrovirus. Erythroid and myeloid hematopoietic colonies derived from GFP-transduced marrow were easily scored for retroviral gene transfer by direct in situ fluorescence microscopy. Clonogenic progenitors expressing increased levels of antifolate drug resistance could be enriched from the GFP-transduced marrow population by fluorescence activated cell sorting of cells expressing high levels of GFP. In vivo, splenic hematopoietic colonies and peripheral blood cells from animals transplanted with GFP-transduced marrow displayed intense fluorescence. These results show that GFP is an excellent marker for scoring and tracking gene-modified hematopoietic cells and for allowing rapid selection and enrichment of transduced cells expressing high levels of the transgene.